Points to Consider in the Manufacture and Testing 

of Monoclonal Antibody Products for Human Use 

U. S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and 	 Research 
February 28,1997 
1  Date:  February 27, 1997 
From:  Kathryn C. Zoon , Ph.D., Director 
Center for Biologics Evaluation and Research 
Food and Drug Administration 
Subject:  Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human 
Use 
To:  Manufacturers of Biological Products and Other Interested Persons 
This Points to Consider (PTC) document has been developed for manufacturers of monoclonal antibody products for 
human use. These Points are not regulations nor are they guidelines, but represent the current thinking that the 
Center for Biologics Evaluation and Research (CBER) staff believe should be considered at this time. This 1997 PTC 
document supersedes the 1994 PTC document of the same title, announced in the Federal Register of August 3, 1994 
(59 FR 39571). 
It is our intention to continuously update and revise this document in order to improve its usefulness. We invite 
your review and comment on the Points. Comments should be identified with the docket number 94D-0259. Two 
copies of any comments should be submitted except that individuals may submit one copy. All comments should be 
addressed to: 
Dockets Management Branch (HFA-305)
 
Food and Drug Administration
 
12420 Parklawn Drive, Room 1-23 

Rockville, MD  20857

 _______-s-_______________ 
Kathryn C. Zoon, Ph.D. 
2  Points to Consider in the Manufacture and Testing 
of Monoclonal Antibody Products for Human Use 
[Docket No. 94D-0259] 
For further information regarding this document, contact: 
Sharon Carayiannis 
Center for Biologics Evaluation and Research (HFM-630) 
Food and Drug Administration 
1401 Rockville Pike, Suite 200N 
Rockville, MD 20852-1448 
301-594-3074 
Submit written comments on this document to: 
Dockets Management Branch (HFA-305) 
Food and Drug Administration 
12420 Parklawn Drive, Room 1-23 
Rockville, MD 20857 
Comments should be identified with the docket number found in the heading of this page. 
Submit written requests for additional copies of this document or any other CBER guidance to: 
Office of Communication, Training, and Manufacturers Assistance (HFM-40) 
Food and Drug Administration 
1401 Rockville Pike, Suite 200N 
Rockville, MD 20852-1448 
Send one self-addressed adhesive label to assist that office in processing your request. 
These documents may also be obtained by mail by calling the CBER Voice Information System at 1-800-835-4709 or 
301-827-1800, or by fax by calling the FAX Information System at 1-888-CBER-FAX or 301-827-3844. 
Persons with access to the INTERNET may obtain these documents using, the World Wide Web (WWW), or 
bounce-back e-mail. For WWW access, connect to CBER at "http://www.fda.gov/cber/cberftp.html". To receive this 
document by bounce-back e-mail , send a message to "ptc__mab@A1.CBER.FDA.GOV". 
3  TABLE OF CONTENTS
 
Note:  Page numbering may vary for documents distributed electronically. 
I. INTRODUCTION..........................................................................................................................................................................6
 
A. BACKGROUND.......................................................................................................................................................................6
 
B. DEFINITIONS...........................................................................................................................................................................6
 
C. FILING INFORMATION.........................................................................................................................................................7
 
II. PRODUCT MANUFACTURE AND TESTING........................................................................................................................8
 
A. GENERAL PRINCIPLES AND DEFINITIONS.....................................................................................................................8
 
B. MANUFACTURE AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES ...............................................8
 
1. Cell lines...............................................................................................................................................................................8
 
2. Production in cell culture.................................................................................................................................................9
 
3. Production in animals or plants .....................................................................................................................................10 

4. Purification ........................................................................................................................................................................10 

5. Characterization of purified unmodified mAb............................................................................................................12 

6. Anti-idiotype vaccines......................................................................................................................................................13 

7.  Monoclonal antibodies conjugated with toxins, drugs, radionuclides or other agents (immunoconjugates).13 
C. QUALITY CONTROL AND PRODUCT TESTING...........................................................................................................15 

1. Cell line qualification.......................................................................................................................................................15 

2. Lot-to-lot quality control monitoring of unprocessed bulk lots and purified bulk lots, and final product 

specifications..........................................................................................................................................................................19 

3. Stability of product...........................................................................................................................................................23 

4. General considerations on quantitation and removal of a retrovirus contaminant...............................................23 

5. General considerations on the design and interpretation of virus clearance studies ..........................................24 

6.  Generic or modular virus clearance studies..............................................................................................................24 

7. Product testing requirements for mAb used as ancillary products.........................................................................25 

D. PRODUCT SAFETY TESTING FOR FEASIBILITY CLINICAL TRIALS IN SERIOUS OR IMMEDIATELY LIFE
THREATENING CONDITIONS................................................................................................................................................26 

1. General considerations ....................................................................................................................................................26 

2. Product safety data needed before the initiation of feasibility trials in serious or immediately life-

threatening conditions..........................................................................................................................................................27 

E. ISSUES RELATED TO MANUFACTURING CHANGES (DEMONSTRATION OF PRODUCT 

COMPARABILITY)....................................................................................................................................................................28 

1. General................................................................................................................................................................................28 

2. In vitro evidence of product comparability ....................................................................................................................28 

3. Animal studies ...................................................................................................................................................................29 

4. Clinical studies to support manufacturing changes...................................................................................................29 

III. PRECLINICAL STUDIES .......................................................................................................................................................29 

A. TESTING CROSS-REACTIVITY OF MAB........................................................................................................................29 

1. In vitro testing for cross-reactivity ..............................................................................................................................30 

2. In vivo testing for cross-reactivity ................................................................................................................................30 

B. PRECLINICAL PHARMACOLOGY AND TOXICITY TESTING....................................................................................31 

1. General considerations ...................................................................................................................................................31 

2. Animal toxicology studies................................................................................................................................................31 

3. Pharmacokinetics and pharmacodynamics ..................................................................................................................32 

4. Preclinical in vivo studies with immunoconjugates..................................................................................................33 

IV. CLINICAL STUDIES ...............................................................................................................................................................34 

A. CLINICAL CONSIDERATIONS FOR PHASE 1 AND 2 STUDIES ................................................................................34 

1. General................................................................................................................................................................................34 

4  2. Dose-setting .......................................................................................................................................................................35 

B. IMMUNOGENICITY: CLINICAL CONSIDERATIONS....................................................................................................37 

1. Monitoring the development of antibodies to mAb......................................................................................................37 

2. Clinical consequences of immunogenicity..................................................................................................................38 

C. PRODUCT-RELATED CONSIDERATIONS FOR PHASE 3 STUDIES ..........................................................................39 

D. ADMINISTRATION OF RADIOLABELED ANTIBODIES ...........................................................................................39 

1. Dosimetry...........................................................................................................................................................................39 

2. Early clinical development of therapeutic radiolabeled mAb....................................................................................41 

3. Adverse events for patients enrolled in trials of therapeutic radiolabeled mAb....................................................43 

4. Clinical development of radiolabeled mAb used as imaging agents.........................................................................43 

V. APPENDIX I: NORMAL HUMAN TISSUES USED IN CROSS-REACTIVITY TESTING..........................................46 

VI. APPENDIX II: MOUSE ANTIBODY PRODUCTION TEST............................................................................................47 

VII. APPENDIX III: ORGANS TO BE CONSIDERED IN DOSIMETRY ESTIMATES .....................................................48 

VIII REFERENCES .........................................................................................................................................................................49 

