Guidance for Industry

Monoclonal Antibodies Used as

Reagents in Drug Manufacturing

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

March 2001
Guidance for Industry

Monoclonal Antibodies Used as

Reagents in Drug Manufacturing

Additional copies are available from:

Drug Information Branch, HFD-210

Center for Drug Evaluation and Research (CDER)

5600 Fishers Lane

Rockville, Maryland 20857

(Tel) 301-827-4573

(Internet) http://www.fda.gov/cder/guidance/index.htm

or

Office of Communications

Training and Manufacturers Assistance, HFM-40

Center for Biologics Evaluation and Research (CBER)

1401 Rockville Pike

Rockville, Maryland 20852-1448

(Fax) 888-CBERFAX or 301-827-3844

(Voice Information) 800-835-4709 or 301-827-1800

(Internet) http://www.fda.gov/cber/guidelines.htm

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

March 2001
TABLE OF CONTENTS

I. INTRODUCTION..................................................................................................................................................................1

II. BACKGROUND....................................................................................................................................................................2

III. PRODUCTION OF MONOCLONAL ANTIBODY REAGENTS .................................................................................2

IV. MONOCLONAL ANTIBODY REAGENTS IN DRUG MANUFACTURING..............................................................4

A. PURIFICATION OF DRUG SUBSTANCE ............................................................................................................................ 4

B. COMPARABILITY............................................................................................................................................................... 5

V. SPECIFICATIONS FOR MONOCLONAL ANTIBODY REAGENTS........................................................................5

A. TESTING OF UNCONJUGATED MONOCLONAL ANTIBODY REAGENTS ....................................................................... 6

B. TESTING OF MONOCLONAL ANTIBODY REAGENTS LINKED TO SOLID SUPPORT .................................................. 6

VI. STABILITY OF MONOCLONAL ANTIBODY REAGENTS .......................................................................................6

REFERENCES ..................................................................................................................................................................................8
GUIDANCE FOR INDUSTRY1

Monoclonal Antibodies Used as Reagents in Drug Manufacturing

I. INTRODUCTION

This guidance is intended to provide recommendations to sponsors and applicants on the use of

monoclonal antibodies (mAbs) as reagents in the manufacture of drug substances2 that are regulated by

the Center for Drug Evaluation and Research (CDER) or the Center for Biologics Evaluation and

Research (CBER). The guidance focuses on the chemistry, manufacturing, and control (CMC) issues

that should be addressed in new drug applications (NDAs), abbreviated new drug applications

(ANDAs), biologics license applications (BLAs), supplements to these applications, or investigational

new drug applications (INDs).

This document presents issues associated with and recommendations on the documentation to support

the use of mAb reagents generated by hybridoma technology or production of recombinant mAb or

their fragments in bacteria, including phage display technology, fungi (yeasts and molds), and nonprimate

animal-derived transfected cell lines. Monoclonal antibodies or their fragments generated by other

methods can present additional concerns. The recommendations provided in this document should be

considered when such materials are used; however, the guidance does not address the particular

method of production of the mAbs or their fragments.

This document does not provide recommendations on mAbs that are used as diagnostics, radiolabeled

imaging agents, or therapeutic products. For a discussion of mAb products for human therapeutic or

diagnostic use please refer to the Points to Consider in the Manufacture and Testing of Monoclonal

 

1

 This guidance has been prepared by the Monoclonal Antibodies Working Group of the rDNA Reagent Technical

Committee of the Complex Drug Substances Coordinating Committee (CDS CC) in the Center for Drug Evaluation and

Research (CDER), with input from the Center for Biologics Evaluation and Research (CBER), at the FDA. 

2

 The term drug substance, which is used throughout the text, is intended to include biological products as defined in

21 CFR 600.3(g).

This guidance represents the Food and Drug Administration's (FDA's) current thinking on this

topic. It does not create or confer any rights for or on any person and does not operate to bind

FDA or the public. An alternative approach may be used if such approach satisfies the

requirements of the applicable statutes and regulations.
2

Antibody Products for Human Use (PTC 1997).3 The recommendations for characterization and

testing for mAbs used as parenteral pharmaceuticals are by necessity stringent, and not all of them are

applicable to mAbs that are used as reagents in drug manufacturing.

II. BACKGROUND

Monoclonal antibodies are immunoglobulin molecules secreted from a population of identical cells (i.e.,

cloned cells). They are homogeneous in structure and binding specificity. In the context of this

guidance, mAb reagents refers to monoclonal antibodies used as reagents in a drug substance

manufacturing process.

The issues related to mAbs used as reagents are somewhat different from those of mAbs used as

parenteral therapeutic agents. For mAb reagents, the primary emphasis is on assessment of the

following:

C Biological safety, in particular the assessment of contamination of the mAb reagent with

adventitious agents and/or process-related impurities from the cell substrate or cell line sources.

