Guidance for Industry

Characterization and Qualification of 

Cell Substrates and Other Biological 

Starting Materials Used in the Production 

of Viral Vaccines for the Prevention and 

Treatment of Infectious Diseases 

DRAFT GUIDANCE 

This guidance is for comment purposes only. 

Submit comments on this draft guidance by the date provided in the Federal Register notice 

announcing the availability of the draft guidance. Submit written comments to the Division of 

Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, 

Rockville, MD 20852. Submit electronic comment to http://www.fda.gov/dockets/ecomments. 

You should identify all comments with the docket number listed in the notice of availability that 

publishes in the Federal Register. 

Additional copies of this draft guidance are available from the Office of Communication, 

Training and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200 N, Rockville, 

MD 20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact the Office of Vaccines Research and 

Review at 301-827-5105. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

September, 2006 
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TABLE OF CONTENTS

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

II. OVERVIEW: CHARACTERIZATION AND QUALIFICATION OF CELL 

SUBSTRATES .............................................................................................................................. 2

A. BACKGROUND ............................................................................................................... 2 

B. PRODUCT-SPECIFIC PARAMETERS INFLUENCING CHARACTERIZATION

AND QUALIFICATION OF CELL SUBSTRATES ..................................................... 3 

1. Vaccine Purity................................................................................................................. 3 

2. Potential Sources of Contamination................................................................................ 4 

3. Quality Design ................................................................................................................ 5 

4. Use of Control-Cell Cultures.......................................................................................... 5 

5. Assay Validation............................................................................................................. 5 

III. CHARACTERIZATION AND QUALIFICATION OF CELL SUBSTRATES, 

VIRAL SEEDS, BIOLOGICAL RAW MATERIALS AND VACCINE PRODUCTION .... 6

A. PROPERTIES OF THE CELL SUBSTRATE............................................................... 6 

1. Properties relevant to cell substrate selection ................................................................. 6 

2. Source ............................................................................................................................. 7 

3. History (including identifying characteristics) and other important characteristics....... 7 

4. Growth Characteristics.................................................................................................... 8 

5. Expression Characteristics.............................................................................................. 9 

6. Susceptibility to adventitious agents............................................................................... 9 

7. Generation of Cell Substrate........................................................................................... 9 

B. CELL BANKING............................................................................................................ 10 

1. Cell Banking Strategies and Methods........................................................................... 10 

2. Qualification of Cell Banks and Primary Cells............................................................. 11 

3. Special Considerations for Primary Cells..................................................................... 12 

4. Special considerations for Diploid Cell Strains............................................................ 13 

5. Special Considerations for Continuous Cell Lines....................................................... 14 

6. Additional considerations for cell lines that are tumorigenic or tumor-derived........... 14 

7. End-of-Production Cells............................................................................................... 15 

C. VIRAL SEEDS ................................................................................................................ 16 

1. Master Viral Seed ......................................................................................................... 16 

2. Working Viral Seed ...................................................................................................... 17 

D. BIOLOGICAL RAW MATERIALS AND ANCILLARY REAGENTS ................... 17 

1. Serum............................................................................................................................ 18 

2. Trypsin .......................................................................................................................... 18 

3. Amino Acids................................................................................................................. 18 

4. Other Biological Reagents............................................................................................ 18 

E. CONSIDERATIONS IN TESTING AT DIFFERENT STAGES OF PRODUCTION

........................................................................................................................................... 19 

1. Cell Banks..................................................................................................................... 19 

2. Pre-Production Cells..................................................................................................... 19 

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3. Pre-filtered Harvest or End-of-Production Cells.......................................................... 19 

4. Control Cells................................................................................................................. 20 

5. Post-Filtered Harvest or Final Bulk .............................................................................. 20 

6. Final Filled Product....................................................................................................... 20 

IV. DESCRIPTION OF QUALITY-CONTROL TEST METHODS ................................. 21

A. TESTING FOR ADVENTITIOUS AGENTS............................................................... 21 

1. In Vivo Tests................................................................................................................. 21 

a. Adult Mice ................................................................................................................ 22 

b. Suckling Mice ........................................................................................................... 22 

c. Guinea Pigs............................................................................................................... 23 

d. Rabbits...................................................................................................................... 24 

e. Embryonated Chicken Eggs...................................................................................... 24 

f. Antibody Production Tests ....................................................................................... 25 

2. In Vitro Tests for Viruses ............................................................................................. 26 

a. Cell Culture Safety Test............................................................................................ 26 

i. Human Diploid Cells............................................................................................ 26 

ii. Monkey Kidney Cells........................................................................................... 26 

b. Transmission Electron Microscopy (TEM) .............................................................. 28 

c. Biochemical Tests for Retroviruses.......................................................................... 28 

d. Infectivity Tests for Retroviruses.............................................................................. 29 

e. PCR or Other Specific in vitro Tests........................................................................ 29 

f. Sourcing and Testing of Bovine-Derived Materials................................................. 30 

g. Testing of Porcine-Derived Reagents....................................................................... 30 

3. In Vitro tests for Non-Viral Agents.............................................................................. 31 

a. Mycoplasma.............................................................................................................. 31 

b. Bacterial and Fungal Sterility ................................................................................... 34 

c. Mycobacteria Testing................................................................................................ 34 

B. TESTING OF CELL PROPERTIES ............................................................................ 35 

1. Tests for Tumorigenicity............................................................................................... 35 

2. Testing for Oncogenicity .............................................................................................. 36 

3. Identity Testing of Cell Substrates................................................................................ 37 

4. Testing for Genetic Stability......................................................................................... 38 

C. OTHER TESTS............................................................................................................... 38 

1. Testing for the Presence of Residual Cells................................................................... 38 

2. Testing for Residual Cellular DNA .............................................................................. 38 

3. General Safety Test (GST)............................................................................................ 39 

V. CONCLUSIONS ................................................................................................................ 39

VI. GLOSSARY........................................................................................................................ 40

VII. REFERENCE LIST........................................................................................................... 44

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Guidance for Industry 

Characterization and Qualification of Cell Substrates and Other 

Biological Starting Materials Used in the Production of Viral 

Vaccines for the Prevention and Treatment of Infectious Diseases 

This draft guidance, when finalized, 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. You can use an alternative approach if the 

approach satisfies the requirements of the applicable statutes and regulations. If you want to 

discuss an alternative approach, contact the appropriate FDA staff. If you cannot identify the 

appropriate FDA staff, call the appropriate number listed on the title page of this guidance.

I. INTRODUCTION 

We, the Food and Drug Administration (FDA), are providing you, manufacturers of viral 

vaccines, recommendations for the characterization and qualification of cell substrates and viral 

seeds used for the production of viral vaccines for human use. These recommendations may be 

used to support a Biologics License Application (BLA) or an application for an Investigational 

New Drug (IND). 

This guidance applies to the production of viral vaccines for the prevention and treatment of 

infectious diseases regulated by the Office of Vaccines Research and Review (OVRR) of the 

Center for Biologics Evaluation and Research (CBER). 

This document is intended to replace the information pertaining to viral vaccines used for the 

prevention and treatment of infectious diseases that we provided in the document entitled “Points 

to Consider in the Characterization of Cell Lines Used to Produce Biologicals,” dated 1993 (Ref. 

1). This document also is intended to supplement recommendations on the production of viral 

vaccines for the prevention and treatment of infectious diseases, provided in International 

Conference on Harmonization (ICH) documents Q5A and Q5D (Refs. 2 and 3, respectively). 

For the production of biological products not covered under this guidance, we recommend that 

you refer to the “Points to Consider in the Characterization of Cell Lines Used to Produce 

Biologicals,” dated 1993 (Ref. 1). 

For the purpose of this document, viral vaccines are a heterogeneous class of preventive, and in 

some cases, therapeutic and medicinal products that when administered are intended to elicit 

immune responses that could prevent and/or lessen the severity of one or more infectious 

diseases. A viral vaccine may be a live attenuated preparation of viruses, inactivated (killed) 

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whole or subunit virions, purified recombinant proteins, including those derived from 

recombinant deoxyribonucleic acid (DNA) in a host cell, synthetic antigens, polynucleotides 

(such as plasmid DNA vaccines), or live viral vectors expressing specific heterologous vaccine 

antigens. It may also be a combination of viral vaccines listed above. Antigens may be 

presented alone or delivered in combination with other antigens, adjuvants, additives and other 

excipients. Therapeutic vaccines for non-infectious diseases (e.g., certain cancer vaccines) and 

monoclonal antibodies used as immunogens (e.g., anti-idiotypic antibodies) are not considered 

here. 

The scope of this guidance document is limited to cell substrates of human or animal origin and 

does not cover characterization of unicellular organisms, such as bacteria or yeast. This 

guidance also applies to the characterization and qualification of viral seeds. A glossary defining 

scientific terms in this guidance is located in Section VI below. 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe FDA’s current thinking on a topic and should be 

viewed only as recommendations unless specific regulatory or statutory requirements are cited. 

The use of the word should in a guidance document means that something is recommended, but 

not required. 

II. OVERVIEW: CHARACTERIZATION AND QUALIFICATION OF CELL 

SUBSTRATES 

A. BACKGROUND 

The number of different cell substrates used in currently licensed vaccines is limited. 

The emergence of new infectious diseases necessitates the need for development of 

new vaccines for agents such as human immunodeficiency virus (HIV), pandemic 

influenza virus strains, severe acute respiratory syndrome (SARS) virus, and agents of 

bioterrorism. In some cases, novel human and animal cell substrates might be needed 

to manufacture certain vaccines, including live attenuated viruses, live viral vectors 

expressing vaccine antigens, inactivated whole or subunit virions, purified recombinant 

proteins, and virus-like particles. Development of new-generation vaccines likely will 

involve a wider variety of cell substrates, both for the isolation of virus seeds and 

vectors, and for vaccine manufacture. Due to the absence or limited experience with 

new cell substrates, manufacturers should develop and apply the best technologies 

available to assure that these new cell substrates are safe. 

Selection of a cell substrate influences the safety and purity of the biological product 

manufactured in it. You should evaluate cell substrates on a case-by-case basis, but 

characterization of any cell substrate used for the development and manufacture of a 

vaccine should address certain general issues that might affect the safety and purity of 

vaccine products manufactured in them. Examples of such issues include karyotype 

and tumorigenic phenotype of the cell substrate, the identity and genetic stability of 

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the cell substrate and virus seeds, and the requirement that the vaccine product be free 

of extraneous infectious microorganisms and potential oncogenic agents. Tests to 

address these general issues affecting characterization and qualification of cell 

substrates are described in Section IV. 

 

Qualification of a biological starting material includes an assessment of the material to 

demonstrate its safe use in manufacturing a final product. The qualification of cells 

intended for use in the manufacture of biologicals includes assessment of the: 

• history and general characteristics of the cells; 

• cell banking system; and 

• characterization of the cells through quality-control testing. 

In some situations, additional validation studies to demonstrate virus removal and/or 

inactivation by the manufacturing process might be necessary. 

B. PRODUCT-SPECIFIC PARAMETERS INFLUENCING 

CHARACTERIZATION AND QUALIFICATION OF CELL SUBSTRATES 

1. Vaccine Purity 

The regulations in 21 CFR 610.13 state that “Products shall be free of extraneous 

material except that which is unavoidable in the manufacturing process described in 

the approved biologics license application.” In 21 CFR 600.3(r), purity is defined 

as the “relative freedom from extraneous matter in the finished product, whether or 

not harmful to the recipient or deleterious to the product.” (see Section VI. 

Glossary). Because live attenuated viruses, whole inactivated virions, or virus-like 

particles often cannot be purified as rigorously as viral subunit vaccines, their 

potential for contamination might be greater than that of subunit vaccines. The 

generation of live viral vaccines often involves cell disruption. In addition, such 

vaccines often are minimally purified and are not subjected to any inactivation 

steps. Comprehensive testing and qualification of the biological starting materials 

and raw materials should be performed, and lot-by-lot testing for adventitious 

agents might be necessary, because it might not be possible for a manufacturer of 

live viral vaccines to validate clearance of any adventitious agents. 

For more highly purified products where viral clearance can be achieved during 

downstream processing, you may place more reliance on process validation (Ref. 

2). For inactivated vaccines, the process you use to inactivate your vaccine virus 

might not inactivate all adventitious agents that might be present (as occurred 

with early inactivated poliovirus vaccines (Ref. 4). Therefore, you should provide 

documentation of your validation for inactivation of adventitious agents. The 

choice of tests and the stages at which the tests are applied will depend on your 

inactivation process. The degree of viral clearance that is feasible might influence 

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the sensitivity of the testing you should perform to demonstrate the absence of 

contaminants in your product. 

You should validate any methods used to inactivate or clear potential viral 

contaminants during production of your vaccine including the starting materials 

used to produce it, as the purity of your product could be affected by the purity of 

reagents and biological raw materials you use to produce the vaccine. For 

example, inactivation of viruses by irradiation of serum could provide additional 

assurance regarding the purity of the final product. Certificates of Analysis 

(COA) for all reagents and biological raw materials used for vaccine production 

should be included in your submission. 

If a seed was exposed to a known adventitious agent or if the passage history of a 

virus seed is unclear, you should purify viral seeds (e.g., by molecular cloning, 

serial passage in medium containing neutralizing antibody directed against the 

adventitious agent, or plaque purification). If your purification method is 

demonstrated to be capable of removing all adventitious agents from a viral seed 

to within an acceptable safety margin, this approach could be used to qualify a 

seed. If you are contemplating performing seed purification, you should discuss 

your proposed methodology and its validation with us as early in the process as is 

feasible. 

