Date: 

Time: 

Location: 

PR I11l11111l1 II 1111 

MEMORANDUM OFMEETING L i 

January 29,2001 

1:OO - 3:OO PM 

12th floor Conference Room 

11 10 Vermont Ave., NW 

Participants: 

Visitors 

Robert Hill 

Charles Morin 

Bertjan Ziere 

Paul Leuflcens 

pthleen Mc Aveney Jones 

Rudaina Alrefd 

Rebecca Edelstein 

Susan Carberry 

George Pauli 

inda Kahl L aulette Gaynor 

'chael DiNovi 

4 SueAnderson 

Me1 Dong 

+ AntoniaMattia 

Hill Research Associates, Inc. 

Morin & Krasny, LLP 

Pharming B.V. 

Pharming B.V. 

HFS-206 

HFS-206 

HFS-246 

HFS-246 

HFS-205 

HFS-206 

HFS-2 15 

HFS-246 

HFS-83 1 

HFS-225 

HFS-207 

Subject: Product Under Development 

The visitors requested the meeting to consult with FDA regarding Pharming's recombinant human - - laetoferrin (rJ3L.F) derived &om transgenic-cows. The -visitors-had met-with-FDA to disams this 

background information. A copy of that material is attached to this memorandum. 

k product on two earlier occasions. Prior to the meeting, the visitors provided a package of 

. . __.. ~ __ 
7 

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SJL 

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Page 2 - January 29,2001, Meeting 

administration, as well as an ingredient for non-infant formula food uses. 

The visitors discussed a proposed 90-day toxicitv study with add-ons intended to address 

reproductive and developmental issues. This prompted discussion fiom FDA about appropriate 

safety testing for Pharming’s product. Dr. Sue Anderson emphasized the importance of using the 

food-grade rather than pharmaceutical-grade product for food-related safety studies. Dr. Linda 

Kahl discussed safety assessment studies comparing rHLF with HLF found in breast milk. Dr. 

Kahl informed the visitors that the “Red Book” provided guidance, not requirements for safety 

testing. She stated that the basic approach to assessing the saf“ of rHLF would be the same for 

evaluating the safety‘of any protein. She recommended determining the real safkty issues 

presented by this substance, and performing studies relevant to these issues. 

Dr. Toni Mattia informed the visitors that she could not chart a path for them because the safety 

of a food ingredient was dependent upon many things. Dr. Mattia recommended examining the 

differences between rHLF and HLF derived fiom breast milk, and evaluating animal and human 

studies on digestion, nutrition and allergenicity which were currently available. She discussed 

standard metabolic studies and endpoints. Dr. Mattia stressed the importance of the diet used in 

studies because of its impact on results. She also discussed the grade of the material used for 

safety studies as compared with the grade of material intended for use in the food product, and 

recommended characterizing mmmaterial for toxicology testing. Dr. Sue Carberry 

emphasized the importance of providing exposure levels. Drs. Pauli and Mattia questioned the 

purpose of the reproduction add-on to the 90-day study. Dr. Mattia stated that 

P dY. 

Dr. Sue Anderson stressed the importance of considering infant physiology and development 

when assessing the safety of rHLF for use in idant formula, and inquired about the feasibility of 

the digestibility of rHLF versus breast milk by infants be considered. Dr. Anderson also informed 

the visitors of the requirements for infah formulas citing the Want Formula Act of 1980 and 

subsequent amendments in 1986, and the Proposed Rule for Current Good Manufacturing 

Practice, Quality Control Procedures, Quality Factors, Notification Requirements, and Records 

and Reports, for the Production of Infant Formula (61 FR 36153). 

s. Drs. Kahl and Anderson recommended that 

The potential allergenicity of Pharming’s product was discussed, due to possible contamhation 

with bovine lactoferrin (BLF), a known food allergen present in cow’s milk. Dr. Kahl also 

discussed issuesr~ated..ta.non-i~~~-fo~ul.a.uses..of rI&-Fy B-ecause _H[T-F is i-unologically . . as . .., 

well as biologically active, it was recommended that Pharming address whether any of these 

effects might be adverse, rather than beneficial, for &me people. 

Dr. Kahl presented an overview of the GRAS notification process, comparing it with the food 

additive petition process. Drs. Kahl and Pauli emphasized that for a food ingredient to be 

considered GRAS, there must be safety data that is generally available plys consensus among 

qualified experts. Dr. Kahl made a recommendation that if the visitors were considering convening 

1 ... ....._ - .. . .. . . . ._ . . __ _. .. __ . -- . _. - . 
a GRAS panel, that the panel should consist of scientists and/or physicians with the appropriate 

expertise, and should be consulted early in the process in order to provide input as to the 

appropriate safety testing. Dr. Kahl emphasized that there was no clear path to proving the dety 

of a food ingredient. 

Kathleen McAveney Jones, Ph.D. 

CC: HFA-224 HFS-200 HFS-207 HFS-215 HFS-225 HFS-246 HFS-831 

FDA participants 

WD: KMcAveneyJones: HFS-206:3/2/01 :Pharming.wpd- ' 

Edited and initialed: LSKahl: HFS-206:3/2/01 

F/T: KMcAveneyJones: HFS-206:3/6/01 

. . __ 

.. . ___ 

... . -.. 

.. .... 

............... 

- . -. ...... - ... .. ... 
5' 

'I 

I 

MEMORANDUM OF CONFERENCE 

Date: July 20,1998 

Time: 1:00 - 230 pm. 

Location: 7th Floor Conference Room 

Participants: 

Vermont Ave. Bldg.,lllO Vermont Ave., NW 

YhitQrx 

Joost van Bree Pharming Group N.V. 

Juha Koivurinta Pharming Group N.V. 

Patrick van Berkel Pharming Technologies B.V. 

Charles Morin Burditt and Radzius 

FDA; 

Nega Bern 

MikaAlewyase 

John Matheson 

Bill Price 

Isabel Chen 

George Pauli 

Jeanette Glover-Glew 

Zofia Olempska-Beer 

J. Eugene LeClerc 

Anita Chang 

Wendy Dixon 

Stephanie McQuillcin 

Lin& Kahl 

Felicia Satchell (by phone) 

NickDuy ; COY phone) 

Sue Anderson (by phone) 

Linda Tonucci (by phone) 

Gillian Robert-Baldo (by phone) 

HFS-206 

HFV-228 

HFV-200 

HFV-200 

HFS-207 

HFS-205 

HFS-207 

HFS-207 

HFS-237 

HFS-225 

HFS-200 

HF'S-206 

HFS-158 

HFS-456 

HFS-456 

HFS-465 

HFS-456 

Subject: Recombinant Human Lactoferrin 

The visitors requested the meeting in order to consult FDA regarding Pharming's 

recombinant human lactofenin (rhLF) derived fiom transgenic cows. The visitors stated that 

while they envisaged other uses in the future grid that they intended to consult the 

appropriate FDA centers regarding these uses; ' rhLF at present is intended for use in infant 

formulas. Prior to the meeting, the visitors provided a package of information consisting of 
Q1/05/.0 1 18 : 16 FAX 2022054422 FDAICFSANIOPDFB 

IC 

4 

e. 

Page 2 - Memorandum of Conference 

a003 

a historical perspective of the company and its development and characterization of rhLF. 

The package also contained draft preclinical study protocols aimed at addressing the safety 

of rhLF for its intended use. 

During the meeting, the visitors presented information regarding the generation of 

the transgenic animals including the genetic constructs that were used. They also 

enumerated the tests aimed at comparing the stsuctural and hctional characteristics of rhLF 

to that of native lactoferrin isolated fiom human milk. The visitors reported that most of the 

studies have been completed ahd that the assays conducted to date show that rhLF and 

natural human lactofenin appear similar except in the relative concentrations of the different 

forins of the protein that are normally 'present due to heterogeneity in N-linked 

gl ycosy lation. 

The preclinical study protocols were discussed both with respect to the product as 

well the dose that is appropriate to use in the studies. We stated that we were in the midst of 

developing guidance for macro food additive testing and that we would be willing to provide 

further guidance.. We suggested that they provide us with a list and purpose of preclinical 

and clinical studies they intend to conduct for our review. We also noted that the July 9, 

1996, issue of the Federal Register contains a proposed rule amending the infant formula 

regulations and that this document includes guidance on clinical studies for infant formulas. 

The visitors inquired regarding the possibility of We of the milk after the removal of 

rhLF in the production of other food products such as cheese; they noted that not all of the 

rhLF can be removed from the milk quantitatively. We stated that assuming that there were 

no safety concerns they would need to discuss with FDA regarding appropriate labeling and 

regarding how the product might be used in standardized foods. 

a 

$$p 

1 We also discussed the proposed premarket notification for generally recognized as 

safe (GRAS) substances including the ba&s for making GRAS determinations, the 

information that should be contained-ga notification, and possible agency respgnses. We 

indicated that FDA is accepting notifications pending finalization of the pro,$%sed rule. 

Finally, we noted that GRAS notification cannot be used in lieu of, and does not replace the 

mandatory premarket notification requirement for infant formulas. 

Nega Beru, PbD. 

CC: HF'S-200 HFS-205 HFS-206 HFS-207 KFS-225 HFS-235 HFS-246 HFS-456 

HFS-158 HFS-247 HTS-13 -HFV-228 HFV-200 

R/D:HFS-206:NBeru:4 18-3097: 7/22/98:Saved as meet0720.98 

a F/t : HFS-206:NBeru:srd: 8/20/98 
Mom G, KRAsm, LLP *734zx 

e SUITE 1600 

201 SPEAR STREET 

SAN FRANCISCO, CALIFORNIA 94105-1635 

e-mall: Icra-rnorQearthllnlrset 

Charles L. Morin 

Leslie T. Krasny 

January 5,2001 

George H. Pauli, PhD (Room 1250) 

Director (HFS-205) 

Division of Product Policy 

Office of Premarket Approval 

Center for Food Safety 

and Applied Nutrition 

Food and Drug Administration 

11 10 Vermont Avenue, N.W. 

' Washington, D. C. 20201 

alephone: (41J) 957-0101 

Facsimile: (415) 957-5905 

CONFIDEN"IAL 

Re: Request for meeting 

Dear Dr. Pauli: 

Thanks very much for returning my phone call and for taking the time to 

discuss aspects of GRASing human lactofenin for use in infant formulas and 

supplementing other foods. This will memorialize the substance of our 

conversation and provide you with additional information. 

First, please find attached a copy of my July 2, 1998 confidential letter to 

you which provided information pertinent to the meeting subsequently held on July 

20, 1998. It covers all of the information we discussed. 
Mom G) KRASNY, LLP 

George H. Pauli, PhD 

Re: Request for meeting 

January 5,2001 

Page 2 of 3 

Second, please find attached a copy of my July 22, 1998 confidential 

memorandum summarizing the substance of the July 20* meeting with FDA. 

Please note that it includes a list of those who were in attendance. 

Thud, we discussed having a meeting towards the end of January (our 

prioritized list of dates for such meeting was as follows: 

1. 1/26 (first choice); 

2. 1/29 (second choice); or 

3. 1/22 (third choice)) 

at from approximately 1:30 - 3:OO p.m. in the afternoon. 

Fourth, the purpose of the meeting is to discuss exactly what preclinical tox 

testing should be done in order to satisfy the needs of both the OPA group and the 

Infant Formula group. As of this date, two studies had been agreed to, i.e.: 

1. 

2. 

the first, a 90-day study in rats; and 

the second, a 90-day study in dogs. 

Please note that both studies are currently drafted to start exposure to the pups 

earlier than is usual and both have a reproductive evaluation added at the end. Are 

these the battery of tests that are still required? 
J 

Mom G) KRAsm, LLP 

i 

George H. Pauli, PhD 

Re: Request for meeting 

January 5,2001 

Page 3 of 3 

Finally, please feel free to invite any FDA employee to the meeting that you 

feel may play an important role in any future GRAS Notification that my client 

may file pertinent to the above-referenced uses. 

As you can appreciate, this entire matter is very confidential; thus, we trust 

that the enclosed information will not be discussed or released, except as necessary 

to prepare FDA personnel for the end of January meeting. 

Please call me if you have any questions. 

Thanks again for your help. 
BURDITT LAW OFFICES G, RADZIUS OF FILE 

SUITE 1600 

201 SPEAR SI’REET 

SAN FRANCISCO, CALIFORNIA 94105-1635 

(415) 951-0101 

G3 FAX: (415) 957-5905 

CHICAGO OFFICE ’ 

sum Mw) 

333 WEST WAU(ER DRIVE 

CHICAGO. IUJNOlS B 

012) 711-6113.1 

GlFAXt.11217nl-66W) . 

WASHINGIDN. D.C. DFFlrr: 

sum 4.50 

In50 M mEpT- N.W. 

WASHINOTON. D.C. M0.W 

tao, -wV.wJn 

GJ FAX: t-mi .~bs7n 

PHILADELPHIA OFFICE: 

sum 7n.l 

lNll WALNUTSTREET 

PHILADELPHIA. PA 19103 

(219 m-.w,o 

0.1 FAX: 13151 77XW17 

July 2, 1998 

George H. Pauli, PhD (HFS-205) 

Director (Room 1250) 

Division of Product Policy 

Office of Premarket Approval 

Office of Programs 

Center for Food Safety & Applied Nutrition 

Food and Drug Administration 

11 10 Vennont Avenue, N.W. 

Washington, D.C. 20201 

.: 

Dear Dr, Pauli: 

HADDONFlI3.D. NJ. OFPKE 

3 SOUTH HADDONAVENUE 

HADDONFlQD. NJ. oRa.3 

I6091 JzIcb6p 

GI FAX: ie091 JS+ih. 

CONFl DENT1 AI. 

Re: Pharming Health Care Products 

Meeting (7/20/98) concerning 

use of recombinant lactoferrin 

Pursuant tL Nega Beds instructions and in preparation for Phanning’s 

meeting with CFSAN on July 20’, I am forwarding to you information concerning 

Phanning Health Care Products (“Pharming”) and its recombinant lactoferrin 

(“rhLF”) product which should serve to background you and your colleagues 

concerning the need for the meeting. Unless I hear differently from you, we will 

anive at 11 10 Vermont Avenue on Monday, July 20* at approximately 1245 pm. 

in preparation for the meeting to be held in your seventh floor conference room 

between 1 p.m. and 2:30 p.m. 

The company .e 

Pharming is a biotech company whose corporate offices are located in 

Leiden (a large, university city), The Netherlands. It was incorporated in 1988. 

Phaming focuses on the research, development, and commercialization of human 0 
Bumm 6 RADZIUS 

1 

George H. Pauli, Director 

Division of Product Policy 

Food and Drug Adminisixation 

Re: Phanning Health Care Products Meeting (7/20/98) 

July 2,1998 

Page 2 

concerning use of lactoferrin 

health care products derived fiom milk, primarily fkom transgenic animals. To tbis 

end, Pharming has developed, and will continue to develop, a proprietary 

production technology using transgenic animals, particularly transgenic dairy 

cattle. Such transgenic animals are generated from a one-cell animal embryo 

whose genetic make-up has been modified in the laboratory via the insertion of 

specifically designed sequences of DNA, so-called gene constructs or transgenes. 

Phanning has developed proprietary transgenes so as to produce transgenic 

animals which, in turn, produce proteins in their milk for use in human health care 

applications. 

In June, 1995, Pharming acquired the Finnish Company, Oy FinnGene Ltd, 

which was subsequently renamed Phanning Oy. As a wholly owned subsidiary, 

Phanning Oy conducts certain research and development activities focused on the 

generation of transgenic cattle. The acquisition served to increase Pharming’s 

commercial flexibility by expanding its scientific and operating base. 

In June, 1996, Pharming established a subsidiary in Belgium, which is 

named Phanning N.V. This subsidiary will focus exclusively on production and 

commercialization of recombinant proteins produced in milk of transgenic rabbits. 

Pharming is currently the leader in the field of production technology using 

transgenic dairy cattle, including having produced the world’s first scientifically 

documented.transgenic dmy calf, Le., the well-known “Herman” the bull. This 

technology creates product opportunities which are otherwise difficult or even 

impossible to address. Transgenic cattle are the production route.of choice for 

complex biomedical proteins which either have to be produced in very large 

quantities at low cost, or which, while representing a small volume, are very 

difficult to produce. In both cases, manageable numbers of transgenic cows suffice 

to produce sufficient product to satisfy market demands. 

Pharming is also pursuing production technology using other transgenic 

animals, such as mice and rabbits. In some instances, manageable numbers of 

000001,009 
George H. Pauli, Director 

’ Division of Product Policy 

Food and Dnrg Administration 

Re: Phaxming Health Care Products Meeting (7/20/98) 

July 2, 1998 

Page 3 

conccrning use of lactofenin 

‘.. I 

BURDITT 6 RADZIUS 

*a: 

these transgenic animals will also suffice to produce sufficient product to satis@ 

market demands. 

Phanning is dedicated to achieving technological excellence and, 

particularly, a leading intellectual propeq position. Cutting-edge technology md 

adequate patent protection are extremely important in the biotechnology industry 

To this end, since 1989, the Company has filed, on a worldwide basis, a number of 

basic patent applications covering a wide range of methods, products and product 

applications in the area of transgenic animal technology. Phanning’s first patent 

was issued in August, 1993. Since then, various other patents have been issued, 

including a basic U.S. patent in April, 1994. This latter event made Pharming the 

first transgenic farm animal company to receive patent protection in a major 

market.. With regard to the other patents, currently Pharming owns or controls 

such patents in the USA, Canada, Europe, Australia and New Zealand. In addition, 

patent applications are also pending in these and many other countries. 

The product (hLF) 

Lactoferrin is the major iron-binding protein in the milk of many 

mammalian species, including humans. Its concentration in mature human milk 

ranges fiom 1-2 gramsfliter. This makes it one of the most abundant proteins in 

human milk. In contrast, the concentration of lactoferrin in mature bovine milk is 

less than 0.1 gramsfliter. 

Several biologkal functions have been ascribed to lactoferrin. The hction 

that is probably most relevant and important to infants consuming human milk is 

lactofd’s ability to regulate bacterial growth. It has been demonstrated that 

lactofenin promotes growth of Bifidobacterium spp. which are the predominant 

organisms of the intestinal flora of healthy infants that are breast-fed. In addition, 

it has been shown that lactoferrin has a strong antibacterial effect on many 

organisms that are potentially pathogenic. In accordance with these observations, 

it is well-known that the intestinal flora of children being breast-fed is dramatically 

oooooi,. 0.kO 
-1 

BURD~ & RADZIUS 

George H. Pauli, Director 

Division of Product Policy 

Food &d Drug Administra tion 

Re: Phanning Health Care Products Meeting (7/20/98) 

July2, 1998 

Page 4 

concerning use of lactoferrin 

different fiom children consuming infant formula. Although the exact composition 

of the intestinal flora is probably regulated by several factors, lactoferrin is 

probably one of the more important regulators. 

Since most infant formulas are derived fiorn bovine milk, they contain very 

little lactofenin. Accordingly, addition of human lactofenin to an infant formula 

would make the formula more closely resemble human milk. However, since 

infants fed with breast-milk consume more than 1 gram of human lactofenin per 

day, the amounts of lactoferrin needed to supplement infant formula have, to date, 

been prohibitively large and unavailable. Classical recombinant-DNA methods are 

not very suitable on a very large scale for producing proteins, such as lactofenin. 

In addition, isolation of the protein Erom other sources, such as pooled human milk, 

is not desirable or practical., I 

Transgenic mice were used to demonstrate the feasibility of producing 

recombinant human lactoferrin in the milk of a different mammal. Human 

lactofenin gene sequences were cloned fiom DNA libraries prepared fiom healthy 

human individuals. These lactofenin sequences were subsequently fused to 

regulatory sequences derived Erom regions of the bovine as 1 -casein gene. These 

regions direct mammar, gland-specific expression of the casein gene. The 

caseidactoferrin gene construct was injected into the pronucleus of fertilized 1 mouse oocytes which were subsequently transferred into recipient animals. Mer 

birth, animals were analyzed for integration of the transgene and, if positive, were 

bred to non-transgenic mice to obtain F 1 -offspring. Milk was collected Erom 

transgenic females and analyzed for the presence of recombinant human 

lactoferrin. In all mice analyzed, such human lactofenin was detectable in the 

milk. In the majority of the cases, expression was higher than the levels observed 

in human milk. No adverse effect on the physiology and health of the lactating 

mother as well as of the pups was demonstrable. 

The protein was subjected to a large number of assays (such as those 

pertinent to N-terminal protein sequencing, determining immunol~gical 
. *** . ' 

* 

Bumm 6 RADZIUS 

i 

George H. Pauli, Director 

Division of Product Policy 

Food and Drug Administration 

Re: Phanning Health Can Products Meeting (7/20/98) 

July 2, 1998 

Page 5 

concerning use of lactoferrin 

characteristics, the ability of the protein to bind to a large variety of ligands, 

purification properties, migration pattern on SDS-PAGE, N-terminal glycosylation, 

and iron-binding) to compare its structural and functional characteristics with those 

of native lactofenin isolated form human milk. It was concluded that the 

recombinant protein was very similar to the human protein. The primary 

difference that was observed between the recombinant and native lactofenin 

appears to relate to a difference in the relative concentrations of the different forms 

of the protein that are normally present due to heterogeneity in glycosylation. 

Expression of the transgene in mice is primarily restricted to the mammary 

gland of lactating females. It was also demonstrated that the size of the transcript 

corresponded precisely with the expected size. The transgene was transmitted to 

the offqring in Mendelian fashion. In the limited number of lines that were 

analyzed, the structure of the transgene appeared to be stable throughout several 

generations. 

Given the foregoing mouse results, Pharming then developed methods to 

produce recombinant human lactofenin in milk of transgenic cows at high levels. 

Transgenic cows are animals that contain, in their genome, one or more copies of a 

gene that is derived fkom another species. .In this case, a gene construct was used 

that directs expression of human lactofenin in the milk of the animal. Since dany 

COWS can produce up to' 12,000 liters of milk per year, a single animal is expected 

to produce at least 10 kilograms of lactofenin per year, depending on the 

expression level occurring in each cow. Therefore, a cow herd of manageable size 

could produce enough lactofenin to supplement infant formda with human 

1 acto fenin. 

Caseidlactofenin gene constructs selected for their ability to function 

efficiently in transgenic mice and to direct high levels of lactofenin expression 

were also used to generate transgenic cattle. Oocytes were derived fkom ovaries of 

slaughtered dairy cows or via OPU technique (ovum pickup) and fertilized in vitro 

with sperm of elite bulls. DNA was injected into one of the pronuclei after which 
BURDXTT & RADZIUS 

i 

George H. Pauli, Director 

Division of Product Policy 

Food and Drug Administration 

Re: Pharming Health Care Products Meeting (7/20/98) 

July 2, 1998 

Page 6 

concerning use of lactoferrin 

embryos were allowed to develop in vitro for another five days. After that period, 

two cells were removed from the embryo and analyzed for the presence of the 

transgene. Positive embryos were transferred non-surgically into the uterus of 

recipient cows (see attached article for additional information). 

Phdg fkt produced recombinant human lactoferrin fiom transgenic 

COW’S milk in 1996. Such rhLF is of excellent quality and has been demonstrated 

- in tenns of biological activity - to be very similar to natural hLF. Pharming now 

has a similar, but growing, production herd of transgenic cows capable of 

producing the necessary quantities of rhLF for testing and commercial use. 

Use of rhLF I 

As indicated above, Phanning desires to commercialize rhLF for use in 

infant formulas intended to more closely simulate human mother’s milk. Given 

hLF’s natural and strong antimicrobial activity, Pharming may, in the future, also 

decide to commercialize rhLF for uses deemed by FDA to be foods associated with 

health claims, medical foods, or drugshiologics. To the extent use concerns health 

claims or medical foods, Pharmjng desires to have CFSAN’s input about such use. 

@rug/biologic use will be discussed at another time with CBER). 

The regulatory interest 

Pharming is now ready to initiate preclinical testing of rhLF. Before doing 

. so, Pharming thought it would be productive for both FDA and itself to thoroughly 

inform FDA as to what has transpired thus far, to discuss its commercial intentions, 

and to discuss the regulatory implications of such intentions, especially the best 

regulatory approach (e.g., GUS affirmation, GRAS notification, a FA petition, or 

other (?)). 

I Preclinical studies 

00000~, uL3 
BURDITT 6 RADZIUS 

George H. Pauli, Director 

Division of Product Policy 

Food and Drug Administration 

Re: P-g Health Care Roducts Meeting (7/20/98) 

July2, 1998 

Page 7 

concerning use of lactofenin 

Phanning intends to initiate three studies to evaluate the safety of its rKF. 

These include an Ames test, a standard 90-day oral study in dogs, and a nonstandard 90-day (plus) oral study in rats. 

Draft protocols for these studies are attached for your review. 

, Phanning would appreciate having CFSAN's input, if any, concerning the 

adequacy of these protocols before they are initiated. 

meeting: 

i. 

2. 

3. 

4. 

The attende'es 

Pharming intends to have the following representatives present at the 

Joost B.M.M. van Bree, PhD 

Vice President, Clinical Development & Regulatory Affairs; 

Juha KoivUrinta 

Vice President, Pharming Holding N.V.; 

Patrick van Berkel, PhD 

Senior Scientist; and 

the undersigned 

all of whom will be prepared to present and discuss in detail the information 

referenced above and outlined on the attached agenda. 

We encourage CFSAN to have present any and all FDA personnel that may 

play a significant role in any btwe hLF regulatory submission sent to CSFAN. 

Such persons might include there with responsibility for infant foxmulas, 
. * 

Bu~~rrr & RADZIUS 

J 

George H. Pauli, Director 

Division of Product Policy 

Food and Drug Administration 

Re: Phanniag Hcalth Care Products Meeting (7/20/98) 

July 2,1998 

Page 8 

conmning use of ]actoferrin 

toxicology, chemistry, microbiology, environmental, regulatory, review and 

supervising. 

Any fbture submission (pertinent to Pharming’s rhLF product) will include 

information which demonstrates, among other things, that the rhLF is sufficiently 

comparable to human derived hLF. Such infomation will also indicate that the 

rhLF ingredient is produced by transgenic cows that incorporate no pathgenic or 

toxicogenic capabilities. It also will indicate that all production methods and 

substances used are appropriate for food use. Finally, the information will indicate 

that the finished hLF ingredient has been thoroughly tested and found to be safe for 

use in human food. 

i As you can appreciate, this entire matter is very confidential; thus, we trust 

that the enclosed information will not be discussed or released, except as necessary 

to prepare FDA personnel for the July 20* meeting. 

Thank you very much for your continuing assistance. If you should have 

questions or need additional information, please let me know. 

n 

Charles L. Morin 

CLM: jkm 

cc: Joost van Bree 

I I 
LAW OFFICES OF 

BURDITT G, RADZIUS 

SUITE 1600 

201 SPEAR STREET 

SAN FRANCISCO, CALIFORNIA 94105-1635 

(415) 957-0101 

C3 FAX: (4U) 951-5905 

WASHINO~DN. O.C. omm PHlUDeLPHlA Om= HADDONRELD. NJ. OFFICE: CHICAGO OFFICE: 

333 WEST WACKER DRIVE ' 1 IUS0 M SIREFT., N.W. 

CHICAGO. ILLINOIS W WASHINGRIN. D.C 200% PHILADELPHIA. PA 19103 

G3FAX(312)781.6630 03 FAX (202) 466-57?7 &FAX: (21S)772-3017 

3 SOUTH HAODON AVENUE 

(-1 42866112 

sum 2600 sum 450 sum 703 

1608 WALNVTSTREFT HADDONRELD. NJ. 08033 

(312)7814633 (102) ~~ (21s) m.3010 03 FAX: (609) 3S4-16S6 

To: Pharming rhLF File 

From: Charles L. Mob 

Re: Meeting (7/20/98) with FDA (CFSAN) 

Date: July 22, 1998 

CONFIDENTIAL 

On Monday, July 20, 1998, the following representatives of Pharming 

1. Joost B.M.M. van Bree, PhD 

Vice President, Clinical Development & Regulatory Affairs; 

2. JuhaKoivurinta 

Vice President, Phaming Holding N.V.; 

3. Patrick van Berkel, PhD 

Senior Scientist; and 

4. Charles L. Morin 

Burditt & Radzius 

met with the following representatives of FDA 

1. Felicia B. Satchel1 (HFS-158) 

Branch Chief 

Food Standards Branch 

CFSAN/OFL/DPEPOFL; 
2. ! t. 

3. 

4. 

5. 

I 

6. 

7. 

8. 

9. 

- 

Stephanie McQuilkin OfFS-200) 

Special Assistant 

Office of Premarket Approval 

CFSAN/OPA; 

George H. Pauli (HFS-205) 

Branch Chief 

Division of Product Policy 

CFSAN/OPA; 

Nega Bern, PhD (HFS-206) 

Team Leader 

Regulatory Policy Branch 

CFS AN/OPA/DPP; 

Wendy J. Dixon (HFS-206) 

cso 

Regulatory Policy Branch 

CFSAN/OPA/DPP; 

Linda S. Kahl (HFS-206) 

Guidelines and Regulations Branch 

CFS AN/OFL/DPEPOFL; 

Isabel S. Chen (HFS-207) 

Scientific Support 

CFS AN/OPA/DPP; 

Jeanette Glover Glew (HFS-207) 

Environmental Scientist 

Scientific Support 

CFSAN/OPA/DPP; 

Zofia S. Olempska-Beer (HFS-207) 

Science/technology 

CFS AN/OFL/DS ATOFL; 

2 
10. 

11. 

12. 

13. 

14. 

15. 

16. 

17. 

18. 

Anita H.C. chang (HFS-225) 

Scientific Support 

CFS AN/OPA/DPP; 

J. Eugene LeClerc, PhD (HFS-237) 

Toxicologist 

Molecular Toxicology Branch 

CFSAN/OPA/DMBRE; 

Nick Duy (HFS-456) 

Regulatory Branch 

CFSAN/OSN/DPEPOSN; 

Gillian L. Robert-Baldo (HFS-456) 

Regulatory Branch 

CFS AN/OSN/DPEPOSN; 

Linda .H. Tonucci (HFS-456) 

Regulatory Branch 

CFSAN/OSN/DPEPOSN; 

Sue A. Anderson (HFS-465) 

Scientific Support 

CFSAN/OSN/DSATOSN; 

John C. Matheson (HFV-200) 

Senior Environmental Scientist 

CWOSC/OSCOD; 

William D. Price, PhD (HFV-200) 

Special Assistant 

Office of Surveillance and Compliance 

CWOSC/OSCOD; and 

Mika G. Alewynse (HFV-228) 

Food Safety 

’ Animal Feeds 

cvM/osc/DAF 

00000100 18 .. 
for the purpose of conveying certain information concerning use of Pharming’s 

rhLF in human foods and discussing the implications of such use. The meeting 

was held in the 7* floor conference room at 11 10 Vexmont Avenue, N.W.; it lasted 

from I p.m. until approximately 2:40 p.m. 

After brief introductory remarks (by CLM) and self introductions by all 

attendees, the presentations evolved as indicated on the attached agenda. Other 

than the information set forth below, the information conveyed to FDA was that 

indicated on the attached copies of overheads. 

The questions asked andor the points discussed were as follows: 

A. 

B. 

! C. 

D. 

Introductions (C.M.) 

No questions 

Overview (J.K) 

No questions 

Characterization efforts (P.B.) 

1. Question: Is the genetic effect seen a result of dominance? 

Answer: Yes. The result duplicates a typical Mendelian 

expectation; thus, 50 percent of the offspring should have the 

transgene for rhLF. 

2. Question: Is the glycosylation that occurs an all or none 

phenomenon? 

Answer: No. The result varies; sometimes it’s all, sometimes it 

differs, and sometimes it’s as little as 5 percent. 

Production process and intended use (J.K.) 

No questions 

4 

000001 Q 019 
E. Pre-clinical testing (JvB) 

Suggestions and Comments: 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

10. 

11. 

12. 

FDA will consider rhLF to be like a macroingredient, given its 

expected consumption level. 

You may not be able to feed the dogs and rats the amounts you 

have indicated in the draft protocols. Feed at the highest level 

technically feasible. 

Take in to account human experience, and adjust the protocols 

accordingly. 

Suggest you do a dose range study (over 2 weeks) fiom which 

you establish the lowest dose that causes an effect. 

It is critical that the characterized, especially as to foreign 

substances. 

It is essential that you be able to explain the impact, if any, of 

the different glycosylation patterns. 

Be able to identi@ qualitatively and quantitatively the nature of 

any impurities, including processing aids. 

It is essential that you demonstrate that digestability is not 

adversely impacted on. 

Purity needs to be adequately identified. 

Safety here should focus on infants, not adults. 

Keep in mind the special place infant formulas occupy in our 

culture. (Hmt! Hint! Hint!) 

If food additives are used in preparation of your product, for 

example as processing aids, be sure that they are used only as 

approved. 
13. J i 

14. 

15. 

16. 

17. 

18. 

i 

19. 

20. 

21. 

22. 

23. 

You should indicate whether infant exposure will be any 

different than that for adults. 

We assume that you intend to use the same exposure level (of 

rhLF) in infant formulas as infants would be exposed to (i.e.’ 

hLF) in mother’s milk. 

As to what product should be pre-clinically tested, try and use 

as downstream a product as reasonable; such testing should 

cover any upstream product. 

FDA suggests that we forward to them a list of the pre-clinical 

tests we intend to conduct for their review. Please also include 

chemistry information as it relates to safety. 

Pharming needs to be able to demonstrate equivalence between 

hLF and rhLF. 

Normal dairy practice procedures may be enough; Pharming 

will need to show that they are. 

Pharming will need to demonstrate that it can control all critical 

aspects of the production of its rhLF product. 

For the proper handling and disposition of animals once culled 

&om the production herd see the CDEWCBER PTC document. 

Pharming will need to show that its rhLF product is biologically 

equivalent to hLF after being pasteurized. 

As to the stability of level of production ofrhLF during the 

lactation period, it was indicated that production approximates 

0.3 to 1.0 during the first week and 0.8-0.9 over the rest of the 

period. 

As to propagation, it was indicated that Pharming only 

generates female transgenics via its transgenic bull. No 

markers are involved. 

! . ._ 

6 
24. As to use of the milk byproducts, they should be able to be 

used, but may require use of labeling infoxmation and may not 

be able to be represented as or in standardized foods. 

25. To date, FDA has received five GUS notifications and has 

responded to three. 

26. Use of a blue ribbon panel is not a substitute for publication but 

may support it. Such a panel should be composed of all assets 

necessary to derive GRASness. 

27. FDA strongly encourages use of the GUS notification process. 

28. Approval of rhLF for use in infant formulas will occur (as 

expected) in two steps - first via GUS notification, and then 

via approval by the infant formula group via a submission ffom 

an infant formula manufacturer). 

29. The narrative portion of the GUS notification needs to be 

thorough and to tell a story - the whole story. 

30. If after receiving a GRAS notification CFSAN wants more 

infomation, it will ask for it. 

3 1. For the new regulations pertinent to clinical testing of infant 

formulas see the July 9, 1996 FR document. (A copy of it is 

attached). 

32. As to the need for an environmental assessment, please note the 

new categories that are now excluded. (A copy of this 

document is attached). Two of these, i.e., numbers 8 and 12, 

may be applicable to Phanning’s product. 

33. Note also. the “extraor“dinary circumstances” exception to the 

subpart 32 (directly above) exemptions. (See subpart C of the 

attached pertinent EA document). 

34. The GRAS notification should include copies of comparison 

dah. 

7 
35. The proposed dog study should include dosing which begins 'e 

just after birth and then for 90 days. 

00000L, 0013 

36. Use at least 4 animals per groups (see Red Book, Appendix II, 

page 45). 

8 
Law Offices Of 

Morin & Associates 

/- 

11" I ll111111 I1 Ill1 

Suite 500 

388 Market Street 

San Francisco, California 94 1 1 1 

Telephone: (415) 957-0101 e-mail: charleslmorin@earthlink.net Facsimile: (4 15) 957-5905 

April 10,2005 

Antonia Mattia, PhD (HFS-255) 

Director 

Division of Biotechnology and 

Office of Food Additive Safety 

Center for Food Safety and Applied 

, Nutrition 

Food and Drug Administration 

5 100 Paint Branch Parkway 

' College Park, MD 20740-3835 

GRAS Notice Review 

OFFICE OF 

FQQD ADDITIVE SAFETY 

Re: Notice of GRAS exemption for human 

lactoferrin derived from the milk of 

transgenic cows expressing a human 

gene encoding human lactoferrin 

GRN 000189 

CFSAN request for information 

Dear Dr. Mattia: 

Pursuant to Mr. Fasano's request, please find attached copies of three of the 

appendices referenced in the above-referenced GRAS Notification, i.e., appendix 

numbers 15, 16, and 18. 

Thank .you in advance for your and your colleagues' efforts on behalf of 

Pharming's notice. 

OOOPO8 
Morin & Associates e 

Antonia Mattia, PhD 

Re: Notice of GRAS exemption.. . April 10,2006 

Page 2 of 2 

rr c 

Charles L. Morin 

Cc: Frans de Loos, PhD 

Project Director (rhLF) 

Pharming Group N.V. 

0 

0 
Charles L. Morin 

Morin & Associates 

385 Market Street, Suite 500 

San Francisco, CA 941 11 8-15 

Ian C. hlunro, PhD, FRCPath, MSc, (Panel Chair) 

Professor 

Department of Nutritional Sciences 

Faculty of Medicine 

University of Toronto 

FitzGerald Building 

150 College Street 

Toronto, Ontario 

CANADA M5S 3E2 

Jeremy H. Brock, ScD, PhD, MSc 

Senior Research Fellow 

Department of Immunology 

University of Glasgow 

Glasgotv 

SCOTLAND G116NT 

F. Jay Murray, PhD 

President 

Murray & Associates 

5529 Penlgia Circle 

San Jose, CA 95138 

Jorge A. Piedrahita, PkD, MSc 

Professor of Genomics 

Department of Molecular Biomedical Sciences 

College of Veterinary Medicine 

North Carolina State University 

4700 Hillsborough Street 

Raleigh, NC 27606 

December, 20,2005 

Expert Panel Opinion Regarding the Generally Reco, onized as Safe (GRAS) 

Status of Pharming’s hLF Product 

1 
@ Panel members were provided with a copy of the GRAS Notification and access to all 

information (including references and appendices) in support of the Notification. The Panel 

independently and collectively reviewed all information provided and met on December 20, 2005 

to consider the information in detail. The Notification contained detailed information on the 

production of transgenic cattle from which Pharniing’s hLF is ultimately obtained. In addition, the 

manufacturing process by which Pharming’s hLF is isolated and purified from the milk of 

transgenic cattle was well-documented in the Notification. The Panel concluded that the process 

of producing transsenic cattle and the hLF manufacturing process did not raise safety concerns. 

The Panel also reviewed the proposed specification €or Pharming’s hLF and confirmed that 

analytical data on three batches of Pharming’s hLF conformed to the specification. The Panel 

critically evaluated the available data supporting the safety of Pharniing’s hLF and noted that 

except for minor differences in glycosylation, hLF derived from transgenic cattle was idential to 

native hLF. It was the opinion of the Panel that hLF derived from transgenic cattle was 

substantially equivalent to native hLF. The Panel also was provided with results of a 14-day 

ge-finding toxicity study on Pharming’s hLF in rats, a GLP 90-day study in rats, and three 

gcnotosicity assays. The data from the 90-day rat study on Pharming’s hLF indicated a NOAEL 

of 2,000 mg/kg body weight/day. 

e 

The Panel also reviewed data in the submission on the potential 

allergenicity/immunotoxicity of Pharrning’s hLF. The Panel was informed that since the product 

was derived from bovine sources, the manufacturer intended to label the product as containing 

milk ingredients. The Panel was statisfied that 1LF derived from transgenic cattle did not present 

any increased risk of allergenicity or imniunotoxicity over conventional milk-derived products. 

The Panel further noted that the manufacturer intended to use hLF in a variety of sports and 

functional foods in an amount not to exceed 100 mg of Pharming’s hLF per serving of such foods. 

These uses result in an estimated total population mean and 90“’ percentile intake of 0.32 and 1.00 

Pharming’s LF/kg body weightlday, respectively. For users only the mean and 90Ih percentile 

intakes are estimated to be 1.91 and 3.95 mg/kg/day, respectively. After reviekving all the 

available information the Panel concluded that Pharming’s hLF derived from transgenic cattle is 

cafe for its intended uses. Thus, the Panel concluded: 

2 
Based on our independent collective critical and in-depth evaluation of the available 

pertinent, scientific (both published and unpublished) and other information, we conclude that 

Pharming’s human lactoferrin - which is derived from the milk of transgenic dairy cattle carrying 

and expressing a human lactoferrin gene - is manufactured in accordance with good dairy 

practices and cGMPs, meets the relevant food grade specifications and, based primarily on 

scientific procedures, is Generally Recognized As Safe (Le., GRAS) for use in food as described 

within the GRAS Notification. 

e 

FRCPath, MSc, (Panel Chair) Date 

Toronto, ON CAN 

Jeremy H. Brock, ScD, PhD, MSc 

University of Glasgow 

Glasgow SCOTLAND 

F. Jay Murray, PhD 

Murray & Associates 

San Jose, CA 95138 

Jorge A. Piedra ita, PhD, hSc 

Raleigh, NC 27606 

North Carolina I late University 

3 
Nederlandse Organisatie voor 

toegepast-natuurwetenschappelljk 

onderzoek /Netherlands Organisation TNO Quality of Life for Applied Scientific Research CQPV Return address Postbus 360. 3700 AJ. Zeist. The Netherlands 

Pharming Group N.V. 

Attn. Dr F. de LOOS 

Archimedesweg 4 

2333 CN Leiden 

I TNetherlands 

Subject 

Expert Opinion 

Statement concerning the bovine glycosylation of Phanning’s hLF 

(chapter 3 “Allergenicity”, paragraph “Glycosylation”). 

Statement: 

Next to: 

- the various observations addressed in the glycosylation paragraph (chapter 3) that the 

bovine glycosylation of Phanning’s hLF is not likely to be a safety factor in respect to 

its immunogenicity (sensitizing potential) in compaxjson to natural hLF, 

there is, moreover, 

- no indication that the bovine glycosylation of Pharming’s hLF will result in clinical 

symptoms of allergy due to cross reactivity with in particular serum IgE-antibodies 

against N-glycans (e.g. IgE antibodies to plant N-glycans of pollen allergic 

individuals). 

This statement is supported by the following observations: 

- It is clear that the glycosylation of Pharming’s hLF is of a mammalian type 

and that, although Pharming’s hLF and native hLF show differences in 

carbohydrate structures, they do not differ in the number and location of the 

glycosylation sites (see chapter 5, 6B). 

Concern in respect to the contribution of glycan epitopes to allergy is mainly 

based on research with plant and invertebrate glycoproteins 

Carbohydrate structures are not generally considered as allergens 

Despite strong in vitro reactivity of IgE antibodies against carbohydrate 

moieties (Cross-reacting Carbohydrate Determinants, CCD) can occur, it is 

clear that they have a poor biological activity (Van der Veen and van Ree, 

1997; Aalberse et al, 2001) 

From a recombinant human lactofemn produced in plants (rice) it was shown 

that, despite 1) two out of the three putative N-glycosylation sites of the 

natural hLF are glycosylated, 2) serum samples of pollen allergic individuals 

with IgE-reactivity to plant glycans showed significant binding to the 

- 

- 

- 

- 

Toxicology and Applied 

Pharinacology 

Location Zeist 

Utrechtseweg 48 

P.O. Box 360 

3700 AJ Zeist 

The Netherlands 

www.tno.nl 

T +31 3069441 44 

F +31 306944777 

infofood@ voeding.tno.nl 

Date 

December 19,2005 

Our reference 

TAP-2005 

E-mail 

Penninks@voeding.tno nl 

Direct dialling 

+31306944564 

Direct fax 

+31306944986 

Project number 

31657.01 03.01 

The Standard Conditions for 

Research Instructions given to TNO 

ns filed at the Registry of the 

District Court and he Chamber of 

Commerce iii The Hague 

shall apply to all instructions given to 1NO. 

the Stmdard Conditions will be sent on 

request. 

. 
Nederlandse Organisatie voor 

toegepast-natuurwetenschappeli]k 

onderzoek/Netherlands Organisation 

for Applied Scientific Research 

Date 

December 19,2005 

Our reference 

TAP-2005 

Page 

212 

recombinant human lactofenin isolated from rice, but negligible binding to 

the natural human lactofemn purified from breast milk, 3) histamine release 

assays demonstrated that the IgE antibodies against plant N-glycans have a 

poor biological activity and are of no or limited clinical relevance. Thus even 

a recombinant lactofemn produced from rice, which has much stronger 

differences in glycosylation than Pharming’s hLF, is regarded as safe in 

respect to allergenicity (GRAS notification 162). 

In conclusion it is considered very unlikely that the bovine glycosylation of 

Pharming’s hLF will result in clinical symptoms of allergy from the consumption of 

foods containing Pharming’s hLF. 

References: 

Van der Veen, M., and R. van Ree. Allergens, IgE, Mediators, Inflammatory 

Mechanisms; Poor Biological activity of cross-reactive IgE directed to carbohydrate 

determinants of glycoproteins. J Allergy Clin Immunol, 100, 327-334,1997. 

Aalberse, R et al. Cross-reactivity of IgE-antibodies to allergens, Allergy 56,478-490, 

a 2o01* 

A 

Dr A.H. Pennin 

TNO Toxicology and Applied Pharmacology 

Dept. Experimental Immunology 
TNO Quality of Life 

Nederlandse Organiratie yoor 

tocgcpast-natuurwctcnschappelijk 

onderroeklNctherlands Orgamtation 

for Applied Scientific Research 

TNO Report p-\s 

Allergenicity prediction of recombinant human 

lactofemne using the database of the Food Allergy 

Research and Resource Program 

Date 

Authors 

Sponsor 

TNO project no. 

Number of pages 

Number of tables 

21 October. 2005 

J.H.M. van Bilsen. Ph.D. 

Pharming Group N.V. 

Archimidesweg 4 

2333 CN Leiden 

31657/01 01.02 

8 

3 

Toxicology and Applied 

Pharmacology 

Location Zeist 

Utrechtseweg 48 

P.O. Box 360 

3700 AI Zeist 

The Netherlands 

www.tno.nl 

P +31 30 694 41 44 

F +31 306957224 

infofoodBvoeding.~no.nl 

p0s.m. 1.3- rhLF archive 

All rights reserved. 

No part of this publication may be reproduced and/or published by print, photoprint, microfilm 

or any other means without the previous written consent of TNO. 

In case this report was drafted on instructions. the rights and obligations of contracting parties 

are subject to either the Standard Conditions for Research Instructions given to TNO. or the 

relevant agreement concluded between thc contracting parties. Submtting the report for 

inspection to parties who have a direct interest is permitted. 

8 2005 TNO 
"NO Report I 

Contents 

Statement 3 

1 Introduction 4 

2 Materials and methods 5 

3 Results 6 

4 Conclusion 8 

218 
TNO Report I 

Statement 

We, the undersigned, hereby declare that this report constitutes a true and complete 

representation of the procedures followed and of the results obtained in this study by 

TNO Quality of Life, and that the study was carried out under our supervision. 

J.HM. van Bilsen, Ph.D 

Project Leader 

Business Unit Toxicology and Applied Pharmacology 

Date: 31 

Product manager 

Business Unit Toxicology and Applied Pharmacology 
c 2 

TNO Report I 

1 Introduction 

Potential allergenicity of recombinant proteins for consumption must be investigated 

before their introduction into the food chain. To asses whether the tested recombinant 

protein is considered to be an allergenic risk, a database search can be performed to 

reveal a level of homology with known allergens that suggests a potential for crossreactivity. 

To evaluate whether recombinant human lactofemne (hLF) has the ability to induce an 

allergy, a BLAST search was performed against the Food Allergy Research and 

Resource Program (FARRP) Protein Allergen database, using the sequence of hLF. 

The FAEUU' Protein Allergen Database contains a comprehensive list (1191 sequence 

entries) of unique proteins of known and putative allergens (food, environmental and 

contact) and gliadins that may cause celiac disease. The 1191 entries were identified by 

searching publicly available protein databases using the Entrez search and retrieval 

system, which is a compilation of a variety of databases including SwissProt, PIR, PRF, 

PDB, and translations from annotated coding regions in GenBank and RefSeq. Search 

tern were the key words "allergen" and "celiac". A few additional entries were 

identified by searching Medline for allergens that have not been entered in a sequence 

database. The strength of the evidence regarding the allergenicity of proteins in the 

database varies greatly. Some entries are from publication of peer reviewed studies 

demonstrating clear clinical cause and effect for some individuals with a history of 

allergy to the source material, to those where the authors of an abbreviated note or a 

sequence database entry claim that protein is an allergen or binds IgE without published 

proof. However, proteins that are merely similar in sequence to an allergen 

(homologues) were not included in the database. 
TNO Report 

2 Materials and methods 

In this search we compared the protein sequence of hLF to entries in the FAW 

database. 

To this end, a complete set of 80-amino acid length sequences (n = 613) derived from 

hLF. together spanning the entire protein, were prepared and individually compared with 

all the amino acid sequences of the entries in the FARRP database. 

The FARRP database utilizes a sequence comparison routine, FASTA (Pearson and 

Lipman, 1988). This version of the FASTA search interface utilizes the FASTA3 

(Pearson, 2000) aIgorithm to evaluate whether the hLF protein sequence is identical to, 

or homologous with known or putative allergens and gliadins in the database. 

Alignments with high identity scores may indicate a potential for allergenic crossreactions. However, there is not sufficient scientific data to establish a simple scoring 

boundary @-score or percent identity), beyond which cross-reactivity is certain, or 

below which cross-reactivity is not possible. 

Based on historical data, cross-reactivity is not likely for proteins with less than 50% 

identity over the entire protein sequence, and is fairly common above 75% identity 

(Aalberse, 2000). 

' According to the FAOiWHO guidelines for allergenicity evaluation of foods derived 

from biotechnology, a query protein is potentially allergenic if it either has an identitiy 

of at least 6 contiguous amino acids or more than 35% sequence similarity over a 

window of 80 amino acids when compared with a known allergen. 

References 

Aalberse, R.C. 2000. Structural biology of allergens. J. Allergy Clin. Immunol. 106:228- 

238. 

o 

o Pearson, W.R. and Lipman, D.J. 1988. Improved tools for biological 

sequencecomparison. Proc. Natl. Acad. Sci. USA 852440-2448. 

Pearson, W.R. 2000. Flexible sequence similarity searching with the FASTA3 program 

package. Methods Mol. Biol. 132:185-219. 

o 

I 
TNO Report I 6/8 

3 Results 

The amino acid sequence of recombinant hLF was provided by the sponsor (Table 1) 

Table 1. hLF derived protein from cDNA sequence 

1 

51 

101 

151 

201 

251 

301 

351 

401 

451 

501 

551 

601 

651 

GRRRRSVQWC AVSQPEATKC FQWQRNMRRV RGPPVSCIKR DSPIQCIQAI 

AENRADAVTL DGGFIYEAGL APYKLRPVAA EWGTERQPR THWAVAWK 

KGGSFQLNEL QGLKSCHTGL RRTAGWNVPI GTLRPFLW GPPEPIEAAV 

ARFFSASCVP GADKGQFPNL CRLCAGTGEN KCWSSQEPY FSYSGAFKCL 

RDGAGDVAFI RESTVFEDLS DEAERDEYEL LCPDNTRKPV DKFKDCHLAR 

VPSHAWARS VNGKEDAIWN LLRQAQEKFG KDKSPKFQLF GSPSGQKDLL 

FKDSAIGFSR VPPRIDSGLY LGSGYFTAIQ NLRKSEEEVA ARRARVVWCA 

VGEQELRKCN QWSGLSEGSV TCSSASTTED CIALVLKGEA DAMSLDGGW 

YTAGKCGLVP VLAENYKSQQ SSDPDPNO RPVEGYLAVA VVRRSDTSLT 

WNSVKGKKSC HTAVDRTAGW NIPMGLLFNQ TGSCKFDEYF SQSCAPGSDP 

RSNLCALCIG DEQGENKCVP NSNERYYGYT GAFRCLAENA GDVAFVKDVT 

VLQNTDGNNN DAWAKDLKLA DFALLCLDGK RKPVTEARSC HLAMAPNHAV 

VSRMDKVERL KQVLLHQQAK FGRNGSDCPD WCLFQSETK NLLFNDNTEC 

LARLHGKTTY EKYLGPQYVA GITNLKKCST SPLLEACEFL RK* 

The FASTA program was used to compare the complete sequence of hLF to the FARRP 

Protein Allergen Database. The best scores are depicted in Table 1. The most significant 

scores are derived from ovotransfemn (chicken) and ovotransfemn precursor) 

Table 2 FASTA search with complete sequence of hLF in FARRP database 

NC31 link Name sw* 2-sc E-value** 

gill 35 12951splW27891~~-CHICK 

git75785 1 lernblCAA26040.II 

gi11707431gblAABO2788.1 I 

gi1217431embICAA43331 .I1 

gi1186391emblCAA33217.1 I 

gi141029591gblAAD01630.1 I 

gil5010501gblAAA 191 62 11 

gi1307934461dbjlBAC76688.1 I 

gi153813231gblAAD42943.1 IAFD91841-1 

gi1216320541gblAAK85129.11 

gill 125581piijjB37330 

gill 182161splP18 1531D7-AEDAE 

Ovotransfemn precursor chicken 

ovotransfemn (chicken) 

HMW glutenin subunit Ax2 

high molecular weightgluteni 

glycinin subunit G3 

ladder protein [Acanthocheil) 

phospholipase A2 inhibitor 

27K protein [Triticum aest) 

2s albumin precursor 

elongation factor [Junipem) 

venom allergen 111 -red importe) 

D7 protein precuror 

2410 

2402 

81 

81 

78 

71 

71 

71 

69 

68 

69 

71 

2801.7 3.6e-151 

2792.4 1.2e-150 

88 8 4.6 

88.7 4.7 

88.6 4.7 

88.6 4.8 

85 9 67 

85.8 6.8 

85.5 7.1 

83.4 9.3 

83.2 9.4 

83.0 9.7 

*Smith-Walerman score 

**Expectation value: The number of different alignements with scores equivalent to or better than S that are 

expected to occur in a database search by chance. The lower the E value, the more significant the score. 
TNO Repon I 

Hits 

718 

Best %ID #hits >35% Full alignment NCBI links 

E-value I %ID I length 

The 80-mer sliding window search revealed that hLF shares significant homology with 

two allergens desribed in the FARRP database: ovotransfemn. precursor (chicken) and 

ovotransfemn (chicken)(Table 2). The best percentage identical amino acids (%ID) in 

80-mer sequence in both hits was about 67%. All 613 overlapping 80-mers from hLF 

showed >35% homology with ovotransfemn (precursor). The percentage identical 

aminoacids in the full alignment (the whole protein, not just 80-mer sequence), was in 

both hits 52%. 

Ovotransfemn precursor (chicken) I 67.55% 1 613 of 613 3.6e-151 I 52.2% I 693 I giI13~1295 

,# Ovotransfemn (chicken) I 66.7% I 613of613 I 1.2e-150 151.9% I 693 I g~ '1757851 

Table 3 SO-mer sliding window search results 
"NO Report I 

4 Conclusion 

From this study, it can be concluded that cross-reactivity between recombinant human 

lactofemn and ovotransfemn (precursor) has to be considered as an allergenic risk. 

Unfortunately, it is currently not possible to define a similarity threshold in allergenicity 

prediction that can truly discriminate between immunologically cross-reactive and noncrossreactive proteins. In most cases experimental studies will be needed to confirm that 

two sequence similar proteins may cause allergic cross-reactions. 
 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: February 1, 2006 

TIME: 3:00 PM 

PHONE NUMBER: 415-957-0101 (initial contact) 

PARTICIPANTS: 

FDA 

Jeremiah Fasano HFS-255 

External 

Charles Morin Morin & Associates 

SUBJECT: Basis of GRAS Claim for GRN 189 

Mr. Morin submitted GRN 000189 to FDA on behalf of Pharming, N.V. The notice 

claims that recombinant human lactoferrin produced in bovine milk is generally 

recognized as safe for use in a variety of foods, on the basis of both scientific procedures 

and common use in food prior to 1958. 

I contacted Mr. Morin to discuss this dual basis for Pharming’s GRAS claim. He 

returned my call shortly thereafter. I explained that we expected GRAS notices to 

include a GRAS determination based either on scientific procedures or on common use in 

food, in accordance with 21 CFR 170.30. I requested that Mr. Morin provide a short 

written statement clarifying the basis of the notifier’s GRAS determination. I noted that, 

as a practical matter, we would expect to evaluate the notice on the basis of scientific 

procedures, since we were not aware of any evidence that recombinant human lactoferrin 

produced in bovine milk had been in common use in food prior to 1958. 

Mr. Morin agreed to consider our request. 

Jeremiah Fasano 

R/D:HFS-255:JMFasano:02/21/06 

F/T:HFS-255:JMFasano:05/25/10 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

DEPARTMENT OF HEALTH & HUMAN SERVICES default Public Health Service 

Food and Drug Administration 

College Park, MD 20740 

February 14, 2006 

Charles L. Morin 

Morin & Associates 

Suite 500 

388 Market Street 

San Francisco, CA 94111 

Re: GRAS Notice No. GRN 000189 

Dear Mr. Morin: 

The Food and Drug Administration (FDA) has received the notice, dated December 29, 2005, that 

you submitted on behalf of Pharming Group N.V., in accordance with the agency’s proposed 

regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally 

Recognized as Safe (GRAS)). FDA received this notice on January 3, 2006, filed it on January 

12, 2006, and designated it as GRN No. 000189. 

The subject of the notice is human lactoferrin derived from the milk of transgenic cows 

expressing a human gene encoding lactoferrin. On February 8, 2006, you clarified the basis for 

the GRAS determination. The notice informs FDA of the view of Pharming Group N.V. that 

their lactoferrin is GRAS, through scientific procedures, for use as an ingredient in sports and 

functional foods at a level of 100 milligrams per serving. 

In accordance with proposed 21 CFR 170.36(f), a copy of the information in the notice that 

conforms to the information described in proposed 21 CFR 170.36(c)(1) is available for public 

review and copying on the homepage of the Office of Food Additive Safety (on the Internet at 

http://www.cfsan.fda.gov/~lrd/foodadd.html). If you have any questions about the notice, contact 

me at 301-436-1173 or jeremiah.fasano@fda.hhs.gov. 

Sincerely yours, 

/s/ 

Jeremiah Fasano 

Division of Biotechnology and

 GRAS Notice Review 

Center for Food Safety

 and Applied Nutrition 
Page 2 - Mr. Morin 

Hard copy cc: GRN 000189 (1 copy) 

Filename: Final GRN 189 Acknowledgement Letter 

R/D:HFS-255:JMFasano:01/26/06 

Init:HFS-255:JGGlew:02/13/06 

Comment:HFS-255:PGaynor:02/14/06 

F/T:HFS-255:JMFasano:02/14/06 
 

 

 

 

 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

Date: April 6, 2006 

Time: 3:00 PM 

Number: 415-957-0101 

Participants: 

FDA 

Jeremiah Fasano HFS-255 

External 

Charles L. Morin Morin & Associates 

Subject: Request for GRN 189 Appendices, Notice of Questions 

Pharming Group, N.V. (Pharming) submitted a notice on December 29 informing FDA that 

recombinant human lactoferrin produced in the milk of transgenic cows is generally recognized 

as safe (GRAS) for use in sports and functional foods at a level of 100 milligrams (mg) per 

serving. 

Mr. Morin, Pharming’s agent, contacted me for an update on the status of the notice. I explained 

that our review was in progress, and that we would appreciate the provision of a number of 

appendices to the notice that were listed but not included with the original submission, namely 

appendix 15 (Pharming’s GRAS panel report), appendix 16 (TNO report concerning 

allergenicity), and appendix 18 (Expert opinion of Dr. A. H. Penniks concerning potential 

allergenicity of Pharming’s LF). Mr. Morin had previously offered to provide any appendices 

desired. 

I also advised Mr. Morin that we had tentatively identified a number of questions regarding the 

notice. While we were still developing clear and concise statements of these questions, we 

considered it appropriate to apprise Pharming of this development. 

Mr. Morin asked what our concerns were. I said that the two foremost in our minds at the 

current time were: 

• the potential for adverse effects of increased human lactoferrin exposure in adults, and 

• the potential for breakdown of self-tolerance to endogenous human lactoferrin. 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

 

Page 2 – April 6, 2006 Memorandum of Teleconference 

I stated that once we had articulated our primary concerns to our own satisfaction, we would be 

willing to discuss the relevant scientific issues with Pharming if the firm was interested in doing 

so. Mr. Morin expressed his preference that the request be received in writing, and said that a 

face-to-face meeting with the company’s scientists might be the most effective way of discussing 

scientific issues associated with the notice. 

Mr. Morin agreed to pass on the requested appendices, and I agreed to contact Mr. Morin when 

we were prepared to communicate our concerns more fully to Pharming. 

Jeremiah Fasano 

R/D: HFS-255:JMFasano:04/06/2006 

F/T: HFS-255:JMFasano:05/26/2010 
 

 

 

 

 

 

 

 

Page 1 of 3 

Fasano, Jeremiah 

From: Fasano, Jeremiah 

Sent: Wednesday, May 17, 2006 10:18 AM 

To: 'charleslmorin@earthlink.net' 

Subject: Offer to meet for discussion on GRN 189 

Mr. MorinWe have completed a preliminary evaluation of GRN 000189 for recombinant human lactoferrin expressed in bovine milk. 

As discussed previously, we are providing some of our concerns in writing for your consideration. This should not be 

considered an exhaustive list, but does represent what we consider significant questions that we have right now. 

Lactoferrin is a known biological response modifier of the immune system. The action of various parts of the immune 

system can be both beneficial and harmful, depending on the abundance and activation of the effector cell or protein 

relative to other immune system components, as well as the duration of the specific immune activity. While beneficial 

effects bear no weight in a GRAS determination, we are concerned about potential adverse effects of lactoferrin 

consumption. These adverse effects would not necessarily appear in every susceptible individual, and would probably not 

become apparent in short term human or animal studies. 

• Lactoferrin has been shown to enhance Type 1 T helper (Th1) cell activity, as well as the release of specific 

cytokines in the gut and systemically following oral administration. We are concerned about lactoferrin's ability, 

through effects on Th1 cells, to potentially exacerbate pro-inflammatory responses by this arm of the adaptive immune 

system. Chronic pro-inflammatory Th1-mediated immune responses might result in the promotion of autoimmune or 

other inflammatory disorders, in the gut or elsewhere, in individuals predisposed to such disorders. 

• Pharming's lactoferrin is distinct from the endogenous lactoferrin of individual consumers with respect to 

o expected differences between the amino acid sequence of the exogenous lactoferrin and the polymorphic 

endogenous lactoferrin alleles present in the general population, and 

o the modification of some species of the exogenous lactoferrin with oligomannose glycans not found on 

endogenous forms. 

Even small structural or biological differences between the native and modified form of a particular protein may have a 

significant impact on that protein's recognition by the immune system and subsequent response. We are concerned that 

Pharming's exogenous human lactoferrin may evoke a nonallergic immune response in susceptible individuals that 

disrupts previous tolerance to endogenous lactoferrin through determinant spreading from alloepitopes, the potential for 

enhanced pro-inflammatory Th1 responses mentioned above, and increased uptake by antigen-presenting cells via the 

mannose receptor. 

Given these concerns, we have questions about the evidence and information presented in the notice. 

• The notice states that lactoferrin is known for its immunomodulatory properties. However, the preclinical studies 

presented in the notice do not address the immunomodulatory activities of lactoferrin. What preclinical evidence 

supports the safety of exogenous lactoferrin for its intended use given its activity as a biological response modifier of 

the immune system? 

• The primate and human studies of oral lactoferrin administration cited in the notice are in small populations for 

relatively short periods of time. Most of the studies with recombinant human lactoferrin focus on efficacy rather than 

safety, and many of the human studies involve subjects with pre-existing medical conditions. Where safety endpoints 

are included, they do not appear relevant to the effects of lactoferrin as a biological response modifier of the immune 

system. Is there clinical evidence that supports the immunological safety of long-term exogenous lactoferrin 

administration at the proposed use level in the general population? 

• The notice provides an acceptable daily intake (ADI) based on the maximal consumption of lactoferrin in human 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

__________________________________________ 

__________________________________________ 

Page 2 of 3 

milk by infants. The infant immune system and gut are different from that of the adult, for example in the infant bias 

towards Th2 responses relative to Th1. Given this, what evidence supports the use of exposure data derived from 

infants in setting an ADI for adults that takes into account lactoferrin's activity as a biological response modifier of the 

immune system? 

• The notice provides an assessment of the potential allergenicity of Pharming's lactoferrin and states that there is no 

evidence to date that anti-lactoferrin antibodies are associated with autoimmune pathology. Other than this statement, 

the notice does not address the potential for adverse non-allergic responses to Pharming's lactoferrin by the adaptive 

immune system as described above. To what extent has Pharming evaluated this risk, and what evidence was used in 

the evaluation? 

While we have tried to state the essence of our concerns here, we believe that we could most effectively convey the 

complexity, significance, and relationships of each concern to the others in a verbal discussion. Such a discussion would 

provide you with an opportunity to clarify any points that were unclear and obtain as much detail as needed in preparing 

your response. We would be willing to have a second discussion with you, potentially including members of your GRAS 

panel, after you have had time to consider the issues we have raised,. In our estimation, these issues are sufficiently 

complex that we do not expect that you will necessarily be prepared to address them all at our first meeting. 

We would be available to meet by phone or in person after May 31st, 2006 to explain our concerns. If this is agreeable, 

please provide us with a few dates (and time of day) that would be best for you and we will confirm if the appropriate FDA 

staff are available. 

 Sincerely,

 Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Note Transition to New Email Address: jeremiah.fasano@fda.hhs.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

Mailing Address: 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

This e-mail is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential, and it should not be 

disseminated, distributed, or copied to persons not authorized to receive such information. If you are not the intended recipient, any dissemination, distribution or copying is strictly 

prohibited. If you think you have received this e-mail message in error, please e-mail the sender immediately at jfasano@cfsan.fda.gov. 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

6/27/2007 
 

 

 

 

 

 

 

Page 3 of 3 

Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Note Transition to New Email Address: jeremiah.fasano@fda.hhs.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

Mailing Address: 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

This e-mail is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential, and it should not be 

disseminated, distributed, or copied to persons not authorized to receive such information. If you are not the intended recipient, any dissemination, distribution or copying is strictly 

prohibited. If you think you have received this e-mail message in error, please e-mail the sender immediately at jfasano@cfsan.fda.gov. 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

__________________________________________ 

Page 1 of 2 

Fasano, Jeremiah 

From: Charles Morin [charleslmorin@earthlink.net] 

Sent: Thursday, June 01, 2006 4:50 PM 

To: Fasano, Jeremiah 

Cc: Frans de Loos 

Subject: Re: FDA-Pharming Discussion - June 29th @ 10 am EST is open 

Dear Dr. Fasano, 

Thank you for your email concerning reservation of June 29th at 10:00 a.m. EST for our meeting concerning hLF for food use. I 

understand that the time and date are a go. As of this date, they are also a go for Pharming. However, as indicated to you earlier, we 

are adding additional expertise (in immunology) to our hLF project team to respond to CFSAN's concerns and hope to be fully 

prepared so as to be able to proceed on June 29th. If we cannot be fully prepared by that date, then we should know by June 12th 

and I will let you know. 

In any case, I will communicate with you in a week or so as to final details. 

Thanks for your help! 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 500 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

Email: charleslmorin@earthlink.net 

----- Original Message -----

From: Fasano, Jeremiah 

To: 'charleslmorin@earthlink.net' 

Sent: Tuesday, May 30, 2006 9:30 AM 

Subject: FDA-Pharming Discussion - June 29th @ 10 am EST is open 

Mr. MorinThe 10 am slot on June 29th works for us - I've reserved it for the necessary FDA personnel. 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

Office of Food Additive Safety 

6/27/2007 
 

 

 

 

 

 

Page 2 of 2 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Note Transition to New Email Address: jeremiah.fasano@fda.hhs.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

Mailing Address: 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

This e-mail is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential, and it should not be 

disseminated, distributed, or copied to persons not authorized to receive such information. If you are not the intended recipient, any dissemination, distribution or copying is 

strictly prohibited. If you think you have received this e-mail message in error, please e-mail the sender immediately at jfasano@cfsan.fda.gov. 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page 1 of 1 

Fasano, Jeremiah 

From: Charles Morin [charleslmorin@earthlink.net] 

Sent: Monday, June 12, 2006 1:48 PM 

To: Fasano, Jeremiah 

Subject: CFSAN Meeting with Pharming 

Follow Up Flag: Follow up 

Flag Status: Completed 

Dear Dr. Fasano, 

This communication makes two requests. First and with regard to the meeting date (currently set for June 29th at 10:00am), as 

anticipated (and as mentioned to you in an email on June 1st) it is taking longer to arrange for additional experts and prepare for the 

meeting than Pharming had hoped. Consequently, so as not to waste CFSAN time and resources, Pharming respectfully requests 

that the meeting date be changed to July 13th (if possible). Pharming apologizes for any inconvenience this request may cause.

 Second, in prior communications with you, you had indicated that (in addition to two major concerns) CFSAN also had some minor 

concerns/questions. If such questions currently exist, please forward a copy of them to us so that Pharming can proceed to respond 

(in writing) to all outstanding questions. 

Thank you for your continuing efforts. 

Best regards. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 500 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

Email: charleslmorin@earthlink.net 

6/27/2007 
dgennix Incorporated O” I11111111 II 11111 1111 

June 27,2006 

Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Room 3044 

University Station 

4300 River Road 

College Park, Maryland 20740 

Re: Safety Concerns Raised by Recombinant Human Lactofemn from Transgenic Cows 

(GRN No. 000189 Submitted by Pharming Group N.V.) 

