Guidance for Industry 
 
 
 
Characterization and Qualification of 

Cell Substrates and Other Biological 

Materials Used in the Production of Viral 

Vaccines for Infectious Disease 

Indications 

 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD) (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852
1448, or by calling 1-800-835-4709 or 301-827-1800, or email ocod@fda.hhs.gov, or from the 
Internet at  
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida 
nces/default.htm. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers listed above. 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research  
[February 2010]  Contains Nonbinding Recommendations  
 
 
TABLE OF CONTENTS  
 
I. 		 INTRODUCTION.........................................................................................................................................1
	 
II. 		 DEFINITION.................................................................................................................................................2
	 
III. 		 CHARACTERIZATION AND QUALIFICATION OF CELL SUBSTRATES, VIRAL SEEDS, 

BIOLOGICAL RAW MATERIALS AND VACCINE INTERMEDIATES ...........................................2
	 
A. 		 PRODUCT-SPECIFIC  PARAMETERS  INFLUENCING  CHARACTERIZATION  AND	
 
QUALIFICATION  OF  CELL  SUBSTRATES ...........................................................................................3
  
1. 	  Vaccine Purity ............................................................................................................................................3
  
2. 	  Potential Sources of Contamination...........................................................................................................4
  
3. 	  Current Good Manufacturing Practices (cGMP) and Cell Substrate Development ..................................4
  
4. 	  Use of Control-Cell Cultures......................................................................................................................4
  
5. 	  Assay Validation.........................................................................................................................................5
  
B.   PROPERTIES  OF  THE  CELL  SUBSTRATE.................................................................................................5
  
1. 	  Properties Relevant to Cell Substrate Selection .........................................................................................6
  
2. 	  Source.........................................................................................................................................................6
  
3. 	  History (including identifying characteristics) and other important characteristics .................................7
  
4. 	  Growth Characteristics ..............................................................................................................................8
  
5. 	  Expression Characteristics.........................................................................................................................8
  
6. 	  Susceptibility to Adventitious Agents..........................................................................................................8
  
7. 	  Generation of Cell Substrate ......................................................................................................................8
  
C.   CELL  BANKING............................................................................................................................................9
  
1.   Cell Banking Strategies and Methods.........................................................................................................9
  
2.   Qualification of Cell Banks  and Primary Cell Cultures...........................................................................10
  
3.   Special Considerations for Primary Cell Cultures...................................................................................11
  
4.   Special Considerations for Diploid Cell Strains ......................................................................................12
  
5.   Special Considerations for Continuous Cell Lines...................................................................................13
  
6.   Additional Considerations for Cell Lines that are Tumorigenic or Tumor-derived .................................14
  
D.   VIRAL  SEEDS..............................................................................................................................................14
  
1.	   Master Viral Seed.....................................................................................................................................15
  
2.	   Working Viral Seed...................................................................................................................................16
  
E.   BIOLOGICAL  RAW  MATERIALS  AND  ANCILLARY  REAGENTS ......................................................16
  
1.	   Serum........................................................................................................................................................16
  
2.	   Trypsin......................................................................................................................................................17
  
3.	   Amino Acids..............................................................................................................................................17
  
4.	   Other Biological Reagents........................................................................................................................17
  
F.   TESTING AT DIFFERENT STAGES OF PRODUCTION..........................................................................17
  
1. 	  Cell Banks.................................................................................................................................................17
  
2. 	  Pre-Production Cells................................................................................................................................17
  
3. 	  Pre-Filtered Harvest or Post-Production Cells........................................................................................18
  
4. 	  Control Cells ............................................................................................................................................18
  
5. 	  Post-Filtered Harvest (Final Bulk)...........................................................................................................18
  
6. 	  Final Filled Product.................................................................................................................................19
  
IV. 		 DESCRIPTION OF TEST METHODS ....................................................................................................19
	 
A.   TESTING  FOR  ADVENTITIOUS  AGENTS ...............................................................................................19
  