5  Points to Consider in the Manufacture and Testing 
of Monoclonal Antibody Products for Human Use 
February 1997 
I. INTRODUCTION 
A. BACKGROUND 
Points to Consider documents provide a flexible approach in which FDA provides and updates its guidance 
on regulatory issues in many areas of drug development. Such documents are particularly useful in the 
rapidly  evolving  field  of  biotechnology-derived  drugs  and  other  biologics.  The  Center  for  Biologics 
Evaluation and Research (CBER) set out to revise the "Points to Consider (PTC) in the Manufacture and 
Testing of Monoclonal Antibody Products for Human Use" with several objectives. An important goal was 
to  facilitate  initial  development  of  monoclonal  antibodies  for  serious  or  life  threatening  indications. 
Additionally,  it  was  felt  that  some  of  the  information  in  the  1994  document  required  updating  and 
streamlining.  Finally, it was necessary to review this document for consistency with current CBER policy 
and with International Conference on Harmonisation (ICH) documents dealing with this category of 
products. This updated document supersedes the 1994 version, and is designed to assist sponsors and 
investigators regarding monoclonal antibody (mAb) product development, including information to submit 
when filing Investigational New Drug Applications ("INDs") and License Applications.  Although this 
document does not create or confer any rights for or on any person and does not operate to bind FDA or 
the public, it does represent the agencys current thinking on monoclonal antibody products for human use. 
For mAb, as for other biologics, certain regulations contained in 21 CFR Parts 200-299 and 600-680 apply 
and should be consulted.  In common with the other PTC, the mAb PTC are not intended to be all-
inclusive. They represent recommendations on how to conduct the clinical development of a product up to 
and after licensure, not checklists of items to be provided before or after phase 1 trials are initiated. 
Specific products which raise issues that are not considered in these "Points" will be evaluated on a case
by-case basis. The discussion on abbreviated product testing for feasibility trials in serious and immediately 
life-threatening conditions and on generic and modular virus clearance studies does not apply to human 
products  made  in  human  cell  substrates.  Consultation  with  CBER  is  strongly  advised  for  sponsors 
considering  the  application  of  abbreviated  testing  policies  to  products  that  have  the  potential  to  be 
contaminated by human pathogens. For aspects of manufacturing and of the production facility that are not 
included  in  this  discussion  or  in  applicable  regulations,  sponsors  should  consult  with  the  Office  of 
Therapeutics Research and Review and the Office of Establishment Licensing and Product Surveillance 
respectively. 
B. DEFINITIONS 
For the purpose of this document, the terms "antibody" and "monoclonal antibody" (mAb) may be used 
interchangeably and refer to  intact immunoglobulins produced by hybridomas, immunoconjugates and, as 
appropriate, immunoglobulin fragments and recombinant proteins derived from immunoglobulins, such as 
chimeric  and  humanized  immunoglobulins,  F(ab')  and  F(ab')   fragments,  single-chain  antibodies, 
2
recombinant  immunoglobulin  variable  regions  (Fvs)  etc.  Recommendations  on  the  manufacture  of 
recombinant  products  are  contained  in  other  PTC  documents  from  CBER  (1,2).  Some  of  these 
recommendations pertaining to recombinant mAb produced in cell substrates other than hybridomas are 
reiterated in this document for convenience of consultation. This document applies to mAb used as 
therapeutic or  in  vivo  diagnostic  agents,  as  well  as  to   ancillary  products,  i.e.  mAb  used  in  the 
manufacture of other products for  in  vivo  use.  The  latter  include  mAb  that  are  used  alone  or  in 
6  conjunction with devices, for example, for ex vivo purging of cells to remove immune or tumor cells, for ex 
vivo cell collection (e.g. hematopoietic stem cells), or for purification of other products intended for  in 
vivo administration. Generally, these mAb should meet the same criteria for safety and freedom from 
adventitious agents as mAb intended for direct administration to patients. Likewise, reagents that are 
commonly used in conjunction with mAb for ex vivo manipulations of cellular products intended for  in 
vivo administration (e.g. complement, DNAase) should meet the same safety standards as mAb intended 
for direct administration to patients. However, in such cases, some procedures for virus inactivation or 
removal may be performed on the downstream product rather than on the mAb or other reagent (see 
II.C.7). Complete information on products that will be used in conjunction with the mAb, such as rabbit 
complement or DNAase, should be submitted before clinical studies begin.  This information can be 
submitted as a part of the original IND submission or in the form of a Master File. 
As used in this document, "cocktails" are defined as two or more mAb administered at a fixed ratio. 
Relevant targets may  include multiple antigens on infectious pathogens and multiple tumor-associated 
antigens.  The rationale for combining the products should be clear and based on the clinical context or 
previous  clinical  experience  with  individual  products.  Lack  of  interference  among  the  mAb  in  the 
combination should be shown and synergistic or additive effects should be characterized.  Dose-ranging 
for each of the components is highly desirable. In some instances, dose-setting may be based on preclinical 
or clinical data that show the necessity or superiority of a particular dose and ratio of mAb in the 
combination. 
As used in this document, "panels" are defined as sets of mAb directed against related antigens from 
which  one  or  more  members  would  be  used  for  an  individual  patient  based  on  target  antigen 
characterization. Such panels could be submitted for approval in a single license application.  Examples of 
panels might include anti-idiotype mAb for lymphoma and mAb directed against different bacterial or viral 
serotypes. Dose-ranging for each mAb would be necessary.  During the phase 3 trials to establish 
efficacy of the entire panel, some clinical experience with each member of the panel should be obtained. 
C. FILING INFORMATION 
It is not necessary to have all of the information discussed in this document available in the initial IND 
submission.  Rather, much of the information may be developed during clinical development, with guidance 
from CBER or other appropriate Centers by means of frequent dialogue. At pre-IND meetings, CBER 
staff may provide guidance in planning clinical development and establishing the format and content of 
initial IND submissions. Such meetings may be particularly useful when the product is a novel molecular 
entity or is produced by a novel process, and when drug development plans are unusually complex. 
The manufacture of mAb that are produced and controlled by similar procedures in the same facility may 
in some cases be documented in a single Master File. This may be particularly helpful when data from 
generic or modular virus clearance studies are used for multiple antibodies that differ only in the variable 
(v) or complementarity-determining region (CDR) and when multiple antibodies are purified by identical 
procedures (see Section II.C.6). 
See references 3 and 4 for information on filing biologics license applications. An Establishment License 
Application is no longer required for mAb intended for in vivo use (3). 
7  II. PRODUCT MANUFACTURE AND TESTING 
A. GENERAL PRINCIPLES AND DEFINITIONS 
Traditionally,  most  mAb  are  produced  by  hybridoma  cell  lines  through  immortalization  of 
antibody-producing cells by chemically-induced  fusion  with  myeloma  cells.  In  some  cases,  additional 
fusions with other lines have created "triomas" and "quadromas". We anticipate an increase in recombinant 
mAb (e.g. chimeric or humanized mAb, single-chain or dimeric Fvs, mAbs derived from phage display 
libraries etc.) and human mAb in the future. These may be produced in animal cell lines (e.g. CHO, 
SP2/0) transfected with recombinant DNA constructs, in human cells (e.g. immortalized lymphoid cells), in 
bacteria, yeast, insect cells etc.  Novel methods of production for mAb or mAb-derived recombinant 
proteins may include insects, plants or transgenic animals,. 
The principles reviewed in Sections II.B. 1 through 4 may be applied, in general to all hybridoma and 
heterohybridoma generated products, regardless of the species of origin. All steps in manufacturing of 
mAb to be used in trials intended to support licensure and of licensed mAb should comply with current 
Good Manufacturing Practices (cGMPs), as appropriate for the stage of product development. 
While manufacturing details and safety issues may be different for different expression systems, some 
general principles can be applied. The establishment of a reliable and continuous source from which the 
antibody can be consistently produced is highly recommended (e. g. master cell banks for cell cultures, 
seed banks for transgenic plants, founder strains for transgenic animals). If transient expression systems 
are used, master vector seed stocks should be generated, and the genetic stability of the expression 
constructs used should be tested. Appropriate in-process testing which takes into consideration the specific 
safety concerns of the expression system used should be instituted. Sponsors are encouraged to consult 
the most recent available versions of the Points to Consider in the Characterization of Cell Lines Used to 
Produce Biologicals, the Points to Consider in the Production and Testing of New Drugs and Biologicals 
produced by Recombinant DNA Technology or the Points to Consider in the Manufacture and Testing of 
Therapeutic Products for Human Use Derived From Transgenic Animals (1, 2, 5), the 1996 CBER/CDER 
Guidance Document on the Submission of Chemistry, Manufacturing and Controls Information for a  
Therapeutic Recombinant DNA-derived Product or a Monoclonal Antibody Product for In Vivo Use (4), 
as well as relevant International Conference on Harmonization (ICH) documents (e.g. 6, 7), if applicable 
to their expression systems. Sponsors considering novel expression systems not specifically covered by 
guidance documents are encouraged to consult with CBER. 
B. MANUFACTURE AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES 
1. Cell lines 
The following information should be provided in the IND or biologics license application: 
a.  Source, name, and characterization of the parent cell line, including any immunoglobulin heavy or light 
chains that it synthesizes and/or secretes, the fusion partner in the case of hybridomas, or the host cell line 
in the case of transfected cells producing recombinant mAb. 
b.  Species, animal strain, characterization, and tissue origin of the immune cell. 
c.  Description of immortalization procedures, if any, used in generating the cell line. 
8  d.  Identification and characterization of the immunogen. A complete biochemical characterization may not 
be possible or necessary in all cases. However, we recommend that as much information as possible be 
gathered on the nature and characteristics of the material used as an immunogen. Such data can be useful 
in choosing appropriate potency assays, as well as in evaluating potential for cross-reactivity and possible 
clinical usefulness. For example, a determinant which is not expressed on the surface of target cells bind 
necrotic cells better than intact cells. 
e.  Description of the immunization scheme. In the case of human mAb, any   in  vitro or  in  vivo 
immunization procedures should be described, as well as any relevant aspects of the subject's medical 
history. 
f.  Description of the screening procedure used. In the case of human mAb, steps performed in order to 
enrich antigen-specific human B cell populations should be described. 
g.  Description of the cell cloning procedures. If changes in cell culture process (e.g. cells adapted from 
serum-containing to serum-free medium) are shown not to affect product quality, it is not necessary to 
reclone the cells or rebank the MCB or WCB. In this context, product quality includes not just the identity, 
purity, potency and pharmacological characteristics of the purified product, but also its safety profile. For 
example, possible changes in types and/or titers of viruses detectable in the unpurified bulk material and 
the ability of the purification process to remove or inactivate them should be addressed). 
h.  For transfected animal or plant cell substrates, as well as for microbial cell substrates (bacteria, yeast), 
a  detailed  description  of  the  vector(s)  and  final  construct(s)  generation,  including  whether  or  not 
extraneous amino acid sequences are introduced into the product as a result of subcloning, and description 
of  transfection/transformation,  screening  and  selection  procedures  (see  refs.  1-6).  Determination  of 
cDNA  sequence(s)  of  the  predominant  transcript(s)  is  acceptable  as  an  indication  of  clonality  of 
transfected cell lines. 
i. For cell culture systems using autonomously replicating vectors (e.g. baculovirus or other transient 
expression systems) a detailed description of the vector system, construct generation, selection, vector 
banking procedures, and infection/transfection procedures should be provided (1-6). 
j. For all cell substrates, description of the seed lot system for establishing and maintaining the master cell 
bank (MCB) and the working cell bank (WCB). 
2.  Production in cell culture 
The following information should be provided in the IND or biologics license application: 
a.  A description of the culture procedures if production is entirely in vitro or if cells are passaged in vitro 
prior to mouse inoculation. 
b.  A description of the culture media used, including certification and testing. Serum additives used in 
hybridoma propagation should be free of contaminants and adventitious agents. 
c.  The steps taken to prevent or control contamination by viruses, bacteria, fungi, mycoplasma and 
transmissible spongiform encephalopaties (TSE) agents. These include, among other things, a description 
of the equipment, transfers, room classification, employee gowning procedures etc. 
9  d.  The acceptance criteria for cells or tissue culture supernatants intended for further manufacture. 
3. Production in animals or plants 
The following information should be provided in the IND or biologics license application: 
a.  A description of the cell line used as the inoculum (if any) should be provided (see 2.a. above). 
b.  Animal care should be in accordance with the NIH Guide for Care and Use of Laboratory Animals. 
For  ascites  production,  the  use  of  specific  pathogen  free  (SPF)  mice  is  recommended.  To  ensure 
manufacture of consistent, high quality ascites for production of mAb, an animal health monitoring program 
should be in place that encompasses quarantine procedures, sentinel animals, and an in-house  health 
surveillance program (including screening for mycoplasma). Frequency of serological testing of sentinel 
mice should be established and is usually based on the incidence of virus contamination. Screening 
programs  for  known  infectious  agents  should  be  updated  to  reflect  advances  in  the  knowledge  of 
infectious diseases. Sponsors should be responsible for the adequacy of screening programs. 
c.  All protocols for ascites production should also incorporate information on: i) species, sex and age of 
animals used; ii) animal supplier; iii) volume of pristane; iv) volume and concentration of cell inoculum; v) 
timing of priming, inoculation, and ascites harvesting; vi) frequency and procedure for ascites harvesting; 
vii) definition of a batch; viii) animal bedding, food and water; ix) number of animals housed together; x) 
environmental conditions under which each procedure takes place and  xi) number of times cells are 
passaged from one animal to another, if applicable. 
d. For production in transgenic animals, the vectors, constructs and procedures used for gene transfer 
should be described. The genetic background and characterization of founder animals, the generation and 
selection of production herds and animal maintenance procedures should be described as well (see ref. 5 
for  details).  Health  monitoring  programs  for  animal  herds  or  colonies  should  be  in  place,  including 
screening for zoonoses known to exist in captive animals of the relevant species in North America. 
Programs for screening and detection of known infectious agents should be tailored for the animal species 
and periodically updated to reflect advances in the knowledge of infectious diseases. Sponsors should be 
responsible for the adequacy of screening programs (see paragraph b above). When initially establishing 
transgenic animal strains, the following considerations should be kept in mind: i) non-transgenic animals to 
be used for breeding or gene transfer procedures should be obtained from closed herds or colonies that are 
serologically well characterized and as free as possible of pathogens of concern for the animal species or 
for humans;  ii)  the  use  of  imported  animals  or  first  generation  offspring  from  imported  animals  is 
discouraged and iii) animals from species in which TSE have been documented should be obtained from 
closed herds with documented absence of dementing illnesses and controlled food sources. 
e. For production using autonomously replicating vectors (e.g. baculovirus) in live insect larvae, larvae 
maintenance procedures should be described in detail, including procedures used to control and monitor 
bioburden. 
f. For production in plants (e.g. transgenic plants or autonomously replicating vectors using plants as 
bioreactors), early consultation with CBER staff is recommended. 
4. Purification 
Purification  schemes  for  mAb  should  be  described  in  the  IND  or  biologics  license  application.  We 
10  recommend that mAb purification schemes incorporate the following characteristics: 
a. Production techniques that will prevent the introduction of and eliminate contaminants, including animal 
proteins and materials, DNA, endotoxin, other pyrogens, culture media constituents, components that may 
leach from columns, and viruses. 
b.  Incorporation of one or more steps known to remove or inactivate retroviruses in excess of the 
endogenous particle load, whenever applicable (see Reference 8 and discussion of virus clearance studies 
in Section II.C.). As a general guidance, we recommend that each purification protocol include at least 
two orthogonal (i.e. based on different mechanisms) robust virus removal steps (see below). Including 
these steps would not obviate the need for virus clearance studies, except in the case of products intended 
for use in feasibility trials in serious or life-threatening conditions (see Section II.D.2.) 
i. Robust virus removal/inactivation steps  are defined as those that have been shown to work 
well under a variety of conditions (e.g. pH or ionic strength of column buffers) with a variety of 
mAb.  Robust steps include low pH, heat, solvent/detergent treatments, and filtration (see Table 
III). Sponsors have the option of providing adequate evidence indicating that a step different from 
these is robust, or is reliably effective for removal/inactivation under the conditions employed. An 
estimate of the efficiency of robust steps in removing virus may be demonstrated by: (a) cross-
referencing master files or reliable scientific literature published in peer-reviewed journals or (b) 
generic or modular clearance studies (see Section II.C.6. for definition). 
c.  Demonstration  of  the  ability  of  the  purification  scheme  to  remove  adventitious  agents  and  other 
contaminants, by means of a clearance study. For some contaminants, e.g. DNA, pristane or protein A, 
such a clearance study, if appropriately carried out, may be an acceptable alternative to routine testing for 
the contaminant. In the case of virus clearance studies, we recommend the use of several model viruses 
encompassing large and small particles, DNA and RNA genomes, as well as chemically sensitive and 
resistant lipid enveloped and non-enveloped strains. Human blood products should be avoided in production 
of other biologicals. When human blood products that may be contaminated with hepatitis C virus (HCV) 
or other infectious agents must be used in production (e.g. as media additives), such schemes should 
include viruses that are acceptable models for HCV, such as bovine viral diarrhea virus (BVDV) or 
Sindbis virus. Retrovirus clearance studies should be performed prior to phase 1 trials, except for products 
intended for use in the setting of serious or life-threatening conditions in feasibility trials (see Section 
II.D.). Clearance studies for other viruses and/or other contaminants should be carried out prior to 
production for phase 2/3 trials and may need to be repeated if the final manufacturing process has 
changed. ICH guidelines are currently being drafted to address in further detail the viral safety evaluation 
of biotechnology products derived from cell lines of human or animal origin. 
d.  Limits  should  be  prospectively  set  on  the  number  of  times  a  purification  component  (e.g.  a 
chromatography  column)  can  be  reused.  Such  limits  should  be  based  upon  actual  data  obtained  by 
monitoring the components performance over time. 
e. As a product is developed, retention samples from each production should be saved under appropriate 
conditions so that side-to-side comparisons may be made to determine product comparability (see Section 
II.E.). 
f. A description of the purification room(s) design features, HVAC and other support systems, equipment, 
transfers and personnel should be provided. Emphasis should be placed on operational features that 
minimize the risk of contamination from the environment or cross-contamination from other products. 
11  5.  Characterization of purified unmodified mAb 
Before  a  mAb  is  studied  in  humans,  a  precise  and  thorough  characterization  of  antibody  structural 
integrity, specificity, and potency should be conducted and described in the IND. The mAb should be as 
free as possible of non-Ig contaminants. A properly qualified in-house reference standard with known 
characteristics, specificity, and potency, and that is stored under appropriate conditions and periodically 
tested to ensure its integrity, should be used for lot-to-lot comparisons. Reference standards should be 
updated as a product evolves but should be finalized by the start of phase 3 trials. Appropriate standard 
operating procedures (SOPs) should be developed for qualification of a new reference standard. 
a.  Structural Integrity 
A combination of SDS-PAGE, IEF, HPLC, mass spectrometry, or other appropriate physicochemical 
methods should be used to show that the purified antibody is not fragmented, aggregated, or otherwise 
modified (e.g. loss of carbohydrate side chains).  Side-by-side comparisons of production lots to the in-
house reference standard should be performed. 
b.  Specificity 
Assays should provide evidence that the binding of the mAb to the target antigen is specific. Once the 
specificity of the antibody is characterized, it should be screened for cross-reactivity with human tissues 
(see Section III.A.). The following are some suggestions on the design of specificity studies: 
i.  Direct binding assays should include both positive and negative antibody and antigen controls. 
At least one isotype-matched, irrelevant (negative) control antibody should be tested. Negative 
antigen controls should include a chemically similar, antigenically unrelated compound, if available 
(e.g. similar chemical nature, size, charge, and charge density). 
ii. Whenever possible, the protein, glycoprotein, glycolipid, or other molecule bearing the reactive 
epitope, should be biochemically defined, and the antigenic epitope, itself, determined. If the 
antigenic  determinant  is  a  carbohydrate,  the  sugar  composition,  linkage,  and  anomeric 
configuration should be established. 
iii. If possible, fine specificity studies using antigenic preparations of defined structure (e.g. 
oligosaccharides or peptides) should be conducted to characterize antibody specificity by means of 
inhibition or other techniques.  For complex biological mixtures, the lots of test antigen and/or 
inhibitors used for direct binding tests should be standardized.  Inhibition of antibody binding by 
soluble antigen or other antibodies should be measured quantitatively. 
iv.  Once the specificity of an antibody has been determined, it is important to quantitate antibody 
binding  activity  by  affinity,  avidity,  immunoreactivity,  or  combinations  of  these  assays,  as 
appropriate. A number of published methods are suitable for measurement of antibody binding 
activity (9, 10). 
c.  Potency Assays and Potency Specifications 
Potency assays are used to characterize the product, to monitor lot-to-lot consistency, and to assure 
stability of the product.  Potency may be measured by a binding assay, a serologic assay, activity in an 
12  animal model, and/or a functional assay performed in vitro or in vivo.  It is desirable that the assay(s) 
bear the closest possible relationship to the putative physiologic/pharmacologic activity of the product and 
be sufficiently sensitive to detect differences of potential clinical importance in the function of the product. 
In particular, when the performance of the antibody depends not only upon antigen binding but also on 
other critical functions, it is desirable that the potency assay(s) measure all such functions. Documentation 
of the potency assay's performance, including sensitivity, intra- and inter-assay variation and robustness, 
should be provided. 
i.  Antibody  binding  activity  may  be  quantitated  by  ELISA,  RIA,  radioimmune  precipitation, 
cytotoxicity, flow cytometry, or any other standard, appropriate method.  Activity should be 
expressed  as  specific  antigen-binding units per mg or  :g  of  antibody.  Product  should  be 
compared to an in-house reference standard.  Appropriate measurements of antibody affinity, if 
established, may be a useful adjunct to other assays.  Parallel line bioassay or a similar, valid 
statistical procedure should be used in calculating potency. 
ii. The potency of a mAb may also be tested by measurement of  in vivo  function in animal 
models, although such assays are often cumbersome and difficult to standardize and should not be 
the sole measure of potency. 
iii. The permissible range of values in potency assays that reflects adequate biological activity of a 
product should be based on experience with a particular antibody.  Ideally, potency assays should 
be correlated with in vivo activity in order to develop control tests which will ensure an effective 
product. This implies that multiple production lots should be used during the clinical development 
program and potency assay results should be correlated with clinical performance. When clinical 
performance is measured by in vitro tests used as surrogates of efficacy, such tests should be 
validated in a phase 3 clinical trial of appropriate design. 
6.  Anti-idiotype vaccines 
The following issues should be addressed for anti-idiotype vaccines: 
a. In the case of an anti-idiotype vaccine (Ab2 vaccine), the Ab2 immunogen should be characterized as 
to the Ab2 type, e.g. classical type (Ab2") or antigen mimic (Ab2$) (11). 
b. Ab2$ vaccines should be shown to be reactive with the appropriate population of human Ab1 (antibody 
to nominal antigen) if such antibodies are available. 
c. The Ab2 preparation should be studied for the appropriateness of response (to target antigen) in 
xenogeneic as well as syngeneic animals (12). 
7.  Monoclonal  antibodies  conjugated  with  toxins,  drugs,  radionuclides  or  other  agents 
(immunoconjugates) 
Immunoconjugates  are  typically  produced  by  chemical  processes  using  specific  reagents  to  link  the 
unconjugated antibody with a non-antibody agent. Alternatively, immunoconjugates can be obtained as 
chimeric recombinant proteins containing non-immunoglobulin and immunoglobulin sequences in the same 
polypeptide chain. In addition to previously discussed recommendations for unconjugated (naked) mAb, 
manufacturers of immunoconjugates should address the following: 
13  a. Construction of the Immunoconjugate. 
A full description of the reagents and the process used to construct an immunoconjugate should be 
submitted, including: 
i.   A description of components such as toxins, drugs, enzymes, and cytokines that are linked to 
the mAb, including: the source, structure, production, purity (including demonstration of freedom 
from adventitious agents), and characterization of all components (if components are purchased, a 
certificate of analysis should be supplied). 
ii.  A description of chemical components, such as linkers and chelating agents, that will be used in 
preparing the immunoconjugate.  These should include documentation of the sources of reagents 
and method of preparation and determinations of residual impurities from synthesis or purification. 
Charts of the synthetic reaction pathways and any relevant published or in-house data concerning 
the toxicity of chemicals used in the production of an immunoconjugate should be provided. 
iii. The average ratio of coupled material to antibody and the number of conjugated moieties per 
antibody should be determined as the first step in establishing lot release criteria for the final 
product and developing the relationship between immunoglobulin substitution number, potency, and 
stability. 
iv.  Products prepared using recombinant DNA technology (e.g., derived from transfected cell 
lines or microbial cell substrates, chimeric, reshaped, complementarity determining region [CDR] 
grafted,  single  chain  Fv  antibodies,  and  recombinant  immunoconjugates)  should  follow 
recommendations  discussed  in  references  1-7,  as  appropriate.  The  stability  of  recombinant 
immunoconjugates  should  be  studied  carefully,  as  such  chimeric  proteins  may  have  altered 
conformational  stability,  solubility  or  tendency  to  aggregate  compared  to  their  component 
polypeptides in their native structures. Loss of specific immunoreactivity due to denaturation or 
formation of aggregates (e.g.  diabodies  formed  by  recombinant  Fvs)  may  lead  to  altered 
pharmacokinetics and/or binding to non-target tissues. 
b. Purity of the Immunoconjugate 
i.  Special care should be taken to ensure that the antibody preparations are as free as possible of 
extraneous immunoglobulin and non-immunoglobulin  contaminants  as  such  contaminants  could 
react with nuclides, toxins or drugs during the construction of the immunoconjugate. 
ii. The amount of free antibody and free components in the final product should be determined 
with limits set for each. Reactive intermediates should be inactivated or removed. 
c.  Immunoreactivity, Potency and Stability of the Immunoconjugate 
Coupling of toxin or drug to an antibody may alter the activity of either component. 
i.  Immunoreactivity before and after coupling should be assessed using appropriate methodology 
(9, 10). 
ii.  Activity of the non-immunoglobulin component of immunoconjugates, should be assessed by a 
potency  assay  whenever  appropriate  (e.g.,  toxins,  cytokines  or  enzymes,  but  not  radio
14  immunoconjugates intended for use in imaging) 
iii.   Limits  on  the  percent  change  in  immunoreactivity  resulting  from  construction  of  the 
immunoconjugate should be established as part of product specifications. 
iv.  The immunoconjugate should be tested for stability  in vitro by incubation in pooled human 
serum at 37o C under sterile conditions. Plasma may be used instead of serum, provided that the 
anticoagulant used does not affect the stability of the immunoconjugate (e.g., chelating agents may 
react with some radioisotopes, heparin may interact with basic proteins, etc.). Aliquots should be 
analyzed at timed intervals for the concentration of intact immunoconjugate and degradation 
products. The conditions under which product stability is evaluated and the positive and negative 
controls used should be fully described. Stability in human serum or plasma is not relevant for 
topically administered immunoconjugates which are demonstrated not to be absorbed into the 
bloodstream. It should be established whether or not such immunoconjugates or their components 
are detectable in plasma after topical administration, and whether or not they elicit an immune 
response. 
d. Specific Issues Related to mAb Coupled to Radionuclides 
The preparation of the radioimmunoconjugate should be performed in a standardized, well-controlled, and 
validated manner.  Methods should be developed to estimate the percent radioactivity in each of the three 
species of concern: free isotope, conjugated mAb, and labeled, non-mAb substances. 
i.  It is recommended that the initial IND submission for a radiolabeled mAb contain analytical 
results  from  two  to  three  radiolabeling  runs  that  demonstrate  the  preparation  of  an 
immunoreactive, sterile, and pyrogen-free product. These radiolabeling runs should be performed 
by the same personnel who will radiolabel the mAb for the study, using the reagents that will be 
used for the study. 
ii.  Radiopharmaceutical grade isotopes should be used when preparing immunoconjugates. The 
sterility and pyrogen-free  nature  of  each  isotope  should  be  documented  by  submission  of  a 
certificate of analysis and letters of cross-reference for manufacturing information. 
iii.  The concentrations of covalently-bound and free isotope in the final product  as  well  as 
residual levels of labeling reagents and their decomposition products should be determined during 
the trial labeling runs. 
iv.  Quality control tests that will be performed before and/or after each patient administration 
should be described. 
v. When appropriate, colloid formation by the radio-immunoconjugate should be determined and 
limits set for it. 
C. QUALITY CONTROL AND PRODUCT TESTING 
1. Cell line qualification 
Qualification of the cell line for production of a mAb to be used as a biologic therapeutic should include 
screening the master cell bank (MCB) and the working cell bank (WCB), at least on a one-time basis, for 
endogenous and adventitious agents utilizing the tests outlined in Table I and described in the Points to 
15  Consider in the Characterization of Cell Lines Used to Produce Biologicals (1). Because the WCB is 
derived from the MCB and propagated for only a few additional passages in tissue culture, abbreviated 
testing for detecting newly introduced contaminants is acceptable. Any virus contaminant should be 
quantified and, wherever possible, identified in order to establish the extent of virus clearance that the 
purification process should achieve (see also Section II.C.5.). The tropism of virus contaminants for 
human cells should be determined by appropriate infectivity assays. In the case of tissue culture or 
fermenter production, end-of-production cells (EPC) should be tested at least once to  evaluate whether 
new contaminants are introduced or induced by the growth conditions.  EPC should also be re-examined 
when there are changes in culture medium or in the scale of production. Cells at the limit of in vitro age 
used for production can be tested in lieu of EPC to allow for possible extensions in the length of time cells 
are kept in culture as manufacturing schemes are developed. The term EPC will be used throughout this 
text for ease of consultation. For cell lines which are known not to be susceptible to infection with 
mammalian viruses (e.g. plant cells, some insect cells), bacterial and fungal sterility testing, and in some 
cases  testing  for  Mycoplasma  or  other  mollicutes  such  as  Spiroplasma,  will  be  the  most  important 
concerns. Consultation with CBER is advised before using these cell substrates. 
16  Table I
 