Dear Dr. Tarantino: 

This letter is to address the claim of Pharming Group N.V. (“Pharming”) in GRN No. 0001 89, 

submitted to FDA’s Center for Food Safety and Applied Nutrition (CFSAN), that recombinant 

human lactoferrin (rhLF) from transgenic cows is generally recognized as safe (GRAS) for use in 

sports and functional foods and drinks. As you know, Agennix is a biopharmaceutical company 

focused on developing protein-based drugs for the treatment of cancer and diabetic ulcers. We 

have significant experience with rhLF from a fermentation process that conforms to current good 

manufacturing process (cGMP) requirements for drugs, and have been conducting clinical trials 

with oral rhLF under Investigational New Drug Applications (INDs) filed with the FDA since 

1996. Agennix recently completed blinded, placebo-controlled Phase I1 clinical trials with rhLF 

that met their primary efficacy endpoints in indications including non-small cell lung cancer and 

diabetic foot ulcers. 

We have carefblly reviewed GRN 000189 and consulted with leading experts qualified by 

scientific training and experience to assess the safety of transgenic cow-produced rhLF for the 

proposed uses. As explained more fully below and in the attached scientific assessments, serious 

concerns and unanswered questions preclude any determination that transgenic cow-produced 

rhLF is GRAS. Indeed, opinions of qualified experts confirm that rhLF is a potent and complex 

bioactive molecule for which extensive clinical investigations of appropriate size and durationfar beyond those described in GRN 0001 89-are warranted to establish safety. Accordingly, we 

respectfully ask that FDA conclude that this notification does not provide a basis for a GRAS 

determination. The scientific assessments and other supporting materials on which this request is 

based are provided in Appendix Volumes 1 and 2. L/ 

- 1 / Appendix Volume 1 provides a detailed assessment of safety concerns raised by the claimed GRAS 

status of rhLF from transgenic cows. Volume 1 also contains letters from Dr. Simon Roger, Dr. Irma van Die 

and Dr. Eugene Weinberg commenting on GRN No. 000189, as well as a copy of Pharming’s web page that 

Eighl Greenwy Plaza Suite 910 Houstorl Texas 77046 Telephone 7131552-1091 Facsimile 731552-0795 0 0 0 1.2 3 
I. THE GRAS STANDARD 

As you are aware, a substance added to food is a “food additive” for which FDA pre-market 

approval is required unless the substance is GRAS or qualifies for another statutory exemption. 

The intended use of a substance is GRAS if it isgenerally recognized, among experts qualified by scientific 

training and experience to evaluate its safety, as having 

been adequately shown through scientific procedures (or, in 

the case of a substance used in food prior to January 1, 

1958, through either scientific procedures or experience 

based on common use in food) to be safe under the 

conditions of its intended use . . . 2/ 

As the statutory language suggests, a GRAS determination may be based either on “scientific 

procedures” or common use in food prior to 1958. A GRAS determination based on scientific 

procedures requires the same quantity and quality of scientific evidence as is required to obtain 

approval of a food additive regulation for the ingredient. 3/ 

Based on the statute, FDA has advised that a GRAS determination requires three elements, all of 

which must be present: 

1. Evidence that a substance is safe for its intended use; 

2. A basis for concluding that such evidence of safety is generally available; and 

3. A basis for concluding that such evidence of safety is the subject of scientific consensus 

- 

among qualified scientific experts. 

FDA refers to the first element as “technical evidence of safety”; the second and third criteria 

collectively constitute the “common knowledge” element of the GRAS standard. 

Technical evidence of safety requires a showing that “there is a reasonable certainty in the minds 

of competent scientists that the substance is not harmful under the intended conditions of use.” 4/ 

This is frequently paraphrased as demonstrating that there is a “reasonable certainty of no harm.” 

The second element, general availability, requires publication of key data or information in peerreviewed scientific journals, general reference materials, textbooks, or other appropriate 

suggests pharmaceutically relevant uses for transgenic cow-produced rhLF. Appendix Volume 2 contains 

copies of the CV’s for the experts contributing to the scientific assessment. 

- 2/ FFDCA $201(s). 

- 3/ 

- 4/ 

21 C.F.R. Q 170.30(b). 

21 C.F.R. Q 170.3(i); 62 Fed. Reg. 18937, 18948 (Apr. 17, 1997). 

_n 

Page 2 of 9 000124 
--- sources. The third element, expert consensus, may be demonstrated by the scientific literature, 

documentation of the opinion of an expert panel, or the pertinent opinion of an authoritative body, 

such as the National Academy of Sciences (NAS), among other references.31 Expert consensus 

does not require unanimity; however, the existence of a “severe conflict’ among experts will 

preclude a GRAS determination.z/ 

11. APPLICATION OF THE GRAS STANDARD TO RECOMBINANT HUMAN 

LACTOFERRIN FROM TRANSGENIC COWS 

Pharming fails on all three counts of the GRAS standard. Specifically, (1) GRN 000189 fails to 

establish that transgenic cow-produced rhLF presents a reasonable certainty of no harm under the 

intended conditions of use; (2) Pharming fails to cite published studies that credibly support the 

safety of rhLF from transgenic cows; and (3) a severe conflict exists between scientists consulted 

by Pharming and numerous highly qualified scientists with specific expertise in lactoferrin, the 

toxicological significance of glycosylation, immunogenicity of recombinant proteins and other 

subjects pertinent to an evaluation of GRN 0001 89. 

Technical Evidence of Safety 

As the attached scientific assessments state, rhLF is a complex molecule with potent biological 

activity for which a rigorous safety assessment is warranted. In a drug context, extensive clinical 

trials and post-market surveillance are needed to adequately assess the safety of a bioactive 

substance such as rhLF because adverse reactions may not be evident absent extended study. In 

a food context involving comparable conditions of use, an even greater assurance of safety is 

essential due to the general availability of the product and absence of direct medical supervision. 

~ 

~~ ,A, 

In GRN 001 89, Pharming asserts that rhLF from transgenic cows is GRAS for use in sports and 

functional foods and drinks at levels not to exceed 100 mgherving. The assertion that rhLF from 

transgenic cows is GRAS is based on (i) claimed substantial equivalence between rhLF from 

transgenic cows, native human lactoferrin and rhLF from a cGMP fermentation process, and (ii) 

the opinion of an expert panel that rhLF from transgenic cows presents no immunotoxicity or 

other safety concerns. As described in the attached assessments, however, Pharming’s analysis 

fails to adequately address numerous important safety issues, including the following: 

0 Differences of potential toxicological significance between transgenic cow-produced 

rhLF and other types of lactoferrin, including native human lactoferrin and rhLF from a 

cGMP fermentation process. 

The glycosylation pattern that is unique to transgenic cow-produced rhLF is of 

particular concern. Glycosylation can have a significant impact on the function and 

safety of proteins, including impacts on pharmacokinetics, immunogenicity and 

- 51 21 C.F.R. 5 170.30@); 62 Fed. Reg. at 18942-43. 

=F.. - 61 62 Fed. Reg. at 18940-43. 

- 71 See 62 Fed. Reg. at 18939. 

Page 3 of 9 000125 
_-= allergic potential, stability, resistance to thermal or enzymatic degradation and 

specific activity. 

0 The absence of relevant studies sufficient to assess the safety of transgenic cow-produced 

rhLF. 

Only one clinical trial with transgenic cow-produced rhLF is referenced in GRN 

0001 89-an unpublished study with six subjects who consumed two acute 52 mg 

doses of transgenic cow-produced rhLF over a 24-hour period. Other clinical trials 

cited by Pharming involved rhLF from a cGMP fermentation process, which differs 

significantly from transgenic cow-produced rhLF. 

0 

0 

lmmunotoxicity concerns. 

The GRAS submission does not adequately address (i) immunotoxicity risks posed by 

major differences between the composition of transgenic cow-produced rhLF and 

native lactoferrin, and (ii) the possibility that administration of recombinant human 

lactoferrin with bovine glycosylation, along with up to 10% contaminating foreign 

proteins and carbohydrates, may induce recognition of rhLF as a foreign protein with 

resulting cross-reactivity to an individual's native lactoferrin. 

Potential induction or exacerbation of autoimmune disease and generation of antilactoferrin antibodies. 

Published literature cited in the GRAS Notice indicates that (i) lactoferrin is a potent 

immunostimulatory molecule known to induce a systemic immune response in both 

animals and humans, (ii) anti-lactoferrin antibodies are associated with a host of 

serious human autoimmune diseases, and (iii) there is animal evidence suggesting that 

lactoferrin might indeed exacerbate autoimmune disease. These concerns raised by 

these literature references are not adequately addressed. 

0 Other risks associated with extended dosing with any rhLF. 

These include risks of iron-overload in susceptible individuals, iron delivery to ironconstrained pathogens, iron delivery to tumors, systemic amyloidosis caused by 

lactoferrin variants, induced changes to immune function, induction of antibiotic 

resistance and viral activation. 

0 An intended daily dose far in excess of exposure to native LF (which is not equivalent to 

rhLF from transgenic cows in any event). 

The intended daily dose for transgenic-produced rhLF cited by Pharming is up to a 

hundred times higher than that resulting from the levels of native LF claimed to be 

present in saliva. 

0 Concerns relating to the manufacturing of rhLF in transgenic cows. .n 

Page 4 of 9 000126 
_-- Highly controlled systems for production, purification and characterization are 

required to ensure the integrity and safety of complex recombinant proteins. The 

production of rhLF in transgenic cows is not sufficiently controlled to allow for a 

consistent and pure final product that is free from potentially harmful impurities, 

degradation products, and contaminants. The genetic stability of the host expression 

system (transgenic cows) has not been established. 

Based on these and other concerns, the experts consulted by Agennix found that GRN 0001 89 

raised substantial issues and unanswered questions that preclude a finding of safety for the 

intended conditions of use. Indeed, these experts believe that rigorous testing, including clinical 

trials of appropriate size and duration, would be required before transgenic cow-produced rhLF 

could be considered safe for addition to the food supply. 

This last point is particularly important. Although many GRAS determinations have been made, 

and should continue to be made, based on an established battery of animal toxicology studies and 

safety factors that establish safe conditions of use, there are some compounds that must 

necessarily be subject to rigorous clinical testing in order to demonstrate a reasonable certainty 

of no harm, as required by the statute. This especially includes molecules such as recombinant 

human proteins with potent biological activities and toxicities that may not be accurately 

reflected in animal models, and immunomodulatory molecules whose full spectrum of activity 

can only be observed following extended administration and surveillance in humans. We believe 

that rhLF, with its potent biological effects demonstrated in Phase I1 clinical trials in cancer and 

diabetic ulcers, is one such substance. 

Past examples of compounds that also warranted significant clinical testing data include artificial 

sweeteners and fat substitutes, so FDA has ample precedent to require such significant clinical 

testing. Even though these other examples were in the context of a food additive petition, the 

legal standard for showing technical evidence of safety, as noted on page 2 above, is exactly the 

same for food additives and GRAS substances. We believe, as a matter of scientific evidence, 

that extensive clinical trials are needed for rhLF in order to adequately investigate the critical 

question of how humans will react to this compound under widespread conditions of long term 

use. The scant clinical evidence referenced in GRN 0001 89 does not even scratch the surface of 

what is needed to meet the statutory standard and protect public health. 

=-- 

The concerns stated above are serious and should preclude GRAS status for any sports or 

functional food or drink application of rhLF from transgenic cows. These concerns are even 

more pressing in light of the perceived therapeutic uses for which transgenic cow-produced rhLF 

might be consumed. Public statements of Pharming indicate that rhLF from transgenic cows will 

be expressly or implicitly promoted for therapeutic uses that are functionally indistinguishable 

from proposed drug uses undergoing critical evaluation by FDA’s Center for Drug Evaluation 

and Research (CDER). Pharming’s website states clearly that “human lactoferrin (hLF) is a 

natural protein that helps to fight and prevent infections and excessive inflammations and 

strengthens the defense system of the human body. . . and has been shown to fight bacteria that 

cause infections of the eye and lungs . . . which makes it a good candidate for a number of 

product applications. Since the protein has the ability to bind iron, is a natural anti-bacterial, antifungal and anti-viral, is an antioxidant and also has immunomodulatory properties, large groups 

of people might benefit from orally administered lactoferrin.” These statements suggest 

~~ 

-. 

Page 5 of 9 0 0 0 12 ‘1 
=- pharmaceutically relevant activities. A copy of Pharming’s web page containing these 

statements is provided in Appendix Volume 1. 

We recognize that the regulatory classification of a product as a “drug” due to an intent to treat, 

prevent, cure, or mitigate disease does not typically factor into an assessment of whether the 

product meets the GUS standard (i.e., assessment of whether a product is a “drug” under 

section 20 l(g) of the Federal Food, Drug, and Cosmetic Act is not usually considered as part of a 

GRAS assessment conducted under section 201(s)). By law, however, the safety of a substance 

that will be added to food is to be assessed in light of its intended use, taking into account its 

“probable consumption.” Based on Pharming’s apparent intent to market rhLF from transgenic 

cows for its pharmaceutical or pharmacological benefits, it is entirely appropriate for CFSAN to 

consider the unique types of harms that may result from individuals consuming rhLF for 

perceived therapeutic effects in potentially unlimited doses for unlimited periods of time. s/ 

Indeed, the law requires consideration of these factors, as GRAS for a food compound must be 

shown “under the conditions of its intended use.” Moreover, a GRAS determination for rhLF 

should require a safety assessment even more rigorous than that required by CDER, to account 

for general availability of the substance without prescription or ongoing medical supervision. 

The Common Knowledge Element-Publication 

Pharming cites no published studies that support the safety of its rhLF from transgenic cows. All 

of the studies cited by Pharming are either unpublished or are not applicable to rhLF from 

transgenic cows, including studies conducted with rhLF from a cGMP fermentation process. 

Further, Pharming’s assertions concerning the substantial equivalence of rhLF from transgenic 

cows to native human lactoferrin or rhLF from a cGMP fermentation process are unfounded in 

light of published information to the contrary concerning such biologically important features as 

glycosylation and specific contaminants. Accordingly, GRN 0001 89 fails to satisfy the second 

element of the GRAS standard-demonstration that key studies and information supporting the 

GRAS determination are generally available to qualified experts. 

The Common Knowledge Element-Severe Conflict Amonp Qualified Experts 

Finally, Pharming clearly fails to satisfy the third element of the GRAS standard-demonstration 

that the safety of the proposed use of rhLF from transgenic cows is the subject of expert 

consensus. Consensus is lacking because more than a dozen experts qualified by scientific 

training and expertise to evaluate the safety of transgenic cow-produced rhLF do not consider it 

to be safe or generally recognized as safe for use in food. 

As discussed in FDA’s GRAS proposal and the pertinent case law, a proponent of a GRAS claim 

bears the burden of establishing expert consensus (i.e., that experts “generally” consider the 

ingredient at issue to be safe). The courts and FDA have interpreted this to mean that, although a 

mere divergence of views will not necessarily preclude GRAS status, as “even properly 

F” - 81 These concerns are exacerbated by the possibility that rhLF may be used disproportionately by 

susceptible groups including immunocompromised individuals, those with systemic infections and infants. 

000128 

Page 6 of 9 
-n conducted studies may produce disagreement,” p/ a “severe conflict” of expert opinion will 

prevent a finding of general recognition. @/ ~ 

There is no bright-line test for identifying what constitutes a “severe conflict,” but courts have 

found a “severe conflict” to exist after evaluating the merits of each situation. In one case, even 

where the proponent of a GRAS claim presented the testimony of seven experts supportive of 

GRAS status, general recognition was found to be lacking in light of persuasive opposing views 

offered by “several” government experts.J/ In another case, “sharply divided testimony” was 

found to present a severe conflict of opinion. u/ Expert testimony critical of general 

recognition in that case suggested that the studies presented did not prove safety or efficacy and 

that the studies were not “well controlled” within the meaning of FDA’s regulations. lJ/ 

Although these and other cases addressing expert consensus involve drug products, the expert 

consensus standard is the same for both food products and drugs. u/ For both food products 

and drugs, determining whether there is a meaningfbl and substantive dispute is key. 

Expert credentials are also important when assessing whether expert consensus exists. In one 

case evaluating the status of a drug for the treatment of various vaginal infections, the court gave 

great weight to the opinions of several chairmen of leading Obstetrics and Gynecology 

departments. The court stated that “it cannot be denied that the affidavits of five of the leading 

doctors in the field which deny general recognition creates more than a ‘mere’ conflict . . . [i]t is 

inconceivable that a drug such as this could be considered generally recognized in the face of 

such learned non-recogni tion.”&/ 

- 91 See, e.g., United States v. Articles of Food and Drug. . . “Coli-Trol80”, 5 18 F.2d 743,746 (5th Cir. 

1975). 

- 101 62 Fed. Reg. at 18939 (citing United States v. Articles of Drug . . . 5,906 boxes, 745 F.2d 105, 1 19 n. 

22 (1st Cir. 1984); United States v. An Article of Drug. . . 4,680 Pails, 725 F.2d 976,990 (5th Cir. 1984); 

Premo Pharmaceutical Lab. v. Unitedstates, 629 F.2d 795, 803 (2d Cir. 1980); CoIi-Trol80,518 F.2d at 746 

(5~ Cir. 1975); United States v. Articles of Drug. . . Promise Toothpaste, 624 F.Supp. 776,782 (N.D. Ill. 

1985), afd 826 F.2d 564 (7” Cir. 1987)). 

- 111 See, e.g., Pails, 725 F.2d at 990 (holding that presentation by the United States of the views of 

“several experts” that a drug was not generally recognized as effective showed a “severe conflict” in the expert 

testimony and precluded general recognition). 

- 121 United States v. An Article ofDrug . . . X-Otag Plus Tablets, 441 FSupp. 105, 113-1 14 (D. Colo. 

1977). 

- 131 Id. at 113. 

141 

GUS standard under section 201(s) of the FFDCA). 

- 151 

See, e.g., 62 Fed. Reg. at 18938-18939 (citing drug and food precedent in discussion of meaning of 

=&-% 

United States v. An Article ofDrug . . . “Mykacer?”, 345 F.Supp. 571 (N.D. Ill. 1972) 

Page 7 of 9 000129 
Finally, the general quality of the evidence on which expert consensus is suggested to be based is 

also relevant. A lack of general recognition was found in one case where expert witnesses knew 

of no studies supporting a finding of general recognition and the manufacturer responded with 

“irrelevant or incomplete studies, expert opinions based on these tests or clinical experience (as 

opposed to clinical studies), and the interested opinions of. . . salesmen.” B/ The court stated 

that general recognition is precluded where there is a “lack of the proper reputation for ... safety 

of the food additive among appropriate experts” or “what reputation there is, is not based on 

adequate studies.” u/ Accordingly, even expert opinions lack persuasive value where the 

underlying evidence is weak or incomplete. 

Agennix, the clear worldwide leader in research, development and production of rhLF, has 

consulted leading international experts on lactoferrin, glycosylation, immunogenicity, 

biosimilars and related subjects relevant to the safety of rhLF from transgenic cows. These 

experts include, among others, a pioneer in the field of lactoferrin research, a founder and 

director of a major center for Medical Glycobiology, and an author of more than 200 papers in 

peer-reviewed journals addressing biotechnology-derived therapeutic proteins (with a recent 

emphasis on biosimilars and the immunogenicity of therapeutic proteins). These highly qualified 

experts have expressed serious concern regarding the safety of rhLF from transgenic cows, 

demonstrating a “severe conflict” of expert opinion. Although the opinions of one or two of 

these experts would be compelling, the opinions of more than a dozen experts concurring in the 

attached scientific assessments unambiguously demonstrates a “severe conflict” that precludes 

GRAS status. 

In summary, the clear lack of scientific consensus that rhLF is GRAS is evidenced by the 

published literature raising legitimate safety questions and by the views of Scientific experts 

whose opinions are expressed in the attached scientific assessments. That so many, and such 

highly qualified, experts have expressed serious concern about the proposed use qualifies as a 

“severe conflict” of expert opinion and precludes GRAS status for rhLF fiom transgenic cows. 

111. CONCLUSION 

After carefully reviewing GRN 000 189 and consulting with leading experts qualified to judge 

the complex safety issues raised by rhLF from transgenic cows, it is our view that Pharming 

presents insufficient data and information to reach any credible conclusion about the safety of 

transgenic cow-produced rhLF in humans. In view of the documented biologic activity of rhLF 

and its ability to induce clinically significant changes in immune function, we believe that 

transgenic cow-produced rhLF has not been shown to be safe for use in food products under the 

anticipated conditions of use, and that there is a severe conflict among qualified experts 

regarding its safety. Accordingly, we ask that FDA respond to this GUS Notice by concluding 

that an adequate basis for a GRAS determination has not been provided. 

- 161 

- 171 Id. at 746. 

“Coli-Troi 80 ”, 5 18 F.2d at 747. 

Page 8 of 9 000130 
Agennix appreciates CFSAN’s consideration of this important information as Pharming’s GRAS 

exemption claim for rhLF from transgenic cows is considered. Please do not hesitate to contact 

us if there are any questions or if additional information would be useful. 

Sincerely, 

Rick Barsky 

Chief Executive Officer 

Cc: Robert Merker, Ph.D. (HFS-255) 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

-- 

Page 9 of 9 
00013% 
- E=- =* 

SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PURIFIED FROM THE MILK OF TRANSGENIC COWS: 

SCIENTIFIC ASSESSMENT OF GRAS NOTICE NO. 000189 

SUBMITTED BY PHARMING GROUP, N.V. 

We have been asked to review data and information presented in GRAS Notice No. 

000 1 89 concerning the safety of recombinant human lactoferrin (rhLF) produced in 

transgenic cows. This Notice asserts that transgenic cow-produced rhLF is generally 

recognized as safe (GRAS) within the meaning of the Federal Food, Drug, and Cosmetic 

Act for use in sports and functional foods at levels not to exceed 100 mglserving. The 

assertion that transgenic cow-produced rhLF is GRAS is based on (i) claimed substantial 

equivalence between transgenic cow-produced rhLF, other forms of recombinantly 

produced human lactoferrins and native human lactoferrin derived from human milk, and 

(ii) opinions of an expert panel that transgenic cow-produced rhLF presents no 

immunotoxicity or other safety concerns. 

RhLF is a complex molecule with potent biological activity for which a rigorous safety 

assessment is warranted. We are aware of Phase I1 clinical trials in which rhLF from a 

cGMP fermentation process was found to meet efficacy endpoints for indications such as 

non-small cell lung cancer. The drug demonstrated promising anti-cancer activity 

although adverse events were observed. Considering the anti-cancer activity observed 

with rhLF, and its early promise as a novel and effective anti-cancer drug, extensive 

clinical trials will be required to evaluate its safety as a drug prior to its being made 

available to patients under a doctor’s prescription. Post-market surveillance will also be 

required to adequately assess its safety following administration to a larger patient 

population. RhLF’s use as a cancer drug involves its use under medical supervision by 

patients who have few alternatives available and who will receive anti-cancer therapy for 

a limited period of time. In contrast, allowing rhLF to be marketed in a food context will 

enable its consumption by a much larger number of people for unlimited periods of time 

and without medical supervision or post-market surveillance. Thus, an even greater 

assurance of safety is required than would be needed prior to rhLF’s approval as an anticancer drug. 

=P% 

The conditions of use for transgenic cow-produced rhLF as described in the Notice are 

assumed to be comparable to likely drug uses because the identified “sports and 

functional food” categories are consumed for perceived effects on bodily structures or 

functions (as opposed to technical effects in food processing). The rhLF will be 

recommended for consumption at substantial dosage levels, and the Notifier has implied 

that the products will be marketed for express or implied benefits of a pharmaceutical 

nature. 

In our expert opinion, the Notice raises substantial issues that preclude a finding of safety 

(i.e., a reasonable certainty of no harm) for the conditions of use, and that warrant further 

investigation, including clinical trials of appropriate size and duration. Of particular 

concern are the absence of adequate clinical trials with transgenic cow-produced rhLF 

-e-.= required to assess differences of possible toxicological consequence between transgenic 

Page 1 000133 
n 

cow-produced rhLF and other forms of lactoferrin, and failure of the Notice to 

sufficiently address a wide range of safety risks, including risks arising from long-term 

exposure to this highly active immunomodulatory agent. We also note that the intended 

daily dose substantially exceeds exposure from native human lactoferrin. This 

assessment addresses the folIowing specific concerns: 

1. 

2. 

3. 

4. 

5. 

Comparison of the intended daily dose of transgenic cow-produced rhLF to 

normal exposure to human lactoferrin , 

Absence of adequate safety studies conducted with transgenic cow-produced 

rhLF, 

Specific glycosylation risks with transgenic cow-produced rhLF, 

Potential long-term immunological risks with any rhLF, and 

Other risks associated with extended dosing with any rhLF. 

We also note what appear to be substantial safety concerns relating to the manufacture of 

transgenic cow-produced rhLF as described in GRAS Notice No. 0001 89. These 

concerns are addressed in detail in Attachment A and include the following 

observations: 

Genetic stability of the host organism is insufficiently characterized or controlled. 

Production and purification processes are not adequately controlled. 

Product characterization including glycosylation, secondary and tertiary structure, 

degradants, aggregation and contaminants is incomplete and does not provide 

assurances that product differences or contaminants will not pose safety risks. 

Long-term stability data are insufficient to guarantee the integrity of the product 

as it is intended for market. 

0 

For these reasons, we conclude that available information fails to establish transgenic 

cow-produced rhLF as presenting a reasonable certainty of no harm under the claimed or 

probable conditions of use. Specific data and information supporting this conclusion are 

presented in detail below. 

1. Comparison Of The Intended Daily Dose Of Transeenic Cow-Produced RhLF to 

Normal Exposure to Human Lactoferrin 

The GRAS Notice states that transgenic cow-produced rhLF is intended for use in sports 

and functional foods at the following doses: 

Product Content: 

Estimated Maximum Daily Consumption: 

100 mg per serving 

2 14 mg per person 

The Notifier bases these estimates on projected 2-day consumption averages for 

representative food products based on USDA data collected as far back as 1994. 

However, these projected consumption levels may substantially underestimate the doses 

received by significant population sub-groups such as athletes or those who might 

perceive a health benefit related to greater consumption of rhLF. Since there is no 

Page 2 000134 
reliable means of limiting the consumption of food products containing rhLF, a safe dose 

must consider the potential for excess consumption by some segments of the population. 

The Notifier’s projected consumption levels are comparable to the levels effectively 

administered to patients in clinical trials supporting rhLF’s intended approval as a 

pharmaceutical drug. These pharmaceutically effective doses were as low as 250 

mg/day. Additionally, the total cumulative dose received over the course of months and 

years must be considered. 

Species (n=) Duration Dose Reference 

Neonatal 24 14 Days 0,10,100,1000 and (Unpublished) 

Rats Per 6000 mg/kg/day t.i.d. Notifier’s rhLF 

group 

Rats 20 91 Days 0,200,600, and 2000 (Unpublished) 

Per mg/kg/day Notifier’s rhLF 

group 

Acute 10 4 hrs 5.0 g/m3 aerosol (Unpublished) 

Inhalation Notifier’s rhLF 

The GRAS Notice further proposes using a bolus of transgenic cow-produced rhLF that 

results in a daily dose that is up to a hundred times higher than the levels of native 

lactoferrin that are normally consumed in saliva. It should also be noted that native 

lactoferrin levels in saliva from adults may be as low as 3.4 &mL, a level ten times 

lower than that assumed by the Notifier (Lentner 198 1). Notwithstanding the Notifier’s 

assertions about the safety of endogenous hLF, it must be emphasized that the oral dose 

of hLF consumed in saliva represents a homeostatic level and that the safety of disrupting 

this homeostatic equilibrium through the introduction of exogenous lactoferrin, whether 

hLF or rhLF, cannot be assumed. 

Location 

Page 38 

Page 38 

Page 42 

Such large doses of transgenic cow-produced rhLF have never been adequately safety 

tested in humans, either in the Notifier’s intended uses or for extended periods of time. 

The pharmacological effects of large doses of transgenic cow-produced human lactoferrin 

are not fully understood and could pose health risks, including those related to significant 

and sustained changes in immune function. 

The GRAS Notice does not present adequate data to address these potential safety risks. 

2. Absence Of Adequate Safetv Studies Conducted With Transgenic Cow-Produced 

RhLF 

GRAS Notice 0001 89 presents inadequate data from safety studies with rhLF produced in 

transgenic cows. The following tables summarize all of the studies conducted with the 

Notifiers’s transgenic cow-produced rhLF cited in the GRAS Notice. 

Page 3 00013’i 
Study 

(Species) 

Allergenicity 

(Guinea Pig) 

Dermal 

Irritation 

(Rabbits) 

Eye 

Irritation 

(Rabbits) 

(n=) Duration Dose Reference Location 

15 24- 48 30% or 10% rhLF in (Unpublished) Page 40 

3 1- 72 hrs 1000 mg topical (Unpublished) Page 41 

hrS saline - topical Notifier’s rhLF 

Notifier’s rhLF 

3 1- 72 hrs 12 mg topical (Unpublished) Page 42 

Notifier’s rhLF 

* In addition to these studies, unpublished data was presented asserting negative results 

from a series of in vitro and in vivo genotoxicity assays. 

Species 

Humans 

The data presented in GRAS Notice 0001 89 are insufficient to substantiate the safety of 

transgenic cow-produced rhLF. While a series of unpublished preclinical studies and 

assays are cited, the GRAS submission references only one human study with the 

Notifier’s transgenic cow-produced rhLF. This unpublished study had only six subjects 

who were administered two acute 52 mg doses over a 24 hour period. This study is not 

sufficient to establish the safety of a highly bioactive compound such as rhLF. The total 

dose level administered in this study amounts to only one half the dose the Notifier 

predicts may be consumed in food in a single day. These data are also inadequate to 

assess the safety risks of long-term rhLF administration. In fact, to demonstrate safety 

one should administer higher than normal doses (rather than lower than normal doses) 

over an extended period to large numbers of people to observe whether there might be 

long-term effects in a subset of the population. 

(n=) Duration Dose Reference Location 

6 24 hrs 52 mg b.i.d. for 1 day (Unpublished) Page 43 

Notifier’s rhLF 

The toxicity and immunogenicity studies performed by the Notifier were conducted in 

rats, guinea pigs and rabbits. These studies are not relevant, since these animals cannot 

mimic the human immune response to human glycoproteins (Descotes 2004). 

The Notifier also incorrectly asserts the safety equivalence of various forms of lactoferrin 

including transgenic cow-produced lactoferrin, natural human lacto fenin, raw human 

milk, rhLF produced by cGMP fermentation techniques in Aspergillus, rhLF produced in 

rice and raw rice grain containing rhLF. There are substantial differences between these 

various compounds and alternate forms of lactoferrin, including differences in 

glycosylation that may present significant health risks, Some of the glycans on 

transgenic cow-produced rhLF and the plant glycans on rice-produced rhLF, for example, 

are known to be both highly allergenic and immunogenic in humans. 

Page 4 
The Notifier asserts that the primary reason that so few clinical and preclinical studies 

have been undertaken to evaluate the safety of lactofernin is because there is “a general 

consensus among experts that hLF has been shown to be safe - via natural exposure - 

and at such high doses that no additional safety evaluation is necessary” (Notifier’s 

Submission page 30). This statement is not correct. There is clearly no scientific 

consensus on the safety of recombinant human lactoferrin. Natural hLF has a minimal 

history of safety testing in humans because the price, at over $3,600 per gram (SigmaAldrich 2006), has precluded any broad-based clinical evaluation. Furthermore, a safety 

claim based on native hLF (which is in itself a compound with different glycosylation 

than rhLF produced in transgenic cows) “via natural exposure” is irrelevant since the 

Notifier’s intended use is based on deliberate external administration, not natural 

exposure. Other than suckling infants, humans normally do not consume human 

lactoferrin in breast milk. 

~~ =-

Lactoferrin is a glycoprotein -- it has both polypeptide backbone and many covalently 

attached carbohydrates. The human lactoferrin that the Notifier is actually referring to as 

its product is a recombinant form made in transgenic cows and therefore has the 

characteristics of bovine glycosylation rather than human glycosylation. The foreign 

glycosylation of a human protein creates, in effect, a new molecule that carries the risk of 

immunotoxicity. Neither infants nor adult humans have ever been naturally exposed to 

recombinant human lactoferrin transgenically produced in cows or to any other form of 

rhLF. 

--+ Based on this faulty presumption of equivalence, the Notifier presents 25 additional 

references (see pages 59-70 of the Notifier’s submission) for various clinical and 

preclinical studies involving alternative forms of lactoferrin. By ignoring the substantial 

differences between its rhLF and other lactoferrin products, the Notifier avoids the fact 

that extremely limited safety studies have been done with its rhLF. The data with 

substantially different lactoferrin products is not relevant to any safety assessment of 

rhLF produced in transgenic cows. 

For example, 11 out of 25 references provided by the Notifier utilize pharmaceutical 

grade rhLF produced by Agennix, Inc. using established fermentation techniques under 

cGMP conditions. Four of these references (Andersen 2004, Hayes 2003, Hayes 2004, 

VAMC 2003) are redundant and cover data generated within the same study. An 

additional 7 of the 25 references (Davidson 1987, Davidsson 1994, Goldblum 1989, 

McMillan 1977, Spik 1982, Davidson 1990, Lindberg 1997) involve the use of natural 

human milk (not pure lactoferrin) and were not safety studies. Finally, the remaining 

studies referenced involved native human lactoferrin, rice grain expressing human 

lactoferrin or rice-produced rhLF, which in itself may present specific safety risks related 

to its plant glycosylation. Thus, these studies presented by the Notifier to assert the 

safety of transgenic cow-produced rhLF are inapplicable. 

In spite of the Notifier’s insistence of equivalence between the alternative forms of 

lactoferrin used to support the safety claims in its GRAS submission, substantial and 

material differences exist between these compounds: 

Page 5 

00013’7 
---: 

0 

0 

Transgenic cow-produced rhLF contains glycans not found on natural hLF (Van 

Berkel2002). 

Transgenic cow-produced rhLF has up to 6 amino acid differences compared to 

native hLF (GUS Notice page 12). 

Transgenic cow-produced rhLF has completely different glycosylation from rhLF 

produced in rice. 

Transgenic cow-produced rhLF has completely different glycosylation from rhLF 

produced by fermentation in Aspergillus. 

Furthermore, as documented by the Notifier, rhLF from transgenic cows is contaminated 

with bovine lactoferrin (GRAS Notice page 18 and 20). The degree of contamination 

varies from batch to batch and could be as much as 10% with bovine lactoferrin being the 

major contaminant. The effect of mixing bovine lactoferrin with rhLF produced in 

transgenic cows as an administered drug is clouded with uncertainty, since no 

combination studies have been performed and the variable combination of the two may 

have an unpredicted result. 

“Biosimilar” Therapeutic Proteins Are Not Identical 

Recombinantly produced “bio~imilars’~, such as these alternative forms of lactoferrin, are 

currently regarded by FDA as different molecules requiring independent safety testing 

and independent regulatory treatment. 

The biological activities of protein therapeutics are invariably closely linked to the 

processes used to make them. The safety, purity, and potency of a biologic therapeutic 

are ensured - to this day - by maintaining the constancy of the result of each step in the 

production process. Analytical science continues to improve. Nevertheless, recombinant 

protein therapeutics cannot be completely characterized, and their behavior in human 

patients cannot be predicted with certainty from a comparison of chemical and biological 

analyses in the same way that “small molecules” can. The current regulatory frameworks 

established by statute are based upon and reflect these fundamental scientific differences. 

Moreover, as a protein becomes larger and more complex, the structural variability and 

the analytical uncertainties increase, further increasing the differences between even 

identical products manufactured using different processes. Other than antibodies, rhLF is 

substantially more complex than most other recombinant therapeutic proteins (Table 1). 

Page 6 

000138 
Table 1. Comparison between Lactoferrin and Other Therapeutic Recombinant 

Proteins 

Insulin 

Amino Acids 52 

Molecular 5,800 

Erythropoietin Interferon G-CSF Growth rhLF 

alpha Hormone 

(Somatotropin) 

165 165 174 191 692 

21,000 19,27 1 18,800 22,000 76,26 1 

Weight 

Disulfide 

Bonds 

Metal 

coordination 

Glycosylation 

Clinical evaluation is not simply the “gold standard” for monitoring the safety and 

efficacy of biologics - it is the sine qua nun of developing and commercializing a 

recombinant protein therapeutic. The human immune system is more sensitive than any 

available analytical method to subtle changes in protein products. Its behavior cannot be 

effectively modeled or predicted based on in vitro analytical data and bioassays. 

3 2 2 2 2 16 

No No No No No Yes 

No Yes No No No Yes 

Changes in glycosylation can have profound effects on the safety and efficacy of 

recombinant proteins and relevant changes can occur even between closely related host 

cell species. FDA has already indicated that “biosimilar” therapeutic proteins (like 

lactoferrin) produced in different expression systems may not be approvable (as 

medicines) without independent clinical evaluation (FDA Draft Guidance for Industry: 

Comparability Protocols - Protein Drug Products and Biological Products - Chemistry, 

Manufacturing, and Controls Information. September 2003, page 8). 

Although definitive guidelines still need to be finalized, there is a general opinion that 

even where products are derived from the same gene and the same host cells using 

identical down-stream processing, large side-by-side clinical comparisons are essential to 

establish whether recombinant proteins are similar (EMEA/CPMP/3097/02 Guidelines). 

This position has recently been made law in the European Union and has several 

implications for transgenic cow-derived human lactoferrin: 

0 GUS designation of a complex recombinant protein like rhLF is by definition 

difficult to establish because data derived from a specific product usually cannot 

be generalized to class level. 

Data not generated with a specific recombinant product as it will be marketed 

cannot typically be used to support the safety of such a product. 

A market authorization of rhLF from a specific manufacturing process should be 

granted only on the basis of extensive safety testing of the product as it is 

manufactured for marketing. 

0 

0 

Page 7 

000139 
In the Notifier’s comments on page 50 of the GRAS Notice, it is implied that the 

glycosylation differences between transgenic cow-produced rhLF and native human 

lactoferrin represent only a minor potential safety issue. In fact, these differences could 

pose a major safety issue. The nature of the glycans on transgenic cow-derived human 

glycoproteins could indeed have catastrophic consequences on the human immune 

system and induce a plethora of immune responses. Unfortunately, as indicated above, 

the Notifier does not report characterization of the glycans on its transgenic cowproduced rhLF, which is highly concerning. A few scientific examples illustrating the 

possible effects of transgenic cow-derived human glycoproteins on human immune 

responses and biology are provided below. 

-??& 

~~ 

Cows glycosylate proteins in their milk in ways that are distinct from human 

glycosylation, such as using N-glycolylneuraminic acid (NeuGc) (“Hanganutziu-Deicher 

antigen“ (Asaoka 1994)), which is a potent antigen, instead of N-acetylneuraminic acid. 

In fact, it is stated in the scientific literature that “In general, normal human tissues yield 

only NeuAc, while other mammals have significant levels of NeuGc. Despite the small 

structural difference between them (the methyl group in NeuAc being substituted by a 

hydroxymethyl group in NeuGc), the two derivatives do not appear to be biologically 

equivalent” (Moreno 1998). Humans lost the gene required for synthesizing NeuGc 

many millennia ago (Varki, 200 l), thus creating the situation where, when attached to a 

human glycoprotein, it is considered as a “foreign” substance that invokes immunity. 

Bovine glycoproteins also contain the LDN or LacdiNAc antigen and the alpha-Gal 

antigen (Gal(alpha)l-3Gal-R), both of which are lacking in human milk glycoproteins 

(Coddeville 1992, Nakata 1993). Both LDN and alpha-Gal carbohydrates are potent 

antigens. They are also expressed by several parasites and are involved in host immunity 

to parasitic infections (Die I and Cummings RD 2006). The alpha-Gal antigen is hrther 

known to cause transplant rejection of non-human derived tissues in people (Chen 2006). 

-- 

The specific glycosylation of human lactoferrin produced in transgenic cows can have 

major consequences on its biological activity and stability. As the Notifier’s own 

scientist published in 2004 (van Veen 2004), glycosylation contributes to proteolytic 

stability of lactoferrin, but more importantly, bovine and human lactoferrin differ in their 

susceptibility to proteolysis based on glycosylation. 

Since human lactoferrin has demonstrated potent biologic activity in humans, it is crucial 

from a safety standpoint to fully understand the impact of changes in glycosylation. On 

page 50 of the GRAS Notice, there is a troubling misstatement by the Notifier’s scientific 

expert who declares that “carbohydrate structures (glycans) are not generally considered 

to be allergens”. This statement is factually incorrect. In reality, carbohydrates are 

considered among the strongest antigens and allergens and many published studies show 

that the types of carbohydrates likely to be found on transgenic cow-derived human 

glycoproteins are potent inducers of antibody responses, including IgE (Leino 2006, 

Ahrazem 2006, Chow 2005). The reason for the allergic responses to animal- and plantderived carbohydrate antigens is beginning to be better understood, and there is 

Page 9 
increasing concern about the safety of carbohydrate antigens of the type found on some 

bovine glycoproteins. 

As previously stated, carbohydrate antigens are highly allergenic. For example, a recent 

review notes that bee venom phospholipase A2 carries an N-glycan containing the a( 1 ,3)- 

linked fucose, and several T-cell clones have been identified from bee venom-sensitized 

individuals that proliferate in response to honey bee PLA2, but not to its nonglycosylated variants, providing evidence for the involvement of N-glycans in T-cell 

recognition (Die I and Cummings RD 2006). The allergenicity of carbohydrate moieties 

has been documented, and the Notifier’s expert’s statement is contradicted by a wealth of 

scientific publications (Breiteneder 2005, Fotisch 200 1 , Betenbaugh 2004). 

Carbohydrate structures on N-glycans and 0-glycans that occur on many glycoproteins 

from non-human origins are associated with significant human immune responses and are 

also probably important in disease pathogenesis in the case of microorganisms 

(Anderson 2003, Die I and Cummings RD 2006, Foetisch 2003, Malandain, 2005, 

Nyame 2004, Showalter 2001). 

Besides being antigenic and allergenic, carbohydrate moieties of glycolipids and 

glycoproteins in plants, animals and humans have very high biological activity and are 

involved in a tremendous range of biological processes including cell-cell adhesion, cellcell signaling, immune regulation, innate immunity, and cell biological phenomena 

including organelle biosynthesis (Bertozzi 200 1 , Engering 2002, Feizi 2000, Freeze 2005, 

Gu 2004, Helenius 2004, Rudd 2004, van Kooyk 2004). 

Thus, the presence of bovine carbohydrate antigens on rhLF produced transgenically in 

cows raises the possibility that they may not only induce immune responses, but may in 

fact interfere with and act as antagonists in regard to the biology of human cells involving 

endogenous carbohydrate moieties. 

c-5 

Finally, and contrary to the Notifier’s conclusions, carbohydrate moieties have 

increasingly been implicated in the immunogenicity of recombinant proteins (Die I and 

Cummings RD 2006, Hermeling 2004, Schellekens and Casadevall2004). It has been 

shown that there are significant issues to address in regard to immunogenicity and 

antibody formation with recombinant proteins, such as insulin, interferon, epotin alfa and 

others. Interestingly, the immunogenicity to recombinant proteins is independent of the 

route of administration (Schellekens 2003). 

Given that the role of carbohydrates as recognition elements in biology is well 

understood, there is no justification to ignore evidence that foreign glycoforms will have 

an effect on transgenic cow-produced rhLF’s safety. To summarize, dismissal of the 

safety risks relating to glycosylation is improper for the following reasons: 

1. Carbohydrate moieties have increasingly been implicated in the immunogenicity 

of recombinant proteins (Schellekens 2004, Hermeling 2004, Die I and 

Cummings RD 2006). Immunogenicity and antibody formation have been noted 

with proteins and glycoproteins, including insulin, interferon, epotin alfa and 

Page 10 000141 
-. 

2. 

other recombinant proteins. Additionally, immunogenicity to recombinant 

proteins is independent of the route of administration. There have been no 

published cases where a change in route of administration completely negated 

immunogenicity (Schellekens 2003). 

The GRAS Notice treats the glycans on transgenic cow-produced rhLF as part of 

a food product rather than structural components of a therapeutic protein vital to 

immune system recognition. RhLF is a biologically active immunostimulatory 

drug that interacts directly with receptors in the gut responsible for regulating 

immune response and inducing maturation of dendritic cells (Varadhachary 2006, 

Varadhachary 2005). Through its effect on the Gut Associated Lymphoid Tissue 

(the largest immune organ in the body), lactoferrin may actually serve as a vector 

to deliver cross-reactive glycans directly to activated antigen presenting and 

immune effector cells. Additional clinical studies are needed to assess these risks 

before any broad safety claims can be made. 

3. The emergence of an antibody response and the breaking of B-cell tolerance 

require prolonged exposure to a recombinant protein, and generally antibodies can 

appear up to one year after chronic treatment (Schellekens 2004). The induction 

of anti-lactoferrin antibodies in people receiving exogenous lactoferrin has been 

documented (Brock 1998) and anti-lactoferrin antibodies have been associated 

with serious autoimmune disease. The induction of anti-lactoferrin antibodies 

could have tremendous consequences by “neutralizing” many other vital functions 

of endogenous lactoferrin, which is a degranulation product of neutrophils 

involved in, among other things, the regulation of inflammation and protection 

against the development of cancer. 

Until robust clinical studies are conducted to determine the effects of long-term exposure 

to bovine glycans delivered by recombinant human lactoferrin to immune cells in the gut, 

no general conclusions can be reached about the safety of transgenic cow-produced rhLF 

as described in GRAS Notice 000 189. 

4. Potential Lone-Term ImmunoloPical Risks with any rhLF 

The Notifier acknowledges a wide variety of biologic activities for human lactoferrin and 

references several studies that elaborate on its anti-viral, anti-microbial, antiinflammatory and immunomodulatory properties (Notifier’s Submission page 28). No 

discussion is included regarding the potential physiological consequences of these 

activities in humans. The Notifier concludes that “most of the biological actions of hLF 

are mediated by the sequestration of iron or by the previously mentioned positively 

charged domain located in the N-terminus” (Notifier’s Submission p. 29). This 

conclusion is incorrect because it pertains only to part of human lactoferrin’s antimicrobial properties. 

Page 11 
Until recently there has been little understanding of the exact mechanism by which 

human lactoferrin mediates its profound anti-inflammatory and immunomodulatory 

activity. Over the last few years, proprietary research conducted with rhLF produced by 

fermentation in Aspergillus, which is under development as an FDA regulated 

pharmaceutical drug for the treatment of non-small cell lung cancer, has revealed that 

human lactoferrin’s mechanism of action is far more complex than previously understood 

and involves all aspects of the molecule’s structure. 

-e- 

~~ 

Based on this research, it has been shown that rhLF is an orally active 

immunomodulatory protein that is not absorbed or systemically bioavailable but that acts 

via the gut and Gut Associated Lymphoid Tissue (GALT) (Figure 1). 

Orally administered rhLF specifically binds receptors on cells lining the upper 

gastrointestinal tract, and induces the production of immunomodulatory cytokines and 

chemokines within the small intestinal Peyer’s Patches (PP), initiating an 

immunostimulatory cascade in the GALT. RhLF induces dendritic cell (DC) maturation, 

and induces the production of key chemokines by enterocytes, including MIP3- 

alphdCCL20, an important chemokine for attracting immature DCs. RhLF also acts 

directly as a chemokine, binding chemokine receptors including IL-8RB (CXCR2) and 

CCR2 and attracting immune cells such as lymphocytes and antigen presenting cells 

(APCs). Oral administration of rhLF also results in the production of key cytokines 

including IL-18 and IFN-gamma in the gut. These cytokines, in concert with the influx 

of immune cells into the PPs, play an important role in stimulating both the innate and the _ -I =- adaptive immunity. 

Oral administration of rhLF in preclinical experiments produced an increase in the total 

cellularity of small intestinal Peyer’s Patches, including an increase in the numbers of 

Natural Killer-T cells and CD8+ T-lymphocytes. RhLF induces the migration of DCs to 

the intestinal Peyer’s Patches and induces DC maturation. The systemic 

immunostimulation induced by orally administered rhLF is demonstrated by an increase 

in NK and CD8+ lymphocyte mediated cytotoxicity, an activation of tumor-draining 

lymph nodes, and immune infiltration into distant tumors. RhLF’s anti-tumor effects are 

lost in animals lacking NK-T or CD8+ cells demonstrating the importance of these 

effector cells in rhLF’s anti-cancer activity. 

Simply stated, oral rhLF binds to specific receptors on gut enterocytes and immune cells, 

resulting in the attraction and activation of key immune cells and the production of key 

immunomodulatory cytokines. This results in the systemic activation of both the innate 

and the adaptive immunity, and the killing of cancer cells. 

Page 12 000143 
-- 

Figure 1: RhLF Mechanism of Action in Cancer 

Oral Talactoferrin 

Mechanism of Action in Cancer 

I 0 Tdactoferrin 0 B Cell I IL-18. IFN, (I Granuioefles 

Infiltration of Distant Tumors and 

Activation of Drainlnp LymDh Nodes 

Agennrzr: Confidential 

The induction of these cytokine and chemokine cascades as well as the systemic 

activation of immune cells has profound implications on the potential safety of long-term 

administration of rhLF, including the possibility of various immune reactions or impacts 

on autoimmunity which can only be assessed by long-term human clinical trials. 

4.1 Immunotoxicity Risks 

Immunomodulatory agents present a distinct risk of immunotoxic effects that require 

careful preclinical and clinical evaluation. A recent review by a noted 

immunotoxicologist summarized these concerns: 

“Immunotoxic effects are divided into four categories: immunosuppression, 

immunostimulation, hypersensitivity and autoimmunity. Each category is 

associated with distinct adverse effects.” 

Historically, concern has primarily focused on immunosuppression and 

hypersensitivity (allergenicity). “However, immunostimulation is also a key 

issue, especially with pharmaceuticals. It is not known whether function assays 

used to predict immunosuppression or hypersensitivity are applicable to the 

Page 13 000144 
prediction of immunostimulation. Most available animal models and assays are 

not valid to assess the potential of immunostimulation, and autoimmunity is not 

predictable at all. Conflicting guidelines and the lack of human data contribute 

further to this situation.” 

“Due to the redundancy of the immune system, a single change is not necessarily 

sufficient evidence for immunotoxicity so that a global assessment of all 

preclinical findings is advisable. Genetic factors also play a major role in immune 

responses. As wide inter-individual variability is unavoidable, sufficient numbers 

of animals, a cautious comparison with both study and historical controls, and the 

possible use of inbred or genetically modified animals are to be considered. 

Marked interspecies differences in the immune system [should] lead to the use of 

more than one animal species and to confirm animal data during clinical trials. 

The inclusion of certain immune endpoints applicable to animals and man is 

therefore essential.” 

“Due to the many adverse effects reported in man, the immunotoxicity potential 

of every new molecular entity should be systematically and specifically 

evaluated.” (Descotes 2004). 

The long-term effects of immunostimulation by rhLF (from any source) have never been 

evaluated, either in animals or in human clinical trials, Given the evolving scientific 

understanding of the consequences of immune system manipulation and the long-term 

potential for unknown immunotoxic effects with immunostimulatory agents, caution is 

warranted. Published FDA Guidelines support this approach stating: 

-- e- 

“Change in an immune function or level of immunological mediator may not 

necessarily appear as an “adverse effect”, but rather as immunostimulation. 

Caution must be exercised in such cases, because a non-specific enhancement of 

the immune response that might be interpreted as a beneficial effect may result in 

suppression of specific immunity against a particular infection. A decision on 

whether a material/device is immunotoxic must rely on the available evidence 

from pre-clinical test results and clinical evaluation, as well as prior history of use 

(Guidance for Industry and FDA Reviewers - Immunotoxicity Testing Guidance, 

May 6, 1999). 

TeGenero Case 

The potential risks to humans posed by immunomodulatory recombinant proteins, and the 

need for robust clinical testing before asserting claims of safety, was recently 

demonstrated in the tragic case of TGN14 12 (TeGenero Immuno-Therapeutics). 

TGNl412 is an immunomodulatory antibody that (like rhLF) stimulates T-cell expansion 

and activation. In spite of a strong preclinical safety record, including testing in primates, 

TGN 14 12 produced severe and unexpected reactions in a Phase I clinical trial involving 

seven healthy volunteers. On March 13,2006, six of the volunteers who received the 

Page 14 000145 
- E\ 

active drug suffered organ failure and violent, life-threatening side-effects after being 

administered TGN 14 12. 

A follow-up investigation by the UK’s MHRA (Medicines and Healthcare products 

Regulatory Agency) determined that: 

0 TeGenero observed the highest standards in developing this drug and that these 

symptoms were both unexpected and unforeseeable. Data previously released 

showed that there was no sign of risk from the pre-clinical tests of TGNl4 12. 

Animal study results showed that there were no drug related deaths in the 

preclinical testing of TGN1412, with just one animal having to be euthanized 

during the trial after suffering severe diarrhea caused by an unrelated bacterial 

infection. 

0 

In a press release on March 17,2006, TeGenero stated, “We are shocked about the 

symptoms we have seen in the (clinical trial) volunteers. Extensive pre-clinical tests 

showed no sign of any risk. We observed strict standards for this clinical test and we 

obtained all required approvals both in Germany and Great Britain. The drug was tested 

extensively in laboratories and has been tested on rabbits and monkeys. We saw no drug 

related adverse events and there were no drug related deaths.” 

This case illustrates that the “consequence of interspecies differences is that no one can 

assure that negative (immunotoxicology) results obtained in rats or dogs or both, will also 

prove negative in man. Thus, animal results have to be confirmed in human beings” 

(Descotes 2004). While this appears to be an extreme case of acute immune reaction, it 

serves as a cautionary note, especially when considering the long-term risks of 

immunostimulation by rhLF, which may not surface in such an immediate or startling 

fashion. 

4.2 Risks of Exacerbating Auto-Immune Disease with RhLF 

A specific immunotoxicological risk of long-term administration of rhLF in humans is 

the potential induction or exacerbation of autoimmune disease and the generation of antilactoferrin antibodies, which are often present in patients with autoimmune disease: 

0 Anti-lactoferrin antibodies have been associated with autoimmune liver 

disease (Ohana 1998). 

0 Anti-lactoferrin antibodies have been associated with inflammatory bowel 

disease (IBD) (Roozendaal 1999). 

Anti-lactoferrin antibodies have been associated with Wegener’ s 

granulomatosis (van der Woude 1985). 

0 

0 Anti-lactoferrin antibodies have been associated with rheumatoid arthritis 

(Locht 2000). 

Page 15 000146 
Anti-lactoferrin antibodies have been associated with systemic lupus 

erythaematosus (Galeazzi 1998). 

0 Anti-lactoferrin antibodies have been associated with autoimmune 

pancreatitis (Okazaki 2000). 

The presence of non-human glycosylation on transgenic cow-produced rhLF raises the 

troubling possibility that the carbohydrates may in fact stimulate immunity to the protein 

itself, thereby generating neutralizing and/or inhibitory antibodies that could block 

functions of the protein. In fact, conjugation of such carbohydrates to so-called carrier 

proteins is the modern way to induce protective immunity to parasites and bacteria 

carrying unusual carbohydrates. This is well documented in a recent review citing 

specific examples of vaccine development using carbohydrate-conjugates to proteins 

(Nyame 2004), and lactoferrin is an extremely effective carrier protein. 

Evidence suggests that administration of bovine lactoferrin in mice can produce a 

systemic immune response (Debbabi 1998, Sfeir 2004), and that oral administration of 

human milk proteins containing 40% lactoferrin resulted in the production of IgG, IgM, 

IgA, and anti-hLF antibodies with spleen sensitization (Yuki 1998). Another published 

study showed that ingestion of human lactoferrin by breast-fed human infants produced 

IgG and anti-hLF antibodies in those subjects (Brock 1998). 

The Notifier's evidence that transgenic cow-produced rhLF will not elicit a similar 

immune response is grossly inadequate. The Notifier relies on a single unpublished 

human safety study conducted by the Notifier in which only six adults were administered 

two 52 mg doses of transgenic cow-produced rhLF in a single 24 hour period. Any 

conclusion of safety based on such minimal clinical data is unwarranted and lacks 

credibility. 

In view of the evidence presented demonstrating that lactoferrin can induce an immune 

response in animal models, as well as in human studies involving infants, the safety 

position taken in the GRAS Notice is unsupportable. Data are not presented on the 

impact of transgenic cow-produced rhLF in non-healthy subjects, who would also likely 

consume rhLF from common consumer products. Nor does the Notice address the 

potential impact on patients with conditions that are known to be associated with antilactoferrin antibodies (autoimmune liver disease, inflammatory bowel disease, Wegener's 

granulomatosis, rheumatoid arthritis, systemic lupus and autoimmune pancreatitis). 

Credible evidence to mitigate the known safety concerns relating to lactoferrin and 

autoimmunity is not presented. 

Regarding the pathological significance of anti-lactoferrin antibodies, no evidence is 

presented contradicting the known risks. Anti-lactoferrin antibodies may be associated 

with inflammation of the colon (Roozendaal 1999). In a mouse model of rheumatoid 

arthritis, collagen-induced arthritis was exacerbated in transgenic mice expressing human 

lactoferrin (Guillen 2002). As indicated above, administration of transgenic cowproduced rhLF might induce anti-lactoferrin antibodies due to the carbohydrates acting as 

Page 16 Oc1014'7 
_-= 

-- 

adjuvants. Thus, there is a real concern about the potential complications of oral 

administration of rhLF containing non-human carbohydrates. 

While there is evidence that orally-ingested lactoferrin can induce a systemic immune 

response and that antibodies to lactoferrin could theoretically be involved in disease 

progression, no scientific data with the Notifier’s rhLF has been presented to mitigate 

these concerns. Rather, the facts, as revealed in published literature indicate that: 

1) Lactoferrin is a potent immunostimulatory molecule that has been shown to induce a 

systemic immune response in both animals and humans, including the induction of 

anti-lactoferrin antibodies; 

2) Anti-lactoferrin antibodies are associated with a host of serious human autoimmune 

diseases; 

3) There is animal evidence that lactoferrin might indeed exacerbate autoimmune 

disease. 

No data are presented to rebut these safety concerns. An unpublished, non-statistically 

significant “safety” study in six subjects cannot be considered indicative, much less 

conclusive. 

Despite the known association of lactoferrin antibodies with serious human autoimmune 

diseases, the GRAS Notice fails to address the potential consequences of long-term 

consumption of transgenic cow-produced rhLF in people with these diseases. Moreover, 

while the potential risk of administering rhLF in a high dose oral bolus is real, no data are 

presented on the effects of such administration in humans. Given the documented risk of 

rhLF-induced autoimmune reactions, a conclusion of safety broad enough to authorize 

mass consumption by the general population in unlimited doses without medical 

supervision can only be based on properly controlled, definitive, long-term clinical trials 

in humans. No such trials are included in the GRAS Notice. 

4.3 Induced Changes to Immune Function 

Studies show that lactoferrin can induce a change in immune system function (Zimecki 

2001), including the induction of a TH1 shift. Transgenic mice over-expressing 

lactoferrin show a prominent TH1 immune shift (Guillen 2002). In vitro studies with 

lactoferrin indicate that it suppresses IL-4 and IL- 10 production in respiratory epithelial 

cells obtained from human patients (Abraham 1992). In vivo rodent studies show that 

orally administered lactoferrin is a potent stimulator of IL- 1 8 production in the gut, and is 

thus a stimulator of IFN-g production (Kuhara 2000, Varadhachary 2004). Enhanced 

IFN-g production is associated with the induction of a THl immune response. 

Because of this potential to inhibit IL-4 and IL-10 and stimulate IL-18 / IFN-g, which 

shifts the immune balance from a TH2 to TH1 response, lactoferrin represents a potential 

Page 17 000148 
risk for people with serious THl -associated diseases like multiple sclerosis, type 1 

diabetes and chronic obstructive pulmonary disease (COPD), among others. 

It has also become clear that oral lactoferrin exerts a potent immunostimulatory effect 

including the production of key cytokines in the gut and in systemic circulation, an 

increase in circulating CD4+ and CD8+ cells and an increase in NK cell activity 

(Varadhachary 2004). Lactoferrin has been shown to be effective in stimulating the 

proliferation of a broad range of immune cells in animals (Artym 2005). In vitro, 

lactoferrin has been shown to activate macrophages (Edde 2001) and has been shown to 

induce immune maturation and proliferation of a range of immune cells (Legrand 2005, 

Shau 1992, Dhennin-Duthille 2000). In confidential research conducted by Agennix, Inc. 

and submitted to FDA (IND BB-11728), it was demonstrated that rhLF is extremely 

effective at inducing maturation of Dendritic Cells, the most important class of antigen 

presentation cells. These biological effects are important for pharmacological 

applications of lactoferrin (including the treatment of cancer). However, the long-term 

effect of chronic immunostimulation, including the possible induction of autoimmune 

diseases, is not known. There have been several recent examples of serious adverse 

events resulting from long-term or acute administration of immunomodulatory agents, 

including most recently Tysabri (Drazen 2005). 

5. Other Risks Associated with Extended Dosing with Anv RhLF 

There are numerous other potential risks associated with the consumption of 

pharmacologically relevant doses of rhLF for extended periods of time by the general 

population, without the necessary premarket clinical testing or postmarket surveillance. 

5.1 Toxicity in Individuals with Iron Overload 

As articulated in a standard hematology textbook (Hoffman 1998 ), “Iron overload 

denotes an excess in total body iron resulting from an iron supply that exceeds 

requirements. Because requirements are limited and humans lack a physiological means 

of excreting excess iron, any sustained increase in intake may eventually result in 

accumulation of iron. . . . When the accumulation overwhelms the body’s capacity for 

safe storage, potentially lethal tissue damage is the result.” The most common form of 

iron overload in the US is hereditary hemochromatosis, occurring in as much as 0.5% of 

the population or as many as 1 million individuals (Edwards 1993, Edwards 1988). Other 

forms, which also affect thousands of patients with substantially higher prevalence within 

specific population subgroups, include thallasemias and refractory anemias. Iron 

overload may also follow increased absorption of dietary iron in some patients with 

chronic liver disease, including those with alcoholic cirrhosis and portacaval shunting 

(Jakobvits 1979). Iron overload can proceed asymptomatically for years, with the patient 

often presenting only after severe tissue damage has already occurred. Liver disease is 

the most common complication of iron overload resulting in hepatomegaly, functional 

abnormalities, fibrosis and eventually cirrhosis (Scheur 1962). Hepatocellular carcinoma 

can be an ultimate complication. Diabetes mellitus is a common complication of all 

Page 18 
forms of systemic iron overload (Stremmell988) occurring in 48% of patients. A variety 

of other complications have been reported including such fatal ones as iron-induced 

cardiac disease causing cardiomyopathy with heart failure, arrythmias or both (Model 

1984). 

-~ -- 

The daily requirement of iron is only - 1 mg in adult men and - 2 mg in pre-menopausal 

adult women. In hereditary hemochromatosis, dysregulation of intestinal iron absorption 

occurs, wherein iron continues to be efficiently absorbed even in the face of substantial 

elevation of body iron stores eventually leading to the major morbidities and mortalities 

associated with the disease. Treatment for patients diagnosed with iron load disorders 

involves iron chelation by either regular phlebotomy or with chelating agents. Patients 

are also counseled to avoid foods rich in iron and avoid any iron containing supplements. 

The dietary concern is of course even more critical in the substantial number of patients 

with undiagnosed disease. 

5.2 Iron Delivery to Iron Constrained Pathogens 

Lactoferrin binds iron with a high avidity across a broad range of pH concentrations and 

its ability to deliver iron is an important biological property of this molecule. In 

experiments with human duodenal mucosa, unlike serum transferrin and ovotransferrin, 

lactotransferrin was able to yield its iron to intestinal tissue in a receptor-mediated 

process (Cox 1979). As has been discussed in the literature as a potential concern, 

administration of lactoferrin with its highly bioavailable iron can accelerate growth by 

pathogenic bacteria and protozoa (Weinberg 1978). An outstanding example of this 

involves infections caused by the enteric pathogen, Vibrio vulnificus, most often acquired 

by eating raw shellfish. When ingested by humans with iron-overload, this organism can 

cause rapidly progressing and fatal bacteremia (Wright 198 1). In mice, the LDSO for an 

inoculum of V. vuZnlJicus drops from 1 O6 in normal mice to an estimated 1.1 organisms in 

iron-loaded animals, an impressive 6 log difference. Similar 5 to 6 log differences also 

have been reported for certain strains of E. coli (Eaton 1982). In humans, traumaassociated sepsis, which has often been linked to the ability of otherwise normal 

commensal bacteria to invade and penetrate the gut mucosal barrier, appears to involve 

catecholamine mediated iron removal from lactoferrin and its acquisition by bacteria 

(Freestone 2002). 

fi 

Microbial colonies tend to be iron constrained (Andrews 2003), and access to a source of 

iron can induce infectious flare-up. In fact, a variety of bacteria have evolved a 

mechanism for acquiring iron directly from human lactoferrin. This mechanism involves 

surface receptors capable of specifically binding LF from the host, removing iron and 

transporting it across the outer membrane. The iron is then bound by a periplasmic ironbinding protein, FbpA, and transported into the cell via an inner membrane complex 

comprised of FbpB and FbpC (Elkins 2004). Iron availability is also critical to the 

virulence of A4 tubercuzosis and other mycobacteria that have also evolved a mechanism 

to acquire iron from lactoferrin (Ratledge 2004, Purdey 2006), as well as a variety of 

other pathogenic organisms. 

Page 19 000150 
The importance of iron levels in regulating bacterial growth is best expressed in the B-. following words of an editorial comment (Shock 2002): 

“We all carry around a dangerous sack of goods-intestines filled with so many 

bacteria that they actually outnumber our own somatic cells. A dab of these lively 

intestinal contents released into the body is sufficient to kill any of us. For this 

reason, the guts are a site of constant and vigilant surveillance. . . . The aerobes 

require iron and this requirement is their Achilles’ Heel and, in some cases, our 

major protection. This is because iron is an exceedingly scarce commodity in 

normal mammalian body fluids.. . Some crafty bacteria have evolved iron-binding 

siderophores, such as desferrioxamine or enterobactin, which enable them to steal 

iron from normally safe iron reservoirs, in some cases even from the iron-binding 

proteins, transferrin and lactoferrin. Once this theft has occurred, bacterial growth 

is enabled and, clinically speaking, it is Katy, bar the door.” 

5.3 Iron Delivery to Tumors 

The role of iron as a growth-regulating factor applies more broadly beyond microorganisms. The growth of tumors is also known to be iron regulated (Weinberg 1983), 

and increased dietary iron has been shown to promote colon tumors in mice (Ilsley 2004, 

Hann 1991). Tumor cells are also known to over-express receptors that bind lactoferrin 

with a high affinity. These lactoferrin receptors have been shown to be up-regulated in 

the presence of iron chelators and to deliver iron to the interior of colon carcinoma tumor 

cells (Mikogami 1995). Thus, there is a risk that pre-cancerous or early stage GI tumors 

could also access iron from lactoferrin to accelerate their growth and metastasis. 

-n 

5.4 Risks of Systemic Amyloidosis Caused by Lactoferrin Variants 

In recent studies, lactoferrin variants have been linked to systemic amyloidosis. 

Amyloidosis is an acquired or hereditary disorder related to protein folding. Fragments 

of proteins that are normally soluble are deposited extracellularly where they accumulate 

and form deposits that interfere with the function of effected tissues or organs (Pepys 

2001). These amyloid deposits have been implicated in the pathogenesis of diseases such 

as Alzheimer’s disease, various prion diseases and type TI diabetes. 

Lactoferrin fragments have specifically been implicated as a cause of amyloidosis 

accompanied by trichiasis (a common vision threatening condition of the eyelid). The 

lactoferrin fragment responsible results from a single change from glutamic to aspartic 

acid near the end of the protein molecule (Ando 2003). The association of lactoferrin 

fragments with amyloidosis is of particular concern because all recombinant forms of 

lactoferrin will contain variants and protein fragments that cannot be fully characterized 

or isolated given current technology (see Attachment A). Given that the cited 

production standards for transgenic cow-produced rhLF are already far below cGMP 

pharmaceutical norms, adequate assurance that potentially pathogenic mutant proteins are 

-4-k not present cannot be provided. 

Page 20 
5.5 Induction of Antibiotic Resistance 

Another of lactoferrin's biological properties is its ability to interact with microbial 

membranes resulting in a variety of effects including depolarization. A recently 

published study demonstrated that exposure to rhLF can induce antibiotic tolerance in 

Pseudomonas aeruginosa, an important pathogen responsible for numerous hospital 

infections (Andres 2005). Other negative bacteriological effects may also be associated 

with lactoferrin. For example, it was recently described that exposure of pathogenic 

streptococci to lactoferrin results in the induction of the streptococcal pyrogenic exotoxin 

A (Kansal2005). 

5.6 Viral Activation 

Oral lactoferrin may be involved in viral transmission and the facilitation of viral 

replication. Lactoferrin has been shown to facilitate replication of HTLV-I by upregulating viral expression (Moriuchi 2001). Human lactoferrin in saliva has also been 

shown to act as a ligand for HHV-8, which suggests that orally administered rhLF could 

serve as a carrier for viral particles (Grange 2005). Moriuchi et a1 show that rhLF 

facilitates the replication of HTLV- 1 in lymphocytes derived from asymptomatic HTLV1 carriers and enables viral transmission to cord blood mononuclear cells (Moriuchi 

200 1). Transient expression assays revealed that lactoferrin can transactivate the HTLV1 long terminal repeat promoter. Thus, lactoferrin may enhance vertical transmission of 

this milk-borne retrovirus, which could affect an extremely vulnerable population where 

even a slight risk is unacceptable. 

.r? 

Conclusion 

This scientific panel has reviewed the evidence and arguments presented in GRAS Notice 

No. 0001 89 and concludes that the safety of transgenic cow-produced rhLF has not been 

established. Arguments and data presented by the Notifier's GRAS submission 

highlight-rather that eliminate-known safety concerns. From published data, 

including data referenced in the GRAS Notice, the following may be reasonably 

observed: 

0 Proposed doses of rhLF are far in excess of those naturally occurring in saliva. 

Numerous published studies and recent clinical trials show that, in large doses, 

rhLF has a potent immunostimulatory effect. Notifier has neither conducted 

studies nor published data showing that large doses are safe in humans. Studies 

with Agennix's cGMP fermentation produced rhLF, which is a distinct product 

with a completely different glycosylation, are not relevant to the establishment of 

safety of rhLF produced in transgenic cows. 

0 Published data show that glycans present on transgenic cow-produced lactoferrin 

can induce a systemic immune response, including the generation of IgE 

Page 21 00015% 
antibodies. IgE mediated immune responses are a serious health risk and, in 

extreme cases, can lead to anaphylaxis, or even death. 