2. Potential Sources of Contamination 

It is important that you identify and examine all potential sources of 

contamination of your product. For example, the viral seed used in the production 

of many licensed viral vaccines is exposed to the following potential sources of 

contamination: the person or animal from which it was isolated; the cells and raw 

materials (e.g., serum or trypsin) used in its isolation and attenuation; materials 

used in banking and propagation of cells for viral seed growth; and other 

materials used during production and filling of the seed. You also should 

consider the species of origin of your cell substrates, viral seeds, and other 

biological starting materials in selecting your tests. You should consider any 

infectious viruses (including those that infect nonhuman species) as potential 

contaminants if there is the possibility of contact with your product at any time 

during development and production. You should test your product at appropriate 

stages of production for contaminating agents. Finally, you should consider the 

possibility of contamination from unusual sources, as exemplified by the reported 

presence of minute virus of mice (MVM) in some lots of recombinant proteins 

(Ref. 5). Testing might be needed to verify the absence of additional 

contaminating agents, particularly those agents whose propagation might be 

supported by your cell substrate. 

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3. Quality Design 

Concepts of quality design consistent with Current Good Manufacturing Practices 

(cGMP) and Good Laboratory Practices (GLP) (see 21 CFR parts 211 and 58) are 

relevant to the selection of suitable cell substrates and viral seeds. If your cell 

substrate or viral isolate was not originally derived under concepts consistent with 

cGMP and GLP practices, additional documentation (e.g., donor and source of 

raw materials), testing and production steps might be necessary to support its use. 

This may include having some virus seeds re-derived or further purified to reduce 

the possibility of adventitious agent contamination. 

4. Use of Control-Cell Cultures 

If you are using primary cell cultures to propagate your vaccine virus, complete 

testing of the primary culture might not be feasible prior to inoculation of virus. 

In this situation, you should produce and test uninfected control-cell cultures that 

are derived in parallel with and handled in the same manner as the production 

culture. Control-cell cultures should be processed simultaneously with your 

production culture, but left uninfected and tested for the presence of adventitious 

agents by direct observation and testing of the cell sheet and by testing the culture 

fluid using appropriate tests (described in Section III.A and Table 1 in Section 

IV.E). Tests for adventitious agents should be performed at times at which you 

would perform similar tests on your manufactured product if it were possible to 

do so. 

Use of control-cell cultures is important when your vaccine virus might interfere 

with the results of in-process testing of the product; for example, when the virus 

cannot easily be neutralized to permit testing for adventitious agents. You should 

propagate control-cell cultures under conditions similar to production for a 

suitable period to allow for possible reactivation and detection of latent or 

endogenous adventitious agents and poorly replicating adventitious agents. You 

should use a culture period of at least 14 days beyond the time of inoculation of 

the production vessels prior to testing. A longer period might be needed to detect 

some agents. Testing of control cells does not always eliminate the need for 

testing end-of-production cells, which might be required to demonstrate the 

absence of agents induced during vaccine manufacture. 

 

5. Assay Validation 

You should demonstrate the reliability of assays or tests used to evaluate your cell 

substrate in the context of intended use. Guidance on appropriate ways to validate 

an analytical assay may be obtained from the ICH Q2A and Q2B documents 

(Refs. 6 and 7, respectively). Further suggestions are provided by the U.S. 

Pharmacopoeia (USP) in Chapter 1225 (Ref. 8). Test methods should be 

validated according to the principles defined in these guidance documents. 

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Certain “compendial” methods might not require full validation, as long as you 

verify their suitability under your actual conditions of use. Verification of 

suitability for your use may include a demonstration of comparability between 

your limits of detection and those for methods described in the published 

literature. 

You should include appropriate statistical analysis in your assay validation. This 

may include assessment of assay accuracy, precision, limits of detection, limits of 

quantification, specificity, linearity and range, ruggedness and robustness, and 

system suitability. Further guidance in this regard may be obtained from the ICH 

Q2A document (Ref. 6). 

III. CHARACTERIZATION AND QUALIFICATION OF CELL SUBSTRATES, 

VIRAL SEEDS, BIOLOGICAL RAW MATERIALS AND VACCINE 

PRODUCTION 

In this section we describe strategies for cell banking, the stages of production and adventitious 

agent testing or characterization recommended at those stages, and the factors that could influence 

your choice of tests. Although your tests should address issues specific to your viral vaccine 

product, the following recommendations represent general guidance. To maximize the value of 

the recommended tests, it is important that you perform each test at the stage of production at 

which contamination is most likely to be detected. 

A. PROPERTIES OF THE CELL SUBSTRATE 

The cell substrate you choose for production of your viral vaccine should be well 

characterized. Your application should address the following issues. 

1. Properties relevant to cell substrate selection 

The safety and efficacy of the vaccine might depend on the characteristics of the 

cell substrate. Ideally, all stages of development of a potential vaccine candidate 

starting with the initial isolation of the viral strain from a screened and suitable 

donor through to a licensed vaccine should be performed using a wellcharacterized cell substrate. 

Assuring that the vaccine virus strain remains stable during passage in cell culture 

is an important component of cell substrate selection. Different cell types may 

apply different selective pressures on the vaccine virus, which could alter its 

sequence and possibly its phenotype. For example, when Sabin poliovirus strains 

are grown in different cells, their likelihood of reversion to neurovirulence is 

different (Ref. 9). If attenuating mutations or other genetic markers (including 

expression of antigens relevant to immune response) of the vaccine virus are 

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known, the influence of serial passage in the cell substrate on retention of these 

markers can be useful in characterizing the genetic stability of the vaccine virus. 

Whatever starting materials are used for the generation of the cell substrate (e.g., 

parent cells or plasmids used for transfection), information about those starting 

materials and any characterization (e.g., sequence of the plasmid) that has been 

performed should be provided. If a sponsor starts with a primary cell to generate 

a novel cell substrate, complete information on donor screening and testing should 

be provided. See Sections III.A.2. for additional information on donor sceeening 

and testing. 

2. Source 

You should provide documentation of the species of origin and the tissue type of 

your cell substrate. You should also provide the medical history of the donor and 

the results of any screening and testing performed on the donor or on samples 

from the donor. Generally, human donors should be screened and tested in a 

manner comparable with whole blood or source plasma donors, as described in 21 

CFR part 640. Animal donors should be screened and tested by applying similar 

concepts. They should be in general good health (except in cases where the cell 

substrate is derived from a tumor) and free from symptoms of infectious diseases. 

Ideally, animals intended to be donors should be quarantined prior to sacrifice or 

obtaining the sample to be used to generate the cell substrate (Ref. 10). Issues 

specific to bovine- and porcine-derived materials are dicussed in sections III.A.2.f 

and III.A.2.g). The species of origin is highly relevant, because different animal 

species have different potential contaminants, and some animal cells might be 

capable of propagating human viruses that are of potential concern as adventitious 

agents. Tissue type is also an important element for you to describe and consider 

in deciding on testing strategies. For example, neuronal cells might harbor latent 

viruses (e.g., herpesviruses) or express infectious prion proteins (PrP) and should 

be evaluated for these potential adventitious agents. Additional considerations 

might apply to cells that are tumor-derived 

3. History (including identifying characteristics) and other important characteristics 

The history of your vaccine cell substrate must be identified according to 21 CFR 

610.18(c)(1)(i) and should include: sources and testing of the raw materials to 

which it was exposed in its passage history; listing of any other agents grown in 

the facilities around the time of cell substrate passage; and the conditions under 

which it was passaged (including adherence to GLPs or cGMPs, if applicable). In 

some cases (e.g., Chinese hamster ovary [CHO] cells and Vero cells), multiple 

related cell lines or cell clones exist, and it is important to specify which of these 

related lines are being used, including the source you obtained it from and the 

passage (or population doubling) level at the time. You should provide 

documentation of all raw materials you used for the entire passage history, but it 

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is particularly important for passages using bovine materials acquired during and 

after 1980, when BSE emerged in the U.K. You should document all known 

manipulations (adaptation, engineering, cloning) performed on the cell substrate 

since original isolation from a primary donor, whether they occurred prior to or 

after your receipt of the cells. 

In addition, you must provide the following information for any cell substrates used 

for the production of viral vaccines (except that under 21 CFR 610.18(c)(3) these 

elements are not mandatory if you use primary cell cultures that are not 

subcultivated or that are subsequently subcultivated for only a very limited number 

of population doublings, in which case FDA still recommends that you provide 

them): 

• results of all identity testing, including information about any 

characteristics (such as cytogenetic characteristics, required by 21 CFR 

610.18(c)(1)(ii)) that could be used to identify the cells; 

• results of all available adventitious agent testing (21 CFR 

610.18(c)(1)(iv)); 

You should also provide the following: 

• age, gender, and species of the donor; 

• donor's medical history and the results of tests performed on the donor 

for the detection of adventitious agents; 

• culture history of the cell line, including methods used for the isolation of 

the tissues from which the line was derived, passage history, medium 

used, and history of passage in animals; 

• documentation of the history of human-derived and animal-derived 

materials used during passage of the cells; and 

• documentation of any genetic material introduced into the cell substrate. 

4. Growth Characteristics 

You must characterize the in vitro growth characteristics (e.g., suspension or 

monolayer cultures, rate of growth) of your cell substrate (21 CFR 

610.18(c)(1)(iii)), except if you use primary cell cultures exempted from that 

requirement by 21 CFR 610.18(c)(3). If the cells have a finite life expectancy, the 

total number of population doubling levels through senescence should be 

determined. You should perform tumorigenicity testing on continuous cell lines. 

A description of the tumorigenic property of cells is required for all diploid and 

non-diploid cells (21 CFR 610.18(c)(1)(ii)), but is not applicable to primary cell 

cultures that are not subcultivated or that are subsequently subcultivated for only a 

very limited number of population doublings (21 CFR 610.18(c)(3)). 

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The microscopic appearance of your cell substrate should be stable from the 

master cell bank to the end-of-production cells. If there are specific markers that 

might be useful in characterizing your cell line (such as marker chromosomes, 

transfected genetic elements, specific surface markers, etc.), these should be 

analyzed for their stability at manufacturing passage levels. 

5. Expression Characteristics 

If your cell substrate is used to produce a recombinant protein, you should 

evaluate its ability to do so as a part of its characterization. This includes 

evaluating the copy number and stability of introduced nucleic acids and the 

quantity and quality of expressed proteins. In some cases, it might be appropriate 

for you to evaluate expression of other genes relevant to the cell phenotype. 

Some cell substrates might express (or might be induced to express) genes of 

endogenous or latent viruses. Such expression should also be described if it is 

associated with a safety concern or could be used to identify the cell. If viral 

sequences are expressed, you might need to assess their infectivity and potential 

interference with adventitious agent testing. 

6. Susceptibility to adventitious agents 

In developing comprehensive and appropriate testing strategies for adventitious 

agents, you should consider the susceptibility of the cell substrate to infection 

with viruses other than the vaccine strain. For example, because the cell 

substrates used for the production of licensed oral poliovirus vaccines (primary 

monkey kidney cells) were susceptible to contamination with certain simian 

viruses, such as SV40 or herpes B virus, specific tests were required to assay for 

these viruses in each lot. On the other hand, because human papillomaviruses 

cannot be propagated in cell culture, we do not generally recommend that 

sponsors assess virus production lot-by-lot for human papillomaviruses once the 

initial cell substrate and viral seed have been demonstrated to be free of these 

agents. The same concepts might be useful for you in developing new cell 

substrates and testing strategies for them. 

7. Generation of Cell Substrate 

You should characterize the cell substrate for the impact that the manipulations 

have had on the characteristics of the cell when you use cell substrates that have 

been generated by in vitro manipulations or engineered to express a viral antigen. 

For example, immortalization of primary cells with viral oncogenes to generate a 

complementing cell line for production of defective viruses or viral vectors will 

inevitably change the phenotype of the cell. Such manipulations might induce the 

expression of latent or endogenous viruses, and/or might cause the cells to 

become tumorigenic. In addition, a cell substrate that has been derived by cell 

cloning might have different characteristics from the parental cell line. Because it 

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is derived from one or a few cells, it might not have characteristics representative 

of the original population from which it was cloned. Alternatively, a clone might 

be selected as the cell substrate because of its particular outlier characteristics, 

such as rapid propagation in culture or adaptation to particular cell culture 

conditions that modify its growth properties to enhance vaccine virus replication 

(e.g., development of suspension cell cultures from adherent cells). It is important 

that you thoroughly evaluate the characteristics of generated or engineered cell 

substrates, as it cannot be assumed that the parental cell characteristics were 

maintained following the manipulations used to generate the production cell 

substrate. 

B. CELL BANKING 

Cell banking assures that an adequate supply of equivalent, well-characterized cells 

exist for production over the expected lifetime of the product. In addition to providing 

a constant supply of biological starting material, cell banking provides you with the 

opportunity to undertake a comprehensive characterization of the cell substrate and to 

minimize the chance of adventitious agent contamination and/or to maximize the 

chance of detection of a contamination. 

1. Cell Banking Strategies and Methods 

Ordinarily, the cell bank system consists of two tiers: a master cell bank (MCB) 

and a working cell bank (WCB), often called a manufacturer’s working cell bank 

(MWCB). The MCB represents a collection of cells of uniform composition 

derived from a single source prepared under defined culture conditions. 