Cell Line Qualification
 
Tests  MCB  WCB  EPC 
Sterility  +  +  + 
Mycoplasma  +  +  + 
Virus
 Adventitious  +  - +
 Species-specific*  +  - -
Retrovirus   +  - + 
Authenticity  +  +  + 
* Tests for rodent, primate, or human viruses (other than retroviruses), as appropriate 
Retrovirus testing is not required for murine hybridomas. All other cell substrates should be tested as described in Section II.C.1.d. 
a.  Cell lines should be free from bacterial and fungal contamination as demonstrated by sterility testing. 
Recommended testing procedures for mycoplasma (cultivable and non-cultivable) are described in Ref. 1. 
b.  Screening for adventitious viruses (other than retroviruses)  should include routine in vivo and in vitro 
tests (1). 
c. Screening for species-specific viruses (other than retroviruses): 
i.  The mouse antibody production (MAP) test for murine cell lines (see Appendix II), the HAP 
test for  hamster lines, and the RAP test for  rat  lines  should  be  used.  In  vivo  testing  for 
lymphocytic choriomeningitis (LCM) virus, including non-lethal strains, is recommended. Testing 
of hamster cell lines should include minute virus of mice (see III.B.1.c.). 
ii. Material that is contaminated with LCM, reovirus, Sendai virus, or Hantaan virus should not be 
used for mAb production. 
iii. Cell lines from non-human primates should be screened for the following: herpes viruses 
(simiae and SA-8), cytomegalovirus (sCMV), encephalomyocarditis virus, simian hemorrhagic 
fever virus (SHF), varicella virus of simians (sVZV), adenovirus, SV-40, monkeypox, rubeola, and 
Ebola virus. Any other zoonotic agents suggested by the cell line derivation history should be 
screened for. 
iv.  Human cell lines should be screened for Epstein-Barr virus (EBV), cytomegalovirus (CMV), 
hepatitis B (HBV) and HCV, human herpes virus 6 (HHV-6), and any other viruses that are 
suggested by the medical history of the donor and type of tissue used to establish the original line. 
Cells from patients who are known to have developed Creutzfeld-Jakob disease (CJD) or other 
TSE, or from persons with two or more genetically related family members with CJD, should not 
be used. 
v.  Heterohybridomas using cells from 2 different species should be tested as appropriate for both 
species of origin. 
17  vi. For cell lines of other species please consult with CBER. 
d.   Retrovirus  testing of cell lines: Retrovirus contamination of cells from different species varies.  The 
following should be considered when designing studies to detect retrovirus: 
i.  Murine cells used to produce monoclonal antibodies should be considered inherently capable of 
producing infectious murine retrovirus. The amount of retrovirus in the unprocessed bulk should be 
quantitated on a series of bulk harvests and shown to be consistent from lot to lot (1). Endogenous 
virus  particle  burden  should  be  determined  at  the  end  of  a  typical  fermentation,  prior  to 
purification, preferably by thin-section EM on material pelleted by ultracentrifugation. Particle 
burden determination is preferable to infectivity assays at this stage of production because it does 
not depend upon the susceptibility to infection of the cell lines used for virus amplification and it 
provides a "worst case scenario" of the level of viral contamination. Thin-section EM also allows 
morphological observation of viruses. Other, novel methods of equal or superior sensitivity and 
general applicability may be acceptable, if appropriately validated. Sufficient retrovirus removal by 
the purification scheme should be demonstrated (see also Section II.C.4.). 
ii.  Rat myeloma cell lines and hybridomas may not express retrovirus (13).The absence of 
detectable retrovirus, however, should be demonstrated by co-cultivation of the test article with a 
cell line(s) susceptible to a wide range of retroviruses combined with a sensitive detection assay, 
including  examination  of  EPC  and  several  production  lots.  If  retrovirus  is  not  detected  by 
infectivity assays or electron microscopy, further clearance studies may not be needed. It is 
suggested that purification schemes for mAb produced by rat cell lines include one or more robust 
retrovirus inactivation or removal step. 
iii.  CHO cell lines express defective retrovirus particles (14).  Whether hamster cell lines express 
infectious retroviruses has not been shown.  Sponsors should demonstrate the lack of infectious 
hamster retroviruses by means of the most sensitive infectivity assays available. These include 
co-cultivation of the test article with a cell line(s) susceptible to a wide range of retroviruses 
combined with a sensitive detection assay.  As a product moves into pivotal clinical trials, it may 
be necessary to make additional attempts to detect potential infectious virus by utilizing a wider 
range of indicator cells, including human cell lines (15, 16). Because of uncertainty about the 
validity of infectivity assays for hamster retroviruses, sufficient retrovirus particle removal by the 
purification scheme should be demonstrated (see also Section II.C.4-5). It is suggested that 
purification schemes for mAb produced by hamster cell lines include one or more robust retrovirus 
inactivation or removal step. 
iv. Hybridomas or transfected clones produced from cells of non-human primate or human origin 
should be examined for the presence of retrovirus.  Generic assays, such as transmission electron 
microscopy  (TEM)  or  reverse  transcriptase  (RT)  can  be  used  to  assess  the  presence  of 
retrovirus.  Other assays may be used, as long as they are appropriately validated.  In addition, all 
primate  cell  lines  should  be  screened  for  simian  immunodeficiency  virus  (SIV),  simian  T 
lymphotropic  virus  (STLV),  Foamy  virus,  human  T  lymphotropic  virus  (HTLV),  and  human 
immunodeficiency virus (HIV).  Cell lines from non-human primates should be additionally tested 
for presence of simian retroviruses (SRV). 
e. Each cell clone generated by stable transfection of widely used parental lines (e.g. CHO) should be 
considered as a new cell line from the standpoint of viral safety. Such clones may have significantly 
18  different genetic characteristics compared to the parental line as a result of the transfection procedure 
itself, the clonal selection process and positional effects due to random integration of vector DNA into the 
cellular genome. Each clone should be screened for retroviruses and for adventitious viruses as described 
above and in Table I. Screening for species-specific viruses can be done once, on the MCB of the 
parental line. 
f.   Authenticity testing should confirm the cell line's species of origin, identity, and lack of cell-line 
cross-contamination. 
2. Lot-to-lot quality control monitoring of unprocessed bulk lots and purified bulk lots, and final 
product specifications 
Quality control monitoring should be performed on each lot of product, as defined in 21 CFR 600.3(x). 
Table II summarizes lot-to-lot product safety testing. 
Table II
 