0 RhLF’s mechanism is far more complex than previously understood and results in 

broad changes to systemic immune function. The safety impact of these immune 

system changes has not been fully evaluated and precludes an assumption of 

safety until long-term clinical trials have been conducted. 

According to published studies, IgE antibody responses and the breakdown of Bcell tolerance can take up to a year of chronic exposure to an allergen to develop, 

making short-term clinical trials completely inadequate for detecting induced 

allergic sensitization or antibody development. Human exposure to transgenic 

cow-produced rhLF has been extremely limited to date and there is no clinical 

data whatsoever on the effects of long-term consumption. 

Anti-lactoferrin antibodies are known to be associated with, and potentially 

exacerbate, a wide range of serious human autoimmune diseases. Furthermore, 

rhLF is known to induce a potent THl immune response. No studies have been 

conducted to determine the consequences of long-term immunostimulation by 

rhLF in people with autoimmune disease. 

Additionally, since lactoferrin is believed to directly interact with immune cells in the 

gut-associated lymphoid tissue, it is possible that, through receptor binding, transgenic 

cow-produced rhLF may act as a vector to deliver allergenic bovine glycans directly to 

immune cells in gut associated lymphoid tissue. Lastly, there are other potential risks, as 

described above, associated with long-term administration of any rhLF, particularly in 

compromised patient populations. 

F-k 

Transgenic cow-produced rhLF presents numerous documented risks. These risks should 

be thoroughly and scientifically evaluated. The potent immunostimulatory activity of 

rhLF warrants large, controlled, long-term clinical safety studies before broadly exposing 

the public to potentially unlimited consumption. In fact, given the potential effects on 

lactoferrin-associated autoimmune diseases and the long period required to develop 

antibodies, rhLF from any source should be administered only under medical supervision. 

In our opinion, to expose the general public to these well-documented risks, without 

credible clinical safety data on the prolonged use of transgenic cow-produced rhLF, is 

both inappropriate and unnecessary. The designation of transgenic cow-produced rhLF 

as GRAS is inappropriate until it has been shown that the known risks (including, without 

limitation, the risks described in this scientific assessment) pose no threat to public 

safety-i.e., a reasonable certainty of no harm. 

Note: None of the experts listed below are affiliated with Agennix or other parties with a 

commercial interest in recombinant human lactoferrin, but they share the concern that a 

GRAS listing prior to the adequate establishment of rhLF’s safety in the larger population 

is inappropriate, and that unsupervised long-term administration of rhLF poses a 

-~ =-., significant risk to the general population. 

Page 22 000153 

. 
Richard D. Cummings, Ph.D. 

Professor of Biochemistry and Molecular 

Biology, Ed Miller Endowed Chair, 

Director - Oklahoma Center for Medical 

Glycobiology, University of Oklahoma Health 

Sciences Center 

~ 

Dr. Arno Kromminga, M.D. 

Director, Immunology 

Institute for Immunology, Clinical Pathology, 

Molecular Medicine (IPM) 

Hamburg, Germany 

Marco van de Weert, PhD 

Associate Professor Biomacromolecules Group, 

Dept. of Pharmaceutics and Analytical 

Chemistry, The Danish University of 

Pharmaceutical Sciences 

David J.A.Goldsmith, MA MB BChir FRCP 

Consultant Nephrologist - Guy's Hospital, 

London, Honorary Senior Lecturer, 

Guy's King's and St Thomas' Hospitals Medical 

School, King's College, London 

~ Simon David Roger, M.D. 

Director of Renal Medicine, 

Chairman, Medical Staff Council, 

Gosford Hospital, NSW Australia 

Nicole Casadevall, M.D. 

Professor of Hematology 

Hapita1 Hatel-Dieu 

Paris, France 

E.D. Weinberg, Ph.D. 

Professor Emeritus of Microbiology, 

Indiana University 

Huub Schellekens, M.D. 

Professor - Faculty Pharmaceutical Sciences, 

Utrecht University Central Laboratory, 

Animal Institute and Department of Innovation 

Studies, Utrecht University 

Netherlands 

Michael Pierce, Ph.D. 

Professor of Biochemistry and Molecular Biology, 

Director - University of Georgia Cancer Center, 

Director - Complex Carbohydrate Research Center, 

University of Georgia 

Sidney E. Grossberg, M.D. 

Walter Schroeder Professor of Microbiology and 

Molecular Genetics, Professor of Medicine, Medical 

College of Wisconsin 

Professor John Axford, DSc MD FRCP 

Chair of Clinical Rheumatology 

Director of The Sir Joseph Hotung Centre for 

Musculoskeletal Diseases, St George's University of 

London 

Irma van Die, PhD 

Department of Molecular Cell Biology & 

Immunology, VUMC Glyco-immunology Group W 

University Medical Center , Netherlands 

Martin K. Kuhlmann, M.D. 

Associate Professor of Medicine and Nephrology, 

Director, Department of Internal Medicine, 

Vivantes Clinical Center - Friedrichshain, Berlin 

Dr. Ashraf Mikhail, MB B Ch, MSc, FRCP 

Consultant Nephrologist 

Morriston Hospital 

Swansea, Wales 

Professor Wolfgang Jelkmann, M.D. 

Dean of Medical Faculty, Institute of Physiology 

University of Luebeck, Germany 

Page 23 000154 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTICE NO. 000189 SUBMITTED BY PHARMING GROUP, N.V. --= 

-~ -- 

The preceding scientific assessment of snfetj issues concerning recombinant human 

lactoterrin produccd it1 transgenic cattle has been provided by and reflects the opinion of: 

Name: 

Page 24 
SAFETY CONCERNS MSED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTICE NO. 000189 SUBMITTED BY PHARMING GROUP, N.V. - --= 

---- 

-"a_ 

Page 25 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFEW 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTTCE NO. 000189 SUBMITTED BY PHARMING GROUP, N.V. 

=e-= 

The preceding scientific assessrnent of safety issues concenung recombinant human 

Lactoferrin produced in transgenic cattle has been provided by and reflects the opinion of 

Signature 

N'ullz: 

Title: 

Page 26 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFEREUN 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GUS - NOTICE NO. 000189 SUBMITTED RY PITARMING GROUP, N.V. -- 

/,I 

Nainc. Sidney E Grossberg, h4.11. :- 

Medical College of \Yisconsln 

Dcpartment of Microbiology and Molecular Genctics 

870 1 \Vateltown Plank Road, CCN2542 

Milwaukee, \VI 53226 

Title: Walter Schroeder Professor of Microbiology and Molecular Genetics 

Professor of Medicine 

-- =- 

Page 27 

000158 
SAFETY CONCERNS RAISED BY RECOMBINANT HUhlAN LACTOFEFWN 

PRODUCED 18 TRANSGENIC COTYS: SCIENTIFIC ASSESSMENT OF GR4S 

NOTICE NO. 000189 SUBMITTED BY PK4RbIING GROUP, S.1'. 

Thc preceding scientific assessiiierit of safety issues concerning recombinant human 

Iactofsrrin produced in transgenic cattle has been provided b!. and reflects the opinion of 

Name: ~~Iu-co van de Weeit 

Title. PhD, Associate Professor 

Page 28 

000159 
SAFETY CONCERNS RAISED BY REC:OMBINANT HIJMAN LACTOFEKKIN 

PRODUCED IN THANSCENIC COWS: SCIEIVTIE’IC ASSESSMENT OF GRAS 

NOTlCE NO. 000189 SUBMITTC:I) BY PHARiMING GROUI’, N.V. 

‘I lie preceding scieiililic asscssrnent of safety issues concerning rccombinant human 

inctoferriri prodttced in rransgciiic cattle has been provided bj, and rctlccts the opinion or. 

f h 

Namc: 

Tirlc: 

/ 

Page 29 
-e-= 

SAFETY CONCERNS RA ISED BY RECOhlBlNANT HlJMAN LACTOFERRIN 

PRODUCED IN TRANSC,ENTC COWS: SCTENTIFIC ASSESShWW’ OF ClLAS 

NOT[C15 NO. UUOlbrc) S(JI MI’I’TED BY PlIARMINC, GROUP. N.V. 

~~ 

The preceding scit‘n(ilic ns:.:ssment of safq issues coricerning recombinant human 

Ioctokc.ri\i ptowd in tran genic cattle has becii providcd by and rrllrcrjThc upiniun uT: 

-. 

-- . -~ 

Page 30 
Page 31 

000162 
SAIF1E"Y CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTICE NO. 000189 SUBMITTED BY PHAKMING GROUP, N.V. 

The preceding scientific assessment of safety issues concerning recombinant human 

lactoferrin produced in transgenic cattle has been provided by and reflects the opinion of: 

JO. y 24 fw 26496 

Signature: 

Name: 

Title: Director of Renal Medicine 

APmf Simon D Roger MD FRACP 

Gosford Hospital 

Holden St 

Gosford 2250 

Australia 

Page 32 

000163 
SAFETY CONCERNS RAISED BY RECOMBINANT mIMAN LACTOFERRIN 

PRODIJCED IN TRANSGENiC COWS: SCIENTIFIC ASSESSMENT OF GR.43 

NOTICE NO. 000189 SUBRl[TT'ED Rk' PNGRMTNC GROUP. 3.k'. 

Netzwerk fijf Gesundheit CmbH 

Klinik fur Inkre Mcdirin - Nepllrolope 

Sekretariat 

bndsbcrger Akc 49 iozqg Rc6:rn 

kl. [ojol $221.1322 Fax -2046 -I-.- __ --- 

Page 33 
-. n- SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODlJCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTICE NO. 000189 SIJRMITTEI) 81’ PIIARMINC; GROlJP, N.V. 

‘l’hc preceding scientific assessiiienf 01’ saf‘cfy issues concerning recombinant human 

lactofcrrin produced in traiisgcriic cattlc has bwi provided by and rcflects tlic opinion of’: 

re% 

Page 34 

000165 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN PRODUCED 

IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GUS NOTICE NO. 000189 

SUBMITTED BY PHARMING GROUP, N.V. 

The preceding scientific assessinent of safety issties concerning recombinant human lactofcrrin 

prodticed in transgenic cattle has been provided by and reflects the opinion of: 

Si g;j t ure : 

Name 

Title: 

Page 35 

Dr. Ashraf Mikhail 

MB.B.Ch. MSc, FRCP 

Consultant Nephrologist, Honorary Senior Tutor 

Morriston Hospital 

Swansea Medical School 

University of Wales, Swnasea 

e.mail:ashraf.mikhail@sn~ansea-tr.wales nhs.uk 

Fax: ##44 1792 703716 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODUCED IN TRANSGENIC COWS: SCXENTIFIC ASSESSMENT OF CRAS 

-- s- NOTICE NO, 000189 SUBMI'XTED BY PHARMING GROUP, N.V. 

The pxceding scientific assessment of safety issues concerning recombinant human 

lactofemn produced in transgenic cattle has been provided by and reflects the opinion of: 

Name: Eugene D. Weinberg, PhD 

Title: Professor Emeritus of Microbiology 

Indian2 University 

Yloornington, IN 47405 
Page 37 

000168 
SAFETY CONCERNS RAISED BY RECOMBINANT HUMAN LACTOFERRIN 

PRODUCED IN TRANSGENIC COWS: SCIENTIFIC ASSESSMENT OF GRAS 

NOTICE NO. 000189 SUBMITTED BY PHARMING GROUP, N.V. 

- 

The preceding scientific assessment of safety issues concerning recombinant human 

lactoferrin produced in transgenic cattle has been provided by and reflects the opinion of: 

Signature : 

Name: Dr. Huub Schellekens 

Title: Professor - Faculty Pharmaceutical Sciences, 

Utrecht University Central Laboratory, 

Animal Institute and Department of Innovation Studies, Utrecht University 

Netherlands 

n. 

Dr. Schellekens is currently traveling. We will 

provide his signature page upon his return. 

Page 38 

000169 
Attachment A to the Scientific Assessment 

Production and Purification Issues 

As the use of recombinant proteins continues to increase, a debate has begun concerning 

the analytical tools used to characterize proteins and the comparability of products 

produced by different manufacturers and processes. Lactoferrin is a complex protein, 

even when compared to products such as insulin, growth hormone and other recombinant 

proteins. In contrast with small molecules, analytical tools are not available for the full 

characterization of complex proteins. It is also increasingly recognized that the 

biological properties of a protein product are highly dependent on the process of 

production. Consequently, a consensus is emerging that efficacy and safety cannot be 

established exclusively by physical and chemical characterization and should always 

include data obtained from studies in humans. 

Complex proteins like lactoferrin differ from small molecules in that they never consist 

of a single type of molecule, but are always heterogeneous mixtures of different isoforms 

and other variants caused by clipping, mis-folding, etc. Complex proteins are also 

relatively unstable. During storage, modifications such as oxidation, de-amidation and 

aggregation are common. These modifications can have a major effect on product safety. =&-% 

Because even the most detailed physical chemical characterization leaves uncertainties 

concerning the biological activities of a protein, the safety of these products needs to be 

ensured by showing: 

1. The genetic stability of the host organism 

2. Consistency of production 

3. Detailed product characterization 

4. Purity of the final product and Iack of viral or prion contamination 

5. Stability of the product through production and distribution 

In these aspects the Notifier’s GRAS petition is highly deficient and this raises serious 

safety concerns. 

Genetic Stability 

Recombinant DNA derived proteins intended for human use can be produced from 

microorganisms, tissue culture or transgenic plants or animals. For fermentation and 

tissue culture production systems, product stability is ensured by using master and 

working cell banks, which allows production by well characterized identical cells. ~ _n. 

~~ 

Page 39 000I’i’0 
Moreover, these cells are further analyzed after the production run to guarantee the 

genetic stability of the production system. 

The Notifier’s recombinant human lactoferrin is produced in transgenic cows. These 

animals have not been cloned and each animal is genetically unique. Consequently, 

genetic stability within generations or through successive generations cannot be assumed. 

The Notifier asserts that only the founder bull (“Max”) has been genetically analyzed, 

and only at the level of lactoferrin m-RNA. There is no genetic evaluation of this 

animal’s offspring and no criteria that the related offspring animals must meet to qualify 

as production animals. Due to the at-random insertion of the human lactoferrin gene into 

the bovine genome, induced changes in protein expression of the milk must also be 

evaluated to assure the integrity of the derived protein. 

The inherent instability of the Notifier’s expression system combined with an inadequate 

level of process controls creates an unacceptable level of production risk for a highly 

biologically active protein such as rhLF. 

Consistency of Production 

Because the biological characteristics of rhLF are highly dependent on the production and 

purification process, consistency must be shown between successive batches. Batch 

testing must encompass the actual batches that will be used for marketing. Batches 

should also be produced and purified at the same scale used for marketing. In general, at 

least five different production batches need to be analyzed using accepted methods for 

protein characterization in order to confirm product comparability and safety. 

_n 

The Notifier presents very rudimentary batch characterization methods and it is unclear 

how and at what scale batches are produced. Based on the data and methods presented, it 

is not clear that the Notifier is able to produce human lactoferrin in a consistent manner. 

Without testing well characterized representative batches, an adequate determination of 

product safety is not possible. 

The criticality of process and formulation to product safety has been illustrated many 

times with recombinant proteins, In one well known example, a relatively minor change 

in the formulation of recombinant human erythropoetin (EPO) resulted the generation of 

antibodies against EPO and the occurrence of potentially fatal pure red cell aplasia’ 

(Casadevall2005). 

Responding to regulatory concerns from EMEA relating to the use of materials of human origin, J&J 

changed its formulation for EPREX by substituting one well-known, thoroughly characterized expander 

(sorbitol) for another (human serum albumin). Routine testing of the new formulation in biological and 

chemical assay systems revealed no cause for concern. After the reformulated product had been marketed, 

some 200 patients developed pure red cell aplasia (PRCA) as a result of neutralizing antibodies they 

generated against the erythropoietin protein in the reformulated product - meaning that their bodies cannot 

produce new red cells in response to the erythropoietin they produce, and they are now dependent on 

I 

--- 

Page 40 
me% 

In a submission to the FDA (Submission to Docket No. 2004N-0355 dated November 11, 

2004) Genentech describes their experience with recombinant human growth hormone. 

Following a relatively minor process change that did not even involve changes in the 

vector or expression strain, the antibody response to the protein changed by over ten-fold. 

Genentech notes that due to complexity of proteins, analytical testing alone could not 

have identified the differences between products despite their immunogenicity varying so 

substantially. 

As Genetech points out: 

“A survey of prescribing information for approved therapeutic proteins shows 

wide variability in their immunogenicity rates, ranging from 0.1% to >50%. 

There is similarly a wide range in the clinical sequelae associated with 

immunogenicity, ranging from no consequence, to anaphylactoid responses, to 

loss of effectiveness, to autoimmune disease. Immunogenicity is also likely to 

vary with type of disease and the co-medications given to patients. People with 

autoimmune diseases, for example, may be expected to have a more prevalent 

immune response to therapeutic proteins than oncology patients who may be on 

other immunosuppressive drugs. . . . Consequently, individual patient populations 

must be separately evaluated for the potential immunogenicity of a drug. A low 

percentage of seroconverting patients treated for one disease is not predictive of 

the seroconversion rate in another patient population, or in the same patient 

population treated with different co-medications.” 

Physical chemical characterization 

Based on the Notifier’s described production process, it cannot be assumed that the 

biological characteristics of Notifier’s rhLF will not change during production scale-up 

and transition from development to final manufacture. 

The efficacy and safety of biologic proteins are highly dependent on the manufacturing 

process. Both FDA and EMEA (European Medicine Evaluation Agency) have 

established detailed guidelines for the evaluation of recombinant proteins following 

manufacturing changes. The Notifier’s manufacturing and quality control processes are 

rudimentary and inadequate for a proper evaluation of complex protein characteristics, 

including biologic activity. Major flaws in the Notifier’s characterization processes 

include the lack of amino acid sequencing, lack of glycosylation pattern identification 

and the failure to characterize isoforms, contaminants and impurities. The Notifier fails 

transfusions. To its credit, J&J moved aggressively, in cooperation with regulatory authorities and 

academic scientists, to determine the cause of the adverse reactions. Yet some years after the relationship 

between the reformulated EPREX and the increased incidence of PRCA was established, the reason the 

reformulated product led to neutralizing antibodies in some patients has not been finally established. Only 

clinical experience was able to reveal and characterize the enhanced risk associated with the reformulated 

product. 

Page 41 
to employ adequate testing to assure protein integrity. Additionally, the Notifier has not -- reported any biological characterization of the product. -- 

The Notifier has not performed much of the typical product characterization that is 

routinely required to establish the safety of recombinant proteins. Provided below are 

some of the product characterization requirements that the FDA has discussed 

confidentially with Agennix: 

o Glycosylation. RhLF has a higher glycosylation content than native human 

lactoferrin and its carbohydrate content is significantly skewed towards mannose 

relative to that of normal human LF. To assure safety, complete characterization 

of the glycosylation is required, including site occupancy, monosaccharide 

analysis and chain-length analysis, and batch to batch consistency demonstrated 

in order to evaluate immunogenicity risks. 

o Aggregates. Protein aggregation should be characterization and a consistent lack 

of protein aggregation established. Aggregation is an important cause of 

immunogenicity since protein aggregates are more rapidly cleared, predisposing 

aggregates to increased phagocytosis and antigen presentation. This includes the 

risk of presentation of epitopes which are normally folded and hence may be not 

be recognized as self-antigens, triggering autoimmunity. 

o Host cell protein and DNA. In order to ensure safety, recombinant proteins 

must have very low levels of host-cell protein and host-cell DNA. Typically any 

contaminants above 1% must be fully characterized and their safety established 

independently. 

o Disulfide bond formation. A sensitive metric to detect and demonstrate the 

extent of disulfide bond formation is required. This is particularly relevant in the 

case of rhLF, since human LF has 16 disulfide bonds. Errors in the disulfide 

linkages can result in alterations in the tertiary structure of the protein potentially 

leading to increased immunogenicity including antibodies against previously 

masked epitopes. 

o Oxidation and Deamidation are important determinants of protein stability and 

the risk of conformational changes that can lead to immunogenicity. 

Although these immunogenicity issues are most often discussed in the context of 

parenterally administered proteins, they are critical for orally administered rhLF as 

well for the following reasons: 

o The gut has a very active mucosal immune system which includes the gut 

associated lymphoid tissue (GALT), the largest immune organ in the body. The 

GALT plays a critical role in the immune surveillance of ingested proteins. 

Page 42 000173 
o Lactoferrin has been shown to stimulate immune cells in the GALT as part of its 

role in modulating the immune system. 

o Dendritic cells, which are the most important antigen presenting cells in the body, 

are known to interdigitate between the gut enterocytes in order to access the gut 

lumen and sample luminal contents including ingested proteins. 

o Orally administered lactoferrin has been described as being transported intact 

through the gut wall, making it available to the systemic immune system. 

Thus, in the absence of adequate characterization of transgenic cow-produced rhLF, 

these immunological safety risks cannot be adequately evaluated and argue against 

any use of rhLF as a food additive. This in itself is sufficient reason to deny GRAS 

status. 

Purification 

Because of the uncertainties concerning rhLF’s biological effects, and to avoid effects 

due to impurities or protein variants, the purity of the final product is highly important. 

The Notifier’s specification currently allows for up to 10% impurities. This is an 

unacceptably high level for a bioactive recombinant protein. It is also essential that these 

impurities be identified and characterized to the fullest extent possible by current 

analytical methods. The Notifier presents no plan for a validated characterization of 

impurities and merely assumes the impurities to be bovine lactoferrin and other whey 

proteins. Such assumptions are not acceptable given the source of the product and the 

non-aseptic condition of harvesting. Since the Notifier’s transgenic cow-produced 

lactoferrin is not aseptic, which is unusual for recombinant proteins, microbial 

contamination remains a significant risk. Further, Notifier does not report LPS levels in 

the rhLF. Since LPS can cause morbidity or even death at high levels in vulnerable 

populations, levels of contaminating LPS are routinely measured in recombinant proteins 

with a requirement that the manufacturing process be controlled to ensure low 

endotoxins. 

Viral Contamination and BSE 

Although cow’s milk is now considered safe regarding the potential transmission of BSE, 

this is the first time genetically modified cows have been used for the production of a 

recombinant human protein intended for human consumption. Due to the random 

insertion of a human gene, including a promoter to direct expression in the mammary 

gland that may alter the excretion function of the gland cells, precautions to avoid prion 

contamination should be evaluated and implemented. The purification process should 

therefore be validated for the removal of possible prion contaminants, as is standard for 

the production of proteins from animal and human sources intended for human use. 

Additionally, because living animals are employed as the source of production, a 

Page 43 000174 
validated method for the removal of conventional viruses is needed. The Notifier’s --- described methods fail to control for these forms of potential product contamination. 

Long-Term Stability 

Recombinant proteins are usually unstable at room temperature. Stability studies are 

essential, not only to support any claim of biological activity, but also to exclude the 

presence of harmful degradation products that have been widely described to occur in 

recombinant proteins during storage. Long term stability testing is particularly vital in 

the Notifier’s case because production and storage are not under sterile conditions and 

because a cold chain is not strictly controlled. Without these controls, there is a 

significant risk that microbial contaminants may destroy or modify the Notifier’s rhLF. 

Stability should not only be tested in real time but also under accelerated conditions. 

Stability analyses should include assays that detect alterations to the protein including 

changes such as aggregation, degradation, structural changes, oxidation and deamidation, 

as well as assays for biological activity. Without proper stability studies, the integrity 

and safety of the Notifier’s marketed product cannot be assured. 

The Notifier only mentions that the product is stable, but provides no definition of 

stability and the techniques to evaluate stability are not elaborated. The lack of these data 

present serious safety questions. 

Page 44 0001’75 
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Page 54 

000185 

SIMON D ROGER MDFRACP Level 1, 37 William Street 

=A Prov No: 457563X Gosford NSW 2250 AUSTRALlA 

NEPHROLOGY & HWERTENSION Tel: 61 243 237977 Fax: 61 243 252522 

June 23.2006 

Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

center for Food Safewand Applied Nutrition 

Food and Drug Admkristration 

Room 3044 

University Station 

51OO Paint &anch Parkway 

CoWege Park, Maryland 20740 

RE: GRAS AodW O00 189 for human bdofenii UCBd in transaenic cows 

-. 

 ear Dr. Tarantino. 

The scienMc rebuttal to GRAS Notice OOO189 raises many questions about the safety of transgenic cowproduced human tactoferrin as a food ingredient l am Writing to independently emphasize and expand 

upon a few of the points made in the sdentific assessment 

First, the impactufgtywsyMm on protdytkacbvity is of major importance and should be emphasaed 

The arteration in glycosytatiocl profiles between the different entities may impact on the on metabolic 

Seoond, the issueofdelay in thedevelopmentofimmurogenicityis of significant medical concern. The fact 

thatthe Natifier's bansgenlcaw-pfaducd human tadDfenin wasonlygiven h 2doseswithin 24 hours 

gives no aedenoe to the amceqtthat it is not immunogenic In the case of-, the development 

of pure fed cell8ptElsiadutttoantCerythropoiedin anbibadiioccurrad months aftelthe ilmtionof 

ttwbmnt oelayed immunogenicity is a serious safetycoclcsmthat b now being considemdin the safety 

~ofbioslmltar mMnant prowls in Europe, Austrafia and the us. 

lastly, the tssue of skle affec& in healthy vdunh-rs versus disease spx%c hdiuals ts very important 

lnanatheraampkred8tedb~ - ittnisbef?ulobservedth8tM~lnduced 

~end~redcegap(asiaare~lbundkrpatients~~~~d~andnotother 

patknts taWng this drug (e.g. AIDS, rheumatoid arthritis, mufti@ myeloma and other oncology patients). 

Given the immummodulating activ#yof human Ladoferrin, an sssessmentofsafety cannot be made 

without ducting a cwbsEantialdinical progrem that indudes both healthyandat-dskpopulations. 

The krdusion oftransgenkawpmduced human ktdenin in food as a wtritbnal ingredient is not 

appropriategiven the very limited dataavailableon itssafety. 

s--- cleanme ratesand henceJphydobgicalactivityprofib. 
~~ 

-- 

ii 

VU medisch centrum Department of Molecular Cell Biology & Immunology 

Datum: June 20, 2006 Uw brief van Telefax +31 20 444 8144 Bijage(n) 

Ons kenmerk Uw kenmerk Telefoon +31 20 444 8 157 

Postadres Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands 

Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Room 3044 

U n ive rs i t y Stat ion 

5100 Paint Branch Parkway 

College Park, Maryland 20740 

RE: GRAS Notice 00189 

-- Dear Dr. Tarantino, 

As a contributor to the rebuttal document submitted in response to GRAS Notice 

000189, I am writing independently to emphasize my agreement with the 

conclusions of this scientific assessment. 

I would also like to provide further support to the arguments made about the 

importance of (lactoferrin) glycosylation. Specifically, I would like to bring to your 

attention an additional example where human and bovine sources of lactoferrin 

differentially interact with immune cells, which may be highly relevant to a safety 

assessment of human lactoferrin produced in transgenic cows. 

Naarding et al., described differences in the interaction of human versus bovine 

lactoferrin with DC-SIGN, a major lectin receptor on immature dendritic cells that 

specifically binds glycans. Whereas human lactoferrin (from SIGMA) shows no 

interaction with DC-SIGN, bovine lactoferrin (from SIGMA) clearly binds to 

dendritic cells via DC-SIGN. Although the authors do not explain the differences 

in binding, this most likely is the result of binding of DC-SIGN to glycans of bLF. 

DC-SIGN is known to contribute to controlling the balance between immune 

activation and tolerance induction (Van Kooyk 2004, Caparros, 2006). If DCSIGN indeed recognizes the glycans on bLF and triggers DC-SIGN function, 

such an interaction may have severe immunological consequences. 

e-mall: im.vandi.e@vumc.nl 

Profdr C.0 Dqkstra 

Prof.dr. G Kraal 

Prof.dr Y van Kooyk 

Prof dr R H J Beekn 

Prof dr C L. Venveii 

z-= 

Or T.K van den Berg 

Dr J vandenBorn 

Dr f M van Die 

Or. T.B.H. Geijtenbeek 

Dr. E W.A. Kamperdijk 

Dr RE Mebius 

Dr C T.M van der Pouw Kraan 

Dr H deVries 

000188 
Given the demonstrated biologic activity of human lactoferrin and its affects an 

immune function, its inclusion in food products without rigorous clinical testing is 

not advised. 

Respectfully. 

Irma van Die, PhD 

Dept. of Molecular Cell Biology 8 Immunology 

VU University Medical Center 

Van der Boechorststraat 7 

1081 BT Amsterdam, the Netherlands 

References 

Caparros E, Munoz P, Sierra-Filardi E, Serrano-Gomez D, Puig-Kroger A, 

Rodriguez-Fernandez JL, Mellado M, Sancho J, Zubiaur M, Corbi AL (2006). DCSIGN ligation on dendritic cells results in ERK and P13K activation and modulates 

cytokine production. Blood 107: 3950-8. 

-- 

- 

Naarding MA, Ludwig IS, Groot F, Berkhout B, Geijtenbeek TB, Pollakis G. 

Paxton WA (2005). Lewis X component in human milk binds DC-SIGN and 

inhibits HIV-1 transfer to CD4+ T lymphocytes. J CIin Invest. 115: 3256-64, 

van Kooyk Y., A. Engering, A.N. Lekkerkerker, I.S. Ludwig, and T.B. 

Geijtenbeek.. "Pathogens use carbohydrates to escape immunity induced by 

dendritic cells." Curr Opin Immunol. 2004 16:488-93. 

e-mail: irn.vandie@vumc,nl 

Prof dr C.D Dijkstra 

Prof.dr G Kraal 

Prof dr Y van Kmyk 

Prof dr RHJ Eeelen 

Prof dr C L. Verweij 

s-- 

Or. T K van den Berg 

Or J van den Born 

Or 1.M van Dle 

Dr. T B.H. GeFjtenbeek 

Dr EW.A Kamperdijk 

Or R.E Mebius 

Or C T M van der Pouw Kraan 

Or H deVries 
June 20,2006 

Laura M. Tarantino, P1i.D. (I 113-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Room 3044 

University Station 

5 100 Paint Branch Parkway 

College Park, Maryland 20740 

RE: Recombinant Human Ixtofei-rin: A Pharmaceutical Product, Not a Nutrient 

Dear Dr. Tarantino, 

Lactofcrrin, a powerful iron-scnvenging defense protein, is present constitutively in 

exocrine sccretions that arc constantly exposed to microbial flora: milk, tears, 

tubotynpanum and nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, 

cervical-vaginal mucus, and scininnl fluid. Additionally, lactoferrin is promptly 

dclivcred by circulating neutrophils to sites of microbial invasion. In only two of the 

fluids listed above (milk and tears) is lactoferrin continuously maintained at high 

concentration. In each of the other fluids listed above lactoferrin is maintained at quite 

low amounts until an actual invasion occurs. 

- 

The high concentration of lactoferrin in human milk suppresses growth in the 

infant gut of such iron-dependent bacteria as Bacteroides, Clostridium, Escherichia, 

Salmonella and Staphylococcus while permitting abundant growth of the relatively 

harmless iron-abstaining Lactobacillus. In humans above the age of infancy, the immune 

lymphatic system has matured so that elevated amounts of the iron-trapping lactoferrin 

are neither necessary nor natural in the intestine. 

In tears, however, moderately high concentrations of lactoferrin are needed 

throughout life to inhibit (together with lysozyme) a broad spectrum of bacterial species. 

Accordingly, the need for antibodies to inhibit ocular bacteria is markedly lessened, thus 

resulting in a decrease in vision-obscuring scarring. 

The proposed inclusion of 100 milligrams of lactoferrin per product serving 

specified in GRN 001 89 would havc no natural function in the gastrointestinal tracts of 

humans above the age of infancy. But its inclusion might have harmful effects in some 

proportion of consumers. Several potential risks that have not been evaluated should be 

considered. 

000190 
1) Orally administered lactoferrin is known to increase total cellularity of the 

small intestine Peyer’s patches, including an increase in the numbers of 

natural killer-T cells and CD8+ T lymphocytes. Possible adverse effects of 

such irnmunostimulation by the continual ingestion of large amounts of 

lactoferrin over long periods of time rcmain to be determined. 

2) Those persons who produce a polymorphic variant of Iactoferrin would be 

expected to regard the ingested protein as a foreign antigen. Antibodies to 

lactoferrin have been reported in persons who have such autoimmune 

conditions as lupus, rheumatoid arthritis, type 1 diabetes, primary sclerosing 

cholangitis, inflammatory bowel disease, and pancreatitis. 

3) Some persons have latent or overt infections caused by virulent microbes that 

express receptors to lactoferrin that enable the pathogens to acquire growthessential iron. Among such pathogens is Helicohacfer pylori, an important 

cause of gastric ulcers and possibly gastric cancer. (The discoverers of this 

pathogen were awarded the 2005 Nobel Prize in Medicine.) Prior to mass 

medication of foods and beverages with lactoferrin, it will be necessary to 

determine if persons negative for the Helicobacter pylori breath test convert to 

positive when fed the protein over a period of time. 

Recommendation 

-=E= Recombinant human lactofenin, a powerful pharmaceutical product, should not 

be distributed to general, non-selected populations in the absence of rigorous tests for 

safety. These tests should ascertain that use of the product will not result in an 

unacceptable rate of toxicity including (1) unbalancing the innate and adaptive immune 

components of intestinal lymphatic tissue, (2) development of allergic hypersensitivities, 

and (3) overgrowth of specific pathogenic microbes such as Helicobacferpyfori. 

Respectfully, 

Dr. Eugene Weinberg 

Professor Emeritus 

Indiana University 

Department of Biology 

Jordan Hall Room 142 

1001 East Third Street 

Bloornington, M 47405 

000191 
000192 
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Human lactderrin 

Human lactofemn @LF) is a natural protein that heips to tight and preuern infection; and evxzsiue intlamm;Riorc arid 

strengthens the defense system of the human bady. The protein ii presenr in jipniticant amiiurd% in numerous human 

biOb3giCal fluids and mucus ;ecretions. including tears and lung secretions. and hai Deer, shoun to tigh tactena that 

imponam role in the defense synem of infants, as well as aduhs haofernn promme: the heath of the ganm-inreninal 

system by impmiring the intestinal microbial balance 

PmJect amu, 

Related pubilomlorls 

Gloszsry cause infections of the eye and lungs. In addition. hLF is presem in iuhst~ntial quarft~lies irl moltds milk arid plays an 

Market opportunity 

bctofemn is a multi-functional protein iunh many beneficial propertiei. which rn3ke~ K a 8JoGd carldidnc for a rlurnbw cjr producz applicmioils ';irlLa the pruteirt has the abiln $1 io 

btnd iron. is a natural anti.bacterial. ami.fungal and arm-viral. IS an antioddam ard aljo ha: irr~rnuoomodulJtor~~ propertier, large group; 01 pec.pie might benetn irom orally 

administered lactofemn 

Phaming has a patem on human lactnfemn imm the Japanese Patern Office, which cover5 the pmduction and punticmiori 01 hLF iwth Pharmincq s tcct~nolcqg!, 35 ~4 8s 0; u5e 

in sports and food fonnulsttonz In Japan, bodine IaLtofemn 1% currrrnly used 31 an 3ddnit.e in taod produc?z and ai a r,mmional ~~ppk~tilnl Jap3n repreieriti a ?igniticart 

markel for recombinant human lactofemn 

Pharming's hLF approach 

Because ot ns unique biologtc~l activrttes. Ptiarming iz developing PI humx Iac?otrmn ar a rood rupplemern urmy K: proteir, prohmior, rechrdqy , Phamlirng'; hunian 

lactofenin is produced fmm the milk of transgenic COINS. a method thm fns t~ni4itjri3I tood dr~ieloprnrrir ucrf WYII a; CUII,': rrtilh ii 8 comm~or~ iiccd :uurce worldwide. Pham-irg 

h3s riled a GRAS (Generally Recognized As Safe)riottficalion for ns hLF loth the LIS FCI& 

The company has mediumsize production tai:iInies 10 supply ns hLF tor further rese~irc h and developmm p!~cp~?eir lr, all~lniun. the compar,,i hx a pannet?-hip imh the Neiv 

Zealand based research instnute .&Research for deadopmerrl or Ks hurnan iacIc8temri Phammg md 4jF:e;earch ir, VIIV in uestc8~, corripmiei drd ir,Rniute: 10 partner tor 

further development of human lactotemn for oral npplications 
Agennix Incorporated 

July 6, 2006 

Robert Merker, Ph.D. (HFS-255) 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

Food and Drug Administration 

Room 3044 

University Station 

4300 River Road 

College Park, Maryland 20740 

Re: Correspondence of June 27, 2006 - Safety Concerns Raised by Recombinant Human 

Lactoferrin from Transgenic Cows (GRN No. 000189 Submitted by Pharming Group 

N.V.) 

Dear Dr. Merker: 

Please find enclosed the signature page for Dr. Huub Schellekens supporting our scientific 

assessment in response to GRAS Notice No. 000189. Per our correspondence of June 27, 2006, 

we mentioned that Dr. Schellekens had been away on travel and that we would submit his 

signature page upon his return. 

Please do not hesitate to contact us if there are any questions or if additional information would 

be useful. 

Sincerely, 

Rick Barsky 

Chief Executive Officer 

Cc: Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Eight Greenway Plaza Suite 910 Houston Texas 77046 Telephone 713/552-1091 Facsimile 713/552-0795 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_______________________ 

Page 1 of 3 

Fasano, Jeremiah 

From: Fasano, Jeremiah 

Sent: Thursday, July 20, 2006 3:17 PM 

To: 'Charles Morin' 

Subject: RE: Conference telephone call concerning Pharming's hLF 

Mr. MorinOf the three dates you proposed for a rescheduled discussion, September 1st (10 am EST) is the 

only one that would work well for FDA. Is this date still workable for you and your client? 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

DBGNR/OFAS/CFSAN/FDA 

jfasano@cfsan.fda.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

From: Charles Morin [mailto:charleslmorin@earthlink.net] 

Sent: Wednesday, July 12, 2006 11:23 AM 

To: Fasano, Jeremiah 

Subject: Re: Conference telephone call concerning Pharming's hLF 

Dear Dr. Fasano, 

Thanks very much for your communication concerning postponement of Thursday's meeting. We look forward to receiving at 

your convenience the date and time of the rescheduled meeting. 

Thanks again for your continuing support. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 500 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_______________________ 

Page 2 of 3 

Email: charleslmorin@earthlink.net 

----- Original Message -----

From: Fasano, Jeremiah 

To: 'Charles Morin' 

Sent: Tuesday, July 11, 2006 8:16 AM 

Subject: RE: Conference telephone call concerning Pharming's hLF 

Mr. MorinI've notified the appropriate FDA personnel of the postponement. I will contact you again 

soon about a date for the rescheduled discussion. 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

DBGNR/OFAS/CFSAN/FDA 

jfasano@cfsan.fda.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

From: Charles Morin [mailto:charleslmorin@earthlink.net] 

Sent: Monday, July 10, 2006 2:40 PM 

To: Fasano, Jeremiah 

Subject: Conference telephone call concerning Pharming's hLF 

Dear Dr. Fasano, 

With regard to GN number 189 (concerning use of Pharming's rhLF in certain foods), currently we are scheduled to 

meet at 10:00 a.m. (E.S.T.) on July 13th to preliminarily discuss CFSAN's initial questions concerning potential 

immunogenicity. Pharming has been very busy over the last several weeks identifying and communicating with 

potential experts (all of whom are associated with academic institutions ) to be added to its expert panel who can 

address CFSAN's questions. Pharming has made significant progress with some of them, but through no fault of 

anyone has not been able to get to the desired point with all of them. Part of the problem faced by Pharming is that 

experts' schedules and vacations and availability were all established in early January for 2006 and some are not 

quickly available. Thus, Pharming is not yet sufficiently prepared to have the initial conference telephone call (on 

Thursday). Accordingly, it respectfully asks that it be postponed. In order to work within the above-identified 

limitations and within the vacation times also set in January for Pharming's employees (please remember that Europe 

basically shuts down the last two weeks of July and all of August) and to assure that Pharming will not have to ask for 

yet another postponement, Pharming respectfully requests that the conference telephone call be postponed to (in 

order of preference):

 1. August 31;

 2. August 30; or 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

Page 3 of 3 

 3. September 1. 

We apologize for any inconvenience caused by the requested postponement. 

Please let me know that the conference telephone call has been postponed and which date can be accommodated 

by CFSAN's schedule. 

Thank you for you continuing assistance. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 500 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

Email: charleslmorin@earthlink.net 

6/27/2007 
Questions Intended 

To Clarify 

Questions Posed By 

CFSAN 

1. When CFSAN uses the term “biological response modifier” does it mean 

anything more (or different) than – in this case – whether oral consumption 

of lactoferrin does, in fact, specifically or nonspecifically increase or 

decrease immune response? 

2. When CFSAN uses the term “adverse effects” does it mean anything more or 

different than harmful to humans? 

3. When CFSAN uses the term “ susceptible individual” it suggests there are 

those more likely to be affected. Does this include everyone or some subset 

of everyone – all of which have an inherent vulnerability? If the latter, 

please specifically identify the subset(s). 

4. CFSAN questions reference – in the context here in question – the utility of 

short term and long term animal (preclinical) studies. Given that it is most 

likely that recombinant human proteins will be immunogenic in animals and 

that the induction of antibody formation in animals is not predictive of a 

potential for antibody formation in humans and that there are currently no 

reliable animal models and no standard test for predicting the 

immunogenicity of proteins in humans, what is the value here to be provided 

by use of preclinical studies to predict immunogenicity? 

5. A CFSAN question raises the subject of the availability of clinical evidence. 

Is this matter being raised merely to obtain, if available, any already existing, 

relevant, clinical evidence or to indicate the possibility of CFSAN requiring 

“long term” or other clinical trial(s). If the latter, does CFSAN believe that it 

has legal authority to require any clinical trial in the context of the matter 

here in question? If so, please identify such authority. 

6. What published information and direct evidence is known to CFSAN that 

indicates that “lactoferrin has been shown to enhance Type 1 T helper (Th1) 

cell activity, as well as the release of specific cytokines in the gut and 

systemically following oral administration? 
7. What published information and direct evidence leads CFSAN to be 

concerned – assuming for the moment oral consumption of lactoferrin has an 

effect on Th1 cells – that such an effect potentially might exacerbate proinflammatory responses? 

8. What published information and direct evidence leads CFSAN to believe 

that “Chronic pro-inflammatory Th1 – mediated immune responses might 

result in the promotion of autoimmune or other inflammatory disorders, in 

the gut or elsewhere, in individuals predisposed to such”? 

9. What published information and direct evidence leads CFSAN to believe 

that “Even small structural or biological differences between the native and 

modified form of a particular protein may have a significant impact on that 

protein’s recognition by the immune system and subsequent response”? 

10. What published information and direct evidence leads CFSAN to believe 

that Pharming’s exogenous human lactoferrin may evoke a nonallergenic 

immune response in susceptible individuals that disrupts previous tolerance 

to endogenous lactoferrin through determinant spreading from alloepitopes, 

the potential for enhanced pro-inflammatory Th1 responses mentioned 

above, and increased uptake by antigen-presenting cells via the mannose 

receptor? 

11. When CFSAN uses the term “determinant spreading from alloepitopes”- as it 

does above - exactly what does it mean? 

12. When CFSAN uses the term “adult” should Pharming believe that CFSAN 

does not mean a human who is merely some specific age (like 18 or 21) but 

rather means a human with a mature gut and immune system? 

13. When CFSAN uses the term “immunological safety” does it mean anything 

more (or different from) than that oral consumption of lactoferrin (at the 

maximum level here in question) does not cause adverse, non-allergenic 

responses by the adaptive immune system? 
 

 

 

 

 

 

 

 

_______________________ 

Page 1 of 3 

Fasano, Jeremiah 

From: Fasano, Jeremiah 

Sent: Thursday, August 17, 2006 12:53 PM 

To: 'Charles Morin' 

Subject: RE: Details of conference telephone call with CFSAN 

Mr. MorinWe're currently expecting the following people on the call: 

-myself 

-Supratim Choudhuri, Toxicology Reviewer 

-Alison Edwards, Chemistry Reviewer 

-Robert Merker, Supervisory Consumer Safety Officer 

-Jeanette Glover Glew, Supervisory Consumer Safety Officer 

-Ron Chanderban, Supervisory Toxicologist 

-Mike DiNovi, Supervisory Chemist 

-Bob Martin, Deputy Division Director, DBGNR 

-Toni Mattia, Division Director, DBGNR 

-Stefano Luccioli, OFAS Medical Officer 

-Kathleen Jones, CFSAN Biotechnology Coordinator 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

DBGNR/OFAS/CFSAN/FDA 

jfasano@cfsan.fda.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

From: Charles Morin [mailto:charleslmorin@earthlink.net] 

Sent: Wednesday, August 16, 2006 3:02 PM 

To: Fasano, Jeremiah 

Subject: Details of conference telephone call with CFSAN 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page 2 of 3 

DBGNR/OFAS/CFSAN/FDA 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740

 Re: Pharming

 rhLF 

Details of conference

 telephone call with CFSAN 

Dear Dr. Fasano,

 With regard to the upcoming conference telephone call (on Friday, September 1 at 10:00 a.m. EST) 

between CFSAN and Pharming, please find below the details I promised. 

Call in directions

 Call in number: (866) 448-6761

 Code: 940357 

Pharming participants

 Those individuals who will be participating on behalf of Pharming include: 

1. Frans de Loos, PhD

 Project Director (rhLF);

 2. Sandra van Wetering, PhD

 Scientist, Molecular Biology and Immunogenics; 

3. Bertjan Ziere, PhD 

Senior Director, Preclinical;

 4. Harrie van Veen

 Muscle Scientist;

 5. Erik Doevendans 

Director, Product Registration;

 6. Anurag Relan, MD

 Director Corporate Development; and

 7. myself.

 Please let me know (e-mail is OK) who will be participating on behalf of CFSAN. 

Questions for CFSAN

 Please find attached a copy of those thirteen questions Pharming intends to ask CFSAN during the 

teleconference. They are intended to clarify the questions CFSAN has asked Pharming so that 

Pharming's response can be as on point and complete as possible.

 After you have had an opportunity to review the forgoing information if you have questions or need 

additional information, please let me know. 

6/27/2007 
 

 

 

 

 

 

 

Page 3 of 3 

 We look forward to a candid and helpful exchange.

 Best regards. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 500 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

Email: charleslmorin@earthlink.net 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page 1 of 2 

Fasano, Jeremiah 

From: Charles Morin [charleslmorin@earthlink.net] 

Sent: Wednesday, August 30, 2006 2:22 PM 

To: Fasano, Jeremiah 

Subject: Details of conference telephone call with CFSAN 

Dear Dr. Fasano,

 On Wednesday, August 16th, I sent you an e-mail concerning those Pharming individuals who will participate 

in Friday's conference telephone call. Unfortunately, I somehow got some of the titles wrong; mea culpa! 

Following are the individuals and their correct titles: 

1. Frans de Loos, PhD

 Director, Business Development Products (rhLF); 

2. Sandra van Wetering, PhD

 Scientist, Biochemistry and Immunochemistry; 

3. Bertjan Ziere, PhD

 Senior Director, Preclinical; 

4. Harry van Veen

 Scientist, Process Development; 

5. Erik Doevendans

 Director, Regulatory Affairs (including product registration); 

6. Anurag Relan, MD

 Director, Corporate Development; and 

7. myself.

 Looking forward to a productive meeting!

 Best Regards. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 1460 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

6/27/2007 
Page 2 of 2 

Email: charleslmorin@earthlink.net 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: September 1, 2006 

TIME: 10:00 AM 

LOCATION: US 2013 

PARTICIPANTS: 

FDA 

Jeremiah Fasano HFS-255 

Supratim Choudhuri HFS-255 

Anna Brown HFS-255 

Jeanette Glover Glew HFS-255 

Alison Edwards HFS-255 

Robert Martin HFS-255 

Stefano Luccioli HFS-205 

Mary Ditto HFS-255 

Michael DiNovi HFS-255 

Carrie Hendricksen HFS-255 

Ronald Chanderbhan HFS-255 

Robert Merker (by phone) HFS-255 

External 

Frans de Loos Pharming 

Sandra van Wetering Pharming 

Bertjan Ziere Pharming 

Harry van Veen Pharming 

Erik Doevendans Pharming 

Anurag Relan Pharming 

Charles Morin Morin and Associates 

SUBJECT: Discussion of GRN 189 status 

Members of the Office of Food Additive Safety met with representatives of Pharming, 

Inc. (Pharming), including Pharming’s agent (Mr. Morin) to discuss the status of GRN 

189 for the use of recombinant human lactoferrin from bovine milk in various foods 

consumed by adults. The specific purpose of the meeting was to ensure that our 

questions, previously conveyed to Pharming in writing, were clearly understood. 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

September 1, 2006 Memo of Teleconference – Page 2 

Pharming had provided some written questions in response, which were also discussed at 

this meeting. 

FDA staff reviewed some general information about food ingredient safety assessment, as 

well as questions posed to the notifier in our correspondence of May 17, 2006. We then 

discussed the specific questions posed by the notifier in their correspondence of August 

16, 2006. 

The meeting concluded with an agreement by Pharming to consider the issues raised by 

FDA staff and follow up with the agency. 

Jeremiah Fasano 

Drafted:HFS-255:10/12/2006 

F/T:HFS-255:1/16/2007 
 

To: Pharming rhLF file 

From: Charles Morin 

Re: Teleconference with CFSAN 

On Friday, September 1, 2006 the following personnel from CFSAN1

, i.e., 

1. Brown, Anna Marie (HFS-820) 

Division of Research and Applied Technology (ONPLDS), 

2. Chanderban, Ron PhD (HFS-255) 

Supervisory Toxicologist, 

3. Choudhuri, Supratim PhD (HFS-255) 

Toxicology Reviewer, 

4. Diho, Mary PhD (HFS-255) 

Consumer Safety Officer, 

5. DiNovi, Mike PhD (HFS-255) 

Supervisory Chemist, 

6. Edwards, Alison PhD (HFS-255) 

Chemistry Reviewer, 

7. Fasano, Jeremiah PhD (HFS-255) 

Consumer Safety Officer, 

8. Glew, Jeanette Glover (HFS-255) 

Supervisory Consumer Safety Officer, 

9. Hendrickson, Carrie PhD (HFS-255) 

Consumer Safety Officer, 

1

 Although Toni Mattia, PhD (HFS-255), Division Director and Kathleen Jones, PhD (HFS-013), Biotechnology 

Coordinator had originally planned to participate in the teleconference, due to conflicts both were unable to attend. 

1 
10. Luccioli, Stefano MD (HFS-255) 

Medical Officer, 

11. Martin, Bob PhD (HFS-255) 

Deputy Division Director, and 

12. Merker, Robert PhD (HFS-255) 

Supervisory Consumer Safety Officer, 

met – via telephone – with the following personnel from Pharming, i.e., 

1. Frans De Loos, PhD 

Senior Director, Business Development Products (rhLF), 

2. Sandra van Wetering, PhD 

Scientist, Biochemistry and Immunochemistry, 

3. Bertjan Ziere, PhD 

Senior Director, Preclinical, 

4. Harry van Veen, MSc 

Scientist, Process Development, 

5. Erik Doevendans, MSc 

Director, Regulatory Affairs (including product registration), 

6. Anurag Relan, MD 

Director, Corporate Development, 

7. Mourad Salaheddine, PhD 

Senior Director, Animal Health and Production, and 

8. myself, 

for the sole purpose of clarifying the substance and scope of the concerns/questions posed in 

CFSAN’s email of May 17, 2006. The teleconference began at 10 a.m. (EST) and ended at 10:51 

a.m. (EST). 

The meeting began with Morin indicating to CFSAN that all seven individuals representing 

Pharming were on the line. Dr. Fasano – who coordinated and chaired the meeting and who is 

2 
coordinating the overall GRAS evaluation of GN 189 – then asked each CFSAN attendee to self 

identify, which each did. 

Dr. Fasano then offered numerous introductory remarks, which included: 

1. that the purpose of the meeting was to identify to Pharming certain questions which 

CFSAN has and to assure that Pharming understands the substance and scope of the 

questions; 

2. that with regard to food ingredient safety evaluation: 

a. the FD&C Act and certain CFR regulations set forth the basic requirements; 

b. safety means that there is a reasonable certainty in the minds of qualified experts that 

a specific substance is not harmful under the intended conditions of use (see 21 CFR 

§ 170.3(i)); 

c. general recognition of safety based upon scientific procedure requires the same 

quantity and quality of scientific evidence as is required to obtain approval of a food 

additive (see 21 CFR § 170.3(b)); 

d. the difference between determining safety of a food additive and a GRAS substance is 

that in the former instance CFSAN decides what is safe and in the latter instance safety 

is determined by qualified experts based on publicly available information; 

e. general recognition requires a consensus by qualified experts based on common 

knowledge throughout the pertinent scientific community; 

f. intended use is a key consideration both with regard to general recognition and 

determining safety; 

g. safety is evaluated (per intended use) via daily consumption (i.e., exposure), assuming 

lifetime (i.e., everyday) exposure; 

h. potential benefits do not weigh into a safety evaluation at all (no risk/benefit analysis 

takes place); 

3 
i. traditionally, when dealing with essentially small molecules which are intended to have 

an effect in the food (and not in the consumer), safety could be assessed via use of an 

evaluation which substantially depended on a set of testing that emanated from, for 

example, the “Red Book”; however, when the substance may (or is intended to) have an 

effect in the consumer, then such use may result in the asking of different questions 

(although asked pursuant to the same reasonable certainty standard); 

j. no “blind check list” of standard tests applies (in each case, one must use all tests 

necessary to demonstrate safety; the actual sets of tests used in any one instance may 

vary from another instance); and 

k. the identity and nature of the substance (within the context of the intend use) is what 

drives the testing; 

3. that with regard to the GRAS program itself: 

a. if a substance and its intended use(s) are not listed in the CFR (as having been approved 

or GRASed by CFSAN), then one needs to assess whether such substance and use(s) 

are GRAS; 

b. if a substance has been in broad use for some time then it may be GRAS based on 

common use in food; 

c. if a substance is – in essence – new, then GRASness must be based on scientific 

procedures; 

d. while one can independently assess GRASness (and not interface with CFSAN), 

CFSAN does operate a voluntary program currently known as the GRAS Notification 

process (which is not an approval process and which emanates from a rule proposed in 

1997) which permits one to obtain a written opinion from CFSAN as to whether 

CFSAN believes one has demonstrated in a given instance GRASness (via the 

information conveyed to CFSAN in the GN); 

e. CFSAN will respond either that it has “no questions” concerning the petitioner’s 

determination of GRASness or that it believes that the evidence submitted does not 

demonstrate (or support) a GRAS determination; 

4 
f. in any safety evaluation, CFSAN considers all of the information in the GN, as well as 

all other information that may be available to it; 

g. CFSAN expects that a GN will disclose all matters (i.e., the good, the bad and the ugly) 

that may be pertinent to a GRAS determination, especially any problematical matters; 

h. use of an expert GRAS panel can often be helpful, especially in serving as a “proxy” 

for the expert community as to whether a general consensus exists; 

i. an expert panel not only can offer technical advice but also serve to provide insight into 

what the expert community is thinking; 

j. the GN evaluation process is generally not an interactive process (usually the decision 

rests solely on the information provided in the GN), but in this case, due to the novelty 

of the substance and its intended use(s), it is productive and helpful to have a certain 

amount of back and forth (thus CFSAN’s questions and this meeting); and 

k. GRASness can be time specific, i.e., information can become available which changes 

a not GRAS status to a GRAS status; 

4. with respect to CFSAN’s list of questions: 

a. lactoferrin is clearly a molecule with multiple modes of action – some of which appear 

to be immunomodulatory; 

b. use of the term “biological response modifier” is intended to convey the thought that 

LF may have – via its motive capability – an effect on one or more of the many 

components of the human immune system (and not just an on or off effect on the entire 

system as a whole); 

c. oral consumption of lactoferrin appears to produce effects both in the gut and 

systemically; 

d. some of these effects appear to be anti-inflammatory, others pro-inflammatory (e.g., 

Th1 cell activity is enhanced, some cytokines (e.g., IL-18, IFNγ, and IL-12) are 

increased in the gut and systemically and may be pro-inflammatory or linked to 

Crohn’s disease or arthritis, and natural killer cell activity may be increased; 

5 
e. there is some evidence in support of species specific interactions – but no clear picture 

has emerged; 

f. there is very limited evidence from some comparative studies on affinity of receptors in 

the small intestine (e.g., bLF is a poor competitor for such receptors vis-à-vis hLF in 

the human gut, whereas the mouse receptor appears to be equally receptive to either 

bLF or hLF – all of which complicates interpretation of this type of scientific 

information); 

g. there exists some good papers from Pharming concerning characterization of rhLF (as 

expressed in cows); clearly Pharming’s product is human lactoferrin; differences appear 

to include differences in glycosylation (i.e., different glycans are attached than to rhLF) 

and differences that exist between the amino acid sequence of rhLF and the different 

amino acid sequences that exist naturally due to allelic variation in humans (do all of 

these variations actually exist in nature?); 

h. with regard to human exposure, the ADI was calculated based on exposure to infants 

(the most extreme exposure) which exposure is very large; however, adults and infants 

vary with respect to the strength and profile of Th1 and Th2 responses which difference 

may be important (and, thus, needs to be considered); in addition, the structure of the 

gut differs in the infant (which may have a more “leaky” gut) and adult (e.g., with 

regard to lymphatic tissues and porosity) and this too needs to be addressed; 

i. because we are talking about the maturation of multiple components (i.e., from infant to 

adult) – both with regard to the gut and the immune system – we are talking about a 

continuum – with the infant at one end and the adult at the other; 

j. with regard to the preclinical studies submitted, they amount to classical tox studies 

which do not focus on the immunomodulatory endpoints here at issue; thus, if other 

pertinent information is available, that would be helpful; 

k. no real validated preclinical models for evaluating potential immunogenicity currently 

exist, but there may be animal models for evaluating activity of hLF (is Pharming 

aware of any such information?); 

6 
l. with regard to the clinical studies, they were short term and on small populations and 

not really related to the immunomodulatory endpoints here at issue; thus, if other 

pertinent information is available – especially with regard to long term exposure – that 

would be helpful; 

m. some of the effects of LF in infants may be different then in adults; therefore, there may 

be a problem with using infant data to establish safe levels in adults (do you have any 

information that could help resolve this situation and indicate whether it is appropriate 

to use the infant data for establishing the safe consumption level for all consumers – 

especially as it relates to functionality); 

n. it is important to consider – given the immunomodulatory effects of lactoferrin – 

whether chronic consumption of LF might cause a pro-inflammatory response or 

exacerbate autoimmune disorders or whether tolerance can be broken by continuous 

exposure to exogenous lactoferrin (especially via, for example, a Th1 increase, since 

there are models in which an increase by a single, type molecule leads to an event – 

such as a Th1 increase (a bias) or alloepitope spreading (i.e., tolerance to one kind of 

allelic hLF and exposure to another); 

o. also consider the difference in glycosylation and the possible increased uptake of 

lactoferrin by mannose receptors in dendridic cells whereby there can result an 

increased presentation to the immune system; and 

p. generally need information concerning “likelihood” that these concerns will or will not 

occur; 

5. with regard to Pharming’s thirteen specific questions pertinent to clarification: 

a. in response to Morin’s general inquiry concerning the sources of CFSAN’s concerns – 

so that all such sources can be responded to – Dr. Fasano indicated that all such 

concerns flow from various (not just one seminal article), pertinent, published, 

scientific articles; thus, an appropriate literature search should reveal all such articles 

(CFSAN would be willing to examine our combined references list and let us know if 

these are important references that have been left off); and 

7 
b. as to the thirteen questions and CFSAN’s remarks they follow (for the sake of clarity, 

the questions are set forth and immediately followed by CFSAN’s comments): 

Question 1 

When CFSAN uses the term “biological response modifier” does it mean anything more 

(or different) than – in this case – whether oral consumption of lactoferrin does, in fact, 

specifically or nonspecifically increase or decrease immune response? 

Response 

No, provided that there is an understanding that the human immune system consists of 

numerous components and, thus, one would be looking to see whether oral consumption of 

lactoferrin – at the levels in question – produces any effect on any one or more of these 

components (and not merely an on or off effect on the system as a monolithic whole, which it 

isn’t). 

Question 2 

When CFSAN uses the term “adverse effects” does it mean anything more or different than 

harmful to humans? 

Response 

 No. 

Question 3 

When CFSAN uses the term “susceptible individual” it suggests there are those more likely 

to be affected. Does this include everyone or some subset of everyone – all of which have an 

inherent vulnerability? If the latter, please specifically identify the subset(s). 

8 
Response 

CFSAN is particularly concerned about evoking or exacerbating any disorder in individuals 

that have a genetic predisposition to autoimmune disorders. This may involve 5-10 percent of the 

general population. These individuals are difficult to identify, whether via screening or otherwise, 

due to the complex nature of their predisposition. 

Question 4 

CFSAN questions reference – in the context here in question – the utility of short term and 

long term animal (preclinical) studies. Given that it is most likely that recombinant human 

proteins will be immunogenic in animals and that the induction of antibody formation in humans 

and that there are currently no reliable animal models and no standard test for predicting the 

immunogenicity of proteins in humans, what is the value here to be provided by use of preclinical 

studies to predict immunogenicity? 

Response 

CFSAN “absolutely agrees.” However, CFSAN wonders whether Pharming has any 

additional information concerning the above-noted effects of lactoferrin. 

Question 5 

A CFSAN question raises the subject of the availability of clinical evidence. Is this matter 

being raised merely to obtain, if available, any already existing, relevant, clinical evidence or to 

indicate the possibility of CFSAN requiring “long term” or other clinical trial(s). If the latter, does 

CFSAN believe that it has legal authority to require any clinical trial in the context of the matter 

here in question? If so, please identify such authority. 

Response 

CFSAN – being aware of some relevant, preexisting, clinical information – merely 

wonders whether Pharming has any such data that speaks to any of the above – noted concerns. 

9 
Questions 6 – 10 

What published information and direct evidence is known to CFSAN that indicated that 

“lactoferrin has been shown to enhance Type 1 T helper (Th1) cell activity, as well as the release 

of specific cytokines in the gut and systemically following oral administration? 

What published information and direct evidence leads CFSAN to be concerned – assuming 

for the moment oral consumption of lactoferrin has an effect on Th1 cells – that such an effect 

potentially might exacerbate pro-inflammatory responses? 

What published information and direct evidence leads CFSAN to believe that “Chronic 

pro-inflammatory Th1 – mediated immune responses might result in the promotion of autoimmune 

or other inflammatory disorders, in the gut or elsewhere, in individual predisposed to such”? 

What published information and direct evidence leads CFSAN to believe that “Even small 

structural or biological differences between the native and modified form of a particular protein 

may have a significant impact on that protein’s recognition by the immune system and subsequent 

response”? 

What published information and direct evidence leads CFSAN to believe that Pharming’s 

exogenous human lactoferrin may evoke a nonallergenic immune response in susceptible 

individuals that disrupts previous tolerance to endogenous lactoferrin through determinant 

spreading from alloepitopes, the potential for enhanced pro-inflammatory Th1 responses 

mentioned above, and increased uptake by antigen-presenting cells via the mannose receptor? 

Response 

Such evidence all emanates from the published literature. 

With regard to question 10, CFSAN is not aware of any direct evidence that indicates that 

lactoferrin will cause the tolerance breakdown referenced above. Its concern arises from certain 

models – such as a mouse model pertinent to experimental auto-immune encephalomyelitis and 

10 
other information (such as that pertinent to the flooding of numerous receptors) – which generally 

indicate that a substance can cause such a breakdown. 

Question 11 

When CFSAN uses the term “determinant spreading from alloepitopes” – as it does above 

– exactly what does it mean? 

Response 

Since there are in nature various slightly different hLFs resulting from different alleles for 

hLF which will activate different T cell epitopes, will receptors for lactoferrin be tolerant to all 

such resulting epitopes or will any one slightly different epitope – which may vary from the 

epitope associated with endogenous lactoferrin – cause a non-tolerant effect. 

Question 12 

When CFSAN uses the term “adult” should Pharming believe that CFSAN does not mean a 

human who is merely some specific age (like 18 or 21) but rather means a human with a mature 

gut and immune system? 

Response 

Yes. CFSAN merely means that since there are known differences between the gut and 

immune systems of an infant and adult (for example, an infant’s gut is a leaky one and its immune 

system is biased towards Th2) and since Pharming has relied on exposure confirmation from 

infants to calculate an ADI for adults, do the known differences make any difference. If so, 

perhaps the ADI needs to be recalculated. 

Question 13 

When CFSAN uses the term “immunological safety” does it mean anything more (or 

different from) than that oral consumption of lactoferrin (at the maximum level here in question) 

does not cause adverse, non-allergenic responses by the adaptive immune system? 

11 
Response 

No. The focus here is not potential allergenicity; rather, it deals with the issues unique to 

this substance and its intended use. 

******* 

Dr. de Loos indicated that the clarifications and questions are clear and very helpful. 

Morin inquired into the schedules of events – including the filing of Pharming’s response, 

CFSAN’s review of Pharming’s response, and a face-to-face meeting at CFSAN – that need to 

take place prior to CFSAN making its decision. Someone at CFSAN indicated that CFSAN would 

be looking for all information to be in the written response and that no meeting would likely take 

place. However, Morin pointed out that such remark differs from the schedule of events – 

including a second meeting – set forth in Dr. Fasano’s email and discussed with and agreed to with 

Dr. Mattia before the filing of Pharming’s GN. It was determined that such prior agreed to 

schedule should prevail and that a scientific meeting could prove very useful. Dr. Fasano raised 

the question of whether such meeting should take place before or after Pharming filed its response. 

Finally, Ms. Glew encouraged Pharming to contact Dr. Fasano if it had any follow up 

questions. 

Dr. Fasano encouraged Pharming to tie all the pieces of its response together so as to set 

forth a unified, compelling story. Also, he encouraged Pharming to carefully tie its response to 

intended use(s). 

12 
 

 

 

 

 

Page 1 of 1 

Fasano, Jeremiah 

From: Charles Morin [charleslmorin@earthlink.net] 

Sent: Monday, September 11, 2006 6:41 PM 

To: Fasano, Jeremiah 

Subject: Details of conference telephone call with CFSAN 

Follow Up Flag: Follow up 

Flag Status: Completed 

Attachments: fasano rhLF_Draft_1[1][1].doc 

 Dear Dr. Fasano, 

 Again, thank you for organizing and chairing the meeting concerning CFSAN’s questions about Pharming’s rhLF. Please find 

attached a copy of my memorandum of the meeting. Although I do not believe you are required to do so, if you choose to review the 

memo and find any errors or omissions, I would appreciate learning of same. 

Best regards. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 1460 

San Francisco, CA 94111 

US 

Phone: (415) 957-0101 

Fax: (415) 957-5905 

Email: charleslmorin@earthlink.net 

6/27/2007
1 ;AM I ll11ll I1 1111 I I 

DEC 26 206 

Law Offices Of 

Morin & Associates 

Suite 1460 

388 Market Street 

San Francisco, California 94 1 1 1 

Telephone: (4 15) 957-0 10 1 e-mail: charleslmorin@earthlink.net Facsimile: (415) 957-5905 

December 22,2006 

Antonia Mattia, PhD (HFS-255) 

Di rector 

Division of Biotechnology and 

GRAS Notice Review 

Office of Food Additive Safety 

Center for Food Safety and Applied 

Nutrition 

Food and Drug Administration 

51 00 Paint Branch Parkway 

College Park, MD 20740-3835 

Re: Pharming Group N.V. 

Notice of GRAS exemption for human 

lactoferrin derived from the milk of 

transgenic cows expressing a human 

gene encoding human lactoferrin 

GRN No. 000189 

Response to request for additional 

information 

Dear Dr. Mattia: 

On December 29, 2005 Pharming forwarded a GRAS Notification to a CFSAN concerning the above-referenced lactoferrin. Such notice was 
Antonia Mattia, PhD 

Re: GRN 189 Response to CFSAN request 

December 22,2006 

Page 2 of 6 

received on January 3, 2006 and filed on January 12, 2006. It has been 

designated GRN No. 000189. 

On May 17, 2006 Dr. Fasano forwarded an email to me indicating 

that CFSAN -had completed its “preliminary evaluation” of GN 189 and was 

forwarding a list of those concerns/questions considered “significant”. Such 

concerns/questions seek additional specific information. All such 

concerns/questions involve certain specified aspects of Pharming’s hLF’s 

potential to induce any adverse, non-allergic response by the adaptive 

immune system. 

In order to adequately respond to such concerns/questions and to 

assure that such response is complete, accurate and represents - at a 

minimum - tHe consensus view of qualified experts, Pharming (among 

other things): 

0 

1. participated in a telephone conference with CFSAN 

personnel on September 1, 2006 during which certain 

clarifications were provided to assure that Pharming 

fully understood the concerns/questions CFSAN i‘s 

seeking responses to; and 

added additional pertinent expertise to its expert panel 

to help address CFSAN’s concerns/questions. 

2. 