Sometimes, a Parent Cell Bank, comprising vials of the progenitor cells to the 

Master Cell Bank, is also maintained. 

The WCB is derived from one or more vials of cells from the MCB, which are 

expanded by serial subculture. The pooled cells are dispensed into individual vials 

and cryopreserved to form the WCB. One or more vials from the WCB are 

generally used for the production of a lot of a vaccine. If cells from more than one 

WCB vial are used, the cell suspensions should be pooled at the time of thawing. 

The population doubling level or passage level of cells used for production should 

not exceed an upper limit based on criteria established by you. 

When using a two-tiered banking system, we generally recommend that you 

perform an extensive characterization of the MCB. Because all WCBs would be 

derived from the well-characterized MCB, your WCBs may be tested in a more 

limited manner, focusing on adventitious agents to which the WCB could have 

been exposed during expansion from the MCB. The use of a cell banking system 

might allow you to perform more limited testing of individual vaccine lots, 

focusing on adventitious agents to which the cells could have been exposed during 

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manufacturing from the WCB. Alternatively, some manufacturers might choose to 

extensively characterize each WCB in lieu of thorough characterization of the 

MCB. The testing strategy chosen will depend on your own circumstance, but you 

should characterize at least one bank extensively. Testing of the MCB will be a 

one-time testing. However, in the absence of thorough characterization of the 

MCB, we may request a thorough characterization on the WCB used to produce 

each lot. 

You may use other cell banking systems, for example, when the original source of 

your cell substrate is limited and/or the need to maintain a low passage level 

restricts expansion of cell numbers. Regardless of what cell banking system you 

use, cells at some level of expansion should be characterized completely. In either 

case, the derivation and cell bank designations should be thoroughly described and 

explained. 

We recommend that all cell banks be stored in either the liquid or vapor phase of 

liquid nitrogen. Storage of cell banks in the vapor phase of liquid nitrogen might 

reduce the potential for cross-contamination. You should document in your 

biologics license application the location, identity, and inventory of individual 

ampoules of cells. Cell recovery or viability data should be documented to 

demonstrate cell stability under the freezing and storage conditions. You should 

store the MCB and WCB in two or more separate areas within the production 

facility as well as at a distant site in order to avoid loss of the cell substrate due to 

local disaster or malfunction. Access to these cell banks by your staff should be 

limited and controlled. 

2. Qualification of Cell Banks and Primary Cells 

The purpose of qualifying cell banks is to demonstrate their suitability for use in 

vaccine production. This section includes recommendations on testing for different 

types of cells and stages of cell banking; they also apply to the use of primary cells. 

Not all categories of cells (primary, diploid, continuous, or tumor-derived) will be 

suitable for all types of vaccines (live, inactivated, recombinant, or highly purified). 

Characterization of a cell bank is dependent on its use, and your particular use 

should guide your determination of which of the recommended testing is necessary 

and whether additional testing might be necessary. Consideration of the choice of 

tests will be based on the assessment of the risks that the cell substrate might 

represent for the product type and its clinical indication. 

Testing to qualify the MCB should be performed directly on the cell bank, except 

when it is more appropriate to test cell cultures derived from the cell bank. 

Tests that you might perform on your MCB include tests for bacteria, fungi 

mycoplasma, and viruses (e.g., in vitro and in vivo testing, specific tests for 

retroviruses, and specific tests for viruses known to exist in the species of origin or 

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that could be acquired during serial passage in cell culture). Other specific tests 

might be warranted, depending on the passage history of the cell substrate. Many 

of the tests in tissue culture or animals were originally promulgated in 21 CFR part 

630 and remained in the regulations until revision of the regulations in 1996 (61 FR 

40153). FDA removed many of the specific tests from the regulations to allow 

manufacturers flexibility in choosing appropriate new tests that might become 

available. Details of these tests are presented in Section IV. If the species from 

which your cells were derived is susceptible to infection with Mycobacterium 

tuberculosis, an appropriate test should be performed for this agent as well. 

Depending on the source species, you may consider specific tests such as the test 

for avian leukosis virus for product produced in avian cells. If you are using 

primary monkey kidney cell cultures, for example, you should test for speciesspecific simian agents, such as SV40, and herpes B virus, and other simian agents, 

such as simian polyomaviruses and simian cytomegalovirus. 

Either the MCB or all animal-derived reagents to which it was exposed should be 

shown not to contain infectious agents that could potentially have been present in 

the source animal. Some of the tests that are relevant in selected circumstances are 

described in Section IV, including the tests for bovine (Section IV.A.2.f) and 

porcine agents (Section IV.A.2.g). Additional considerations are described for 

serum in Section III.D.1 and for trypsin in Section III.D.2. In the case of bovine 

serum, the issue of TSE agents will need to be considered. Finally, testing should 

be performed in most circumstances to determine if the cells produce retroviruses 

or retrovirus particles. This testing is described in Sections IV.A.2.c and IV.A.2.d. 

Extensive characterization of the MCB should be done once and should also 

address the considerations in Section III.B.1. Methods that may be used for 

identity testing of the MCB are discussed in Section IV.B.3.

3. Special Considerations for Primary Cells 

Primary cells are obtained directly from the tissues of healthy animals. Major 

successes in the control of viral diseases, such as poliomyelitis, measles, mumps, 

and rubella, were made possible through widespread use of vaccines prepared in 

primary cell cultures, including those from chicken or duck embryos, the kidneys 

of monkeys, and other tissues. Cultures of monkey kidney cells have been used for 

the production around the world of IPV and OPV for prophylaxis of poliomyelitis 

for almost 50 years. Cultures of primary chick embryo fibroblasts or chicken eggs 

continue to be used to manufacture safe and effective influenza, measles, and 

mumps vaccines. 

Primary cells are more likely to contain adventitious agents than banked, wellcharacterized cells. This concern with primary cells is mitigated by rigorous 

qualification of source animals and primary cell substrates. Animals from which 

primary cultures are established should be from specific-pathogen-free closed 

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flocks, herds, or colonies, when feasible. The term “closed” refers to the 

maintenance of a group (flock, herd, and colony) free from introduction of new 

animals (new genetic material). Animals that are not from closed flocks, herds, or 

colonies should be quarantined and thoroughly evaluated for a period sufficient to 

detect signs of disease or infection before introducing them into the flock, herd, or 

colony. Animals should be screened serologically for appropriate adventitious 

agents to determine their suitability as a source for the primary cell substrate. 

Animal husbandry practices should be described in the application. 

Embryonated chicken eggs used as the source of chick embryo tissue for the 

propagation of viral vaccines should be derived from flocks certified to be free of 

Salmonella pullorum, avian tuberculosis, fowl pox, Rous sarcoma virus, avian 

leucosis virus, reticuloendotheliosis virus, and other adventitious agents pathogenic 

for chickens. Certificates of Analysis (COA) should be submitted for 

documentation. If eggs are procured from flocks that are not certified, tests should 

be performed to demonstrate freedom of the vaccine from such agents. 

Control cells are often used with primary cell substrates and are described in more 

detail in Section II.B.4. 

4. Special considerations for Diploid Cell Strains 

Diploid cell strains are established from primary cell cultures by expansion and cell 

banking. These types of cells have a finite life span and are not immortal like cell 

lines. Diploid cells usually retain a diploid or near diploid karyotype, a 

characteristic that also differs from cell lines, which are generally aneuploid or nondiploid. 

You should determine the karyotype of your cell source, as it might be helpful to 

establish identity and to characterize a cell strain. Such analyses will establish the 

diploid character of your cells and determine its freedom from contamination with 

other cell lines. It might also be useful to monitor the genetic stability of the 

diploid cell strain throughout production. Other tests of identity (see Section IV) 

might be appropriate. 

Animal tumorigenicity testing is not needed if you are using genetically 

unmodified diploid cell strains, such as MRC-5 and WI-38, and FRhL-2, because 

their extensive previous characterization and well-defined non-tumorigenic 

phenotype satisfy the requirement in 21 CFR 610.18(c)(1)(ii) that they be described 

with respect to tumorigenicity. However, tumorigenicity testing is needed for a 

novel diploid cell substrate because such testing is needed to satisfy the 

requirement in 21 CFR 610.18(c)(1)(ii) that you describe its tumorigenicity. 

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5. Special Considerations for Continuous Cell Lines 

Some continuous cell lines, including Vero cells and CHO cells, have been used as 

substrates for licensed biologicals. Cell lines might have biochemical, biological, and 

genetic characteristics that differ from primary or diploid cells (e.g., they are typically 

aneuploid and have accumulated genetic changes). Because the mechanism by which 

most cell lines become immortal is generally unknown, and because some cell lines 

form tumors when inoculated into immunodeficient rodents, there are additional 

concerns for continuous cells lines compared with diploid cells, including potential 

adventitious agents and residual DNA. 

There has been considerable discussion internationally on general criteria for the 

acceptability of products prepared in cell lines (e.g., hormones, blood components, 

viral vaccines). These products should be purified to be free (see Section VI. 

Glossary) of adventitious agents and residual cells and should have low levels of 

cell-substrate DNA. When potential biological activity of residual cell substrate 

DNA is a concern, you should introduce procedures that extensively remove or 

degrade DNA. If you are considering the use of cell lines, we encourage you to 

develop and evaluate efficient methods for the purification of your product as an 

essential element of any product development program. 

Because certain cell lines might express endogenous viruses (e.g., retroviruses), tests 

capable of detecting such agents should be carried out on cells grown under 

production conditions (See Section IV.A. Testing for Adventitious Agents). If 

specific contaminants have been identified as endogenous agents in the MCBs and 

WCBs you are using, it is essential that you demonstrate their clearance (inactivation 

and/or removal) to a defined level by the purification procedures you use in 

production. You may obtain further guidance on this topic from the ICH Q5A 

document (Ref. 2). 

Under 21 CFR 610.18(c)(ii), you must describe cell lines with respect to 

tumorigenicity. Cell lines could acquire tumorigenic properties with increasing 

passage levels. It is therefore important that you limit the passage level of the cells 

used in production and that you characterize cells at or beyond this end-of-production 

limit. The maximum end-of-production passage level should be based on data 

derived from production cells expanded under comparable or analogous conditions to 

the production conditions. Cells from either the MCB or the WCB may be expanded 

for this evaluation. Tumorigenicity testing should be performed at or beyond this 

level. If you increase your defined limit for end-of-production, you should support 

the safety of this with data from cells that have been expanded to a passage level or 

population doubling level that is equal to or greater than your newly proposed end-ofproduction level. 

6. Additional considerations for cell lines that are tumorigenic or tumor-derived 

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You should perform additional testing if your cell lines are tumorigenic or derived 

from a tumor. You should assess cell lines that are tumorigenic or tumor-derived 

for potential oncogenic viruses and oncogenic substances (including nucleic acids), 

which could be associated with induction of a neoplastic process in a vaccine 

recipient. Test strategies for potential oncogenic viruses or oncogenic substances 

may be determined case-by-case, depending on the tissue type, source species, 

passage history, and extent of knowledge about the transforming event(s). (See 

section IV.B.1 for a discussion of tumorigenicty testing and section IV.B.2 for a 

discussion of oncogenecity testing.) 

In cases where you know the transforming event (for example, if the cells were 

transformed by a known oncogene), your testing should demonstrate that your final 

product is free of the transforming agent. For example, if adenovirus sequences are 

used to transform a primary human cell to produce a cell line (e.g., 293 cells), then 

testing should demonstrate that the final product is free of the introduced viral 

sequences. Similarly, if a virus is used to transform cells, that virus and its genetic 

material should not be detectable in the final product using an assay with sensitivity 

sufficient to provide assurance of safety. Tumorigenic or tumor-derived cell lines 

for which the mechanism of transformation is unknown will require additional 

testing to ensure the absence of potential transforming and oncogenic agents. You 

should consult with CBER on which tests you should perform and which methods 

you should follow. 

The testing recommended for qualification of cell substrates and cell banks should 

also be applied to rodent cell lines. However, because most rodent cell lines used 

for the production of biologicals are known to be tumorigenic, it is considered 

unnecessary to test rodent cell lines for tumorigenicity. A presumption is made that 

they will be tumorigenic, and the considerations described above apply. In 

addition, rodent cell lines are presumed to be capable of producing endogenous 

retroviruses. Assessment of the quantity and type of retroviruses produced should 

be performed. Infectivity assays for retroviruses are also recommended. You 

should only use rodent cell lines if your product can be sufficiently purified to 

demonstrate levels of viral clearance that assure the final product is not 

contaminated with retroviral particles. You may obtain additional guidance on 

viral clearance validation from the ICH Q5A document (Ref. 2). 

7. End-of-Production Cells 

You should characterize cells from the actual end-of-production of one or more lots 

or cells that are expanded to or beyond the end of production passage level. Such 

cells are referred to as End-of-Production Cells (EOPC). You should demonstrate 

the stability of the cell substrate characteristics using your EOPC. Your 

characterization should include growth characteristics, tumorigenic phenotype, 

expression of endogenous viruses, stability of expression of the inserted or 

engineered genes, and genetic stability. 