Lot-to-lot product safety tests
 
Tests  Unprocessed  Purified Product  Final Product 
Bulk 
Sterility  + *  +  + 
Mycoplasma  +  - -
Virus
 Adventitious   +  - -
Species-specific  +  - -
Retrovirus   +  - -
Polynucleotide  - +  -
Endotoxin  - - + 
*Bioburden testing with acceptable limits is sufficient at this stage. 
  In vitro testing with three indicator cell lines should be performed routinely for non-ascites material. In vivo testing is generally done 
once for non-ascites material but should be repeated when production methods change. 
 MAP, RAP or HAP testing for ascites only.. 
Quantitation of retrovirus (preferably by TEM) in the unprocessed bulk is important for murine hybridomas. For other hybridomas, 
generic assays for detection and quantitation of retrovirus, such as TEM coupled with appropriate co-cultivation assays are important 
if MCB or EPC are positive. 
a.  Unprocessed Bulk Lots 
i.  There should be set limits for bacterial bioburden in unprocessed bulk material.  If bioburden 
testing of pooled ascites harvests shows the presence of viable contaminants, they should be 
quantified, and allowable limits for bacterial contamination should be set based on manufacturing 
experience.  The identity of the bacterial species should be determined on a periodic basis and 
whenever  the  allowable  limits  for  contamination  have  been  exceeded.  Filtration  of  ascites 
19  harvests through a # 0.45 :m filter prior to storage is recommended (see also II.C.2.a.ii.). 
ii.  Tests  for  cultivable  and  non-cultivable  mycoplasma  should  generally  be  performed  on 
unprocessed bulk hybridoma supernatants, prior to any clarification by filtration (1).  The filtration 
of unprocessed bulk ascites through a 0.45  :m filter followed by storage at  #-60o C prior to 
testing for mycoplasma is acceptable if samples of unfiltered material are retained for testing. If 
mycoplasma contamination of animals or unpurified bulk ascites or hybridoma supernatants is 
detected, these should not be used or processed further. 
iii.   In vitro virus testing with three indicator cell lines (e.g. Vero, MRC5, 3T3) should be 
performed routinely.  In vivo testing is generally done once (as part of cell line qualification, 
Section  II.C.1)  but  should  be  repeated  when  production  methods  change  (1).  Bioreactors 
containing hamster cells can become contaminated with minute virus of mice that may escape 
detection in routine in vitro assays. MAP testing or PCR testing for this virus appear to be more 
sensitive.  In all cases, the frequency of monitoring should be specified in SOPs and justified based 
on actual experience when using continuous production in contrast to batch production. When 
contamination with a particular virus is encountered in a facility, consideration should be given to 
modifying the routine testing program in order to detect that virus. 
iv. Species-specific virus testing should be performed (see Table II and II.C.1.c.). 
v.  Murine retrovirus contamination should be quantitated routinely for bulk ascites harvests.  This 
may not be necessary if the sponsor can provide data showing that little variation exists in the 
concentration  of  mAb  and  retroviral  load  of  their  bulk  ascites  over  several  consecutive 
manufacturing runs, and the purification scheme used can remove substantially more than the 
highest load observed. If ascites production uses different groups of mice, periodic serologic 
monitoring for species-specific viruses should be performed on each group prior to their use for 
producing ascites. For tissue culture harvests, retrovirus contamination should be quantitated on 
three clinical grade production lots in order to establish the level of virus contamination for the 
specific cell line and manufacturing process (1) (see II.C.4).  This quantitation of retrovirus should 
be done preferably by generic assays such as TEM or alternatively by sensitive infectivity assays 
(see also Section II.C.1.d.). Quantitation should be repeated when changes in tissue culture media, 
duration or scale of culture are made. 
vi.     For  hybridomas  of  non-murine  origin  or  other  cell  substrates,  see  section  II.C.1.d.  for 
appropriate assays to determine whether retrovirus is present.  In those cases where MCB or 
EPC are positive for retrovirus, each lot of unpurified bulk should be examined for detection and 
quantitation of retrovirus by generic assays, such as TEM, coupled with appropriate co-cultivation 
assays. 
b.  Purified Bulk Lots (Drug Substance) 
In addition to lot-to-lot safety testing summarized in Table II, routine testing on purified bulk lots of 
unmodified and modified mAb product should include the following determinations (for discussion of 
immunoconjugates see Section II.B.7.): 
i. If a cell bank containing a known infectious agent is used, CBER staff should be consulted 
before proceeding with development, and clearance studies should be conducted to demonstrate 
the  removal/inactivation  of  this  agent  by  the  mAb  purification  scheme.  Testing  for  murine 
20  retrovirus  during  clearance  studies  should  employ  infectivity  assays  which  detect  ecotropic 
recombinant  murine  retrovirus  (ERV)  and  the  polytropic  or  mink-cell  focus-forming  murine 
retrovirus (MCF). The infectivity assay should be comprised of an amplification period on a cell 
line sensitive to infection by these murine retroviruses (for example, Mus dunni cells, 17) coupled 
with  an  appropriate  indicator  assay  (for  example,  PG4  S+L-  assay  for  MCF  virus, 
immunofluorescence assay with appropriate antibodies for detection of ERV). Assays which do 
not rely on infectivity, such as PCR-based assays, may be substituted, provided they have been 
validated for sensitivity and specificity, and that their results are correlated with those of infectivity 
assays. The first consecutive 3-5  lots  of  purified  bulk  should  be  tested  to  confirm  that  the 
contaminant was removed by the purification scheme. For cell lines containing viruses or virus-like 
particles, the absence of detectable virus should be confirmed for purified bulk by means of 
suitable methods having high specificity and sensitivity for the detection of the virus in question. 
For the purpose of marketing authorization, data from at least 3 lots of purified bulk manufactured 
at pilot-plant or commercial scale should be provided. However, for cell lines for which the 
endogenous particles have been extensively characterized, such as CHO cells, and adequate 
clearance has been demonstrated, it is not usually necessary to assay for the presence of non
infectious particles in purified bulk. Should a human infectious agent be identified in the cell bank, 
every lot of purified product should be tested and consultation with CBER staff is recommended 
before extensive product development. 
ii. Chemical purity including the residual amounts of extraneous animal proteins, e.g., albumin, 
immunoglobulin or other contaminants in the final product. An SDS-PAGE analysis, under both 
reducing  and  non-reducing  conditions,  of  increasing  amounts  of  purified  material  should  be 
provided.  Generally,  silver  staining  methods  are  more  sensitive  but  less  quantitative  than 
Coomassie blue for SDS-PAGE. 
iii.   Molecular integrity, including the presence of aggregated, denatured or fragmented product. 
iv.  Immunoglobulin class or subclass, if used as a test of identity. 
v.  IEF pattern of the antibody (or its heavy and light chains) in each bulk lot with comparison to 
the in-house reference standard. 
vi. Sterility. 
vii.  Lot-to-lot testing for DNA content, prior to any excipient addition, is recommended as a way 
to monitor purification efficiency and reproducibility. DNA content in the final product should be 
as low as possible, as determined by a highly sensitive method. Low cell viability at harvest may 
contribute to high DNA levels in unprocessed bulk. It is suggested that, whenever possible, the 
final product contain no more than 100 pg cellular DNA per dose (18). It is suggested that a 
method with a sensitivity of 10 pg be used to determine DNA levels (2). An appropriately 
conducted clearance study for DNA removal may be an acceptable substitute for lot-to-lot testing. 
viii.  Tests for detection and quantitation of potential contaminants or additives (e.g., antibiotics, 
other  media  components,  host  cell  proteins,  chromatography  reagents,  preservatives,  or 
components that may be leached from affinity chromatography columns such as protein A). 
Whenever possible, contaminants introduced by the recovery and purification process should be 
below detectable levels using a highly sensitive analytical method. However, for many of these 
potential  contaminants,  depending  upon  their  potential  for  toxicity  or  immunogenicity,  an 
21  appropriately conducted clearance study may be an acceptable substitute for lot-to-lot testing. For 
products intended for marketing, at least 3 exhibit lots should be tested to confirm the removal of 
the contaminant(s) for which clearance studies have been conducted. Such clearance studies for 
product contaminants may have to be repeated when manufacturing schemes are changed. We 
recommend that antibiotics, particularly penicillin or other beta lactams, not be used. However, if 
they  are  used,  their  removal  must  be  demonstrated  by  an  adequate  clearance  study.  The 
acceptability  of  trace  contaminants  that  cannot  be  removed  by  standard  methods  should  be 
discussed with CBER prior to the submission of an IND.  Pristane, if used in the propagation of 
ascites fluid, should be shown to be undetectable by a sensitive test. 
ix. A brief description of the formulation process and areas to be used for it should be provided. 
This description should incorporate information on the processing area, support systems, personnel 
and product transfers in sufficient detail to highlight the design or operational features utilized to 
minimize contamination or cross-contamination. 
c.  Final-Filled Product (Drug Product) 
The following tests should be performed on the contents of final containers from each filling of product as 
defined in 21 CFR 600.3(y). In certain situations (e.g. user-radiolabeling), special approaches to final 
container testing may need to be developed on a case-by-case basis after discussion with CBER: 
i. Protein quantity. 
ii. Potency (21 CFR 610.10). 
iii. Purity (21 CFR 610.13). Electrophoretic migration of the product in both the native and 
reduced states on polyacrylamide gels with comparison to the in-house reference standard can be 
used as a test for purity. 
iv.  Sterility (21 CFR 610.12). 
v. A test for endotoxin. The Limulus Amebocyte Lysate (LAL) assay may be an acceptable 
equivalent method as allowed in 21 CFR 610.9 when validated by the rabbit pyrogen test as 
described in 21 CFR 610.13.  A U.S. licensed test system should be used to perform the LAL 
assay.  Comparative testing should be repeated when LAL lots made by a different manufacturer 
are used. Conditions necessary for comparative testing of the rabbit pyrogen and LAL assay 
procedures should be discussed with CBER on a case-by-case basis.  See also "Guidelines on 
Validation of the LAL Test as an End-Product Endotoxin Test for Human and Animal Parenteral 
Drugs, Biological Products, and Medical Devices", December , 1987 (19). 
vi.  An appropriate identity test  (21 CFR 610.14). 
vii.  Moisture (21 CFR 610.13) testing, when appropriate. 
viii.  Preservative (21 CFR 610.15) testing, when appropriate. 
ix.  Excipients, when appropriate 
x.  pH, when appropriate. 
22  xi. The areas used for final fill of drug products should follow the recommendations provided in 
the Guidelines on Sterile Drug Products Produced by Aseptic Processing (20). For information 
on validation of equipment used in aseptic processing, refer to the  Guidelines for Submitting 
Documentation for Sterilization Process Validation in Applications for Human and Veterinary 
Drug Products (21). 
3.  Stability of product 
Product stability should meet the demands imposed by the clinical protocol. Accelerated stability testing 
data may be supportive but do not substitute for real-time  data for product approval and labeling. 
a.  A stability testing program should be developed that includes tests for physico-chemical integrity (e.g., 
fragmentation  or  aggregation),  potency,  sterility,  and,  as  appropriate,  moisture,  pH  and  preservative 
stability,  at  regular  intervals  throughout  the  dating  period.  See  also  "Guideline  for  Submitting 
Documentation for the Stability of Human Drugs and Biologics" (22) and the relevant ICH document (7). 
For products that are in clinical trials, significant changes that occur during storage should be reported to 
CBER. For license applications, and preferably prior to each stage of development, stability tests that 
support the proposed dating period should be performed on the final-filled product, using the container and 
closure configuration intended for distribution. For biologics license applications, storage of production 
intermediates (e.g. unpurified bulk, purified bulk) also should be supported by stability testing. 
b. Stability tests for assuring biological activity (e.g. quantitative in vitro potency assays) should include a 
manufacturer's in-house reference standard. Whenever possible, a single lot of test antigen (e.g. purified 
antigen, cells or tissue) should be used throughout the study.  A quantitative potency assay(s) should be 
used to permit a meaningful comparison of activities. 
c.  Accelerated stability studies, i.e. stability testing after storage at temperatures exceeding routine 
storage temperatures, may help to identify and establish which tests are stability-indicating.  Specific 
parameters that indicate stability should be monitored by trend analysis on every lot on the stability testing 
program. 
4. General considerations on quantitation and removal of a retrovirus contaminant 
The amount of any retrovirus contaminant in the unprocessed bulk product should be estimated. If a 
retrovirus is detected, any possible or suspected tropism for human cells should be explored (e.g. by co-
cultivation assays).  This should be followed by demonstration of removal of the contaminant, using the 
contaminant itself or a representative analogue of the known contaminant (e.g. a model retrovirus) in a 
model purification system (1). 
Unprocessed bulk supernatant concentrates or ascites should be assayed prior to any manipulation other 
than clarification by low speed centrifugation, unless it can be shown that virus testing would be made 
more sensitive by initial partial processing.  We recommend that retroviruses contaminating ascites or 
supernatants produced by rodent cell lines be quantitated by TEM of concentrated samples of supernatant. 
If TEM results are negative, it should be assumed that the titer of retrovirus is equivalent to the lowest 
limit of detection (1x106/ml).  Ascites and supernatants produced by non-rodent or hybrid cell substrates 
should be assayed as described (see Section II.C.2.b.vi.). Demonstration that retroviruses are removed or 
inactivated by the purification scheme should occur prior to phase 1 studies, except for mAb intended for 
use in feasibility trials in serious or life-threatening conditions (see Section II.D.). References 1, 2 and 8, 
23  and paragraphs 5 and 6 below, also discuss the design of studies to demonstrate retrovirus removal. The 
goal of such studies is to demonstrate that the purification process is able to remove substantially more 
virus than is estimated to be present in a single dose equivalent of starting material. 
5. General considerations on the design and interpretation of virus clearance studies 
These considerations are provided here solely for guidance purposes in the design of virus clearance (i.e., 
removal/inactivation) studies. 
a. General Experimental Design 
The objective of a virus clearance study is to provide a quantitative estimate of the level of virus reduction 
provided by the removal/inactivation procedures. Thus, the study should be designed, conducted and 
analyzed  in  a  manner  that  will  provide  accurate  information  to  reliably  assess  the  ability  of  these 
procedures to remove/inactivate viruses.  The study, including virus infectivity assays, should be designed 
according to good scientific practice to yield data with accuracy and precision that are amenable to 
statistical analysis.  Each clearance study should include an appropriate control experiment performed in 
parallel to the experimental condition to assess virus inactivation caused by experimental manipulation 
(dilution, concentration, filtration, storage etc.). Any observed difference should be used to adjust the virus 
reduction/inactivation values for each removal/inactivation procedure.  It is preferable that the study be 
conducted under current Good Laboratory Practices (21 CFR, Part 58). 
b. Statistics 
Virus clearance studies should be supported by appropriate statistical analysis demonstrating that the study 
is  valid  and  reliable.  Virus  infectivity  assays  used  to  quantitate  the  virus  titer  should  be  sensitive, 
reproducible and conducted with sufficient replicates to demonstrate statistical accuracy. Sufficient sample 
volumes should be tested to ensure that there is a high probability of detecting virus in the sample if 
present.  The experimental variability present in virus titration should be determined in virus infectivity 
assays  and  reported  using  confidence  intervals.  Within  assay  variability  should  be  calculated  using 
conventional means (standard deviation, standard error of the mean, etc.).  Between assay variation can 
be monitored by the inclusion of an appropriate virus reference preparation run in parallel with unknown 
samples. The 95% confidence intervals for within assay variation should be established and reported for 
each virus infectivity assay, and should be in the order of  " 0.5 log  of the mean. The quantitative 
10
estimate of virus reduction for each procedure should be reported as the reduction factor. This is defined 
as the log  of the ratio of the virus load in the starting material to the virus load in the post reduction 
10
material (log   (volume x virus titer of starting material / volume x virus titer of post-reduction material)). 
10
In general, this virus reduction factor should be based upon the amount of virus detected in the spiked 
starting material. The 95% confidence interval for each reduction factor is calculated from the 95% 
confidence interval of the virus infectivity assays at the beginning (+s) and end (+a) of each procedure 
using the formula  "   a 2+b2 . Such confidence intervals should be calculated whenever possible in 
studies of clearance using relevant or specific model viruses (with respect to viruses actually detected in 
the cell line as opposed to non-specific model viruses). The reduction factors for each procedure are 
summed to calculate an overall reduction factor for the entire process.  Additivity of reduction factors 
assumes that different steps are independent, i.e. have different mechanisms of action so that virus forms 
(mutant, aggregates etc.) escaping one step do not have a higher likelihood of also escaping the other. 
Reduction factors from two steps with the same mechanism of action (e.g. two incubations at low pH) are 
not necessarily additive. Due to the high intrinsic variability of infectivity assays, reduction factors of one 
log or less are considered negligible and are not included in the overall reduction factor. 
6.   Generic or modular virus clearance studies 
24  a. A generic clearance  study  is one in which virus removal and inactivation is demonstrated for several 
steps in the purification process of a model antibody.  These data may then be extrapolated to other 
antibodies following the same purification and virus removal/inactivation scheme as the model antibody. 
b. A modular clearance study  is one that demonstrates virus removal or inactivation of individual steps 
during the purification process (column chromatography, filtration, pasteurization, solvent/detergent, low 
pH, etc).  Each module in the purification scheme may be studied independently of the other modules. 
Different model mAb may be used to demonstrate viral clearance in different modules, if necessary.  If 
the purification process of a product mAb differs at any of the virus removal or inactivation modules from 
the model mAb, this module must be studied independently from the model.  The other, identical modules 
in the procedure may be extrapolated to the product mAb. 
c.  Applicability:  For monoclonal antibodies manufactured at one site, generic or modular clearance data 
may be extrapolated to other monoclonal antibodies of the same species and H and L chain class and, 
generally, subclass, derived from the same source (e.g. ascites or tissue culture) and cell substrate. 
Generic and/or modular clearance will apply only when the mAb have similar biochemical properties and 
are purified by identical methods. Particular attention should be paid to column elution buffer conditions, 
including pH and ionic strength, the sequence of columns, protein concentration, dwell times, flow rate, 
pressure, temperature, and potential problems associated with scale-up, at all steps of virus inactivation 
and removal. In some cases, sponsors may demonstrate virus removal/inactivation for a particular module 
at two different values of a given parameter (e.g. ionic strength, dwell time, temperature) and use any 
values  of  that  parameter  falling  within  this  range.  In  order  to  apply  generic  or  modular  clearance 
algorithms to a specific product, it is necessary to determine the virus load in the unpurified bulk for each 
specific product using at least 3 lots (see Section II.C.2.). This information is then used to determine 
whether  the  total  reduction/inactivation  factor  provided  by  the  purification  procedure  ensures  that 
substantially more virus is removed or inactivated than the estimated unpurified bulk titer. For example, 
generic clearance might apply to a series of murine mAb of different specificities but of the same H and L 
chain isotypes that are purified in an identical manner, or to a series of humanized mAb of the same H and 
L chain isotype but with different CDRs, that are purified by identical methods. The concepts of generic 
and modular virus clearance studies do not apply to products of entirely human origin or to products that 
have the potential to be contaminated by human pathogens. Consultation with CBER is advised before 
applying generic or modular virus clearance studies. 
d. Virus Clearance Master Files:  A sponsor purifying mAb for a variety of applications or a manufacturer 
producing mAb for a variety of sponsors may submit a Master File containing the data demonstrating virus 
removal or inactivation for different purification schemes.  This Master File may then be cross-referenced 
in INDs/IDEs or license applications using mAb purified by these schemes if applicability criteria are met. 
7. Product testing requirements for mAb used as ancillary products 
Testing requirements for mAb used as ancillary products (see I.B. for definition) vary depending on the 
clinical  indication  and  the  stage  of  product  development.  From  a  safety  standpoint,  they  should  be 
characterized in the same way as products intended for in vivo administration. However, production steps 
which follow purification of the mAb can be used as part of the virus removal/inactivation scheme. These 
include, for example, conjugation of mAb to solid phases for affinity purification, sanitation of affinity 
columns etc. The concepts of generic and modular clearance studies apply to ancillary products as well. 
Purity of the ancillary product may not be as critical as for products intended for in vivo administrations, 
provided that the performance of the ancillary product in the production of the final product is acceptable 
25  and reproducible, limits are set for impurities and the nature of impurities is known. Leaching of mAb or 
impurities from the mAb preparations into the final product should be taken into consideration in testing the 
final  product,  as  appropriate.  Removal  of  mAb  or  impurities  from  the  mAb  preparation  may  be 
demonstrated  by  means  of  a  clearance  study.  Labeling  for  the  final  product  may  need  to  carry 
precautionary statements about potentially toxic or immunogenic residual impurities. 
D.  PRODUCT  SAFETY  TESTING  FOR  FEASIBILITY  CLINICAL  TRIALS  IN  SERIOUS  OR 
IMMEDIATELY LIFE-THREATENING CONDITIONS 
1. General considerations 
The extent of product testing necessary before a particular clinical trial is initiated depends on the source 