1. 	  In Vivo Tests .............................................................................................................................................20
  
a. 	  Adult Mice............................................................................................................................................................ 20
  
b. 	  Suckling Mice ...................................................................................................................................................... 21
  
c. 	  Guinea Pigs .......................................................................................................................................................... 21
  
d. 	  Rabbits.................................................................................................................................................................. 22
  
e. 	  Embryonated Chicken Eggs ................................................................................................................................. 22 
 
f. 	  Antibody Production Tests ................................................................................................................................... 23 
 
2. 	  In Vitro Tests for Viruses..........................................................................................................................24
  
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a.   Cell Culture Safety Tests...................................................................................................................................... 24
  
   Human Diploid Cells....................................................................................................................................... 24
  
   Monkey Kidney Cell Line (usually Vero cells) ................................................................................................ 24
  
b.   Transmission Electron  Microscopy (TEM) .......................................................................................................... 26
  
c.   Biochemical Tests  for Retroviruses...................................................................................................................... 26 
 
d.   Infectivity Tests  for Retroviruses ......................................................................................................................... 27
  
e.   PCR or Other Specific In Vitro Tests ................................................................................................................... 27
  
f.   Sourcing and Testing of Bovine-Derived Materials ............................................................................................. 28
  
g.   Testing of Porcine-Derived Reagents...................................................................................................................29
  
3.   In Vitro Tests for Non-Viral Agents..........................................................................................................29
  
a.   Mycoplasma/Spiroplasma .................................................................................................................................... 29 
 
b.   Bacterial and Fungal Sterility ............................................................................................................................... 32
  
c.   Mycobacteria Testing ........................................................................................................................................... 32
  
B.   TESTING  OF  CELL  PROPERTIES .............................................................................................................33 
 
1.   Tests for Tumorigenicity...........................................................................................................................33
  
2.   Testing for Oncogenicity ..........................................................................................................................35
  
3.   Identity Testing of Cell Substrates............................................................................................................35
  
4.   Testing for Genetic Stability.....................................................................................................................36
  
C.   OTHER  TESTS.............................................................................................................................................36
  
1.   Testing for the Presence of Residual Cells ...............................................................................................36
  
2.   Testing for Residual Cellular  DNA...........................................................................................................37
  
3.   General Safety Test (GST)........................................................................................................................37
  
V.   GLOSSARY.................................................................................................................................................38
	 
VI.   REFERENCE LIST ....................................................................................................................................42
	 
 
ii  Contains Nonbinding Recommendations 
 
 
Guidance for Industry 

 
Characterization and Qualification of Cell Substrates and Other 

Biological Materials Used in the Production of Viral Vaccines for 

Infectious Disease Indications 

 
 
This guidance represents the Food and Drug Administrations (FDAs) 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, FDA, are providing you, manufacturers of viral vaccines, guidance for the characterization 
and qualification of cell substrates, viral seeds, and other biological materials used for the 
production of viral vaccines for human use. 
 
This guidance applies  to the development of viral vaccines for the prevention and treatment of 
infectious diseases that are regulated by the Office of Vaccines Research and Review (OVRR) of 
the Center for Biologics Evaluation and Research (CBER) under section 351 of the Public Health 
Service (PHS) Act (42 U.S.C. 262). 
 
Cell substrates are cells used to produce vaccines.  The scope of this guidance document is 
limited to cell substrates of human or animal (including insect) origin and does not cover 
characterization of unicellular organisms, such as bacteria or yeast.  In this document, cell 
substrates are categorized as primary (cells, including eggs, derived directly from an animal 
source and that are not stored as cell banks), diploid (cells with a normal or near-normal 
karyotype and that are stored as cell banks prior to use in vaccine manufacture), or continuous 
(cells that are immortal and do not undergo senescence).  This guidance also applies to the 
characterization and qualification of viral seeds and other biological materials (including vaccine 
intermediates) used in vaccine manufacture.   
 