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C. VIRAL SEEDS 

The methods used to bank stocks of vaccine viral seeds are similar to those used in cell 

banking. As with cell banks, you should document passage history and derivation 

history of viral seeds. Your description should include donor screening, testing and 

donor medical history. Any manipulation of the viral phenotype, such as coldadaptation, development of temperature-sensitivity, or attenuation of virulence, should 

be well documented and described. Any genetic manipulations, such as reassortment 

or recombination, should also be well documented and described, including 

determining the nucleic acid sequences and sourcing of each biological starting 

material (e.g., plasmids, parental viruses). 

These vaccine virus banks are commonly referred to as the master viral seed (MVS) 

and working viral seed (WVS). Viral seeds should be stored in liquid nitrogen and in 

more than one location within a manufacturing facility or at a distant site for security 

reasons. You should assess your viral seed for its growth characteristics on your 

production cell substrate, tissue tropism, genetic markers, viability during storage, 

genetic stability through production, attenuation (if applicable), and its absence of 

adventitious agents. If attenuation or derivation is achieved by passage through 

different cell types from different species your viral seed should be assessed for 

absence of adventitious agents from all species that they might have been exposed to 

from isolation, through passage, and during production, including those that might be 

present in the raw materials used at each of these stages. 

1. Master Viral Seed 

You should extensively characterize your MVSs. In addition, you should 

demonstrate the stability of genotype and phenotype for a number of passages 

beyond the level used in your production. For example, if your MVS is considered 

to be at passage 47, your WVS at passage 48, your production cultures inoculated 

with WVS at passage 49, and your production process allows for only a single 

round of viral replication, then it would be appropriate for you to assess stability of 

your vaccine virus at passages 47 through 51 (5 passages). 

You should perform tests for identity (which could necessitate sequencing the 

entire vaccine virus), bacterial and fungal sterility, the presence of mycoplasmas, 

Mycobacterium tuberculosis (if appropriate), adventitious viruses (in vitro and in 

vivo tests), the viral phenotype (e.g., tissue tropism, attenuation properties, 

temperature sensitivity), and genetic stability. You should also consider specific 

tests for agents that might be present in the seed due to its passage history. 

In some cases, the virulence and the broad host range of your vaccine virus might 

complicate in vitro and in vivo adventitious agent testing. Testing for adventitious 

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agents might then require neutralization of viral seed. Preferably, neutralizing 

antibodies should be monoclonal and prepared in a species other than the cells in 

which the MVS was prepared. In addition, due to the potential for crossneutralization of adventitious human viruses, neutralizing antibodies should 

generally not be prepared from human or primate serum. It is important that you 

demonstrate that the neutralization procedure does not interfere with detection of 

adventitious viruses. Sometimes, it is not possible to efficiently neutralize a viral 

seed. In such cases, you may choose alternative strategies, including testing 

smaller quantities of seed, subculture onto fresh target cells in the in vitro 

adventitious agent test, or introduction of additional tests (e.g., PCR, Antibody 

Production assays (Ref. 11). 

Assessment of neurovirulence is often appropriate, and we recommend that you 

consult with CBER on appropriate animal models, methods, and scoring systems 

for this assessment before you initiate such studies. For viruses that are 

neurovirulent or might revert to neurovirulence (e.g., polioviruses), it might be 

necessary to assess neurovirulence not only on the MVS or an end-of-production 

passage level virus stock, but also on the product lot-by-lot. 

2. Working Viral Seed 

You may subject working virus seeds (WVSs) to less rigorous characterization than 

the MVSs from which they were derived. Once you have demonstrated your MVS 

to be free of adventitious agents from the species to which your vaccine virus had 

been exposed during its isolation and passage history, then you only need to show 

that your WVS is free of adventitious agents from the species you used to generate 

the WVS (e.g., production cells and raw materials used in propagation and 

processing). 

 

D. BIOLOGICAL RAW MATERIALS AND ANCILLARY REAGENTS 

You should test your raw materials and ancillary reagents and certify them for use in 

your vaccine production, including during the production of your cell banks and viral 

seeds. Raw materials derived from animals or humans should be tested and certified to 

be free from adventitious agents of the species from which they are derived. You 

should document whether you are relying on the vendor's COA or whether you are 

testing the raw materials. 

If your documentation for the raw materials used in the passage of your cell substrate is 

not adequate, you should test the cell substrate itself for adventitious agents of the 

relevant species. For human-derived raw materials and reagents, your documentation 

should include sourcing from appropriately screened and tested donors (using 21 CFR 

640.3 and 640.63 as guidelines) or use of products that are already licensed for human 

use. Testing and sourcing of bovine-derived materials and special considerations for 

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porcine-derived materials are discussed in Section III. All biological raw materials 

should be free of adventitious agents, including bacterial and fungal agents, 

cultivatable and non-cultivatable mycoplasmas, mycobacteria, and viruses. If your 

process includes removal or inactivation of potential infectious contaminants from 

biological raw materials, you should validate this process. 

1. Serum 

The serum used in vaccine production (including the production of the cell banks 

and viral seeds), such as bovine serum used in culture medium or human serum 

albumin used to stabilize vaccine virus, should be tested and certified. Human 

serum albumin should be from a source licensed for human use in the U.S. or 

derived from donors who have been appropriately screened and tested (21 CFR 640.3 

or 640.63). Bovine serum should be free of adventitious agents, including bacterial 

and fungal agents, mycoplasmas, mycobacteria, and bovine viruses (Section 

IV.A.2.f). 

Additionally, you should consult with CBER to ensure that you take the 

appropriate actions to minimize BSE risk. 

2. Trypsin 

You should clearly identify the species from which the trypsin used in your vaccine 

production (including the production of the cell banks and viral seeds) is derived. 

If bovine trypsin is used, the concerns identified in Section IV.A.2.f, “Testing and 

Sourcing of Bovine-Derived Materials,” apply. If porcine trypsin is used, it should 

be tested in accordance with the recommendations described in Section IV.A.2.g, 

“Testing of Porcine-Derived Reagents." 

3. Amino Acids 

You should document the source of the amino acids used in growth medium or in 

production. You should also consult with CBER to ensure that you take the 

appropriate actions to minimize BSE risk. 

4. Other Biological Reagents 

You should control and test other biological reagents used during manufacturing, 

such as transferrin, insulin, or other growth factors used in growth medium, as 

appropriate for their species of origin. If derived from humans, the ancillary 

product should either be licensed in the U.S. or derived from donors for whom 

appropriate screening and testing have been applied (if appropriate, comparable 

with blood or source plasma donors, see 21 CFR 640.3 or 640.63). 

Under 21 CFR 610.15(c), you may not add penicillin to the production substrate of 

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viral vaccines. Also, other beta-lactam antibiotics should not be present in 

production cell cultures. 

E. CONSIDERATIONS IN TESTING AT DIFFERENT STAGES OF 

PRODUCTION 

See Appendix 1 for a list of tests to consider at each stage of production for a viral 

vaccine. 

1. Cell Banks 

Qualification of cell banks is discussed in Section III.B. 

2. Pre-Production Cells 

An identity test may be performed on cells directly prior to production. 

3. Pre-filtered Harvest or End-of-Production Cells 

In general, the stage at which adventitious agents are most likely to be found is the 

stage at which the most extensive adventitious agent testing should be performed 

for each product. For many viral vaccines, the pre-filtered harvest is the stage of 

manufacture that is most concentrated and at which the least processing has been 

performed. For these reasons, this might be the best stage of production for testing 

for adventitious agents. 

In addition to testing he viral or vaccine bulk harvest for cultivatable mycoplasmas, 

as required under 21 CFR 610.30 requires, you should also test the viral or vaccine 

bulk harvest for non-cultivatable mycoplasmas (and spiroplasmas, if appropriate), 

and adventitious viruses by in vitro and in vivo methods. This might include a test 

for hemadsorbing viruses. If the production process is capable of inducing 

expression of retroviruses, PCR-based RT testing or infectivity studies might also 

be recommended at this stage. If appropriate, a test or tests for Mycobacterium 

tuberculosis should be performed. 

As discussed for the MVS (Section III.C.1), if the assay system used for in vitro or 

in vivo adventitious virus testing is capable of supporting replication of the vaccine 

virus, and if the vaccine virus replicates to levels that interfere with the adventitious 

agent tests, then it might be necessary to neutralize the vaccine virus prior to 

performing these tests. If neutralization is difficult to achieve, the other 

considerations in Section III.C.1 might be applicable. 

If cells survive the production process (e.g., if the vaccine virus does not result in a 

lytic infection), end-of-production cells may also be tested for adventitious agents. 

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If multiple harvests are performed for a single vaccine lot, testing should be 

performed on each individual harvest in order to avoid dilution of a potentially 

contaminated harvest with uncontaminated harvests. 

4. Control Cells 

Recommendations for testing of control cells are discussed in Section II.B.3 and 4. 

5. Post-Filtered Harvest or Final Bulk 

The post-filtered harvest or final bulk should be tested for bacterial and fungal 

sterility. Other testing might be appropriate for the safety, identity, purity, potency, 

and quality of the final bulk. The appropriate test will depend on the product. 

These include testing for levels of residual cellular proteins and cellular nucleic 

acids. If the cell line used to produce the vaccine is known to produce viruses other 

than the vaccine virus, the clearance of these viruses should be demonstrated in the 

production processes. Guidance regarding viral clearance may be sought from the 

ICH Q5A document (see Ref. 2). If processes are not validated for clearance of 

residual cells, a test might be recommended at this stage. 

6. Final Filled Product 

 

Under 21 CFR 610.12, the sterility of bulk and final filled biologics must be 

demonstrated by specific tests listed in that section, except for products listed in 

subsection (g), including products granted an exemption under subsection (g)(4)(ii). 

Such an exemption may be granted for oral products, but those products should still 

be assessed by the microbial limits test for bioburden (Ref. 13). Under 21 CFR 

610.11a, each lot of inactivated influenza vaccine must be assayed for endotoxin in 

comparison to a reference preparation provided by FDA. Other final filled 

products should also be assessed for endotoxin levels. In addition, a general safety 

test for detecting extraneous toxic contaminants, under 21 CFR 610.11, is required 

on final filled containers, except as described in 21 CFR 610.11(g). Other tests 

should be performed to assess the safety, identity, purity, potency, and quality of 

the final filled product. These tests are determined case-by-case depending on the 

product and should be described in your IND and BLA submissions. 

When downstream processing provides clearance, concentration or dilution of the 

product, the final filled product stage might not be the best stage for performing 

certain tests (adventitious agents).

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IV. DESCRIPTION OF QUALITY-CONTROL TEST METHODS 

A. TESTING FOR ADVENTITIOUS AGENTS 

Your biological starting materials should be characterized to ensure that they are free 

from extraneous infectious organisms such as bacteria, fungi, cultivatable and noncultivatable mycoplasmas and spiroplasma, mycobacteria, viruses, rickettsia, 

protozoa, and the agent(s) responsible for transmissible spongiform encephalopathies 

(TSEs), where possible. For a substance to be considered free of a contaminant, your 

assay should demonstrate, at a predefined level of sensitivity, that a certain quantity 

of the substance is free of that contaminant. Alternatively, a validated process that is 

known to remove a contaminant to a defined level may be used to demonstrate the 

absence of that contaminant. If an adventitious agent is known to be present in your 

cell substrate or viral seed, then you should demonstrate that your production process 

is sufficiently robust to eliminate or inactivate the agent within an appropriate margin 

of safety. You should avoid exposure of your product to agents that are known to be 

infectious for humans (other than the vaccine virus) and/or to agents for which there 

are no appropriately sensitive validated testing procedures. You should provide 

documentation indicating that you have appropriate and sufficient controls in place to 

avoid such exposure. 

Many of the recommended tests described later in this document were originally 

promulgated in 21 CFR part 630 but were removed when the regulations were revised 

in 1996 (61 FR 40153). FDA removed many of the specific tests from the regulations 

to allow manufacturers flexibility in choosing appropriate new tests that might 

become available. For each of the suggested adventitious agent tests, alternatives 

such as those recommended by the World Health Organization (WHO) or the 

European Pharmacopoeia (EP) might be considered if justified with data showing 

sensitivity comparable to the recommended test. Different inoculation strategies for 

testing cell bank lysates and viral harvests may be acceptable when justified in the 

context of the entire testing program. 

The appropriate test for adventitious agents will vary depending on a variety of 

factors, including the origin of the cell substrate and its history. We encourage 

manufacturers to consult with us regarding the appropriate tests. See sections II and 

III for additional information on factors to take into account in selecting appropriate 

tests. 

1. In Vivo Tests 

In the development of viral vaccines, in vivo adventitious agent testing includes 

inoculation of adult and suckling mice and inoculation of embryonated chicken 

eggs. Animal antibody-production tests may be performed when the potential for 

exposure to rodent viruses exists, for example, through exposure to rodents or 

rodent cells. These tests also may be performed in other species to detect species-

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specific viruses. For example, inoculation of rabbits or guinea pigs may also be 

used to detect additional agents, when appropriate. 

a. Adult Mice 

This test detects adventitious viruses including lymphocytic choriomeningitis 

virus (LCMV), coxsackieviruses, flaviviruses, and rabies virus. 

Each of at least 20 adult mice weighing 15-20 grams should be inoculated 

intraperitoneally with 0.5 mL and intracerebrally with 0.03 mL of the material 

to be tested. The mice should be observed daily for 21 days. Each mouse that 

dies after the first 24 hours of the test, or is sacrificed because of illness, 

should be necropsied and examined for evidence of viral infection by gross 

observation and intraperitoneal and intracerebral inoculation of appropriate 

homogenized tissue into at least five additional mice, which should each be 

observed daily for 21 days. The material may be used only if at least 80% of 

the originally inoculated mice and at least 80% of each subsequently 

inoculated group of mice remain healthy and survive the observation period, 

and if none of the mice show evidence of a transmissible agent or other viral 

infection, other than agents known to be a component of the tested material 

(i.e., vaccine strains of virus, when relevant). 