and nature of the product, the stage of product development and the clinical indication. Abbreviated testing 
needs described in this Section (II.D.) apply to feasibility clinical trials in serious or immediately life-
threatening conditions for which no effective alternative treatment exists. Abbreviated testing does not 
apply to human products made in human cell substrates, but may apply to recombinant products made 
from transfected human genes, depending on the cell substrate.  Consultation with CBER is strongly 
advised for sponsors considering the application of abbreviated testing in this setting to products that have 
the potential to be contaminated by human pathogens. For the purpose of this document, the following 
definitions should be considered: 
i) Feasibility clinical trials. These are pilot studies whose objectives include, among others, early 
characterization of safety and initial proof of concept in a specific patient population. These trials 
are limited in scope, and are generally conducted in a single center, with a small number of 
patients (e.g. 5-20). These trials cannot be used by themselves to support licensure of a product. 
Studies conducted in normal volunteers are not included in this definition. 
ii) An immediately life-threatening condition is "a stage of a disease in which there is a 
reasonable likelihood that death will occur within a matter of months or in which premature death 
is likely without early treatment" (21 CFR 312.34). 
See 21 CFR  312.34 and the Federal Register vol. 52, No. 99 (May 1977) for a discussion of serious or 
life-threatening conditions. Application of abbreviated testing requirements to serious conditions which are 
not immediately life-threatening as defined in 21 CFR 312.34 will depend upon an assessment of the 
potential  risks and benefits to the patient(s). Factors that should be considered in such a risk-benefit 
analysis include, among others: i) the nature and manufacture of the product; ii) the nature and severity or 
stage of the disease; iii) the anticipated effect(s) of product administration (e.g., diagnosis, palliation or 
cure); iv) the availability of comparable or satisfactory alternate treatments  v)  characteristics  of  the 
patient population (e.g. age, response to previous therapy); vi) the number of patients involved and vii) the 
design of the clinical trial (e.g. patient follow-up, safety monitoring etc.). Pre-IND consultation with CBER 
staff is strongly recommended for sponsors planning to use the abbreviated testing described below for 
serious but not immediately life-threatening diseases. The guiding principle for these trials is that sufficient 
information should be provided before testing in human subjects to assure that patients and their contacts 
will not be put at unacceptable risk. Informed consent issues for these trials are discussed in Section 
IV.A.1.e. The limited testing described below should not be used to support development beyond the stage 
of feasibility trials. Therefore, sponsors are encouraged to plan for additional testing and characterization 
as  described  in  Section  II.C.  when  they  intend  to  pursue  advanced  clinical  development  and  seek 
licensure. In designing the purification process, it is advisable to include at least two orthogonal robust virus 
inactivation/removal steps. This would further reduce the testing necessary to begin initial clinical trials. 
26  2. Product safety data needed before the initiation of feasibility trials in serious or immediately 
life-threatening conditions 
a. Sterility (bacteria and fungi)  testing should be performed on the final product. Three vials of final 
product should be tested. Routine methods in use in the sponsor's hospital accredited clinical diagnostic 
laboratory can be used for these tests.  Mycoplasma and endotoxin testing are strongly encouraged. 
b. In vitro and in vivo testing for adventitious viruses : If the unpurified bulk product is free of adventitious 
viruses by in vitro and in vivo tests or if the purification scheme does include at least two orthogonal 
robust steps (see Table III), these tests are not required. 
Table III shows ranges of retrovirus removal (expressed in decimal logs) that might be expected with 
various robust inactivation/removal steps and is presented to aid manufacturers in the design of mAb 
purification schemes. 
Table III 
Inactivation Step  Reported log virus removal 
pH ##  3.9  3-4 
heat  4 
solvent/detergent  5 
filtration (15-40 nm)  4-8 
If the mAb is produced as an ascites fluid and the mice used have been MAP tested and found free of 
species-specific viruses, adventitious virus testing of the final product is not necessary. When testing is 
necessary, we recommend that a minimum amount of product equal to 3 maximum human doses be used 
for these tests.  For the purpose of these trials, MAP testing can be limited to known human pathogens: 
Hantaan, LCM, Reovirus and Sendai virus. For mAb produced in primate cell lines or in non-murine cell 
lines whose potential for contamination by human pathogenic viruses in unknown, the cell lines or the EPC 
or the unpurified bulk product should be tested as described in Section II.C.1. c and d. 
c. Murine retrovirus testing  of the final product is needed on the final-filled product only when the antibody 
is produced in a murine cell substrate and the purification scheme does not include at least two robust 
orthogonal virus inactivation/removal steps (see Table III). We recommend that a minimum amount of 
product equal to 3 maximum human doses be used for these tests. Testing of the final product, when 
necessary, should be done by a highly sensitive infectivity assay, such as amplification in Mus dunni cells 
followed by detection in PG4 cells or by other sensitive means as outlined in II.C.2.b.vi. 
d. MAb used as ancillary products (see I.B. for definition) in feasibility clinical trials in serious or life-
threatening conditions.  Two cases can be distinguished: 
i. When the final product can be tested and the results are available prior to administration (e.g. 
purified recombinant proteins or cells that can be stored frozen), safety testing can be carried out 
on the final product itself, as determined during review of the final product. 
27  ii. When the final product is administered prior to or without any safety testing and/or processing, 
testing of the mAb should be performed as described above in paragraphs D.2. a. through c. In 
this case, amounts of mAb comparable to those used in one run of final product purification should 
be used. 
The same safety considerations apply to complement, DNAase and other biological reagents used as 
ancillary products for cell depletion in conjunction with mAb. 
E. ISSUES RELATED TO MANUFACTURING CHANGES (DEMONSTRATION OF PRODUCT 
COMPARABILITY) 
1. General 
Changes in the product manufacturing scheme frequently occur during clinical development of mAb. 
Sponsors should develop a plan for demonstrating that the products made by the old and new schemes are 
comparable, particularly when preclinical or clinical data developed prior to the production changes will be 
used to support further clinical trials and/or marketing applications (23). Similar considerations apply in the 
case of significant scale up in the manufacturing process (with or without modification of the general 
manufacturing scheme) implemented during or after completion of phase 3 trials. 
When changes in manufacturing occur during early clinical development, plans for evaluation of product 
comparability should be incorporated into product development strategies. Such plans should be discussed 
with CBER and, when appropriate, submitted to CBER for review (see ref. 23). 
In-process specifications may be affected by manufacturing changes or process scale-up and appropriate 
revisions should be undertaken.  Similarly, process validations (e.g., virus clearance studies, removal of 
contaminants or leachables) for all affected steps should be repeated after any significant manufacturing 
change.  In the case of process scale-up, it is recommended that, whenever possible, a column geometry 
and a ratio between sample volume and bed volume as close as possible to that of the original process be 
maintained. This is particularly important for those steps (e.g., size-exclusion chromatography) where 
these parameters are critical to the chromatographic process.  Ref. 23 should be consulted for more 
details on demonstrating product comparability. 
2. In vitro evidence of product comparability 
In general, when a product is obtained by a modified or scaled-up manufacturing scheme, the results of a 
rigorous physico-chemical characterization and in vitro functional comparison (see Product Manufacture 
and Testing, Section II.B.) will dictate whether additional data (e.g., pre-clinical and/or clinical data) will 
be needed. A protocol for demonstrating physico-chemical, immunochemical and biological comparability 
of two products should prospectively define acceptable variation in the results of individual assays and 
acceptance criteria for product comparability. For quantitative assays (specific biological assays) accurate 
estimates of inter and intra-assay variations should be provided.  Assays with high intrinsic variability are 
poorly suited for the evaluation of product comparability. Comparisons should test a number of separate 
product lots in parallel in order to demonstrate the reproducibility of the new manufacturing scheme. An in 
vitro biochemical characterization of mAb comparability should include a side by side comparison of the 
two products by a number of different techniques. Properly stored retention samples from previous lots 
should be used for such side by side comparison. A list of techniques could include SDS-PAGE under 
reducing and non-reducing conditions, Western blot, size-exclusion analytical chromatography, reverse 
phase  high  performance  liquid  analytical  chromatography,  isoelectrofocusing,  mass  spectrometry,  an 
28  analysis  of  glycosylation  including  carbohydrate  content  and  composition,  peptide  mapping  or  other 
appropriate tests. In vitro functional comparison should include assays aimed at the characterization of the 
biological function of the antibody (e.g., binding, cytotoxicity, epitope modulation, etc.). Whenever possible, 
a comparison of the affinity constants of the two products is highly recommended. 
3. Animal studies 
Depending on the quality of the data and the type of  in vitro assays, the nature of the manufacturing 
change and the types of product differences observed or anticipated, a program of comparative testing 
(pharmacokinetics, etc.) in appropriate animal models may be considered in lieu of human clinical data 
when biochemical testing shows differences or cannot exclude significant differences in two products. In 
some cases, pharmacokinetic studies are complementary to  in vitro studies. Pharmacokinetic studies in 
animals may be informative, even in the absence of the target antigen, depending upon the question to be 
addressed and the expected contribution of antigen binding to the biodistribution of specific mAb in 
humans. The extent of animal toxicity testing that may be needed to assess comparability will depend upon 
the safety profile of each specific product, the magnitude of the changes in manufacturing, the presence or 
absence of detectable differences in purity, structure or  in vitro activity. Sponsors are encouraged to 
discuss plans for comparative testing of the two products in animals with CBER or to submit proposal for 
such testing to CBER for review and comment.  The proposed program should be appropriate in view of 
biochemical data and include statistical considerations. 
4. Clinical studies to support manufacturing changes 
Comparative clinical evaluation of the products produced by different or scaled-up manufacturing schemes 
may be needed in certain situations: 
a.  Product activity cannot be adequately characterized by analytical testing. 
b.  Biochemical or biological testing show differences in the products. 
c.  Animal testing reveals significant pharmacokinetic or other differences in the products. 
d. The formulation of the product has been changed in a way that can affect its bioavailability. The latter 
changes generally dictate a need for clinical pharmacokinetic studies. 
Pharmacokinetic, safety and/or efficacy data may be required depending upon the nature and magnitude 
of the observed changes in the biochemical and or biological properties of the product. 
Additional information on product comparability testing can be found in ref. 23. 
III. PRECLINICAL STUDIES 
A. TESTING CROSS-REACTIVITY OF MAB 
When the same or related antigenic determinant is expressed on human cells or tissues other than the 
intended target tissue, binding of the antibody to this tissue may be observed.  Non-target tissue binding 
may  have  serious  consequences,  particularly  when  pharmacologically  active  antibodies  or  cytotoxic 
immunoconjugates  are  used.  Accordingly,  cross-reactivity  studies  with  human  tissues  (or  cells  if 
29  applicable) should always be conducted prior to phase 1 to search for cross-reactions or non-target tissue 
binding. In the special case of bispecific antibodies, each parent antibody should be evaluated individually, 
in addition to testing of the bispecific product. 
1. In vitro testing for cross-reactivity 
Human  cells  or  tissues  are  presently  surveyed  immunocytochemically  or  immunohistochemically. 
Appropriate newer technologies should be employed as they become available and validated. 
a.  Reactivity of the antibody or immunoconjugate should be determined with the quick-frozen adult tissues 
listed in Appendix I. Surgical samples are preferred.  Post-mortem samples are acceptable with adequate 
tissue preservation. Tissues from at least three unrelated human donors should be evaluated in order to 
screen for polymorphism.  The effect of fixatives on tissues that are known to be positive should be 
evaluated to ensure that the target antigen is preserved during tissue processing. 
b. In special situations it may be appropriate to assay cross-reactivity on representative cultured cell lines, 
stem cells, and embryonic/fetal tissue. 
c.  Several concentrations of the product should be tested.  The ability to detect cross-reactions may 
depend on antibody concentration.  Antibody affinities as well as expected achievable peak plasma 
concentrations should be considered when choosing the proper concentrations for tissue binding studies. 
An "ideal" concentration for these studies may be the lowest mAb concentration that produces maximum 
(plateau) binding to the target antigen.  An attempt should also be made to compare the ratio of specific 
binding to target tissue to specific binding to cross-reactive tissue.  Because non-specific as well as Fc
mediated binding may be observed, it should be distinguished from specific cross-reactions using inhibition 
assays with purified antigen, when available. 
d.  Positive  and  negative  controls  are  essential  for  interpreting  study  results.  Controls  confirm  the 
acceptable condition of the tissues and adequacy of the assay.  Anti-transferrin receptor mAb may be a 
useful positive control, since transferrin receptor is a common and abundant molecule on the surface of 
growing normal and tumor cells. 
e. If a conjugated, chemically modified antibody or antibody fragment is to be used clinically, it should be 
tested in that form if at all feasible.  The substitution of antibodies of similar specificity for cross-reactivity 
testing is discouraged. 
f. When cross-reactions are encountered and there is a reason to suspect genetic polymorphism of the 
target  antigen,  studies  should  be  expanded  to  a  larger  panel  of  tissues  to  better  characterize  this 
polymorphism. 
g. A comparison of in vitro cross-reactivity in tissues from different species is important in determining 
the most relevant animal for subsequent toxicology studies. 
2. In vivo testing for cross-reactivity 
Cross-reactivity of a monoclonal antibody with non-target human tissues should dictate a comprehensive 
in vivo investigation in animals, when appropriate models are available. This finding, particularly with 
cytolytic immunoconjugates or antibodies with ADCC activity, generally indicates the desirability of more 
extensive preclinical testing, including studies in more than one animal species over a range of doses and 
30  repeat dose animal studies. Localization to non-target tissues should be kept in mind when designing 
clinical trials. 
B. PRECLINICAL PHARMACOLOGY AND TOXICITY TESTING 
1.  General considerations 
a. Preclinical safety testing of mAb is designed to identify possible toxicities in humans, to estimate the 
likelihood and severity of potential adverse events in humans, and to identify a safe starting dose and dose 
escalation,  when  possible.  Preclinical  testing  concerns  surrounding  mAb  products  include  their 
immunogenicity,  stability,  tissue  cross-reactivity,  and  effector  function(s).  Species  differences  may 
complicate the design and interpretation of preclinical studies. CBER recognizes that animal models 
expressing the antigen of interest or a closely related, highly cross-reactive epitope are not always 
available. Pharmacokinetic and pharmacodynamic properties of mAb that are dependent upon specific 
antigen binding may not be evident in animal studies conducted in species which do not express the antigen 
of interest. In some cases, xenograft models can be developed by introducing cells expressing the antigen 
of interest into immunodeficient mice (e.g.  SCID or nude mice). Such models can provide information on 
specific targeting of desired cells, especially with radiolabeled mAb or immunoconjugates. Transgenic 
models expressing the antigen of interest are another possibility, if available.  Whenever they are available, 
parallel models which explore the effects of mAb against the animal homolog of the antigen of interest can 
be informative. In vivo activity models have proven valuable in providing data which support a rationale 
for the proposed product use and in defining safety and toxicity. Animal disease models are available to 
study the effects of mAb on many inflammatory and autoimmune diseases, and allograft rejection. The 
extent of preclinical safety testing and the results of such testing will influence safety considerations for 
initial clinical trials (e.g. starting dose, dose escalation scheme, etc.). 
b. Preclinical testing schemes should parallel to the greatest extent feasible those anticipated for clinical 
use with respect to dose, concentration, schedule, route, and duration.  The range of doses selected for 
study should include at least one dose that is equivalent to and one dose that is a multiple of the highest 
anticipated clinical dose, with appropriate adjustments for interspecies differences in body size.  A broad 
dose range should be explored. The highest doses tested should elicit adverse effects, whenever possible. 
Dose ranges are best established with a minimum of three doses.  The linearity and overall shape of the 
dose response curve should also be defined by investigation of several doses and dosing intervals. If 
changes in manufacturing and/or formulation are made subsequent to conduct of preclinical studies, the 
decision to repeat some or all preclinical studies should depend on an assessment of the impact or likely 
impact of these changes on the product (see Section II.E.). 
2. Animal toxicology studies 
When planning toxicity testing for mAb, the following should be considered: 
a.  If the test article is an unconjugated antibody and there is no animal model of disease activity or animal 
that carries the relevant antigen, and cross-reactivity studies with human tissues are clearly negative, 
toxicity testing may not be necessary. 
b.  When a relevant animal model is available, an attempt should be made to study the dose-dependence of 
pharmacodynamic effects.  The use of a broad range of doses, including high doses may allow a better 
prediction of the therapeutic index. 
c.  The  properties  of  a  relevant  antigen  in  the  animal  should  be  comparable  to  those  in  humans  in 
31  biodistribution, function, and structure.  For example, studies of CD34+ progenitor cells in the baboon are 
useful  because  the  same  cell  fractions  in  both  species  express  the  CD34+  antigen  and  produce 
hematopoietic engraftment.  Absolute equivalence of antigen density or affinity for the mAb, however, is 
not necessary for an animal model to be useful.  Differences in binding, for example, may be compensated 
for by alterations in the dose or dosing frequency.  Differences between the animal and human in antigen 
number, the affinity of a mAb for the antigen, or the cellular response to mAb binding, should be identified. 
This will allow more accurate extrapolation of safe human starting dose and estimation of the margin of 
safety. 
d.  Routine assessments of mutagenicity are not generally needed for mAb. 
e.  Reproductive and developmental studies including teratogenicity in an appropriate animal species should 
be carried out in instances in which the product is intended for repeat or chronic administration to women 
of childbearing potential. Results of such peri- and post-natal developmental studies should be submitted 
for marketing approval. Evaluation of male fertility, when appropriate, should be completed before phase 3 
trials. 
3. Pharmacokinetics and pharmacodynamics 
A pharmacokinetic model may aid in the interpretation of preclinical activity and toxicity, and in the 
recommendation of an appropriate dosing regimen and thereby improve the design of clinical trials.  Such 
studies  should  aim  at  determining  pharmacokinetic  and  pharmacodynamic  endpoints.  Of  particular 
importance to the selection of clinical dosage is determining the relationship activity to area under the 
curve (AUC) of tissue or blood concentration over time. In considering the relationship of activity to AUC, 
factors related to the pharmacodynamics of the monoclonal antibody should be used in evaluating potential 
clinical effects.  These factors include pathophysiologic status, threshold for effects as well as molecular 
events like the rates of association and dissociation for the site of action. Studies of biodistribution may 
provide the initial evidence for inappropriate tissue targeting by a mAb or explain toxicities that are 
observed in animals.  Interpretation of data should consider species of origin, isotype, whether the mAb is 
an  intact  immunoglobulin,  a  fragment  or  an  immunoconjugate,  method  of  labeling,  stability  of  the 
immunoconjugate, level of antigen expression in the recipient, binding to serum proteins, and route of 
administration. Even if antigen is expressed in an animal model, the mAb may bind the human target 
antigen  and  its  animal  counterpart  with  different  affinities.  MAb  half-life  may  also  be  affected  by 
glycosylation, susceptibility to proteases, presence of circulating antigen, and host immune response. The 
presence of antibodies to the product may alter biodistribution and elimination. In some cases, informative 
pharmacokinetic  studies  may  be  obtained  in  animal  models  which  do  not  express  specific  antigenic 
determinants,  depending  upon  the  role  played  by  antigen  binding  in  product  biodistribution, 
biotransformation and excretion. 
a.  Selection of the animal species for pharmacokinetic and pharmacodynamic testing should be guided by 
the following considerations: 
i.  Preference should always be given to study of a mAb in an animal model in a species that 
shares a cross-reactive or identical target antigen with humans, whenever such a species is 
available. For unconjugated mAb directed at human antigens not expressed in animal models or 
foreign antigens (bacterial, viral, etc.), studies in animal species lacking the target antigen may not 
be necessary unless they are designed to address manufacturing issues (see Section II.E. on 
Product Comparability). 
ii. Study of non-human primates is appropriate for unconjugated mAb when there are antigen 
32  binding data that indicate that primates are the most relevant species. 
iii.  Normal rodent and murine xenograft models should be critically evaluated for their likelihood 
of predicting accurately human pharmacokinetic behavior of mAb. Xenograft models may be 
more useful in evaluating the ability of mAb to bind to human tumors in vivo. 
b.  Changes  in  manufacturing  or  formulation  may  result  in  significant  changes  in  biological  activity. 
Therefore, it is recommended that the material used in the preclinical studies be manufactured using the 