Such latter, total, pertinent expertise - that is, the individuals who are 

expert in immunology (and related matters) - now include those who have 

already contributed to Pharming’s GN, i.e., 

1. Jeremy H. Brock, ScD, PhD, MSc; 

Senior Research Fellow 

Department of Immunology 

U n iversi ty of Glasg ow 
Antonia Mattia, PhD 

Re: GRN 189 Response to CFSAN request 

December 22,2006 

Page 3 of 6 

(Dr. Brock is a chemist/ micro-biologist/immunologist who is 

currently a Senior Research Fellow at the University of 

Glasgow in the Department of Immunology and who has a 

long and distinguished research career concerned with ironbinding proteins - especially with regards to infection and 

immunity - which includes the study and publication of much 

of what is known about lactoferrin) and 

2. Andre H. Penninks, PhD 

Senior (Immuno) Toxicologist 

Division of Experimental Immunology 

Department of Toxicology and Applied Pharmacology 

Department of Immunotoxicology 

University of Ut recht 

TNO; and --, 

(Dr. Penninks is trained in experimental immunology and 

immunotoxicology, teaches immunotoxicology and cell 

pathology, and has spent a lifetime researching the effects of 

compounds, especially food-related substances), 

as well as (new members): 

1. Charles 0. Elson, MD 

Professor of Medicine and Microbiology 

Vice-Chair for Research 

Department of Medicine 

Director, Inflammatory Bowel Disease Center 

Senior Scientist, Multipurpose Arthritis Center 

University of Alabama at Birmingham 

(Dr. Elson is trained in medicine and, in particular, 

gastroenterology (especially mucosal immunology) and has a 

1 

1, 

j 

ii 

li 

/! 
Antonia Mattia, PhD 

Re: GRN 189 Response to CFSAN request 

December 22,2006 

Page 4 of 6 

long and distinguished research career involving numerous 

aspects of the gastrointestinal immune system (especially as 

that relates to IBD and oral tolerance) and is associated with 

numerous professional associations and committees and 

ed ito ria I boards); 

2. Cathryn R. Nagler, PhD 

Associate Professor of Pediatrics (Immunology) 

Center for Immunology and Inflammatory Disease 

Division of Rheumatology, Allergy and Immunology and 

Center for the Study of Inflammatory Bowel Disease 

Massachusetts General Hospital and Harvard Medical School 

(Dr. Nagler is an expert in immunology specializing in 

various immunology-related research topics, including 

inflammatory bowel disease, Crohn's disease, ileitis and 

colitis, immunological related mechanisms and 

autoimmunity. She has studied the immune response, 

induction of tolerance, and the consequences of 

breaking tolerance as induced by dietary agents for 

years. She is associated with various professional, 

committee and editorial entities); and 

3. Hubertus F.J. Savelkoul, PhD 

Professor and Chairman 

Department of Cell Biology and Immunology 

Wageningen University 

(Dr. Savelkoul is a cell biologisthmmunologist who 

specializes in basic and applied immunology, 

immunoregulation, immune assays, regulatory T cells, 

cytokines and allergy. In addition to chairing his 

department, he has published 255 peer-review articles 

I; 

I' 

i 

! 

j 
I’ 

Antonia Mattia, PhD 

Re: GRN 189 Response to CFSAN request 

December 22,2006 

Page 5 of 6 

and serves on various boards, in societies and on 

editorial boards). 

All of these individuals together with numerous Pharming personnel - 

including those with an expertise in immunology - have worked together for 

several months to prepare the attached response to CFSAN’s questions. It 

concludes - after considerable discussion of pertinent information - that 

Pharming and its experts: 

are of the opinion that when all of the pertinent, direct, scientific 

evidence is considered as a whole, a fair evaluation of it 

demonstrates to a reasonable certainty that Pharming’s exogenous 

lactoferrin will not induce any adverse, non-allergic response by the 

adaptive immune system and - when combined with the information 

in Pharming’s GN - demonstrates to a reasonable certainty that 

Pharming’s product is not deleterious and generally recognized as 

safe for human consumption at I00 mg per product serving. 

a 

After you and your colleagues have had an adequate opportunity to 

review the attached information, if you determine that CFSAN has no 

further questions regarding Pharming’s and its expert panel’s 

determination, Le., that the above-referenced lactoferrin is GRAS under the 

intended conditions of use, please forward to me an “Agency Response 

Letter”. If, however, you have additional questions (including concerns), 

please let me know and we can arrange - pursuant to my 

conversation/agreement with you - to meet with you and your colleagues to 

discuss such remaining questions/concerns. 

Finally, as agreed to at the start of this project, Pharming has not 

attached copies of all of the references. However, if you need any of them, 

(and they are all set forth on the reference list), please let me know and I 

can have them to you by next day (if not before via email). 

!’ 

I 

!! 

/, 

’! 

I: 
Antonia Mattia, PhD 

Re: GRN 189 Response to CFSAN request 

December 22,2006 

Page 6 of 6 

Thank you in advance for your and your colleagues’ efforts. 

cc ce ly, c 

Charles L. Morin 

cc: Frans de Loos, PhD 0 

i: 

Senior Director, Business Development 

Pharming Group N.V. 
-' 
Content 

Table of Contents / 

Pharming’s response 

I. Introduction 

A. 

B. 

C. Pharming utilized appropriate, professional 

Pharming sought out direct evidence ......................... 1 

Pharming consulted with pertinent expertise .............. 2 

opinion ....................................................................... 3 

CFSAN’s concern (its second bullet point) ................. 5 

The substance in question ............................. 5 

II. Concerns and Responses 

A. 

B. Pharming’s response 

1. 

2. Endogenous and exogenous human 

lactoferrin ....................................................... 6 

Both lactoferrins are almost entirely. 

the same ............................................. 6 

The extent to which both Iactoferrins 

are different ......................................... 8 

(1 .) With regard to respective amino 

(2.) With regard to glycosylsation . 10 

The importance of the single 

difference ........................................... 1 I 

3. Determinant spreading ................................. 13 

Enhanced pro-inflammatory Thl response ... 15 

cells via the mannose receptor ..................... 16 

CFSAN’s concern (its first bullet point) .................... 16 

a. 

b. 

acid sequences ....................... 8 

c. 

4. 

5. Increased uptake by antigen-presenting 

C. 

D. Pharming’s response 

1. Background information concerning the 

adaptive immune system and Thl and Th2 

cells .............................................................. 17 i 

4. 

I 
h 2. Enhancement otTh1 cell activity and 

release of specific cytokines 

a. Thl cell activity ................................. 18 

b Release of specific cytokines ............ 21 

3. Effect of oral administration of hLF on 

autoimmune or other inflammatory 

disorders 

a. Autoimmunity ................................... 22 

b Inflammatory disorders .................... 25 

E. CFSAN's concern (its third bullet point) .................. 28 

F. Pharming's response ............................................ 28 

G CFSAN's concern (its fourth bullet point) ................. 30 

H. Pharming's response ........................................... 30 

I. 

J Pharming's response .............................................. 32 

K. 

CFSAN's concern (its fifth bullet point) .................... 31 

CFSAN's concern (its sixth bullet point) ................. 33 

L Pharming's response ........................................... 34 

111. Conclusion ......................................................................... 34 

IV. Expert Panel's Statement ................................................... 35 

C. References .~ 

Attachment 1 (entitled: Background information concerning the 

adaptive immune system and Thl and Th2 cells) 

Attachment 2 (which summarizes and critiques, when appropriate, 

all of the preclinical and clinical studies which evaluate the biological response modification abilities, if any, of lactoferrin when 

orally consumed) 

Attachment 3 (experts' CVs) 
'i 

I 
Pharming’s Response 

To CFSAN’s Concerns 

Regarding 

Its Lactoferrin’s Potential 

* To Induce 

Any Adverse, Non-allergic Response 

By The 

Adaptive Immune System 

1. Introduction 

Before and after the September 1, 2006 telecon,aence w th CFS, I 

(to assure that Pharming fully understood the concerns of CFSAN that 

Pharming needed to address with regard to the potential of its exogenous 

lactoferrin to cause any adverse, non-allergic, immunological response by 

the adaptive immune system), Pharming did a number of things to assure 

that this document is fully and professionally responsive. Such things are 

discussed below in detail (in highlighted subparts). 

_. 

A. Pharming sought out direct evidence 

Dr. Tarantino, Director, Office of Food Additive Safety, has correctly 

indicated (see, e.g., Tarantino response - dated November 28, 2005 - to a 

scientific question raised in connection with GRN000049), that any decision 

(whether made by CFSAN or a notifier) made with regard to a GRAS 

1 
Notification is legally required to be based on “direct evidence” and not on 

mere “hypothesis” or on mere “speculation”. Such a requirement is intended ~ 

a B. Pharming consulted with pertinent expertise 

Notwithstanding that Pharming has in-house expertise in immunology, 

Pharming also sought out and utilized additional, qualified expertise in 

immunology in preparing this response and to serve on its expert panel (as 

expanded). Such total expertise was used to assure that the information 

referred to above was as complete as possible and to provide the best 

possible expert insight into and opinion concerning the meaning of such 

information. 

General recognition based upon scientific procedures - as is the case here - requires the same quantity and quality 

The list of references initially considered was much longer than the list of references attached to this response. The 

1 

of scientific evidence as is required to obtain approval of a food additive. (21 CFR §170.30(b)). 

initial list included all - even remote - possibilities; the final list includes only those determined - after carehl 

review -to be really pertinent. 

2 
C. Pharming utilized appropriate, professional opinion 

When preparing this response, Pharming required - pursuant to pertinent 

legal requirements - that certain proof requirements be met. First, it 

required that this response be primarily and adequately based - as the law 

requires - on publicly available (Le., both peer-reviewed and published), 

established, direct, scientific e~idence.~ (21 CFR §I 70.30(b)). As the law 

mandates, use of, for example, secret information is - by definition - 

unacceptable. Of course, other information might be used as secondary, 

supplemental, corroborating evidence - but not as direct evidence. Second, 

qualified experts (as a result of their pertinent training and expertise) were 

consulted to assure that all pertinent, direct evidence was identified and 

considered. Such experts were also utilized to review the pertinent evidence 

and to determine its meaning and to help formulate and confirm the 

conclusions that could reasonably and accurately be derived from it. Third, 

Pharming required - as does the law - that such meanings and conclusions 

emanate from a general recognition, i.e., a consensus, of the expert^.^ (21 

USC $321 (s)). Finally, Pharming required that meanings and conclusions - 

including with regard to safety - be based (as the law requires) on 

reasonable ~ertainty.~ As pertinent regulations make unequivocally clear 

with regard to the meaning of reasonable certainty: 

General recognition of safety through scientific procedures shall ordinarily be based upon published studies which 

may be corroborated by unpublished studies and other data and confirmation. (21 CFR 4 170.30@)). 

The law also requires that the safety factors utilized to evaluate safety also be generally recognized. 

(21 CFR §170.3(i)(3)). 

Safety means that there is a reasonable certainty in the minds of competent scientists, ie., qualified experts, that a 

specific substance is not harmful under the intended conditions of use. (21 CFR §170.3(i)). 

3 
It is impossible in the present state of scientific knowledge to establish 

with complete certainty the absolute harmlessness of the use of any 

substance. 

(21 CFR §170.3(i)). Thus, absolute certainty is not required. Nor is proof 

beyond a reasonable doubt. Nor is one required to prove a negative (which, 

of course, is impossible). Nor does reasonable certainty amount to mere gut 

feeling. Rather, reasonable certainty is achieved when qualified experts 

determine in consensus fashion - pursuant to generally recognized 

principles of safety - that it is generally recognized among such experts that 

a fair evaluation of the direct evidence indicates that there is convincing 

evidence a substance is not harmful under the intended conditions of use. 

Following, then, is Pharming’s response - including the identification 

and meaning of, and the conclusions emanating from the pertinent, direct 

evidence. For the convenience of the reader, such response is set forth in 

the following format - CFSAN’s concerns are individually set forth in the 

order in which they appear as bullet points in CFSAN’s email (with one 

exception)6 and then immediately followed by Pharming’s response (usually 

in highlighted subparts). In addition, since Pharming cannot practically know 

the extent to which any reader of this response may or may not be a 

qualified expert on the subject matter and in the spirit of attempting to make 

this response as useful to all readers as possible (including, especially, any 

non-expert), Pharming has, from time to time, throughout the response 

included some very basic information, especially as it relates to definitions of 

0 

terms. Pharming trusts that the inclusion of such information will not offend 

anyone, especially any qualified expert. 

‘6 CFSAN’s concern about the identity of “Pharming’s lactoferrin“ (i.e., CFSAN’s second bullet point) is set forth first 

since it is important to the response to clearly establish at the outset exactly what substances we are all discussing. 

4 
a 

II. Concerns And Responses 

Following are CFSAN’s concerns (as set forth in its email of May 17, 

2006) and Pharming’s responses. 

A. CFSAN’s concern (its second bullet point): 

Pharming’s lactoferrin is distinct from the endogenous lactoferrin of 

individual consumers with respect to expected differences between the 

amino acid sequence of the exogenous lactoferrin and the polymorphic 

endogenous Iactoferrin alleles present in the general population, and the 

modification of some species of the exogenous lactoferrin with oligomannose 

glycans not found on endogenous forms. Even small structural or biological 

differences between the native and modified form of a particular protein may 

have a significant impact on that protein’s recognition by the immune system 

and subsequent response. We are concerned that Pharming’s exogenous 

human lactoferrin may evoke a nonallergic immune response in susceptible 

individuals that disrupts previous tolerance to endogenous lactoferrin 

through determinant spreading from alloepitopes, the potential for enhanced 

pro-inflammatory Th 1 responses mentioned above, and increased uptake by 

antigen-presenting cells via the mannose receptor. 

B. Pharming’s response: 

1. The substance In question 

At the outset, it seems important to emphasize that this entire 

discussion should be about the substances which are the focus of 

Pharming’s GRAS Notification - that is, Pharming’s and native human 

5 om7 I 1 
lactoferrin. Thus, much of what is in the GRAS Notification and this 

response is only about human lactoferrin (sometimes referred to in this 

response as hLF). However, Pharming recognizes that human Iactoferrin is 

only one in a broad set of mammalian Iactoferrins (including, especially, 

bovine lactoferrin - sometimes referred to in this response as bLF)’; 

accordingly, Pharming has also - from time to time - included in its response 

information about other lactoferrins because such information is helpful in 

establishing a broader context of safety of human lactoferrin. 

2. Endogenous and exogenous human lactoferrin 

’ CFSAN has expressed a concern that Pharming’s exogenous 

Iactoferrin is structurally significantly different from the polymorphic, 

endogenous Iactoferrin produced naturally by the individual consumers 

comprising the U.S. population and that such structural differences may 

have a significant impact on the way in which such exogenous lactoferrin is 

recognized and responded to by the human immune system. Accordingly, 

what follows is a discussion of the extent to which both lactoferrins are 

identical, the extent to which both lactoferrins are different, and the 

importance of any difference. 

a. Both lactoferrins are almost entirely the same 

As discussed in considerable detail in Pharming’s GRAS Notification 

(please see, e.g., pages 4-5, 12-1 3, and 32-34), both Pharming’s exogenous 

’ To the extent hLF is considered to be a “known biological response modifier” (KBRM) of the human immune 

system, bLF must also be considered a IU3RM. bLF has been determined to be GUS and at a level equivalent to the 

level being requested in Pharming’s GN. 

6 
I 

lactoferrin (“rhLF”) and endogenous Iactoferrin (“hLF”) are overwhelmingly I 

i identical. As a reminder, such identicalness extends to the fact that both 

lactoferrins: 

1. are the same metal-binding, glycoprotein, Le., hLF (Thomassen, 

2. have the same amino acid sequence and composition based on 

2005; van Berkel, 2002; Anderson, 1989); 

the nucleic acid sequence pertinent to the allelic variation seen in 

the normal population (see, GN, subsection Ill(C)(l)(e)); 

3. have the same N-terminal protein sequence (van Berkel, 2002); 

4. have the same three-dimensional, protein structure (Thomassen, 

2005); 

5. are N-glycosylated (van Berkel, 2002); 

6. have the same number and location of glycosylation sites (van 

7. show the same chromatographic profiles upon analytical Mono S 

8. have the same core-molecular weight (although overall molecular 

weight slightly differs - Pharming’s hLF is slightly lower - due to 

the differences in the carbohydrate moieties attached to the 

lactoferrin core) (van Berkel, 2002); 

9. show the same tryptic degradation kinetics, Le., digestibility (van 

Veen, 2004); 

IO. have the same iron-binding and iron-release properties (van 

Berkel, 2002); and 

11. are equally effective against experimental infections with 

Veen, 2004); 

analysis (van Berkel, 2002); 

multidrug-resistant S. aureus and K. pneumoniae in mice (van 

Berkel, 2002). 

7 
Thus, from the point of view of considering any difference which actually 

makes any significant difference, Pharming’s lactoferrin is identical to 

endogenous lactoferrin - except for the difference that is discussed below. 

b. The extent to which both Iactoferrins are different 

CFSAN questions whether Pharming’s lactoferrin differs from 

endogenous lactoferrin in two, different ways. Each is discussed below. 

(I .) With regard to respective amino acid sequences 

CFSAN first questions whether the amino acid sequence of Pharming’s 

exogenous lactoferrin is structurally different from that of endogenous 

Iactoferrin.* As Pharming’s GN explains (see pages 12-13) and as 

discussed below, the two lactoferrins are not really different. 

Careful comparison of the ten, published, amino acid sequences of 

endogenous Iactoferrin demonstrates that such naturally-occurring 

sequences may naturally differ from one another in six instancesg, i.e., in 

amino acid positions 4, 11, 14, 29, 413, and 561. In each such natural 

instance, the amino acid present is one of only two possibilities. Thus, there 

exists a well-known and well-documented naturally-occurring range of amino 

acid variation in endogenous lactoferrin. 

Specifically, CFSAN asserts that “Pharming’s lactoferrin is distinct fi-om the endogenous lactoferrin of individual 8 

consumers with respect to expected differences between the amino acid sequences of the exogenous lactoferrin and the 

polymorphic endogenous lactoferrin alleles present in the general population.” 

Only four of these instances have been scientifically confmed, Le., at amino acid positions 4, 1 1,29 and 561. (Van 

Veen, 2004). The other two, Le., positions 14 and 413, have not yet been confmed. Pharming’s lactoferrin has - with 

regard to these latter two amino acid positions - the same amino acids as reported in 9 of the 10 above-referenced 

amino acid sequences. (See pages 12 and 13 of Pharming’s GN). It is possible that the latter two differences may not 

be real. 

9 

8 
Pharming’s lactoferrin does not differ from but rather exactly duplicates 

this range, Le., it is no more different from such range than any one of the 

ten, known endogenous lactoferrins. In each of the six, above-referenced 

amino acid positions, Pharming’s lactoferrin incorporates exactly the same 

one of two possible amino acids, as does any one of the ten endogenous 

lactoferrins. Thus, there exists no real difference here between what occurs 

endogenously and what occurs exogenously. (Please note that with regard 

to all other amino acid positions, they are all identical). 

There is no scientific evidence whatsoever that an individual producing 

one of the above-referenced endogenous lactoferrins reacts - 

immunologically speaking - differently when exposed to any one of the other 

a bove-refe ren ced en dog eno us 1 act ofe rri ns. I ndeed , extensive and Ion g -te rm 

human experience demonstrates just the opposite. For example, infants 

when consuming mother’s milk are exposed - in the vast majority of 

instances - to an endogenous hLF variety that differs from their own and all 

without adverse, immunological reaction, probably due to oral tolerance” 

and anergy”. It should be noted that these daily exposure levels far exceed 

the daily exposure level being requested in Pharming’s GRAS Notification, 

i.e., I00 mg/product serving.’* In addition, patients - of all varieties and 

ages - who receive transfusions of blood products, e.g., fresh, frozen 

plasma, are routinely exposed to an endogenous lactoferrin that differs from 

lo The term “oral tolerance” is defined as the suppression of systemic humoral and cell-mediated immune responses 

to an antigen after the oral administration of that antigen, due to anergy of antigen-specific T cells or the production of 

immunoregulatory mediators such as transforming growth factor+ or interleukin-1 0. Oral tolerance is a physiological 

mechanism for preventing immune responses to food antigens. For a thorough discussion of how tolerance is 

established, etc., see Iweala ,2006 or Faria, 2005. 

The term “anergy” is defined to mean a state of unresponsiveness to antigenic stimulation. Lymphocytic anergy 

(also called clonal anergy) is the failure of clones of T or B cells to react to antigen, and this may be a mechanism of 

maintaining immunologic tolerance to self antigens. In clinical practice, anergy refers to a generalized defect in T 

cell-dependent cutaneous delayed-type hypersensitivity reactions to common antigens. (Abbas, 2006). 

discussed in a later section. (See, section J). 

The issue concerning the impact of the differences between the adult and infant gut and immune system are 

9 
their own. This lactoferrin - present in the plasma at varying concentrations 

from 42-202 pg/mI - is predominantly derived from degranulating 

neutrophils. (Scott, 1989). Moreover, patients who receive such transfusions 

commonly have ongoing inflammatory reactions, e. g., trauma. Even so, 

such very numerous, systemic exposures to these exogenous Iactoferrins in 

these patients have not been reported to have led to any known, adverse, 

immunological event. Oral exposure to human Iactoferrin should be even 

less potentially immunogenic than this type of exposure. Since Pharming’s 

exogenous human lactoferrin only duplicates endogenous, human Iactoferrin 

(with regard to amino acid sequence), one would also expect such 

exogenous lactoferrin not to induce any adverse, immunological event (as a 

result of its amino acid sequence). And there in no evidence that it could or 

does. 

(2.) With regard to glycosylation 

As CFSAN correctly notes, Pharming’s exogenous Iactoferrin does 

differ from endogenous lactoferrin with regard to the type of carbohydrate 

structures that are attached at each of the three glycosylation sites. 

However, that is the extent of their structural differences (as Pharming’s GN 

discusses at pages 4 and 33), since both lactoferrins incorporate the same 

number and location of glycosylation sites and both utilize these I 

glycosylation sites in the same fashion. (van Veen, 2004). 

I 

I 

t 

With regard then to the specific glycans attached at each of the 

glycosylation sites, the only glycans attached to the glycosylation sites of 

natural hLF (from human milk) are N-linked, complex-type glycans. (van 

Berkel, 2002; Spik, 1982). In addition to the complex, N-linked glycans that 

10 
are attached to the endogenous lactoferrin glycosylation sites, Pharming’s 

exogenous lactoferrin also bears oligomannose and/or hybrid-type, N-linked 

glycans (van Berkel, 2002) - as one would expect, since the distribution and 

structures of attached glycans is species-, tissue-, cell type- and proteinspecific. (James, 1995; Opdenakker, 1993). Furthermore, the complex, Nlinked glycans of Pharming’s hLF contain N-acetylgalactosamine next to 

galactose, which is typical for N-linked glycoproteins produced in bovine 

milk, such as bovine lactoferrin. (Van den Nieuwenhof, 1999; Coddeville, 

1992). Finally, the glycans of Pharming’s hLF contain less fucose compared 

to natural hLF. (van Berkel, 2002). However, as a result of crystallography 

studies, it has been determined that - despite the differences in N-linked 

glycosylation - the three-dimensional structure of Pharming’s hLF and I 

natural hLF are identical. (Thomassen, 2005). 

Thus, the attached glycan-related differences then are the only known 

structural differences that exist between endogenous lactoferrin (from human 

milk) and Pharming’s exogenous lactoferrin. 

c. The importance of the single difference 

At this point, the key question becomes: Does the above-described 

difference (with regard to exactly what glycan is attached at each of 

Iactoferrin’s three glycosylation sites) make any real difference with regard to 

the ability of Pharming’s exogenous lactoferrin to disrupt previous tolerance 

to endogenous Iactoferrin? The direct evidence indicates that it does not. 

The mere fact that a difference exists - as here - between two forms 

of a molecule (one of which naturally occurs - in this case in human milk - 

and the other of which differs from that naturally-occuring form only with 

11 
regard to the kinds of glycans attached at each of the glycosylation sites) 

does not - by itself - amount to direct evidence that such difference will 

affect the latter molecule’s potential immunogenicity. For example, please 

note that endogenous hLF (from human milk) and endogenous hLF (from 

human neutrophils) also differ in their respective glycosylation patterns. 

(Derisbourg, 1990). The glycan associated with neutrophilic hLF is not 

fucosylated - thus, it resembles the glycan pattern of human serum 

transferrin. (Spik, 1994). However, such difference in glycosylation pattern 

does not affect hLF’s function with respect to isoelectric point, stability of the 

iron-saturated form, rate of clearance, or antigenicity. (Derisbourg, 1990; 

Moguilevsky, 1985). 

And there exists another, even more relevant, well-known example, 

which demonstrates that consumption of a differently glycosylated lactoferrin 

does not lead to any adverse consequences with regard to immune 

response or any interruption of tolerance. The example, of course, involves 

the human consumption of bLFI3 which is long known to be safe (and at 

levels far exceeding the level here at issue, Le., 100 mg per product serving) 

as a result of a long and well-documented history of safe use (and by 

humans of every variety, including age, race and ethnic background). 

Since Pharming’s exogenous lactoferrin and bLF are both produced by 

the bovine mammary gland which determines the type of glycosylation (in 

this case, a mammalian type of glycosylation) and since, similarly to 

Pharming’s hLF, bLF bears oligomannose-type glycans and complex-type 

l3 Bovine lactofemn (bLF) is also - like hLF - an iron-binding glycoprotein (of about 80 ma) which is similar in 

structure and function compared to its human homologue. (Nuijens, 1996). The amino acid sequence of bLF (which 

contains 689 amino acids) shows 69% homology with WF. (Pierce, 1991). The sequence of bLF contains five 

possible N-linked glycosylation sites. Four sites, i.e., Asn 233, 368,476, and 545, are always utilized (Spik, 1994) 

while the fifth (Asn 28 l), located in the N-lobe, is glycosylated in about 30% and 15% of the molecules in bovine 

colostrums and mature milk, respectively. (van Veen, 2002; Wei, 2000; Yoshida, 2000). 

t 

i 

I 

i 

I 

I 

i 

i 

I 

, 

12 
glycans with N-acetylgalactosamine next to galactose (Coddeville, 1992) 

and since historical human consumption of bLF at or exceeding the level of 

consumption of hLF being proposed in Pharming’s GN has not resulted in 

any reported, adverse, immunological events, one would not expect that 

consumption of Pharming’s exogenous Iactoferrin would induce any adverse, 

immunological event. And there is no direct evidence that it does - 

absolutely none. 

Of course, under certain circumstances, it may be possible that a 

specific difference in glycosylation pattern may make a significant difference 

in the way in which a specific glycosylated protein will be recognized by the 

human immune system. (Cobb, 2005). But in the specific instance at hand, 

the single difference that exists between Pharming’s human Iactoferrin and 

I 

I 

I 

endogenous human lactoferrin is hardly a difference which might lead to an 

adverse effect. Finally and not least importantly, neither Pharming nor its 

experts are aware of any direct evidence that indicates that there is any 

protein to which humans are tolerant - including bLF and hLF - which will 

induce any adverse, immune response merely as a result of a difference in 

glycosylation. Therefore, it is extremely unlikely that such difference will alter 

the normal way in which Pharming’s hLF is recognized and processed. 

3. Determinant spreading 

With regard to the potential for determinant spreading from 

alloepitopes, Pharming and its experts believe that such event is unlikely to 

occur in the situation involving consumption of Pharming’s hLF. An epitope 

is any molecular structure that can be recognized by the immune system. 

Epitopes, or the antigen from which they are derived, can be composed of 

13 
protein, carbohydrate, lipid, nucleotide, or a combination thereof. (Abbas, 

2006). It is through recognition of foreign, or non-self, epitopes that the 

immune system can identify and destroy pathogens. T-cells are known to 

respond only to linear epitopes, Le., peptide fragments (usually 8 or 20 

amino acids in length) digested from the native protein, that are presented in 

association with major histocompatability complex (MHC) molecule^.'^ 

(Abbas, 2006). An epitope is considered linear, if the target of the immune 

response is apparent in the series of adjacent amino acids without any 

requirement for secondary or tertiary structure (folding) as would occur in a 

native protein. Thus, any discussion of glycosylation is irrelevant to linear 

peptide fragments which are the only entity which determines T-cell 

response and, thus, T cell tolerance. Moreover, neither Pharming nor its 

experts are aware of any evidence showing that a mere difference in 

glycosylation would alter epitope spreading or that oral tolerance can be 

disrupted by the introduction of a differently glycosylated version of the 

same, native protein. 

I 

In addition, although single amino acid substitutions have been 

reported to alter epitope spreading resulting in increased immune response, 

the amino-acid substitutions in Pharming’s Iactoferrin mirror those in 

endogenous lactoferrin in the general population. 

Therefore, while it is true that polymorphisms present in Pharming’s 

lactoferrin can differ from those in the endogenous lactoferrin for a given 

individual, such naturally-occurring, amino acid substitutions - which fall 

within the range of variation that can be found in a normal population - are 

l4 A major histocompatibility complex molecule is defined to mean a heterodimeric membrane protein encoded in 

the major histocompatibility complex (MHC) locus that serves as a peptide display molecule for recognition by T 

lymphocytes. Two structurally distinct types of MHC molecules exist. Class I MHC molecules are present on 

nucleated cells, bind peptides derived from cytosolic proteins, and are recognized by CD8’ T cells. Class I1 MHC 

molecules are restricted largely to professional antigen-presenting cells, macrophages, and B lymphocytes, and bind 

peptides derived from endocytosed proteins, and are recognized by CD4’ T cells. (Abbas, 2006). 

14 
considered not to be immunogenic and, therefore, of little or any risk. 

Moreover, since T cells recognize only linear peptide epitopes, the concern 

about the effect, if any, of glycosylation is likely to be irrelevant to the 

discussion of T cell tolerance. 

4. Enhanced pro-inflammatory Thl response 

As indicated in Pharming’s GN, there is already a fairly sizeable 

endogenous lactoferrin production that occurs in humans as a result of 

human lactoferrin being produced in salivary glands and in intestinal mucosa 

(and elsewhere). Therefore, ingestion of Pharming’s human Iactoferrin 

would simply supplement an already existing endogenous protein. Humans 

are already tolerant to human lactoferrin and bovine lactoferrin and once 

mucosal tolerance is established, it is quite difficult to “break it. For 

example, a recent study looking at chronic ingestion of foreign proteins by 

humans (Zivny, 2001) showed that the major response to chronic antigen 

feeding is T-cell anergy (the major mechanism of tolerance to chronic 

antigen feeding) even though there are low titers of antibodies to dietary 

proteins present in secretions and serum, such as ovalbumin, bovine 

gammaglobulin and soy proteins. These anergic, antigen-specific T cells 

actively contribute to maintenance of homeostasis in the intestine in the face 

of massive antigen challenge. (Zivny, 2001). This is why significant 

consumption of bovine lactoferrin does not result in any breakage of 

tolerance to bLF and why the same significant consumption of Pharming’s 

lactoferrin will not disrupt any tolerance to endogenous lactoferrin. Indeed, 

one would expect Pharming’s Iactoferrin to be even less immunostimulatory 

and more tolerogenic than bovine lactoferrin. 

15 
Finally, Pharming and its experts believe that it is very unlikely that 

consumption of Pharming’s lactoferrin would result in perturbation of 

intestinal barrier function. (Dickenson, 1998). 

5. Increased uptake by antigen-presenting cells via the mannose 

receptor 

Although it may be theoretically possible that the differences in 

glycosylation between Pharming’s lactoferrin and endogenous lactoferrin 

could result in increased lactoferrin uptake by an antigen presenting cell 

(APC) via mannose receptors in such a manner that the Thl response is 

potentiated, Pharming is not aware of any direct evidence to support this. 

On the contrary, uptake by a mannose receptor appears to lead to an antiinflammatory response, rather than a Th 1 response. (Chieppa, 2003). 

Furthermore, the mannose-type glycans present in Pharming’s lactoferrin 

are also present in bovine lactoferrin, which is already GRAS and is not 

reported to give rise to harmful, Thl responses. Finally, Pharming is not 

aware of any direct evidence demonstrating that differential glycosylation 

alters antigen uptake and potentiates immune reactivity for native proteins. 

In conclusion, the risk of disruption of previous tolerance to endogenous 

Iactoferrin via any increased uptake of Pharming’s lactoferrin by APCs via 

the mannose receptor is considered remote. 

C. CFSAN’s concern (its first bullet point): 

Lactoferrin has been shown to enhance Type 1 T helper (Thl) cell 

activity, as well as the release of specific cytokines in the gut and 

16 
systemically following oral administration. We are concerned about 

Iactoferrin’s ability, through effects on Th 1 cells, to potentially exacerbate 

pro-inflammatory responses by this arm of the adaptive immune system. 

Chronic pro-inflammatory Thl -mediated immune responses might result in 

the promotion of autoimmune or other inflammatory disorders, in the gut or 

elsewhere, in individuals predisposed to such disorders. 

D. Pharming’s response: 

1. Background information 

concerning 

the adaptive immune system 

and 

Thl and Th2 cells 

Since the term “adaptive immune system” and, in particular, an 

understanding of the activities engaged in by Thl and Th2 cells are critical to 

CFSAN’s concerns and Pharming’s response, it seems appropriate - at this 

specific point - to provide some helpful background inf~rmation‘~ concerning 

what such term and activities entail - so as to promote common 

understanding. Since such information is quite basic and, therefore, not 

particularly helpful to a “qualified expert”, it has been set forth in a standalone attachment. (See, Attachment 1). Notwithstanding its basic nature, 

l5 Such information - over 6 pages of it - is not set forth with numerous quotes because almost all of it comes fiom 

two, authoritative, sources, i.e., two widely-respected and widely-used medical school textbooks by two widelyrespected immunologists - specifically, that by Abbas, Abul K. (at UCSF Medical School) and Lichtman, Andrew H. 

(at Harvard Medical School) entitled: Basic Immunology: Functions and Disorders of the Immune System, Second 

Edition, Saunders Elsevier (Phil, PA) (2006) and that by Abbas, Abul K. (at UCSF Medical School) and Lichtman, 

Andrew H. (at Harvard Medical School) entitled: Cellular and Molecular Immunology, Fifth Edition, Saunders 

Elsevier (Phil, PA) (2005). These sources were used because they are widely respected and represent the consensus, 

established viewpoint of qualified experts. The authors are to be credited for the information presented - including 

that appearing in many of the footnotes (in particular, the definitions). 

17 
however, the information is important and is, therefore, intended as a part of \ 

Pharming's response. 

2. Enhancement of Thl cell activity and release of specific cytokines 

a. Thl cell activity 

Recent studies inconsistently suggest that lactoferrin has 

immunoregulatory properties influencing both innate and acquired immunity. 

(See, review by Fischer, 2006). In particular, it has been suggested that 

lactoferrin influences T cell maturation, proliferation and differentiation into Thelper I (Thl') or T-helper 2 (Th2) cells. Thl and Th2 cells are two 

functional subsets of Th- or CD4-positive T cells, whose function depends 

upon the specific types of cytokines that are generated. (Rafiq, 2000; 

Mosmann, 1996; Abbas, 1996). CD4-positive Thl cells produce IFNy and 

IL-2, but not IL-4 or IL-5, and drive cellular immunity to attack viruses and 

other intracellular pathogens; conversely, CD4-positive Th2 cells produce IL4, IL-5 and IL-13, but not IFNy or IL-2, and drive humoral immunity that upregulates antibody production to attack extracellular organisms. Whereas 

Thl cells are known as important producers of IFNy, other cell types are also 

able to produce IFNy, including (in particular) NK cells and nonpolarized 

memory T cells. (Ye, 1995; Biron, 1999). It is important to note that 

increased IFNy production does not necessarily reflect increased Thl cell 

I 

1 

I 

I 

I 

activity . , 

I 

The establishment of the Thl/Th2 balance is determined early during i 

immune responses and depends on many factors including antigen 

structure, the functional status of antigen-presenting cells (APCs), the 

strength of T cell activation, the presence of cytokines, co-stimulatory signals I 

18 
and the microenvironment. (Rafiq, 2000). Both Thl and Th2 cells negatively 

cross-regulate the function of one another through their respective cytokines. 

(Romagnani S, 1994; Maggi, 1992). Furthermore, it should be noted that IL18, frequently reported as being upregulated upon lactoferrin oral 

administration, does not skew Th responses towards a Thl response. 

Rather, Thl responses are highly dependent on and stimulated by IL-12. 

Once Thl cells are polarized, then IL-18 can act on them to enhance IFNy 

production. IL-18 also enhances IFNy production of NK cells. Thus, 

production of IL-18 does not correlate to induction of Thl responses. 

(Nakanishi, 2001 ; Okamura, 1998). 

Regarding oral administration of lactoferrin, most of the data comes 

from orally administrated bovine lactoferrin (bLF) rather than human 

lactoferrin (hLF). Since there is sufficient evidence indicating that both 

proteins are comparable in structure and function (Baker, 2000; Nuijens, 

1996), the effects observed on the immune system as a result of either bLF 

or hLF administration have been used as model for oral administration of 

Pharming’s hLF. 

Review of the available, scientific literature16 concerning oral 

administration of lactoferrin indicates that there are contradictory results with 

respect to the evidence showing that Iactoferrin affects proliferation and 

differentiation of T cells into Thl and Th2 cells17. The induction of either Thl 

or Th2 biased immune responses by lactoferrin is complex as the observed 

l6 These preclinical and clinical studies - involving oral administration of lactoferrrin - include those by Artym 

(2003), Haversen (2003), Hayes (2005), Iigo (2004), Ishii (2003), Kruzel(2006), Kuhara (2006), Kuhara (2000), 

Nakajima (1999), Sfeir (2004), Takakura (2006), Takakura (2004), Tanaka (1999), Togawa (2002), Ueno (2006), 

Varadhachary (2004), Wakabayashi (2006), Wakabayashi (2004), Wakabayashi (2003), Wakabayshi (2002), Wang 

(2000), Zimecki 2006), Zimecki (2005), Zimecki (1998) and Zimecki (1995). Other, older studies also have been 

conducted. Their findings are all referred to in one or more of the studies referred to in the above-referenced list. All 

of the above-referenced studies are hlly cited and summarized in Attachment 2. 

” However, please note that Zimecki, et al. reported that lactoferrin inhibits proliferation and cytokine production by 

Thl cells -but not Th2 cells. (Zimecki, 1996). 

19 
effects appear to be, at least in part, dependent on the mode of lactoferrin 

delivery and on whether any ongoing inflammatory or immune response is 

occurring. (For a review of all pertinent studies, see Fischer, 2006). Based 

on the available data, Pharming and its expert panel concluded that the 

evidence for orally administered lactoferrin eliciting a positive CD4' Thl 

biased response is not convincing. This is because most studies suggest a 

change in Thl cell activity based on alterations in cytokine levels, in 

I 

particular IFNy levels, but did not identify the cell-type responsible for the 

cytokine production. As mentioned above, increased IFNy production does 

not specifically indicate increased Thl cell activity. More likely, it indicates 

enhanced NK cell activity. In addition, the information is not convincing 

because some papers show potential Thl responses (Le., IFNy secretion) 

within a few days. However, there is a critical time element involved in that it 

takes weeks for Thl and Th2 cells to become firmly polarized. (Murphy, 

1996). Even in culture, where one can create an optimal environment, it 

takes at least a week - and usually 2-3 weeks - to generate CD4' Thl and 

Th2 cells. (Perez, 1995). 

Finally and not least importantly, even if - for sake of argument - oral 

consumption of human lactoferrin were to enhance Thl responses, that 

would not necessarily be deleterious. First of all, there is nothing in the 

direct evidence that demonstrates that lactoferrin given orally enhances any 

pathologic Thl responses'*. On the contrary, there is evidence from a rat 

colitis model and other rat and mouse studies that demonstrate that oral 

Guillen (2002) did report increased severity of collagen-induced arthritis in transgenic mice expressing human 

lactoferrin associated with an apparently enhanced Thl response. However, this conclusion was based on cytokine 

levels which, as argued elsewhere, do not automatically imply a Thl response, and the continuous and chronic 

systemic exposure in this model is quite different from the oral exposure envisaged in humans. In contrast to these 

results, the same group earlier demonstrated that periarticular injection of hLF in mouse models of autoimmune 

arthritis and septic arthritis demonstrated significant treatment benefits. (Guillen, 2000). 

20 
consumption of lactoferrin inhibits a pathologic Thl response via 

upregulation of IL-I 0 and inhibition of IFN-y. (Zimecki, 2006; Takakura, 2006; 

Togawa, 2002). 

b. Release of specific cytokines 

With respect to increased release of specific cytokines in the gut 

and/or systemically following oral administration of lactoferrin, various animal 

studies generally reported only local changes in the expression/production of 

both Thl (e.g., IFNy, IL-2) and Th2 (e.g.,IL-4, IL-IO) cytokines. (Wang, 

2000; Kuhara, 2000; ligo, 2004; Wakabayashi, 2006; Varadhachary, 2004). 

In addition, various animal studies indicate that oral lactoferrin administration 

might increase both local and systemic IL-I8 levels. (ligo, 2004; 

Wakabayashi, 2004; Kuhara, 2006; Hayes, 2005). Pharming’s expert panel 

believes, however, that the effect of IL-18 will occur locally and not 

systemically. Regarding the systemic levels of IL-I 8, oral administration of 

lactoferrin at doses up to 9 gram per day in human adults with solid tumors 

only resulted in a 15% increase of circulating IL-I 8, which is considered very 

low. (Hayes, 2005). More importantly, in this study no serious adverse 

events were reported and Iactoferrin was well-tolerated by all subjects at a 

dosage of 150 mg/kg/day - which is very significantly higher than the level of 

maximum daily consumption that Pharming proposes in its GRAS 

notification. In another study, a transient increase of IL-18 was observed in 

serum of hepatitis C patients receiving Iactoferrin at an oral dosage of 600 

milligrams per day for 12 months. (Ishii, 2003). However, the data showed 

large variation and the observed increase of IL-18 decreased again after 3 

months to baseline levels. Taking all such information into account, 

Pharming and its experts believe that to the extent cytokines are reported to 

21 
be released upon oral administration of lactoferrin, such reports do not 

indicate a consistent pattern of enhancement. 

In conclusion, it is Pharming’s and its experts’ opinion that, based on 

the available data, there is not convincing evidence that demonstrates to a 

reasonable certainty that lactoferrin specifically enhances Th 1 responses or 

can significantly increase systemic cytokine levels over time. In contrast, 

there is sufficient evidence that lactoferrin enhances innate immune 

responses in the gut, e.g., by increasing IL-18 produ~tion’~ (most likely 

locally, not systemically) and by increasing NK cell activity, both of which are 

considered beneficial rather than deleterious. Indeed, there is no direct 

evidence Uhat increasing innate function is in any way detrimental; rather, 

such increased function is considered beneficial. 

3. Effect of oral administration of lactoferrin on autoimmune 

or other inflammatory disorders 

a. Autoimmunity 

T cell responses to antigens are classified on the basis of the amount 

and kind of cytokines produced. Using this classification, T cell responses in 

MHC-class-l I-restricted autoimmune diseases appear to be predominantly of 

the Thl type. (Rosloniec, 2002). Thus, Pharming understands CFSAN’s 

concern to be about whether oral administration of lactoferrin enhances Thl 

responses and, thus, whether same could lead to the onset or enhancement 

of autoimmune diseases. Although the mechanisms of autoimmunity are not 

Lactoferrin has been shown to enhance IL- 18 production by intestinal epithelial cells, thus enhancing the innate 

immune response. Human intestinal epithelial cells have been shown to condition human dendritic cells along a noninflammatory ThZlike pathway, rather than towards Thl responses. (Rimoldi, 2005). 

22 
yet sufficiently understood, the concern of CFSAN is considered possible but 

highly unlikely by experts consulted by Pharming. First (and, perhaps, most 

importantly), there is a growing body of scientific evidence that indicates that 

orally administered lactoferrin significantly inhibits and/or diminishes and/or 

improves ((rather than initiates or enhances) autoimmune diseases. (See, 

e.g. , Kruzlel, 2006 (orally administered lactoferrin causes reduction of 

clinical signs of multiple sclerosis in patients - in parallel to normalization of 

cytokine production by peripheral blood cells); Zimecki, 2006 (orally 

administered Iactoferrin significantly diminished the clinical symptoms of 

experimental autoimmune encephelomyelitis in Lewis rats); and Togawa, 

2002 (oral administration of lactoferrin significantly reduced colitis in rats))*’. 

Second, it is very possible that Thl cells are not even involved in 

autoimmune diseases. Rather, such diseases may well be induced by the 

recently discovered T-helper 17 subset. (Hue, 2006; Yen, 2006). Third, as 

already discussed above, the evidence that orally administered lactoferrin 

elicits a Thl biased response or potentiates a pre-existing Thl-mediated 

immune response is considered not well-established. Fourth, hLF is 

naturally expressed in saliva and the gastro-intestinal tract; thus, humans 

have a significant daily naturally-occurring exposure to hLF2’. For instance, 

the intake of lactoferrin from saliva alone is about 20 mg/day. (Tanida, 2003). 

Consequently, humans are tolerant to hLF. Once oral tolerance has been 

established, it is very hard to disrupt, even in patients with chronic 

0 

2o See also, two other studies showing similar results, i.e., Guillen, 2000 (which study demonstrated that periarticular 

injection of hLF in mouse models of autoimmune arthritis and septic arthritis demonstrated significant treatment 

benefits) and Zimecki, 1995 (which study demonstrated that intraperitoneal injection of bLF in mice significantly 

inhibited autoinunune hemolytic anemia). 

Such daily, natural exposure also emanates from lactoferrin produced and released by or in, for example, mothers’ 

milk, neutrophils and various mucosa. Indeed, as Pharming’s GN indicates (at pages 26 and 28), human lactoferrin is 

virtually ubiquitous throughout the human body. 

23 
stimulation of the immune system. (Zivny, 2001). Moreover, the oral 

administration of an autoantigen has been shown to be beneficial in the 

treatment (of various experimental, autoimmune diseases and this method of 

inducing immune non-responsiveness has currently been applied to the 

prevention and treatment of human autoimmune diseases. (See reviews by 

Wardrop, 1999; and Sosroseno, 1995). Fifth, although there is extensive 

reporting on the presence of autoantibodies against lactoferrin, there is no 

evidence that these antibodies play any role in the pathology of autoimmune 

diseases. There is a large body of scientific literature on antilactoferrin autoantibodies as a component of antineutrophil cytoplasmic antibodies (ANCA). 

(See review by Malenica, 2004). In addition, individuals with a wide range of 

autoimmune conditions have anti-lactoferrin autoantibodies. Despite this 

large body of scientific literature on these antibodies, there is no evidence 

showing them to have any role in the etiology,of autoimmune disease, and 

there is a general consensus among qualified experts that they are an 

epiphenomenon. Furthermore, all individuals possess low but detectable 

amounts of circulating and mucosal human lactoferrin. Therefore, it is 

considered highly unlikely that oral administration of human lactoferrin, even 

to an individual with an ongoing autoimmune disease, would increase 

autoantibody levels. Even if oral lactoferrin were to increase the level of such 

antibodies, it would be clinically irrelevant, Le., unlikely to have any impact 

on disease pathogenesis. 

Anti-lactoferrin autoantibodies have not been shown to be 

involved in the pathogenesis of any disease. In contrast, there is data that 

autoantibodies in general may help clear and degrade autoantigens, thus 

reducing T cell sensitization to them. (Mizoguchi, 1997). It should also be 

pointed out that there have been multiple trials in which autoantigens were 

24 
fed to patients with autoimmune diseases to see if this might ameliorate the 

disease. For example, these trials have fed human insulin to autoimmune 

diabetics, collagen to rheumatoid arthritis patients, and myelin proteins to 

patients with multiple sclerosis. These trials have not shown any consistent 

benefit to the patients; however, there were no deleterious effects from 

autoantigen feeding and this was done in substantial numbers of individuals. 

(Faria, 2005). 

In conclusion, Pharming and its experts believe that it is highly unlikely 

that oral cionsumption of Pharming’s lactoferrin at the level here in question 

would lead to the development or the perpetuation or enhancement of an 

autoimmune response. 

b. Inflammatory disorders 

As discussed above, there is sufficient evidence that lactoferrin 

enhances innate immune responses in the gut. It is CFSAN’s concern that 

this may lead to promotion of inflammatory disorders in the gut. Pharming 

understands this concern, particularly as it relates to inflammatory bowel 

disease (IBD), a term which commonly incorporates ulcerative colitis (UC) 

and Crohn’s disease (CD). Both diseases are chronic inflammatory 

conditions of the gut in which Crohn’s disease may affect any part of the 

gastrointestinal tract, whereas UC mainly affects the colon. In IBD, there 

appear to be multiple levels of immune responses, including innate, adaptive 

and regulatory immune responses. There is emerging literature that innate 

immune defects can contribute to the development of IBD. (See, e.g., 

Beckwith, 2005; Hugot, 2001 ; Ogura, 2001 ). However, neither Pharming nor 

its experts are aware of any scientific evidence that supports the idea that a 

25 
a 

0 

a 

low-level Thl response or enhancement of the innate immune response, 

even on a chronic basis, would be detrimental or trigger IBD. In contrast, 

Iactoferrin has been repeatedly shown to enhance the production of IL-18 by 

intestinal epithelial cells (see Attachment 2), thereby increasing innate 

immunity, which is considered beneficial rather than deleterious for 

susceptible individuals. This beneficial enhancing of innate immunity has 

been confirmed in a recent open label trial in patients with Crohn’s disease 

who received granulocyte-macrophage colony-stimulating factor (GM-CSF). 

(Dieckgraefe, 2002). GM-CSF is a cytokine involved in enhancement of the 

qualitative function of various immune cells, and stimulates the expansion 

and differentiation of haemopoetic progenetors. (Armitage, 1998). The 

results showed an enhancement of the intestinal innate immune response 

resulting in an amelioration of the disease. 

Even with regard to individuals who have a “leaky” gut2*, such as can 

be found in inflammatory bowel disease, orally administered exogenous 

Iactoferrin is simply supplementing large endogenous production of 

lactoferrin in alimentary secretions. There are low levels of antibodies to 

various foods in intestinal secretions and serum, but there is no evidence 

that these have any detrimental effect. There is also no evidence that 

immunologic reactions to food have any adverse effect in inflammatory 

bowel disease or that any foods exacerbate inflammatory bowel disease. 

In contrast to the concern that orally administered lactoferrin may 

impact negatively on inflammatory bowel disorders, there is a growing body 

of scientific evidence - as Zimecki et al. point out - that demonstrates just 

the opposite, i.e., that orally consumed lactoferrin exhibits “distinct antiinflammatory properties.” (Zimecki, 2006). Such conclusion - the authors 

To the extent that the “leaky” gut concept exists - and such concept is not generally recognized - it generally refers 22 

to the movement of molecules with a molecular weight of less than 1000 daltons. 

26 
indicate - is supported by a growing number of studies incorporating a 

number of models “including experimentally induced bowel inflammation in 

rats (Togawa et al., 2002), autoimmune disorders in mice (Zimecki et al., 

1995; Guillen et al., 2000), experimental endotoxemia in mice (Kruzel et al., 

2002), and inflammatory reactions to Mycobacterium bovis (Zimecki et al., 

1994).” (Zimecki, 2006; see also Haverson, 2003 which reported on the 

anti-inflammatory effects of hLF in an experimental colitis model in mice). In 

all such models, lactoferrin exhibited significant anti-inflammatory properties. 

Moreover, lactoferrin induces TGF-P production which is widely 

considered an anti-inflammatory cytokine. (Zimecki, 2005; Ward, 2002). 

Since TGF-P is an anti-inflammatory cytokine associated with the induction 

of antigen-specific regulatory T cells and such cells produce TGF-P or IL-IO, 

these cells can inhibit the induction of inflammatory responses. In particular, 

these cytokines suppress lFNy production and activity from activated Thl 

cells. Lactoferrin can even exhibit strong anti-inflammatory effects in 

dexamethasone-induced acute colitis in a mouse model. (Haverson, 2003). 

In further contrast to suggesting that human lactoferrin - a substance 

native to humans - might be responsible for either autoimmune or other 

inflammatory disorders, there is a growing body of scientific evidence 

showing that defects in innate immunity can lead to an abnormal adaptive 

immune response, some of which are manifest by autoimmune disease. A 

good example of this is the non-obese diabetic (NOD) mouse, which has 

some well-defined defects in innate immune responses. Stimulation of the 

NOD innate system by a variety of means blocks the development of the 

autoreactive T cell response to islet cells and, thus, prevents diabetes. In 

inflammatory bowel disease there is emerging literature that innate immune 

defects can contribute to the development of IBD. (Korzenik, 2006). For 

27 
example, a colitis susceptibility gene has been identified which appears to 

function by regulating innate immunity. (Beckwith, 2005; Hugot, 2001 ; 

Ogura, 2081). In addition (and as mentioned above), there is a trial in which 

GM-CSF has been administered to patients with Crohn’s disease to enhance 

their innate immunity and, thus, ameliorate their disease. (Diekgraefe, 

2002). Thus, autoimmune or chronic inflammatory diseases are more likely 

I 

I 

to result from deficient innate immune cytokine production or function. 

Of course, there is no scientific evidence that suggests - let alone 

demonstrates - that orally consumed human lactoferrin induces any 

deficiency in any innate immune mechanism. In fact, orally consumed 

human lactoferrin does just the opposite, Le., it enhances innate immunity, 

which is deemed beneficial. 

E. CFSAM’s concern (its third bullet point): 

The inotice states that Iactoferrin is known for its immunomodulatory 

properties. However, the preclinical studies presented in the notice do not 

address the immunomodulatory activities of lactoferrin. What preclinical 

evidence supports the safety of exogenous lactoferrin for its intended use 

given its activity as a biological response modifier of the immune system? 

F. Pharming’s response: , 

Fortunately, there are numerous, published, preclinical studies and 

other published information which are pertinent to and evaluate the potential 

immunogenicity of lactoferrin. Most of the preclinical studies have already 

been discussed above. Ordinarily, Pharming would review all such studies 

28 
at this juncture; however, because almost have been recently reviewed 

by Fischer, 2006 (the review was published in June), it need not do so again 

here. (Holwever, all such studies are cited and summarized in Attachment 2. 

Such attachment is intended as a part of Pharming’s response). 

Fischer et at. reviewed 80 different studies and related publications 

pertinent to lactoferrin’s potential immunoregulatory properties - especially 

as they relate to lactoferrin’s ability to regulate Thl and Th2 responses. 

After discussing the findings of all such studies and information, Fischer et 

al. concluded that lactoferrin does not induce any adverse, non-allergic 

immune responses - via either the innate or adaptive immune defense 

mechanisms. More specifically, the authors concluded that: 

1. Iactoferrin causes a Thl polarization in diseases in which the 

ability to control infection or tumor relies on a strong Thl 

response; 

2. lactoferrin also reduces the Thl component to limit excessive 

3. Iactoferrin provides protection against Thl - or Th2-induced 

diseases, such as autoimmune or allergic diseases, through 

correction of the ThITTh2 imbalance. 

Thus, consistent with Pharming’s and its experts’ assessment, Fischer et at. 

also concluded that the available information indicates that oral consumption 

of lactoferrin - even at levels exceeding the level of use here at issue - 

results in only beneficial immunological effects. 

Finally, since it is very difficult - despite numerous attempts - to find a 

mucosal adjuvant among substances likely to be orally consumed, Pharming 

inflammatory response; and 

23 There are a few preclinical studies on Pharming’s reference list which do not appear on the reference list attached to 

the Fischer article. Since such studies supply only information like that already reviewed by Fischer, they do not alter 

the scope of the substance discussed or the conclusions reached by Fischer, et al. 

29 
and its exlperts believe that it is very unlikely that further preclinical testing of 

Pharming’s Iactoferrin at the daily level here at issue and even for longer 

periods of exposure would result in any demonstration that Pharming’s 

lactoferrin is able to induce - via oral consumption - any adverse 

immunomodulatory effect. 

1 

G. CFSAN’s concern (its fourth bullet point): 

The primate and human studies of oral lactoferrin administration cited 

in the notice are in small populations for relatively short periods of time. 

Most of the studies with recombinant human lactoferrin focus on efficacy 

rather than safety, and many of the human studies involve subjects with preexisting medical conditions. Where safety endpoints are included, they do 

not appear relevant to the effects of lactoferrin as a biological response 

modifier of the immune system. Is there clinical evidence that supports the 

immunological safety of long-term exogenous lactoferrin administration at 

the proposed use level in the general population? 

H. Pharming’s response: 

To date, no clinical studies have been performed in healthy volunteers 

in whom the long-term safety of exogenous applied lactoferrin has been 

investigated. The primary reason for this is that there is general consensus 

among experts that hLF has been shown to be so safe - via natural 

exposures - and at such high doses that no additional safety evaluation is 

necessary. However, there are a few clinical studies that have investigated 

the immunological consequences of long term oral administration of 

I 

30 
Iactoferrin in diseased adults. In one study, 36 patients with chronic hepatitis 

C and orally administered bLF (600 mg/day for 12 months) showed a 

transient increase in serum IL-18 levels (pg/mI range) that peaked after 3 

months and gradually returned to base-line. (Ishii, 2003). The authors 

concluded that the effect of bLF administration was limited to 3 months 

which suggest that prolonged administration results in adaptation. During 

this study lactoferrin was co-administered with active (live) Bifidobacterium 

longurn. These bacteria are known to be potent immunomodulatory 

"probiotic" bacteria, which makes it impossible in this study design to 

distinguish the effects of lactoferrin, if any, from those of the bifidobacteria. In 

another study, 199 subjects were evaluated for safety and efficacy in a 

randomized, double-blind, placebo-controlled trial using bovine lactoferrin 

fed orally (1.8 gramlday for 12 weeks) to chronic hepatitis C patients. The 

authors concluded that bLF treatment was well-tolerated and no serious 

toxicities were observed. (Ueno, 2006). I 

Taken together, based on these data and the fact that exogenous 

administration of lactoferrin would supplement an already substantial amount 

of endogenous lactoferrin, it is Pharming's and its experts' opinion that it is 

extremely unlikely that long-term, exogenous lactoferrin administration (at 

the level here in question) would be detrimental when consumed by either 

the general population or by individuals with pre-existing conditions. 

1. CFSAN's concern (its fifth bullet point): 

The notice provides an acceptable daily intake (ADI) based on the 

maximal consumption of lactoferrin in human milk by infants. The infant 

immune system and gut are different from that of the adult, for example in 

31 
the infant bias towards Th2 responses relative to Thl. Given this, what 

evidence supports the use of exposure data derived from infants in setting 

an AD1 for adults that takes into account lactoferrin’s activity as a biological 

response modifier of the immune system? 

J. Pharming’s response: 

The infant immune system and gut are, indeed, different from those of 

an adult - in both instances the infant system is less mature than that of the 

adult. Both situations should result in an infant being significantly more - not 

less - vulnerable to the deleterious effects of orally consumed substances 

than adults who are able to tolerate significantly more. Indeed, infants 

tolerate very large quantities of bLF with no problem. With regard to those 

adults who are deemed “predisposed” or “susceptible”, please see the 

discussions set forth above in Sections II B and D. 

The notice actually provides not one but various approaches to 

determine, based on safety parameters, the maximum daily exposure of 

human lactoferrin that is safe for oral consumption, one of which is indeed 

based on the maximal consumption of natural lactoferrin in human milk 

calculated for infants. (GRN000189 subsection V(A)(3)). Other approaches 

are based on preclinical studies, mainly in rats and rhesus monkeys, and 

clinical studies in both infants and adults with natural or recombinant human 

lactoferrin. (See subsections V(A)(6), V(H)(l )(b)(l-3), V(H)(2)(a-b)). 

The maximum acceptable daily oral exposure of humans to human 

lactoferrin found in the evaluation of available data was obtained from infants 

- which consumption level was equivalent to, at least, 266 mg (and perhaps 

32 
as high as 3077) hLF/kg/day which was based on a daily consumption of at 

least 2 gram hLF. 

In this respect, it should be noted that such values have also been 

obtained for adults, as the highest oral consumption of hLF given to adults 

was 250 mg hLF/kg/day which corresponded to an oral dosage of I5 gram 

hLF. (Andersen, 2004). In animals, even higher oral dosages of hLF were 

tested; the highest dose evaluated and considered safe was 6000 

mg/kg/day. (See, section V(H)( 1 )(b)( 1 )). 

More importantly, the oral safety of Pharmings hLF, as proposed for 

use as an ingredient in sports and functional foods at 100 mg per product 

serving, is based on the NOAEL of 2000 mg of Pharming’s hLF per kg BW 

as described in section V(1)(2). Based on this NOAEL, all estimated daily 

intakes (see section GRNOOOl89 V(1)(2)) are all well under 1/100th of the 

NOAEL - even at the 90th percentile consumption level. Accordingly, the 

consumption levels should not pose any safety risk to any consumer. In 

addition, the safety level is fully supported by the safety levels emanating 

from the natural exposure to native hLF and from the preclinical and clinical 

studies of various hLF products (as discussed in subsections V(H)( l)(b)(3) 

and V(H)(2)(b)), which include both adults and infants. 

K. CFSAN’s concern (its sixth bullet point): 

The notice provides an assessment of the potential allergenicity of 

Pharming’s lactoferrin and states that there is no evidence to date that antiIactoferrin antibodies are associated with autoimmune pathology. Other than 

this statement, the notice does not address the potential for adverse nonallergic responses to Pharming’s lactoferrin by the adaptive immune system 

33 
as described above. To what extent has Pharming evaluated this risk, and 

! what evidence was used in the evaluation? I 

L. Pharming’s response: 

The extent to which Pharming has considered and evaluated the risk, if 

any, that oral consumption of Pharming’s lactoferrin - at the level proposed 

in its GN - might induce some adverse response by the adaptive immune 

system has been thoroughly discussed in the preceding responses to bullet 

points numbers 1-5. Such responses indicate that any such risk is, at worst, 

very unlikely, and at best, practically non-existent. 

111. Conclusion 

Pharming and its experts are of the opinion that when all of the 

pertinent, direct, scientific evidence is considered as a whole - and as 

specifically discussed above in this response and in Attachments 1 and 2 - a 

fair evaluation of such evidence demonstrates to a reasonable certainty that 

Pharming’s exogenous lactoferrin will not induce any adverse, non-allergic 

response by the adaptive immune system. This discussion and conclusion - 

when combined with the information in Pharming’s GN - demonstrate to a 

reasonable certainty, Pharming and its experts believe, that Pharming’s 

human lactoferrin product is not deleterious and generally recognized as 

safe for human consumption by all individuals at 100 mg per product serving. 

34 
a 

a 

I. 

IV. Expert Panel’s Statement 

We, the undersigned, are the qualified experts asked by Pharming to 

participate in responding to CFSAN’s concerns (as set forth in its May 17, , 

2006 email to Charles L. Morin, Pharming’s regulatory counsel). Copies of 

our curriculum vitae are attached. 

In response to Pharming’s request, we (among other things): 

1. agree to participate as qualified experts; 

2. assisted in identifying pertinent information, especially 

direct scientific evidence; 

3. reviewed much of such information; 

4. reviewed Pharming’s GRAS notification; 

5. provided initial assessments of CFSAN’s concerns; 

6. responded to numerous written and oral questions presented 

by Pharming; 

7. reviewed and commented on non-final drafts of Pharming’s 

response to CFSAN; and 

8. reviewed and accepted Pharming’s final draft of its response 

to CFSAN. 

Accordingly, that which is in the response as received by CFSAN amounts to 

not only Pharming’s response, but also our expert view. 

More specifically, we believe, in summary: 

I 

1. that with regard to the amino acid sequence of Pharming’s 

exogenous lactoferrin there is no scientific evidence whatsoever 

that an individual producing one of the above-referenced 

endogenous lactoferrins reacts - immunologically speaking - 

35 
differently when exposed to any one of the other abovereferenced endogenous lactoferrins; consequently, since 

Pharming’s human lactoferrin only duplicates endogenous 

lactoferrin (i.e., falls within the range of variation that can be 

found in a normal population), we believe that a reasonable, 

qualified expert would also expect such exogenous lactoferrin 

not to induce any adverse, immunological event and we believe 

that there is virtually no risk that it will; 

2. that with regard to the specific glycosylation pattern (including 

any attached oligomannose glycans) of Pharming’s exogenous 

lactoferrin there is no demonstrated reason to believe that such 

pattern of glycans - either individually or collectively - would 

induce any adverse immunological event and we think it very 

unlikely that it would; 

3. that with regard to previous tolerance being disrupted via 

determinant spreading from alloepitopes we are not aware of any 

evidence showing that a mere difference in glycosylation would 

alter epitope spreading or that oral tolerance can be disrupted by 

the introduction of a differently glycosylated version of the same, 

native protein; therefore, we believe it is very unlikely that 

Pharming’s lactoferrin could or would induce such an event; 

4. that with regard to previous tolerance being disrupted via 

enhanced pro-inflammatory Th 1 responses there is nothing in 

the direct evidence that demonstrates that lactoferrin given orally 

36 
- especially at the fairly small level here in question, i.e., 100 

mg/product serving - enhances any pathologic Thl responses 

and we believe it very unlikely that it could or would; 

I 

5. that it is very unlikely that consumption of Pharming’s 

lactoferrin would result in perturbation of intestinal barrier 

function ; 

6. that with regard to previous tolerance being disrupted via 

increased uptake by antigen-presenting cells via the mannose 

receptor we are not aware of any direct evidence demonstrating 

that differential glycosylation alters antigen uptake and 

potentiates immune reactivity for native proteins; thus, we 

believe the risk of disruption of previous tolerance to endogenous 

lactoferrin via any increased uptake of Pharming’s Iactoferrin by 

APCs via the mannose receptor to be remote; 

7. that it is highly unlikely that oral consumption of Pharming’s 

lactoferrin at the level here in question would lead to the 

development or the perturbation or enhancement of an 

autoimmune response - either in the general population or in 

one predisposed to inflammatory disorders; 

8. that there is no scientific evidence that immunologic reactions 

to food have any adverse effect on inflammatory bowel disease 

or that any foods exacerbate inflammatory bowel disease; thus, 

we think it very unlikely that oral consumption of Pharming’s 

37 
I 

lactoferrin at the level here in question could or would lead to the 

development, perturbation or enhancement of any inflammatory 

disorder; 

9. that there is no scientific evidence that suggests - let alone 

demonstrates - that orally consumed human lactoferrin induces 

any deficiency in any innate immune mechanism; in fact, orally 

consumed human lactoferrin does just the opposite, i.e., it 

enhances innate immunity, which we deem beneficial; 

IO. that to the extent oral consumption of lactoferrin at the level 

here at issue might induce local production of cytokines we 

believe such production would result in beneficial - not adverse - 

effects; 

11. that the available scientific evidence indicates that oral 

consumption of lactoferrin - even at levels exceeding the level 

here at issue (Le., 100 mgkerving) - results in only beneficial 

i mmu nolog ical events; 

12. that it is very unlikely that further preclinical testing of 

Pharming’s lactoferrin would result in any demonstration that 

Pharming’s Iactoferrin is able to induce - via oral consumption 

(especially at the level here at issue) - any adverse 

immunomodulatory effect; and 

38 
13. that it is extremely unlikely that long-term, exogenous 

Iactoferrin consumption (at the level here in question) would be 

I 

detrimental when consumed by either the general population or 

by individuals with pre-existing conditions. 

\ 

Accordingly, we are of the opinion that when all of the pertinent, direct, 

scientific evidence is considered as a whole, a fair evaluation of it I 

demonstrates to a reasonable certainty that Pharming’s exogenous 

lactoferrin will not induce any adverse, non-allergic response by the adaptive 

immune system and - when combined with the information in Pharming’s 

GN - demonstrates to a reasonable certainty that Pharming’s product is not 

deleterious and is generally recognized as safe for human consumption at 

Date I ci J 11/06 

Jeremy H. Brock, ScD, PhD, MSc 

Senior Research Fellow 

Department of Immunology 

University of Glasgow 

Charles 0. Elson, MD 

Professor of Medicine and Microbiology 

Vice-Chair for Research 

Department of Medicine 

Director, Inflammatory Bowel Disease Center 

Senior Scientist, Multipurpose Arthritis Center 

University of Alabama at Birmingham 

39 
Cathryrn R. Nagler, P d 

Associate Professor of Pediatrics (Immunology) 

Center for Immunology and Inflammatory Disease 

Division o§ Rheumatology, Allergy and Immunology and Center for the Study 

Harvard Medical School 

Date -JLcun,/b~ 20; 2~ 6 

AndrtS H. Penninks, PhD 

Senior (Immuno) Toxicologist 

Division of Experimental Immunology 

Department of Toxicology and Applied Pharmacology 

TNO; and 

Department of Immunotoxicology 

University of Utrecht 

Hubertus F.J. Savelkoul, PhD 

Professor and Chairman 

Department of Cell Biology and Immunology 

Wagenineen Universitv U U d 

40 

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I 8 
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9 
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IO 
I 

7 

. .. J 
Background Information' 

Concerning 

The adaptive immune system 

and 

Thl and ThZ cells 

The human immune system is comprised of a collection of 

specific cells, tissues, and molecules that mediate protection against 

- under normal conditions - entities (such as pathogens) deemed to 

be foreign, Le., non-self. The immune response is the coordinated 

reaction of the above-referenced cells and molecules to such foreign 

entities. 

To the extent that one is focusing - as here - on that part of the 

immune system that responds to and protects against foreign entities 

that enter the body through mucosal surfaces - such as those found 

in the gastrointestinal tract, Le., the gut - one is focusing on the 

mucosal immune system. This system is comprised of collections of 

lymphocytes* and antigen-presenting cells3 in the epithelia4 and 

' Such information - over 6 pages of it - is not set forth with numerous quotes because almost all of it 

comes from two, authoritative, sources, Le., two widely-respected and widely-used medical school 

textbooks by two widely-respected immunologists - specifically, that by Abbas, Abul K. (at UCSF Medical 

Schoo1)'and Lichtman, Andrew H. (at Harvard Medical School) entitled: Basic Immunology: Functions and 

Disorders of the Immune System, Second Edition, Saunders Elsevier (Phil, PA) (2005) and that by Abbas, 

Abul K. (at UCSF Medical School) and Lichtman, Andrew H. (at Harvard Medical School) entitled: 

Cellular and Molecular Immunology, Fifth Edition, Saunders Elsevier (Phil, PA) (2005). These sources 

were used because they are widely respected and represent the consensus, established viewpoint of 

qualified experts. The authors are to be credited for the information presented - including that appearing in 

the footnotes. 

* A lymphocyte is a cell type found in the blood, lymphoid tissues, and virtually all organs, that expresses 

receptors for antigens and mediates immune responses. Lymphocytes include B and T cells (the cells of 

adaptive immunity) and natural killer (NK) cells (the mediators of some innate immune responses). 

An antigen-presenting cell (APC) is a specialized cell that displays peptide fiagments of protein 

antigens, in association with major histocompatibility (MHC) molecules on its surface, and activates 

antigen-specific T cells. In addition to displaying peptide-MHC complexes, APCs must also express 

1 - 
lamina propria5 of mucosal surfaces. It includes intra-epithelial 

e 

a 

lymphocytes - mainly T cells6 - and organized collections of 

lymphocytes - often rich in B cells7 - below mucosal epithelia. 

Host defense mechanisms consist of “innate immunity” and 

“adaptive immunity”. Innate immunity (a pre-existing or native 

immunity) mediates the initial protection against infectious, foreign 

entities (but not noninfectious foreign entities) and relies on 

mechanisms that exist before infection. These mechanisms are 

capable of rapid responses and react in essentially the same way to 

repeat infections. The innate immune system includes - as its first 

line of defense against invading microbes - epithelial barriers, 

specialized cells and natural antibiotics present in epithelia and - 

when a microbe does breach epithelia and enter either a tissue or the 

circulation - phagocytes* (including neutrophilsg and macrophages”), 

~ ~~ 

costimulatory molecules to optimally activate T lymphocytes. Located in the epithelium, APCs serve to 

capture antigens and transport them to peripheral lymphoid tissues - such as regional lymph nodes. 