C Performance characteristics of the mAb reagent during drug substance manufacture (e.g., avidity

and specificity for the target molecule).

C Potential presence of residual amounts of the mAb reagent in the final drug substance and/or

drug product.

The recommendations in this guidance apply to the use of mAb reagents in the drug substance

manufacturing process where the mAb reagent is used to purify the drug substance. The extent of

characterization required for the mAb reagent depends on the nature of the steps that follow use of the

mAb, and thus will vary among submissions. While many CMC concerns regarding the use of mAb

reagents are unique to biotechnology-produced reagents, the general concepts expressed in the FDA

Guideline for Submitting Supporting Documentation in Drug Applications for the Manufacture

of Drug Substances (FDA 1987) also apply. An early and continued dialogue between the applicant

and the Agency is encouraged to discuss the data that should be submitted to support the use of the

mAb reagent.

III. PRODUCTION OF MONOCLONAL ANTIBODY REAGENTS

 

3

 This document is available on the Internet at http://www.fda.gov/cber/points.htm.
3

The sponsor or applicant should submit information (e.g., production process, specification) to support

the use of the mAb reagent or a letter of authorization (LOA) to a drug master file (DMF) that contains

this information.

A description of the mAb manufacturing process should be provided. The description is used to assess

the potential impact on the biological safety, quality, and purity of the drug substance and/or drug

product. The mAb reagent should be adequately characterized and its identity, purity, and structural

integrity should be assessed, as these factors are vital to its efficient and uninterrupted performance

during production of drug substances (see section IV). Reagents that have not been fully characterized

for viral safety should not be introduced into facilities where biologics and drugs from mammalian cell

culture are produced because of the potential for cross-contamination. Additional recommendations

relating to mAb reagents are:

• For mAb reagents prepared using hybridoma propagation, serum additives in culture media

should be free of contaminants and adventitious agents.

• Manufacturers should use bovine-derived materials only from cattle that were born, raised, and

slaughtered in countries that are free of BSE (bovine spongiform encephalopathy).4

The predominant concern with the use of mAb reagents in drug substance manufacture is the 

introduction of adventitious agents (e.g., viruses, bacteria, fungi, mycoplasma) and/or process-related

impurities (e.g., protein and DNA contaminants, column leachables, media components) into the drug

substance. Of particular concern are those that are not removed during drug substance manufacture

steps after the introduction of the mAb reagent. In many instances, the extent of the cell bank safety

characterization and the clearance studies for adventitious agents and/or process-related impurities

should follow the established standards for mAbs intended for human use (see PTC 1997, sections II.B

and C). A reduced level (i.e., less than recommended in PTC 1997) of testing of cell banks and/or

validation of the procedures used to remove or inactivate adventitious agents and/or process-related

impurities during purification of the mAb may be appropriate under certain circumstances, with

justification. Early dialogue with the Agency is encouraged when a reduced level of testing and/or

validation is planned. A reduced level can be justified when, for example:

C The drug product is terminally sterilized.

 

4

 A list of countries affected by BSE or those that have a substantial risk associated with BSE (due to a lack of

implementation of an adequate surveillance program) can be found on the Internet at

http://www.aphis.usda.gov/NCIE/country.html.
4

C The use of the reagent is followed by adequate steps for the removal and/or inactivation of the

adventitious agents and/or process-related impurities. In this instance, the overall assessment of

the removal and/or inactivation process can take into account validation data from steps in the

manufacture of the reagent and manufacture of the drug substance.

C Processing steps downstream of the reagent include extremes of pH or organic solvents, and

there are reliable data in the scientific literature that the extremes remove and/or inactivate

adventitious agents and/or process-related impurities.

C The mAb reagent is produced in an expression system in which human infectious agents do not

propagate (e.g., plants, bacteria, fungi, insect cultures).

IV. MONOCLONAL ANTIBODY REAGENTS IN DRUG MANUFACTURING

A major use of mAb reagents is in the purification of drug substance by mAbs attached to a solid

support (e.g., immunoaffinity chromatography). Issues relating to and recommendations on the

information to submit in support of the use of mAb reagents in the purification process are discussed

below. The information that should be submitted to support other uses of mAb reagents in drug

manufacture will depend on the use and are not discussed in this guidance. Sponsors or applicants with

questions on documentation to support other uses of mAb reagents are encouraged to contact the

Agency.