same procedures as used or intended for use in manufacturing material for clinical trials.  In some cases it 
may be appropriate to modify the components of the formulation for preclinical testing. For example, 
substitution of the homologous animal serum albumin for human serum albumin that is used as a carrier 
will prevent the formation of anti-albumin antibodies in animal studies and thereby increase the relevance 
of preclinical testing. 
c. Pharmacokinetic parameters should be defined using one or more assay methods (e.g., a radiolabeled 
mAb  should  be  assayed  by  ELISA  and  by  measurement  of  radioactivity).  In  the  case  of 
immunoconjugates of any type, intact conjugate should be distinguished from free mAb and free ligand 
(e.g.  toxin,  drug,  or  radionuclide).  Pharmacokinetic  parameters  that  are  most  important  for  product 
characterization, as well as most useful for determining product comparability include T , C , T  and 
max max 1/2
AUC. 
d.  The development of anti-immunoglobulin antibodies greatly complicates study and interpretation of the 
effects of repeat dosing in animals.  Murine antibodies are non-immunogenic in mice but are immunogenic 
in humans, making it difficult to extrapolate the results of repeat dose studies in mice to planned repeat 
dose  administration  in  humans.  The  reciprocal  problem  will  occur  with  fully  human,  chimeric  or 
"humanized" mAb.  Repeat dose studies in rodents in this case may be of little value. 
4.  Preclinical in vivo studies with immunoconjugates 
a. Immunoconjugates should be tested for stability in vivo. 
i. Individual components of an immunoconjugate should be measured during pharmacokinetic and 
tissue distribution studies in animals and compared to the distribution of unconjugated antibody. 
ii.  The target tissues for the various components and the potential toxicities that they may cause 
should be established. 
b. Immunoconjugates containing radionuclides, toxins, or drugs should undergo animal toxicity testing even 
when the target antigen is not present in an animal species, because of possible conjugate degradation or 
activity in sites that are not the result of mAb targeting. Depending upon the nature of the components of 
the immunoconjugate and the stability of the conjugate itself, separate studies of the components may be 
warranted. The toxicity profile of each component should adequately describe the incidence and severity 
of  possible  adverse  effects.  Results  should  be  correlated  closely  with  studies  of  conjugate  stability. 
Studies of the immunoconjugate should be performed in a species with the relevant target antigen or 
disease model, whenever available and generally in rodents if a target antigen-positive species is not 
available.  Toxicity testing of free toxin or nuclide may be performed in a different species. 
c.  For immunoconjugates containing radionuclides: 
33  i. Animal biodistribution data may be used for initial human dose estimation. 
ii.  Animal models that express the targeted antigen,  whenever such models are available, are 
more likely to reveal the effects of antigen "sinks" or tissues with unexpected antigen expression 
on biodistribution and/or toxicity. 
iii.  Xenograft  models  may  evaluate  tissue  targeting  and  antigen  non-specific 
radioimmunoconjugate distribution problems, but are not helpful at identifying areas of normal 
tissue cross-reactivity. 
iv. An adequate number of animals should be studied to achieve radiation dose estimates with an 
acceptable coefficient of variation (usually less than 20%). 
v.  There should be complete accounting of the metabolism of the total dose of administered 
radioactivity and an adequate number of time points to determine early and late elimination phases. 
vi.  Radioimmunoconjugates should be tested for stability  in vitro by incubation in serum or 
plasma (see Section II.C.7.). Methods should be developed to estimate the percent radioactivity in 
each of the three species of concern: free isotope, conjugated mAb, and labeled, non-mAb 
substances. 
IV. CLINICAL STUDIES 
MAb administered to humans have usually been well-tolerated.  Instances of serious or fatal adverse 
events have generally resulted from intended or unintended binding of mAb to specific antigens.  These 
events  emphasize  the  importance  of  screening  tissues  for  mAb  cross-reactivity,  particularly  when 
relevant-antigen animal models are not available. The results of preclinical tests may alert physicians to 
potential toxicities and may indicate that more conservative dosing schemes are justified during dose 
escalation. 
A. CLINICAL CONSIDERATIONS FOR PHASE 1 AND 2 STUDIES 
1. General 
a.  Different approaches to Phase 1 and 2 studies may be warranted depending on the nature of the mAb. 
Initial studies of therapeutic mAb in phase 1 are generally escalation studies of single-doses of the mAb. 
The goal should be to determine a presumed optimal biologic dose (OBD) that is usually defined by 
pharmacokinetics or pharmacodynamic measurements (e.g., degree of antigen binding or saturation or 
target  blood  levels,  determined  on  the  basis  of  preclinical  studies)  and,  where  appropriate,  by  the 
tolerability to the agent (e.g., the maximally tolerated dose [MTD]) (24-26). In the case of a therapeutic 
unconjugated mAb, studies that identify the MTD may not be necessary.  Instead, determination of a 
presumed OBD may be a more appropriate goal.  Immune activation, when relevant to the mechanisms of 
action or toxicity of the mAb, should be evaluated. In the case of radiolabeled therapeutic mAb or 
immunotoxins, undesired tissue targeting and release of conjugate due to degradation are major safety 
concerns.  Patients  receiving  immunotoxins  should  be  monitored  for  capillary  leak  syndrome  and  for 
hepatic, renal, and muscle toxicities. 
Some antibody-specific side effects are more likely to occur with certain subclasses of immunoglobulins. 
34  These antibodies (e.g., human IgG1 and IgG3, mouse IgG2a) are more likely to activate complement or 
activate antibody-dependent cell-mediated toxicity (ADCC) via their Fc regions, leading to lysis of bound 
cells.  MAb may also cause desired or adverse effects by blocking or inducing functions of target cells 
(e.g., cytokine release syndrome following stimulation of T-cell receptors by anti-CD3). 
b.  In general, subjects in clinical trials of therapeutic and diagnostic products, including mAb, should be 
representative  of  the  population  targeted  for  eventual  product  use.  Because  of  the  potential 
immunogenicity of mAb, healthy volunteers may not be appropriate candidates for phase 1 trials.  The 
nature of the mAb, the target antigen, and the proposed clinical application should be considered before 
deciding to enroll healthy volunteers in a trial.  Situations in which healthy volunteers might be used in early 
trials include the following: 
i.  When the risks of studying a new agent initially in the index population are too high, such as 
when the index population expresses abnormally high levels of antigen (raising specific toxicity 
concerns) or when the index population may be particularly vulnerable to toxicity because of 
serious illness or significant organ dysfunction. 
ii.  When the index population is so ill that safety data are confounded and difficult to interpret. 
When healthy volunteers are considered for inclusion in initial studies of a mAb, the informed consent 
should reflect the absence of direct medical benefit. For healthy volunteers, as for patient volunteers, the 
informed consent process should also illustrate the potential immediate and long-term risks of receiving 
xenogeneic proteins.  These include possible toxicity, allergic reactions, and, in the case of murine and 
other non-human mAb, potential future inability to receive or benefit from a diagnostic or therapeutic mAb 
because of the development of an immune response against the foreign protein. 
c.  Sponsors and investigators should carefully consider whether single doses of the mAb, multiple doses 
of the mAb in a single course, or multiple courses of therapy will be most likely to optimize benefit over 
risk. Concomitant therapies or repeat administration of the mAb may alter its safety and efficacy profiles. 
Changes in antigen mass (e.g., due to binding and clearance, or to antigen modulation by the mAb) and 
immune responses to the mAb, for example, may prevent extrapolation of single dose data to multiple-dose 
schedules.  Furthermore, repeat administrations in the face of an antibody response against the therapeutic 
agent may lead to toxicity and/or loss of therapeutic benefit. 
d. Subjects with prior parenteral exposure to xenogeneic proteins or with a history of xenogeneic protein 
allergies should be excluded from phase 1 studies of mAb products that have been derived from the same 
or a closely related species. 
e. Informed consent issues in feasibility clinical trials in serious or immediately life-threatening conditions: 
If  applicable,  informed  consent  forms  for  these  trials  should  clearly  state  the  following  in  language 
understandable to the patients: 
i. only a limited characterization of the processes used to prepare the product for their ability to 
remove endogenous or exogenous infectious/toxic agent(s) was performed, and 
ii. there may be potential health risks, including hitherto unknown risks, derived from exposure to 
such agents if they are present in the product. 
2. Dose-setting 
35  a.  Whenever possible, the selection of the phase 1 starting dose should be based on safety and toxicity 
information derived from testing in a relevant animal model. When extrapolating from animal doses to 
human doses, information about the relative affinity of the mAb for the human antigen as compared to its 
animal analogue may be of great value. The target in vivo dose or concentration range should be based 
both on in vitro studies of cells for which antibody-antigen affinity and functional activity (e.g., immune 
modulation, cytotoxicity) have been measured, as well as on study of a relevant-antigen animal model, if 
available, to assess in vivo activity.  If animal studies are judged to be impossible or of no relevance and 
initial in vivo studies are to be performed in humans, testing should begin at a low dose that is based on 
extrapolation from tissue culture studies or from available information gathered in clinical trials of a similar 
mAb. 
b.  Initial studies of radioimmunotherapeutic mAb should also employ escalating single-doses of the mAb, 
with the lowest and highest doses based on animal dosimetry and on the projected tolerance of normal 
organs  to  radiation.  Both  the  elimination  half-life  of  the  mAb  and  the  elimination  half-life  of  the 
radioactivity should be characterized. 
c.  If a multiple-dose regimen of a mAb is anticipated, multiple-dose schedules should be explored in late 
phase 1 or phase 2 trials, after basic data on toxicity, peak levels, clearance, distribution, and biologic 
effects are available from single-dose studies.  The time required for recovery from the biologic effects of 
single doses (e.g., immune recovery after CD4+ cell depletion or modulation, return of bone marrow 
function after radioimmunotherapy) should also be well understood prior to initiation of multiple-dose 
regimens.  The rationale for dosing schedules should be provided. The rationale should be based on dose 
tolerance, available pharmacokinetic and pharmacodynamic data in humans, and on relevant animal models 
of safety and efficacy. Modified dosing regimens to compensate for a high antibody response against the 
agent or circulating antigen may need to be studied. Pharmacokinetic studies to determine the relationships 
of human anti-mAb antibody titers and circulating antigen levels with organ distribution, clearance, and 
toxicity may be necessary. 
d.  Before repeat administration of a radioimmunotherapeutic or immunotoxin, the investigator should 
characterize all organ toxicities and pathology resulting from single dose administration. The timing of 
recovery  from  all  toxic  effects  should  be  determined.  Intra-patient  dose  escalation  may  confound 
interpretation of safety data because it may be difficult to determine whether toxic effects (e.g., to bone 
marrow) are due to prolonged therapy or to increased dosing levels.  Intra-patient dose escalation may be 
appropriate if no toxicity is seen at the initial dose levels or if it is possible to use initial safe "test" doses 
and if cumulative toxicity is deemed unlikely.  If intra-patient dose escalation is performed, consideration 
should be given to threshold and carryover effects, as well as to the reversibility of clinical and laboratory 
adverse events. 
e.  Design  of  pharmacokinetic  studies  should  include  consideration  of  the  species  in  which  the 
immunoglobulin is produced, the immunoglobulin class and subclass, and the structure of the antibody (e.g., 
whole mAb, Fab fragment) or immunoconjugate.  A relevant study population will have  the appropriate 
antigen and antigen mass.  If antigen mass is likely to alter the bioavailability of the mAb, this should be 
determined in pharmacokinetic studies so that its impact on dose setting and on stratitifcation and analysis 
of later trials can be considered. Aside from obtaining estimates of common pharmacokinetic parameters, 
pharmacokinetic studies may also be very useful in situations in which the comparability of different 
products or formulations is to be determined (see Sections II.E. and III.A.). Pharmacokinetic studies 
optimally include the following: 
36  i.  Determination of plasma concentration profiles, distribution, and clearance of the mAb. 
ii.  Determination of  doses for further study based on dose-concentration effect relationship and 
correlation with desired concentrations estimated from in vitro studies. 
iii. Determination of peak and trough mAb levels and elimination rate constants. 
iv.  Determination  of  the  organs  and  sites  where  the  mAb  is  distributed,  metabolized,  and 
eliminated. 
v.  Determination  of  the  fate  of  immunoconjugates  by  assaying  the  whole  molecule  and  its 
components. 
vi. Investigation of the relationships between the elimination rate and the method of administration, 
antigen  load,  and  presence  of  a  circulating  antigen  or  of  an  antibody  response  against  the 
therapeutic agent. 
B. IMMUNOGENICITY: CLINICAL CONSIDERATIONS 
Monitoring of antiglobulin titers and immune activity is of great importance in evaluating the safety and 
efficacy of mAb and in designing protocols involving repeat administration.  Immune responses to mAb 
may have little or no effect, or may interfere significantly with the safety and/or efficacy of a mAb. 
1.  Monitoring the development of antibodies to mAb 
Depending on the source of the mAb, assays for anti-immunoglobulins will need to be developed to detect 
human anti-mouse antibodies (HAMA), human anti-rat antibodies (HARA), human anti-human antibodies 
(HAHA), human anti-chimeric antibodies (HACA), and anti-idiotype antibodies.  As appropriate for the 
mAb,  assays  should  be  developed  to  detect  human  immunoglobulins  directed  against  humanized  or 
primatized antibodies, immunonuclides and immunotoxins, their individual components (e.g., ricin), and 
neoantigens formed by the linked antibody/toxin/nuclide. 
a.  The timing of sample collection for evaluating the presence of an anti-mAb antibody should take into 
account whether the mAb is intended to be given as single or as multiple doses. Titers of the anti-mAb 
antibody should always be established at baseline to account for pre-existing antibodies (including anti-
globulin or anti-conjugate antibodies, when appropriate), and also before readministration of the mAb. 
Post-administration samples may be drawn early (e.g., two weeks after administration), but should also be 
drawn at  later times (e.g., at six-eight weeks). 
b. The assay(s) used to detect the anti-mAb antibody should be standardized to the greatest  extent 
possible.  Aliquots of a "reference" preparation of antibody, e.g. anti-mouse antibody for a HAMA assay, 
with defined specificity from a human or primate source should be aliquoted and frozen to facilitate future 
intra- and inter-study comparisons.  The reference preparation can also be used to establish a standard 
curve for routine testing. The assay(s) should be validated by establishing sensitivity, specificity, precision, 
and accuracy.  Inhibition or competition studies with both negative and positive controls should be used to 
demonstrate that the assay detects antibodies to the mAb product. Studies should assess the range of 
reactivity of normal individuals and should evaluate potential interference by serum components such as 
bilirubin and lipids. 
37  c.  The specificity of the immune responses to the mAb should be identified and characterized in a sample 
of patients. These studies should establish whether the responses are generated against a heavy-chain 
isotype  determinant,  a  light-chain,  constant  (C)  region,  variable  (V)  region,  idiotypic  epitope(s), 
immunoconjugate, or neoantigen.  These data will demonstrate whether it is possible to use an anti-mAb 
antibody test with broad specificity (e.g., detecting human antibodies reactive with antigens of both heavy 
and light chain constant regions of all foreign immunoglobulin classes), or whether a more restricted 
anti-mAb antibody test that is idiotype-specific is necessary.  In certain instances the anti-mAb antibody 
assay should include the actual mAb product as the detection antigen. 
d.  The choice of the appropriate assay for anti-mAb antibody depends on the proposed use and labeling of 
the product.  Development and validation of the assay should accompany the clinical development of the 
new mAb. The results of the anti-mAb antibody testing should be correlated with product efficacy and 
adverse events. 
e.  A license application submitted for a mAb to be administered in a repeated dosing regimen should 
include a clinically available, validated test that reliably measures human antibody responses to the mAb, if 
an anti-mAb response may affect the safety, efficacy or dosing of the product. If a commercially available 
HAMA  (or  other  anti-mAb  antibody)  test  kit,  is  available,  it  may  be  used  provided  it  has  been 
demonstrated to reliably detect antibody response against the new mAb product. In most cases, humanized 
mAb require an assay specific for the product itself. If no appropriate anti-mAb antibody test is available, 
a properly validated test system should be developed by the sponsor. 
2.  Clinical consequences of immunogenicity 
a.  When a patient is found to have developed an antibody response against the therapeutic or diagnostic 
mAb, adverse events should be anticipated and appropriate precautions taken.  MAb are generally given in 
facilities where acute resuscitative care is immediately available.  The use of non-hospital settings for 
mAb administration (e.g., clinic or home) should be justified by clinical safety data. Vital signs should be 
observed closely for at least one hour after completion of the mAb administration.  The possibility of 
delayed adverse effects from immune responses to mAb should be considered and reflected in the trial 
design, including appropriate clinical and laboratory testing. 
b. Anaphylaxis, anaphylactoid and other immune reactions 
i.  True  IgE-mediated  anaphylaxis  to  whole  mouse  immunoglobulins  is  infrequent.  It  is 
theoretically possible that anti-human allotype responses of an allergic nature could occur but they 
have not been reported to be of clinical importance to date.  If the mAb is conjugated to chelating 
agents or toxins, the likelihood of allergic reactions may be greater.  In all cases, repeated 
administration  of  a  mAb  increases  the  likelihood  of  a  hypersensitivity  response.  Immediate 
hypersensitivity reactions may range from mild to severe. Skin testing is not advised because it is a 
poor predictor of sensitivity to mouse immunoglobulins and can cause sensitization. 
ii.  Infusional reactions such as chills, rigors, aches, and low grade fever, are common during or 
immediately  following  mAb  administration  (the  incidence  is  approximately  5%  with  several 
antibodies).  The mechanism of these reactions is not clear. The frequency and intensity of such 
reactions can often be controlled by using slower infusion rates or by pre-medication. 
iii.  Serum sickness is unusual following mAb administration but has been described. Unlike 
anaphylaxis and infusional reactions, which occur during or immediately after antibody treatment, 
38  serum sickness is delayed by several hours. The correlation between circulating levels of soluble 
antigen and immunocomplex-mediated adverse events such as serum sickness should be explored 
if such adverse events are observed. 
c.  Anti-mAb antibodies can interfere directly with some antibody-based clinical tests for antigens, such as 
CA-125 and CEA, by binding to the murine detection antibody.  Indirect interference with diagnostic 
assays is theoretically possible if mAb administration induces anti-idiotypic responses that mimic the 
antigen.  When appropriate, evidence for either type of interference should be systematically sought using 
well designed in vitro studies.  Ideally, attempts should be made to circumvent such interference and 
alternative clinical assays should be validated. 
d.  When subjects are selected for testing a mAb, the risk that future therapy with a monoclonal antibody 
may be compromised by elicitation of an antibody response against the therapeutic agent or other mAb 
should be considered and reflected in the informed consent form. 
C. PRODUCT-RELATED CONSIDERATIONS FOR PHASE 3 STUDIES 
When planning manufacturing changes or scale-up programs during phase 3 clinical trials, sponsors should 
consider that product comparability may have to be demonstrated (see ref. 23 and Section II.E.). This may 
or may not require additional clinical studies depending upon the adequacy of preclinical data (see ref. 23 
and Section II.E.). Thus, it is suggested that scale -up programs and contemplated changes in product 
manufacture  be anticipated prior to the initiation of phase 3 trials. Sponsors should study a number of 
separate product lots during drug development to demonstrate that a safe and effective product can be 
prepared reliably. 
D.  ADMINISTRATION OF RADIOLABELED ANTIBODIES 
1. Dosimetry 
Grade 3 and grade 4 organ toxicities have been reported with therapeutic radioisotopes. Therefore, 
dosimetry  estimates  for  human  subjects  are  required  prior  to  the  initiation  of  phase  1  studies.  The 
dosimetry  estimates  should  be  developed  with  simulation  models  utilizing  an  appropriate  diagnostic 
radioisotope label on the antibody. If no diagnostic radiolabel for the antibody is available for simulation, 
animal  studies  with  the  therapeutic  radiolabel  may  be  utilized  for  dosimetry  estimates.  The  actual 
dosimetry data for the therapeutic radiolabeled antibody, itself,  should be acquired concurrent with the 
initial phase 1 study and reported prior to the initiation of a phase 2 study. 
For diagnostic radiolabeled antibodies, as for the therapeutic isotopes, the investigator should provide 
estimates of the organ dosimetry prior to the first phase 1 study. Final dosimetry calculations from human 
studies should be completed prior to the submission of the license application. 
a. General considerations: Sufficient data from animal or human studies should be submitted to the IND, to 
allow a reasonable calculation of radiation-absorbed dose to the whole body and to critical organs upon 
administration to a human subject [21 CFR 312.23(a)(10)(ii)]. See Appendix III for a list of organs to be 
included in dosimetry estimates. 
The amount of radiation delivered by internal administration of radiolabeled antibodies should be calculated 
by internal radiation dosimetry.  The absorbed fraction method of radiation dosimetry is described in two 
systems [21 CFR 361.1 (b)(3)(iv)]: 
39  i. The Medical Internal Radiation Dose (MIRD) Committee of the Society of Nuclear Medicine 
ii. The International Commission on Radiological Protection (ICRP). 
The investigator should specify which methodology is used.  The mathematical equations used to derive 
the radiation doses and the absorbed dose estimates should be provided.  Sample calculations and all 
pertinent assumptions should be listed and submitted. 
Safety hazards for patients and health care workers during and after administration of the radiolabeled 
antibody should be identified, evaluated, and managed appropriately. 
b. Calculation of radiation dose to the target organ 