The epithelia are the coverings of the internal and external surfaces of the human body. They consist of 

cells joined by small amounts of cementing substances. Epithelium is classified into types on the basis of 

the number of layers deep it is and the shape of the superficial cells comprising each layer. 

’ The lamina propria is the connective tissue coat of a given membrane just deep to the epithelium and 

basement membrane. 

T cells are the cell type that mediates cell-mediated immune responses in the adaptive immune system. T 

lymphocytes mature in the thymus, circulate in the blood, populate secondary lymphoid tissues, and are 

recruited to peripheral sites of antigen exposure. They express antigen receptors (T cell receptors) that 

recognize peptide fragments of foreign proteins bound to self major histocompatibility complex molecules. 

Functional subsets of T lymphocytes include CD4’ helper T cells and CD8’ cytolytic T lymphocytes. 

B cells are the only cell type capable of producing antibody molecules and, therefore, the central cellular 

component of humoral immune responses. B lymphocytes, or B cells, develop in the bone marrow, and 

mature B cells are found mainly in lymphoid follicles in secondary lymphoid tissues, in bone marrow, and 

in low numbers in the circulation. 

7 

* Phagocytic cells are responsible for ingesting and destroying foreign matter such as microorganisms or 

debris via a process known as phagocytosis, a process analogous to cellular digestion, usually using 

lysosomes (a membrane-bound, acidic organelle abundant in phagocytic cells which contains proteolytic 

enzymes that degrade proteins derived mainly from the extracellular environment and which is involved in 

2 
NK cells”, several plasma proteins (including those made by the 

complement system’2), and cytokine~’~ (largely made by 

mononuclear phagocyte^'^) that regulate and coordinate many of the 

activities of the cells of innate immunity. Different mechanisms of 

innate immunity may be specific for molecules produced by different 

classes of microbes. Finally, in addition to providing early defense 

the class 11 major histocompatibility complex (MHC) pathway of antigen processing) which cany potent 

enzymes that digests cell components such as other lipids or proteins. Phagocytes are extremely useful as 

an initial immune system response to tissue damage. 

A neutrophil is the most abundant circulating white blood cell, also called a polymorphonuclear 

leukocyte (PMN), which is recruited to inflammatory sites and is capable of phagocytosing and 

enzymatically digesting microbes. 

lo A macrophage is a tissue-based phagocytic cell derived from blood monocytes, which plays important 

roles in innate and adaptive immune responses. Macrophages are activated by microbial products, such as 

endotoxin, by molecules such as CD40 ligand, and by T cell cytokines such as interferon-y Activated 

macrophages phagocytose and kill microorganisms, secrete proinflammatory cytokines, and present 

antigens to helper T cells. Macrophages may assume different morphologic forms in different tissues, 

including the microglia of the central nervous system, Kupffer cells in the liver, alveolar macrophages in 

the lung, and osteoclasts in bone. 

A natural killer (NK) cell is a subset of bone marrow-derived lymphocytes, distinct from B and T cells, 

that function in innate immune responses to kill microbe-infected cells and to activate phagocytes by 

secreting interferon-y. NK cells do not express clonally distributed antigen receptors like immunoglobulin 

or T cell receptors, and their activation is regulated by a combination of cell surface stimulatory and 

inhibitory receptors, the latter recognizing self MHC molecules. 

’* The complement system is a system of serum and cell surface proteins that interact with one another 

and other molecules of the immune system to generate important effectors of innate and adaptive immune 

responses. There are three pathways of complement activation that differ in how they are initiated. The 

classical pathway is activated by antigen-antibody complexes, the alternative pathway by microbial 

surfaces, and the lectin pathway by plasma lectins that bind to microbes. Each complement pathway 

consists of a cascade of proteolytic enzymes that generate inflammatory mediators and opsonins and leads 

to the formation of a lytic complex that inserts in cell membranes. 

11 

Cytokines are secreted proteins that function as mediators of immune and inflammatory reactions. In 

innate immune responses, cytokines are produced by macrophages and NK cells and, in adaptive immune 

responses, mainly by T lymphocytes. 

l4 A mononuclear phagocyte is a cell with a common bone marrow lineage whose primary function is 

phagocytosis. These cells function as antigen-presenting cells in the recognition and activation phases of 

adaptive immune responses and as effector cells in innate and adaptive immunity. Mononuclear phagocytes 

circulate in the blood in an incompletely differentiated form called monocytes, and once they settle in 

tissues they mature into cells called macrophages. 

13 

3 
against infection, innate immune responses enhance adaptive 

immune responses against infectious agents. 

In contrast, adaptive immunity (also called acquired immunity) 

develops more slowly than innate immunity and mediates the later - 

even more effective - defense against foreign entities. When such 

entities actually invade tissues (Le. , pass through epithelial barriers), 

such invasion stimulates adaptive immunity - which, of course, 

adapts to the presence of any invading, foreign entity. This form of 

immunity is mediated by lymphocyte^'^ and their products, such as 

antibodies. In contrast to innate immunity, adaptive immunity is 

characterized by exquisite specificity for distinct macromolecules 

(including those that are non-infectious) and “memory,” which is the 

ability to respond more vigorously to repeated exposures to the same 

microbe. Whereas the mechanisms of innate immunity recognize 

structures shared by classes of microbes, the cells of adaptive 

immunity, namely lymphocytes, express receptors that specifically 

recognize different substances produced by microbes as well as noninfectious molecules. These substances are called antigens? 

Adaptive immune responses are only triggered if microbes or their 

antigens pass through epithelial barriers and are delivered to 

lymphoid organs where they can be recognized by lymphocytes. 

l5 These lymphocytes, i.e., T cells and B cells, originate in the tissues of the immune system referred to 

above. These tissues consist of the generative (or “primary” or “central”) lymphoid organs, i.e., the bone 

marrow and the thymus, in which T and B lymphocytes mature and become competent to respond to 

antigens, and the peripheral (or ”secondary”) lymphoid tissues, Le., the lymph nodes, the spleen, and the 

mucosal and cutaneous immune systems, in which adaptive immune responses are initiated. 

An antigen is a molecule that binds to an antibody or a T cell antigen receptor (TCR). Antigens that bind 

to antibodies include all classes of molecules. TCRs only bind peptide fragments of proteins complexed 

with major histocompatibility molecules; both the peptide ligand and the native protein from which it is 

derived are called T cell antigens. 

4 
Adaptive immune responses generate mechanisms that are 

specialized to combat different types of infections. For example, 

antibodies function to eliminate microbes in extra-cellular fluids, and 

activated T lymphocytes eliminate microbes living inside cells. 

Adaptive immune responses often use the cells and molecules of the 

innate immune system to eliminate microbes, and adaptive immunity 

functions to greatly enhance the antimicrobial mechanisms of innate 

immunity. For instance, antibodies (a component of adaptive 

immunity) bind to microbes, and these coated microbes avidly bind to 

and activate phagocytes (a component of innate immunity), which 

ingest and destroy the microbes. By convention the terms “immune 

system” and “immune response” usually refer to adaptive immunity. 

There are two types of adaptive immunity, called “humoral 

immunity” and “cell-mediated immunity”, that are mediated by 

different cells and molecules and are designed to provide defense 

against extracellular microbes and intracellular microbes, 

respectively. Humoral immunity is mediated by proteins called 

antib~dies’~, which are produced by cells called B lymphocytes. 

Antibodies are secreted into the circulation and mucosal fluids, and 

they neutralize and eliminate microbes and microbial toxins that are 

present in the blood and in the lumens of mucosal organs, such as 

the gastrointestinal tract. One of the most important functions of 

a 

An antibody is a type of glycoprotein molecule, also called immunoglobin (Ig), produced by B 

lymphocytes, that binds antigens, often with a high degree of specificity and high affinity. The basic 

structural unit of an antibody is composed of two identical heavy chains and two identical light chains. 

Amino-terminal variable regions of the heavy and light chains form the antigen binding sites, whereas the 

carboxy-terminal constant regions of the heavy chains functionally interact with other molecules in the 

immune system. In any individual, there are millions of different antibodies, each with a unique antigenbinding site. Secreted antibodies perform various effector functions, including neutralizing antigens, 

activating complement, and promoting phagocytosis and destruction of microbes. 

17 

5 A 
antibodies is to stop microbes that are present at mucosal surfaces 

and in the blood from gaining access to and colonizing host cells and 

connective tissues. In this way, antibodies prevent infections from 

ever getting established. Antibodies do not have access to microbes 

that live and divide inside infected cells. Defense against such 

intracellular microbes is called cell-mediated immunity because it is 

mediated by T lymphocytes (or T cells). Some T lymphocytes 

activate phagocytes to destroy microbes that have been ingested by 

the phagocytes into phagocytic vesicles. Other T lymphocytes kill 

any type of host cells that are harboring infectious microbes in the 

cytoplasm. The antibodies produced by B lymphocytes are designed 

to specifically recognize extracellular microbial antigens, whereas T 

lymphocytes recognize antigens produced by intracellular microbes 

and presented on the cell surface by MHC proteins. Another 

important difference between B and T lymphocytes is that most T 

cells recognize only these microbially-derived, protein antigens, 

whereas antibodies are able to recognize many different types of 

microbial molecules, including proteins, carbohydrates, and lipids. 

With regard to the above-referenced helper T they are 

the functional subset of T lymphocytes whose main effector functions 

are to activate macrophages in cell-mediated immune responses and 

promote B cell antibody production in humoral immune responses. 

These effector functions are mediated by secreted cytokines and by T 

cell CD40 ligand binding to macrophage or B cell CD40. Most helper 

T cells express the CD4 molecule. 

CD4+ T cells are called helper T cells because they help B lymphocytes to produce antibodies and help 

phagocytes to destroy microbes. 

6 
Finally, two functional subsets of helper T cells are of key 

importance to CFSAN’s concerns and Pharming’s response. The 

first, PHI cells, secrete a particular set of cytokines (discussed in 

0 

I 

7 

Preclinical And Clinical Studies 

Concerning 

Oral Administration Of Lactoferrin 

Which Are Pertinent 

To The Concerns Raised By CFSAN 

Concerning Pharming’s GRAS Notification 

Artym, J., Zimecki, M., Paprocka, M., and Kruzel, M.L. Orally 

administered lactoferrin restores humoral immune response in 

immunocompromised mice. lmmunol Lett. 89, 9 (2003). 

Artym et al. reported that in cyclophosphamide 

immunocompromised mice (specifically with inhibited humoral 

immune responses) oral administration of bLF (at 0.5 percent of 

drinking water for five weeks) was shown to restore the humoral 

response, as measured by elevated T (including CD4’ T cells) 

and 6 cells, macrophage content, and the proliferative 

response of splenocytes. 

Haversen, L. A., Baltzer, L., Dolphin, G., Hanson, L. A., and MattsbyBaltzer, 1. Anti-inflammatory activities of human lactoferrin in acute 

dextran sulphate-induced colitis in mice. Scand. J. Immunol. 57, 2 

(2003). 

Haversen et al. investigated the anti-inflammatory effects 

of orally administered human lactoferrin in the course of 

experimental colitis induced in mice by giving five percent 

dextran sulfate in the drinking water. Mice were given hLF 

twice a day at a dose of 2 mg until the end of the experiment, 

i.e., the killing of the mice after 2 or 7 days of DX exposure. 

The findings reported by the authors were as follows: 

I. significantly delayed and partly reduced appearance 

of occult blood in the stool and macroscopic rectal 

bleeding ; 

2. significantly less pronounced shortening of the colon; 

3. significantly diminished IL-1 f3 levels in the blood; 

4. a significantly lower crypt score pertinent to the distal 

1 
part of the colon; 

5. significantly reduced numbers of CD4 cells, F4/AD 

positive macrophages and TNF-a-producing cells 

(detected via i m m u no h ist oc hem ist ry ) in the d ist a I 

colon; and 

6. a reduction of IL-10 producing cells in the middle 

colonic submucosa. 

Based on these findings, the authors concluded that hLF has 

significant anti-inflammatory effects in the colon. 

Hayes, T.G., Falchook, G.F., Varadhachary, G.R., Smith, D.P., Davis, 

L.D., Dhingra, H.M, Hayes, B.P., and Varadhachary, A. Phase 1 trial 

of oral Iactoferrin alpha in refractory solid tumors. Invest New Drugs 

(2005). 

Hayes et ai. investigated the immunomodulatory effect of 

rhLF in ten patients with progressive advanced solid tumors 

who had failed conventional chemotherapy. The patients were 

orally administered rhLF in doses ranging from 1.5 to 9.0 grams 

per day (Le., approximately 25-150 mg/kg/day), using a 2 

weeks on, 2 weeks off schedule. Following such 

administration, significant levels of rhLF were not detected in 

circulation; however, a “small” Le., 15 percent, but statistically 

significant, increase in circulating IL-18 was observed. Such 

increase was not dose dependent and “far smaller” than the 

increase in intestinal IL-18. The authors indicated that these 

concentrations are consistent with other studies showing much 

lower levels of circulating IL-18 levels as compared to the 

increase in levels of intestinal IL-18. 

This study contains no data indicating any effect on Thl 

cells. 

ligo, M., Shimamura, M., Matsuda, E., Fujita, K., Nomoto, H., Satoh, 

J., Kojirna, S., Alexander, D.B., Moore, M.A., and Tsuda, H. Orally 

administered bovine lactoferrin induces caspase-1 and interleukin-I 8 

in the mouse intestinal mucosa: a possible explanation for inhibition 

of carcinogenesis and metastasis. Cytokine 25, 36 (2004). 

2 
e 

a 

e 

ligo et al. studied the effects of orally administered bLF to 

mice - either as a single dose (300 mg/kg) or as the same 

single dose for seven consecutive days. The authors 

concluded that bLF: 

1. “markedly elevated” IL-I 8 levels in the small intestine; 

2. “significantly increased” caspase-I activity and IFNy 

in the small intestine (but not IFNy in serum); and 

3. “markedly enhanced” response caspase-I activity and 

IL-18 levels in peritoneal macrophages. 

However, the authors did not identify the source of the IFNy. 

Finally, the authors indicated that bLF’s effects seemed to be 

confined to the intestinal mucosa. 

Ishii, K., Takamura, N., Shinohara, M., Wakui, N., Shin, H., Sumino, 

Y., Ohmoto, Y., Teraguchi, S. , and Yamauchi, K. Long-term follow-up 

of chronic hepatitis C patients treated with oral lactoferrin for 12 

months. Hepatol Res. 25, 226 (2003): 

lshii et al. reported in a fairly recent, “long term” study in 

36 patients with chronic hepatitis C that orally administered bLF 

(600 mg/day for 12 months) showed a rapid, significant 

increase in serum IL-18 levels (in the pg/mI range) that peaked 

after 3 months and gradually returned to base-line (even though 

significant oral administration of bLF continued for another 9 

months). No such effect occurred in the control group. No 

significant increases in IL-10 levels were observed. Also, the 

number of Thl cells in the peripheral blood increased, although 

not significantly, and returned to baseline after 3 months. 

Kruzel, M.L., Artym, J., Chodaczek, G., Kocieba, M., Kochanowska, 

I., Kruzel, T. and Zimecki, M. Effects of lactoferrin on stress-related 

immune dysfunction in mice and humans. Proceedings of the qfh 

International Whey Conference, Chicago, pp. 121-1 32 (2006). 

Kruzel et al. (the same group otherwise known as Zimecki 

et al.) investigated the effects of orally administered bovine 

lactoferrin (bLF) on the cellular and humoral immune responses 

in mice subjected to immobilization stress (IS). Here, the 

3 
authors demonstrated that long-term IS (5 d) induced significant 

suppression of cellular and humoral immune responses in CBA 

mice. This suppression was attenuated by bLF administered to 

mice in drinking water as determined by antibody-forming cells 

and delayed-type hypersensitivity (DTH). Conversely, bLF 

lowered elevated DTH responses in mice exposed to short-term 

IS (5 h only) on the day of elicitation of the DTH reaction. To 

evaluate the effect of bLF on stress-related autoimmune 

disorders, the production of selected cytokines in multiple 

sclerosis (MS) patients was evaluated. Treatment of MS 

patients (n = 6) with bLF (50 mgld) administered orally for 7 

consecutive days resulted in a significant increase in inducible 

interleukin 10 production by leukocyte cultures stimulated with 

Ii po pol ysaccharide and ph yto hemagg I u t in i n compared with 

reduced responses in placebo-treated control patients with 

fatigue syndrome. Conversely, interferon gamma production 

was reduced 4-fold in MS patients with minor changes in the 

placebo group. Together, these findings, the authors indicated, 

revealed that the medical benefit of bLF in stress-related and 

autoimmune disorders may be in part due to differential 

regulation of interleukin 10 and interferon gamma production. 

Kuhara, T., Yamauchi, K., Tamura, Y., and Okamura, H. Oral 

administration of lactoferrin increases NK cell activity in mice via 

increased production of IL-18 and type I IFN in the small intestine. J 

Interferon Cytokine Res 26, 489 (2006). 

Kuhara et al. investigated the immunomodulatory effects 

of bLF (suspended in saline) orally administered (via a gastric 

tube) to mice for 7 days at a dose of 30, 100, 300, or 1000 

mg/kg body weightlday. The findings reported by the authors 

included: 

1. 

2. 

3. 

a significant increase (in a dose-dependent 

manner) in the percentage of leukocytes that 

were NK cells in both peripheral blood and 

the spleen; 

enhanced IFNy production by NK cells; 

increased NK cell migration (after using 

intraperitoneal injection of poly (I:C) to in4 
duce NK cell trafficking into the peritoneum); 

4. an increase of IL-18 in the portal circulation; and 

5. increased expression of IFN-a and IFN-P in 

Payer’s patches and mesenteric lymph nodes. 

The authors also reported that bLF - in IL-18 knockout mice - 

did not increase the numbers of NK cells, although NK cell 

cytotoxic activity and poly( I:C)-induced trafficking activity were 

enhanced. Collectively, these results - the authors concluded 

- demonstrate that “orally administered bLF stimulates 

intestine-associated immune functions.” 

Kuhara, T., ligo, M., Itoh, T., Ushida, Y., Sekine, K., Terada, N., 

Okamura, H. , and Tstuda, H. Orally administered Iactoferrin exerts 

an antimestastic effect and enhances production of IL-18 in the 

intestinal epithelium. Nutr. Cancer 38, 192 (2000). 

Kuhara et al. studied the effects of orally administered 

bLF to mice (in a tumor metastasis model) at doses of 100 or 

300 mg/kg/day for seven consecutive days. The authors 

reported finding: 

1. “augmented” CD4’, CD8+, and asialoGM1’ cells 

in the spleen and peripheral blood; 

2. “markedly increased” CD4’ and CD8’ cells in 

the small intestine epithelium; 

3. “enhanced production” of IL-I8 in the intestinal 

epithelial cells; and 

4. “significantly augmented” NK activity. 

Administration of bLF did not increase serum levels of 

bLF; thus, the authors indicated that bLF probably has its effect 

in the gastrointestinal tract. The authors also concluded that 

bLF did not have any “direct effects” in the study. Finally, the 

authors indicated that bLF may enhance “mucosal immunity”. 

This study did not measure any Thl responses. 

Nakajima, M., Iwamoto, H., Shirasawa, T., Miyauchi, H., Takatsu, Z., 

Yamazaki, N., Teraguchi, S. , and Hayasawa, H. Oral administration 

cells cultured with concanavalin A or lipopolysaccharide. Biomed. ~ 

of lactoferrin enhances the productions of lFNy and IL-10 in spleen 

0 

3 
Res., 20, 27 (1999). e 

Nakajima et al. investigated the effects of orally 

administered bovine lactoferrin (bLF) on cytokine productions 

of spleen cells. Spleen cells from BALB/c mice (female only) 

were cultured with concanavalin A 1 to 4 days after the oral 

administration of bLF (at 3 mg/mouse). Concentrations of IFNy 

in the supernatants were enhanced by bLF feeding, while those 

of IL-4 were not. In contrast to bLF, an oral administration of f3- 

lactoglobulin or pepsin hydrolysate of bLF failed to show the 

enhancement. When stimulated by anti-CD3 antibody, IFNy 

production by CD4+ T cells fractionated from spleen cells was 

augmented by the oral administration of bLF. On the other 

hand, in response to lipopolysaccharide (LPS), spleen cells 

from the mice fed bLF secreted enhanced levels of IL-10 - “IL10 being known to be an immuno-suppressive cytokine 

secreted by Th2 cells or monocytes which inhibits other 

cytokine productions such as IFN-y, IL-2, TNF-a, IL-I and IL-8, 

and suppresses the antigen-presenting capacity of monocytes 

through down-regulation of class II molecules”. Levels of IFNy 

secretion from the spleen cells were not affected. While IL-10 

production in response to LPS by CDI IC+ cells from spleen 

cells was promoted by bLF feeding, the cytokine secretion from 

CDI 1 b+ cells was not affected. 

Sfeir, R.M., Dubarry, M., Boyaka, P.N., Rautureau, M., and Tome, D. 

The Mode of Oral Bovine Lactoferrin Administration Influences 

Mucosal and Systemic Immune Responses in Mice. J. Nutr. 134, 

403 (2004). 

Sfier et al. reported that oral administration (i.e., via 

gastric intubation, single buccal doses, or continuous doses of 

bLF in the diet, but not via addition of bLF to the drinking water) 

of bLF (I00 mg/day for 4 weeks) to mice resulted in a biased 

mucosal and systemic T-cell response towards a Th2 response. 

In contrast, the “less natural gastric intubation” also promoted 

Th I -type responses. 

Unfortunately, all that was measured in this study - with 

regard to Thl- and Th2-type cytokines - were the cytokines 

6 
themselves in spleen cells. The CD4' T cells were not isolated 

and studied separately; thus, one cannot conclude from the 

information provided by this study whether - in fact - there 

were either CD4' Thl and/or CD4' Th2 responses generated. 

Takakura, N., Wakabayashi, H. Yamauchi, K. and Takase, M. 

Influences of orally administered lactoferrin on IFNy and IL-I 0 

production by intestinal intraepithelial lymphocytes and mesenteric 

lymph-mode cells. Biochem. Cell Biol. 84, 363 (2006). 

Takakura et al. investigated the influences of orally 

administered bovine lactoferrin (bLF) on cytokine production by 

intestinal intraepithelial lymphocytes (IEL) and mesenteric 

lymph-node (MLN) cells, especially T cells. Bovine lactoferrin 

or bovine serum albumin (control) was administered to mice 

(via intragastric intubation at 500 mg/kg/day) once daily for 3 d. 

After 24 h from the last administration, IEL of the jejunum and 

ileum and MLN cells were isolated. These cells were cultured 

with and without the anti-T-cell-receptor antibody, and then the 

culture supernatants were assayed for cytokines with ELISA. 

Oral bLF did not affect the ratio of T cell subpopulations in IEL 

and MLN; however, bLF enhanced both interferon IFN-y and 

IL-10 production by unstimulated IEL and by IEL stimulated with 

the ap T cell receptor but not with the y6 T cell receptor. bLF 

aOso enhanced both IFN-y and IL-10 production by stimulated 

and unstimulated MLN cells. The production level of IFNy by 

MLN cells was correlated with that of IL-IO. The authors 

indicated that these results suggest that oral bLF enhances the 

production of both Thl -type and ThZTTr-type cytokines in the 

small intestine of healthy animals. 

Takakura, N., H. Wakabayashi, H. Ishibashi, K. Yamauchi, S. 

Teraguchi, Y. Tamura, H. Yamaguchi, and Abe, S. Effect of orally 

administered bovine lactoferrin on the immune response in the oral 

candidiasis murine model. J Med Microbiol 53, 495 (2004). 

Takakura et al. investigated the effect of orally 

administered bLF (in drinking water - equivalent to 0.5 g/kg 

BW/day - administered continuously from day I before the 

7 A 
infection) to mice “immunosuppressed with prednisolone 1 day 

before and 3 1 

’C 

e 

study lasted 7 

1. 

2. 

3. 

The authors 

enhancement 

days after” being infected with Candida. The 

lays. The authors reported the following results: 

bLF prevented the reduction in the numbers of 

peripheral blood leukocytes (PBLs) on day 1 

and cervical lymph node (CLN) cells on days 1, 

5 and 6; 

increased production of lFNy and TNF-a by 

CLN cells; and 

a significant increase in the production of IFNy 

and TNF-a on day 6. 

concluded that these results may indicate 

of the number of leukocytes and their cytokine 

responses in regional lymph nodes. 

Tanaka, K., Ikeda, M., Nozaki, A., Kato, N., Tsuda, H., Saito, S., and 

Sekihara, H. Lactoferrin inhibits hepatitis C virus viremia in patients 

with chronic hepatitis C: a pilot study. Jpn. J. Cancer Res. 90, 367 

(1 999). 

a Tanaka et al., having recently found that in vitro 

administered bLF effectively prevented hepatitis C virus (HCV) 

infection in cultured human hepatocytes (PH5CH8), in this 

report investigated (in a “pilot study”) the hypothesis that bLF 

inhibits HCV viremia in patients with chronic hepatitis C. Eleven 

patients with chronic hepatitis C received an 8-week course of 

bovine Iactoferrin (1.8 or 3.6 g/day). At the end of lactoferrin 

treatment, a decrease in serum alanine transaminase and HCV 

RNA concentrations was apparent in 3 (75%) of 4 patients with 

low pretreatment serum concentrations of HCV RNA. However, 

7 patients with high pretreatment concentrations showed no 

significant changes in these indices. (See, Ueno, 2006 for a 

summary of the published report of the full clinical trial 

conducted after this pilot study). 

Togawa, J., Nagase, H., Tanaka, K., Inamori, M., Umezawa, T., 

Nakajima, A., Naito, M., Sato, S., Saito, T., and Sekihara, H. 

Lactoferrin reduces colitis in rats via modulation of the immune 

8 
system and correction of cytokine imbalance. Am. J. Physiol. a Gastrointest. Liver Physiol. 283, GI 87 (2002). 

Togawa et at. reported that, in a TNBS-induced colitis 

model in rats, it has been shown that oral administration of bLF 

reduces colitis in rats via modulation of the immune system and 

correction of cytokine imbalance. More specifically, oral 

administration of bLF (at 200 mg/kg/day for, essentially, a 

lifetime) resulted in decreased levels of the pro-inflammatory 

cytokines TNF-a, IL-I 8 and IL-6, whereas the anti-inflammatory 

cytokines IL-4 and 11-10 levels - both cytokines known to 

promote a Th2 response - were significantly increased. The 

authors concluded that bLF exerts a protective i.e., antiinflammatory, effect against colitis in rats. 

Ueno, H., Sato, T., Yamamoto, S., Tanaka, K., Ohkawa, S., Takagi, 

H., Yokosuka, O., Furuse, J., Saito, H., Sawaki, A., Kasugai, H., 

Osaki, Y., Fujiyama, S., Sato, K., Wakabayashi, K. and Okusaka, T. 

Randomized, double-blind, placebo-controlled trial of bovine 

Iactoferrin in patients with chronic hepatitis C. Cancer Sci 971 105 

(2006). 0 

Ueno et al. (including Tanaka, K.) investigated the 

efficacy of orally administered bovine lactoferrin (bLF) in 

patients with chronic hepatitis C. The patients with chronic 

hepatitis C randomly received either oral bLF at a dose of 1.8 g 

daily for 12 weeks, or an oral placebo. The primary endpoint 

was the virologic response, defined as a 50% or greater 

decrease in serum HCV RNA level at 12 weeks compared with 

the baseline. The secondary endpoint was the biochemical 

response, which was defined as a 50% or greater decrease in 

the serum alanine aminotransferase (ALT) level at 12 weeks 

compared with the baseline. The study involved 199 

subjectdpatients. bLF treatment was well-tolerated and no 

serious toxicities were observed. A virologic response was 

achieved in 14 of 97 patients (14.4%) in the bLF group, and 19 

of 101 (18.8%) in the placebo group. There was no significant 

difference in virologic response rates between the two groups 

(-4.4%, 95% confidence interval -14.8, 6.1). In addition, bLF 

9 
intake did not have any favorable effect on the serum ALT level. 

The virologic responses were not different between two groups 

in any subgroup analysis. The authors concluded that orally 

administered bLF does not demonstrate any significant efficacy 

in patients with chronic hepatitis C. 

Varadhachary, A., Wolf, J.S., Petrak, K., O'Malley Jr., B.W., Spadaro, 

M., Curcio, C., Forni, G., and Pericle, F. Oral Iactoferrin inhibits 

growth of established tumors and potentiates conventional 

chemotherapy. Int. J. Cancer 11 1 , 398 (2004). 

Varadhachary et al. demonstrated - in both tumor-bearing 

and normal (Le., na'ive) mice - that oral administration of rhLF 

(at 300 mg/kg /day) for three consecutive days resulted in (1 .) 

significantly increased production of the active form of IL-I 8 in 

the intestinal tract mucosa, Le., in the intestinal epithelial cells 

(consistent with the fact, the authors noted, that the gut is the 

primary site of lactoferrin activity since lactoferrin is not 

absorbed (systemic bioavailability c 1 %) and the lactoferrin 

receptors are present in the gut epithelium), (2.) systemic NK 

cell activation, i.e., enhanced cytotoxicity of splenic NK cells, 

and (3.) a significant increase in the relative percentage of 

CD8' and CD3'1 CD8' cells. 

Please note that rhLF also was reported to have 

increased IL-18 in nude mice that lacked T cells. Please also 

note that the authors measured for but found no statistically 

significant increase in CD4' T cells. This study contains no 

data indicating any effect on Thl cells. 

Finally, this study also incorporated use of bovine 

Iactoferrin (as a test article) exactly as rhLF had been used. 

The results for bLF were exactly the same as for rhLF. 

Wakabayashi, H., Takakura, N., Yamauchi, K., and Tamura, Y. 

Modulation of Immunity-Related Gene Expression in Small Intestines 

of Mice by Oral Administration of Lactoferrin. Clinical and Vaccine 

Immunology 13, 239 (2006). 

10 
Wakabayashi et al. initially confirmed an 

immunomodulatory effect of bLF by observing changes in the 

number of cells in the leukocyte subsets in the peripheral blood 

and spleens of mice 1 day after oral administration (via gavage) 

of bLF (in solution equivalent to 2.5g/kg BW/day for 1 day). 

Then the authors developed a quantitative reverse 

transcription-PCR method for 20 immunity-related genes of 

antimicrobial proteins, pattern recognition receptors, cytokines, 

and lymphocyte mobilization-related proteins, and assessed the 

expression of these genes in the small intestines of mice 2 

hours after administration of water, bovine serum albumin 

(BSA), or bLF. Expression of the bLF gene was lower in mice 

administered bLF than in mice administered water or BSA, 

implying a negative-feedback control. Expression of gamma 

interferon (IFN-y) and interleukin-I 0 (IL-I 0) was lower in both 

BSA- and bLF-administered mice than in water administered 

mice, suggesting a nonspecific effect of protein ingestion. 

Expression of NOD2, IFN-P, and IL-Up40 was higher with bLF 

administration than with water or BSA administration. The 

expression levels of these three genes were correlated. This 

study indicated, the authors concluded, that oral administration 

of bLF modulates the small intestinal expression of genes 

closely related to the host defense in a specific or a nonspecific 

manner. 

Wakabayashi, H., Kurokawa, M., Shin, K., Teraguchi, S., Tamura, Y., 

and Shiraki, K. Oral lactoferrin prevents body weight loss and 

increases cytokine responses during herpes simplex virus type I 

infection of mice. BioSci. Biotechnol. Biochem. 68, 537 (2004). 

Wakabayashi et al. reported that oral administration of 

bLF (a 1.5 percent solution fed ad libitum) for 10 days to mice 

then exposed to herpes simplex virus-I infection resulted in 

significantly increased serum IL-18 levels (but only on day 9), 

whereas no increase was observed in non-infected animals. 

bLF treatment also resulted in significantly increased 

splenocyte production of IL-12 and IFNy - but only on day 5; 

however, the source of the cytokines was not identified. bLF 

11 
consumption did not significantly affect levels of RANTES, MIPla, MIP-2, TNA-a or IL-10. 

This study provides no direct demonstration of any Thl 

response after viral infection. 

I 

Wakabayashi, H., Takakura, N., Teraguchi, S., and Tamura, Y., 

Lactoferrin feeding augments peritoneal macrophage activities in 

mice intraperitoneally injected with inactivated Candida albicans. 

Microbiol. Immunol. 47, 37 (2003). 

Wakabayashi et al. investigated the effect on peritoneal 

macrophage activities of bLF orally administered (I 5%) in 

water (ad libitum) to mice intraperitoneally injected with 

inactivated Candida albicans as a priming agent generating a 

local inflammation. Oral administration lasted for 14 days post 

priming. The authors reported that bLF administration: 

1. slightly increased the number of peritoneal exudate 

cells; 

2. significantly enhanced the production of superoxide 

anion and nitric oxide by peritoneal macrophages at 

day 7; and 

3. significantly enhanced IFNy at day 9 and IL-12 at day 

day 5, but not TNF-a or IL-10. 

The authors concluded that the results indicated that bLF 

augmented the activities of macrophages and such effects may 

be related to enhanced cytokine levels. 

Wakabayashi, H., Takakura, N., Yamauchi, K., Teraguchi, S., Uchida, 

K., Yamaguchi, H. and Tamura, Y. Effect of lactoferrin feeding on the 

host antifungal response in guinea-pigs infected or immunised with 

Trichophyton mentagrophytes. J Med Microbiol 51,844 (2002). 

Wakabayashi et al. investigated the effect of bLF orally 

(gavage) administered (twice a day at a daily bLF dose of 

2.5g/kg BW) on, among other end points, the immune system in 

guinea pigs infected or immunized with Tryptophyton 

mentagrophytes. Animals received bLF for either 1 or 2 weeks. 

Among the results, the authors reported that: 

12 
1. bLF administration caused no significant effects on 

either phagocytic activity or reactive oxygen production of 

blood neutrophil polymorpho-nuclear leukocytes in either 

infected or noninfected animals; and 

2. in the bromo-deoxyuridine incorporation assay, the 

stimulation index was significantly higher for mononuclear 

cells (MNC) dereived from bLF-treated animals. 

The authors concluded that bLF may act by modulating MNC 

function. 

Wang, W.P., ligo, M., Sato, J., Sekine, K., Adachi, I., and Tsuda, H. 

Activation of intestinal mucosal immunity in tumor-bearing mice by 

Iactoferrin. Jpn. J. Cancer Res. 91, 1022 (2000). 

Wang et. al. investigated the activation of intestinal 

mucosal immunity in tumor-bearing mice. Mice were orally 

administered 300mg/kg/day of bLF from day 11 for three days. 

The authors indicated that their study demonstrated that oral 

administration of bLF resulted in: 

1. “strong increases” in CD4’ and CD8’ T-cells, as 

well as asialoGM1’ cells in lymphoid tissues and 

lamina propria of the small intestine; 

2. “significantly increased” IgM’ and IgA’ B cells in 

lamina propria of the small intestine; 

3. “significantly increased” CD8’ cells and 

significantly decreased asialoGM 1’ cells in the 

colon; 

“increased production of IL-18, IFN-y and caspase1 in the mucosa of the small intestine; 

“particularly high levels” of IL-18 in the epithelial 

cells of and induced lFNy presenting cells in the 

small intestine; and 

caspase-1 being induced in the epithelial cells of 

the small intestine. 

The authors concluded that such results may be important for 

elevation of intestinal mucosal immunity. 

4. 

5. 

6. 

13 
Unfortunately, the authors did not indicate which cell type 

was producing the IFNy. Thus, this study does not necessarily 

provide evidence of any Thl effect. 

Zimecki M., Kociqba M., Chodaczek G., Houszka M., Kruzel M. 

Lactof err i n a me1 i or at es symptoms of ex per i menta I en ce p h a I o my el it i s 

in Lewis rats. J. Neuroimmuno. (2006). 

Zimecki et al. investigated the effects of oral 

administration of lactoferrin (bLF) on experimental autoimmune 

encephalomyelitis (EAE) in Lewis rats. bLF was given in 

drinking water (as a 0.25% solution) beginning the day of 

elicitation of EAE or with a seven-day delay. The authors 

reported that I actofe rri n treat men t 

1. led to a “significant acceleration’’ of the recovery 

process; 

2. “normalized” cell number of the inguinal lymph nodes 

of untreated EAE rats (which were almost 3 times 

higher as compared with central, naive rats) after 

I actofe r ri n treat men t ; 

3. decreased elevated serum concentrations of TNF-CI 

and TNF-P (Le., Thl proinflammatory cytokines) to 

those values found in controls; and 

4. reduced (evaluated via histological analysis of the 

spinal cords) the number and size of inflammatory 

foci. 

As a consequence of the above-referenced findings, the 

authors concluded that - as others have shown - lactoferrin 

has the ability to inhibit autoimmune disorders - in this case, 

diminution of the clinical symptoms of EAE. This study 

confirmed other recent findings by the authors, i.e., that orally 

administered lactoferrin caused reduction of clinical signs of MS 

in patients - in parallel to normalization of cytokine production 

by peripheral blood cells. (Kruzel, 2006). 

Zimecki, M., Artym, J., Chodaczek, G., KocSba, M. and Kruzel, M. 

Effects of Iactoferrin on the immune response modified by the 

immobilization stress. Pharma. Reports 57, 81 1 (2005). 

14 
e Zimecki et al. investigated the effects of orally 

administered bovine Iactoferrin (bLF) on the cellular and 

humoral immune responses in mice subjected to immobilization 

stress (IS). First, the authors demonstrated that long-term IS 

induced significant suppression of cellular and humoral immune 

responses in CBA mice. Then the authors indicated that the 

suppression was attenuated by bLF given to mice in drinking 

water as determined by the number of antibody-forming cells 

(AFC) in the spleen and the magnitude of delayed type of 

hypersensitivity (DTH). On the other hand, bLF lowered the 

elevated DTH response in mice exposed to short-term IS (5 h 

only) on the day of elicitation of the DTH reaction. The authors 

also reported that bLF up-regulated spontaneous transforming 

growth factor beta (TGF-P) production in the cultures of 

mesenteric lymph node cells derived from short-term stressed 

mice. Finally, the authors indicated that this study represents 

the first report on the regulatory effect of bLF on the immune 

response modified by the psychic stress and is consistent with 

other reports on antinociceptive and analgesic actions of bLF in 

experimental animals. 

Zimecki, M., Wlaszczyk, A., Cheneau, P., Brunel, A.S., Mazurier, J., 

Spik, G., and Kubler, A. lmmunoregulatory effects of a nutritional 

preparation containing bovine Iactoferrin taken orally by healthy 

individuals. Arch. Immunol. Ther. Exp. (Warsz) 46, 231 (1 998). 

Zimecki et al. reported on a study performed in 17 healthy 

volunteers during which individuals consumed 40 mg of bLF per 

day for ten days. Such exposure resulted in (during treatment) 

a 100 percent increase in the level of immature cell forms, a 

significant decrease in the percentages of eosinophils and 

monocytes, and a marked increase (from 33-42 percent) in the 

numbers of lymphocytes. Unfortunately, the type of 

lymphocytes (either Thl or Th2 cells) was not specified; thus, 

this study does not necessarily provide support for any Thl 

response. The authors concluded that bLF may be applied in 

the clinical setting “to improve the immune status of patients.” 

15 
Zimec,. 

the incidence of positive Coombs' test in New Zealand Black mice. 

Arch. Immunol. Ther. Exp. 43, 207 (1995). 

Wieczorek, Z., Mazurier, J., Spik, G. Lactoferrin lowers 

Zimecki et al. indicate in this publication that New Zealand 

Black (NZB) mice treated for a prolonged period (i.e., 329 days) 

with bovine lactoferrin (bLF) (given intraperitoneally at doses of 

2, 10 or 50 pg 3 timeslweek) exhibit a decreased frequency of 

positive Coombs' reaction. The authors reported that this effect 

was dose dependent and best pronounced at a dose of 50 

pg/dose, In addition, the authors reported that incubation of 

peritoneal cells with bLF resulted in a decreased number of 

cells recognizing Hb antigen on autologous erythrocytes. 

Consequently, the authors concluded that the data indicated 

that bLF may be of therapeutic value in treatment of 

autoimmune disorders - in particular, in inhibiting autoimmune 

hemolytic anemia. 

16 


 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 
 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: March 9, 2007 

TIME: 2:30 PM. 

NUMBER: 415-957-0101 

PARTICIPANTS: 

FDA 

Jeremiah Fasano 

Antonia Mattia 

HFS-255 

HFS-255 

External 

Charles Morin Morin & Associates 

SUBJECT: Discussion of public hearing on human lactoferrin 

Dr. Mattia and I contacted Mr. Morin to alert him to our interest in fostering public 

discussion of issues related to the use of human lactoferrin in foods. Mr. Morin is the 

agent for GRN 189, submitted by Pharming, Inc. (Pharming), which describes certain 

food ingredient uses of human lactoferrin from bovine milk. We noted that in FDA's 

response to a previous notice from a different firm that also involved human lactoferrin 

(GRN 162), the agency had stated its intent to engage the wider scientific community in 

consideration of the complex scientific issues raised by the notice. Though Pharming's 

notice was distinct from GRN 189, in our view some of the issues raised by each notice 

were related. For that reason, we thought it appropriate to keep Pharming informed about 

our ongoing efforts to engage the wider scientific community. We explained that we were 

currently considering a proposal for a Part 15 hearing as a vehicle for this engagement. 

Mr. Morin requested an opportunity for a broader selection of Pharming representatives 

to hear and discuss FDA's plans at a future date. We agreed to this request. 

Jeremiah Fasano 

R/D:HFS-255:JMFasano:03/09/2007 

Init:HFS-255:AMattia:06/01/2010 

F/T:HFS-255:JMFasano:06/01/2010 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

 

 

 

 

 

_______________________ 

Page 1 of 2 

Fasano, Jeremiah 

From: Fasano, Jeremiah 

Sent: Wednesday, April 04, 2007 10:46 AM 

To: 'charleslmorin@earthlink.net' 

Subject: RE: April 16th teleconference 

Mr. MorinThank you for arranging the line. I've reserved the time slot for the CFSAN participants. Dr. Mattia 

and Dr. Merker are well-briefed and can handle it without me if necessary, but I will participate if I 

can. 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

DBGNR/OFAS/CFSAN/FDA 

jfasano@cfsan.fda.gov 

Phone: 301-436-1173 

Fax: 301-436-2964 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

From: Charles L. Morin [mailto:charleslmorin@earthlink.net] 

Sent: Tuesday, April 03, 2007 5:55 PM 

To: Fasano, Jeremiah 

Subject: April 16th teleconference 

Dear Dr. Fasano,

 This will memorialize the information I left on your voice mail concerning the future teleconference 

between CFSAN and Pharming. Pharming has now confirmed the availability of all its participants and, 

thus, can now represent that all can participate on Monday, April 16th starting at 10:30 a.m. (EST). Each 

such participant as well as those participating on behalf of CFSAN can connect to the teleconference on 

Monday by dialing 1 (866) 448-6761 and then, when asked to do so, by dialing the dial-in code, i.e., 

940357. 

 Please let me know if there is anything else we need to do prior to our teleconference on April 

16th .

 Best regards. 

6/27/2007 
 

 

Page 2 of 2 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 1460 

San Francisco, CA 94111 

US 

Phone: (415) 957.0101 

Fax: (415) 957.5905 

6/27/2007 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: April 16, 2007 

TIME: 10:30 AM 

PARTICIPANTS: 

FDA 

Antonia Mattia HFS-255 

Robert Merker HFS-255 

Jeremiah Fasano HFS-255 

External 

Charles L. Morin Morin and Associates 

Frans de Loos Pharming 

Bertjan Ziere Pharming 

Sanda van Wetering Pharming 

Harrie van Veen Pharming 

Anurag Relan Pharming 

Charles Elson University of Alabama at Birmingham 

SUBJECT: Discussion of Lactoferrin Public Hearing Proposal 

Members of the Division of Biotechnology and GRAS Notice Review met with representatives of 

Pharming, including the firm's agent (Mr. Morin) and a member of the GRN 189 GRAS panel 

(Dr. Elson). We wished to discuss our proposal for a public hearing on the use of recombinant 

human lactoferrin (rhLF) in food. 

We began by explaining that although the proposed Part 15 public hearing was prompted in part 

by the submission of GRN 189, the evaluation of GRN 189 and the public meeting were distinct 

processes that were not dependent on each other. Nevertheless, we considered that both Pharming 

and the agency would benefit if Pharming had the opportunity to learn about the kinds of 

questions we were proposing to ask at the hearing. 

The proposed hearing would focus entirely on scientific issues rather than the regulatory status of 

rhLF. The issues are currently as follows: 

 Evaluation of rhLF idenitity with respect to 

 bLF in terms of bioactivities (in various species, including humans) 

 hLF in terms of antigenicity (perception of rhLF as a nonself protein potentially 

leading to concerns about autoimmunity) 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

 

 

 

 

 

 

 

 

 

April 16, 2007 Memorandum of Teleconference – Page 2 

 The significance of existing exposures to hLF and their utility in a food safety assessment 

of rhLF 

 infant oral exposure to hLF in human milk 

 adult oral exposure to hLF from exocrine secretions such as saliva 

 The consequences of long-term exposure to an exogenous immunomodulator such as 

rhLF 

With respect to the last issue, we explained that we were interested in what sorts of studies, 

species, and endpoints might or might not be appropriate for this kind of substance and exposure 

scenario, and the scientific reasoning for any given position. 

Pharming noted that the issues we had described were complex and relatively specific and that a 

third party would require substantial background information to understand them clearly. 

Pharming then asked about the scope of the hearing and whether their ingredient would be 

conflated with other ingredients. We explained that the hearing was intended to explore general 

issues raised by rhLFs as a class, but that any related GRAS determination would continue to be 

on a case-by-case basis. Pharming would have the opportunity to clearly identify any relevant 

distinctions between its own product and other rhLFs if the firm chose to give a presentation. 

Finally, we agreed that a public hearing notice on this topic would require clarity and specificity. 

We invited Pharming to submit sample text. At a minimum, this would help identify gaps in our 

draft text. 

We then stated that while we intended to hold the meeting in the summer of 2007, at this point the 

public meeting was still a proposal. As such, it would require clearance by the Center and FDA 

and we could make no guarantees about the timing or exact content of the meeting notice. 

Finally, we explained that the GRAS notification process is not intended to be lengthy or 

iterative, but that we had offered Pharming the opportunity to provide additional information 

because of the novelty of the subject matter from a scientific and regulatory perspective. We 

considered that Pharming had provided a substantive amendment to the original notification, and 

that we would not be requesting any additional information from the firm. We reminded 

Pharming that we had not yet arrived at a conclusion of ‘no questions’ for GRN 189 and that all 

potential outcomes (including ‘no questions,’ ‘no basis,’ or ‘withdrawal without prejudice’ 

responses) were still possible. We also reiterated that the outcome of our evaluation was not 

formally connected to the results of the proposed public meeting and that our evaluation could be 

completed prior to or following the public meeting. However, we would contact Pharming prior 

to issuing any letter as a courtesy. 

Pharming stated that they appreciated the opportunity for dialogue and would pass suggested 

language to us soon. 

Jeremiah Fasano 

R/D:HFS-255:JMFasano:04/17/2007 

Init:HFS-255:RIMerker:04/18/2007 

Comment/Init:HFS-255:AMattia:04/18/2007 

Edit:HFS-255:JMFasano:05/24/2010 

F/T:HFS-255:JMFasano:05/24/2010 
 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: May 29, 2007 

TIME: 12:40 P.M. 

NUMBER: 415-957-0101 

PARTICIPANTS: 

FDA 

Jeremiah Fasano HFS-255 

External 

Charles Morin Morin & Associates 

SUBJECT: Discussion of information relating to recombinant human lactoferrin 

Mr. Morin called me regarding the participation of Pharming, whom he represents, in a 

planned public hearing on the safety of recombinant human lactoferrin. 

Mr. Morin first stated that Pharming wished it to be clear that when the ‘biological 

activities’ of lactoferrin were referred to as a subject of inquiry at the hearing, in this 

context this phrase meant immunological activities rather than generic toxicity. 

Mr. Morin also stated that Pharming was investigating, and planning to introduce, two 

additional kinds of evidence at the hearing. The first was the significance of the longestablished practice of “wet-nursing” in which infants consumed nonparental hLF. 

Second, there is limited experience with adult consumption of nonparental human milk 

and thus hLF. In light of this evidence, Mr. Morin suggested that parts of the public 

hearing notice referring to consumption of nonparental hLF be carefully phrased. 

I thanked Mr. Morin for his suggestions and information and we concluded the call. 

Jeremiah Fasano 

R/D:HFS-255:JMFasano:05/29/2007 

F/T:HFS-255:JMFasano:05/26/2010 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 








 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 





















































































































 

From: Charles L. Morin [mailto:charleslmorin@earthlink.net] 

Sent: Thursday, November 15, 2007 5:58 PM 

To: Brackett, Robert 

Subject: Request for a meeting 

Dear Dr. Brackett, 

 Please find attached an e-copy of a letter whose formal version is on its 

way to you via FedEx for delivery Friday, November 16th. I will contact you early 

next week to try to make arrangements for a meeting date which is convenient 

for all interested parties. 

 Best regards. 

Charles L. Morin 

Morin & Associates 

388 Market Street, Suite 1460 

San Francisco, CA 94111 

US 

Phone: (415) 957.0101 

Fax: (415) 957.5905 









_________________________ 

-----Original Message----- 

From: Levitt, Joseph A. [mailto:JALevitt@HHLAW.com] 

Sent: Thursday, December 13, 2007 3:43 PM 

To: Tarantino, Laura M 

Cc: Landa, Michael; Masoudi, Gerald F 

Subject: Memorandum on Why Section 912 is NOT Retroactive 

Laura-- 

This follows up on my earlier letter and our telephone conversation 

concerning FDA's implementation of Section 912 of the FDA 

Amendments Act of 2007. One question you said was being 

discussed was whether Section 

912 was retroactive in its application. We have since researched the 

point, and the case law is very clear: There is a strong presumption 

that Congress intends all statutes to apply only PROSPECTIVELY. 

Accordingly, if Congress wants a statute to apply retroactively, it 

must say so expressly. Because Section 912 has no such express 

statement, it must be applied prospectively only. 

Attached is a memorandum that summarizes the case law on this 

point, including Supreme Court cases. Please share with the Office of 

Chief Counsel for use in developing your implementation plan. We 

hope this assists the agency in dismissing any concern about 

retroactivity, and instead allows you to apply your attention to 

Section 912's prospective application, particularly with respect to 

recombinant human lactoferrin. 

Please let me know if you have any additional questions on this 

subject. 

Best regards, 

 Joe 

Joseph A. Levitt, Esq. 

Hogan & Hartson L.L.P. 

555-13th Street, NW 
Washington, DC 20004 

202/637-5759 

202/637-5910 (fax) 

This electronic message transmission contains information from this 

law firm which may be confidential or privileged. The information is 

intended to be for the use of the individual or entity named above. If 

you are not the intended recipient, be aware that any disclosure, 

copying, distribution or use of the contents of this information is 

prohibited. 

If you have received this electronic transmission in error, please 

notify us by telephone (+1-202-637-5600) or by electronic mail 

(PostMaster@HHLAW.COM) immediately. 
AM I1111111 111111 II 1111 

Law Offices Of 

Morin & Associates 

Suite 1460 

388 Market Street 

San Francisco, California 941 11 

Telephone: (415) 957-0101 e-mail: charleslmorin@earthlink.net Facsimile: (415) 957-5905 

December 26,2007 

Laura M. Tarantino, PhD (HFS-200) 

Director (Room 3044) 

Office of Food Additive Safety 

Center for Food Safety and Applied 

Food and Drug Administration 

4300 River Road 

College Park, MD 20740 

Nutrition 

Re: Pharming Group NV 

Notice of GRAS exemption for human 

lactoferrin derived from the milk of 

transgenic cows expressing a human gene 

encoding human lactoferrin 

GRN No. 000189 

Follow up to 12/11/07 meeting 

Dear Dr. Tarantino: 

This letter is being forwarded to you since during our recent meeting you 

indicated that future communications should be with you. Please make it available to 

your colleagues as you deem appropriate. 

Thanks very much to you and your FDA colleagues for providing Pharming with 

an opportunity to discuss matters pertinent and important to obtaining a final 

determination - on the merits - concerning its GRAS Notification, i.e., GRN No. 000189. 

cir, 
Laura Tarantino, PhD 

Re: GRN 189 

December 26,2007 

Page 2 of 6 

We appreciated the opportunity to discuss the status of CFSAN’s review and its 

progress towards a “no questions” letter. 

These are summarized and memorialized below in highlighted subparts 

During the meeting, several important (indeed, critical) matters were discussed. 

Information pertinent to Pharming’s GN 

Repeatedly over the last almost nine months, Pharming has been informed that 

CFSAN has no further questions for Pharming with regard to the need for any additional 

information pertinent to Pharming’s GN - that is, that Pharming has supplied all that is 

required to meet Pharming’s legal obligation of providing sufficient scientific information 

to support its GN. During the meeting this position was again affirmed by CFSAN 

personnel. 

Pharming has been repeatedly encouraged by CFSAN to keep its GN file 

updated, and Pharming has updated from time to time its GN file. To that end, please 

find attached additional updating information. 

1 ~* 

The current status of Pharming’s GN 

Contrary to what Pharming has been repeatedly told over the last several 

months, i e., that nothing was impeding progress on Pharming’s GN except for obtaining 

final, go ahead approval from upper FDA management for conduct of a Part 15 hearing, 

we were very surprised to learn during the meeting that all progress on Pharming’s GN 

had stopped some time ago because it is believed by, at least, some at CFSAN that 

Pharming’s product cannot successfully make it through and out of the gauntlet posed 

bi 
x 

Laura Tarantino, PhD 

Re: GRN 189 

December 26,2007 

Page 3 of 6 

by Section 912 and, therefore, there is no point in wasting CFSAN resources on 

completing the GN when Pharrning's product will never be able to be introduced into 

interstate commerce. This very critical acknowledgement warrants several responses. 

First and most importantly, CFSAN has no legal authority to significantly deviate 

from the express legal duty imposed on it with respect to receiving, reviewing, and 

making a final determination on the merits of a GRAS Notification. The duties imposed 

on CFSAN expressly emanate from 21 CFR !j 170.36. Failure to act in a reasonable 

fashion on a GN amounts to unlawful conduct and arbitrary and capricious conduct in 

violation of the Administrative Procedures Act. Accordingly, Pharming expects that from 

this time forward, Pharming's GN will receive the attention and action it is legally entitled 

Second, Section 912 in no way prohibits CFSAN from acting in accordance with 

Section 170 36 or other APA-related legal requirements. Indeed, Section 912 has 

nothing to do with the regulatory process involved when GRASing a substance. 

Section 912 requirements do not even arise unless and until: 

1 a qualifying substance has been added; 

2 to a food; 

3. which food has been introduced, 

4 into interstate commerce. 

\. to. 

Then and only then does the regulatory status of the qualifying substance come into 

question. Of course, to the extent that any such substance - at that time - is found to 

meet any of the eight, express exceptions (to the general prohibition rule of Section 

91 2), then such substance would not be prohibited from being introduced into interstate 

commerce. 
Laura Tarantino, PhD 

Re: GRN 189 

December 26,2007 

Page 4 of 6 

Third, any attempt to currently apply Section 912 to Pharming’s hLF product 

would be arbitrarily and capriciously premature for two reasons. First, until and unless 

the many, key words andlor phrases used in Section 912 and left undefined are defined 

(perhaps in a guidance document), no reasonable person could apply Section 912 to 

any actual, factual scenario. Second, unless there exists an actual factual scenario to 

which Section 91 2 requirements can be applied, any hypothetical scenario is 

speculative and not ripe for adjudication Accordingly, CFSAN needs to deal with 

Section 912 matters only after an actual, factual scenario arises which triggers Section 

912’s provisions. 

Finally, as indicated to you during the meeting, receiving CFSAN’s no questions 

letter in response to Pharming’s GN is very important to Pharming even if Pharming 

were never to introduce its hLF product into interstate commerce in the United States. 

Needless to say, introduction of a new product on a world-wide basis is a very 

complicated, multi-faceted process. The strategy for doing so should be left to the 

sponsor, while regulatory bodies should respond as required by law to the regulatory 

implications actually and when raised by such strategy. 

“r. .* 

Need for a hearing 

CFSAN has indicated - although less vigorously recently - that it may need 

some sort of public hearing before it can make a final determination on Pharming’s GN. 

Although Pharming does not object per se to meaningful public participation when it can 

be of real value, in this case Pharming believes no public hearing is necessary. To date 

(and over the last approximately 30 years), more than 200 products - including drugs, 

devices and food substances to be added to food - emanating from numerous 
k. 

Laura Tarantino, PhD 

Re: GRN 189 

December 26,2007 

Page 5 of 6 

transgenic platforms (all incorporating use of recombinant technology) have been via 

various types of submissions reviewed and authorized by FDA for use in interstate 

commerce. Not one, including numerous, injectable, human proteins has required 

any public hearing before being authorized for use by FDA. To date, CFSAN has not 

cited to Pharming any adequate reason(s) for altering this well-established and longfollowed precedent. If CFSAN feels the need for additional, qualified expert advice, it 

should, for example, bring in for a day a group of qualified experts and let them consider 

whether the scientific opinions conveyed by Pharming and its independent, qualified 

experts to CFSAN are indeed correct and representative of the consensus view among 

qualified experts 

%. With regard to additional qualified expert input, please note the information 

contained in the attached update document. It includes, among many other pieces of 

information, additional reviews by qualified expert groups from around the world 

concerning the impact of consuming hLF As you will see, they all agree with 

Pharming’s experts’ assessment that such consumption does not pose any unique risk 

of untoward event to those doing the consuming. In addition and very importantly, such 

document establishes that the proposed daily consumption level of hLF pales in 

comparison to the huge, natural, background of hLF that an individual can be exposed 

to on a daily level for, perhaps, a lifetime. 

For all the foregoing reasons, Pharming believes that no hearing is necessary 

and that CFSAN currently has before it all the information - including various opinions 

of numerous, eminently-qualified experts - it needs to make a final decision on 

Pharming’s GN. 

Pharming trusts that there will be no more delay in dealing with Pharming’s GN. 

It looks forward to hearing from you by the end of January (or sooner, if possible) as to 

*,‘“* 
Laura Tarantino, PhD 

Re: GRN 189 

December 26,2007 

Page 6 of 6 

exactly what process is now to be applied to Pharming’s GN and what timelines will be 

followed for applying and reaching an end to such process. 

Thank you again for your and your colleagues’ efforts to resolve the current, 

rather lengthy stall associated with the GN If at any time I can be of assistance to you 

with regard to any aspect of this matter, please do not hesitate 

n 

to contact me. 

m L. Morin 
Pharming, NV 

Updating Information 

Pertinent To 

GRN No. 000189 

Which Involves 

Production Of 

Recombinant Human Lactoferin 

From 

Transgenic Cows 

December 26,2007 
Table Of Contents 

Contents Page 

A. Introduction 1 

B. Subareas of discussion 

1. Subarea 1 (concerning the precedential value emanating 

from the GRASing of hLF) 

2 Pharming update 

a. Introduction 

b. Identification of bovine lactoferrin 

c. bLF’s and hLF’s ability to induce immunological effects 

d. bLF’s safety 

(1 .) Long term 

(2.) GRAS status 

e. Safety of hLF 

(1 .) Introduction 

(2.) hLF naturally occurs in humans 

(3.) Long term history of exposure to hLF 

(4.) Regarding host organism 

(5.) Regarding “scientific procedures” evidence 

(6.) Regarding clinical evidence 

(7.) Regarding assessment of qualified experts 

(8.) Regarding ability to induce adverse immunological 

effects 

(a,) Thl cell activity 

(b.) Release of specific cytokines 

(c.) Effect of oral administration of hLF on auto1 

1 

2 

2 

2 

3 

3 

3 

4 

5 

5 

5 

6 

6 

7 

8 

9 

9 

10 

13 

i 
., x 

. -. 

immune or other inflammatory disorders 

((a,) Introduction) 

((b.) Autoimmunity) 

((c.) Inflammatory disorders) 

(d.) Conclusion 

f. Conclusion 

3. Subarea 2 (concerning potential antigenicity of hLF) 

4. Pharming update 

a. Introduction 

b. comparability of the two lactoferrins 

(1 .) Introduction 

(2.) Both lactoferrins are almost entirely the same 

(3.) How the lactoferrins are different 

(a,) With regard to their respective amino 

acid sequences 

(b.) With respect to their respective glycosylation patterns 

(c.) The importance of the differences 

c. Potential for adverse effects 

(1 .) Introduction 

(2.) Determinant spreading 

(3.) Enhanced pro-inflammatory Thl response 

(4.) Increased uptake by antigen-presenting cells 

via the mannose receptor 

(5.) Conclusion 

5. Subarea 3 (concerning exposure to exogenous hLF) 

6. Pharming update 

a. Introduction 

b. Fetal exposure 

c. Infant exposure 

(1 .) Via human milk from one's mother 

14 

14 

14 

17 

19 

23 

23 

24 

24 

24 

24 

25 

26 

26 

29 

30 

32 

32 

32 

33 

34 

35 

35 

35 

35 

36 

40 

40 

ii 
(2.) Via human milk from a female other than one’s 

mother 

(3.) Via clinical applications 

d. Adult exposure 

e. Conclusion 

7. Subarea 4 (concerning oral exposure to endogenous hLF) 

8. Pharming update 

a. Introduction 

b. Exposure via the oral route 

c. Conclusion 

9. Subarea 5 (concerning helpfulness of exposures to hLF) 

10. Pharming update 

11. Subarea 6 (concerning safety criteria) 

12. Pharrning update 

a. lnroduction 

b. Scientific criteria 

c. The adequacy of the “criteria” 

d Application of the criteria to products of biotechnology 

e. No need for additional “criteria” 

C. Conclusion 

42 

43 

43 

48 

49 

49 

49 

49 

53 

53 

54 

54 

55 

55 

55 

60 

61 

62 

63 

iii 
Pharming, NV 

Updating Information 

Pertinent To 

GRN No. 000189 

Which Involves 

Production Of 

Recombinant Human Lactoferrin 

From 

Transgenic Cows 

A. Introduction 

From time to time Pharming - via the encouragement of CFSAN - has 

forwarded additional information pertinent to its GRAS Notification (i.e., GRN No. 

000189 which pertains to certain specified food uses of human lactoferrin 

derived from the milk of transgenic cows expressing a human gene encoding 

human lactoferrin) to keep such GN updated. What follows is more of such 

information. For the convenience of the reader, it has been subdivided into those 

subareas which have been the bases of discussion with CFSAN over the last 

approximately nine months. 

B. Subareas Of Discussion 

1. Subarea 1 (concerning the precedential value emanating from the GRASing of 

bLF) 

The scientific literature indicates that bovine lactoferrin and human 

lactoferrin have very similar biological activities (with regard to their ability to 

1 
induce immunological effects). Bovine lactoferrin has been significantly, safely, 

and long-consumed by both infants and adults either in purified form (as a 

functional food or as an ingredient added to numerous foods) or via the drinking 

of cow’s milk. To what extent can this long history of safe, human consumption of 

bovine lactoferrin contribute to assessing the safety of transgenically produced 

human lactoferrin when added to and consumed in certain foods? 

2. Pharming Update 

a. Introduction 

This subarea raises several subsidiary subareas. Each of these is 

identified below and then responded to. 

b. Identification of bovine lactoferrrin 

The term “lactoferrin” refers to a specific glycoprotein found in, among 

other tissues, the milk of almost all mammals. (Nuijens, 1996; Lonnerdal, 1995). 

Bovine lactoferrin (Le., “bLF) is the version of lactoferrrin found in the milk of 

cows. (Nuijens, 1996). Bovine lactoferrin is - like human lactoferrin (i.e., “hLF) - 

an iron-binding glycoprotein (of about 80 kDa) which is substantially similar in 

structure and function compared to its human homologue, Le., hLF. (Nuijens, 

1996). The amino acid sequence of bLF (which contains 689 amino acids) 

shows 69% homology with hLF. (Pierce, 1991). The sequence of bLF contains 

five possible N-linked glycosylation sites (as compared to three such sites in the 

sequence of hLF). Four sites, Le., Asn 233, 368, 476, and 545, are always 

utilized (Spik, 1994) while the fifth (Asn 281), located in the N-lobe, is 

glycosylated in about 30% and 15% of the molecules in bovine colostrums and 

mature milk, respectively (van Veen, 2002; Wei, 2000; Yoshida, 2000). (For 

2 
additional, extensive technical information concerning bLF see GRAS Notices 

0042,0067,0077 and 0130). 

c. bLF’s and hLF’s ability to induce immunological effects 

Bovine lactoferrin and human lactoferrin have both been extensively 

studied - especially over the last two decades and often in the same study - for 

their potential to induce immunological effects. Indeed, more than 100 such 

studies have been published in the scientific literature and are directly referenced 

in this update document.’ Pharming generally agrees that such studies indicate 

that bLF and hLF have very similar biological activities (with regard to their ability 

to induce immunological effects). However, to be as precise as possible, one 

should note that a careful reading of all such studies also indicates that: 

1. bLF induces - qualitatively or quantitatively - certain immunological 

effects not induced by hLF; and 

2. hLF does not induce any immunological effect not also induced by 

bLF 

Thus, bLF is somewhat more biologically active than hLF. (More about such 

activity in subpart (7)). 

d. bLF’s safety 

(1.) Long term 

Milk from cows and other dairy products have been significantly, 

consistently, and long-consumed by populations all over the earth for well over 

2,000 years. (See, e.g,, GRN Nos. 42 and 77). As sources became more 

available and safety became assured, increasing numbers of individuals 

consumed increasing amounts of milk and dairy products. Today, in the United 

’ In addition, over 200 other such studies are lnduectly referenced in the referenced, published articles. 

Since they are older studies and duplicative of the substance contained in the referenced studies, they are 

not directly referenced. 

3 
States, so long as milk and dairy products are derived from locations following 

good dairy practices and are properly pasteurized, they are broadly viewed as 

being generally recognized as safe - based on, at least, long history of common 

use as food. 

Current, daily consumption of milk and dairy products (and, thereby, bLF) 

in the United States amounts to the following: 

Age Groups 

1-12 

13-19 

2 20 

MDP (q/dl* bLF (mq/d13 

Ave. 90% Ave. 90% 

396 73 1 40 74 

377 747 38 75 

240 500 24 50 

(GRAS Notice 0042/0077). Thus, persons consuming milk and dairy products 

are regularly and for a lifetime exposed to from 24-75 mg bLF/day (with such 

exposure decreasing by about half, Le., from a max of 75 to a minimum of 24, 

over one’s life). 

This level of long-term exposure to bLF is not known to be associated with 

any adverse, immunologically-related effects. (See, e.g., GRAS Notices 0042, 

0067,0077, amd 0130). 

(2.) GRAS status 

In addition to the above-described, long history of safe consumption of 

bLF - via the consumption of cow’s milk and other dairy products - bLF (as a 

stand alone substance derived from milk) has been repeatedly reviewed by FDA 

as to whether it is generally recognized as safe (“GRAS”) (for certain intended 

use@)) and repeatedly determined by FDA to be GRAS. (See FDA “no 

questions” letters - dated 8/14/01, 10/23/01, 8/21/03, and 5/27/04 - in response 

* MDP = milk and dairy products 

One gram or ml ofmilk contains 0.1 mg bLF. (Batth, 1997). 3 

4 
to GRAS Notice numbers 000067, 000077 and 000130). In each such instance, 

GRASness was based primarily on “scientific procedures”, Le., on animal toxicity 

and genotoxicity studies, which demonstrated that bLF exposure levels up to 

2,000 mglk BW/day were neither toxic nor genotoxic. (See, e.g., FDA letter - 

dated 8/14/01 - in response to GRN 000077 at page 2). In addition, GRASness 

was based on results from human studies in which (1.) infants were exposed to 

dose levels ranging from 1.4 mg/d (0.3 mgkg BW/d) to 2.9 g/d (1.0 glkg BWld) 

and study durations from 11 days to 5 months and (2.) adults were exposed to 

dose levels ranging from 100 mg/d (1.7 mg/kg BW/d) to 3.6 g/d (60 mglkg BW/d) 

and study durations from single dose to 8 weeks. (See, e.g., GRAS Notice 

0042/0077). In about half of these studies (Le., 5 of 12), subjects were exposed 

to both bLF and hLF (but not at the same time, i.e., in separate arms). Finally, 

GRASness was also based on an expert panel of immunologists’ determination 

that lactoferrrin is highly unlikely to induce either an allergic response or any 

autoimmune disease, especially in adults. (Id. at page 3). Accordingly, FDA 

authorized two uses of bLF as an “antimicrobial” agent and, importantly, for “use 

as an ingredient in sports and functional foods at a level of 100 milligrams per 

product serving”. (See, GRAS Notices 000067, 000077, and 000130). 

e. Safety of hLF 

(1.) Introduction 

Assessing the safety of hLF (including the safety of long-term 

consumption) - like assessing the safety of bLF - is dependent on several 

subassessments. Each of these is identified and discussed below. 

(2.) hLF naturally occurs in humans 

Notwithstanding that bLF does not naturally occur in humans, is 

approximately 31 percent different than hLF in its amino acid sequence, has 

5 
a 

,. 

more glycosylation sites (i.e., two more) than does hLF, and induces (at a 

minimum) the same immunological effects as hLF, bLF has been appropriately 

deemed by FDA to be GRAS - and at the exact daily exposure level and uses 

that Pharming is seeking for its hLF product. In contrast, hLF is native to humans 

and - as discussed in significant detail in subsequent sections - is present both 

endogenously and exogenously at significant levels far in excess of that being 

sought (via Pharming’s GRAS Notice) as a daily exposure level; thus, one would 

not expect hLF to induce any safety concerns beyond those induced by bLF. 

And there is no direct, scientific evidence that hLF does. 

(3.) Long history of exposure to hLF 

As discussed in very significant detail in the updating information pertinent 

to subareas 3 and 4, virtually all humans have significant and lifelong exposure to 

both endogenous and exogenous hLF from about week 12 in utero onward. As 

also discussed, such long-term exposure is at levels significantly higher than 

those long-term exposure levels to bLF. Accordingly, if lesser levels of exposure 

to bLF are sufficient to determine that bLF is GRAS (at the same level of daily 

exposure and use as is being requested by Pharming), then significantly greater 

levels of long-term exposure to hLF should suffice to determine that hLF (at the 

same levels GRASed for bLF) is GRAS. 