When this test is used to evaluate cell substrates, a lysate of cells and their 

culture medium (equivalent to 107 cells per mL) should be inoculated. When 

virus seeds are tested, it might be necessary to neutralize the vaccine virus 

prior to inoculation into mice. If the vaccine virus needs to be neutralized in 

order to perform a valid test, the source of the antiserum and its influence on 

test results should be considered; in general, antiserum used for neutralization 

should not be of human or simian origin or from the same cell type used for 

production of the vaccine virus. 

b. Suckling Mice 

This test detects adventitious agents including many human viruses, such as 

coxsackievirus types A and B (type B is also detectable in cell culture) and 

other picornaviruses such as polioviruses and echoviruses, alphaviruses, 

bunyaviruses, (including phleboviruses and nairoviruses), arenaviruses, 

flaviviruses, rabies, and herpesviruses (such as herpes simplex virus). This 

test can also detect many murine agents. 

Each of at least 20 suckling mice less than 24 hours old should be inoculated 

intraperitoneally with 0.1 mL and intracerebrally with 0.01 mL of the material 

to be tested. The mice should be observed daily for at least 14 days. Each 

mouse that dies after the first 24 hours of the test, or is sacrificed because of 

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illness, should be necropsied and examined for evidence of viral infection by 

gross observation and intraperitoneal and intracerebral inoculation of 

appropriate tissue into at least five additional mice, which should each be 

observed daily for 14 days. In addition, a blind passage (via intraperitoneal 

and intracerebral inoculation into at least 5 additional mice) should be made of 

a single pool of the emulsified tissue (minus skin and viscera) of all mice 

surviving the original 14-day test. The material may be used only if at least 

80% of the originally inoculated mice and at least 80% of each group of 

subsequently inoculated mice remain healthy and survive the entire 

observation period and if none of the mice show evidence of a transmissible 

agent or other viral infection, other than agents known to be a component of 

the tested material (i.e., vaccine strains of virus, when relevant). 

When this test is used to evaluate cell substrates, a lysate of cells and their 

culture medium (equivalent to 107

 cells per mL) should be inoculated. When 

virus seeds are tested, it might be necessary to neutralize the vaccine virus 

prior to inoculation into mice. If the vaccine virus needs to be neutralized in 

order to perform a valid test, the origin of the antiserum and its influence on 

test results should be considered as noted above. 

c. Guinea Pigs 

This test detects Mycobacterium tuberculosis and adventitious viruses 

including paramyxoviruses (including Sendai virus), reoviruses, and 

filoviruses. 

Each of at least 5 guinea pigs each weighing 350-450 grams should be 

inoculated intraperitoneally with 5 mL and intracerebrally with 0.1 mL of 

each material to be tested. The animals should be observed daily for at least 

42 days. Each animal that dies after the first 24 hours of the test, or is 

sacrificed because of illness, should be necropsied. All remaining animals 

should be sacrificed and necropsied at the end of the observation period. The 

material may be used only if at least 80% of the originally inoculated animals 

remain healthy and survive the observation period and if none of the animals 

shows evidence of a transmissible agent or other viral infection, other than 

agents known to be a component of the tested material (i.e., vaccine strains of 

virus, when relevant). 

When this test is used to evaluate cell substrates, a lysate of cells and their 

culture medium (equivalent to 107 cells per mL) should be inoculated. When 

virus seeds or production harvests are tested, it might be necessary to 

neutralize the vaccine strain prior to inoculation into animals. 

 

In vitro methods, such as culture and PCR, are also acceptable for identifying 

Mycobacterium tuberculosis when validated. 

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d. Rabbits 

This test detects simian herpes B virus. 

Each of at least 5 healthy rabbits each weighing 1500-2500 grams should be 

inoculated intradermally in multiple sites with a total of 1.0 mL of the material 

to be tested and subcutaneously with 2.0 mL of the material to be tested. The 

animals should be observed daily for at least 30 days. Each animal that dies 

after the first 24 hours of the test, or is sacrificed because of illness, should be 

necropsied. The material may be used only if at least 80% of the originally 

inoculated animals remain healthy and survive the observation period, and if 

none of the animals show evidence of a transmissible agent or other viral 

infection, including lesions at the site of inoculation, other than agents known 

to be a component of the tested material (i.e., vaccine strains of virus, when 

relevant). 

When this test is used to evaluate cell substrates, a lysate of cells and their 

culture medium (equivalent to 107 cells per mL) should be inoculated. When 

virus seeds are tested, it might be necessary to neutralize the vaccine strain 

prior to inoculation into animals. 

Alternative tests for herpes B virus might also be acceptable if justified. 

e. Embryonated Chicken Eggs 

This test detects adventitious agents including: 

• by the allantoic route: orthomyxoviruses (influenza virus) and 

paramyxoviruses (mumps, measles, parainfluenza viruses), 

alphaviruses, and vesiculoviruses; and 

• by the yolk sac route: herpesviruses, poxviruses, rhabdoviruses, as 

well as rickettsiae, mycoplasmas, and bacteria. 

A sample volume, equivalent to at least 100 doses, or 10 mL, whichever 

represents a greater volume, should be used in egg testing. At least 10 

embryonated eggs, 10 to 11 days old, should be inoculated by the allantoic 

route using 0.5 mL per egg. Following incubation at 35oC for 72 hours, the 

allantoic fluids should be harvested, pooled, and passaged by the same route 

into fresh, embryonated eggs, 10 to 11 days old, using 0.5 mL per egg and 

incubated at 35°C for 72 hours. Both the initial pool and the passaged harvest 

should be tested for the presence of hemagglutinating agents with red cells 

from guinea pigs, humans (type O), and an avian species. The tested material 

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passes the test if all the embryos appear normal and there is no evidence of 

hemagglutinating agents. 

At least 10 additional embryonated eggs, 6 to 7 days old, should be inoculated 

by the yolk sac route using 0.5 mL per egg. Following incubation at 35°C for 

at least 9 days, the yolk sacs should be harvested and pooled. A 10% 

suspension of yolk sacs should be passaged by the same route into the yolk 

sacs of fresh embryonated eggs, 6 to 7 days old, using 0.5 mL of inoculum per 

egg and incubated at 35°C for at least 9 days. The material passes the test if 

all the embryos in both the initial test and the passage appear normal. 

When this test is used to evaluate cell substrates, a lysate of cells and their 

culture medium (equivalent to 107 cells per mL) should be inoculated. When 

virus seeds are tested, it might be necessary to neutralize the vaccine strain 

prior to inoculation into animals. If vaccine virus needs to be neutralized in 

order to perform a valid test, then antisera used for neutralization should not 

be of human, simian, or avian origin. 

f. Antibody Production Tests

Antibody production tests are performed in the species-appropriate model 

(often rodents such as hamsters, rats or mice, but sometimes chickens) when 

the potential exists for exposure to rodent viruses. The test article is 

inoculated into specific-pathogen-free (SPF) animals that are subsequently 

tested for antibodies to specific agents. Agents detected in hamster antibody 

production (HAP) test include: lymphocytic choriomeningitis virus (LCMV), 

pneumonia virus of mice (PVM), reovirus type 3 (Reo3), Sendai virus, and 

simian virus 5 (SV5). The agents detected in rat antibody production (RAP) 

test include: Hantaan virus, Kilham rat virus (KRV), LCMV, mouse 

adenovirus, mouse encephalomyelitis virus (Theilers, GDVII), PVM, rat 

coronavirus (RCV), Reo3, sialodacryoadenitis virus (SDAV), Sendai virus, 

and Toolan virus (HI). The agents detected in mouse antibody production 

(MAP) test include: ectromelia virus, mouse rotavirus (EDIM) Hantaan virus, 

LCMV, lactic dehydrogenase virus (LDM), minute virus of mice (MVM), 

mouse adenovirus (MAV), murine cytomegalovirus (MCMV), mouse 

encephalomyelitis (Theilers, GDVII), mouse hepatitis virus (MHV), PVM, 

polyoma virus, Reo3, Sendai virus, thymic virus, and K virus (a mouse 

pneumonitis virus related to polyomaviruses).

A specific in vivo test for LCMV may be warranted when specific concerns 

about LCMV exist.. This in vivo test involves intracerebral inoculation of 

mice used in the MAP test with live virulent LCMV to detect interference 

from non-lethal strains. Animals infected during the MAP test with nonvirulent strains of LCMV will survive this challenge. If you use alternative 

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methods, such as PCR, you should provide data showing sensitivity 

comparable to that of the suggested test. 

Antibody tests such as “Test for Extraneous Agents Using Chicks” (Ref. 14), 

might also be useful in certain cases where the vaccine virus interferes with in 

vitro culture assays for adventitious virus testing. 

2. In Vitro Tests for Viruses 

a. Cell Culture Safety Test 

Cell culture safety tests can detect a variety of adventitious viruses that 

include cytopathic viruses, hemadsorption viruses, and hemagglutinating 

viruses. You should select cells appropriate for such tests and relevant to 

potential exposures of your materials including: 

 

• monolayer cultures of the same species and tissue type as that used 

for production; 

• monolayer cultures of human diploid cells; and 

• monolayer cultures of monkey kidney cells. 

Human diploid cells and monkey kidney cells are commonly used and are 

often susceptible to infection by many of the viruses listed below. Specialized 

cell culture testing might be needed to detect growth of other viruses. 

i. Human Diploid Cells 

Human viruses: herpesviruses (including herpes simplex virus, 

varicella zoster virus, and cytomegalovirus), adenoviruses, 

coronaviruses, reoviruses, alphaviruses, rubella, flaviviruses, rabies, 

enteroviruses, certain strains of hepatitis A virus, poliovirus, coxsackie 

B virus, echovirus, rhinoviruses, orthomyxoviruses, and 

paramyxoviruses. 

Simian viruses: SCMV (simian cytomegalovirus). 

ii. Monkey Kidney Cells 

Human viruses: enteroviruses, coxsackie B viruses, echoviruses, 

orthomyxoviruses, paramyxoviruses, herpes simplex virus, poxviruses, 

polyomaviruses, rotavirus, alphaviruses, rubella, flaviviruses, rabies 

viruses, vesiculoviruses, filoviruses, influenza viruses, bunyaviruses 

(including phleboviruses and nairoviruses), arenaviruses, and 

reoviruses, polioviruses, rhinoviruses, adenoviruses (some strains). 

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Simian viruses: herpes B virus. 

 

Methods: 

 

An appropriate volume should be inoculated onto monolayer cultures of at 

least 3 cell types (see preceding discussion for appropriate cell types). The 

sample to be tested should be diluted as little as possible. The cell cultures 

should be observed for at least two weeks. After two weeks of observation, 

supernatants or lysates are subcultured onto fresh cells and observed for at 

least an additional two weeks. This subculture onto fresh cells might help to 

distinguish between non-specific CPE and virus-induced CPE, as toxic effects 

of the initial specimen or length in culture will be diluted, whereas virusinduced CPE should be amplified. If the possibility of contamination with 

human or simian cytomegaloviruses is a consideration, the first set of human 

cell cultures should be observed for at least four weeks unless a shorter 

incubation period is shown to be equally sensitive. A test is considered 

satisfactory only if there is no evidence of adventitious agents and at least 

80% of the cultures are available for observation at the end of the observation 

period. 

The volume to be tested should be determined based on your total culture or 

batch size and the number of cells you require to produce a dose of vaccine. 

Generally, you should use 107

 cells or cell lysate equivalent to that number of 

cells when testing the cell substrate. When it is not possible to directly test 

cells or cell lysates, a corresponding quantity of cell culture supernatant 

should be tested. Historically, 500 doses or 50 mL of neutralized vaccine 

harvest, pool, or bulk (whichever represented more virus) were used as the 

inoculum onto each indicator cell culture. Under some circumstances, where 

neutralizing the vaccine strain has been difficult, smaller amounts were tested. 

When virus seeds are tested, it might also be necessary to neutralize the 

vaccine strain prior to inoculation onto indicator cells. 

You may assess virus replication in cell-culture tests by various means, 

including development of cytopathic effect. If appropriate for your product, 

testing for hemadsorption or hemagglutination at the end of the observation 

period could facilitate the detection of non-cytopathic viruses. Hemadsorption 

refers to the ability of infected cells to bind to red blood cells (RBCs), while 

hemagglutination refers to the ability of infected cell supernatants to 

agglutinate RBCs. The test for hemadsorbing and hemagglutinating viruses is 

generally performed at the end of the observation period using guinea pig, 

chicken, and human type O RBCs, which are added independently to different 

portions of the cell cultures or to aliquots of the supernatants at 2-8°C for 30 

minutes, and observed for hemadsorption and hemagglutination, respectively. 

This is followed by incubation with RBCs for 30 minutes at room 

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temperature, followed by observation for hemadsorption/hemagglutination. 

Red blood cells from rhesus monkeys have occasionally been used for 

detection of certain respiratory viruses.