Investigators should determine the following, based on the average patient: 

i. The amount of radioactivity that accumulates in the target tissue/organ. 
ii. The amount of radioactivity that accumulates in tissues adjacent to the target tissue/organ. 
iii. The residence time of the radioactive mAb in the target tissue/organ and in adjacent regions. 
iv. The radiation dose from the radioisotope, including the free radioisotope and any daughter 
products generated by decay of the radioisotope. 
v.  The  total  radiation  from  bound,  free,  and  daughter  radioisotopes  associated  with  the 
radioimmunoconjugate,  based  upon  immediate  administration  following  preparation  and  upon 
delayed administration at the end of the allowed shelf life. 
c. Maximum absorbed radiation dose 
The amount of radioactive material administered to human subjects should be the smallest radiation dose 
that is practical to perform the procedure without jeopardizing the benefits obtained. 
i.  The amount of radiation delivered by the internal administration of radiolabeled antibodies 
should be calculated by internal radiation dosimetry using both the MIRD and ICRP systems.  The 
higher estimate of the absorbed dose determined from either of these systems should be used in 
the radiation dosimetry safety assessment. 
ii.  Because  of  known  or  expected  toxicities  associated  with  radiation  exposure,  dosimetry 
estimates should be obtained as delineated in IV.D.1.a and b. 
iii. Calculations should be provided that anticipate changes in dosimetry that might occur in the 
presence of diseases in organs that are critical in radioimmunoconjugate metabolism or excretion 
(e.g., renal dysfunction causing a larger fraction of the administered dose to be cleared via the 
hepatobiliary system or vice versa). 
iv. Possible changes in dosimetry resulting from patient to patient variations in antigen mass should 
also be considered in dosimetry calculations (e.g., a large tumor mass may result in a larger than 
40  expected radiation dose to a target organ from a radiolabeled anti-tumor mAb). 
v. The mathematical equations used to derive the estimates of the radiation dose and the absorbed 
dose should be provided.  Sample calculations and all pertinent assumptions should be listed. 
vi. Calculations of dose estimates should be done assuming freshly labeled material to account for 
maximum amount of  label as well as at the maximum shelf life of the radiolabeled antibody to 
allow for the upper limit of radioactive decay contaminants and should: (a) Include the highest 
amount of radioactivity to be administered; (b) Include the radiation exposure contributed by other 
diagnostic procedures such as roentgenograms or nuclear medicine scans that are part of the 
study; (c) Be expressed as Gray (Gy) per megaBequerel (MBq) or per millicurie of radionuclide; 
and (d) Be presented in a tabular format and  include doses of individual absorbed radiation  for 
the target tissues/organs and the organs listed in Appendix III. 
2.  Early clinical development of therapeutic radiolabeled mAb 
a. Evaluations that should be conducted prior to Phase 1 studies. 
Prior to phase 1 studies, investigators of therapeutic applications of radiolabeled antibodies should conduct 
the following evaluations for the average adult to be entered into the study: 
i. The therapeutic radiolabeled antibody should be evaluated for in vitro stability and composition 
of the radioactive material to be administered. The expected and acceptable levels of the percent 
of  free  radioisotope,  the  percent  of  radioisotope  bound  to  immunoreactive  antibody,  and  the 
percent of bound radioisotope to non-immunoreactive antibody should be established.  Calculations 
of the estimates should be at the maximum planned shelf life of the radiolabeled antibody to allow 
for the upper limit of radioactive decay contaminants and should be based upon the maximum dose 
of radioactive material to be administered to patients. 
ii. The expected biodistribution and routes of clearance of the administered radiolabeled antibody 
dose fractions in tissues/organs should be defined. 
iii. The expected biodistribution and routes of clearance that might occur in the presence of 
diseases in organs that are critical in radioimmunoconjugate metabolism or excretion should be 
described. 
iv. The expected biodistribution and routes of clearance that might occur in the presence of 
immune responses (e.g., HAMA, HAHA, HARA) should be described. 
With reference to the radioactive fractions of the administered radiolabeled antibody dose and the 
patterns of biodistribution, the following issues should be addressed: 
v. From the biodistribution estimation, the expected residence time of the radiolabeled antibody 
fractions in the target tissues/organs and non-target tissues/organs should be determined. 
vi.  Based  on  the  estimated  residence  times  in  each  organ,  the  radiation  exposure  for  each 
tissue/organ should be estimated. 
vii.  Based  on  the  radiation  exposure  for  each  tissue/organ,  the  potential  toxicity  should  be 
41  described. 
viii. Based on the potential radiation toxicity to tissues/organs, toxicity monitoring protocols should 
be developed and incorporated into the clinical trial. 
ix. If the study has increasing doses of radioactive materials (e.g., a study of the maximum 
tolerated dose), the radiation exposure for tissues/organs and the associated potential toxicities 
should be estimated for each radiation dose level. 
b.  Selection of patients for phase 1 trials of therapeutic radiolabeled mAb. 
Patients  should  be  entered  into  phase  1  trials  with  therapeutic  radiolabeled  antibodies  only  after 
consideration of the following: 
i. To reduce the potential for alterations in biodistribution, patients enrolled in early studies should 
not have prior or concurrent exposure to investigational or approved antibodies. 
ii. Patients should be evaluated for immune response to the appropriate species of monoclonal 
antibody (e.g., HAMA, HARA).  Patients demonstrating evidence of immune response should 
generally be excluded from phase 1 studies. 
iii.  For  proper  evaluation  of  potential  adverse  events  in  early  studies,  patients  should  be  in 
adequate health to allow follow-up for three months without adjunctive chemotherapy or radiation 
therapy. Generally, patients should have a Karnofsky score greater than 70. 
iv. Consultation with CBER is strongly recommended when considering the inclusion of pediatric 
patients in early radiolabeled antibody trials. 
c.  Study design issues for phase 1 studies of therapeutic radiolabeled mAb 
Phase 1 studies should be designed to address the following points: 
i. The prepared therapeutic radiolabeled antibody should be evaluated for in vivo stability. The 
previously estimated expected and acceptable levels of the percent of free isotope, the percent of 
bound radioisotope to immunoreactive antibody, and the percent of bound radioisotope to non
immunoreactive antibody should be confirmed and demonstrated to be reproducible.  Calculations 
should be with the routine shelf life of the prepared radiolabeled antibody and based upon the 
maximum dose of radioactive material administered to patients. 
ii. The pharmacokinetics and biodistribution in the patient population should be studied. 
iii. The residence times with radiation dosimetry for tumor (when applicable), tissues, and organs 
should be determined. 
iv. The pattern of toxicity, its relationship to administered dose and the organs of concern for 
acute and delayed radiation injury should be established. 
v. Any apparent evidence of response of tumor to the administration of the radiolabeled antibody 
should be documented. 
42  vi. The trial design should incorporate patient imaging with a diagnostic radiolabel on the antibody 
to confirm the expected biodistribution and residence times prior to the administration of the 
therapeutically radiolabeled antibody. 
3. Adverse events for patients enrolled in trials of therapeutic radiolabeled mAb 
The mechanism for follow-up of patients and reporting of adverse events should be described in the 
protocol prior to initiation of the trial. Complete evaluation and reporting of the adverse events potentially 
associated with the therapeutically radiolabeled antibody should be assured. If patients are referred to their 
attending physicians during the follow up phase, the investigator should plan and control the follow up of 
the treated patients for complete and timely reporting of adverse events potentially associated with the 
administration of the therapeutic radiolabeled antibody. 
Patients removed from a trial should be continued in follow up for three months.  All adverse events during 
that time interval should be reported, even if they are not thought to be related to the administered 
radiolabeled antibody. 
4. Clinical development of radiolabeled mAb used as imaging agents 
a.  Prior to the initiation of phase 3 studies 
Investigators  of  diagnostic  applications  of  radiolabeled  antibodies  should  collect  stability,  safety  and 
pharmacokinetic information for the average adult expected to be entered into phase 3 studies. 
i. The expected percent of free radioisotope, the expected percent of radioisotope bound to 
immunoreactive  antibody,  and  the  percent  of  the  radioisotope  bound  to  non-immunoreactive 
antibody should be determined.  If unlabeled antibody is to be administered in conjunction with 
radiolabeled antibody, the ratio and amounts of the labeled and unlabeled antibody should be 
defined. 
ii. The biodistribution of the administered radiolabeled antibody dose fractions in tissues/organs 
should be delineated, and from the biodistribution, the expected residence time of the radiolabeled 
antibody fractions in the target tissues/organs and non-target tissues/organs should have been 
estimated.  The routes of clearance of the radiolabeled antibody should be determined. 
iii. The changes in pharmacokinetics of the radiolabeled antibody with organ impairment, antigen 
load in the circulation, and tumor burden should be evaluated.  The potential for clearance artifact 
to degrade patient imaging should be explored. 
iv. Estimates of appropriate imaging times and techniques should be developed.  Adjunctive 
imaging aids (e.g., enemas, emptying of the urinary bladder) should be evaluated. 
v. Evidence of image quality should be gathered. The ability of the radiolabeled antibody to image 
known and/or occult disease should be documented.  These data should be compared to imaging 
data obtained using standard diagnostic techniques whenever possible.The incidence of false 
positive localization of the radiolabeled antibody and the incidence of misinterpretation of the 
images to produce false positive and false negative interpretations should be explored. Disease 
specific factors (e.g., stage of disease, tumor burden, and co-morbid illness) should be evaluated 
for impact on technical procedures in the imaging protocol (e.g., time of imaging). 
43  vi. Phase 2 trials should be designed to define the appropriate patient populations for phase 3 
trial(s), to define the technical procedures used for imaging in the anticipated patient populations 
and to identify potential clinical utility of the test to be further explored in later studies. 
vii. Multiple clinical sites should be employed in phase 2 studies to assess the reproducibility of the 
imaging techniques, and of the preparation and administration of the radiolabeled antibody. 
b.  Pivotal efficacy studies of radiolabeled mAb used as imaging agents 
CBER staff should be consulted for review of and comments on Phase 3 protocol(s), prior to the initiation 
of the phase 3 study(ies).  The following elements should be incorporated into each clinical protocol: 
i.  A prospectively defined and detailed primary efficacy endpoint and analytical plan. 
ii.  A study population consisting of those patients for whom the imaging agent is intended after 
licensure.  The performance and utility of an imaging test may vary substantially based on the 
stage, extent, or severity of the disease, determined in part by the results of other diagnostic tests. 
Therefore, the study population and subpopulations to be analyzed should be carefully defined in 
terms of stage of disease as well as in terms of diagnostic tests performed and test results prior to 
study imaging.  The protocols should be designed to assess the imaging performance and the utility 
in the populations. 
iii.  A  plan  for  acquisition  and  storage  of  imaging  data  for  radiolabeled  antibody  in  the 
confirmatory studies. 
iv.  A prospective plan for evaluation of imaging performance: 
(a). On-site image interpretation and reporting should be defined and documented.  To the 
extent possible, the information available to the on-site  reader  should  be  defined  by 
protocol and recorded on the case report forms. 
(b). The off-site image interpretation should be the basis of the principal analysis of 
imaging performance in the phase 3 clinical trial.  The off-site image interpretation and 
reporting of all radiolabeled antibody image findings and all confirmatory imaging should 
be defined and documented prospectively.  The information available to the off-site reader 
should be defined by protocol and recorded on the case report form. In general, the off-
site  reader  should  have  little  information  beyond  the  entry  criteria  of  the  study  and 
specifically should  not be aware of the on-site reading, the results of other diagnostic 
tests, or patient outcome data. 
(c). Planned sample size should be sufficient to determine imaging performance measures 
to a predetermined precision (i.e., 95% confidence interval width).  Imaging performance 
may  vary  with  the  stage,  extent,  and/or  site  of  disease  as  defined  by  pre-imaging 
evaluations, and this should be accounted for prospectively in planning analysis of imaging 
performance.  To  determine  performance,  imaging  results  should  be  compared  with 
another  indicator  of  disease,  usually  results  of  standard  imaging,  biopsy,  exploration, 
patient follow-up or some combination of these. 
44  (d).  If the planned use of the test is in conjunction with other diagnostic tests, its imaging 
performance should be determined and reported in groups of patients defined by the 
results of the other tests. For example, in some cases it will be important to know the 
imaging performance in patients with positive CT scans and the imaging performance in 
patients with negative CT scans. 
v.  A prospective plan for evaluation of clinical utility: 
(a).When an agent has a significant incidence of false negative and false positives or 
significant toxicities, it is particularly important that the clinical utility be assessed to 
determine whether the value of the diagnostic information outweighs the potential adverse 
consequences of incorrect information or the toxicities. In this context, clinical utility 
means the extent to which information obtained by use of the mAb agent in a defined 
clinical setting can be expected to contribute to outcome, to contribute to the convenience 
or appropriateness of patient management, or to provide accurate prognostic information. 
(b). Based on phase 2 and other data, the protocol should indicate the specific manner in 
which clinical utility is to be explored.  The following issues should be addressed: 
(1) The stage and severity of the disease in which the test is to be indicated 
should be specified. 
(2) The protocol should specify whether the test is to be used in conjunction with 
or  in  lieu  of  other  diagnostic  tests.  Because  radiolabeled  mAb  image  by  a 
mechanism distinct from that of radiopharmaceuticals or diagnostic devices, the 
information obtained from a monoclonal antibody image may be complementary to 
that obtained by those means.  For example, a mAb agent may not be as sensitive 
overall as an accepted standard test, but may be able to detect disease accurately 
under conditions where the standard technique fails. 
(3)  How the various results of the test are hypothesized to be clinically useful 
(for  management  or  prognosis)  should  be  clearly  delineated.  For  example: 
positive results together with a positive CT scan are sufficiently diagnostic to 
avoid further diagnostic evaluation including biopsy; positive results are useful to 
guide biopsy; positive uptake is predictive of response to a specific therapeutic 
modality. 
45  V. APPENDIX I: NORMAL HUMAN TISSUES USED IN CROSS-REACTIVITY TESTING 

