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

__________________________________________________________________

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



				Kathryn C. Zoon, Ph.D.

_____________________________________________________________________________


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".

__________________________________________________________________________

TABLE OF CONTENTS

Note:  Page numbering may vary for documents distributed
electronically.

I. INTRODUCTION
   A. BACKGROUND
   B. DEFINITIONS
   C. FILING INFORMATION
II. PRODUCT MANUFACTURE AND TESTING
   A. GENERAL PRINCIPLES AND DEFINITIONS
   B. MANUFACTURE AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES
       1. Cell lines
       2. Production in cell culture
       3. Production in animals or plants
       4. Purification
       5. Characterization of purified unmodified mAb
       6. Anti-idiotype vaccines
       7. Monoclonal antibodies conjugated with toxins, drugs,
          radionuclides or other agents (immunoconjugates)
   C. QUALITY CONTROL AND PRODUCT TESTING
       1. Cell line qualification
       2. Lot-to-lot quality control monitoring of unprocessed
          bulk lots and purified bulk lots, and final product
          specifications
       3. Stability of product
       4. General considerations on quantitation and removal of
          retrovirus contaminant
       5. General considerations on the design and interpretation
          of virus clearance studies
       6. Generic or modular virus clearance studies
       7. Product testing requirements for mAb used as ancillary
          products
   D. PRODUCT SAFETY TESTING FOR FEASIBILITY CLINICAL TRIALS
      IN SERIOUS OR IMMEDIATELY LIFE-THREATENING CONDITIONS
       1. General considerations
       2. Product safety data needed before the initiation of
          feasibility trials in serious or immediately
          life-threatening conditions
   E. ISSUES RELATED TO MANUFACTURING CHANGES (DEMONSTRATION
      OF PRODUCT COMPARABILITY)
       1. General
       2. In vitro evidence of product comparability
       3. Animal studies
       4. Clinical studies to support manufacturing changes
III. PRECLINICAL STUDIES
   A. TESTING CROSS-REACTIVITY OF MAB
       1.  In vitro testing for cross-reactivity
       2. In vivo testing for cross-reactivity
   B. PRECLINICAL PHARMACOLOGY AND TOXICITY TESTING
       1. General considerations
       2. Animal toxicology studies
       3. Pharmacokinetics and pharmacodynamics
       4. Preclinical in vivo studies with immunoconjugates
IV. CLINICAL STUDIES
   A. CLINICAL CONSIDERATIONS FOR PHASE 1 AND 2 STUDIES
       1. General
       2. Dose-setting
   B. IMMUNOGENICITY: CLINICAL CONSIDERATIONS
       1. Monitoring the development of antibodies to mAb
       2. Clinical consequences of immunogenicity
   C. PRODUCT-RELATED CONSIDERATIONS FOR PHASE 3 STUDIES
   D.  ADMINISTRATION OF RADIOLABELED ANTIBODIES
       1. Dosimetry
       2. Early clinical development of therapeutic radiolabeled
          mAb
       3. Adverse events for patients enrolled in trials of
          therapeutic radiolabeled mAb
       4. Clinical development of radiolabeled mAb used as imaging
          agents
V. APPENDIX I: NORMAL HUMAN TISSUES USED IN CROSS-REACTIVITY
               TESTING
VI. APPENDIX II: MOUSE ANTIBODY PRODUCTION TEST
VII. APPENDIX III: ORGANS TO BE CONSIDERED IN DOSIMETRY ESTIMATES
VIII REFERENCES

______________________________________________________________________

Points to Consider in the Manufacture and Testing
for Monoclonal Antibody Products for Human Use
February 1997


I.  INTRODUCTION

Note:  This ASCII version does not contain all tables and other
       formatted items.  A PDF version which includes all
       tables is available on CBER's internet site.  

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 agency's 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')sub2 fragments,
single-chain antibodies, 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 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).


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.

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.

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 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 component's 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.

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 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
    microgram 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 (Ab2alpha) or antigen mimic (Ab2beta) (11).

b.  Ab2beta 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:

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-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 37C 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 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.

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Table I   Cell Line Qualification

NOTE:  Table I is not available in this ASCII version.
A PDF version of the document is available on CBER's internet
page.
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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.

   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 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.

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Table II    Lot-to-lot product safety tests

NOTE:  Table II is not available in this ASCII version.
A PDF version of the document is available on CBER's
internet page.
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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
   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 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 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 shouldbe 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.

   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 (1x10 to the 6 per 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, 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 log10 of the mean.  The
quantitative estimate of virus reduction for each procedure should
be reported as the reduction factor.  This is defined as the log10
of the ratio of the virus load in the starting material to the virus
load in the post reduction material (log10  (volume x virus titer of
starting material / volume x virus titer of post-reduction
material)).  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:
   +/- the square root of (a squared + b squared).
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

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 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.

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.

*****************************
NOTE:  Table III is not available in this ASCII version.  An MSWord
version of the document is available on CBER's internet page.  A
printed copy is available by mail or FAX by calling 1-800-835-4709.
*****************************


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.

   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 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 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 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 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 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 Tmax, Cmax, T1/2 and 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:

   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.  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

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:

   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.

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, 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)]:

   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 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 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.

   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).

   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.

      (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.

______________________________________________________________________


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)

_________________________________________________________________

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

_________________________________________________________________


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

______________________________________________________________________

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