(4.) Regarding host organism 

It may be obvious, but since it is of a critical nature it should be noted that 

both bLF and - in this specific instance - Pharming’s hLF are produced by the 

same host organism, i.e., a cow, and appear in the same, naturally-occurring, 

bovine product, Le., cow’s milk. As Pharming’s GRAS Notice discusses in 

significant detail, the transgenic cows in question are treated just like dairy cattle 

pursuant to good dairy practices and are no different than ordinary dairy cattle 

except that they contain a single, extra gene - in this case responsible for 

6 
U” 

c ,- 

producing hLF (GRAS Notice 0189). Such cattle do not represent any identified 

risk beyond those that have been identified over decades and which are currently 

adequately dealt with via compliance with good dairy practices. In addition, the 

milk emanating from such transgenic cattle is identical to milk emanating from 

normal dairy cattle except for the presence of hLF. Such presence has been 

shown - see next section - to be of no risk to either the dairy cows or to an 

individual consuming up to 2000 mg hLF/kg BW/day. Thus, the fact that the hLF 

here in question emanates from a transgenic source presents - in this specific 

instance - no additional, novel risks. 

(5.) Regarding “scientific procedures” evidence 

Bovine lactoferrin was determined by its sponsors and FDA to be GRAS 

based on “scientific procedures” (e.g , see GRAS Notice 0042/0077; see also, 

GRAS Notices 0067 and 0130) More specifically, the safety of bLF was 

evaluated in a series of published animal toxicity and genotoxicity studies4 which 

demonstrated that consumption of bLF at levels up to 2000 mglkg BW/day given 

up to 13 weeks produced no adverse effects. (See “no questions” letter from FDA 

- dated 08/14/01 - to sponsor of GRAS Notice 0077). With one exception 

(discussed at length in subsection 7 that follows), FDA determined that such 

testing was adequate since it issued a “no questions” letter. 

Pharming also is primarily relying on “scientific procedures” to 

demonstrate that its hLF is GRAS. It too tested its hLF via published, animal 

toxicity and genotoxicity studies - although such testing was considerably more 

extensive than that conducted in support of bLF. Such testing demonstrated 

that hLF was also not genotoxic and also did not produce any adverse effects 

when consumed up to 2000 mglkg BW/day. (GRAS Notice 0189). 

Pharming also included other published information emanating from 

Rhesus monkey studies which indicated that hLF was safe to consume up to 79 

mgkg BW/day. (GRAS Notice 0189, page 31). 

‘ Clinical studies are discussed in the next section. 

7 
(6.) Regarding clinical evidence 

The sponsor of GRAS Notices 0042 and 0077 (pertinent to use of bLF) 

also provided human clinical information supporting the GRAS status of bLF. 

(GRAS Notice 0042, pages 85-91). Such studies - which in about half the 

instances (as indicated above) also included separate evaluation of hLF - 

exposed subjects as follows: 

Infants 

Doses -from 1.4 mglday (0.3 rnglkglday) to 2.9 glday (1 .O glkglday) 

Duration - from 11 days to 5 months 

Adults 

Doses -from 100 mglday (1.7 mglkglday) to 3.6 glday (60 rnglkglday) 

Duration -from a single dose to 8 weeks 

FDA found these studies to be sufficient, since it issued a “no questions” letter. 

Despite the fact that humans are naturally exposed to significant quantities 

of hLF from in utero to death (Le., from 50-3100 mglkg BWlday during the first 12 

months of life and from 10-200 mglkg BWlday during the rest of life (see 

subparts 6, 8 and 10 infra)),Pharming also included clinical information in its GN 

in support of the safety of hLF. Such twenty-six studies exposed subjects as 

follows: 

Infants 

Doses - from 700 mglday (150 mglkglday) to about 5 glday (1538 

mglkglday) 

Duration -from 1 day to 21 days 

Adults 

Doses -from 52 mglday (0.87 mglkglday) to 15 glday (250 mglkglday) 

Duration -from 1 day to 42 days 

As can be seen by comparing the two sets of doses and durations pertinent to 

bLF and hLF: 

8 
“i . 1. the number of clinical studies in support of hLF are significantly 

more numerous; 

2. the hLF doses to which infants and adults were exposed were 

significantly greater; and 

3. the durations were longer for infants for bLF and about the same 

in adults for hLF and bLF. 

Accordingly, if the GN’s pertinent to bLF contained enough clinical information, 

then the GN pertinent to hLF certainly contains enough clinical information. 

(7.) Regarding assessment of qualified experts 

The GN’s pertinent to bLF and hLF both initially included professional 

assessments by qualified experts. Eventually both also included professional 

assessments by qualified experts with respect to the potential of either substance 

to induce immunologically-related effects and the meaning of any such effects. 

The latter assessment pertinent to bLF was one page long and consisted only of 

conclusions. In contrast, Pharrning’s comparable assessment was 40 pages 

long and included both extensive, in-depth, scientific discussion and expert 

conclusions. In addition, such latter assessment was twice updated. Since the 

former assessment pertinent to bLF was deemed to be sufficient, presumably 

Pharming’s assessment will also be deemed, at the very least, sufficient. 

(8.) Regarding ability to induce adverse immunological 

effects 

With regard to both hLF and bLF (since both are known to induce 

essentially the same immunological5 effects at the same oral dosages), the 

Since the term “immunological” incorporates numerous terms - such as “innate immunity”, “adaptive 

immunity”, “T-cells”, “cytokioes”, and many more, an understanding of these terms and the manner in 

which they are internelated is critical to Phanning’s update document. Thus, it seems appropriate - at this 

specific point - to provide some helpful background information concerning what such terms and their 

related activities entail - so as to promote common understanding. Since such information is quite basic 

and, therefore, not particularly helpful to a “qualified expert”, it has been set forth in a stand-alone 

9 
I..+- 

'*- 

question has been raised - primarily by research conducted over ten years ago - 

as to whether any of the effects induced are adverse effects. Presumably the 

answer is no since FDA determined that bLF is GRAS. Since hLF is intended for 

the same uses at the same ingestion level as bLF was GRASed for, presumably 

it also does not induce any adverse, immunological effects. 

Nevertheless, for the sake of thoroughness, each of the 

immunological-related questions asked about the potential for either bLF or hLF 

to induce any adverse, immunological effect are set forth below and then 

answered via reference to current, direct, and consensus, scientific evidence. 

(a.) Thl cell activity 

The pertinent, scientific literature inconsistently suggests that lactoferrin 

has irnmunoregulatory properties influencing both innate and acquired immunity. 

(See, e.g., the comprehensive review by Fischer, 2006). In particular, it has 

been suggested that lactoferrin influences T cell maturation, proliferation and 

differentiation into T-helper 1 (Thl) or T-helper 2 (Th2) cells. Thl and Th2 cells 

are two functional subsets of Th- or CD4-positive T cells, whose function 

depends upon the specific types of cytokines that are generated. (Rafiq, 2000; 

Mosmann, 1986, Abbas, 1996). CD4-positive Thl cells produce IFNy and IL-2, 

but not IL-4 or IL-5, and drive cellular immunity to attack viruses and other 

intracellular pathogens; conversely, CD4-positive Th2 cells produce IL-4, IL-5 

and IL-13, but not IFNy or IL-2, and drive humoral immunity that up-regulates 

at(achment. (See, Pharming response, Attachment 1). Notwithstanding its basic nature, however, the 

information is important and is, therefore, intended as a part of Pharming's update document. Such 

information - over 6 pages of it - is not set forth with numerous quotes because almost all of it comes from 

two, authoritative, sources, i.e , two widely-respected and widely-used medical school textbooks by two 

widely-respected immunologists ~ specifically, that by Abbas, Abul K. (at UCSF Medical School) and 

Lichtman, Andrew H (at Harvard Medical School) entitled Basic Immunology. Functions and Disorders 

of the Immune System, Second Edition, Saunders Elsevier (Phil, PA) (2006) and that by Abbas, Abul K. (at 

UCSF Medical School) and Lichtman, Andrew H. (at Harvard Medical School) entitled Cellular and 

Molecular Immunology, Fifth Edition, Saunders Elsevier (Phil, PA) (2005) These sources were used 

because they are widely respected and represent the consensus, established viewpoint of qualified experts. 

The authors are to be credited for the information presented - including that appearing in many of the 

footnotes (m particular, the definitions). 

10 
k. 

antibody production to attack extracellular organisms. Whereas Thl cells are 

known as important producers of IFNy, other cell types are also able to produce 

IFNy, including (in particular) NK cells and nonpolarized memory T cells. (Ye, 

1995; Biron, 1999). It is important to note that increased IFNy production does 

not necessarily reflect increased Thl cell activity. 

The establishment of the ThliTh2 balance is determined early during 

immune responses and depends on many factors including antigen structure, the 

functional status of antigen-presenting cells (APCs), the strength of T cell 

activation, the presence of cytokines, co-stimulatory signals and the 

microenvironment. (Rafiq, 2000). Both Thl and Th2 cells negatively crossregulate the function of one another through their respective cytokines. 

(Romagnani S, 1994; Maggi, 1992). Furthermore, it should be noted that IL-18, 

frequently reported as being upregulated upon lactoferrin oral administration, 

does not skew Th responses towards a Thl response. Rather, Thl responses 

are highly dependent on and stimulated by IL-12. Once Thl cells are polarized, 

then IL-18 can act on them to enhance IFNy production. IL-18 also enhances 

IFNy production of NK cells. Thus, production of IL-18 does not correlate to 

induction of Thl responses. (Nakanishi, 2001; Okamura, 1998). 

Regarding oral administration of lactoferrin, most of the data comes from 

orally administrated bovine lactoferrin (bLF) rather than human lactoferrin (hLF). 

Since there is sufficient evidence indicating that both proteins are comparable in 

structure and function (Baker, 2000; Nuijens, 1996), the effects observed on the 

immune system as a result of either bLF or hLF administration have been used 

as model for oral administration of Pharming’s hLF. 

Review of the available, scientific literature6 concerning oral administration 

of lactoferrin indicates that there are contradictory results with respect to the 

These preclinical and clinical studies - involving oral administration of lactofemin - include those by 

Artym (2003), Haversen (2003), Hayes (2005), Iigo (2004), Ishii (2003), Kruzel(2006), Knhara (2006), 

Kuhara (2000), Nakajima (1999), Sfeir (2004), Takakura (2006), Takakura (2004), Tanaka (1999), Togawa 

(2002), Ueno (2006), Varadhachary (2004), Wakabayashi (2006), Wakahayash (2004), Wakabayasbi 

(2003), Wakabayshi (2002), Wang (ZOOO), Zimecki (2006), Zmecki (2005), Zmecki (1998) and Zmecki 

(1995) Other, older studies also have been conducted. Their fmdings are all referred to in one or more of 

the studies referred to in the above-referenced list. All of the above-referenced studies are fully cited and 

summarized in Pharming’s response, Attachment 2. 

6 

11 
evidence showing that lactoferrin affects proliferation and differentiation of T cells 

into Thl and Th2 cells'. The induction of either Thl or Th2 biased immune 

responses by lactoferrin is complex as the observed effects appear to be, at least 

in part, dependent on the mode of lactoferrin delivery and on whether any 

ongoing inflammatory or immune response is occurring. (For a review of all 

pertinent studies, see Fischer, 2006). Based on the available data, Pharming 

and its expert panel concluded that the evidence for orally administered 

lactoferrin eliciting a positive and adverse CD4' Thl biased response is not 

convincing. This is because most studies suggest a change in Thl cell activity 

based on alterations in cytokine levels, in particular IFNy levels, but did not 

identify the cell-type responsible for the cytokine production. As mentioned 

above, increased IFNy production does not specifically indicate increased Thl 

cell activity. More likely, it indicates enhanced NK cell activity. In addition, the 

information is not convincing because some papers show potential Thl 

responses (i.e., IFNy secretion) within a few days. However, there is a critical 

time element involved in that it takes weeks for Thl and Th2 cells to become 

firmly polarized. (Murphy, 1996). Even in culture, where one can create an 

optimal environment, it takes at least a week - and usually 2-3 weeks - to 

generate CD4' Thl and Th2 cells. (Perez, 1995). 

Finally and not least importantly, even if - for sake of argument - oral 

consumption of human lactoferrin were to enhance Thl responses, that would 

not necessarily be deleterious. First of all, there is nothing in the direct evidence 

that demonstrates that lactoferrin given orally enhances any pathologic Thl 

responses'. On the contrary, there is evidence from a rat colitis model and other 

However, please note that Zmecki, et al. reported that lactoferrin mhibits proliferation and cytokine 

production by Thl cells - but not Th2 cells. (Zimecki, 1996). 

Guillen (2002) did report increased seventy of collagen-induced *tis in transgexnc mice expressing 

human lactofenin associated with an apparently enhanced Thl response. However, this conclnsion was 

based on cytokine levels which, as argued elsewhere, do not automatically imply a Thl response, and the 

continuous and chronic systemic exposure in this model is quite different fiom the oral exposure envisaged 

in humans. In contrast to these results, the same group earlier demonstrated that periarticular injection of 

hLF in mouse models of autoimmune arthritis and septic arthritis demonstrated significant treatment 

benefits. (Guillen, 2000). Also in sharp contrast are the many, preclinical results reported by Zimecki et al. 

which only demonstrated very beneficial results in immunosuppresssed and autoimmune animals when fed 

lactoferrin. (Zmecki, 2007) 

12 
%”, Y rat and mouse studies that demonstrate that oral consumption of lactoferrin 

inhibits a pathologic Thl response via upregulation of IL-10 and inhibition of IFNy. (Zimecki, 2006; Takakura, 2006; Togawa, 2002). In addition, there is 

considerable evidence that - to the extent lactoferrin influences T-cell maturation, 

proliferation, and differentiation - it does so only beneficially. (Fischer, 2006). 

(b.) Release of specific cytokines 

With respect to increased release of specific cytokines in the gut andlor 

systemically following oral administration of lactoferrin, various animal studies 

generally reported only local changes in the expressionlproduction of both Thl 

(e.g., IFNy, IL-2) and Th2 (e.g.,lL-4, IL-IO) cytokines. (Wang, 2000; Kuhara, 

2000; ligo, 2004; Wakabayashi, 2006; Varadhachary, 2004). In addition, various 

animal studies indicate that oral lactoferrin administration might increase both 

local and systemic IL-18 levels. (ligo, 2004; Wakabayashi, 2004; Kuhara, 2006; 

Hayes, 2005). Pharming’s expert panel concludes, however, that the effect of IL18 will occur locally and not systemically. Regarding the systemic levels of IL-18, 

oral administration of lactoferrin at doses up to 9 gram per day in human adults 

with solid tumors only resulted in a 15% increase of circulating IL-18, which is 

considered very low. (Hayes, 2005). More importantly, in this study no serious 

adverse events were reported and lactoferrin was well-tolerated by all subjects at 

a dosage of 150 mglkglday -which is very significantly higher than the level of 

maximum daily consumption that Pharming proposes in its GRAS Notification. In 

another study, a transient increase of IL-18 was observed in serum of hepatitis C 

patients receiving lactoferrin at an oral dosage of 600 milligrams per day for 12 

months. (Ishii, 2003). However, the data showed large variation and the 

observed increase of IL-18 decreased again after 3 months to baseline levels. 

Taking all such information into account, Pharming and its experts conclude that 

to the extent cytokines are reported to be released upon oral administration of 

lactoferrin, such reports do not indicate a consistent pattern of enhancement or 

any adverse effect. 

13 
In conclusion, it is Pharming’s and its experts’ opinion that, based on the 

available data, there is not convincing evidence that demonstrates to a 

reasonable certainty that lactoferrin specifically enhances any adverse Thl 

responses or can significantly increase any adverse, systemic cytokine levels 

over time. In contrast, there is sufficient evidence that lactoferrin enhances innate 

immune responses in the gut, e.g., by increasing IL-18 productiong (most likely 

locally, not systemically) and by increasing NK cell activity, both of which are 

considered beneficial rather than deleterious. Indeed, there is no direct evidence 

that increasing innate function is in any way detrimental; rather, such increased 

function is considered beneficial. (Fischer, 2006). 

(c.) Effect of oral administration of hLF on autoimmune or other 

inflammatory disorders 

((a.) Introduction) 

Finally, this presentation considers whether oral consumption of human 

lactoferrin induces any adverse effect(s) with regard to autoimmunity or other 

inflammatory disorders. Both areas of potential effects are addressed below. 

((b.) Autoimmunity) 

T cell responses to antigens are classified on the basis of the amount and 

kind of cytokines produced. Using this classification, T cell responses in MHCclass-11-restricted autoimmune diseases appear to be predominantly of the Thl 

type. (Rosloniec, 2002). Thus, Pharming understands the potential autoimmune 

concern to be about whether oral administration of lactoferrin enhances Thl 

responses and, thus, whether same could lead to the onset or enhancement of 

autoimmune diseases. Although the mechanisms of autoimmunity are not yet 

sufficiently understood, the concern here is considered highly unlikely by experts 

Ladoferrin has been shown to enhance IL-18 production by intestinal epithelial cells, thus enhancing the 

innate immune response. Human intestinal epithelial cells have been shown to condition human dendritic 

cells along a non-inflammatory TbZ-lie pathway, rather than towards Thl responses. (Rimoldi, 2005). 

14 
consulted by Pharming. First (and, perhaps, most importantly), there is a 

growing body of scientific evidence that indicates that orally administered 

lactoferrin significantly inhibits and/or diminishes and/or improves (rather than 

initiates or enhances) autoimmune diseases. (See, e.g., Zimecki, 2007 (orally 

administered lactoferrin only induces numerous, beneficial” effects in various 

studies conducted in immunosuppressed and autoimmune mice and rats); 

Kruzel, 2006 (orally administered lactoferrin causes reduction of clinical signs of 

multiple sclerosis in patients - in parallel to normalization of cytokine production 

by peripheral blood cells); Zimecki, 2006 (orally administered lactoferrin 

significantly diminished the clinical symptoms of experimental autoimmune 

encephelomyelitis in Lewis rats); and Togawa, 2002 (oral administration of 

lactoferrin significantly reduced colitis in rats))”. Second, it is very possible that 

Thl cells are not even involved in the pathogenesis of autoimmune diseases. 

Rather, such diseases may well be induced by the recently discovered T-helper 

17 subset. (Hue, 2006; Yen, 2006). Third, as already discussed above, the 

evidence that orally administered lactoferrin elicits an adverse, Thl -biased 

response or potentiates a pre-existing Thl-mediated immune response is 

considered highly unlikely. Fourth, hLF is naturally expressed in saliva and the 

gastro-intestinal tract; thus, humans have a significant daily naturally-occurring 

exposure to hLF’*. For instance, the intake of lactoferrin from saliva alone is 

about 20 mg/day. (Tanida, 2003). Consequently, humans are tolerant to hLF. 

Once oral tolerance has been established, it is very hard to disrupt, even in 

patients with chronic stimulation of the immune system. (Zivny, 2001). 

Moreover, the oral administration of an autoantigen has been shown to be 

beneficial in the treatment of various experimental, autoimmune diseases and 

this method of inducing immune non-responsiveness has currently been applied 

Io For a discussion of all such benefits, see pages 18-22 of this update document. 

I’ See also, two other studies showing similar results, i.e., Guillen, 2000 (which study demonstrated that 

periarticular mjection of hLF in mouse models of autoimmune arthritis and septic arthritis demonstrated 

significant treatment benefits) and Zmecki, 1995 (which study demonstrated that intraperitoneal injection 

of bLF in mice significantly inhibited autoimmune hemolytic anemia). 

human miUc, neutrophls and various mucosa. Indeed, as Phanning’s GN indicates (at pages 26 and 28 of 

Attachment I), human lactofenin is virtually ubiquitous throughout the human body. 

Such daily, natural exposure also emanates kom lactoferrin produced and released by or in, for example, 

15 
‘dr. 

n,.- 

to the prevention and treatment of human autoimmune diseases. (See reviews 

by Wardrop, 1999; and Sosroseno, 1995). Fifth, although there is extensive 

reporting on the presence of autoantibodies against lactoferrin, there is no 

evidence that these antibodies play any role in the pathology of autoimmune 

diseases. There is a large body of scientific literature on antilactoferrin autoantibodies as a component of antineutrophil cytoplasmic antibodies (ANCA). 

(See review by Malenica, 2004). In addition, individuals with a wide range of 

autoimmune conditions have anti-lactoferrin autoantibodies. Despite this large 

body of scientific literature on these antibodies, there is no evidence showing 

them to have any role in the etiology of autoimmune disease, and there is a 

general consensus among qualified experts that they are an epiphenomenon, 

i.e., unrelated to treatment, disease activity, duration of disease, or disease 

extent. (Malenica, 2004; Chikazawa, 2000; Guillen, 1998; Roozendal, 1998; and 

Audrain, 1996). Furthermore, all individuals possess low but detectable amounts 

of circulating and mucosal human lactoferrin. Therefore, it is considered highly 

unlikely that oral administration of human lactoferrin, even to an individual with an 

ongoing autoimmune disease, would increase autoantibody levels. Even if oral 

lactoferrin were to increase the level of such antibodies, it would be clinically 

irrelevant, i.e., unlikely to have any impact on disease pathogenesis or severity. 

Anti-lactoferrin autoantibodies have not been shown to be involved 

in the pathogenesis of any disease. In contrast, there is data that autoantibodies 

in general may help clear and degrade autoantigens, thus reducing T cell 

sensitization to them. (Mizoguchi, 1997). It should also be pointed out that there 

have been multiple trials in which autoantigens were fed to patients with 

autoimmune diseases to see if this might ameliorate the disease. For example, 

these trials have fed human insulin to autoimmune diabetics, collagen to 

rheumatoid arthritis patients, and myelin proteins to patients with multiple 

sclerosis. These trials have not shown any consistent benefit to the patients; 

however, there were no deleterious effects from autoantigen feeding and this 

was done in substantial numbers of individuals. (Faria, 2005). 

16 
‘.b. In conclusion, Pharming and its experts conclude that it is highly unlikely 

that oral consumption of Pharming’s lactoferrin at the level here in question 

would lead to the development or the perpetuation or enhancement of an 

autoimmune response. 

((c.) Inflammatory disorders) 

As discussed above, there is sufficient evidence that lactoferrin enhances 

innate immune responses in the gut Thus, it is a potential concern that this may 

lead to promotion of inflammatory disorders in the gut. Pharming understands 

this concern, particularly as it relates to inflammatory bowel disease (IBD), a term 

which commonly incorporates ulcerative colitis (UC) and Crohn’s disease (CD). 

Both diseases are chronic inflammatory conditions of the gut in which Crohn’s 

disease may affect any part of the gastrointestinal tract, whereas UC mainly 

affects the colon. In IBD, there appear to be multiple levels of immune 

responses, including innate, adaptive and regulatory immune responses. There 

is emerging literature that innate immune defects can contribute to the 

development of IBD. (See, eg., Beckwith, 2005; Hugot, 2001; Ogura, 2001). 

However, neither Pharming nor its experts are aware of any scientific evidence 

that supports the idea that a low-level Thl response or enhancement of the 

innate immune response, even on a chronic basis, would be detrimental or 

trigger IBD. In contrast, lactoferrin has been repeatedly shown to enhance the 

production of IL-I 8 by intestinal epithelial cells, thereby increasing innate 

immunity, which is considered beneficial rather than deleterious for susceptible 

individuals. This beneficial enhancing of innate immunity has been confirmed in 

a recent open label trial in patients with Crohn’s disease who received 

granulocyte-macrophage colony-stimulating factor (GM-CSF). (Dieckgraefe, 

2002). GM-CSF is a cytokine involved in enhancement of the qualitative function 

of various immune cells, and stimulates the expansion and differentiation of 

haemopoetic progenetors. (Armitage, 1998). The results showed an 

17 
enhancement of the intestinal innate immune response resulting in an 

amelioration of the disease. (See also, Fischer, 2006). 

Even with regard to individuals who have a “leaky” gutI3, such as can be 

found in inflammatory bowel disease, orally administered exogenous lactoferrin is 

simply supplementing large endogenous production of lactoferrin in alimentary 

secretions. There are low levels of antibodies to various foods in intestinal 

secretions and serum, but there is no evidence that these have any detrimental 

effect. There is also no evidence that immunologic reactions to food have any 

adverse effect in inflammatory bowel disease or that any foods exacerbate 

inflammatory bowel disease. 

In contrast to the concern that orally administered lactoferrin may impact 

negatively on inflammatory bowel disorders, there is a growing body of scientific 

evidence - as Zimecki et al. point out -that demonstrates just the opposite, Le., 

that orally consumed lactoferrin exhibits “distinct anti-inflammatory properties.” 

(Zimecki, 2006). Such conclusion - the authors indicate - is supported by a 

growing number of studies incorporating a number of models “including 

experimentally induced bowel inflammation in rats (Togawa et al., 2002), 

autoimmune disorders in mice (Zimecki et al., 1995; Guillen et al., 2000), 

experimental endotoxemia in mice (Kruzel et al., 2002), and inflammatory 

reactions to Mycobacterium bovis (Zimecki et al., 1994).” (Zimecki, 2006; see 

also, Zimecki, 2007 which reported on the anti-inflammatory effect of hLF in rats 

and mice; Fischer, 2006 which reported on the anti-inflammatory effects of hLF in 

mice; and Haverson, 2003 which reported on the anti-inflammatory effects of hLF 

in an experimental colitis model in mice). In all such models, lactoferrin exhibited 

significant anti-inflammatory properties. 

Moreover, lactoferrin induces TGF-P production which is widely 

considered an anti-inflammatory cytokine. (Zimecki, 2005; Ward, 2002). Since 

TGF-P is an anti-inflammatory cytokine associated with the induction of antigenspecific regulatory T cells and such cells produce TGF-P or IL-10, these cells can 

l3 To the extent that the “leaky” gut concept exists - and such concept is not generally recognized - it 

generally refers to the movement of molecules with a molecular weight of less than 1000 daltons. 

18 
a, inhibit the induction of inflammatory responses. In particular, these cytokines 

suppress IFN-y production and activity from activated Thl cells. Lactoferrin can 

even exhibit strong anti-inflammatory effects in dexamethasone-induced acute 

colitis in a mouse model. (Haverson, 2003). 

In further contrast to suggesting that human lactoferrin - a substance 

native to humans - might be responsible for either autoimmune or other 

inflammatory disorders, there is a growing body of scientific evidence showing 

that defects in innate immunity can lead to an abnormal adaptive immune 

response, some of which are manifest by autoimmune disease. A good example 

of this is the non-obese diabetic (NOD) mouse, which has some well-defined 

defects in innate immune responses. Stimulation of the NOD innate system by a 

variety of means blocks the development of the autoreactive T cell response to 

islet cells and, thus, prevents diabetes. In inflammatory bowel disease there is 

emerging literature that innate immune defects can contribute to the development 

of IBD. (Korzenik, 2006). For example, a colitis susceptibility gene has been 

identified which appears to function by regulating innate immunity. (Beckwith, 

2005; Hugot, 2001; Ogura, 2001). In addition (and as mentioned above), there is 

a trial in which GM-CSF has been administered to patients with Crohn’s disease 

to enhance their innate immunity and, thus, ameliorate their disease. 

(Dickgraefe, 2002). Thus, autoimmune or chronic inflammatory diseases are 

more likely to result from deficient innate immune cytokine production or function. 

Of course, there is no scientific evidence that suggests - let alone 

demonstrates - that orally consumed human lactoferrin induces any deficiency in 

any innate immune mechanism. In fact, orally consumed human lactoferrin does 

just the opposite, Le., it enhances innate immunity, which is deemed beneficial. 

(See, e.g., Zimecki, 2007; and Fischer, 2006). 

(8.) Conclusion 

Fortunately, there are now numerous, published, preclinical and clinical 

studies and other information - most of which have been reviewed above - 

19 
"- which are pertinent to and evaluate the potential immunomodulatory ability of 

hLF. Thus, the primary question arising at this time from such studies and 

information with regard to the safety of hLF is: What do all such studies and 

information currently indicate about such potential? 

Clearly, the growing body of evidence indicates that lactoferrin can 

induce a broad range of immunological effects with regard to the manner in 

which a human immunologically reacts given the exposure to an invading 

pathogen or other substance. Early on, some thought that such evidence might 

indicate that lactoferrin may have a causal role in human disease (such as 

Crohn's disease, irritable bowel syndrome or autoimmune disorders); thus, they 

asked- Are such effects possibly adverse to the individual? More recent 

evidence increasingly indicates that lactoferrin also has a well-established ability 

to induce various, clearly beneficial effects. Thus, to some the evidence in the 

aggregate may seem confusing - perhaps even inconsistent. Fortunately, all of 

the above-referenced studies and information have been recently and thoroughly 

reviewed for the purposes of determining what exactly all such evidence currently 

indicates. Qualified experts now conclude - as discussed in greater detail below 

-that oral consumption of lactoferrin does not induce detrimental effects; rather, 

its numerous effects function only in beneficial - but differing - manners. 

Fischer et al l4 recently reviewed approximately 80 different studies and 

related publications pertinent to lactoferrin's potential immunoregulatory 

proper tie^'^ - especially as they relate to lactoferrin's ability to regulate Thl and 

Th2 responses. (Fischer, 2006)16. After discussing the findings of all such 

studies and information, Fischer et al concluded that lactoferrin does not induce 

any adverse, non-allergic immune responses - via either the innate or adaptive 

immune defense mechanisms. More specifically, the authors concluded that: 

.", ^. 

I' Fischer et al. are a group of experts in nutrition and immunology that is ~ when not involved in a visiting 

professorship - employed as a group hy the French National Institute of Agronomy in Panis to investigate 

aspects of the physiology of nutrition and the alimentary canal. 

Is The Fischer paper was peer reviewed prior to publication. 

l6 There are a few preclinical studies on Pharming's reference list which do not appear on the reference list 

attached to the Fischer article. Since such studies supply only information like that already reviewed by 

Fischer, they do not alter the scope of the substance discussed or the conclusions reached by Fiscber, et al. 

20 
1. under nonpathogenic conditions, lactoferrin functions to set up 

the immune system - by, for example, influencing T-cell 

maturation, proliferation and differentiation into Thl and Th2 

cells - so as to be able to effectively respond to the conditions 

described immediately below; 

2. under infectious disease conditions, lactoferrin affords 

protection by inducing a Thl polarization in diseases in which the 

ability to control infection or tumor relies on a strong Thl 

response; 

3. under inflammatory conditions, lactoferrin functions as an antiinflammatory by reducing the Thl component to limit excessive 

inflammatory response; and 

4. under noninfectious disease conditions, lactoferrin provides 

protection against Thl- or Th2-induced diseases, such as 

autoimmune or allergic diseases, through correction of the 

cytokine Thl/Th2 imbalance. 

Thus, consistent with Pharming’s and its experts’ assessment, Fischer et al. also 

concluded that the available information indicates that oral consumption of 

lactoferrin - even at levels exceeding the level of use here at issue - results in 

only beneficial immunological effects. 

Zimecki et aI.l7 also published a review of the implications of the ability of 

lactoferrin to influence immunoregulatory function - especially as it relates to 

their preclinical work involving immunosuppressive and autoimmune animals. 

(Zimecki, 2007; see also, Artym, 2003; Artym, 2003; Zimecki, 2999; and Baveye, 

1999). After reviewing the pertinent information, the group concluded that oral 

administration of lactoferrin. 

1. accelerates reconstruction of immune system function (including 

restoring innate and acquired, antigen-specific cellular, and 

Zmecki et al. are a group headed up by Dr. Michael Zmecki, an immunologist (who is Chief, 17 

Laboratory of lmmunobiology and Chief, Department of Experimental Therapy ~ both at the Ludwik 

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences) and which 

group also includes experts in several other medical-related discipllnes and which group has tested, 

evaluated, and published more articles concerning lactofenin than, perhaps, any other entity. 

21 
‘r humoral immune response) in instances of immunosuppresssion; 

development; 

2. accelerates counteraction (i.e , inhibition) of autoimmune disease 

3. normalizes the ration of major blood cell types; 

4. lowers clinical scores of disease and diminished 

pathohistological changes; and 

cytokines 

5. reduces serum levels of both pro- and anti-inflammatory 

Thus, like Fischer et al. and Pharming’s experts , Zimecki et ai. found no adverse 

effects induced by the oral consumption of human lactoferrin. 

Kruzel et al.” also reviewed the information - pertinent to the effects that 

oral consumption of lactoferrin has on the immune system. (Kruzel, 2007). They 

reported that - contrary to inducing any adverse effects - lactoferrin functions: 

1. as a frontline defense protein involved in protection and 

prevention; 

2. as a mediator that bridges innate and adaptive immune functions 

to achieve successful outcome; 

3. to protect cells from oxidative injury; and 

4. most importantly, as an immune sensor directing specific 

immune responses to achieve immune homeostasis. 

Thus, Kruzel et al. - like numerous, other qualified experts (including Pharming’s 

qualified experts) who have carefully and thoroughly evaluated the potential of 

lactoferrin to induce adverse immunological effects - concluded that lactoferrin 

does not induce adverse immunological effects. 

Finally, since it is very difficult - despite numerous attempts - to find a 

mucosal adjuvant among substances likely to be orally consumed, Pharming and 

its experts conclude that it is very unlikely that further preclinical or clinical testing 

/. i 

Is Dr. Marian bel is an immunologist who is currently a visiting professor in the Department of 

Integrative Biology and Pharmacology at the University of Texas, Health Science Center (Houston, Texas). 

Her group includes experts in immunology, pathology and microbiology. She is also part of the Zimecki 

group referenced above. 

22 
.. 

of Pharming's lactoferrin at the daily level here at issue and even for longer 

periods of exposure would result in any demonstration that Pharming's lactoferrin 

is able to induce -via oral consumption - any adverse immunomodulatory effect. 

f. Conclusion 

All of the scientific information set forth above - including long-term data - 

indicates that bLF is not only safe for consumption at the levels here in question 

but also GRAS. Accordingly, FDA has repeatedly determined that it is GRAS. 

Based upon structural and functional similarities, one would expect that hLF 

would be as safe as bLF (if not more so since it is native to humans). All of the 

scientific information provided in support of hLF - which is considerably more 

than that provided for bLF - indicates that hLF - like bLF - is safe for ingestion at 

the same levels and is also GRAS. Thus, the history of safe consumption of bLF 

is - in effect - also the history of the safe consumption of hLF. 

3. Subarea 2 (concerning potential antigenicity of hLF) 

Human lactoferrin in its native forms and human lactoferrin in its 

transgenically-produced forms are virtually, but not absolutely, identical. With 

regard to the differences, since human lactoferrin in the normal population has 

four polymorphic sites, differences between exogenous and endogenous human 

lactoferrin can arise because of different polymorphic alleles in the exogenous 

and endogenous molecules. Additionally, another difference can arise as a 

characteristic arising from the transgenic organism in which the protein is 

expressed, i.e., transgenically-produced proteins will be glycosylated in a way 

characteristic for the expression system and, thus, the resulting glycosylation 

pattern may differ from its native form. Due to these known differences between 

native and transgenically-produced human lactoferrin, to what extent would such 

differences be expected to impact on the potential antigenicity of oral exposure to 

transgenically-produced human lactoferrin? 

23 
4. Pharming Update 

a. Introduction 

At the outset, it seems important to emphasize that this entire presentation 

is primarily about the two substances (here in question) about which Pharming 

has expertise and which are the focus of Pharming’s GRAS Notification -that is, 

Pharming’s and native human la~toferrin‘~. Thus, much of what is in the GFWS 

Notification and this presentation is only about human lactoferrin (sometimes 

referred to in this presentation as hLF). However, Pharming recognizes that 

human lactoferrin is one in a broad set of mammalian lactoferrins (including, 

especially, bovine lactoferrin - sometimes referred to in this presentation as 

bLF)“; accordingly, Pharming has also - from time to time - included in its 

updating documents information about other lactoferrins because such 

information is helpful in establishing a broader context of safety of human 

lactoferrin. 

b. Comparability of the two lactoferrins 

(1.) Introduction 

This subarea of discussion considers whether Pharming’s exogenous 

lactoferrin is structurally significantly different from the polymorphic, endogenous 

lactoferrin produced naturally by the individual consumers comprising the U.S. 

population and, if so, whether any such structural difference may have a 

This information may or may not be applicable to other versions of human lactoferrin. While there are 

bkely to be very significant similarities between versions of bLF produced fiom transgenic sources, there 

may also be differences - in some cases, a difference that makes a difference ~ thus, each version must be 

safety assessed based on its own particulars. 

To the extent hLF is considered to he a “hown biological response modifier” (KBRM) of the human 

immune system, bLF has also been demonstrated to the same extent to be a KBRM. (See, subarea 

B(2)(e)(8)). bLF has been determined to be GRAS and at a level equivalent to the level being requested in 

Pharming’s GN. 

19 

20 

24 
-- significant, negative impact on the way in which such exogenous lactoferrin is 

recognized and responded to by the human immune system. Accordingly, what 

follows is a discussion of the extent to which both lactoferrins are identical, the 

extent to which both lactoferrins are different, and the importance of any such 

difference. 

(2.) Both lactoferrrins are almost entirely 

the same 

As thoroughly discussed in considerable detail in Pharming’s GRAS 

Notification (please see, Attachment 1, pages 4-5, 12-13, and 32-34), both 

Pharrning’s exogenous lactoferrin (“rhLF”) and endogenous lactoferrin (“hLF) are 

overwhelmingly identical. As a reminder, such identicalness extends to the fact 

that both lactoferrins: 

1. are the same metal-binding, glycoprotein, i.e., hLF (Thornassen, 

2005, van Berkel, 2002; Anderson, 1989); 

2 have the same amino acid sequence and composition based on the 

nucleic acid sequence pertinent to the allelic variation seen in the 

normal population (see, GRN No. 00189, subsection III(C)(l)(e)); 

3. have the same N-terminal protein sequence (van Berkel, 2002); 

4. have the same three-dimensional, protein structure (Thomassen, 

2005); 

5. are N-glycosylated (van Berkel, 2002); 

6. have the same number and location of glycosylation sites (van Veen, 

2004); 

7. show the same chromatographic profiles upon analytical Mono S 

analysis (van Berkel, 2002); 

8. have the same core-molecular weight (although overall molecular 

weight slightly differs - Pharming’s hLF is slightly lower - due to the 

differences in the carbohydrate moieties attached to the lactoferrin 

core) (van Berkel, 2002); 

25 
9. show the same tryptic degradation kinetics, Le., digestibility (van 

Veen, 2004); 

10 have the same iron-binding and iron-release properties (van Berkel, 

2002); and 

11. are equally effective against experimental infections with multidrugresistant S. aureus and K. pneumoniae in mice (van Berkel, 2002). 

Thus, from the point of view of considering any difference which actually makes 

any significant difference, Pharming’s lactoferrin is identical to endogenous 

lactoferrin -except for the difference that is discussed below. 

(3.) How the lactoferrins are different 

CFSAN initially questioned whether Pharming’s exogenous, human 

lactoferrin differs from endogenous human lactoferrin in two, different ways. 

Each is discussed below. 

h 

((a.) With regard to their respective amino acid sequences) 

CFSAN first questioned whether the amino acid sequence of Pharming’s 

exogenous lactoferrin is structurally different from that of endogenous 

lactoferrin.” As Pharming’s GN explains (see pages 12-13) and as discussed 

below, the two lactoferrins are not really different. 

Careful comparison of the ten, published, amino acid sequences of 

endogenous lactoferrin demonstrates that such naturally-occurring sequences 

may naturally differ from one another in six instances , i.e., in amino acid 22 . 

Specifically, CFSAN questioned whether “Pharming’s lactofemn is distinct from the endogenous 

lactofenin of individual consumem with respect to expected differences behveen the amino acid sequences 

of the exogenous lactoferrin and the polymorphic endogenous lactofemn alleles present in the general 

population.” 

22 Only four ofthese instances have been scientlfically confirmed, i.e., at amino acid positions 4, 11,29 

and 561. (van Veen, 2004). The other two, Le., positions 14 and 413, have not yet been confirmed. 

Phammg’s lactofenin has - with regard to these latter two amino acid positions - the same amino acids as 

reported in 9 of the 10 above-referenced amino acid sequences. (See pages 12 and 13 of Pharming’s GN, 

Attachment 1) It is possible that the latter two differences may not be real. Thus, FDA’s question refers to 

four. 

21 

26 
positions 4, 11, 14, 29, 413, and 561. In each such natural instance, the amino 

acid present is one of only two possibilities. Thus, there exists a well-known and 

well-documented naturally-occurring range of amino acid variation in 

endogenous lactoferrin. 

Pharming’s lactoferrin does not differ from but rather exactly duplicates 

this range, Le., it is no more different from such range than any one of the ten, 

known endogenous lactoferrins In each of the six, above-referenced amino acid 

positions, Pharming’s lactoferrin incorporates exactly the same one of two 

possible amino acids, as does any one of the ten endogenous lactoferrins. Thus, 

there exists no real difference here (with regard to amino acid sequence) 

between what occurs endogenously and what occurs exogenously. (Please note 

that with regard to all other amino acid positions, they are all identical). 

There is no scientific evidence that an individual producing any one of the 

above-referenced, naturally-occurring, endogenous lactoferrins reacts - 

immunologically speaking - differently when exposed to any one of the other 

above-referenced endogenous lactoferrins. Indeed, extensive and long-term 

human experience demonstrates just the opposite. For example, infants when 

consuming human milk are exposed - in the vast majority of instances - to an 

exogenous hLF variety that differs from their own endogenous variety and all 

without adverse, immunological reaction, probably due - assuming, for sake of 

argument, that the infant‘s immune system even recognizes the exogenous 

variety as being different - to oral tolerancez3 andlor anergf4. It should be noted 

that the daily exposure levels to exogenous hLF that an infant naturally 

experiences (Le., 48-3077 mg hLFlk BWld) far exceed the daily exposure level 

The term “oral tolerance” is defined as the suppression of systemic humoral and cell-mediated unmune 

responses to an antigen after the oral admimistration of that antigen, due to anergy of antigen-specific T 

cells or the production of immunoregulatory mediators such as transforming growth factor-p or interleukin10. Oral tolerance is a physiological mechanism for preventing immune responses to food antigens. For a 

thorough discussion ofhow tolerance is established, etc., see Iweala ,2006 or Faria, 2005. 

The term “anergy” is defined to mean a state of unresponsiveness to antigenic stimulation. Lymphocytic 

anergy (also called clonal anergy) is the failure of clones of T or B cells to react to antigen, and this may be 

a mechamsm of maintaining immunologic tolerance to self antigens. In clinical practice, anergy refers to a 

generalized defect in T cell-dependent cutaneous delayed-type hypersensitivity reactions to common 

antigens. (Abbas, 2006). 

W 

24 

27 
being requested in Pharming’s GRAS Notification, i.e., 100 mglproduct serving 

(or 1.91-3.95 mg hLF/k BW/d).25 In addition, patients - of all varieties and ages - 

who receive transfusions of blood products, e.g., fresh, frozen plasma, are 

routinely exposed to an exogenous human lactoferrin that differs from their own. 

This exogenous lactoferrin - present in the plasma at varying concentrations 

from 42-202 pglml - is predominantly derived from degranulating neutrophils. 

(Scott, 1989). Importantly, patients who receive such transfusions commonly 

have ongoing inflammatory reactions, e. g., trauma; even so, such very 

numerous, systemic exposures to these exogenous lactoferrins in these patients 

have not been reported to have led to any known, adverse, immunological event. 

Oral exposure to human lactoferrin should be even less potentially immunogenic 

than this type of very intimate, blood-to-blood exposure. Since Pharming’s 

exogenous human lactoferrin only duplicates endogenous, human lactoferrin 

(with regard to amino acid sequence), one would also expect such exogenous 

lactoferrin not to induce any adverse, immunological event (as a result of its 

amino acid sequence) And there in no evidence that it could or does 

Finally and not least importantly, in nature, there are numerous, naturallyoccurring, mammalian lactoferrins (i.e., iron-binding glycoproteins) all of which 

are similar in structure and function compared to their human homologue (See, 

e.g., Nuijens, 1996). Why would one expect that oral consumption of Pharming’s 

exogenous hLF -whose amino acid sequence only duplicates that which occurs 

naturally in endogenous human lactoferrin - would induce any adverse 

immunological event when bLF - whose amino acid sequence is 31 percent 

different that endogenous human lactoferrin - does not and is deemed GRAS, 

even when consumed long-term at significant levels (Le., at least at levels 2 100 

mglproduct serving)? Of course, the answer is that one should not have any 

such expectation because there is no direct, scientific evidence to support such 

expectation. 

The issue concerning the impact of the differences between the adult and infant gut and immune system 25 

was discussed in an earlier section. (See, subpart IIIB2e4) 

28 
((b.) With regard to their respective glycosylation patterns) 

As CFSAN correctly notes, Pharming’s exogenous lactoferrin does differ 

from endogenous lactoferrin with regard to the type of carbohydrate structures 

that are attached at each of the three glycosylation sites. However, that is the 

extent of their structural differences (as Pharming’s GN, Attachment 1, discusses 

at pages 4 and 33), since both lactoferrins incorporate the same number and 

location of glycosylation sites and both utilize these glycosylation sites in the 

same fashion. (van Veen, 2004). 

With regard then to the specific glycans attached at each of the 

glycosylation sites, the only glycans attached to the glycosylation sites of natural 

hLF (from human milk) are N-linked, complex-type glycans. (van Berkel, 2002; 

Spik, 1982). In addition to the complex, N-linked glycans that are attached to the 

endogenous lactoferrin glycosylation sites, Pharming‘s exogenous lactoferrin 

also bears oligomannose andlor hybrid-type, N-linked glycans (van Berkel, 2002) 

- as one would expect, since the distribution and structures of attached glycans 

is species-, tissue-, cell type- and protein-specific. (James, 1995; Opdenakker, 

1993). Furthermore, the complex, N-linked glycans of Pharming’s hLF contain Nacetylgalactosamine next to galactose, which is typical for N-linked glycoproteins 

produced in bovine milk, such as bovine lactoferrin. (Van den Nieuwenhof, 1999; 

Coddeville, 1992) Finally, the glycans of Pharming’s hLF contain less fucose 

compared to natural hLF. (van Berkel, 2002). However, as a result of 

crystallography studies, it has been determined that - despite the differences in 

N-linked glycosylation - the three-dimensional structure of Pharming’s hLF and 

natural hLF are identical. (Thornassen, 2005). 

Thus, the attached glycan-related differences then are the only known 

structural differences that exist between endogenous lactoferrin (from human 

milk) and Pharming’s exogenous lactoferrin. 

29 
((c.) The importance of the differences) 

At this point, the key focus becomes: Does the above-described 

difference (with regard to exactly what glycan is attached at each of lactoferrin’s 

three glycosylation sites) make any real difference with regard to the ability of 

Pharming’s exogenous lactoferrin to disrupt previous tolerance to endogenous 

lactoferrin? The direct evidence indicates that it does not. 

The mere fact that a difference exists - as here - between two forms of a 

molecule (one of which naturally occurs endogenously - in this case in human 

milk - and the other of which @e., the exogenous form) differs from that 

naturally-occuring form only with regard to the kinds of glycans attached at each 

of the glycosylation sites) does not - by itself - amount to direct evidence that 

such difference will affect the latter molecule’s potential immunogenicity. For 

example, please note that endogenous hLF (from human milk) and endogenous 

hLF (from human neutrophils) also differ in their respective glycosylation 

patterns. (Derisbourg, 1990). The glycan associated with neutrophilic hLF is not 

fucosylated - thus, it resembles the glycan pattern of human serum transferrin. 

(Spik, 1994). However, such difference in glycosylation pattern does not affect 

hLF’s function with respect to isoelectric point, stability of the iron-saturated form, 

rate of clearance, or antigenicity. (Derisbourg, 1990, Moguilevsky, 1985). 

And there exists another, just as relevant, well-known example, which 

demonstrates that consumption of a differently glycosylated lactoferrin does not 

lead to any adverse consequences with regard to immune response or any 

interruption of tolerance. The example, of course, involves the human 

consumption of bLFZ6 which is long known to be safe (and at levels far exceeding 

26 Bovine lactofernin (bLF) is also -like hLF - an iron-binding glycoprotein (of about 80 ma) which is 

similar in structure and function compared to its human homologue. (Nuijens, 1996). ‘Ike amino acid 

sequence ofbLF (which contains 689 amino acids) shows 69% homology with hLF. pierce, 1991). The 

sequence of bLF contains five possible N-lied glycosylation sites, Le., two more than hLF. Four sites, 

i.e., Asn 233,368,476, and 545, are always utilized (Spik, 1994) while the fifth (Asn 281), located in the 

N-lobe, is glycosylated in about 30% and 15% of the molecules in bovine colostrums and mature milk, 

respectively. (van Veen, 2002; Wei, 2000; Yoshida, 2000) 

30 
the level here at issue, Le., 100 mg per product serving) as a result of a long and 

well-documented history of safe use (and by humans of every variety, including 

age, race and ethnic background). (See, subarea B(2)). Since Pharming's 

exogenous lactoferrin and bLF are both produced by the bovine mammary gland 

which determines the type of glycosylation (in this case, a mammalian type of 

glycosylation) and since, similar to Pharming's hLF, bLF bears oligomannosetype glycans and complex-type glycans with N-acetylgalactosamine next to 

galactose (Coddeville, 1992) (but more of the same since bLF has 5 attachment 

sites as compared to hLF's 3) and since historical human consumption of bLF at 

or exceeding the level of consumption of hLF being proposed in Pharming's GN 

has not resulted in any reported, adverse, immunological events, one would not 

expect that consumption of Pharming's exogenous lactoferrin would induce any 

adverse, immunological event. And there is no direct evidence that it does - 

absolutely none. 

Of course, under certain circumstances, it may be possible (and 

documented) that a specific difference in glycosylation pattern may make a 

significant difference in the way in which a specific glycosylated protein will be 

recognized by the human immune system. (Cobb, 2005). But in the specific 

instance at hand, the single difference that exists between Pharming's human 

lactoferrin and endogenous human lactoferrin is not recognized (Le , 

documented) as a difference which leads to an adverse, immune response. 

Finally and not least importantly, neither Pharming nor its experts are aware of 

any direct evidence that indicates that there is any protein to which humans are 

tolerant - including bLF and hLF - which will induce any adverse, immune 

response merely as a result of a difference in glycosylation. Therefore, it is 

extremely unlikely that such difference will alter the normal way in which 

Pharming's hLF is recognized and processed. 

31 
d. Potential for adverse effect(s) 

(1.) Introduction 

There has been some speculation that Pharming’s human lactoferrin 

might - via the oral route of exposure - possibly induce certain adverse, 

immunological effects due either to differences in different polymorphic alleles or 

in glycosylation pattern. These speculations are addressed below. 

(2.) Determinant spreading 

With regard to the potential for determinant spreading from alloepitopes, 

Pharming and its experts conclude that such event is very unlikely to occur in the 

situation involving consumption of Pharming’s hLF. An epitope is any molecular 

structure that can be recognized by the immune system Epitopes, or the antigen 

from which they are derived, can be composed of protein, carbohydrate, lipid, 

nucleotide, or a combination thereof. (Abbas, 2006). It is through recognition of 

foreign, or non-self, epitopes that the immune system can identify and destroy 

pathogens. T cells are known to respond only to linear epitopes, Le., peptide 

fragments (usually 8 or 20 amino acids in length) digested from the native 

protein, that are presented in association with major histocompatibility complex 

(MHC) molecules ’’ (Abbas, 2006). An epitope is considered linear, if the target 

of the immune response is apparent in the series of adjacent amino acids without 

any requirement for secondary or tertiary structure (folding) as would occur in a 

native protein. Although single amino acid substitutions (which result in a 

different linear epitope) have been reported to alter epitope spreading resulting in 

increased immune response, the amino-acid substitutions in Pharming’s 

’’ A major histocompatibility complex molecule is defined to mean a heterodmeric membrane protein 

encoded in the major histocompahbility complex (MHC) locus that serves as a peptide display molecule for 

recognition by T lymphocytes Two structurally distinct types of MHC molecules exist. Class I MHC 

molecules are present on nucleated cells, bind peptides derived fiom cytosolic proteins, and are 

recognized by CD8’ T cells. Class I1 MHC molecules are restricted largely to professional antigenpresenting cells, macrophages, and B lymphocytes, and bind peptides derived fiom endocytosed protems, 

and are recognized by CD4’ T cells. (Abbas, 2006). 

32 
lactoferrin exactly mirror those in the various forms of endogenous lactoferrin in 

the general population. Thus, no "recognizable" difference results and no 

epitope spreading or disruption of oral tolerance would be expected. 

Any discussion of glycosylation - with regard to determinant spreading - 

is irrelevant to linear peptide fragments which are the only entity which 

determines T cell response and, thus, T cell tolerance. Moreover, neither 

Pharming nor its experts are aware of any evidence showing that a mere 

difference in glycosylation would alter epitope spreading or that oral tolerance 

can be disrupted by the introduction of a differently glycosylated version of the 

same, native protein. 

Therefore, while it is true that polymorphisms present in Pharming's 

lactoferrin can differ from those in the endogenous lactoferrin for a given 

individual, such naturally-occurring, amino acid substitutions - which fall within 

the range of variation that can be found in a normal population - are considered 

not to be immunogenic and, therefore, of little or any risk. Moreover, since T 

cells recognize only linear peptide epitopes, the concern about the effect, if any, 

of glycosylation is likely to be irrelevant to the discussion of T cell tolerance. 

(3.) Enhanced pro-inflammatory Thl 

response 

As indicated in Pharming's GN, there is already a fairly sizeable 

endogenous lactoferrin production that occurs in humans as a result of human 

lactoferrin being produced in salivary glands and in intestinal mucosa (and 

elsewhere). Therefore, ingestion of Pharming's human lactoferrin would simply 

supplement an already existing endogenous protein. Humans are already 

tolerant to human lactoferrin and bovine lactoferrin and once mucosal tolerance 

is established, it is quite difficult to "break" it. For example, a recent study looking 

at chronic ingestion of foreign proteins by humans (Zivny, 2001) showed that the 

major response to chronic antigen feeding is T cell anergy (the major mechanism 

33 
kr of tolerance to chronic antigen feeding) even though there are low titers of 

antibodies to dietary proteins present in secretions and serum, such as 

ovalbumin, bovine gammaglobulin and soy proteins. These anergic, antigenspecific T cells actively contribute to maintenance of homeostasis in the intestine 

in the face of massive antigen challenge. (Zivny, 2001). This is why significant 

consumption of bovine lactoferrin does not result in any breakage of tolerance to 

bLF and why the same significant consumption of Pharming’s lactoferrin will not 

disrupt any tolerance to endogenous lactoferrin. Indeed, one would expect 

Pharrning’s lactoferrin to be even less imrnunostimulatory and more tolerogenic 

than bovine lactoferrin. 

Finally, Pharming and its experts conclude that it is very unlikely that 

consumption of Pharming’s lactoferrin would result in perturbation of intestinal 

barrier function. (Dickenson, 1998). 

(4.) Increased uptake by antigen-presenting 

cells via the mannose receptor 

Although it may be theoretically possible that the differences in 

glycosylation between Pharming’s lactoferrin and endogenous lactoferrin could 

result in increased lactoferrin uptake by an antigen presenting cell (APC) via 

mannose receptors in such a manner that the Thl response is potentiated, 

Pharming is not aware of any direct evidence to support this. On the contrary, 

uptake by a mannose receptor appears to lead to an anti-inflammatory response, 

rather than a Thl response. (Chieppa, 2003). Furthermore, the mannose-type 

glycans attached to the backbone of Pharming’s lactoferrin are also attached - 

but in even greater number - to the backbone of bovine lactoferrin, which is 

deamed GRAS by CFSAN and is not reported to give rise to harmful, Thl 

responses. Finally, Pharming is not aware of any direct evidence demonstrating 

that differential glycosylation alters antigen uptake and potentiates immune 

reactivity for native proteins. In conclusion, the risk of disruption of previous 

34 
Cbr tolerance to endogenous lactoferrin via any increased uptake of Pharming’s 

lactoferrin by APCs via the mannose receptor is considered remote. 

(5.) Conclusion 

5. Subarea 3 (concerning exposure to exogenous hLF) 

Describe the daily, long term, Le., greater than 90 days, oral exposure that 

a human has to exogenous human lactoferrin. 

6. Phaming Update 

a. Introduction 

Perhaps obvious to anyone reasonably informed is the fact that an infant? 

has considerable, long-term, oral exposure to exogenous29 human lactoferrin 

during the approximately 12 months3’ that an infant is consuming - on a daily 

basis - significant quantities of human milk Perhaps not so obvious is the fact 

that a human in utero also experiences such exposure over a period of 

approximately eight months or that significant numbers of children, adolescents, 

or adults may also have similar exposures. Information pertinent to all of these 

exogenous hLF exposures is discussed below. 

For purposes of this updating document, the term “infant” is used to mean a young child from birth to 12 28 

months of age. (See, e.&, Lane, 2005). 

29 This response deals only with information concerning long term, oral, human exposure to “exogenous” 

human lactoferrin, it does not address such exposures to “endogenous” human lactoferrin. Such latter 

exposures are addressed in the next response ’’ The Amencan Academy of Pediatrics -the US entity recognized as the most qualified to establish gold 

standard medical practices pertinent to infants - recommends that infants be fed human milk for 12 months. 

(See, e.g., AAP, 2007). 

35 
h;." b. Fetal exposure 

Fetal exposure to exogenous hLF occurs throughout the entire pregnancy, 

and results from several different exposure scenarios. The first kind of exposure 

to exogenous hLF begins very early in utero - via diffusion - and results from 

embryonic invasion of and contact with the maternal decidua (Le., the uterine 

lining). (King, 2003). At this stage of development, there is no alimentary canal 

or circulatory system, thus what internal exposure to hLF later occurs in the 

formed alimentary canal cannot occur at this stage; nevertheless, diffusion at this 

early stage results in the same kind of internal exposure to hLF as later occurs 

via real oral exposure as a result of the alimentary canal being then in place. 

During such early exposure via diffusion, human LF ranges from being 

significantly present early on, to increasing during the first trimester, to upwards 

of 95ug hLF/g of decidual tissue at term. (King, 2003; Niemela, 1989). The 

second kind of exposure to exogenous hLF results from the formation of the 

umbilical cord. Via this feeding (oral-like) mechanism the fetus is exposed -also 

via diffusion -to a continuous bathing of tissues by a maternal plasma containing 

approximately 200 ng hLF per ml of plasma. (Pacora, 2000). Finally, the last 

type of exposure occurs via fetal immersion in and ingestion of hLF in amniotic 

fluid. (Jauniaux, 2000). 

With regard to hLF exposure from amniotic fluid, please recall that during 

the third week of pregnancy the amniotic cavity forms and surrounds the embryo. 

(See Fig. 1 for details). 

36 
Figure 1 

k 

Diagram showing the different anatomical barriers inside the fxst himester gestational sacs. U = uterine; P 

=placenta; UC = umbilical cord; ECC = exocoelomic cavity; SYS = secondary yolk sac; AC = amniotic 

cavity; AM = amniotic membrane. (As appears in Jaumanx, 2000). 

By the IOth week of gestation the amniotic cavity contains approximately 30 ml of 

amniotic fluid and peaks to as much as 1 liter after 34-37 weeks. (Sadler, 2000). 

During the first trimester, hLF measures c 2 nglml of amniotic fluid. (Jauniaux, 

2000). After 20 weeks, the level of hLF significantly rises (to a mean of 3000 

nglml amniotic fluid) and remains high until term. (Jauniaux, 2000; Pacora, 2000; 

Niemaela, 1989, Figure 2). 

37 
Figure 2 

IO@. 

Em- 

- 

2 5wo- - E 

Iwoe 

”r- 

:r 

.. 

..t.: 

Table 1 

Fetal Exposure to hLF in Bathing Amniotic Fluid 

Age (weeks) Total AF (ml) ng hLF/ml AF mg hLFkdd” 

20 350 800 0.45 6.22 

38-40 900 3000 3.35 8.96 

Amniotic Fluid volumes fiom (Sadler.2000) 

Concentrations of hLF in AF estunated fiom (Pacora, 2000) 

Fetal weights fiom @. 492 Appendix 2 and Fig. 8.1 p. 91 in O’Rahilly, 2001) 

Continuity of amniotic fluid with the intestinal lumen occurs at three weeks 

gestation when the buccophatyngeal membranes ruptures forming the future 

mouth. (Weaver, 1991). By 7 to 8 weeks, the cloacal membrane ruptures, 

forming the future anus. (Weaver, 1991). From 11 weeks gestation, effective 

fetal swallowing is developed and is continuously recycling the amniotic fluid 

through the gastrointestinal tract into the amniotic cavity and back through the 

primordial mouth. (Weaver, 1991). By 12 weeks, the ability to absorb is present 

This figure equals an average case scenario of maximum exposure. 31 

38 
(Milla, 1991), including intact proteins and lipids. (Weaver, 1991). After this time, 

the amount of hLF ingested in amniotic fluid progressively increases until birth. 

(See, Table 2 below.) 

Table 2 

Fetal Ingestion of hLF in Amniotic Fluid (“AF”) 

Gestational Ape WKS) 

20 38-40 

AF PO ml/day 13-16 450 

Ng hLFlml AF 800 3000 

wt (kg) 0.450 3.350 

Tot. hLF nglday 11,600 1,350,000 

Mg hLF/day .0116 1.35 

Mg hLF/k BW/day 0.03 0.40 

Amniotic Fluid volumes fiom (Sadler.2000) 

Concentrations of hLF in AF fiom (Pacora, 2000) 

Fetal weights fiom @. 492 Append= 2 and Fig. 8.1 p. 91 in O’Rahilly, 2001) 

Amniotic Fluid swallowed fiom (pritchard, 1966) 

There are no documented adverse consequences of fetal exposure to 

increasing levels of hLF throughout pregnancy. In fact, necessary benefit is 

implied by increased hLF when the integrity of natural barriers are threatened or 

breached (e.g., during villitis, intrauterine infection, invasion of the embryo into 

the decidua) and via the constant presence of hLF in the external environment 

(i.e., in the decidua, amniotic fluid, vernix caseosa, and maternal cervical mucus) 

39 
and ingested environment (Le., in the amniotic fluid, vernix caseosa, colostrum, 

and breast milk) of humans as they transition from fetus to ne~born~~~~~~~~.~

c. Infant exposure 

During the first twelve months of life, an infant encounters significant, longterm, oral exposure to exogenous human lactoferrin via one or more of three 

different, exposure scenarios. Each is discussed below. 

(I.) Via human milk from one’s mother. 

The typical daily exposure of an infant - over the first 12 months of life -to 

hLF via the consumption of human milk - especially that of an infant‘s own 

mother - is dependent on three factors, Le., the amount of human milk 

consumed per day, the amount of hLF in such daily-consumed human milk, and 

the weight of the infant at the time such milk is being consumed 

The concentration of hLF in human milk varies very significantly during the 

human period of lactation. (Nagasawa, 1972; Lonnerdal, 1976; Mathur, 1990; 

Nuijens, 1996). During the first few days postpartum -when human colostrum is 

being expressed - hLF concentration may approach 10 mg/ml by day two and 

then taper off by day seven to about 5 mglml (Nuijens, 1996, Davidson, 1987). 

Please note that amniotic fluid sampled before 20 weeks gestation supports the growth of bacteria (e g., 

E coli) but remains consistently bacteriostatic or bacteriocidal after 36 weeks gestation coinciding with the 

rise in hLF in the thiid trimester. pacora, 2000; Schlievert, 1975). 

33 There is increased immunohistochemical staining for hLF with chronic mflammation of placental villi 

(“haler, 1993). 

In a study of lactoferrin in amniotic fluid and suppression of IL-6 production in intrauterine infechon, 

Otsuki, 1999 suggests that lactofenin could be useful in the treatment of chorioamnionitis. 

35 Vernix caseosa is a complex proteolipid unique to humans. In utero, the vernix caseosa detaches fiom 

fetal skin and is ingested with the amniotic fluid At birth, it covers the infant and is similar to colostrum 

and breast milk in derivation. Akiobi, 2004 demonstrated hLF m vernix caseosa by immunostaining and 

Western analysis. The authors conclude, “The third trimester fetus subsequently swallows a complex 

mixture of detached vernix, pulmonary surfactant, and amniotic fluid long before the forgut encounters 

breast milk. There is, therefore, considerable functional and structural synergism between the prenatal 

biology of the vernix caseosa and postnatal biology of breast milk.’’ 

32 

34 

40 
Thereafter, the concentration continues to decline during mid lactation, eventually 

persisting at about 2 mglml in mature human milk. (Schanbacher, 1993). 

For purposes of determining the maximum, natural exposure to hLF 

during infancy (the time during an entire human life of highest, natural exposure 

to hLF), let one assume the worst (or the highest exposure), i e., that the 

concentration of hLF reaches 10 mghl in colostrum during the first few days of 

infancy. At this point, an average36 infant's weight would be approximately seven 

pounds (or 3.25 kilograms, assuming 1 kilogram per 2.2 pounds). (Lane, 2005; 

US. EPA, 2002). Later on, when hLF concentration drops to 2 mglml, the 

infant's weight would be between approximately 16 pounds at six months and 21 

pounds at 12 months (or 7 53-9.84 kilograms, assuming 1 kilogram per 2.2 

pounds). (Lane, 2005; U.S. EPA, 2002). At the 90th percentile3', an infant would 

consume an average of 1000 ml per day during early infancy (months 1 and 2) 

and mid infancy (months 3-6). (U.S. EPA, 2002). As Table 3 below shows, this 

consumption could equate to a typical daily exposure (over the first 6 months of 

an infant's life) of 2 266 mg hLF per kilogram of infant body weight and (over the 

first year of an infant's life) of from 48-3077 mg hLF per kilogram of infant body 

weight3'. 

Table 3 

Daily Infant Exposure to hLF 

Total hLF 

Consumed Total hLF 

Potentially Per Day Per Human Amt.Of 

hLF In 

Weight Consump. Human Consumed Kilogram Of 

(Per day) Milk Per Day Body Weight 

3077 mglkg 

BW 

266 mglkg 

BW 

Milk 

Age 

Ibs. I 000 mi IO mglml I 0,000 mg 

2 mglml 2,000 mg 

Newborn (3.25 k) 

Six 16.57 Ibs. looo 

months (7.53 k) 

36 Average weight is used here (instead of the 90* percentile weight) in order to establish worst case, i.e , reasonably highest exposure per kilogram of infant body weight. 

37 Consumption at the 90* percentile is used here in order to establish worst case, Le., reasonably highest 

exposure per kilogram of infant body weight. 

formula. (GN 000042/77, page 255). 

Even for mfants not consuming human milk, they consume ffom 105-245 mg bLF/day from infant 

41 
Twelve 21 Ibs. 

months (9.5 k) 

Importantly, the 

incorporates 21 single 

500 ml 2 mglml 1,000 mg 48 mglkg BW 

gene for human lactoferrin in the normal population 

nucleotide polymorphisms - nine of which generate a 

variant, but normal, amino acid. (Teng, 2006). The frequency of these normal 

variants can range from 1-43 percent. (Teng, 2006). Thus, practically speaking, 

given the large number of infants at any one time in the US population, most 

infants are exposed long-term to a variety of exogenous human lactoferrin that is 

different from the endogenous hLF being produced by any one of such infants. 

Finally, there are approximately 4.2 million infants born each year in the 

United States (NCHS, 2004), of which approximately forty percent are still being 

breast fed at six months. (CDC, 2004). Thus, each year approximately 1.68 

million infants are involved in the above-discussed, six-month (long-term) 

consumption pattern pertinent to exogenous hLF. 

k 

(2.) Via human milk from a female other than one’s 

mother 

There are numerous instances3’ in which an infant‘s mother cannot 

provide - for various potential reasons - an adequate supply of human milk. In 

such instances, substitute human milk can often be obtained either from a “wet 

nurse’” or from a commercial source (such as the not-for-profit “Human Milk 

Bank Association of North America” (HMBANA)41 or the for-profit “Prolacta”). 

When such alternative sources of human milk are resorted to, infants 

consume such milk at the same rate and for the same, long-term period as 

described (in subpart 1) above. Such consumption will often expose an infant to 

39 Such instances are estimated to be 2 10 percent. (HMBANA, 2005). 

to, at least, the time of the Code of Hammurabi, i.e., 2250 BC. 

4’ HMBANA consists of ten North American sites that collect, process and distribute human milk. It will 

distribute approxmately 900,000 ounces in 2007. (HMBANA, 2007). 

A wet nurse is a woman who breastfeeds a non-related infant or adult. This common practice dates back 

42 
an exogenous form of hLF that differs from the form of endogenous hLF being 

produced by the infant.42 

(3.) Via clinical applications 

The majority of banked human milk is used for clinical applications in 

infants. (See, e.g , HMBANA, 2007). The most common diagnosed conditions 

which banked human milk is used to treat include: premature births, allergies, 

respiratory disorders, Gllbowel problems, and jaundice. (E.g., see HMBANA, 

2005) When such milk is used, it is consumed at daily rates and for long periods 

of time like those set forth in subpart I above. As noted above, such 

consumption often exposes an infant to several exogenous forms of hLF that 

differ from the form of endogenous hLF being produced by the infant. 

Table 4 below sets forth some of the published studies pertinent to longterm exposure to exogenous hLF by infants. 

Table 4 

Clinical Long-term Exposure To Exogenous hLF By Infants 

Studv Age Indication Duration Total ml DBM ma hLF mu hLFlku BWld 

(Arnold. 1995) 6wk- GER 11.5m. 175-350.000 350-700,000 210-307 

IZmos Seizures 

P n e u m o n I a 

In utero drugs 

(Asquith, 1987) 

Term infants 9-27 wk Food allergy, FTT 4 5 m. 67,000 134,000 132-306 

3-6 m Maternal illness 3 m 55,000 110,000 162-376 

3-6 m ALL 3m 45,000 90,000 133-308 

d. Adult exposure 

There is also considerable human experience with regard to long-term 

consumption of hLF beyond infancy. For example, suckling beyond infancy 

42 Since HMBANA pools its sources of human milk (up to a maximum of20 donors per 300 ml of human 

milk), infants receiving human milk 60m HMBANA would he exposed to multiple versions of hLF. 

(Asquith, 1987; HMBANA, 2007). 