1.  Adrenal 
2.  Bladder 
3.  Blood cells 
4.  Bone Marrow 
5.  Breast 
6.  Cerebellum 
7.  Cerebral cortex 
8.  Colon 
9.  Endothelium 
10.  Eye 
11.  Fallopian tube 
12.  Gastrointestinal tract 
13.  Heart 
14.  Kidney (glomerulus, tubule) 
15.  Liver 
16.  Lung 
17.  Lymph node 
18.  Ovary 
19.  Pancreas 
20.  Parathyroid 
21.  Pituitary 
22.  Placenta 
23.  Prostate 
24.  Skin 
25.  Spinal cord 
26.  Spleen 
27.  Striated muscle 
28.  Testis 
29.  Thymus 
30.  Thyroid 
31.  Ureter 
32.  Uterus (cervix, endometrium) 
46  VI. APPENDIX II: MOUSE ANTIBODY PRODUCTION TEST 
The following tests for murine viruses (mouse antibody production test (27) should be performed on any 
MCB and EPC derived from murine cell lines and on all lots of mAb derived from mouse ascites fluid: 
1.  Ectromelia 
2.  EDIM 
3.  GD VII virus 
4.  Hantaan virus 
5.  LCM virus, including challenge for non-lethal strains 
6.  LDH-elevating virus 
7.  Minute virus of mice 
8.  Mouse adenovirus 
9.  Mouse encephalomyelitis 
10.  Mouse hepatitis 
11.  Mouse salivary gland (murine CMV) 
12.  Pneumonia virus of mice 
13.  Polyoma 
14.  Reovirus type 3 
15.  Sendai 
16.  Thymic virus 
47  VII. APPENDIX III: ORGANS TO BE CONSIDERED IN DOSIMETRY ESTIMATES
 
1.  all target organs/tissues 
2.  bone 
3.  bone marrow 
4.  liver 
5.  spleen 
6.  adrenal 
7.  kidney 
8.  lung 
9.  heart 
10.  urinary bladder 
11.  gall bladder 
12.  thyroid 
13.  brain 
14.  gonads 
15.  gastrointestinal tract 
16.  adjacent organs of interest 
48  VIII REFERENCES 
1. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, CBER, FDA, 
1993. 
2.  Points  to  Consider  in  the  Production  and  Testing  of  New  Drugs  and  Biologicals  Produced  by 
Recombinant DNA Technology, FDA, CBER, April 10, 1985 and Supplement to the Points to Consider in 
the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology: 
Nucleic Acid  Characterization and Genetic Stability, CBER, FDA, April 6, 1992. 
3. Elimination of Establishment License Application for Specified Biotechnology Products and Specified 
Synthetic Biologics. Final rule. (May 14, 1996; 61 Federal Register 24227). 
4. Guidance for Industry for the Submission of Chemistry, Manufacturing and Controls Information for a 
Therapeutic Recombinant DNA-derived Product or a Monoclonal Antibody Product for In Vivo Use, 
CBER/CDER, FDA, August, 1996. 
5. Points to Consider in the Production and Testing of Therapeutic Products for Human Use Derived from 
Transgenic Animals, CBER, FDA, 1995. 
6. International Conference on Harmonisation; Final Guideline on Quality of Biotechnological Products: 
Analysis of the Expression Construct in Cells Used for Production of r-DNA Derived Protein Products, 
(February 23, 1996; 61 Federal Register 7006). 
7.  International  Conference  on  Harmonisation;  Final  Guideline  on  Stability  Testing  of 
Biotechnological/Biological Products, (July 10, 1996; 61 Federal Register  36466). 
8. Marcus-Sekura,  CJ.  Validation  of  removal  of  human  retroviruses.  In  Developments  in  Biological 
Standardization, volume 75, "International Symposium on Virological Aspects of the Safety of Biological 
Products",  edited by the International Association of Biological Standardization, pp. 133-143, Karger, New 
York, New York, USA, 1991. 
9. Rubinstein, LJ and Stein, KE. Murine immune response to the Neisseria meningitidis Group C capsular 
polysaccharide. J Immunology 1988; 141:4352-56. 
10. Lindmo, T, Boven, E, Cuttitta, F, et al. Determination of the immunoreactive fraction of radiolabeled 
monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Meth 1984; 
72:77-89. 
11. Nisonoff, A, Lamoyi, E. Implications of the presence of an internal image of the antigen in anti-idiotype 
antibodies: possible application to vaccine production. Clin Immunol Immunopathol 1981;21:397-406. 
12. Stein, KE and Epstein, S. [letter] Mol Immunol 1991;28:193. 
13.  Burtonboy,  G,  Delferriere,  N,  Bodeus,  M,  and  Mousset,  B.  Absence  of  Viral  Particles  in  Rat 
Hybridomas. An Ultrastructural Study In Rat Hybridomas and Rat Monoclonal Antibodies (Ed. H. Bazin), 
pp. 465-473, CRC Press, Boca Raton, Florida, USA. 
14. Dinowitz, M, Lie, YS, Low, MA, et al. Recent studies on retrovirus-like particles in Chinese hamster 
49  ovary  cells.  In  Developments  in  Biological  Standardization,  volume  76,  "Continuous  Cell  lines,  An 
International  Workshop  on  Current  Issues",  edited  by  the  International  Association  of  Biological 
Standardization, pp. 201-207, Karger, New York, New York, USA, 1992. 
15.  Miller,  DG  and  Miller,  AD.  Tunicamycin  treatment  of  CHO  cells  abrogates  multiple  blocks  to 
retrovirus infection, one of which is due to a secreted inhibitor. J Virol 1992;66:78-84. 
16. Eglitis, MA, Kadan, MJ, Wonilowicz, E, Gould, L.  Introduction of human genomic sequences renders 
CHO-K1 cells susceptible to infection by amphotropic retroviruses. J Virol 1993;67:1100-1104. 
17. Lander, M R and Chattopadhyay, S K, A Mus dunni cell line that lacks sequences closely related to 
endogenous murine leukemia viruses and can be infected by ecotropic, amphotropic, xenotropic and mink 
cell focus-forming viruses. J Virol. 1984;52:695-698. 
18. Report of a WHO Study group (1987). Acceptability of cell substrates for production of biologicals. 
World Health Organization Technical Report Series 747. 
19. Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for 
Human  and  Animal  Parenteral  Drugs,  Biological  Products,  and  Medical  Devices, 
CDER/CBER/CDRH/CVR, FDA, December, 1987. 
20. Guidelines on Sterile Drug Products Produced by Aseptic Processing, Center for Drugs and Biologics, 
FDA, June, 1987. 
21.  Guidance  for  Industry  for  the  Submission  Documentation  for  Sterilization  Process  Validation  in 
Applications for Human and Veterinary Drug Products, CDER/CVM, FDA, November, 1994. 
22. Guideline for Submitting Documentation for the Stability of Human Drugs and Biologics, Center for 
Drugs and Biologics, FDA, February, 1987. 
23. FDA Guidance Concerning Demonstration of Comparability of Human Biological  Products, Including 
Therapeutic Biotechnology-derived Products, CBER, FDA, April, 1996. 
24.  Goldsmith,  MA,  Slavik,  M,  Carter,  SK.  Quantitative  prediction  of  drug  toxicity  in  humans  from 
toxicology in small and large animals. Cancer Res 1975;35:1354-64. 
25. Collins, JM, Zaharko, DS, Dedrick, RL et al. Potential roles for preclinical pharmacology in phase I 
trials. Cancer Treat Rep 1986; 70:73-80. 
26. EORTC Pharmacokinetics and metabolism group: pharmacokinetically-guided dose escalation in phase 
I clinical trials. Eur J Clin Oncol 1987; 23:1083-87. 
27. Nicklas, W, Kraft, V, and B Meyer. Contamination of transplantable tumors, cell lines, and monoclonal 
antibodies with rodent viruses. Lab Anim Sci 1993;43: 296-300. 
50