The test for hemadsorbing and hemagglutinating viruses may also be 

performed at the end of production, directly on cells used for production or on 

control cell cultures. Historically, one quarter to one third of the control 

vessels were tested for hemadsorption viruses. 

b. Transmission Electron Microscopy (TEM) 

TEM can detect viral particles in a cell substrate, including those from 

endogenous retroviruses. Under some circumstances, it might be appropriate 

to pre-treat cells with chemical or inducing agents to activate production of 

endogenous or latent viruses (Ref. 15). While TEM is fairly insensitive, it is a 

generic assay that can detect adventitious agents of many types. TEM can 

also be used to estimate the concentration of viral particles to support 

validation of viral clearance. Methods include negative staining and thin 

section. A discussion of these methods is provided in Bierley et al. (Ref. 16). 

c. Biochemical Tests for Retroviruses 

Retrovirus testing using reverse transcriptase (RT) assays should be 

performed on cell-free culture medium to detect retroviruses. RT assays can 

detect any retrovirus, as all retroviruses encode and contain RT. You should 

test cell substrates, viral seeds, and/or harvests of all viral vaccines produced 

in mammalian cell substrates (or that use or include mammalian-derived 

materials) using a highly sensitive PCR-based RT (PBRT) assay. If you plan 

to use conventional RT assays, you should provide a strong justification for 

using a less sensitive assay. 

You should validate the performance of the PBRT for your product, especially 

with regard to the lower limit of detection for both manganese- and 

magnesium-dependent retroviral RTs, the specificity, and the reproducibility 

of the assay. The lower limit of detection should be comparable with the 

published literature (Ref. 17). If you obtain a positive result with the assay, 

you might need to perform infectivity studies to demonstrate whether the 

source of the positive result is an infectious retrovirus. The Center for 

Biologics Evaluation and Research (CBER) can provide you with further 

information on infectivity studies upon request. 

The stage of manufacture at which you should perform the PBRT test is 

dependent on the manufacturing process of your product. For example, 

products manufactured from primary cells might need to be assessed lot-bylot. In cases when control cell cultures are set up in parallel with production 

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cells (Section II.B.4), PBRT testing of cell-free medium from the control cell 

culture should be performed. Products manufactured from an established 

PBRT-tested cell bank and PBRT-tested viral seed, generally, do not need to 

be tested lot-by-lot. 

CBER recognizes that some products and reagents have RT activity that does 

not to represent adventitious infectious retroviruses (e.g., some avian cell 

substrates, such as chick embryo fibroblasts, chick embryo dermal cells, and 

eggs). For avian cells that have RT activity, you should perform testing to 

assure the absence of major avian retroviruses. Major avian retroviruses 

include exogenous retroviruses, such as avian leucosis virus (ALV), avian 

sarcoma virus, and reticuloendotheliosis virus, and infectious endogenous 

retroviruses, such as RAV-0. You may perform this testing directly on the 

cell substrate. In some cases, careful documentation of the health of a specific 

pathogen free flock might be adequate to assure the absence of these viruses. 

CBER can provide you with further information upon request. 

d. Infectivity Tests for Retroviruses 

For non-murine retroviruses, infectivity testing on appropriate indicator cells 

(selected for their susceptibility to different retrovirus types) might be 

appropriate. Such testing strategies would be determined case-by-case and 

should be discussed with CBER before implementation. You should test 

rodent cell substrates and viral seeds that are potentially contaminated with 

murine retroviruses for infectivity using Mus dunni cells or other highly 

susceptible cells and/or in S+

L- assays (Ref. 18). S+L- assays may be done on 

feline PG4 cells (to detect amphotropic viruses), mink cells (to detect 

xenotropic viruses) or murine D56 cells (to detect ecotropic viruses). You 

may use a broadly reactive monoclonal antibody to detect viruses cultured in 

these assays. Infectivity tests that are capable of detecting ALV (Ref. 19) and 

other avian retroviruses might be appropriate for cell substrates of avian 

origin. Often, infectivity tests may be augmented with sensitive RT assays. 

Under some circumstances, for example when tumorigenic cell substrates are 

proposed for use, it might be appropriate for you to pre-treat cells with 

chemical agents known to induce reactivation or replication of endogenous or 

latent viruses. 

e. PCR or Other Specific in vitro Tests 

In cases where viruses cannot be readily grown in culture, PCR might be the 

most effective tool to assess a cell substrate for contamination with such 

viruses. Due to the specificity of PCR, you might need to perform multiple 

PCR assays in order to detect the full range of viruses detectable in a single 

more general biological assay. You should consider the tissue source and 

medical history of the donor from which the cell line was derived in 

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determining what testing is appropriate. Specific PCR tests for human 

pathogens that you should consider include assays for hepatitis A, B, and C 

viruses, enteroviruses, human HIV-1, HIV-2, HTLV-1, HTLV-2, circoviruses, 

parvovirus B19, papillomaviruses, human polyomaviruses, human 

adenoviruses, Epstein-Barr virus, human cytomegalovirus, and human herpes 

viruses 6, 7, and 8. Specific tests for simian viruses that could infect humans 

include simian polyomaviruses (e.g., SV40), simian foamy virus (SFV), 

simian immunodeficiency virus (SIV), simian retrovirus (SRV), and simian Tcell lymphotropic virus (STLV). You also should consider using PCR assays 

that detect several agents using degenerate or consensus primers provided the 

sensitivity of these assays is sufficient to assure product safety. 

Although less sensitive, you may consider the use of Southern blot 

hybridization analysis of DNA extracted from the cell substrate or tissue 

culture to assess for contamination with some viruses. Either PCR or 

Southern hybridization may be used to detect non-cytopathic agents in tissue 

culture tests. 

f. Sourcing and Testing of Bovine-Derived Materials 

You should source bovine-derived reagents in a manner that minimizes the 

risk of contamination from bovine spongiform encephalopathy (BSE). We 

recommend that you consult with CBER for more information. 

We recommend that you test bovine-derived reagents using assays at least as 

sensitive as the cell culture and fluorescent antibody testing recommended in 

9 CFR 113.53 and 113.47. We recommend the use of Vero cells and bovine 

cell culture, such as bovine turbinate cells. Agents of concern from bovine 

sources include: bovine parainfluenza Type 3, infectious bovine 

rhinotracheitis virus, rabies virus, reovirus, bovine adenoviruses, bovine 

respiratory syncytial virus, bovine parvovirus, bluetongue virus, and bovine 

viral diarrhea virus (including non-cytopathic strains). 

We may request testing for other potential contaminants of bovine materials 

depending upon exposure history and species of origin of a cell substrate or 

reagent including circoviruses, bovine herpesviruses, bovine polyomavirus, 

bunyaviruses, and bovine retroviruses (such as bovine immunodeficiency 

virus and bovine leukemia virus). Some of these agents might be detected in 

cell culture, while others might require additional tests. 

g. Testing of Porcine-Derived Reagents 

The testing required for porcine adventitious viruses in products intended for 

use in animals (9 CFR 113.47) represents our minimum recommendations for 

products intended for use in humans. Agents of concern from porcine cells 

listed in 9 CFR 113.47(b)(1) and (6) include: porcine parvovirus, bovine viral 

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diarrhea virus (BVDV), reovirus, rabies, porcine adenovirus, transmissible 

gastroenteritis virus (TGE), and porcine hemagglutinating encephalitis virus. 

At a minimum, you should test porcine trypsin for porcine parvovirus. 

Other porcine agents that might be of concern, depending upon exposure 

history and species of origin of your cell substrate or reagent, include 

circoviruses, enteroviruses, porcine respiratory and reproductive syndrome 

virus (PRRS), porcine cytomegalovirus, porcine influenza viruses, 

pseudorabies virus, swine pox virus, swine fever virus (hog cholera virus, 

African), vesicular stomatitis virus (VSV) (Ref. 20), Nipah virus and porcine 

retroviruses. 

3. In Vitro tests for Non-Viral Agents 

a. Mycoplasma 

All licensed viral vaccines produced in cell substrates (and similar products) 

should be tested to ensure the absence of mycoplasma contamination. For 

most such products, testing is performed on the virus seed and/or master cell 

banks, cell substrate, and a representative portion of each working cell stock 

used for manufacture of the product. Each lot of product harvest concentrate 

should be tested prior to further processing, e.g., prior to clarification, 

filtration, purification, and inactivation, unless testing at this stage of the 

manufacturing process is not feasible. Under 21 CFR 610.30, for virus 

vaccines produced from in vitro living cell cultures, you must test each virus 

harvest pool for mycoplasma prior to clarification or filtration in the case of 

live virus vaccines and prior to inactivation in the case of inactivated virus 

vaccines. Prior to testing, the product harvest concentrate sample must be 

stored either between 2 and 8 ̊C for no longer than 24 hours or at -20 ̊C or 

lower if stored for more than 24 hours. If stored for longer than 24 hours, we 

recommend that you store the sample at -60 ̊C or lower to allow for greater 

recovery of mycoplasma. 

Although mycoplasma testing prescribed in 21 CFR 610.30 specifies agar and 

broth media culture procedures, we now know that some fastidious strains of 

mycoplasma, especially Mycoplasma hyorhinis, might not be detected by the 

agar and broth media procedures, and therefore you should include an 

indicator cell culture procedure to detect such strains. We consider the testing 

described below to be acceptable alternatives to the method prescribed in 21 

CFR 610.30. However, you must seek and obtain approval for these 

modifications in accordance with 21 CFR section 610.9(b). Additional 

information regarding mycoplasma testing is available (Ref. 21). 

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Testing for Mycoplasma using both Alternative Agar and Broth Media,

and Indicator Cell Culture Procedures

• Agar and Broth Media Procedure 

(1) Each lot of agar and broth medium should be free of antibiotics 

except for penicillin, and each lot of medium should be examined 

for mycoplasma growth-promoting properties. To demonstrate the 

capability of the media to detect known mycoplasma contaminants, 

use the mycoplasma cultures specified below in (3)(i) as positive 

controls. 

(2) (i) Inoculate no less than 0.2 mL of the test sample (e.g., 

product harvest concentrate sample) in evenly distributed amounts 

over the surface of 2 or more agar plates of 1 medium formulation. 

(ii) Inoculate no less than 10 mL of the product harvest concentrate 

sample into a flask containing 50 ml of broth medium which is 

incubated at 36 ± 1° C. 

(iii) Test 0.2 mL of the broth culture on the 3rd, 7th, and 14th days 

of incubation by subculture onto 2 or more agar plates of the same 

medium formulation as that used above in (i). 

(iv) Incubate 2 of the initial isolation plates and 2 each of the three 

subculture plates in a 5 to 10 % carbon dioxide in nitrogen and/or 

hydrogen atmosphere containing less than 0.5% oxygen during the 

test incubation period. 

(v) Incubate all culture agar plates for no fewer than 14 days at 36 

± 1°C and observe them microscopically at 100 times 

magnification or greater for growth of mycoplasma colonies. 

(3) (i) Include in each test at least 2 known mycoplasma species or 

strains as positive controls, 1 of which should be a dextrose 

fermenter (i.e., M. pneumoniae strain FH or equivalent species or 

strains) and 1 of which should be an arginine hydrolyzer (i.e., M. 

orale strain CH19299 or equivalent species or strain). Positive 

control cultures should be not more than 15 passages from 

isolation and should be used in a standard inoculum of 100 colonyforming units (CFU) or 100 color-changing units (CCU) or less. 

(ii) Include uninoculated agar medium as a negative control. 

(4) Interpret the results of the procedure according to the 

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specification detailed in this section under Interpretation of 

Results. 

• Indicator Cell Culture Procedure 

(1) Using a Vero cell culture substrate, pre-test the procedure by 

using the mycoplasma cultures specified below in (3) (i) as 

positive controls to demonstrate the capability of the cell substrate 

to detect known fastidious mycoplasmal contaminants. An 

equivalent indicator cell substrate might be acceptable if data 

demonstrate at least equal sensitivity for the detection of known 

mycoplasmal contaminants. 

(2) (i) Inoculate no less than 1 mL of the test sample (e.g., product 

harvest concentrate sample) to 2 or more indicator cell cultures 

grown on cover slips in dishes or equivalent containers. 

(ii) Incubate the cell cultures for 3 to 5 days at 36 ± 1°C in a 5% 

carbon dioxide atmosphere. Examine the cell cultures for the 

presence of mycoplasmas by epifluorescence microscopy using a 

DNA-binding fluorochrome, such as bisbenzimidazole or an 

equivalent stain. 

(3) (i) Include in each test two known mycoplasma species or 

strains as positive controls (i.e., M. hyorhinis strain DBS 1050, M. 

orale strain CH19299, or equivalent species and strains), using an 

inoculum of 100 CFU or 100 CCU or fewer. 

(ii) Include as a negative control a non-infected indicator cell 

culture. 

(4) Interpret the results of the procedure according to the 

specifications detailed in this section under Interpretation of 

Results. 

• Interpretation of Results 

(1) For the agar and broth media procedure, compare the 

appearance of the media inoculated with the product to that of the 

positive and negative controls. 

(2) For the indicator cell culture procedure, using 600 times 

magnifications or greater, compare the microscopic appearance of 

the cultures inoculated with the product to that of the positive and 

negative cell controls. 

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(3) Marked cytopathic effects or nuclear chromatin fragmentation 

caused by vaccine virus infection that affect the interpretation of 

the results can be minimized by using a specific neutralizing viral 

antiserum or a nonpermissive cell culture substrate. The antisera 

should also be added to the positive and negative controls. 