43 
".*, 

commonly occurs throughout the world, depending on cultural norms and 

available resources. (Wickles, 1953; Ford, 1945 (as cited in 

www.qeocities.com/HotSprings/Spa/3156/history. htm?200712); Ploss, 1935); 

thus, for centuries, hLF has been ingested by non-infants via milk from wet 

nurses (Hill, 1987). Such consumption has only increased with the advent of 

banked donor milk since about 1910. (Wickles, 1953). In addition, provision of 

human breast milk to the very old or infirm has been practiced, by Asian cultures, 

for centuries. (Baumslag, 1987). Total numbers of non-infant individuals 

ingesting hLF (in human milk) are impossible to ascertain given the universal 

acceptance of human milk as a safe, familiar food and the informal use of wet 

nurses or banked milk by families. Indeed, such practices are so commonplace 

that governmental entities do not keep any count of them. 

The significant increase in milk banking (Sakamoto, personal 

communication, 2007; HMBANA website, 2007) and the more recent emergence 

of recombinant hLF (rhLF) and the ever-increasing awareness among experts of 

the safety and significant nutritional/clinical utility of hLF has significantly 

extended the nutritionallclinical use of hLF in human milk to treat a wide range of 

pathology in humans from premature birth through adulthood.43 Such 

clinicallnutritional applications include, but are not limited to: feeding problems, 

food intolerance, food allergies, prematurity, chronic diarrhea, Hirschsprung's 

43 The majority of banked human milk is donated for the feeding of sick premature infants (Shoto, 

personal communication July 3,2007 who provided a copy of the HMBANA 2005 Diagnoses Chart 

prepared by Linda Gonzales San Jose Mothers' Milk Bank). Prioratiiing banked milk distribution ranks 

recipients i?om most to least critical in the following order; 1) Premature infants, sick; 2) Premature infants, 

well, ; 3) Infants < 12 months with medical conditions likely to respond to donor milk therapy; 4) 

Individuals > 12 months old, likely to respond to donor milk therapy; 5) Research contracts for clinical use 

in well-designed studies; 6) Individuals > 12 months with chronic medical conditions, high normal 

functioning and low dose need; 7) Individuals > 12 months with chronic medical conditions, high normal 

functioning and high dose need; 8) Individuals > 12 months with chronic medical conditions, low level 

functioning, and low dose need; 9) Individuals > 12 months with chronic medical conditions, low level 

function, and high dose need; 10) Infants for short -term use, no specific medical condition, and 11) 

Laboratory reserch (milk that cannot be used for human consumption due to drugs used by donor, lack of 

complete testing of the donor, or age of the milk. (Tully, 2002). Some Umted States nurseries, including 

those at the University of Kentucky and at the Wilmigton Delaware Medical Center have even used 

banked human milk as a standard initial feed for premature infants (Arnold, 1990). The practice is more 

routine in the U.K., Australia and Sweden, hut published studies only cite exposures up to six weeks and, 

therefore, were not included in the discussion of chronic (2 90 days exposure). (Boyd, 2007; Arnold, 2002; 

Lucas,1990). 

44 
disease, congenital anomalies of the mouth or gastrointestinal tract, necrotizing 

enterocolitis, short gut syndrome, immune deficiency such as IgA deficiency, 

chromosomal anomalies, in utero druglalcohol exposure, chronic renal failure, 

dialysis, postoperative cardiac/gastrointestinal/cleft liplpalate repair, irritable 

bowel syndrome, inborn errors of metabolism, cancers, candidiasis, graft versus 

host disease, liver transplant, Hepatitis C, chemotherapy induced mucositis, 

esophagitis, gastrointestinal ulceration, infectious diarrhea, inflammatory bowel 

disease, the elderly, sepsis, pediatric burn cases, and bronchopulmonary 

dysplesia. (Hayes, 2007; Lactation Education Resources Website www.leron1inelMilkBankinq.htm 6/22/07; Williams, 2007; Boyd, 2007; Rehmeyer, 2006; 

Wang, 2006; Zimecki, 2005, Tully, 2004; Updegrove, 2004; Troost, 2003; Arnold, 

2002; Playford, 2000; Morley, 2000; Schanler, 1999; Wiggins, 1998; Merhav, 

1995; Anderson, 1993; Lucas, 1990; Trumpler, 1989; Asquith, 1987; and 

Rangecroft, 1978). For a summary of some of the nutritionallclinical, significant 

and long-term applications of human milk see Table 5 Recombinant forms of 

human lactoferrin are also currently in clinical trialM. Published clinical trials so 

far demonstrate only tolerance and safety of rhLF even when administered to the 

most vulnerable human populations and even following long-term exposure to 

significant doses. Some examples of clinical applications and significant, longterm exposures are discussed below. 

Table 5 

Clinical Long-term Exposure To Exogenous hLF By Adults 

Study Aae Indication Duration Total ml OBM ma hLF total mq hLFk BWId 

CTullv. 

. 2004) 618m Fn 12m 247.320 494.640 143 I. 

GER 

Malrotation 

GI surgery 

Food intolerance 

(Arnold, 1995) 6-30m FTT 24 m. 

Food intolerance 

FP solid food 

(Tully, 1990) 4 y IgA deficiency 23 m 84,000 168,000 213 3 

Food allergies 

As of August 25,2005, rhLF has been administered to over 500 people and appears safe and well 44 

tolerated as reported on the Agennix website, www.agennix.com. 

45 
(Arnold, 1990) School Acrodermatitis 72 m 394,200 788.400 8 8-17 1 

age enteropathica 

ALL = acute lymphocytic leukemia; FP = feeding problem; F’TT =failure to thrive; GER = gastroesophageal reflux; 

DBM = donated banked milk 

None of the above case histories report adverse events secondary to prolonged use of donor human mik, 

but in some instances, did indicate weight loss if tapered too fast from donor human milk. (Arnold, 1995). 

A recent study at Baylor specifically examined the safety of oral 

recombinant hLF (rhLF) administered to 10 adult patients with metastatic, 

refractoly cancer. (Hayes, 2006). Recombinant hLF was administered 14 days 

continuously alternating with 14 days off up to a maximum total of 105 days of 

rhLF consumption. Recombinant hLF was well-tolerated without evidence of 

hematological, hepatic, or renal toxicity. The most common symptom was mild 

diarrhea controlled by over the counter drugs which affected only 2 patients (one 

patient taking 1500 mg/day, one patient taking 9000 mglday). One patient taking 

4500 mg rhLF/day experienced 4-6 loose stools per day. There was no 

correlation between dose of rhLF and severity of diarrhea and any other 

complaint. Other constitutional symptoms noted are those commonly found in 

cancer patients (weakness, nausea, fatigue, vomiting, constipation, nasal 

congestion, taste perception with only patient complaining per symptom). The 

range of total doses of rhLF received over the longest study period of 105 days 

appears in Table 6. 

Table 6 

Clinical Long-term Exposure To Exogenous hLF By Adults 

Study Age # davs me rhLFid me rhLFk BW/d 

(Hayes,2006) median 

63 yr 

105 1500 mg 25 

105 4500 mg 75 

105 9000 mg 150 

46 
Oral admlnistration of rhLF did not increase serum rhLF after 1 dose or following 14 consecutive days of 

dosing. Biologic activity of the rhLF was established by demonstrating an increase in serum and gut IL-IS. 

A randomized, multi-center, double-blind, placebo-controlled study of 110 

chemo-naive patients with advancedlmetastatic, non-small cell, lung cancer was 

presented at the 2006 meeting of the American Society for Clinical Oncology. 

(Wang, 2006). Half of the patients received chemotherapy 

(carboplatinlpaclitaxel) and oral rhLF for 1-3 cycles (35 days per cycle). The 

other 55 patients received chemotherapy alone. Treatment duration and total 

dose of rhLF appear in Table 7 

Table 7 

Studv # daw me rhLF/d rn, rhLFik BW/d 

(Wang, 2006) 35 3000 50 

70 3000 50 

105 3000 50 

Oral rhLF was well-tolerated with no drug-related adverse effects. 

Patients with renal cell carcinoma have been treated for 14 days on 

4500mg twice daily alternating with 14 days off for a maximum of 34 months. 

(Hayes, 2007). This schedule resulted in 476 days of recombinant hLF exposure 

at 9000mg/day, for a total exposure of 4,284,000 mg per patient and a daily 

exposure of 150 mg rhLFlk BW. The same group of investigators recently 

changed to continuous daily rhLf, 1500 mg twice daily to treat refractory patients 

with head and neck cancer. Thus far, the longest exposure is 4 months (120 

days) at 3000 mg rhLF per day (or 50 mg rhLF/k BW), for a total exposure of 

360,000mg per patient. (Hayes, personal communication, 2007). Most patients 

experienced no adverse events, but those who did had only minimal symptoms, 

including occasional mild diarrhea or nasal stuffiness which may or may not be 

related to rhLF. 

47 
e. Conclusion 

".. '' 

As the foregoing information clearly indicates, it is definitely not the case 

that humans are not orally exposed long-term to exogenous hLF. Rather, longterm, oral exposure to exogenous hLF represents the norm. Indeed, such oral 

exposure begins very early in utero, continues to expand during the entire 

pregnancy (to hLF levels approximating 9 mg hLF/kg BW/day), continues to 

further expand during the first 12 months of life (to hLF levels of from 48-3077 mg 

hLF/kg BW/day) and may continue after infancy - from time to time - in large 

numbers of individuals for long-term periods -to include long-term exposures of 

from 9-150 mg hLFlkg BW/d, all of which exposures are deemed by qualified 

experts to be normal and safe. The above-repotted clinical studies only further 

support this conclusion. 

Moreover, such exposures almost universally expose the consuming 

individual to a form or forms of hLF that differ from hidher own. Again, all in 

normal and safe manner. 

Finally, many such long-term, oral exposures are to individuals who are 

members of the most vulnerable populations (including the premature, the very 

old, infants, those with impaired immunity and/or impaired mucosal barriers 

secondary to use of drugs, or disease (including cancer, overwhelming infection 

or autoimmune disease), or inflammation), yet such exposures do not cause 

deleterious effects and are, in fact, broadly deemed safe and beneficial. And, 

most importantly, rather than such exposures causing any adverse, 

immunological effects, the actual effects observed and recorded represent only 

beneficial or positive irnrn~no-effects~~ (even in the most vulnerable) such as 

decreased incidence hospitalization duration, less infections, decreased 

necrotizing enterocolitis, and increased growth 

45 For a comprehensive discussion of these effects, see pages 18-21 supra and Zimecki, 2007; -el, 2007; 

and Fischer, 2006. 

48 
hc 

k.r 

7. Subarea 4 (concerning oral exposure to endogenous hLF) 

Describe the daily, long term, oral exposure that an adult human has to 

endogenous human lactoferrin resulting from, for example, saliva or other 

gastrointestinal secretions. 

8. Pharming Update 

a. Introduction 

Endogenous human lactoferrin is naturally, continuously, and significantly 

present ubiquitously in human tissues throughout the entire human body 

throughout life. (Attachment 1, page 26; Nuijens, 1996). This discussion, 

however, focuses only on that portion of the above-referenced endogenous 

human lactoferrin that results from saliva and other gastrointestinal secretions 

and, thus, is the result of oral exposure. 

b. Exposure via the oral route 

Within a twenty-four hour period, significant quantities of endogenous 

human lactoferrin are secreted into the alimentary canal from one or more of 

several sources. 

Perhaps the most obvious source of hLF is via the ingestion of saliva,46 

which begins to be produced (with hLF) in the fetus from 20 weeks gestation on. 

(Reitamo, 1981). In the adult oral cavity, saliva is produced under two, different, 

flow rate conditions, Le., under unstimulated and stimulated conditions; each 

condition results in a different amount of saliva entering the alimentary canal. 

(Marino, 2003). The total daily amount of saliva introduced from both conditions 

46 Saliva is the clear, alkaline, somewhat viscid secretion secreted by the parotid, sublmgual, submaxillary, 

and smaller mucus glands of the human mouth. It serves to moisten and soften the food, keep the mouth 

moist, and contains, among many other substances, lactofemin. @orlands, 2003; Troost, 2002; Nuijens, 

1996). 

49 
is approximately 1500 milliliters per day (Yamada, 2003). Both flow-rates have 

been measured. Unstimulated saliva flows at a rate of approximately 0.5 mllmin. 

(Tanida, 2003). This basal rate typically produces approximately 720 ml saliva 

per day and contains from 1947 micrograms of hLF per milliliter of saliva. 

(Tanida, 2003). Accordingly, approximately 14-34 milligrams (or more) of hLF 

could be introduced into the alimentary canal per day via production of 

unstimulated saliva Stimulated saliva flows at a rate of 1.5 to 2.4 mllmin. (Lin, 

2001). This results in production of from 2.16-3 46 liters of saliva per day. Such 

saliva contains from 3.4 to 7.1 micrograms of hLF per ml of saliva. (Lin, 2001). 

Accordingly, from 2.4 to 5.1 milligrams of hLF could be ingested each day from 

stimulated saliva. Thus, one could ingest up to 39 mg endogenous hLF per day 

from oral consumption of saliva. 

Distal to the tongue, the next source of endogenous hLF in the alimentary 

canal is the stomach4'. In the fetal stomach, endogenous hLF first appears in 

mononuclear cells, presumably granulocytes, after 16 weeks gestation. 

(Reitamo, 1981). In the adult stomach, immunocytochemistry demonstrates 

lactoferrin expression in the gastric glands in the cardia (the region where the 

esophagus inserts into the stomach), and in the body (the largest region of the 

stomach between the lesser and greater curvatures) and in the antrum (that 

portion of the stomach between the body and the pylorus). (Luqmani, 1991). 

Within gastric glands, chief (peptic) cells in particular, strongly stain for hLF. 

(Luqmani, 1991). Chief cells are protein-secreting exocrine cells structurally 

similar to pancreatic acinar cells. (Weaver, 1991). Within the stomach, hLF may 

also be derived from neutrophils. In the absence of neutrophils, I e., when hLF is 

coming only from gastric glands, hLF measures approximately 0.12 

nglmicrogram of antral mucosa tissue protein and 0.08 nglmicrogram of body 

mucosa tissue protein (Nakao, 1997). With mild neutrophil infiltration of the 

mucosa, hLF measures 0.33 nglmicrogram in the antral mucosa tissue protein 

and 0.26 nglmicrogram in the body mucosal tissue protein. (Nakao, 1997). In 

47 Please note that the tissues comprising the esophagus do not secrete any hLF into the alimentary canal. 

(Luqmani, 1991) 

50 
instances of significant neutrophil infiltration, e.g , during inflammation 

accompanying H. pylori infection, the foregoing hLF levels significantly increase 

to 2.7 ng/microgram of antral mucosal tissue protein and 2.61 nglmicrogram of 

body mucosal tissue protein. (Nakao, 1997). There exists approximately 6400 

milligrams of total protein in the gastric mucosa of the entire stomach. 

(Yamanaka, 1974). Of this amount, approximately eighty percent (Le., 5120 

milligrams) is associated with the body and antrum of the stomach, and 

approximately 75 percent (or 3840 milligrams) of that amount is from the antrum. 

All of this information - in the aggregate - indicates that the stomach provides (in 

non-pathogenic situations) - at a minimum - approximately ,4545 mg of hLF at 

any one time. No data is available indicating the maximum amount of hLF 

provided in any 24-hour period from the gastric mucosa; however, it is 

reasonable to assume that such 24-hour figure is multiple times the 4.5 mg cited. 

In addition, hLF is also present in the mucus layer which coats the gastric 

mucosa. No information is available on how much hLF resides within such 

mucus layer. (Clamp, 1986) 

Within the duodenal lumen, endogenous hLF originates from the 

duodenum (i e., the first approximately 10 inches of the small intestine), the liver 

and the pancreas. The duodenum is the first portion of the small intestine 

connecting the pyloric stomach proximally and intestinal jejunum distally. Biliary 

and pancreatic ducts empty into the duodenum. (Weaver, 1991). Fetal 

expression of hLF occurs after 13 weeks gestation in the liver and small intestine, 

and after 20 weeks gestation in the pancreas. (Reitamo, 1981) In health, 

endogenous hLF immunolocalizes to enterocytes at the villus tips of the 

duodenum and jejunum (Tedeschi, 1987). In diseases such as celiac disease 

and postenteritis syndrome, endogenous hLF is increased in villus tips and 

crypts. (Tedeschi, 1987). During acute cholera, for example, gene expression for 

endogenous hLF in the duodenum is increased and has been interpreted as 

enhanced innate defense during acute infection. (Flach, 2007). 

With regard to the hLF contribution from the liver which appears in bile 

(which originates in the liver, is stored and concentrated in the gall bladder, and 

51 
is secreted into the duodenum), basal hLF levels in bile have been measured in 

studies of inflammatory bowel disease where control subjects were postcolectomy patients cured of ulcerative colitis. Here, the mean hLF concentration 

was 1.2 mglL. In active ulcerative colitis, hLF concentration was 2.8 mglL. 

Crohn’s disease patients in remission were also considered a control population; 

in these patients, hLF measured 1.1 mglL. (Pereira, 1998). In active Crohn’s 

disease, three fold higher concentrations of biliary hLF occur. (Pereira, 1998) 

Given that approximately 500 ml of bile is secreted daily (Yamada, 2003)., 

production of biliary hLF (in health) approximates 0.6 mg per day and (in 

inflammatory bowel disease) 3.1 mg per 24 hours. 

In the pancreas, hLF immunolocalizes to the acinar cells bordering the 

lumen and are contained within zymogen granules and endoplasmic reticulum. 

(Lechene de la Porte, 1981; Colomb, 1976). Pancreatic hLF secretion is 

stimulated by a meal or administration of cholecystokinin alone or in combination 

with secretin, but not by secretin alone and parallels amylase secretion. 

(Hegnhoj, 1986). Basal pancreatic secretions obtained at the ligament of Treitz 

measured pancreatic hLF in healthy volunteers at 0.7 microgramslml of 

pancreatic secretions. (Brugge, 1988) Given that the pancreas secretes 

approximately 1500 mllday, the pancreas produces 1050 micrograms (or 1.1 mg) 

hLF per day. (Yamada, 2003). 

Endogenous hLF throughout the small intestine has been measured in 

whole gut lavage fluid (Le., WGLF, a nonabsorbable liquid preparation routinely 

given to subjects to cleanse the bowel prior to colonoscopy or colorectal surgery 

or used for research purposes to measure ongoing production of intestinal 

proteins). (Kayazawa, 2002). “The rate of fluid passage along the gut during 

lavage is approximately 1 Uh (0.017 Umin), so the estimated daily loss (mg/day) 

of a certain substance can be obtained from the following equation: 24 (L) x the 

concentration in WGLF (mglL).” (Kayazawa, 2002). By this formula, daily hLF 

production was calculated to be approximately 21.5 mglday (24 x 1000 x 0.89) 

with a > 40 fold increase in active ulcerative colitis (Le., 890 mg/day) and a > 30 

fold difference in active Crohn’s disease (i.e., 686 mglday). 

52 
Estimates of colonic hLF are available from determinations of fecal hLF. 

Uchida et al. measured hLF using an enzyme - linked immunosorbent assay 

(ELISA). (Uchida, 1994). They found the fecal concentration of hLF to be 

approximately 0.75 microgram hLF/g feces. (Uchida, 1994) Since humans 

excrete approximately 200 g of feces per day, that would yield about 150 

micrograms hLF/ day in feces (most of which is produced in the colon). 

c. Conclusion 

The foregoing information indicates that on a daily basis humans are 

naturally and normally exposed - via the alimentary canal - to significant 

quantities of endogenous hLF emanating from various internal sources. This is 

not surprising since virtually all other tissues of the human body are also 

constantly exposed to varying levels of endogenous hLF. More specifically, 

under non-disease (Le., normal) conditions, during a twenty-four hour period and 

day after day (for a lifetime) one can be exposed to (conservatively estimated) up 

to 87 milligrams of endogenous human lactoferrin in the alimentary canal. In 

some normal cases, such exposure can be even greater. Moreover, during times 

of disease, such exposure can - as indicated above - increase by 30-40 fold. 

Thus, in certain instances, daily exposure to endogenous lactoferrin can 

significantly exceed 1 gram per day - indeed, possibly up to (again 

conservatively estimated) almost 3.5 grams. Most importantly, all such 

exposures are deemed - by the human body - to be natural, non-harmful, and 

beneficial. 

9. Subarea 5 (concerning helpfulness of exposures to hLF) 

Can the known safety of the exposures to human lactoferrin referenced in 

subareas 3 and 4 above be used to help address the safety of any daily, long 

term, oral exposure of infants or adults resulting from oral consumption of 

53 
e-. 

transgenically-produced human lactoferrin? If yes, to what extent? If not, why 

not? 

IO. Pharming Update 

Given the updates set forth to subareas 3 and 4 above, it is clear that the 

long-term exogenous and endogenous oral exposures to hLF that are 

documented in the informational updates to subareas 3 and 4 can be used to 

help address the safety of Pharming’s human lactoferrin. As set forth in the 

updates to subareas 3 and 4, long-term, daily, oral exposure to exogenous 

human lactoferrin can approximate 48-3077 mg/kg BW in the first 12 months of 

life and 9-150 mglkg BW thereafter for long periods and long-term, daily, oral 

exposure to endogenous, human lactoferrin can approximate 1.45-58 mglkg 

BW. Together, these two types of oral exposures can approximate long-term, 

daily, oral exposures to hLF of (conservatively estimated) approximately 50-31 00 

mglkg BW during the first 12 months of life and approximately 10-200 mglkg BW 

during the rest of life after infancy - all of which the human body deems to be 

safe, non-harmful exposures Such daily, long-term, oral exposures far exceed 

any exposure that would result from consuming Pharming’s hLF, I e., 1.91 mglkg 

BWlday (goth percentile user). (See Pharming’s GRAS Notice, Attachment 1, 

pages 52-58). 

11. Subarea 6 (concerning safety criteria) 

Are the traditional, scientific criteria long-used by CFSAN to assess the 

long-term safety of substances to be added to food adequate to assess the long 

term exposure to such a substance when it may exhibit immunomodulatory 

properties? If so, explain why. If not, describe what other criteria are needed and 

why. 

* . 

54 
4aa 

12. Pharming Update 

a. Introduction 

From a practical point of view the foregoing subarea can be subdivided 

into two subsidiary subareas, Le., (1 .) Whether there currently exists scientific 

criteria pertinent to assessing the safety of substances to be added to food 

(including GFWS substances)? and (2.) Whether such criteria -to the extent they 

currently exist - are sufficient to assess the long-term exposure to such a 

substance when it may exhibit known immunomodulatory properties? These two 

subareas are addressed below. 

b. Scientific criteria 

Numerous scientific criteria exist today for assessing the safety of 

substances appearing in or that have been added to food. They have been 

developed over the last 101 years since passage of the Food and Drugs Act of 

1906. Following is a summary of those criteria and how they came to be 

1. 1906-1958 

Prior to 1958, the terms “food additive” and “GRAS substance” did not - 

from a legal point of view - formally exist, i.e., neither were separate categories 

under either the Food and Drugs Act of 1906 or the Food Drug and Cosmetic Act 

of 1938. Rather, such “added substances” - like all components of food -were 

regulated under the adulteration provisions of the law. In any particular instance, 

FDA paid close scrutiny to the individual safety characteristics of a given 

substance and then determined whether such characteristics amounted to a 

“poisonous” or “deleterious” substance. Safety characteristics were determined 

and evaluated - as they are today - via application of evolving and (at any given 

time) then applicable criteria, Le., analytical and testing methodologies. 

55 
2. The 1958 Food Additives Amendment 

1.“ 

Prior to 1958 numerous difficulties, uncertainties and problems arose 

when FDA attempted to regulate substances - of all kinds -to be added to food. 

(Such difficulties, etc. are best described at length in C. Dunn’s “Legislative 

Record of the 1958 Food Additives Amendment”, S Rep No 2422 and HR Rep 

No 2284, 851‘~ Cong 2d Sess (1958)). After extensive debate, Congress 

unanimously passed the 1958 Food Additives Amendment to the FD&C Act and 

same was signed into law on September 6, 1958. 

The Amendment created numerous legallscientific criteria pertinent to 

evaluating the safety of substances to be added to food. These included that: 

1. the term “food additive” was defined (see 21 USC § 321 (s)); 

2. the term “generally recognized as safe” was defined (see 21 USC § 

321(s)); 

3. both food additives and GRAS substances must be shown to be 

“safe” for each intended use (see 21 USC § 321(s)), 

4. substances to be added to or appearing in food can be found to be 

adulterated if not either prior approved or generally recognized as 

safe (see 21 USC § 342); 

5. substances to be added to or appearing in food can be unsafe if 

poisonous or deleterious and injurious to health, filthy, putrid, 

decomposed, or otherwise unfit for food (see 21 USC § 342); 

6. food additives and GRAS substances not used pursuant to the 

specific requirements of the pertinent regulation can be found to be 

adulterated (see 21 USC § 342); and 

7. no food additive may be approved (or remain approved) if FDA finds 

that such substance induces cancer in men or animals (see 21 USC 

§ 348(c)(WA)). 

These criteria form the initial core elements of evaluating safety and have been 

successfully applied now for approximately five decades. 

56 
3. Regulatory enactments 

In 1977 numerous regulatory criteria were promulgated to flesh out the 

prior-enacted core criteria for evaluating food additives and GRAS substances. 

These include that: 

1. GRAS substances can be found to be GRAS either via “experience 

based on common use in food” or “scientific procedures” (21 USC § 

321(s) and 21 CFR § 170.3(9 and (h)); 

2. the term “common use in food” means a substantial history of 

consumption of a substance for food use by a significant number of 

consumers (21 CFR § 170.3(9); 

3. the term “scientific procedures” is an evolving standard that means 

those human, animal, analytical, and other scientific studies, whether 

published or unpublished, appropriate to establish the safety of a 

substance (21 CFR § 170 3(h)); 

4. the term ”safe or safety” means that there is a reasonable certainty in 

the minds of competent scientists that the substance is not harmful 

under the intended conditions of use. It is impossible in the present 

state of scientific knowledge to establish with complete certainty the 

absolute harmlessness of the use of any substance. Safety may be 

determined by scientific procedures or by general recognition of safety. 

In determining safety, the following factors shall be considered: 

(1) The probable consumption of the substance and of any substance 

formed in or on food because of its use; 

(2) The cumulative effect of the substance in the diet, taking into 

account any chemically or pharmacologically related substance or 

substances in such diet; and 

(3) Safety factors which, in the opinion of experts qualified by 

scientific training and experience to evaluate the safety of food 

”r J 

57 
and food ingredients, are generally recognized as appropriate 

(emphasis added) (21 CFR 3 170.3(i)); and 

5. general recognition of safety shall be determined in accordance with 21 

CFR 5 170.30 which requires, among other things, that: 

a. general recognition of safety may be based only on the views of 

experts qualified by scientific training and experience to evaluate 

the safety of substances directly or indirectly added to food; 

b. the basis of such views may be either (1) scientific procedures or 

(2) in the case of a substance used in food prior to January 1, 1958, 

through experience based on common use in food; 

c. general recognition of safety requires common knowledge about 

the substance throughout the scientific community knowledgeable 

about the safety of substances directly or indirectly added to food; 

d. general recognition of safety based upon scientific procedures shall 

require the same quantity and quality of scientific evidence as is 

required to obtain approval of a food additive regulation for the 

ingredient ; 

e. general recognition of safety through scientific procedures shall 

ordinarily be based upon published studies which may be 

corroborated by unpublished studies and other data and 

information; and 

f. a GRAS substance should: 

(1 .) generally comply with any applicable food grade specifications 

of the Food Chemicals Codex; 

(2.) perform an appropriate function in the food or food-contact article in which it is used; and 

(3.) be used at a level no higher than necessary to achieve its 

intended purpose in that food or, if used as a component of a 

food-contact article, at a level no higher than necessary to 

achieve its intended purpose in that article (21CFR 3 170.30). 

58 
4. Publication of the “Red Book” 

In 1982, FDA published the “Red Book - a 240 page document formally 

entitled: “Toxological Principles for the Safety Assessment of Direct Food 

Additives and Color Additives Used in Food”. This document was intended: 

1. to establish the boundaries of “reasonable certainty”; 

2. to set forth appropriate guidance regarding the scientific criteria to be 

used in assessing safety; 

3. to establish a complete assessment system; 

4. to ensure a cost-effective system; 

5. to establish a flexible framework; and 

6. to encourage use by all of the criteria-incorporating assessment 

system. 

Such comprehensive document includes appropriate criteria, guidelines for 

conduct of studies (Le., scientific procedures), standards for assessing testing 

results, decision elements and model testing protocols. 

In 1993, FDA published the second version of the Red Book. It consists of 

235 pages. It amounted a significant updating of the prior version - consistent 

with changing scientific standards - and was intended to indicate that a 

submission conforming to the Red Book‘s recommendations would provide 

sufficient scientific information to evaluate a substance’s safety for the 

regulated intended use@). It included, among many other protocols, testing 

pertinent to immunological end points. 

Finally, in 2000, a third version of the Red Book - now called 

“Toxicological Principles for the Safety Assessment of Food Ingredients” - was 

published. It too amounted to a significant revision and is an ongoing process. 

Importantly, this third version was made applicable also to assessing the safety 

of GRAS substances. 

59 
“I 5. White House policy 

Due primarily to concerns about lack of coordination and uncertainty as to 

the manner in which biotechnology was going to be regulated in the United 

States, the Reagan Administration formed - in 1984 - an interagency working 

group to study, to establish, and to coordinate the federal government‘s 

regulatory policy pertinent to biotechnology. The group included at least thirteen 

member agencies, including FDA. Importantly, the group was to determine, 

among other things, whether the then current regulatory requirements were 

adequate for regulating biotechnology. The initial results of the working group 

were published on December 31, 1984. (See, 49 FR 50856 (1984)) This 

document was updated on December 14, 1985. (See, 50 FR 47174 (1985)). 

Also in 1985 - specifically on October 31’‘ - the President established the 

Biotechnology Science Coordinating Committee (“BSCC”), a scientific 

coordination body. (See, 50 FR 47174 (1985)). Importantly, the BSCC’s 

responsibilities included establishing a common scientific approach to regulating 

biotechnology. 

Finally, in June of 1986 a very lengthy, final notice was published 

indicating how - pursuant to White House requirements, specifically the Office of 

Science and Technology policy (“OSTP”) - a number of Federal entities, 

including FDA, would regulate biotechnology. (See, 51 FR 23302 (1986)). 

Importantly, such notice stated that the then existing regulatory framework was 

deemed adequate and flexible enough to oversee modern biotechnology 

applications. Further, it made clear that no new laws, regulations or other 

regulatory burdens - such as new criteria - were necessary in order to 

adequately regulate such biotechnology. 

c. The adequacy of the “criteria” 

With regard to whether existing “criteria” for evaluating the safety of 

substances to be added to or that otherwise may become a component of food 

60 
\.._I_ 

\. r 

are “adequate”, such question has been legally answered since 1986 - that is, 

upon publication of the above-references OSTP notice. Such notice indicated 

that the answer is yes. 

As a practical matter, as discussed below, such question has also been 

repeatedly answered in the affirmative by the approximately last thirty years of 

experience of federal entities authorizing use of biotechnology-related products in 

interstate commerce. 

d. Application of the “criteria” to products of biotechnology 

The above - referenced OSTP notice applies, of course, to all “criteria” 

pertinent to evaluating the safety of all products emanating from biotech sources 

regulated by federal entities - including FDA. To date, hundreds of products that 

“may” exhibit immunoregulatory properties have been reviewed by various US 

entities (such as EPA, USDA and FDA) and authorized for introduction into 

interstate commerce. All such reviews were conducted pursuant to the abovereferenced criteria or very similar criteria coordinated by the BSCC. 

More significantly, FDA has used virtually the same criteria to review and 

approve numerous drugs (and biologics) - for over twenty-five years - all of 

which were human proteins intended to be injected (the most potentially 

impactive route of exposure) and all of which were potentially immune 

modulators. In every case, such criteria were deemed adequate for evaluating 

the safety of such products. No new criteria were needed, although FDA has 

often provided pertinent guidance documents to indicate how such criteria would 

be applied to biotech products. 

Even more specifically, CFSAN has been applying - without untoward 

incident - the criteria here in question (and even their predecessor criteria) to 

biotech products since 1990. All such products are proteinaceous in nature and 

might be immunomodulatory. The very first such product, i.e., chyrnosin 

(otherwise known as the active enzyme rennin) has been found to be GRAS 

when produced via three, different transgenic sources. (21 CFR § 184.1685). 

61 
Since this product, CFSAN has reviewed and authorized (Le., found to be GRAS) 

via the criteria here in question numerous biotech enzyme products (as well as 

numerous enzyme products emanating from non-transgenic sources) both under 

the GRAS Affirmation process and the GRAS Notification process. All such 

products have been safely used since being found to be GRAS. 

Finally and not least importantly, the criteria here in question were 

successfully applied to (and, thus, found to be adequate) three lactoferrin (also 

proteinaceous) products, Le., bovine lactoferrin (see, GRN Nos. 67, 77 and 130). 

Especially with regard to GRN No. 77 whose intended uses are identical to those 

currently before CFSAN in GRN No. 189, the exact same immunomodulatory 

properties were before CFSAN since bLF induces every immuno-related effect 

that hLF does - and very often via the same testing common to both (testing that 

includes over 300 studies all cited in Pharming’s GN documents). If the criteria 

here in question were deemed adequate by CFSAN for assessing the safety - 

including immunomodulatory properties - of bLF (and they were), then by 

definition they must be deemed adequate for assessing the safety of hLF under 

identical, requested pattern of use. There is no rational, scientific basis for 

concluding otherwise. 

e. No need for additional criteria 

The fact that the criteria here in question were successfully applied - as 

discussed above - to the bLF-related GRAS Notifications indicates that no new 

additional criteria are needed. The bLF-related GRAS Notifications, especially 

GRN No. 77, raised the very same potential immunomodulatory issue(s) raised 

by Pharming’s hLF GRAS Notification. No new questions have been raised by 

CFSAN with regard to Pharming’s GRAS Notification that were not also raised 

when reviewing and assessing the safety of bLF Thus, such criteria should be 

deemed adequate as is. 

62 
C. Conclusion 

As CFSAN has repeatedly acknowledged, Pharming has answered all 

questions posed to it via the scientific information forwarded to CFSAN. Such 

information includes - both qualitatively and quantitatively - more, better, and 

more current scientific information than has ever been submitted in any GRAS 

Notification to CFSAN. Pharming believes that such extensive information - as 

supported by numerous qualified experts, including those discussed above but 

unconnected to Pharming - demonstrates consistent with all pertinent 

assessment criteria that Pharming's hLF is GRAS for its intended uses and that 

such information is more than sufficient for CFSAN to base its review on Thus, 

Pharming encourages CFSAN to complete its review in the near future based on 

such information and to issue a "no questions" letter. 

63 
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16 










_ Agennix Incorporated i~ - ~ CA,-) L 

April 24, 200 8 

rA 

Andrew C . von Eschenbach, M .D. 

Commissioner of Food and Drugs 

Food and Drug Administration, HF-1 

5600 Fishers Lane 

Rockville, MD 20857 

Re: Request for Meeting 

I am writing on behalf of Agennix, Inc . ("Agennix") to seek a meeting 

with you and relevant senior staff concerning a submission by Ventria Biosciences, 

Inc . (Ventria) to find its rice-derived recombinant human lactoferrin ("rhLF" ) 

substance to be Generally Recognized as Safe (GRAS) for use in foods . We have 

learned through the FDA's public calendar that you have recently met with Ventria 

representatives on this subject. In light of that meeting, and because we have filed 

scientific objections with FDA's Center for Food Safety and Applied Nutrition 

(CFSAN) relating to the GRAS status of this substance, we wanted to be sure that 

we could communicate those concerns to you directly . 

Agennix is a small Houston-based biotechnology company . Agennix 

has been developing recombinant human lactoferrin ("rhLF") as a pharmaceutical 

drug since 1996 under FDA's IND process . Agennix is currently preparing to enter 

Phase III clinical trials with rhLF in advanced non-small cell lung cancer (NSCLC), 

for which Agennix has received Orphan Drug designation from the FDA, and has 

been granted Fast Track designation by the FDA for both first-line combination 

therapy and third-line monotherapy. Agennix has completed double-blind, placebocontrolled Phase II clinical trials supporting both of these NSCLC indications, and 

both of these trials met their primary endpoints . Agennix has also received 

approval of an SPA for the first-line NSCLC indication . In advanced renal cancer, 

Agennix is preparing for a Phase IIb trial, and has been granted Orphan Drug 

designation by the FDA for this indication as well . 

Based on our experience in testing rhLF as a pharmaceutical product, 

we believe there are significant scientific reasons why it would not be appropriate to 

market this compound in food products . Agennix has assembled a panel of experts 

who have expressed their opinion that Ventria's rhLF is not GRAS and submitte d 

Eight Greenway Plaza Suite 910 Houston Texas 77046 Telephone 713/552-1091 Facsimile 73/552-0795 
Letter to Andrew C . von Eschenbach, M.D. Page 2 of3 

that information to CFSAN. We believe the potential availability of rhLF to the 

general population through food is also unwise, in light of the lack of medical 

supervision and pharmaceutical-grade good manufacturing practices . Similar 

concerns exist regarding a related GRAS notification submitted by Pharming, Inc . 

for its rhLF product . 

We would plan to focus our discussion on the scientific issues 

presented with this GRAS issue. Accompanying me in my capacity as Board Chair 

of Agennix would be Rick Barsky, CEO of Agennix, Atul Varadhachary, M .D ., Ph.D., 

COO of Agennix, and two or three outside experts, depending on availability . We 

will also be accompanied by our outside legal counsel, Joe Levitt of Hogan & 

Hartson, LLP, who would plan to speak briefly to the legal issues involving Section 

912 of the recently enacted Food and Drug Administration Amendments Act of 2007 

("FDAAA"), with the intention to update you on discussions he has had with FDA 

Chief Counsel Gerry Masoudi on this subject. 

As a former FDA Commissioner myself, I am most respectful of your 

time and recognize the many scheduling challenges . Towards that end, we have 

listed below a series of dates that we could be available over the coming months . 

Please let me know either by e-mail (frankcosmos@aol.com) or by telephone (301-

908-3182) which of these dates would be acceptable . 

Thank you very much for your consideration and I look forward to 

meeting with you . 

Available Dates: 

June 24-27 

July 1-2 

August 19, 27-2 9 

September 2-3, 16, 23-24, 26, 3 0 

Cc: Susan Winkler, RPh, Esq. 

Chief of Staff, FDA 

Stephen Sundlof, D .V.M., Ph.D. 

Director, CFSAN 
Letter to Andrew C . von Eschenbach, M .D. Page 3 of 3 

Michael Landa 

Deputy Director for Regulatory Affairs, CFSAN 

Laura Tarantino, Ph .D. 

Director, Office of Food Additive Safety, CFSAN 

Gerald Masoudi, Esq. 

Chief Counse l 

Richard Barsky, CEO 

Agennix, Inc. 

Atul Varadhachary, M.D ., Ph.D ., COO 

Agennix, Inc. 

Joseph Levitt, Esq . 

Hogan & Hartson, LLP 
Page 1 of I 

Kaarina Marajh AdWgt : I LB Agennix System# :182737311NET8014 

8 Greenway Pfau, suite 910 Accountt: S `••`•• . .. 

Delivery Address Bar Cod e Houston, TX 77046 

W inmr~ uI "UI SHIP TO: 713-552-1091 BILL SENDER Ref # Regulatory Andrew von Eschenbach Invoice # 

Food and Drug Administration HF-1 PO # 

Dept # 5600 Fishers Lan e 

Rockville, MD 20857 

MON - 28APR A2 

TRK# OR, 7910 5255 4921 PRIORITY OVERNIGHT 

20857 

MD-U S XC OBTA IAD 
D~PARTMENT OF HEALTH & HUMAN SERVICES 

Food and Drug Admi r ration 

Rockville MD 20857 

June 4, 200 8 

rank E. Young, M.D., Ph.D. 

hairman 

gennix, Incorporated 

Vight Greenway Plaza 

uite 91 0 

ou on, ~X 7704 6 

r 

am pleased to accept your request to meet with you to discuss Ventria Biosciences 

ncorporated's submission that requests the agency to find its rice-derived recombinant 

human lactoferrin substance to be generally recognized as safe (GRAS) for use in foods . 

The meeting is confirmed for September 24, 2008, at 1 p .m., at the Parklawn Building, 

Room 14-68, Rockville, Maryland . Please contact Ms. Pam Pisner, my special assistant, 

at 301-827-2410, to make the necessary arrangements . 

As a public servant, I may be called away at the last minute . Should such a situation 

arise, you will be notified, and I will make every attempt to find an FDA representative to 

serve as a substitute . , 

An#w C. von Eschenbach, M .D. 

Commissioner of Food and Drugs 
:\Wp\Brian\meeting request\8-2946 Frank Young .doc 

Drafted by B. Botsford 5/29/08 

Edited by V . Jackson 5/29/08 

B. Clarke 5/30/08 

Cc: HF-1 (2) 

HF-40 
i 

OFFICE OF THE COMMISSIONER 

CORRESPONDENCE SUMMARY SHEE T 

ct: A confirmation letter to Dr. Youn g 

C t ol Number : 8-2946 Date: May 29, 2008 

A ion Required : Commissioner Signature 

S a /Back round: 

A tt r confirming the Commissioner will meet with Dr . Young to discuss a submission by Ventria Bioscie n 

IN . egarding its rice-derived recombinant human lactoferrin substance to be GRAS for use in foods. The 

m eti g is confirmed for September 24, 2008, at 1 p.m., at the Parklawn Building, Room 14-68, Rockville, 

Mrvlnd 

P~p ored By : B. Botsford Reviewed/Cleared By: V. Intranet: Yes[[]] No[[]] 

Jackso 

: B. Botsford n ec Contact Correspondence Analyst: B . Botsford 

Office of the Commissioner 

Review/Clearance 

se hand carry to next addressee 

R Date COMMENTS : ie er Signature 

D ty oellmer, HF-40 ~ S oa 044- '61 

M W nckler, HF- 1 

M Pi ner, HF- 1 

D V Eschenbach, HF-1 / .1 + ~_A / ~ 

ument Signature Date : 

AFTER SIGNATURE RETURN TO EXECUTIVE SECRETARIAT HF-40 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

DEPARTMENT OF HEALTH AND HUMAN SERVICES 

FOOD AND DRUG ADMINISTRATION 

MEMORANDUM OF TELECONFERENCE 

DATE: July 3, 2008 

TIME: 3:30 PM 

PARTICIPANTS: 

FDA 

Jeremiah Fasano 

Antonia Mattia 

Laura Tarantino 

William McConagha 

HFS-255 

HFS-255 

HFS-200 

HF-22 

External 

Charles L. Morin 

Frans de Loos 

Samir Singh 

Morin & Associates 

Pharming 

Pharming 

SUBJECT: Status of GRN 189 

FDA staff met with representatives of Pharming, Inc. (Pharming), including Pharming's agent, 

Mr. Morin, to discuss the status of the firm's notice for the use of recombinant human lactoferrin 

from bovine milk as an ingredient in food, GRN 189. 

We explained that FDA's review of GRN 189 had identified a number of significant issues related 

to the immunological properties of Pharming's ingredient. We had been unable to resolve these 

issues on the basis of the information provided by the notice and its amendments. 

Pharming and FDA discussed these issues, and FDA agreed to provide them in writing. We stated 

our view that unless these issues could be resolved in a relatively short period of time, it would be 

appropriate to move towards closure of the notice. We noted that Pharming could choose to 

withdraw GRN 189 any time without prejudice to future submissions. 

FDA concluded by reaffirming the agency's willingness to engage in continued dialogue on the 

issues we had identified. 

Jeremiah Fasano 

R/D:HFS-255:JMFasano:07/03/2008 

Init:HFS-255:AMattia:06/01/2010 

F/T:HFS-255:JMFasano:06/01/2010 

OFFICE SURNAME DATE OFFICE SURNAME DATE OFFICE SURNAME DATE 

FILE 

COPY 
 

 

 

GRN 189 – Outstanding Questions Summary – Page 1 

Summary – Outstanding Questions Identified by CFSAN Regarding GRN 000189 

Four lines of evidence addressing the safety of the intended use of Pharming’s human 

lactoferrin (hLF-b) can be identified within the data and information presented in GRN 

000189. These are: 

1. infant consumption of hLF in human milk; 

2. endogenous exposure to hLF secreted by the body in the alimentary canal; 

3. bLF as a more potent equivalent of hLF; and 

4. published studies with hLF and bLF in both animals and humans. 

FDA continues to have questions about the adequacy of each line of evidence as a basis for a 

GRAS determination for the intended use of hLF-b. These questions concern hLF-b’s 

immunomodulatory effects1

 when the substance is consumed chronically by the general 

population. 

1. FDA questions whether infant consumption of hLF in human milk is an appropriate 

model for demonstrating the safety of hLF in adults. The infant immune system is 

qualitatively different from the mature immune system and these differences may alter 

the risk of adverse immune-mediated effects resulting from the consumption of hLF. The 

notice does not contain evidence supporting the premise that the immunological effects of 

hLF in infants are equivalent to those in adults. 

2. FDA questions whether endogenous hLF secreted by the body in the alimentary 

tract is an appropriate physiological model for exposure to hLF in food. Pharming 

has not provided information to demonstrate that low-level background secretion is 

physiologically equivalent to consumption of a larger quantity of hLF in food. 

Furthermore, we do not agree that endogenous hLF levels in unhealthy individuals are an 

appropriate point of reference for safe consumption of hLF by the healthy general 

population. 

3. FDA questions whether a history of consumption of bLF in food is sufficient to 

demonstrate the safety of hLF for Pharming’s intended use in food. There is 

evidence suggesting that bLF and hLF are functionally distinct in the context of in vivo 

consumption by humans, as acknowledged in public statements attributed to Pharming. 

The notice does not provide evidence that differences in the complex functionality of 

these proteins are not relevant from the perspective of a safety assessment for chronic 

consumption by the general human population. 

1 During its evaluation, FDA also raised questions about the potential for adverse hLFspecific autoimmune effects as a consequence of consumption of an exogenous human 

protein. It remains unclear to FDA that the potential for disruption of specific immune 

tolerance in the context of this intended use is well understood. 
GRN 189 – Outstanding Questions Summary – Page 2 

4. FDA questions whether studies with hLF and bLF cited by Pharming are sufficient 

to demonstrate the safety of hLF for Pharming’s intended use in food. The cited 

studies are almost entirely therapeutic and do not have endpoints appropriate to a food 

safety assessment. Furthermore, the notice does not provide evidence that the limited 

number of food toxicology studies cited employ animal models and endpoints suitable for 

assessing the safety of chronic consumption of an immunomodulatory human protein. 

We note that immune-mediated consequences of chronic modulation of the adult immune 

system by hLF consumption do not appear to be well-studied in a food safety context. 

As you know, there was a recent session on the use of human protein food ingredients at the 

Summer Meeting of The Toxicology Forum. Although we mentioned during the session that it 

had been prompted in part by specific submissions to FDA, we did not focus on these 

submissions (including your notice) or discuss them. It was not the intent of the session to 

evaluate specific products. Speakers were selected both on the basis of expertise in issues related 

to the use of human proteins in food as well as availability. The session included multiple 

speakers with therapeutic backgrounds because existing experience with human proteins appears 

to be limited outside this area. As noted during the session, the therapeutic experience was 

discussed not because it was directly applicable to food, but in order to see what considerations 

might or might not be relevant. 

We believe that the session accomplished its primary goal of promoting discussion and awareness 

with respect to the topic of human protein food ingredients. Given the complexity and crossdisciplinary nature of the issues and the limited time available during the session, we hope that 

the dialogue will continue more broadly in and beyond the food safety community. 
 

 

 

 

 

 

 

 

 

__________________________________________ 

Page 1 of 1 

Fasano, Jeremiah 

From: Fasano, Jeremiah 

Sent: Thursday, July 17, 2008 11:31 AM 

To: 'charleslmorin@earthlink.net' 

Subject: Summary of outstanding questions re: GRN 189 identified in July 3 phone call 

Attachments: Outstanding Question Summary - GRN 189.pdf 

Mr. MorinPer your request, we're providing a brief list of the issues we discussed on July 3rd, 2008 with 

respect to GRN 189. We have also briefly addressed the recent session on human protein food 

ingredients at The Toxicology Forum. I hope you will find the document useful. 

Regards-

-Jeremiah Fasano 

Jeremiah Fasano, Ph.D. 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Phone: 301-436-1173 

Fax: 301-436-2964 

Email: jeremiah.fasano@fda.hhs.gov 

Mailing Address: 

HFS-255 

5100 Paint Branch Parkway 

College Park, MD 20740 

This e-mail is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential, and it should not be 

disseminated, distributed, or copied to persons not authorized to receive such information. If you are not the intended recipient, any dissemination, distribution or copying is strictly 

prohibited. If you think you have received this e-mail message in error, please e-mail the sender immediately at jeremiah.fasano@fda.hhs.gov. 

7/17/2008 



A Biopharmaceutical Company Developing Dr

for Cancer and Diabetic Ulcers 

FDA Commissioner’s Meeting 

January 16, 2009 
Meeting Attendees 

On Behalf of Agennix, Inc: 

• Frank E. Young, M.D., Ph.D, Chairman of the B

• Rick G. Barsky, Chief Executive Officer • Richard D. Cummings, Ph.D, Emory University 

Medicine 

• Arno Kromminga, Ph.D, IPM BIOTECH • Michael P. Sherman, M.D., Southern Illinois Un

School of Medicine 

• Atul Varadhachary, M.D., Ph.D, President and • Joseph A. Levitt, Counsel to Agennix 
Introduction 

Frank E. Young, M.D., Ph.D. Chairman of the Board Agennix, Inc. Rick G. Barsky Chief Executive Officer Agennix, Inc. 
Topics to be Covered 

1. Recombinant human lactoferrin (rhLF) as a Pr

Cancer Therapy 

2. Scientific Concerns with Food Uses of rhLF 

• Glycosylation of Proteins • Immunology • Pediatric/Neonatal Issues 

3. Toxicology Forum Summary 4. Legal Issues Involving rhLF GRAS Petitions 

• “Severe conflict” among qualified experts 

5. Applicability of 

§ 912 of FDAAA to rhLF 
Glycosylation of Proteins 

Richard D. Cummings, Ph.D. William Patterson Timmie Professor Chair of the Department of Biochemistry Emory University School of Medicine 
Academic Appointments 

• William Patterson Timmie Professor and Chair, Dept

Biochemistry, Emory University School of Medicine 

• Director and Founder, Glycomics Center at Emory U

School of Medicine 

• George Lynn Cross Distinguished Research Profess

Biochemistry and Molecular Biology 

• Ed Miller Endowed Chair in Molecular Biology; Profe

Biochemistry and Molecular Biology, University of OHealth Sciences Center 

• Director and Founder, Oklahoma Center for Medical 

Glycobiology 
Scientific Publications 

• Co-Editor of First Textbook on Glycobiology – 1st E

of Essentials of Glycobiology (1999); 2nd Edition 

published in 2008 

• Published over 180 peer-reviewed articles, over 30 

review articles, eleven textbook chapters, and own27 different U.S. patents 
Modern Expansion of the Central Dogma of Biolo

Post-Translational Modifications Greatly “Amplify” Genomi

Glyc

Glycans 

Proteome 

Genome 

Polypeptide 
Glycans in Glycoproteins from Hum

and Plants are Very Different 

Differ in Composition of Sugars, Linkage of Suga

and Overall Glycan Structures 

See new textbook “Essentials of Glycobiology”–2nd Edition (

A., Cummings, R.D., Esko, J., Freeze, H., Stanley, P., Bertozz

and Etzler, M., Eds), Cold Spring Harbor Laboratory Press, I
These Differences in Glycans Between Humans a

Contribute to Antigenicity and Allergenicity of Gly

“Complex carbohydrates are potent inducers of Th2 responcarbohydrate antigens (Ags) can stimulate the production odifferent classes of glycan-specific antibodies (Abs), includassociated IgG but also non-specific IgE.” “Plants, helminths, and other invertebrates such as insects snails share “common” glycan determinants that are not fohumans.... Such glycan antigens, as well as non-human “sspecific” glycan antigens, are highly immunogenic and reprmajor focus for the host immune response..” 

Quoted from a review by Drs. Irma van Die and Richa

Cummings (2006) Glycans Modulate Immune Respons

Helminth Infections and Allergy, in Parasites and Allergy 

Capron and F. Trottein), pp. 91-112, Karger Publishers, Ba
Plant Food Allergies (including Peanut 

is to Proteins and Glycoproteins 

“During the initial exposure, which may occur in utero, during breast-feearly childhood, antibodies of the IgE isotype, which are highly specific on the surface of the food allergen (usually proteins or glycoproteins)

elaborated. The propensity to produce IgE antibodies against commonplace substhallmark of the allergic diathesis. The factors underlying this propensity remain incompletely understood to include exposure to allergens as well as a genetic predisposition. Exposure through mucosal surfaces, such as those of nasal passages respiratory and gastrointestinal tracts, seems to increase the risk of sewhereas parenteral exposure, such as through subcutaneously adminimmunotherapy, seems to increase the likelihood of tolerance. The threshold for sensitization most likely differs among patients.” 

Quoted from a review by Dr. Adrian Long (2002) The Nu

Bolts of Peanut Allergy, New Engl. J. Med. 346:1320-1
Other Allergies to Proteins and Glycop

“Kolarich and Altmann (18) recently described the major glycspecies of Ara h 1 [Note: this is the major glycoprotein allergethe peanut Arachis hypogaea], as Man(3(-4))XylGlcNAc(2), a 

glycan containing a 

β1-2 xylose attached to the proximal m

the glycan core.” “Individuals with IgE-mediated allergy to bee venom and planor foods have been shown to make specific IgE to these stru“Bardor et al. (28) also found that 25–50% of non-allergic indmade a humoral immune response to these epitopes.” 

Quoted from a paper by Shreffler, et al (2006) The 

Major Glycoprotein Allergen from Arachis 

hypogaea, Ara h 1, is a Ligand of Dendritic CellSpecific ICAM-Grabbing Nonintegrin and Acts as a 

Th1 Adjuvant in Vitro, J. Immunol. 177:3677-3685. 
Bovine and Human Protein 

Glycosylations are Different 

Carbohydrates are considered among the strongest antigens and allergens. • Bovine milk-protein glycosylation includes N-glycolylneuraminic acid (Neu

("Hanganutziu-Deicher antigen" (Asaoka 1994)) which is different from the acetylneuraminic acid glycosylation. 

• NeuGc is a potent antigen in humans; when attached to a human glycopro

considered as a “foreign” substance that invokes immunity. 

• Bovine glycoproteins also contain LDN and alpha-Gal antigens, which are 

human-milk glycoproteins (Coddeville 1992, Nakata 1993). LDN and alphexpressed by several parasites) are involved in host immunity to parasitic I and Cummings RD 2006) and are potent antigens. 

• The types of carbohydrates likely to be found on transgenic cow-derived h

glycoproteins are potent inducers of antibody responses, including IgE (LeAhrazem 2006, Chow 2005). 

• Bovine-type glycosylation of human lactoferrin (“foreign” glycosylation) is 

immunologically different from bovine glycosylation of bovine lactoferrin. 

Bovine-type glycosylation of human lactoferrin 

is also of significant safety concern 
Summary 

• There is clearly a pressing need to make recombinant glycoprotein

therapies. 

• However, it is now well recognized that the source of glycoprotein 

glycosylation contribute greatly to the biological activity and the imand allergenicity of the products. 

• Great care must be taken to avoid undesirable immune effects tha

introduced by producing recombinant human glycoproteins in non-cells, especially in plants. 

• It should be considered that the addition of altered carbohydrates t

recombinant glycoprotein [made in either rice (as for Ventria) or in Pharming)] is akin to introducing modified amino acids, which we acause alarm and concerns in terms of biosafety and bioactivity. 

• Thorough product testing through multi-year clinical trials with app

numbers of subjects of different genders, age groups, and ethnicitiappear to be the only mechanism to ensure product safety to the g

• Neither Ventria nor Pharming have conducted the testing needed t

these risks, and their products are not GRAS for use in foods. 
Immunogenicity of 

recombinant human Lactoferr

PD Dr. Arno Kromminga IPM BIOTECH Hamburg, Germany 
Experience and Credentials 

• Director of IPM BIOTECH in Hamburg, Germany • Summa cum laude Ph.D. from University of Münster • Research at Albert Einstein College of Medicine (NY) • Over 15 years experience in clinical autoimmunity and 

immunogenicity of biopharmaceuticals 

• Co-founder European Immunogenicity Platform (EIP) • My group was one of the first to establish a robust and se

assay for the detection of binding and neutralizing antibodagainst erythropoietin 

• Faculty member at University of Kiel, habilitation in Immun

• Official Certificate as Clinical Immunologist • Frequently published scientific articles, journals, book cha

presenter at international conferences 
Purpose 

I am here to comment on the: • Appropriateness of adding recombinant human 

lactoferrin to food products from the position of mspecialty in immunogenicity 

• To present some aspects of causes and conseq

of immunogenicity of recombinant human therapproteins including lactoferrin 

My starting point: • Most, if not all, recombinant therapeutic proteins 

the potential of inducing an immune response 
Immunoreactivity against Biopharmac

Class Drug Indication Antibodies anti-Il 2 R 

anti-TNF

α

Immune suppression 

RA, M. Crohn 

Receptors CD4 

CD20 

HIV 

NHL, RA, SLE 

Cytokines Interleukin 2 

Interleukin 3 

Cancer Cancer 

Interferons Interferon 

α 2a 

Interferon 

β

HCV 

Multiple Sclerosis 

Enzymes Factor VIII 

DNase 

Hemophilia 

Cystic Fibrosis 

Hormones Insulin 

HGH Erythropoietin 

Diabetes 

Growth 

Anemia 
Causes of Immunogenicity 

Structural Differences • Amino acid sequence • Post-translational 

modification 

• Chemical changes Manufacturing characteristics • Contaminations and 

impurities 

• Formulation • Storage 

Administratio• Route • Frequency • Dosage Others • Immune sta• Genetic bac• Assay form
Risk Management 

Risk = Probability x Impact 

Due to structural differences between the exogenous and elactoferrin, e.g. glycosylation pattern, the likelihood of antiboinduction is moderate. However, the cross-reactivity with enlactoferrin may contribute to the onset or exacerbation of a autoimmune disorder. Furthermore, antibodies that neutraliphysiological immune stimulatory function of endogenous lamay lead to an immune-suppressive status. Therefore, the clinical impact of anti-lactoferrin antibodies is considered to be high and the risk of immunogenicity is high
IgEs Against Carbohydrate Moie

Lead to Anaphylactic Reaction

Chung et al, N Engl J Med, 2008;358:110
Intestinal Lymphoid System 

Aside from all of its other functions, the gastrointestinal tra lymphoid organ (gut-associated lymphoid tissue or GAL
Mortality in Acute Pancreatitis 

after Oral Administration of Probi

Besselink et al, The Lancet, 2008; 371.651-6
Immune Cell Circuits Modulated by Pr

Shida et al, Trends Immunol, 2008; 29.565
Lactoferrin 

• Antibodies against recombinant human lactoferrin may cro

with the endogenous lactoferrin causing an immune impaneutralizing the action of endogenous lactoferrin. 

• Antibodies against lactoferrin are thought to be associated 

some autoimmune diseases. 

• Although it is not known yet whether auto-antibodies agai

lactoferrin have a direct pathogenic effect, these antibodiecontribute to a clinical exacerbation of a pre-existing autodisorder. 

• There is a recent report about the high prevalence of IgE 

against glycans of rice-derived lactoferrin. It should be notis not a general rule that IgE antibodies against glycans frhuman expression systems have no clinical impact. Receshown that IgE against the carbohydrate structure of the m

antibody cetuximab lead to anaphylactic reactions. 
Summary 

• All recombinant human therapeutic proteins have the pote

being immunogenic independent of the route of administr

• The risk of immunogenicity should be assessed by detaile

thorough risk management. 

• Antibodies against rh-lactoferrin may cross-react with end

lactoferrin causing an impaired immune response and poassociation with other autoimmune diseases. 

• Anti-rhLF IgEs may lead to immune mediated allergic ant

reactions with severe anaphylactic consequences. 

• Neither Ventria nor Pharming have conducted the necess

testing to properly assess this risk, and their products are for use in foods. 
rhLF Produced in Rice 

Concerns for Infants and Children 

Michael P. Sherman, M.D., F.A.A.PProfessor of Pediatrics Southern Illinois University School of Medicine 
Training and Experience 

Training and Practice – Pediatrics/Neonatolo

University of Michigan and University of Califo

1969 - 1981 

University of California, Los Angeles 

UCLA Medical Center 

Associate Professor of Pediatrics 

1982 - 1993 

University of Kansas, KU Medical Center 

Professor and Director of Neonatology 

1993 - 1996 
Training and Experience 

Baylor College of Medicine 

Texas Children’s Hospital 

Professor of Pediatrics (1996 – 1998) 

University of California, Davis 

UC Davis Children’s Hospital 

Professor and Chief of Neonatology (1998 – 200

Professor Emeritus (2005 – present) 

Southern Illinois University School of Medicine 

St. John’s Children’s Hospital 

Professor of Pediatrics (2005 – present) 
rhLF in Neonates – Discussion Ov

• Neonates, infants and children are vulnerable human subjec

additional immunological risks when receiving rhLF for gastr

• Antibodies directed against chimeric rice- or bovine-derived 

cross-react with endogenous or therapeutically administered with other proteins present in rice- or bovine-based rhLF form

• Notwithstanding a published report describing rice-based rh

currently no credible evidence that rhLF administration provito children with infantile gastroenteritis 

• Breast-Milk Lactoferrin is a Natural Peptide Antibiotic and 

Immunomodulatory Protein. IT IS NOT A NUTRIENT and shtreated as a food 

• rhLF is not Generally Recognized As Safe (GRAS) in childre
Elevated Risk of Antibody Formation in 

• Neonates, infants and older children with gastroenteritis h

macromolecular opening of intestinal barriers to foreign an

toxins 

• The degree and duration of intestinal opening during intes

illnesses increases exposure to antigens [or toxins] 

• Intestinal opening can facilitate the formation of IgM or IgG 

that can cause: 

- hives - anaphylaxis 

• There can also be formation of IgE antibodies that can cau

- eczema - respiratory symptoms (i.e., wheezing) 

• There is also a risk of developing cross-reacting antibodie

rice- or cow- milk-based proteins that may react with endotherapeutic LF 
Effectiveness in Gastroenteritis

A Study Using LF & Lysozyme in R

• A report suggests a benefit when rhLF + rhlysozy

combination is used to treat infantile gastroenter

- The duration of diarrhea was reduced to 3.7 in the LF/L

vs. 5.2 days in control group [WHO] 

- By 48 hours, a solid stool was achieved in 85% of LF/L

treated children vs. 69% in controls 

- The researchers report but do not qualify a reduction in 

in treated compared to the control groups 

• Careful scientific analysis of the article does not 

the conclusion that rhLF improves outcomes in tpatients or allay safety concerns 
Concerns with Pediatric rhLF Study 

• A small number of children received treatment with rice-produced r

limited dosing duration 

• Methodological concerns with the published study: 

- No pre-defined primary endpoint that was quantitative; no information r

size and power calculations 

- The reported endpoints are not clinically significant and there is no evid

morbidity was reduced 

- Subjects did not receive adequate follow-up 

• Separate published reports of IgE cross-reactivity against rice-deriv

were not evaluated in this study 

• Use of a combined “control group” had half the cases of ORS know

compared to the ORS base received by all the infants in the rhLF/rhgroup” 

• No ability to separate out effects of rhLF versus rhLZ • Difficult to draw conclusions relating to the safety or potential effica

this patient population 
Lactoferrin is a Milk Defense Pro

• Lactoferrin plays an important role in host defense • Lactoferricin produced from Lactoferrin 

- Released by action of pepsin at pH ~2 in the sto- Lactoferricin is a potent microbicide and binds e

• Lactoferrin fragments identified in urine of

human infants – play a role in intestinal and systemimmune priming; there is also an anti-inflammatory 

• Lactoferrin is not a nutritional protein; it is inapprop

add LF to infant formula because LF is a therapeubiologic agent that is still under human-subject investigation in infants and children 
Conclusion from the Peruvian 

Pediatric Study 

Clinical trials are required to ascertain thsafety and effectiveness of rice- and bovderived recombinant human lactoferrin wtreating infants and children. The long-term (multi-year) trials should b• Well-designed • Multi-centered • Well-controlled 
Summary 

• There is an enhanced risks of antibody formation in pediatri

populations. 

• Autoimmune diseases and other immunogenicity concerns 

greater threat to infants and children with developing immunparticularly in neonates or in infantile gastrointestinal infectigut permeability can be further increased. 

• The published Peruvian trial does not establish a safe pedia

rice-derived rhLF or adequately demonstrate a benefit durin

• Human lactoferrin in breast milk is not a nutrient, but a natu

antibiotic and immunomodulatory protein 

• rhLF cannot be considered Generally Recognized As Safe 
Toxicology Forum Summary 

Atul Varadhachary, M.D., Ph.D. President and COO Agennix, Inc. 
The Toxicology Forum Annual Summer Meeting J

Human Protein Food Ingredients 

Chairperson: Antonia Mattia, FDA Key speakers: 

Daniela Verthelyi, FDA Jeremiah Fasano, FDA Rafael Ponce, ZymoGenetics Gopi Shankar, Centocor Research and Development, Inc. Richard Goodman, University of Nebraska Marian Kruzel, University of Texas 

Overall Conclusion: In addition to the known risks associated withuman proteins, there are significant unknowns. These safety concadequately addressed before recombinant proteins can be conside

GRAS. The concerns expressed regarding the immunological and other risk

with recombinant proteins are consistent with those previously articuexpert panel even though neither Agennix nor any of its experts had involvement in this Toxicology Forum panel. 
Concerns Articulated by the Speakers 

• Immunogenicity, cross-reactivity, auto-immunity, and allergenicity 

concerns even with orally administered proteins. Dr. Goodman: “The protein in the stomach is digested and peptide

processed and presented through antigen presenting cells ... Dep

local tissue environment the T cell [may] provide tolerance to that protein. However, under different circumstances, such as a differecytokine profile, T cells produced [will] drive T cell-mediated reactwhich can be deleterious” Dr. Verthelyi: “just because the route is oral [one cannot] eliminate of inducing antibodies.” 

• Recombinant proteins differ from their native counterparts and phy

context in many different ways which can pose additional safety rDr. Shankar, “How do you really know that the human protein is ayour body is making it? Are we really manufacturing it the way the manufacturing it? I don’t know.” 
Speakers at the Toxicology Forum Co

• Apparent safety in animal trials is no guarantee of safety in human

only be evaluated in appropriately sized long-term clinical trials. 

Dr. Shankar: “When we are talking about mostly human proteins, iimmunogenicity [nonclinical animal studies] are generally not usefu

reacts to a human protein differently from a human. It is just as sim

• The consensus from the meeting appears to be that there are sign

concerns to using recombinant proteins as food. Dr. Fasano: “We don’t have a lot of experience with human proteiningredients. This is sort of a new trend or issue.” Dr. Verthelyi: “That is pretty much the bottom line: The potential cointroducing recombinant human proteins is unknown. There are ma

Thus, it appears clear that the use of a recombinant hum

in food is not Generally Recognized as Safe (GRA
Legal Issues 

Joseph A. Levitt Hogan & Hartson, LLP Counsel to Agennix 
Legal Issue regarding GRAS 

• The GRAS standard requires a consensus among qualifie

regarding safety of rhLF 

• Although unanimity is not required, a “severe conflict” amo

qualified experts precludes finding of GRAS 

• Agennix has provided FDA with the opinions of 14 promin

physicians and scientists that rhLF is not GRAS for its inte

• These expert opinions create a “severe conflict” on the ke

affecting GRAS evaluation 

• This reason alone requires denial of both Ventria’s and Ph

GRAS notifications 
Section 912 of FDAAA 

• Law intended to ensure safety and protect dru

development, when drug development came f

• rhLF meets all necessary criteria of Section 91

none of the specific exceptions apply. 

• Agennix submitted comments to FDA docket; 

Ventria and Pharming did not. 

• Conclusion: Section 912 prohibits rhLF from b

added to food. 
Conclusions 

FDA should not permit recombinant human lactofe

added to foods: 

• Scientific basis for GRAS in food has not been 

demonstrated – long-term clinical studies are ne

• “Severe conflict” among qualified experts preclu

GRAS finding as matter of law 

• Section 912 of FDAAA prohibits addition of rhLF 

These scientific and legal bases are clear and com
EXECUTIVE SUMMARY 

AGENNIX PRESENTATION TO FDA COMMISSIONER 

JANUARY 16, 2009 

I. Main Points 

FDA should not permit recombinant human lactoferrin (rhLF) to be added to foods 

because: (a) the scientific basis for GRAS status in food has not been demonstrated—longterm clinical studies are needed; (b) a “severe conflict” among qualified experts exists and 

precludes a finding of GRAS, as a matter of law; and (c) Section 912 of the FDAAA prohibits 

addition of rhLF to foods. 

II. Background: Recombinant Human Lactoferrin is a Promising Cancer Therapy 

Agennix is the pioneer innovator of rhLF as a pharmaceutical. Phase II trials for non-small 

cell lung cancer (NSCLC) and renal cell carcinoma (RCC) indications were conducted and each met 

its primary endpoint. The Phase III program for NSCLC is underway and Agennix has received 

Fast Track Designation from FDA for two NSCLC indications. If approved as a drug, rhLF would 

meet major unmet medical needs. 

III. Significant and Unresolved Scientific Concerns Demonstrate that rhLF is Not GRAS 

for Use in Foods 

A. Risks specifically associated with the glycosylation of rhLF from rice and cows 

Comprehensive studies characterizing the long-term safety risks related to exposure to 

foreign glycans are necessary before any consensus on its safety can be reached. Glycosylation is 

of particular concern because glycans in glycoproteins in human, plants, and animals are very 

different. They differ in composition of sugars, linkage of sugars, and overall glycan structures. It 

is now well recognized that the source of glycoprotein and the type of glycosylation contribute 

greatly to the biological activity and the immunogenicity and allergenicity of the products. 