A. Purification of Drug Substance

The drug substance purification processes should be described in the application. The drug

substance manufacturer should establish a specification for the incoming mAb reagent, and

perform testing before using the reagents in the manufacturing process. In addition to identity

testing for the incoming mAb reagent, drug substance manufacturers should carry out additional

testing (e.g., binding activity, adventitious agents) to ensure that the reagent will perform as

intended. Affinity and specificity studies are recommended to assess whether the characteristics

of a mAb reagent are optimal for targeted binding to the appropriate substrate during the

manufacture of the drug substance.

Leaching of mAb or impurities from the solid support into the final product should be 

considered when specifications are established for the drug substance. The amount of column

leachables is not uniform over the column lifespan and depends on several factors (e.g., length

of storage, solutions used in the regeneration and/or sanitization steps, column operating

parameters). A variety of methods can be used to test for leachables such as sampling the

buffer flow-through prior to the load of the drug substance intermediate, in-process testing of

the intermediate bulk, or testing the final drug substance. Alternatively, if documentation is
5

available that the production steps that follow the use of the reagent mAb reduce the maximum

amount of column leachables to appropriate levels, this documentation can be provided in lieu of

routine testing for leachables.

Data on the ability of the affinity column to achieve the intended purity under specified working

conditions should be submitted. The stability of the mAb reagent during use, the column

performance, and the microbial contaminants should be monitored during production of drug

substance and documented by the drug substance manufacturer. Tests and acceptance criteria

for residual mAb should be included in the specifications for drug substances processed with

mAb reagents. Residual mAb should be monitored by sensitive and specific assay (e.g.,

enzyme-linked immunosorbent assay (ELISA)). 

B. Comparability

Changes in the mAb supplier or changes in the manufacturing process of mAb or solid support

are considered to be drug substance manufacturing process changes that can have an effect on

the biological safety and effectiveness of the drug substance and, consequently, the final

product. In cases where significant changes have been implemented in the mAb manufacturing

process that may change the purity or the performance of the reagent (e.g., specificity, avidity,

microbiological safety), appropriate product comparability testing should be performed. The

guidance document entitled FDA Guidance Concerning Demonstration of Comparability of

Human Biological Products, Including Therapeutic Biotechnology-Derived Products

(1996) contains a discussion of comparability testing for mAbs used parenterally. 

Comparability testing for mAb reagents should focus mainly on the performance characteristics

of the reagent and its purity and stability. This is particularly important when changes in the

reagent manufacture are likely to have an impact on the biological safety, purity, quality, or

stability of the drug substance and/or drug product.

V. SPECIFICATIONS FOR MONOCLONAL ANTIBODY REAGENTS

Specifications for the mAb reagents should be provided. A certificate of analysis (COA) should be

available for each individual reagent lot. For monoclonal antibodies linked to a solid support, COAs

should be provided for both forms, unconjugated and linked. A copy of a representative COA should

be provided. 

The COA should provide the test results, including those for adventitious agents, expiration date, and a

disclaimer statement in large bold lettering: REAGENT USE ONLY; NOT INTENDED FOR

HUMAN USE.
6

A. Testing of Unconjugated Monoclonal Antibody Reagents

Tests to adequately characterize the unconjugated mAb reagent typically include:

C Identity (e.g., reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) pattern, isoelectric focusing (IEF) profile)

C Purity (e.g., high performance liquid chromatography (HPLC), SDS-PAGE, capillary

electrophoresis)

C Protein concentration

C Binding to the target molecule

C pH

C Microbial and/or bacterial endotoxin limits, as appropriate

C Preservatives, as appropriate

B. Testing of Monoclonal Antibody Reagents Linked to Solid Support

Tests for mAb reagents linked to solid support should include, at minimum, the following:

C Physical characteristics (e.g., mean particle size, matrix structure)

C Concentration of mAb (e.g., milligrams of mAb per gram of resin)

C Specific binding capacity at recommended temperature and buffer ranges

C Amount of leaching of mAb

C Microbial and/or bacterial endotoxin limits, as appropriate

C Preservatives, as appropriate

VI. STABILITY OF MONOCLONAL ANTIBODY REAGENTS
7

The mAb manufacturer should perform real-time stability studies of unconjugated and conjugated mAb.

 Based on these studies, the mAb manufacturer should determine and provide an expiry date for each

lot of mAb reagent. Stability indicating tests should focus on performance and physical integrity of the

mAb reagent. Either the drug substance manufacturer or the reagent manufacturer should provide data

supporting the in-use chemical stability of the column and mAb reagent using the recommended storage

buffer, regeneration and/or cleaning solutions under specific time and temperatures.
8

REFERENCES

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human

Use, FDA, 1997 (PTC 1997).

Guideline for Submitting Supporting Documentation in Drug Applications for the Manufacture

of Drug Substances, FDA, 1987.

FDA guidance for industry on Demonstration of Comparability of Human Biological Products,

Including Therapeutic Biotechnology-Derived Products, FDA, 1996.