(4) The product is considered satisfactory for manufacture if both 

the agar and/or broth media procedure and the indicator cell culture 

procedure show no evidence of mycoplasma contamination (i.e., 

no growth) and thus resemble the negative control(s) for Agar and 

Broth and Indicator Cell Procedures. 

(5) If mycoplasmas are recovered, you should perform confirmatory 

testing to establish the species 

Testing for Mycoplasma Using PCR-based Assays 

PCR-based assays may be used to detect mycoplasma, provided that such an 

assay can be shown to be comparable to the agar and broth media procedure 

and the indicator cell culture procedure. In some cases, culture-based 

procedures cannot be used due to an inability to completely neutralize vaccine 

viruses, thus necessitating the use of PCR-based assays to test for mycoplasma 

in these products. 

b. Bacterial and Fungal Sterility 

You must perform the test for bacterial and fungal sterility described in 21 

CFR 610.12 or an equivalent method that fulfills the requirements of 21 CFR 

610.9. 

c. Mycobacteria Testing 

You should use the culture methods for detection of mycobacteria described 

in the next paragraph to test your culture filtrate in its most concentrated form. 

The previously described guinea pig tests have also been used to detect 

mycobacteria. If you use alternative tests, including PCR assays, they should 

be shown to be sufficiently sensitive to test for the presence of mycobacteria. 

A 2.0 mL sample of the filtrate should be inoculated onto Lowenstein – 

Jensen’s egg medium or other media demonstrated to be equally capable of 

supporting growth. An appropriate positive-control test should be conducted 

simultaneously with the sample under test, and the test should be shown to be 

capable of supporting the growth of small numbers of the mycobacteria. All 

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the test vessels should be incubated at a suitable temperature for a period of 6 

weeks under conditions that will prevent drying of the medium, after which 

the cultures should be examined for evidence of mycobacterial colonies. The 

filtrate is satisfactory for use if the test shows no evidence of mycobacteria. 

B. TESTING OF CELL PROPERTIES 

1. Tests for Tumorigenicity 

Tumorigenicity is defined as the process by which cells form tumors when 

inoculated into animals (generally a syngeneic, an immunosuppressed allogeneic or 

an immunosuppressed xenogeneic host) (see Section VI. Glossary). 

Tumorigenicity is a characteristic of the immortalized cells themselves, rather than 

of agents or components present in them.

Tumorigenic cells have not traditionally been used for the production of 

prophylactic viral vaccines, primarily because of theoretical concerns that 

components within tumorigenic cells could induce tumors in vaccine recipients. 

These concerns include the potential presence of exogenous agents, such as 

oncogenic viruses, and the potential risk from endogenous materials, such as 

endogenous viruses or oncogenic nucleic acids. In addition, intact human cells 

derived from human tumors have been shown to form tumors in allogeneic 

humans.

The goal in tumorigenicity testing is to determine whether your cell substrate is 

capable of forming tumors after inoculation into animals. The TPD50 (tumorproducing dose in 50% of animals) and capacity to form metastases are 

characteristic properties of a cell line, and these characteristics might be used to 

further define the tumorigenic phenotype of a cell line. Considerations associated 

with tumorigenicity testing include: 1) choice of appropriate animal models; 2) 

definition of a positive result; 3) determination of the appropriate duration of 

testing; 4) determination of appropriate numbers of cells to be tested; and 5) 

selection of appropriate controls. 

You should use an animal model that is known to be susceptible to tumor formation 

by tumorigenic cells. Because immunocompromised adult and newborn rodents 

are relatively sensitive for revealing a tumorigenic phenotype, you should consider 

these animal models. Thus, the most commonly used animals for tumorigenicity 

testing are nude (nu/nu) mice because they are T-cell deficient. Newborn nude 

mice appear to be more susceptible to tumor formation than adult nude mice (Ref. 

22), suggesting that newborn nude mice might be the best choice to use when 

identification of a weakly tumorigenic phenotype is important. You might choose 

to use another animal model if it has been shown to have comparable sensitivity to 

the nude mouse model. 

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Regardless of the test system you use, the animals should be observed and palpated 

at regular and frequent intervals for the formation of nodules at the site of injection. 

Any nodules formed should be measured in two dimensions. We consider 

progressive tumor formation at the site of injection a positive result. Some cell 

types might cause tumors at distant sites as well (Ref. 22), and these should be 

reported. Animals with nodules that begin to regress during the period of 

observation should not be sacrificed, as nodules that spontaneously regress do not 

represent progressively growing tumors and are not indicative of a tumorigenic 

phenotype. You may euthanize any animals with tumors before the end of the 

study if the tumor interferes with the comfort of the animal. 

You should perform a necropsy on each animal when it dies, is euthanized, or at the 

end of the observation period. The necropsy should include examination for gross 

evidence of tumor formation at the site of inoculation and in other organs such as 

lymph nodes, lungs, brain, spleen, kidneys, and liver. All tumor-like lesions, 

detectable regional lymph nodes, the site of inoculation, and the lungs are to be 

examined histologically. In some cases, it might be necessary for you to use 

molecular or immunological methods to distinguish cell-substrate-related from 

spontaneous tumors. 

The inoculum per animal should consist of 107 reference cells or test cells 

suspended in 0.2 mL (0.1 mL for newborn animals) volume of serum-free medium 

administered by the subcutaneous route. You should inoculate at least ten animals, 

each with 107 test cells that are at or beyond the end-of-production passage level 

and at least ten with your positive control tumor cells. At least 9 out of 10 animals 

injected with positive control cells (e.g., HeLa cells or other cells with comparable 

tumorigenicity) should show progressively growing tumors in order for your test to 

be valid. 

Selection of the appropriate duration of testing requires that you balance the 

increased sensitivity that might be obtained using a longer test, against the 

likelihood of false-positive results due to spontaneous tumor formation. Weakly 

tumorigenic cells might require between 4 and 7 months to form tumors in nude 

mice. Thus, extended observation periods might be necessary in some cases. 

CBER can provide you with further information on tumorigenicity testing and the 

appropriate observation periods. 

2. Testing for Oncogenicity 

Oncogenicity is defined as the process by which agents immortalize cells and 

endow them with the capacity to form tumors (see Section VI. Glossary). It is 

important to ensure that a cell substrate does not contain potentially oncogenic 

components that could contaminate your product. If your vaccine is 

manufactured in a cell substrate that was derived from a tumor, or that has 

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developed a tumorigenic phenotype through an unknown mechanism, it might 

carry a higher theoretical risk of containing oncogenic substances. 

If the presence of an oncogenic virus is suspected because of the cell phenotype or 

the origin of your cell substrate, it might be appropriate for you to perform 

oncogenicity testing in animals using lysates of the cell substrate. For cell 

substrates with a tumorigenic phenotype, it might be appropriate for you to 

perform oncogenicity testing in animals using DNA from the cell substrate, in 

order to provide assurance that residual DNA is non-oncogenic. Oncogenicity 

testing might also be appropriate for products with high quantities of residual 

cellular DNA. Existing assays for the presence of oncogenic agents in certain cell 

substrates might not be adequate to provide sufficient assurance of the safety of 

your vaccine for clinical use. You should consult with CBER if you have any 

questions on the necessity of these tests and the methods to be followed. 

3. Identity Testing of Cell Substrates 

Identity testing is required by the general requirements for laboratory controls (21 

CFR 211.160(b)) and the general standards for biological products (21 CFR 

610.18(b)). Identity tests assure that a licensed viral vaccine is as stated on the 

manufacturer’s label. Identity tests also provide extra assurance in facilities or 

laboratories that are propagating multiple cell lines or viruses. It is important that 

identity tests be performed on cells used and generated in manufacturing. Some of 

the tests you may use for identity testing are described in the paragraphs that 

follow. 

Biochemical tests such as PAGE analysis is one method to determine the species 

from which the cell substrate was derived. Comparison of the migration patterns 

of the cellular enzymes malate dehydrogenase, glucose-6-phosphate 

dehydrogenase, nucleoside phosphorylase and lactate dehydrogenase is another 

method (Ref. 23). 

Cytogenetic analysis can be used to determine the karyotype of the cell substrate as 

a means of identifying the species, gender, ploidy, and, grossly, genetic stability. 

You should examine 100-200 metaphase cells. However, it might be appropriate 

for you to examine more, depending on the heterogeneity of the karyotypes of your 

product. 

DNA fingerprinting is the most commonly used test of genetic identity and has 

been used to document cell origin and to determine whether cell contamination has 

occurred (Ref. 24). Specific methods for this purpose can be found in Stacey, et al. 

(Ref. 25). 

Tests such as Southern blot hybridization, PCR, growth under selective conditions, 

expression of a reporter gene or other gene of interest, etc., may be applied to 

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distinguish an engineered cell line both from the parent cell line and from other cell 

lines. Some examples can be found in Stacey, et al., (Ref. 26). You may also use 

other tests that provide information relevant to identity of cells, such as HLA 

typing. 

4. Testing for Genetic Stability 

You should establish processes and controls that ensure manufacturing consistency. 

This concept extends to the genetic stability of your cell substrate from the 

establishment of the master cell bank through and perhaps beyond the end of 

production. For an engineered cell line, the inserted gene of interest should remain 

intact and at the same copy number, and be expressed at equivalent levels 

throughout production. Also, a diploid cell strain should remain diploid 

throughout. If such characteristics are not stable, then you might be requested to 

show that the instability does not adversely impact manufacturing or product 

consistency. You may obtain further guidance on this topic from the ICH Q2B 

document (Ref. 7). For methods to assess a cell substrate’s genetic stability, you 

may find guidance in the ICH Q5B and Q5D documents (Refs. 27 and 3, 

respectively). 

C. OTHER TESTS 

1. Testing for the Presence of Residual Cells 

You should test your final vaccine product for the presence of residual cells. 

Processes, such as filtration, should be implemented and validated to ensure that 

intact cells are not present in the final product. Validation that residual cell 

removal processes are robust is important for immortalized cells. Determining the 

extent to which intact cells are cleared by these processes is an important part of 

this validation. 

2. Testing for Residual Cellular DNA 

Residual DNA might be a risk to your final product because of oncogenic and/or 

infectivity potential. There are several potential mechanisms by which residual 

DNA could be oncogenic, including the integration and expression of encoded 

oncogenes or insertional mutagenesis following DNA integration. Residual DNA 

also might be capable of transmitting viral infections if retroviral proviruses, 

integrated copies of DNA viruses, or extrachromosomal genomes are present. 

The risks of oncogenicity and infectivity of your cell-substrate DNA can be 

lessened by decreasing its biological activity. This can be accomplished by 

decreasing the amount of residual DNA and reducing the size of the DNA (e.g., 

by DNAse treatment or other methods) to below the size of a functional gene. 

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Chemical inactivation can decrease both the size and biological activity of DNA. 

If DNA removal, digestion, or inactivation is undertaken, you should validate 

your methods. 

You should measure the amount and size distribution of residual DNA in your 

final product. For widely used human diploid cell strains, such as MRC-5 and 

WI-38 cells, measurement of residual DNA might be unnecessary because we do 

not consider residual DNA from these human diploid cells to be a safety issue. 

We might require limitation of the amount of residual DNA, depending on the 

potential risks associated with that DNA, for human diploid or primary cell types 

for which there is less experience. You should limit residual DNA for continuous 

non-tumorigenic cells, such as low-passage Vero cells, to less than 10 ng/dose for 

parenteral inoculation as recommended by WHO (Ref. 28). If you are using cells 

with tumorigenic phenotypes or other characteristics that give rise to special 

concerns, more stringent limitation of residual DNA quantities might be needed to 

assure product safety. 

3. General Safety Test (GST) 

The requirements of a GST are described in 21 CFR 610.11. For vaccines, an 

exemption to the GST may be requested, as specified in 21 CFR 610.11(g)(2). 

V. CONCLUSIONS 

This guidance document provides to you, general advice on the selection, characterization, 

and qualification of cell substrates and viral seeds used to manufacture these products. We 

recognize that this is a complicated and challenging area. We welcome the opportunity to 

discuss cell substrate and adventitious agent issues with you, as early as feasible in your 

development of viral vaccines. 

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VI. GLOSSARY 

Because different guidance documents might use the same or similar terms differently, the 

following glossary is provided to define the terminology used in this document. 

1. ADVENTITIOUS AGENT: A microorganism (including bacteria, fungi, 

mycoplasma/spiroplasma, mycobacteria, rickettsia, viruses, protozoa, parasites, TSE 

agent) that is inadvertently introduced into the production of a biological product. 

2. ANCILLARY PRODUCT: Products that are used in the manufacture or production of a 

biological product that may or may not end up as part of the final product. Examples 

include: insulin, transferrin, growth factors, interferon, interleukins, other proteins, drugs 

or chemicals like dimethyl sulfoxide. 

3. ANEUPLOID: Having an atypical number of chromosomes which is not an integer 

multiple of the haploid number. 

4. CELL BANK: Vials of cells of uniform composition (although not necessarily clonal) 

derived from a single tissue or cell, aliquoted into appropriate storage containers, and 

stored under appropriate conditions, such as the vapor phase of liquid nitrogen. 

5. CELL LINE (CL): Cells that have been propagated in culture since establishment of a 

primary culture and survival through crisis and senescence. Such surviving cells are 

immortal and will not senesce. Diploid cell strains have been established from primary 

cultures and expanded into cell banks but have not past through crisis and are not 

immortal. [The ATCC uses the abbreviation CCL to signify their Certified Cell Lines.] 