Great care must be taken to avoid undesirable immune effects that can be introduced by 

producing recombinant glycoproteins in non-natural host cells. Thorough product testing through 

multi-year clinical trials with appropriate numbers of subjects of different genders, age groups, and 

ethnicities would appear to be the only mechanism to ensure product safety to the general public. 

B. Risks of immunogenicity and allergenicity with rhLF from rice and cows 

Most, if not all, recombinant therapeutic proteins have the potential of inducing an immune 

response in humans. Antibodies against rhLF may cross-react with the endogenous lactoferrin 

causing an immune impairment by neutralizing the action of endogenous lactoferrin. Antibodies 

against lactoferrin are thought to be associated with some autoimmune diseases. Although it is not 

known yet whether auto-antibodies against lactoferrin have a direct pathogenic effect, these 

antibodies may contribute to a clinical exacerbation of a pre-existing autoimmune disorder. AntirhLF IgEs may lead to immune-mediated allergic anti-drug reaction with severe anaphylactic 

consequences. 

Insufficient human data have been presented to resolve the safety concerns relating to 

immunogenicity, induction of anti-lactoferrin antibodies and exacerbation of autoimmune diseases 

that may be associated with anti-lactoferrin antibodies. 
C. Risks associated with feeding rhLF to young children, including infants 

Vulnerable populations including neonates, infants and young children with gastroenteritis 

face additional immunological risks when receiving rhLF. Antibodies against chimeric rice- or 

bovine-derived rhLF could have cross-reactivity to endogenous or therapeutically administered LF 

or against other proteins in a rice- or cow’s milk-based formulation administered to infants and 

children. Breast-milk Lactoferrin is a natural peptide antibiotic and immunomodulatory protein. It 

is not a nutrient and should not be treated as a food. 

The trial conducted by Ventria in South America in a relatively small number of children 

receiving very short term administration did not establish the safety of the pediatric use of ricederived rhLF, nor did it provide credible evidence that rhLF administration provides a benefit to 

children with infantile gastroenteritis. In fact, the study was so poorly designed and conducted that 

no meaningful conclusions can be drawn. Well-designed and well-conducted randomized, multiyear clinical studies are needed to adequately assess safety. 

IV. Experts at Toxicology Forum Meeting Support Conclusion that rhLF is not GRAS 

Scientific experts at the Toxicology Forum concluded that, in addition to the known risks 

associated with recombinant human proteins, there are significant unknowns. These safety 

concerns must be adequately addressed before recombinant proteins can be considered to be GRAS 

in food. In particular, these experts found: (1) immunogenicity, cross-reactivity, auto-immunity, 

and allergenicity remain major concerns; (2) immunological concerns exist with both oral and 

parenteral routes of administration; (3) recombinant proteins differ from their native counterparts 

and physiological context in many different ways which can pose additional safety risks; and (4) 

apparent safety of human proteins in animal trials is not a meaningful indicator of safety in humans. 

The concerns expressed regarding the immunological and other risks associated with 

recombinant proteins are consistent with those previously articulated by Agennix experts, even 

though neither Agennix nor its experts played any role in this meeting. 

V. “Severe Conflict” Among Qualified Experts Precludes Finding of GRAS 

Agennix has contacted 14 prominent, highly qualified scientific and medical experts 

who all believe there are significant, unresolved safety issues and that rhLF has not been 

shown to be safe for its intended food uses. Moreover, as mentioned above, experts at the 

Toxicology Forum who have not had any contact with Agennix expressed the same views. 

This unequivocally demonstrates that a “severe conflict” exists among qualified experts. This 

reason alone requires denial of GRAS status, as a matter of law. 

VI. Section 912 of the FDAAA Prohibits rhLF from Being Added to Food 

This law was intended to ensure product safety and protect drug development, when 

drug development came first. rhLF meets all the necessary criteria of Section 912, and none of 

the specific exceptions apply. Agennix submitted comments to the FDA docket supporting 

this position, while Ventria and Pharming did not submit comments disagreeing. Section 912 

should prohibit rhLF from being added to foods. 

- 2 - 
 

------------------------------------------------------------------------------

From: Levitt, Joseph A. 

To: Tarantino, Laura M; 

cc: Fasano, Jeremiah; Cheeseman, Mitchell A; 

Subject: Background Materials from FDA Commissioner Meeting with Agennix 

Date: Friday, January 23, 2009 2:16:38 PM 

Attachments: Agennix FDA Meeting Jan 16 2009 slides.pdf 

ExecutiveSummaryfromFDACommissionerMeeting.pdf 

Laura--

Attached are copies of the slide deck and the two-page Executive Summary that Agennix presented last Friday to 

the FDA Commissioner and agency staff. As you know, Mitch Cheeseman attended from OFAS. I wanted to be 

sure that these materials were added to the relevant OFAS files for both the Ventria and the Pharming GRAS 

notices. I am copying Jeremiah Fasano so he will have these directly. 

Thank for your continued attention to this matter. 

Best regards,

 Joe 

Joseph Levitt, Partner 

HOGAN & HARTSON LLP 

Columbia Square, 555 Thirteenth Street, NW, Washington, DC 20004 

direct +1.202.637.5759 | tel +1.202.637.5600 | fax +1.202.637.5910 

jalevitt@hhlaw.com | http://www.hhlaw.com 

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============================================================================== 
 

 

 

 

 

 

 

Agennix Incorporated 

8 Greenway Plaza, Suite 910 

Houston, Texas 77046 

Telephone (713) 552-1091 

Facsimile (713) 552-0795 

March 9, 2009 

Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Room 3044, University Station 

5100 Paint Branch Parkway 

College Park, Maryland 20740 

Re: Request for Legal Conclusion that Recombinant Human Lactoferrin from 

Transgenic Cows GRN No. 000189 Submitted by Pharming Group N.V. is not 

Generally Recognized as Safe (GRAS) based on a “Severe Disagreement” among 

Qualified Experts 

Dear Dr. Tarantino: 

On behalf of Agennix, Inc. (Agennix) 1/, we write to urge the Food and Drug 

Administration (FDA) to reach the legal conclusion that recombinant human lactoferrin (rhLF) 

from transgenic cows is not generally recognized as safe (GRAS) for use in sports and functional 

foods and drinks, due to a “severe disagreement” among qualified experts as to whether it is safe 

for these food uses. For that reason alone, GRN No. 189, submitted by Pharming Group N.V. 

1/ Agennix is a Houston, Texas-based biotechnology company and is the pioneer innovator of 

recombinant human lactoferrin as a pharmaceutical product. Agennix began clinical testing of rhLF in 1996 

under the FDA’s investigational new drug (IND) program. Agennix has completed blinded, placebocontrolled Phase II clinical trials with rhLF that met their primary endpoints in indications including non-small 

cell lung cancer and diabetic foot ulcers. In advanced renal cell carcinoma (RCC), rhLF has also been 

successfully tested in a Phase II open label trial to evaluate its effects in patients whose disease had progressed 

after receiving at least one prior regimen of systemic therapy. Additionally, the Company has initiated an 

NIH-funded, randomized, placebo-controlled, multi-center Phase II trial in patients with severe sepsis. 

Agennix obtained FDA Orphan Drug designation for rhLF for indications including graft versus host disease 

(Aug. 2003), non-small cell lung cancer (Aug. 2007), and renal cell carcinoma (Sept. 2006). Agennix also 

obtained Fast Track designation from the FDA for two different non-small cell lung cancer (NSCLC) 

indications (first-line in combination with chemotherapy [Sept. 2006] and third-line as monotherapy [Oct. 

2007]), and has started Phase III trials. Agennix obtained approval of a Special Protocol Assessment (SPA) 

from the FDA for its first-line trial of rhLF in combination with chemotherapy in NSCLC patients (Dec. 2007). 
 

 

 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 2 

(“Pharming”) to FDA’s Center for Food Safety and Applied Nutrition (CFSAN), should be 

denied—based solely on legal grounds. 

Agennix has already filed extensive scientific comments regarding significant, 

unresolved safety issues with the use of rhLF in food. 2/ That submission was supported by the 

opinions of 15 prominent scientific and medical experts that rhLF is not GRAS for these food 

uses. These scientific and medical experts are from disciplines directly applicable to the safety 

assessment of rhLF—including the fields of glycobiology, immunology, and medicine. 

Moreover, these scientific and medical experts are leaders in their respective fields, based on 

their many years of experience, prestigious academic posts, extensive publications, and 

numerous positions on government panels and editorial boards. They are regularly sought after 

as speakers at national and international conferences precisely because they are thought leaders 

whose opinions are highly respected. 

More recently, the 2008 Annual summer meeting of the Toxicology Forum included an 

expert panel discussion on the use of human proteins as food ingredients. The panel, which was 

chaired by Dr. Antonia Mattia, included scientific experts from the FDA, academia and industry. 

These scientific experts concluded that, in addition to the known risks associated with 

recombinant human proteins, there are significant unknowns. According to these experts, these 

safety concerns must be adequately addressed before recombinant proteins can be considered to 

be GRAS in food. In particular, these experts found: (1) immunogenicity, cross-reactivity, autoimmunity, and allergenicity remain major concerns; (2) immunological concerns exist with both 

oral and parenteral routes of administration; (3) recombinant proteins differ from their native 

counterparts and physiological context in many different ways which can pose additional safety 

risks; and (4) apparent safety of human proteins in animal trials is not a meaningful indicator of 

safety in humans. The concerns expressed by the Toxicology Forum panel regarding the 

immunological and other risks associated with recombinant proteins are consistent with those 

previously articulated by our experts, even though neither Agennix nor any of its experts had any 

involvement in this Toxicology Forum panel. 

Today’s submission is tantamount to a “motion for summary judgment” because there are 

no material facts in dispute (i.e., it is a matter of record that there are two independent groups of 

experts expressing views diametrically opposed to those articulated by the Pharming expert panel) 

and so the Agency may rightfully decide this issue as a matter of law. Furthermore, this letter is 

based solely on the third prong of the GRAS test—namely, that there be a consensus among 

qualified experts that the food ingredient is safe. 3/ We are asking FDA to determine, as a matter 

2/ Agennix submitted to CFSAN its original Scientific Assessment on GRN No. 000189 on June 27, 

2006. 

3/ This letter does not rely on either of the first two prongs of the GRAS test—namely, that there be 

technical evidence of safety and that the data relied upon be publicly available. Those prongs are addressed in 

previous comments filed by Agennix to the scientific staff in CFSAN. Because all three prongs are required 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 3 

of law, that rhLF from transgenic cows is not GRAS for use in sports drinks, functional foods, 

and any other food uses because Pharming has failed to demonstrate that there is a scientific 

consensus among qualified experts that the substance is safe. 

The law is clear: a substance must meet all three prongs of the GRAS test to qualify as 

generally recognized as safe. An Agency determination that any one of the three elements is not 

met eliminates the need to evaluate and resolve the other two. As described below, Pharming so 

clearly fails to meet its burden of establishing a scientific consensus among experts that its 

GRAS notification for its transgenic cow-produced rhLF must be denied on this basis alone. 4/ 

I. The GRAS Standard Requires A Consensus Among Qualified Experts 

As you are aware, a substance added to food is a “food additive” for which FDA premarket approval is required unless the substance is GRAS or qualifies for another statutory 

exemption. The intended use of a substance is GRAS if it is— 

generally recognized, among experts qualified by scientific training and 

experience to evaluate its safety, as having been adequately shown 

through scientific procedures (or, in the case of a substance used in food 

prior to January 1, 1958, through either scientific procedures or experience 

based on common use in food) to be safe under the conditions of its 

intended use . . . 5/ 

As the statutory language states, a GRAS determination may be based on “scientific 

procedures.” FDA has advised that a GRAS determination based on scientific procedures 

requires three elements: 

1. Evidence that a substance is safe for its intended use; 

2. A basis for concluding that such evidence of safety is generally available; 

and 

3. A basis for concluding that such evidence of safety is the subject of scientific 

consensus among qualified scientific experts. 

for a GRAS determination, the Agency does not need to reach a conclusion on the first two prongs if FDA 

determines, as a matter of law, that the third prong of expert consensus is not met. 

4/ Should the FDA agree that there is a severe disagreement among qualified experts, not only would 

there be no need for FDA to reach a conclusion on the complex scientific issues surrounding its technical 

evidence of safety, but FDA also would not have to reach a conclusion on the effect of Section 912 of the Food 

and Drug Administration Amendments Act of 2007 (FDAAA) on Pharming’s GRAS notification (see letter of 

October 31, 2007 from Joseph A. Levitt, Counsel to Agennix). 

5/ FFDCA § 201(s). 
 

 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 4 

FDA refers to the first element as “technical evidence of safety”; the second and third 

criteria collectively constitute the “common knowledge” element of the GRAS standard. All 

three elements must be demonstrated or the GRAS notice is considered incomplete. 6/ Further, 

the common knowledge elements of scientific consensus and publication apply to all of the 

evidence that is the basis for the determination of safety. 7/ 

Technical evidence of safety requires a showing that “there is a reasonable certainty in 

the minds of competent scientists that the substance is not harmful under the intended conditions 

of use.” 8/ This is frequently paraphrased as demonstrating that there is a “reasonable certainty 

of no harm.” The second element, general availability, requires publication of key data or 

information in peer-reviewed scientific journals, general reference materials, textbooks, or other 

appropriate sources. 9/ Although we believe that Pharming also fails on the first two counts of 

the GRAS standard, this submission is limited to the third prong of the GRAS standard—the 

common-knowledge element of scientific consensus among qualified experts. 

II. A Scientific Consensus Does Not Exist If There Is A “Severe Disagreement” Among 

Qualified Scientific Experts 

It is well-settled law that a “consensus” of qualified experts does not exist if there is a 

“severe disagreement” among such experts as to whether the food ingredient is safe for its 

intended use. The very fact that Agennix has identified 15 prominent, highly qualified scientific 

and medical experts who all believe there are significant, unresolved safety issues and that rhLF 

has not been shown to be safe for its intended uses, unequivocally demonstrates that a “severe 

disagreement” exists on this pivotal point. Further, the concerns expressed by the independent 

expert panel convened by the Toxicology Forum regarding the immunological and other risks 

associated with recombinant proteins are consistent with those previously articulated by our 

experts, even though neither Agennix nor any of its experts had any involvement in this 

Toxicology Forum panel. Accordingly, Pharming has failed to demonstrate that the safety of its 

proposed uses of rhLF produced from transgenic cows is the subject of expert consensus. 

6/ 62 Fed. Reg. at 18937, 18948 (Apr. 17, 1997) (stating “A notice summary that fully describes the 

technical evidence of safety, but does not provide a basis to conclude that the technical evidence is generally 

available and accepted [by experts], would be incomplete”). 

7/ Id. 

8/ 21 C.F.R. § 170.3(i); 62 Fed. Reg. at 18948. 

9/ 21 C.F.R. § 170.30(b); 62 Fed. Reg. at 18942-43. 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 5 

FDA’s 1997 proposed rule on “Substances Generally Recognized as Safe” provides clear 

guidance on criteria for the basis of concluding expert consensus, 10/ and that the existence of a 

“severe conflict’ among experts will preclude a GRAS determination. 11/ 

As discussed in FDA’s GRAS proposal and the pertinent case law, a proponent of a 

GRAS claim bears the burden of establishing expert consensus (i.e., that experts “generally” 

consider the ingredient at issue to be safe). The courts and FDA have interpreted this to mean 

that, although a mere divergence of views will not necessarily preclude GRAS status (as “even 

properly conducted studies may produce disagreement” 12/) a “severe conflict” of expert opinion 

will prevent a finding of general recognition. 13/ 

Although there is no bright-line test for identifying what constitutes a “severe conflict,” 

courts have readily found a “severe conflict” to exist after evaluating the facts at hand. In one 

case, even where the proponent of a GRAS claim presented the testimony of seven experts 

supportive of GRAS status, general recognition was found to be lacking in light of persuasive 

opposing views offered by “several” government experts. 14/ In another case, “sharply divided 

testimony” was found to present a severe conflict of opinion. 15/ Expert testimony critical of 

general recognition in that case suggested that the studies presented did not prove safety or meet 

other criteria contained in FDA’s regulations. 16/ Another court failed to find a consensus where 

there was a “sharp difference of opinion” between experts regarding the methods and results of 

the available studies. 17/ Although these and other cases addressing expert consensus involve 

drug products, the expert consensus standard regarding safety is exactly the same for both food 

10/ 62 Fed. Reg. at 18948-49. 

11/ See 62 Fed. Reg. at 18939 (citing United States v. An Article of Drug . . . 4,680 Pails, 725 F.2d 976, 

990 (5th Cir. 1984); Premo Pharma. Labs. v. United States, 629 F.2d 795, 803 (2d Cir. 1980). Significantly, 

according to the Proposed Rule, “an ongoing scientific discussion or controversy about safety concerns . . . 

would make it difficult to provide a basis about the safety of a substance for an intended use.” Id. at 18949. 

12/ See, e.g., United States v. Articles of Food and Drug . . . “Coli-Trol 80,” 518 F.2d 743, 746 (5th Cir. 

1975). 

13/ 62 Fed. Reg. at 18939 (citing United States v. Articles of Drug . . . 5,906 boxes, 745 F.2d 105, 119 n. 

22 (1st Cir. 1984); 4,680 Pails, 725 F.2d at 990; Coli-Trol 80, 518 F.2d at 746 (5th Cir. 1975); United States v. 

Articles of Drug . . . Promise Toothpaste, 624 F. Supp. 776, 782 (N.D. Ill. 1985), aff’d 826 F.2d 564 (7th Cir. 

1987)). 

14/ See, e.g., Pails, 725 F.2d at 990 (holding that presentation by the United States of the views of 

“several experts” that a drug was not generally recognized as effective showed a “severe conflict” in the expert 

testimony and precluded general recognition). 

15/ United States v. An Article of Drug . . . X-Otag Plus Tablets, 441 F. Supp. 105, 113-114 (D. Colo. 

1977). 

16/ Id. at 113. 

17/ Premo Pharma. Labs., 629 F.2d 795 at 804. 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 6 

and drugs. 18/ For both food products and drugs, the key is whether there is a “severe 

disagreement” of views among qualified experts. 

As described further below, these judicial characterizations of “sharply divided 

testimony” and “sharp difference of opinion” perfectly describe the current case—i.e., whether 

rhLF is generally recognized as safe for its intended food uses. The experts presented by 

Pharming express one view, and the experts presented by Agennix, as well as the independent 

experts at the Toxicology Forum that have never consulted with Agennix, express the very 

opposite view. Indeed, it is hard to imagine a scenario where the experts are any more “sharply 

divided.” In such cases, the courts have consistently found that expert consensus does not exist, 

and FDA should reach the same conclusion here. 

Expert credentials play an important role when assessing whether expert consensus exists. 

In one case evaluating the status of a drug for a particular treatment, the court gave great weight 

to the opinions of several chairmen of leading medical departments from that specialty area. The 

court stated that “it cannot be denied that the affidavits of five of the leading doctors in the field 

which deny general recognition creates more than a ‘mere’ conflict . . . [i]t is inconceivable that a 

drug such as this could be considered generally recognized in the face of such learned nonrecognition.” 19/ 

Once again, the court has very much described the current case. As detailed below, and 

reinforced in the collection of expert CVs already on file with CFSAN, the 15 scientific and 

medical experts presented by Agennix have national and international stature. They hold 

prestigious academic posts, direct cutting-edge scientific and medical centers, serve on important 

governmental committees, and publish extensively in leading journals. In short, they are 

quintessential examples of “leading doctors [and scientists] in the field” so that a finding of 

GRAS is virtually precluded “in the face of such learned non-recognition.” 

Agennix, the clear worldwide leader in research, development and production of rhLF, 

has consulted leading national and international experts on lactoferrin and issues relevant to the 

safety of rhLF from transgenic cows. These experts are primarily from the fields of: (a) 

glycosylation/glycobiology; (b) immunology; and (c) medicine. Included among these are 

experts who have conducted research directly with recombinant human lactoferrin, so they have 

first hand knowledge of its safety profile. These 15 highly-qualified experts have expressed 

serious and specific concerns regarding the safety of the Notifier’s proposed uses of rhLF from 

transgenic cows, demonstrating a “severe conflict” with the expert opinions and conclusions 

submitted by Pharming. We feel strongly that all of our experts are qualified to opine on various 

issues related to the GRAS status of rhLF from cows and their credentials speak for themselves. 

18/ See, e.g., 62 Fed. Reg. at 18938-18939 (citing drug and food precedent in discussion of meaning of 

GRAS standard under section 201(s) of the FFDCA). 

19/ United States v. An Article of Drug . . . “Mykocert,” 345 F. Supp. 571, 575 (N.D. Ill. 1972). 
 

 

 

 

Laura M. Tarantino, Ph.D. 

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These are notable opinion leaders in various fields of science and medicine expressing widelyheld safety concerns. 

The credibility of the Agennix experts is only strengthened by the concordance between 

their views and those articulated by the Toxicology Forum’s independent expert panel. The 

Toxicology Forum is a highly respected organization with a focus on the scientific underpinnings 

of toxicology. The panel assembled by the Forum included experienced individuals representing 

a broad range of stakeholders including CFSAN, CDER, academia and industry. 

We believe that the opinions of Agennix’s experts and those of the expert panel convened 

by the Toxicology Forum — as contrasted to those of Pharming’s experts — demonstrate there is 

a “severe disagreement” among qualified experts and that there is no “consensus” of the 

scientific community on the safety of rhLF for its intended uses. 

III. Agennix has Provided the Opinions of 15 Prominent Physicians and Scientists that 

Cow-based Recombinant Human Lactoferrin is Not GRAS. 

Agennix has provided FDA with the opinions of 15 prominent physicians and scientists 

that rhLF is not GRAS for its intended uses. These experts were selected based on their 

recognized subject matter expertise, professional reputation, and experience in areas that have a 

high degree of relevance to the safety, biologic activity, and mechanism of action of rhLF, 

including glycobiology, immunology, and medicine. These experts include renowned professors 

at universities in the United States, Europe and Australia, chairs of their respective departments 

or groups, directors of scientific or medical centers, and practicing physicians. Collectively, they 

have published over 1,500 scientific articles, abstracts or book chapters, including a number of 

studies on recombinant human lactoferrin. The background and experience of each of these 15 

experts may be summarized as follows: 

1. Richard D. Cummings, Ph.D.: Dr. Cummings is a preeminent leader in the field 

of glycobiology with over 30 years of research and academic experience. He is William 

Patterson Timmie Professor and Chair of the Department of Biochemistry at Emory University 

School of Medicine. He and his research labs have made numerous significant discoveries and 

contributions at the forefront of this emerging field. Dr. Cummings founded and directed two 

major centers for glycobiology at Emory University School of Medicine and the University of 

Oklahoma Health Sciences Center. He is co-editor of the first textbook on glycobiology. Dr. 

Cummings has published over 170 peer-reviewed articles, over 30 review articles, eleven 

textbook chapters, and owns 27 different U.S. patents. 

Dr. Cummings is an internationally known lecturer and speaker on issues related to 

glycobiology. He has been an invited speaker of over 125 organizations and institutions. He has 

organized or chaired various national and international meetings and symposia on glycomics. He 
 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 8 

is a former President of the Society of Glycomics and is active in numerous professional 

societies. Dr. Cummings has been awarded various prestigious research fellowships from the 

National Institutes of Health (NIH) and National Science Foundation. He has served in an 

editorial capacity on ten different scientific journals. Dr. Cummings and his labs have been the 

recipient of seven current and seventeen prior NIH research grants, and twelve other research 

grants from various public and private institutions. He has provided government service in many 

different roles as an NIH reviewer, panel member, and study section member. 

2. James Michael Pierce, Ph.D.: Dr. Pierce is a Professor of Biochemistry and 

Molecular Biology at the University of Georgia and Director of the University of Georgia Cancer 

Center. He is a tenured professor with over 25 years in academia. Dr. Pierce’s research focuses 

on the function of complex carbohydrates in human health with an emphasis on cancer 

progression and diagnosis. He has conducted extensive research in the area of glycobiology. He 

is the editor of the Handbook of Glycomics and an officer in the Society of Glycobiology. Dr. 

Pierce’s work has been supported by the NIH and the National Cancer Institute. He has 

published over 65 peer-reviewed articles. He has served as a reviewer for various NIH, NCI, and 

American Cancer Society study sections and project reviews. Dr. Pierce is also a reviewer at 

leading publications including Nature, Biochemistry, Gene, Glycobiology, Glycoconjugate 

Journal, and the International Journal of Cancer. Dr. Pierce has been an invited speaker or 

lecturer at over 70 major seminars/symposia in the U.S. and abroad. He also holds eleven U.S. 

patents. 

3. Irma van Die, Ph.D.: Dr. van Die is Head of the Glycoimmunology Group in the 

Department of Molecular Cell Biology & Immunology at Vrije University Medical Center in 

Amsterdam. She has written over 100 publications in the areas of glycobiology and immunology 

and has been a professor for over fifteen years. Dr. van Die has done extensive work for various 

sections of the Netherlands Organization for Scientific Research (NWO), the Dutch government 

organization that funds research at top universities and institutes. She is a regular reviewer for 

major journals including: European Journal of Biochemistry, Glycoconjugate Journal, 

Glycobiology, Journal of Biological Chemistry and a grant reviewer for the NWO. She has been 

a board member and is the current Secretary of the Dutch Society of Glycobiology, and is a 

member of various other professional societies. Dr. van Die’s research is supported by numerous 

major public and private grants. Research at her glycoimmunology department has made a 

significant contribution to the present understanding and knowledge of glycan function. 

4. Hubertus Schellekens, M.D.: Dr. Schellekens is a professor of Pharmaceutical 

Sciences at Utrecht University in the Netherlands. Dr. Schellekens has written more than 200 

peer-reviewed journal articles concerning the preclinical development of biotechnology-derived 

therapeutic proteins. His most recent work focuses on immunogenicity of therapeutic proteins 

and biosimilars. He is the editor-in-chief of Biotherapy. Dr. Schellekens is very active in the 

Netherlands Commission on Genetic Modification (COGEM), serving as chairman of several 

subcommittees. COGEM provides scientific advice to the government on the risks to human 
 

 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 9 

health and environment regarding the production and use of bioengineered compounds. He also 

serves as an expert in rDNA pharmaceuticals for the European Medicines Agency (EMEA), and 

as chairman of the Dutch Society of Microbiology’s Committee for Biological Safety and deputy 

chair of its Committee on Biotechnology in Animals. 

5. Arno Kromminga, Ph.D.: Dr. Kromminga serves as Director of Immunology at 

the Institute for Immunology, Clinical Pathology, and Molecular Medicine (IPM) in Hamburg, 

Germany. IPM’s work focuses on resolving immunogenicity issues by antibody detection 

against biopharmaceuticals using a broad range of methods and different assay formats. It is 

dedicated to the development, validation and application of innovative methods in molecular and 

immunological diagnostics including immunogenicity. He is an international leader in the field 

of immunology. Dr. Kromminga has written over 25 publications and presented over 40 lectures 

at major symposia around the world. 

6. John Axford, D.Sc., M.D., FRCP: Dr. Axford is the Chair of Clinical 

Rheumatology and Director of The Sir Joseph Hotung Centre for Musculoskeletal Disorders at 

St. George’s University of London. He has an active rheumatology clinical practice and his 

academic research focuses on glycoprotein oligosaccharide characterization in rheumatic 

diseases and how sugar profiles are associated with specific disease entities. He has served on 

the editorial board of six journals including Glyconjugate Journal and Journal of Therapeutic 

Biotechnology and is a reviewer for seven journals. Dr. Axford has written 18 textbooks and 

chapters, including Glycoimmunology I and II, and “Glycobiology & Medicine” and won the 

British Medical Association Book of the Year commendation for Medicine in 1997. He has also 

written over 125 articles and been an invited presenter at twenty international symposia. Dr. 

Axford has served as a Past President, Secretary and Council Member of the Royal Society of 

Medicine, coordinating its Clinical Immunology and Allergy Section. He has been awarded 

several prestigious research fellowships, including a Fulbright Scholarship. Dr. Axford has 

received numerous private and government grants for research in areas including glycosylation 

and glycoimmunology studies. 

7. David J.A. Goldsmith, M.D.: Dr. Goldsmith is a Consultant Nephrologist at 

Guy’s Hospital in London and an Honorary Senior Lecturer at Guy’s King’s and St. Thomas’ 

Hospitals Medical School at King’s College in London. Dr. Goldsmith has over 20 years of 

medical teaching experience. He serves on the editorial board of four journals including the 

Journal of Nephrology and as a regular reviewer for seven journals. His publications include ten 

books and chapters, 172 papers, and 22 letters. Dr. Goldsmith has also presented abstracts at 

over 70 national and international meetings. He has received numerous private and public 

research grants. He is an Honorary Secretary and Trustee of the UK Renal Association, a 

Member of the Executive Council of the European Renal Association, and a Medical Advisor to 

the UK National Kidney Federation. 
 

 

 

 

Laura M. Tarantino, Ph.D. 

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8. E.D. Weinberg, Ph.D.: Dr. Weinberg is Professor Emeritus in Microbiology at 

Indiana University and the Scientific Advisory Board Chair for the Iron Disorders Institute. He 

was a professor for over 40 years for more than 15,000 students. He has published over 150 

research papers or book chapters. Two of his papers have been designated as Benchmark Papers 

in Microbiology. Dr. Weinberg has presented thirty-six invited lectures at national and 

international meetings and attended over forty invited seminars throughout the world. 

Dr. Weinberg has conducted important research on lactoferrin over decades and is 

particularly qualified to advise on the safety issues related to human use of rhLF. Three of his 

publications include: “Human lactoferrin: a novel therapeutic with broad spectrum potential,” 

“Therapeutic potential of human transferrin and human recombinant lactoferrin,” and “The 

therapeutic potential of lactoferrin.” 

9. Sidney E. Grossberg, M.D.: Dr. Grossberg is Walter Schroeder Professor of 

Microbiology and Molecular Genetics and Professor of Medicine at the Medical College of 

Wisconsin. He served as Chairman of the Department of Microbiology for thirty-one years and 

has been a medical professor for over fifty years. Dr. Grossberg has been published in over 170 

peer-reviewed publications. He has served as an advisor or reviewer for the National Cancer 

Institute, National Institute of Allergy and Infectious Diseases, the World Health Organization, 

and the National Board of Medical Examiners. His expertise includes microbiology and 

immunology. 

10. Marco van de Weert, Ph.D.: Dr. van de Weert is a professor in the Department 

of Pharmaceutics and Analytical Chemistry and is Biomacromolecules Group Leader at the 

Danish University of Pharmaceutical Sciences. He has been a professor for six years and focuses 

his research on protein formulation and drug delivery. He has written over 20 publications and 

three book chapters. Dr. van de Weert is a regular reviewer for scientific journals, including the 

European Journal of Pharmaceutical Sciences, the Journal of Pharmaceutical Sciences, and the 

International Journal of Pharmaceutics. He is also a member of the European Working Party on 

Biosimilars, the group that advises the European Medicines Agency on issues related to 

comparability testing for follow-on biologics and any other clinical and non-clinical matters 

relating directly or indirectly to the safety and efficacy of biosimilar therapies. 

11. Wolfgang E.B. Jelkmann, M.D.: Dr. Jelkmann is Professor of Physiology and 

Director of the Institute of Physiology at the University of Luebeck in Germany. He has over 

thirty years of academic medical experience and focuses his research on the production and 

action of inflammatory cytokines and hemopoietic growth factors, with an emphasis on 

erythropoietin. Dr. Jelkmann has written over 120 original publications, over fifty book chapters 

and reviews, and edited three books regarding the pathophysiology, pharmacology, molecular 

biology and clinical use of erythropoietin. He is on the editorial board of six journals. 
 

 

 

 

Laura M. Tarantino, Ph.D. 

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12. Martin K. Kuhlmann, M.D.: Dr. Kuhlmann is an Associate Professor of 

Medicine and Nephrology and Director of Internal Medicine - Nephrology at Vivantes Clinical 

Center-Friedrichshain in Berlin. He has been a professor of nephrology for fifteen years, with 

research focusing on various issues related to hemodialysis, peritoneal dialysis, and 

cytoprotection from ischemic/toxic renal injury. Dr. Kuhlmann is a reviewer for fourteen 

different scientific journals. He has written thirty peer-reviewed publications, over forty review 

articles, and has been an invited presenter at over 100 international conferences and symposia. 

13. Simon D. Roger, M.D.: Dr. Roger is a renal physician and Director of 

Nephrology at Gosford Hospital in Australia and a Clinical Associate Professor in the 

Department of Medicine and Health Sciences at Newcastle University. He has written over forty 

publications and a book chapter. Dr. Roger’s research focuses on the management of 

anemia/chronic kidney disease, erythropoietin use and renal failure, and biosimilars. 

14. Ashraf I. Mikhail, M.D.: Dr. Mikhail is a renal physician at Morriston Hospital 

and Senior Clinical Tutor at Swansea University in Wales. Dr. Mikhail’s main areas of research 

include the impact of introducing biosimilar epoetins on the quality of anemia management in 

hemodialysis patients and the role of cytokines in modulating the response to erythropoietin 

therapy. He has published fourteen articles in peer-reviewed journals and two book chapters. 

15. Nicole Casadevall, M.D.: Dr. Casadevall is Professor of Hematology at Saint 

Antoine Hospital in Paris. Her areas of research have centered on hemodialysis with special 

emphasis on erythropoiesis, erythropoietin, and myeloproliferative and myelodyspastic 

syndromes. She has served as a member of the Medical Committee for the French Health 

Products Safety Agency (AFSSAPS) and as Scientific President of the French Society of 

Hematology. 

Agennix sought out these experts solely for the purpose of obtaining an independent 

evaluation of the safety of rhLF for use in food. In doing so, Agennix sought a broad range of 

perspectives and experience including: (1) topical experts from research and academia (experts 

who are leaders in their respective fields and who are familiar with state-of-the art in these fields); 

(2) seasoned medical professionals in academia (physicians from teaching hospitals and/or 

medical professors); (3) practicing medical doctors (providing a perspective from frontline 

clinicians); and (4) experts in proteins, in general, and recombinant human lactoferrin, in 

particular. The mix was selected to provide both technical and practical depth. 

Although the opinions of just a few of these experts would be compelling, the opinions of 

this broad array of experts concurring in their scientific assessments unambiguously 

demonstrates a “severe conflict” that precludes GRAS status. 

IV. These Expert Opinions Create a “Severe Disagreement” with those of Pharming’s 

Experts on the Key Issues Affecting GRAS Evaluation. 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

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The opinions of 15 prominent experts submitted by Agennix, supported by the 

independent opinion of the Toxicology Forum’s expert panel, quite clearly demonstrate there is a 

“severe disagreement” among experts regarding whether the use of transgenic cow-produced 

rhLF in sports drinks, functional foods and other food uses is safe. These experts have raised 

legitimate concerns regarding important, unanswered safety questions as well as regarding the 

safety of long-term use of rhLF in food. Thus, given that these experts have expressed an 

opinion diametrically opposed to that offered by Pharming’s experts, a consensus definitely does 

not exist in the medical and scientific communities. 

The expert opinions provided by Agennix raise concerns in a number of areas, 

particularly concerning: (1) risks specifically associated with the glycosylation of rhLF from 

cows; (2) risks of immunogenicity and allergenicity with rhLF from cows; and (3) risks related 

to iron exposure. 20/ These 15 prominent scientific and medical experts have all endorsed the 

entire Scientific Assessment submitted to FDA on June 27, 2006. The summary below 

highlights particular expertise that certain experts bring to each of the major issues presented. 

Fundamental to the concerns raised by these experts is the genuine opinion and belief that 

the safety of this compound cannot be established in the absence of appropriately powered longterm human clinical studies. Of particular concern was the need to determine (again through 

appropriately powered long-term human clinical studies) rhLF’s safety in uniquely vulnerable 

patient populations, including children and immunocompromised subjects such as those with 

autoimmune disease. Moreover, the opinions of these experts, many of whom have been 

evaluating this particular issue since 2005, have not wavered during the intervening 3 years. 

1. Risks specifically associated with the glycosylation of rhLF from cows. 

Our experts strongly disagree with Pharming’s experts on whether the safety profiles of 

transgenic cow-derived lactoferrin and native human lactoferrin are equivalent and whether the 

structural differences and major changes in glycosylation patterns can pose significant, long-term 

health risks. We have consulted some of the most prominent leaders in the field of glycobiology 

(Dr. Cummings, Dr. Pierce, and Dr. Schellekens) who concluded that the data presented in 

Pharming’s GRAS notice did not substantiate the safety of transgenic cow-produced rhLF. 

Rather, comprehensive studies characterizing the long-term safety risks related to exposure to 

foreign cow glycans are necessary before any consensus on its safety can be reached. 

Pharming’s rhLF product has the characteristics of bovine glycosylation rather than human 

glycosylation and substantial and material differences exist between these compounds. The 

glycosylation issue is of particular concern, according to these experts, because Pharming’s rhLF 

consists of allergenic bovine glycans attached to a human protein sequence. As these experts 

20/ These experts have also expressed concern about the absence of adequate safety studies conducted 

with rhLF from transgenic cows; and other risks associated with extended dosing with any rhLF. 
 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 13 

have explained in submissions to CFSAN, evaluation of the safety of bovine glycans and of 

human lactoferrin separately does not replace the need to evaluate the safety of rhLF that 

combines bovine glycosylation with the human protein sequence. Rather, these experts believe 

that the novel structure of bovine glycosylated human lactoferrin may create new risks relating to 

the recombinant protein’s processing and recognition by the human immune system that can only 

be adequately assessed by long-term human clinical studies with the cow-derived recombinant 

protein. 

Dr. Cummings is one of the preeminent scholars on glycosylation and the resulting effect 

on the function of and safety of therapeutic proteins. As noted above, he holds the prestigious 

position as the William Patterson Timmie Profession at the Emory University School of 

Medicine, where he also chairs the Department of Biochemistry. He founded and directed two 

major centers for glycobiology at leading universities. He has published over 170 peer-reviewed 

articles and is co-editor of the first textbook on Glycobiology. He is also a former President of 

the Society of Glycomics. In short, any “who’s who” in the field of glycobiology would start 

with Dr. Cummings. 

Dr. Pierce is also a prominent expert on glycobiology and carbohydrates. He has spent 

25 years in academia and is currently a Professor of Biochemistry and Molecular Biology at the 

University of Georgia and is Director of the University’s Cancer Center. He has published over 

65 peer-reviewed articles, is the editor of the Handbook of Glycomics and is a reviewer for 

several leading scientific journals, including Nature and Glycobiology. 

Dr. Schellekens is a physician and a professor of pharmaceutical sciences at Utrecht 

University in the Netherlands. He has extensive experience on the effect of glycosylation on the 

immunogenicity of proteins. He has published more than 200 peer-reviewed journal articles and 

is editor-in-chief of Biotherapy. He serves as an expert to the European Medicines Agency and 

is chairman of the Dutch Society of Microbiology’s Committee for Biological Safety. 

In the individual and collective opinions of these experts, there is no justification for 

Pharming’s experts to ignore evidence that foreign glycoforms may have an effect on the safety 

of rhLF from transgenic cows. Further, our experts disagree with Pharming’s experts’ basic 

assertions that carbohydrates are not generally considered allergens and have poor biological 

activity. Moreover, according to our experts, risks related to bovine-derived glycans including 

IgE-mediated responses may even be amplified by the administration of transgenic cowproduced lactoferrin which could serve as a vector to deliver cross-reactive bovine glycans 

directly to immune cells in the gut. 

These unresolved safety issues present an “ongoing scientific discussion or controversy 

about safety concerns” as stated in the Agency’s 1997 Proposed Rule, that should clearly stand in 

the way of establishing the safety of an intended use. The impeccable credentials of our 

glycosylation experts should solidify the validity of their opinions and preclude a finding of 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 14 

scientific consensus, as established by the Mycocert court. In that case, it was “inconceivable” 

that a substance could be GRAS given the “learned non-recognition” of several chairmen of 

leading specialty medical departments. 21/ Here, too, we have a severe disagreement among 

prominent experts and a sharp difference of opinion on the key issue of the potential 

consequences of the glycosylation of transgenic cow-derived rhLF. Failure of these learned 

experts to recognize Pharming’s rhLF as GRAS is demonstrative of a “severe disagreement” in 

the scientific community. 

2. Risks of immunogenicity and allergenicity with rhLF from transgenic cows. 

Drs. van Die, Kromminga, and Schellekens are well-known and esteemed experts in the 

field of immunology, and Drs. Weinberg and van de Weert are notable researchers who have 

addressed protein immunogenicity in their published work. These experts all strongly disagree 

that Pharming has provided sufficient human data to resolve the safety concerns of 

immunogenicity, induction of anti-lactoferrin antibodies and exacerbation of autoimmune 

diseases that are associated with anti-lactoferrin antibodies. Indeed, these experts believe that 

Pharming and its experts may be basing their conclusions on a dated understanding of the 

mechanism and activity of human lactoferrin. Our experts have evaluated several published, 

peer-reviewed studies conducted by Agennix which have further strengthened their conclusion 

that long-term human studies are needed to accurately understand the actual safety profile of 

rhLF. 

Dr. van Die is Head of the Glycoimmunology Group in the Department of Molecular Cell 

Biology & Immunology at Vrije University Medical Center in Amsterdam. She has been a 

professor for over 15 years and has written over 100 publications in the areas of glycobiology 

and immunology. She is a regular reviewer for major scientific journals and is a grant reviewer 

for the Netherlands Organization for Scientific Research. She is a former board member and 

current Secretary of the Dutch Society of Glycobiology. 

Dr. Kromminga is Director of Immunology at the Institute of Immunology, Clinical 

Pathology, and Molecular Medicine in Hamburg, Germany, where he focuses on resolving 

important immunogenicity issues. He is an international leader in the field and has written over 

25 publications and presented over 40 lectures at major symposia around the world. 

Dr. Schellekens’ extensive scientific and medical expertise is summarized in the previous 

section. Dr. Weinberg has researched lactoferrin for decades and has noted immunogenicity and 

other risks from lactoferrin administration in scientific publications. Dr. van de Weert’s research 

and publications on the development of protein-based drugs include relevant concerns relating to 

immunogenicity. 

21/ United States v. An Article of Drug . . . “Mykocert”, 345 F. Supp. 571, 575 (N.D. Ill. 1972). 
 

 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 15 

Our experts strongly disagree with Pharming’s experts’ assertion that possible allergenic 

properties of rhLF cannot be the basis to deny a GRAS petition. Known allergenic/immunogenic 

properties are a significant safety concern and should be questioned when determining if a 

substance is GRAS. Our experts also believe there is a further increase in immunogenicity and 

allergenicity risk in the context of patients with conditions known to be associated with antilactoferrin antibodies (autoimmune liver disease, inflammatory bowel disease, Wegener’s 

granulomatosis, rheumatoid arthritis, systemic lupus, and autoimmune pancreatitis). Our experts 

have provided the equivalent of the “sharply divided testimony” from the X-Otag case on the 

issue of whether exogenous lactoferrin can cause allergenic responses in humans. 22/ According 

to our experts, long-term studies are the only credible way to identify and quantify health risks 

associated with immunogenicity and allergenicity. Our experts fervently believe that the 

conclusions reached in Pharming’s GRAS Notice are not supportable. Thus, an ongoing 

scientific controversy clearly exists on these issues, and the experts from Pharming and Agennix 

maintain an unresolved and severe conflict of opinion on these subjects. 

3. Risks related to iron exposure. 

Dr. E.D. Weinberg is Professor Emeritus in Microbiology at Indiana University and the 

Scientific Advisory Board Chair for the Iron Disorders Institute. As such, he is a renowned 

expert in the field of iron-related disorders. He has also conducted research with lactoferrin itself, 

and so he is well-versed in the potential for lactoferrin to impact individuals with iron-related 

disorders. 

 According to Dr. Weinberg and our other experts, dosing of pharmacologically active 

rhLF for extended periods of time can result in toxicity in individuals with iron overload. Iron 

overload can proceed asymptomatically for years, with the patient often presenting only after 

severe tissue damage has already occurred. Lactoferrin binds with a high avidity across a broad 

range of pH concentrations and its ability to deliver iron is an important biological property of 

the molecule. Iron delivery to iron-constrained pathogens in the gut is also a concern since a 

variety of bacteria have developed a mechanism for acquiring iron directly from human 

lactoferrin. Tumor cells are known to over-express receptors that bind lactoferrin with high 

affinity. Thus there is a risk that pre-cancerous or early stage GI tumors might also access iron 

from lactoferrin to accelerate their growth and metastasis. 

These iron-related concerns have not been addressed adequately in Pharming’s GRAS 

Notice and our experts strongly disagree with the assessment of Pharming’s experts that the 

safety of transgenic cow-produced rhLF has been established. Thus, an ongoing scientific 

controversy clearly exists on these issues, and the experts from Pharming and Agennix maintain 

an unresolved and severe conflict of opinion on these subjects. 

22/ United States v. An Article of Drug . . . X-Otag Plus Tablets, 441 F. Supp. 105, 113-114 (D. Colo. 

1977). 
 

 

 

 

Laura M. Tarantino, Ph.D. 

March 9, 2009 

Page 16 

In summary, the clear lack of scientific consensus that rhLF is GRAS is evidenced by the 

compelling opinions of these 15 prominent scientific and medical experts, further supported by 

the independent opinion of the expert panel convened by the Toxicology Forum. These experts 

raise legitimate questions about the safety of transgenic cow-based rhLF. That so many, and 

such highly qualified, experts have repeatedly expressed serious concern about the proposed uses 

of rhLF demonstrates a “severe conflict” of expert opinion and precludes GRAS status for rhLF 

from transgenic cows. 

III. Conclusion 

Based on Pharming’s failure to demonstrate that there is a scientific consensus among 

qualified experts that rhLF from transgenic cows is GRAS, we are asking CFSAN to determine, 

as a matter of law, that rhLF from transgenic cows is not GRAS for use in sports drinks, 

functional foods or other food uses. 

Agennix appreciates CFSAN’s consideration of this important information as Pharming’s 

GRAS notification for rhLF from cows is considered. Please do not hesitate to contact us if there 

are any questions or if additional information would be useful.

 Sincerely, 

 Rick Barsky 

Chief Executive Officer 

cc: Jeremiah Fasano (HFS-255) 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 

Stephen F. Sundlof, D.V.M., Ph.D. (HFS-001) 

 Director, CFSAN 

Michael M. Landa (HFS-001) 

Deputy Director for Regulatory Affairs, CFSAN 

Jeff Senger (GCF-1) 

FDA Deputy Chief Counsel 
 

------------------------------------------------------------------------------

From: Levitt, Joseph A. 

To: Fasano, Jeremiah; 

cc: Tarantino, Laura M; 

Subject: Agennix "Summary Judgment" Letter on GRN 189 

Date: Monday, March 09, 2009 2:55:20 PM 

Attachments: GRAS Sum Judgment Pharming.pdf 

Dear Jeremiah --

Attached is an electronic copy of a letter being delivered to your office tomorrow. The letter requests that FDA 

reach the legal conclusion that recombinant human lactoferrin (rhLF) from transgenic cows, GRN No. 000189 

submitted by Pharming, Inc, is not Generally Recognized as Safe (GRAS) based on a “severe disagreement” among 

qualified experts. 

Agennix has approached this as being tantamount to a legal motion for summary judgment because there are no 

material facts in dispute (i.e., it is a matter of record that there are two groups of experts expressing diametrically 

opposing views) and so the Agency may rightfully decide this issue as a matter of law. Furthermore, this letter is 

based solely on the third prong of the GRAS test—namely, that there be a consensus among qualified experts that the 

food ingredient is safe. Agennix is asking FDA to determine, as a matter of law, that rhLF from transgenic cows is not 

GRAS for use in foods solely because Pharming has failed to demonstrate that there is a scientific consensus among 

qualified experts that the substance is safe. 

This letter is substantially the same as one filed last August in connection with GRN No. 000235, except that this 

letter contains an additional reference to the discussion held last summer at the Toxicology Forum meeting on 

transgenic proteins. 

Like the previous letter, this letter also notes that, if FDA were to take this approach, then it would obviate the need to 

address the rhLF issue in the context of Section 912 of the FDAAA. 

Please let me know if you have any questions. FYI, I am sending similar notes to others named as cc's on the letter. 

Best regards,

 Joe 

Joseph Levitt, Partner 

HOGAN & HARTSON LLP 

Columbia Square, 555 Thirteenth Street, NW, Washington, DC 20004 

direct +1.202.637.5759 | tel +1.202.637.5600 | fax +1.202.637.5910 

jalevitt@hhlaw.com | http://www.hhlaw.com 

"EMF <HHLAW.COM>" made the following annotations. 

This electronic message transmission contains information from this law firm which 

may be confidential or privileged. The information is intended to be for the use of 

the individual or entity named above. If you are not the intended recipient, be 

aware that any disclosure,

copying, distribution or use of the contents of this information is prohibited. 

If you have received this electronic transmission in error, please notify us by 
telephone (+1-202-637-5600) or by electronic mail (PostMaster@HHLAW.COM) immediately. 

============================================================================== 
 

Agennix Incorporated 

May 28, 2009 

Laura M. Tarantino, Ph.D. (HFS-200) 

Director, Office of Food Additive Safety 

Center for Food Safety and Applied Nutrition 

Food and Drug Administration 

Room 3044, University Station 

5100 Paint Branch Parkway 

College Park, MD 20740 

Dear Dr. Tarantino: 

Attached is a summary of the scientific discussion by an expert panel convened at 

last summer’s Toxicology Forum meeting concerning the safety of human proteins added 

to foods. This panel concluded that there are too many unresolved scientific questions to 

find human proteins to be Generally Recognized As Safe (GRAS) until extensive clinical 

trials have been conducted. 

Specifically, these experts found: 

(1) Immunogenicity, cross-reactivity, auto-immunity, and allergenicity remain 

major concerns. 

(2) Immunological concerns exist with both oral and parenteral routes of 

administration. 

(3) Recombinant proteins differ from their native counterparts and 

physiological context in many different ways which can pose additional 

safety risks. 

(4) Apparent safety of human proteins in animal trials is not a meaningful 

indicator of safety in humans. 

We believe these findings and conclusions are directly relevant to FDA’s 

consideration of the GRAS status of recombinant human lactoferrin (rhLF)—specifically, 

rice-based rhLF (GRN-235), submitted by Ventria Biosciences, and transgenic cowproduced rhLF (GRN-189), submitted by Pharming, Inc. The expert panel’s discussion 

and conclusions reinforce the findings and conclusions of Agennix’s experts that rhLF is 

not GRAS for use in any food products. 

Importantly, the Toxicology Forum panel was convened with no involvement by, 

nor even prior knowledge of, Agennix. Therefore, the concordance between the findings 

of the Toxicology Forum experts and Agennix’s experts is even all the more compelling. 

Eight Greenway Plaza Suite 910 Houston Texas 77046 Telephone 713/552-1091 Facsimile 713/552-0795 
Page 2 of 2 

These findings reinforce the views expressed by Agennix experts that rhLF is not 

GRAS, as a matter of science. Furthermore, these expert views are diametrically opposed 

to those expressed by Ventria’s and Pharming’s experts, thereby greatly exacerbating the 

severity of the conflict among qualified experts, and conclusively making rhLF not 

GRAS, as a matter of law. 

We ask that a copy of this submission be placed in files of both GRN-235 and 

GRN-189. We again call upon FDA to reach the conclusion that rhLF is not GRAS for 

use in any food products. 

 Sincerely, 

 Rick Barsky 

Chief Executive Officer 

Attachment 

cc: Jeremiah Fasano (HFS-255) 

Consumer Safety Officer 

Division of Biotechnology and GRAS Notice Review 
 

Toxicology Forum – Summary of expert discussion on the use of 

Recombinant Human Proteins as Food Ingredients (July 2008) 

The Toxicology Forum is an international, nonprofit organization that is devoted to conducting 

open dialogues among various segments of society concerned with problems in toxicology. 

Views are exchanged among experts from regulatory and health agencies, industry, 

academia, policymakers, and public interest groups to arrive at a balanced view of the topics 

discussed [http://www.toxforum.org/content]. 

The 2008 Annual summer meeting of the Toxicology Forum included an expert-panel discussion 

on the use of human proteins as food ingredients. The panel included scientific experts from the 

FDA, academia and industry.1 These scientific experts concluded that, in addition to the known 

risks associated with recombinant human proteins, there are significant unknowns that directly 

impact a safety assessment. According to these experts, these safety concerns must be 

adequately addressed before recombinant proteins can be considered to be GRAS in food. The 

concerns expressed by the Toxicology Forum panel regarding the immunological and other risks 

associated with recombinant proteins are consistent with those previously articulated by 

Agennix’s experts, even though neither Agennix nor any of its experts had any involvement in 

this Toxicology Forum panel. 

A brief summary of the Toxicology-Forum discussions, arranged according to four key topics, is 

given below. Summaries of expert comments were taken from "Speaker Edits" for Session I of 

the Toxicology Forum meeting, on which each speaker's version starts on page 1, so multiple 

speakers may appear to have the same page reference. Statements from these individual experts 

have been paraphrased in some places to make the style of the narrative more consistent, but are 

intended to be as close to the actual statements as possible. 

(1) Immunogenicity, cross-reactivity, auto-immunity, and allergenicity remain major 

concerns. 

The first key scientific point made by the Toxicology Forum experts was a reminder that 

immunogenicity, cross-reactivity, auto-immunity and allergenicity are all major concerns with 

recombinant proteins. Thus, most, if not all, recombinant therapeutic proteins have the potential 

of inducing an immune response in humans. Agennix has argued previously that this is of 

particular concern with chimeric molecules like rice- or cow-derived recombinant human 

lactoferrin (rhLF) which combine a human-protein sequence with non-human glycosylation. 

Antibodies against rhLF may cross-react with the endogenous lactoferrin, causing an immune 

impairment by neutralizing the action of endogenous lactoferrin. Antibodies against lactoferrin 

are thought to be associated with some autoimmune diseases. Although it is not known yet 

1 David Longfellow, Ph.D., President, The Toxicology Forum; Jeremiah Fasano, Ph.D., FDA; Richard 

Goodman, Ph.D., University of Nebraska; Marian Kruzel, Ph.D., University of Texas; Antonia Mattia, 

Ph.D., FDA; Rafael Ponce, Ph.D., ZymoGenetics; Gopi Shankar, Ph.D., Centocor Research and 

Development, Inc.; Daniela Verthelyi, Ph.D., M.D., FDA. Dr. Mattia moderated the discussion. 

 1 
whether auto-antibodies against lactoferrin have a direct pathogenic effect, these antibodies may 

contribute to a clinical exacerbation of a pre-existing autoimmune disorder. Anti-rhLF IgEs may 

also lead to immune-mediated allergic anti-drug reaction with severe anaphylactic consequences. 

The points made by the Toxicology Forum experts on this topic include the following: 

 It is important to recognize that “All exogenous proteins, including therapeutic ones, have the 

potential to cause antibody formation.” (Dr. Shankar, quoting Dr. Huub Schellekens, an 

Agennix expert, page 2). 

 One can also have different reactions occurring simultaneously with the same protein so 

there is really not an all or none phenomenon. If one tried to identify at an individual cellular 

level who is really tolerant, totally tolerant, to all proteins of a certain class, one would find 

mixed reactions and would conclude that nobody is totally tolerant. (Dr. Goodman, page 4) 

 Antigenicity or immunogenicity of a substance is innate to it. Further, antigenicity is relative 

to the host, and is modulated by several factors, including the accompanying substances in a 

drug product, such as the formulants and impurities. Immunogenicity effects range from no 

adverse side effects to severe adverse reactions. The main concern with the immunogenicity 

of food-based proteins is allergenicity. Other concerns include consequences due to crossreactivity of antibodies against that particular food protein or against other therapeutic 

products. Cross-reactivity with endogenous proteins is another concern. (Dr. Shankar, page 

2) 

 Immunological mediators involved extend beyond antibodies. In addition to the effect of 

antibodies, it is also cell-mediated immune responses and innate immunity and inflammation 

that are of concern. (Dr. Verthelyi, page 1) 

 The available evidence, however imperfect, indicates that food allergy has increased two- to 

four-fold in the past 20 years. Why? This is not known. When one thinks about all of the 

complexity of this, one can imagine that it might be hard to predict what is going to happen. 

Further, complex matrices such as specific emulsions are being used. These may be taken up 

differently than soluble antigen. There have been changes in food processing treatments that 

will lead to aggregation of proteins, glycation, or denaturation. Those can impact how the 

antigen is processed. The different effects may be mostly in terms of elicitation, rather than 

sensitization or tolerance. (Dr. Goodman, page 3) 

 (2) Immunological concerns exist with both oral and parenteral routes of administration. 

Agennix has also previously presented its experience with orally administered rhLF, including its 

presence in and its ability to modulate the Gut-Associated Lymphoid Tissue (GALT), the largest 

lymph organ in the body. RhLF has a direct effect on immature dendritic cells, the key antigenpresenting cell in the body, inducing their maturation. (Rosa 2008; Spadaro 2008) Following 

oral administration, rhLF has been shown to recruit immature DCs to the GALT, increase 

cellularity of the Peyer’s patches, induce systemic activation of the innate and adaptive 

immunity, activate distant tumor-draining lymph nodes, and induce effector-cell infiltration into 

distant tumors with immune-dependent tumor killing. Thus, oral administration of rhLF is 

associated with a robust immunological response, which is not surprising considering the 

 2 
GALT’s importance to the body’s immune defense. Agennix has also previously presented part 

of the extensive published data relating to the immunological effects of orally administered 

proteins. For example, studies with OVA, which has been cited by one of the Notifiers as an 

example of an orally tolerogenic protein, have shown that a cytotoxic T-lymphocyte response 

can be induced by oral administration and indeed trigger the onset of autoimmune disease, 

leading the investigators to conclude that “the intragastric route of antigen administration does 

not necessarily provide a default mechanism for tolerance induction” (Blanas 1996, Blanas 1999, 

Blanas 2000). 

Statements made by the Toxicology Forum experts confirmed that these immunological concerns 

exist not just with parenteral routes of administration, but with oral administration as well, and 

contradict suggestions by the Notifiers of GRN 189 and GRN 235 that immunological concerns 

do not apply to orally administered proteins. 

 Potential consequences of introducing recombinant human proteins are unknown. There are 

many questions. (Dr. Verthelyi, page 4) 

 Such proteins should be considered as biological response modifiers and not as human food 

ingredients. (Dr. Adamson, page 6 in Dr. Ponce “Speaker Edits”) 

 Just because the route is oral, one cannot eliminate the possibility of inducing antibodies. 

Further, the status of the recipient may play a role. For example, if there were a GI 

inflammation, the absorption of the proteins may be different. If the immune system of the 

recipient is suppressed or immature, that may result in a different response. (Dr. Verthelyi, 

page 6) 

 One gets antigen exposure and uptake through the airway, the nose or in the oral area. The 

stomach probably doesn’t present much antigen, but in the lumen of the intestine, there is a 

tremendous immune system that is not as simple as many like to think. Some proteins or 

peptides are absorbed, go through the hepatic portal system and can flow through this way 

and be exposed to different immune cells in a variety of organs such as liver, spleen and 

peripheral lymph nodes. Some antigen can come through the M cells of the Peyer’s patch and 

eventually become picked up by dendritic cells and go to the mesenteric lymph nodes. Some 

antigen will be picked up directly by dendritic cells (these cells directly sample out of the 

lumen with processes reaching into the lumen), and the antigen-loaded dendritic cells can go 

through the lymphatics. These antigen-presenting cells probably are exposing different T-cell 

populations in this environment that lead to IgA and IgG production, rather than to allergenic 

T cells that will lead to IgE. There are also epithelial cells that can take up and actually 

present antigen through either the lymphatics or capillary system. So, there are different 

systems, and they may lead to different outcomes. There are conditions such as inflammatory 

systems where you are going to set up T cells that are effectors or helpers that may drive IgE. 

There are conditions where some individuals have high dose tolerance, a lot of antigen 

presented, and maybe some people are allergic because they were not dosed with highenough doses quickly enough in order to get deletion or anergy of the antigen-specific Th 2 

cells. This is a theory, but maybe there are people who will never become tolerant to some 

proteins. I would suggest that we do not yet understand some of the critical factors in terms 

of which individuals will become spontaneously tolerant, which individuals will be 

 3 
sensitized, which foods they will become tolerant to or sensitized to or why. That is an 

important fact that we have to live with. (Dr. Goodman, page 6) 

 (3) Recombinant proteins differ from their native counterparts and physiological context in 

many different ways which can pose additional safety risks. 

Arguments presented below are especially important to the safety assessment of rhLF. Ventria 

and Pharming have each suggested that exposure to endogenously produced lactoferrin, 

including that produced in human milk and saliva, serves as an adequate basis for establishing 

the safety of exogenously administered rhLF. As already discussed, recombinant proteins, 

especially chimeric molecules with non-human glycosylation, can be treated very differently by 

the body. Beyond this, there are significant differences in terms of the types of exposure. Human 

exposure to lactoferrin from human milk is typically limited to the very young when the 

physiological context can be very different from that of older children or adults. Lactoferrin is 

also present in saliva but can be present at doses as low as 3.4 microgram/mL (Lentner 1981) 

and is presented continuously in a very different physiological context. Thus, it would not be at 

all appropriate to rely on the safety of native human lactoferrin expressed in milk or saliva to 

establish the safety of rhLF. 

The Toxicology Forum experts explicitly articulated the point that recombinant proteins, in 

addition to differing structurally from their native counterparts, can also be presented in ways 

that can differ significantly from the normal physiological context of the native protein. These 

non-physiological presentations can themselves pose additional safety risks. 

 For example, we try to make milk for babies similar to that of their mothers, easily digestible 

and so on, but maternal milk has a host of hormones, cytokines, chemokines, all sorts of 

things. These will modulate the response to the proteins present in milk, and putting those 

same proteins in a different context may result in a different response. (Dr. Verthelyi, page 2) 

 Properties of proteins affect allergenicity. Stability to processing and digestion in the GI 

(gastrointestinal) tract makes a difference. If the protein gets unfolded or falls apart, it may 

have a higher propensity to have an immune response against it. The abundance of a protein: 

most food allergens actually comprise less than 1% of the protein content of foods, especially 

nuts and seeds and so on, whereas the highly abundant proteins, such as enzymes and so on, 

from leaves and other fruits and so on are less often reported as allergens. Interaction with 

lipid structures and aggregation: Food processing causes a propensity of food proteins to 

aggregate via association with lipids, and it is known that aggregation of proteins tends to 

give a greater incidence of immunogenicity. (Dr. Shankar, page 7) 

 How does one really know that the human protein is as human as your body is making it? 

Are we really manufacturing it the way the Almighty is manufacturing it? I don’t know. (Dr. 

Shankar, page 5) 

 The problem is that in all expression systems, we have the glycosylation that is provided by 

the system in which it is expressed. So how do you make it really 100% compatible, not only 

at the level of protein, but also at the level of the glycoprotein? (Dr. Kruzel, page 3) 

 4 
 Aggregates, from our point of view, are a very important factor in eliciting an immune 

response, both because they can change the way that the proteins are processed by cells, such 

as macrophages and dendritic cells, and because repetitive epitopes can activate B cells in the 

absence of specific T cells. Then there are a host of post-translational modifications: 

glycoforms, truncations, oxidation, deamidation, cystilation. All of those can contribute to 

product immunogenicity in their own way, either by revealing epitopes that were before 

hidden or by facilitating aggregation and so on. (Dr. Verthelyi, page 2) 

(4) Apparent safety of human proteins in animal trials is not a meaningful indicator of safety 

in humans. 

The crux of the position that Agennix has been communicating is that biological activity of 

human recombinant proteins cannot be adequately captured by testing in heterologous species. 

This is particularly true of immunomodulatory molecules whose full spectrum of activity cannot 

be accurately reflected in animal models and can only be fully observed following extended 

administration and surveillance in humans. Agennix believes that rhLF, with a low proteinsequence homology to its rodent counterpart (Pentecost et al. 1987; van Veen et al. 2002) and 

biological effects demonstrated in clinical trials, including Phase II trials in patients with cancer 

and diabetic foot ulcers, is one such substance. Although many GRAS determinations have been 

made, and should continue to be made, based on an established battery of animal toxicology 

studies and safety factors that establish safe conditions of use, this approach should not be 

applied to recombinant human proteins that must by necessity be subject to rigorous clinical 

testing in order to demonstrate a reasonable certainty of no harm, as required by the statute. 

The need for human clinical trials to support the safety of administering a human protein was 

also emphasized by the experts in the Toxicology Forum meeting. These experts concluded that, 

although animal studies are helpful in terms of an overall safety assessment in humans, they 

cannot substitute for the conduct of human clinical studies as a necessary part of any safety 

assessment of human proteins, including rhLF. 

The last point below made by Dr. Shankar is especially important, and reinforces the Agennix 

position that adequately powered, long-term human clinical studies are needed to properly assess 

the safety of rhLF in humans as part of the food supply. 

 The use of human proteins in conventional foods raises a number of intriguing scientific 

questions, such as the adequacy of the existing animal models, the validity of data 

extrapolation from homologous proteins and the immunological significance of small 

variations in the homology of the proteins or in post-translational modifications. (Dr. Mattia, 

page 1) 

 It has already been mentioned that animal studies do not predict immunogenicity in humans. 

Moreover, negative results do not abrogate the need for human immunogenicity testing. That 

is what that means. Just because we don’t see a response in animals, it does not mean that we 

are not going to need to look at whether this is immunogenic in humans. On the other hand, 

positive results do not necessarily predict that it is going to be immunogenic in humans 

either. But this does not make animal models irrelevant. It may be very useful to look at what 

would happen were the animals to develop or were the humans to develop antibodies to that 

protein. What are the potential clinical consequences? What is the risk? Lastly, the absence 

 5 
 

of effect on immunogenicity in animal models does not ensure that the structure is the same 

or that the factor does not impact on immunogenicity. We have had several companies come 

and say, “We have changed this and it hasn’t changed immunogenicity in animals. It 

shouldn’t change immunogenicity in humans.” That is not the case. (Dr. Verthelyi, pages 3 

and 4) 

 It is artificial to talk of individual syndromes. The whole focus of immunotoxicity associated 

with immunostimulation is context, meaning what are the cellular players, in what tissues, at 

what levels are these various cytokines being expressed, and then one will see the diverse 

nature of the toxicity that will be elaborated in the host. (Dr. Ponce, page 2) 

 When one looks at the picture of allergic responses in the human or in a mouse model, one 

can have a mixed response, and it is not easy to predict. Rodents rarely have allergies. So, 

they don’t necessarily prove to be the best model. (Dr. Goodman, page 2) 

 Clinical studies, obviously, are required. They support the safety and efficacy of the drug. 

(Dr. Shankar, page 4) 

Conclusions 

The Toxicology Forum panel concluded that there are significant risks attendant with the use of 

recombinant human proteins in foods. These include the risks that have been well characterized, 

such as immunogenicity, as well as additional lesser known or unknown risks that need to be 

better understood. The consensus was that these safety concerns must be adequately addressed 

through human clinical trials before recombinant proteins (such as rhLF) can be considered to be 

GRAS. 

Agennix believes that the scientific concerns articulated by the expert panel convened by the 

Toxicology Forum, independent of any involvement by Agennix or its experts, make it 

abundantly clear that the use of recombinant human lactoferrin cannot be considered to be GRAS 

for use in any human food products at this time. 

References 

Blanas E, Heath WR., Oral administration of antigen can lead to the onset of autoimmune 

disease. Int Rev Immunol. 1999;18(3):217-28. Review. 

Blanas E, Carbone FR, Allison J, Miller JF, Heath WR., Induction of autoimmune diabetes by 

oral administration of autoantigen. Science. 1996 Dec 6;274(5293):1707-9. 

Blanas E, Davey GM, Carbone FR, Heath WR. A bone marrow-derived APC in the gut 

associated lymphoid tissue captures oral antigens and presents them to both CD4+ and CD8+ T 

cells. J Immunol. 2000 Mar 15;164(6):2890-6. 

Lentner C, (Ed.). (1981). Geigy scientific tables (Eighth ed., Vol. 1). Basel: Ciba Geigy. 

 6 
Pentecost BT, C.T. Teng, Lactotransferrin is the major estrogen inducible protein of mouse 

uterine secretions, J. Biol. Chem. 262 (1987) 10134–10139. 

Rosa G, Yang D, Tewary P, Varadhachary A, and Oppenheim JJ. Lactoferrin acts as an alarmin 

to promote the recruitment and activation of antigen presenting cells and antigen-specific 

immune response. Journal of Immunology, 2008, 180(10):6868-76 . 

Spadaro M, Caorsi C, Ceruti P, Varadhachary A, Forni G, Pericle F, and Giovarelli M. 

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human 

dendritic cells. FASEB Journal, 2008, 22:2747-2757. 

van Veen HA, Geerts ME, van Berkel PH, Nuijens JH. Analytical cation-exchange 

chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin. 

Anal Biochem. 2002 Oct 1;309(1):60-6. 

 7 
 

------------------------------------------------------------------------------

From: Levitt, Joseph A. 

To: Tarantino, Laura M; 

cc: Fasano, Jeremiah; 

Subject: Summary of Toxicology Forum Expert Discussion on Human Proteins being Added to Food 

Date: Thursday, May 28, 2009 6:49:29 PM 

Attachments: Tox Forum Submission.pdf 

Dear Laura--

Attached please find a letter from Agennix, Inc., enclosing a summary of the Toxicology Forum expert discussion 

held last summer on the subject of human proteins being added to food. Even though Agennix had no 

participation in, or even prior knowledge of, this expert discussion, the views expressed there are remarkably 

consistent with those expressed by Agennix with respect to GRN 235 and GRN 189 in the context of the agency's 

GRAS review of rice-based and cow-based recombinant human lactoferrin (rhLF) -- particularly, that there are too 

many unanswered scientific questions and that human clinical studies are needed to properly assess its safety. 

Accordingly, this provides yet additional support for Agennix's position that neither rice-based nor cow-based rhLF 

is GRAS, either as a matter of science or as a matter of law. 

We would ask that a copy of this letter and attachment (within the same PDF file) be added to both GRN 235 and 

GRN 198, and that the agency consider its contents as part of the GRAS review process. We will send hard 

copies to Jeremiah Fasano tomorrow. 

Best regards,

 Joe 

Joseph Levitt, Partner 

HOGAN & HARTSON LLP 

Columbia Square, 555 Thirteenth Street, NW, Washington, DC 20004 

direct +1.202.637.5759 | tel +1.202.637.5600 | fax +1.202.637.5910 

jalevitt@hhlaw.com | http://www.hhlaw.com 

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