6. CHARACTERIZATION: Determination of the properties of a cell substrate or cell bank. 

7. CONTROL CELLS: Cells that are split off from the production culture and maintained 

in parallel under the same conditions and using the same reagents (e.g., culture medium) 

in order to perform quality control tests on cells that have not been exposed to the vaccine 

virus (which may interfere with some tests). 

8. DIPLOID: Having the expected number of chromosomes for a species, (i.e., two of each 

autosomal chromosome and two sex chromosomes). 

9. ENDOGENOUS VIRUS: A virus whose genome is present in an integrated form in a 

cell substrate by heredity. Endogenous viral sequences may or may not encode for an 

intact or infectious virus. 

10. END-OF-PRODUCTION CELLS (EOPC): Cells harvested at the end of a production 

run or cells cultured from the MCB or WCB to a passage level or population doubling 

level comparable to or beyond the highest level reached in production. 

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11. END-OF-PRODUCTION PASSAGE LEVEL: The maximal passage level achieved 

during manufacturing at final vaccine harvest. Cells may be evaluated at this level or 

beyond. 

12. FINAL BULK: The stage of vaccine production directly prior to filling of individual 

vials. 

13. FREE OF and FREEDOM FROM: For a substance to be considered free of a 

contaminant, an assay must demonstrate that a defined quantity of the substance to be 

negative for that contaminant to a defined level of sensitivity. The level of assay 

sensitivity is defined by the choice of assay and can be determined experimentally using 

standardized reagents. Alternatively, a validated process that is known to remove a 

contaminant to a defined level may be used to demonstrate freedom from that 

contaminant. 

14. HARVEST: At the end of vaccine virus propagation in cell culture, material is collected 

from which vaccine will be prepared. This material may be the culture supernatant, the 

cells themselves (often in disrupted form), or some combination thereof. 

15. IMMORTALIZATION: The process by which cells with finite lifespan (e.g., primary 

cells, diploid cell strains) are converted to those with infinite lifespan. 

16. LATENT: A virus that is present in a cell, without evidence of active replication, but with 

the potential to reactivate, is considered to be microbiologically latent. 

17. MANUFACTURER'S WORKING CELL BANK (MWCB) OR WORKING CELL 

BANK (WCB): A cell bank derived by propagation of cells from MCB under defined 

conditions and used to initiate production cell cultures on a lot-by-lot basis. 

18. MASTER CELL BANK (MCB): A bank of a cell substrate from which all subsequent 

cell banks used for vaccine production will be derived. The MCB represents a 

characterized collection of cells derived from a single tissue or cell. 

19. MASTER VIRUS SEED (MVS): A viral seed of a selected vaccine virus from which all 

future vaccine production will be derived, either directly, or via Working Virus Seeds. 

20. ONCOGENICITY: The property of certain biological agents (e.g., viruses) or materials 

(e.g., nucleic acids) that are capable of immortalizing cells and endowing them with the 

capacity to form tumors. Oncogenicity is distinct from tumorigenicity (See 

Tumorigenicity). 

21. PARENTAL VIRUS: Virus that has been manipulated in some manner to generate a 

viral seed with characteristics needed for production. 

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22. WHO Technical Report Series 878, 1998 

23. PARENT CELL BANK: A few vials consisting of cells from which the Master Cell 

Bank was derived. Parental Cells may be manipulated to derive a cell substrate with 

desired characteristics. 

24. PASSAGE LEVEL: The number of times, since establishment from a primary cell 

culture, a culture has been split or re-seeded. 

25. POPULATION DOUBLING LEVEL: The number of times, since establishment from a 

primary cell culture, a culture has doubled in number of cells. 

26. PRIMARY CELLS: Cells placed into culture immediately after an embryo, tissue, or 

organ is removed from an animal or human and homogenized, minced, or otherwise 

separated into a suspension of cells. Primary cells may be maintained in medium, but are 

not passaged (split). 

27. PURITY: Relative freedom from extraneous matter in the finished product, whether or 

not harmful to the recipient or deleterious to the product. (21 CFR 600.3(r)) 

28. QUALIFICATION: Determination of the suitability of a cell substrate for manufacturing 

based on its characterization. 

29. TUMORIGENIC: A cell type is tumorigenic if it forms tumors when inoculated into 

animals (generally a syngeneic, an immunosuppressed allogeneic, or an 

immunosuppressed xenogeneic host). These tumors may be at the injection site or a 

different site and may also metastasize to other sites. 

30. NON-TUMORIGENIC: A cell type is non-tumorigenic if it is shown not to form tumors 

in appropriate animal models. 

31. TUMORIGENICITY: Tumorigenicity is the process by which immortalized cells form 

tumors when inoculated into animals (see Tumorigenic). Tumorigenicity is distinct from 

Oncogenicity (See Oncogenicity). 

32. TUMORIGENICITY TESTING: An assay/test that determines whether or not 

immortalized cells are tumorigenic when injected into animals. 

33. VALIDATION: Validation defines the performance characteristics of an analytical 

procedure, based on the demonstration that the procedure is suitable for its intended 

purpose or use. Validation of a process is the determination of what characteristics the 

process is capable of performing and the demonstration that the process uniformly 

performs to defined characteristics. Validation is generally performed in accordance with 

the relevant ICH guidelines. 

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34. VIRAL CLEARANCE: The combination of the physical removal of viral particles and 

the reduction of viral infectivity through inactivation. 

35. VIRUS PRE-SEED: A few vials consisting of vaccine virus from which the MVS was 

derived. 

36. VIRUS SEED or VIRAL SEED: A live viral preparation of uniform composition 

(although not necessarily clonal) derived from a single culture process, aliquoted into 

appropriate storage containers, and stored under appropriate conditions. 

37. WORKING CELL BANK: See “Manufacturer’s Working Cell Bank (MWCB)”. 

38. WORKING VIRUS SEED (WVS): A viral seed derived by propagation of virus from 

the MVS under defined conditions and used to initiate production cell cultures lot-by-lot. 

 

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VII. REFERENCE LIST 

1. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, July 

12, 1993. 

2. The International Conference on Harmonisation (ICH) Guidance, Q5A: Viral Safety 

Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal 

Origin, (63 FR 51074; September 24, 1998). 

3. ICH Guidance, Q5D: Derivation and Characterisation of Cell Substrates Used for 

Production of Biotechnological/Biological Products, (63 FR 50244; September 21, 1998). 

4. Goffe, A.P., Hale J, and Gardner, P.S.: Poliomyelitis Vaccine, Letters to Editor. Lancet 

1961; 1:612. 

5. Garnick R.L., Raw Materials as a Source of Contamination in Large-Scale Cell Culture, 

Dev Biol Stand. 1998; 93:21-29. 

6. ICH Guideline, Q2A: Text on Validation of Analytical Procedures, (60 FR 11260, March 

1, 1995). 

7. ICH Guideline, Q2B: Validation of Analytical Procedures: Methodology, (62 FR 27463, 

May 19, 1997. 

8. U.S. Pharmacopoeia (USP) Chapter 1225, Reference 25, pp. 2256-59. 

9. Rezapkin G.V., et al., Microevolution of Sabin 1 strain in vitro and genetic stability of oral 

poliovirus vaccine, Virology. 1994 202(1): 370-378. 

10. Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues 

Concerning the Use of Xenotransplantation Products in Humans, April 2003. (see also § 

610.18(c)(1)(iii)). 

11. European Pharmacopoeia, 4th Edition, Section 2.6.6. 

12. Transmissible Spongiform Encephalopathies Advisory Committee, October 25, 2001. 

13. U.S. Pharmacopoeia, Chapter 61, Reference 25. 

14. European Pharmacopoeia, 4th Edition, Test for Extraneous Agents Using Chicks, Section 

2.6.6. 

15. Khan, A.S., Muller, J., and Sears, J.F. 2001. Virus Research 79:39-45. 

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16. Bierley, et al., Dev. Biol. Stand., 1996. 

17. Arnold et al., One-step fluorescent probe product-enhanced reverse transcriptase assay, 

Biotechniques 1998, 25: 98-106. 

18. Maudru and Peden, Adaptation of the fluorogenic 5'-nuclease chemistry to a PCR-based 

reverse transcriptase assay. Biotechniques 1998, 25: 972-975. 

19. Lovatt, et al., High throughput detection of retrovirus-associated reverse transcriptase 

using an improved fluorescent product enhanced reverse transcriptase assay and its 

comparison to conventional detection methods. J Virol Methods 1999, 82: 185-200. 

20. Sears, J.F. and Khan, A.S., Single-tube fluorescent product-enhanced reverse transcriptase 

assay with Ampliwax (STF-PERT) for retrovirus quantitation. J Virol Methods 2003; 

108(1):139-42. 

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

Human Use, February 28, 1997. 

22. European Pharmacopoeia, 4th Edition, Section 2.6.4. 

23. Leman, A.D., et al., “Diseases of the Swine”, 1992, 7th Edition. 

24. Barile, M.F. and Rottem, S., Mycoplasmas in Cell Culture, Rapid Diagnosis of 

Mycoplasmas, I. Kahane and A. Adoni (ed.) Plenum Press, New York, p.155-193, 1993. 

25. Sordat, B., et al., Invasive Growth and Dissemination of Human Solid Tumors and 

Malignant Cell Lines Grafted Subcutaneously to Newborn Nude Mice, Proceedings of the 

Second International Workshop on Nude Mice p. 313-326, 1977. 

26. Stacey, G.N., et al., Authentication of Animal Cell Cultures by Direct Visualization of 

Repetitive DNA, Aldolase Gene PCR and Isoenzyme Analysis, Biologicals 1997; 25:75-

85. 

27. Stacey, G.N., et al, DNA Fingerprinting Transforms the Art of Cell Authentication, Nature, 

1992; 357:261-262. 

28. Stacey, G.N., et al., The Quality Control of Cell Banks Using DNA Fingerprinting, EXS. 

1991; 58:361-70. 

29. Stacey G.N., Phillips, P., Quality Assurance for Cell Substrates, Dev Biol Stand. 1999; 

98:141-51. 

30. ICH Guidelines, Q5B: Quality of Biotechnological Products: Analysis of the Expression 

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Construct in Cells Used for Production of r-DNA Derived Protein Products, (61 FR 7006, 

February 23, 1996). 

31. World Health Organization (WHO) Technical Report. Series 878, 1998 

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Appendix 1 

TABLE 1. Example of a Testing Scheme for Manufacture of a Viral Vaccine. 

Multi-stage testing with a variety of different assays is used to assure product safety and ensure 

product quality. This includes testing all the biological materials in vaccine manufacture, 

especially the initial components, which are the virus seed and the cell substrate. Different stages 

for testing are selected based on the maximum likelihood of adventitious agent detection and 

effect on product quality. This includes the Harvest and Final Virus Seed; the Master Cell Bank 

and Control Cell Culture (in certain cases); and the Vaccine Bulk and Final product. Additional 

tests may be included on a case-by-case basis depending upon the cell substrate and other 

biological materials as wells as production conditions for vaccine manufacture. 

Table 1: 

VIRUS SEED: 

 Harvest Sterility 

Mycoplasma/Spiroplasma 

Mycobacteria 

Appearance 

Identity 

Potency/Activity 

Infectious titer 

 In vitro adventitious agent testing 

In vivo adventitious agent testing 

Bovine viruses (if applicable) 

Porcine viruses (if applicable) 

RT assay 

Specific agent testing (if applicable) 

 Final Sterility 

Identity 

Potency/Activity 

Infectious titer 

Attenuation (if applicable) 

Stability of phenotype/genotype (if 

applicable) 

CONTROL CELL CULTURES: Observation 

Hemadsorption 

 Sterility 

Mycoplasma/Spiroplasma 

RT assay 

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In vitro adventitious agents 

In vivo adventitious agents 

Bovine viruses (if applicable) 

Porcine viruses (if applicable) 

Tests for specific agents (as applicable) 

ALV (if applicable) 

Mycobacteria 

CELL BANKS (Diploid Cell Strains or 

Continuous Cell Lines): 

 Master Cell Bank Identity 

Sterility 

Mycoplasma/Spiroplasma 

In vivo adventitious viruses 

In vitro adventitious viruses 

TEM 

Bovine viruses (if applicable) 

Porcine viruses (if applicable) 

Tests for specific agents (as applicable) 

Tumorigenicity (except rodent cells) 

Oncogenicity (if applicable) 

RT assay 

Mycobacterium (if applicable) 

Antibody testing (MAP, HAP, RAP, if 

applicable) 

S+L- assay for retrovirus (if applicable) 

VACCINE BULK: Sterility 

Mycoplasma/Spiroplasma 

Mycobacteria 

Appearance 

Identity 

Potency/Activity 

Infectious titer 

In vitro adventitious agent testing (or on 

control cells) 

In vivo adventitious agent testing (or on 

controls cells) 

Bovine viruses (or on control cells) (if 

applicable) 

Porcine viruses (or on control cells) (if 

applicable) 

RT assay 

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Specific agent testing (if applicable) 

FINAL FILLED PRODUCT: Sterility 

Mycoplasma/Spiroplasma 

Endotoxin 

General Safety Test (unless you receive an 

exemption under 21 CFR 610.11(g)) 

Identity 

Appearance 

Residual cellular protein concentration 

Residual cellular DNA concentration 

Potency/Activity 

Stability 

Residual serum, antibiotics, benzonase, 

ancillary products (as applicable) 

pH 

Osmolality 

 49