This guidance finalizes the draft guidance entitled 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, dated September 
2006 (71 FR 57547). In addition, this document replaces the information pertaining to viral 
vaccines 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 
1   
 Contains Nonbinding Recommendations 

 
 
Biologicals, dated 1993 (Ref. 1). This document also supplements the 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). 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe FDAs 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.	 	 DEFINITION 
 
For the purpose of this document, viral vaccines are a heterogeneous class of preventive, and in 
some cases, therapeutic 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.  These 
products include live attenuated preparations of viruses, inactivated (killed) whole or subunit 
virions, purified recombinant proteins, synthetic antigens, or live viral vectors expressing 
specific heterologous vaccine antigens. 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.  A glossary defining scientific terms in this guidance is 
located in Section V below. 
 
III. 		 CHARACTERIZATION AND QUALIFICATION OF CELL SUBSTRATES, 
VIRAL SEEDS, BIOLOGICAL RAW MATERIALS AND VACCINE 
INTERMEDIATES 
 
Selection of a cell substrate influences the safety and purity of the biological product 
manufactured in it.  The evaluation (e.g., the particular tests and methodologies chosen) should be 
specifically tailored to the cell substrate and vaccine product to be developed to assure vaccine 
safety and purity. FDA recognizes that technology in this area is evolving.  This section describes 
general parameters to be considered in the characterization of cell substrates, viral seeds, 
biological starting materials and vaccine intermediates.  To maximize the value of the 
recommendations in this document, it is important that you perform each test at the stage of 
production at which contamination is most likely to be detected. 
 
Tests to characterize and qualify cell substrates, biological materials, vaccine seeds, and vaccine 
intermediates are described in Section IV.   
  
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 Contains Nonbinding Recommendations 

 
 
A.		  PRODUCT-SPECIFIC PARAMETERS INFLUENCING 
CHARACTERIZATION AND QUALIFICATION OF CELL 
SUBSTRATES 
 
1.   Vaccine Purity 
 
The regulations, in 21 CFR 610.13, state in part 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.  
 
Live attenuated viruses, whole inactivated virions, or virus-like particles often 
cannot be purified as rigorously as viral subunit vaccines; as a consequence, the 
potential for contamination is greater than that of subunit vaccines.  Generation of 
live viral vaccines often involves cell disruption, which may add cellular 
components to the vaccine bulk.  In addition, such vaccines often are minimally 
purified and are not subjected to inactivation steps.  Comprehensive testing and 
qualification of the biological starting materials and lot-by-lot testing for 
adventitious agents are particularly important because it is not possible for a 
manufacturer of live viral vaccines to validate the process for clearance of 
adventitious agents. 
 
In contrast, for more highly purified products where viral clearance can be 
demonstrated during downstream processing, you can place more reliance on 
process validation (Ref. 2).  For inactivated vaccines, the concern is that the 
process used to inactivate the vaccine virus may not inactivate all adventitious 
agents potentially present (as occurred with early inactivated poliovirus vaccines 
[Ref. 4]). Therefore, you should validate your process for inactivation of 
adventitious agents using different model viruses (Ref. 2).  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 may influence the sensitivity of the 
testing you should perform to demonstrate the absence of contaminants in your 
product. 
 
If a viral vaccine seed was exposed to a known adventitious agent or if the 
passage history of a virus seed is unclear, you should purify the viral seed (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 planning to perform seed purification, 
you should discuss your proposed methodology and its validation with us as early 
as is feasible. 
 
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2. 	  Potential Sources of Contamination 
 
It is important that you identify and examine all potential sources of 
contamination of your product.  For example, a viral seed could be 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 should also consider the species of origin of your cell substrates, viral 
seeds, and other biological starting materials in selecting your tests to ensure the 
absence of contaminants.  Furthermore, 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 or cell substrate at any time 
during development or production.  Retroviruses may be either endogeneous (i.e., 
encoded by the cell substrate genome)  or exogenously acquired.  Retrovirus 
testing should address the possibility that either type of retrovirus could 
contaminate a product.  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).  The 
susceptibility of the cell substrate to infection by agents of potential concern can 
influence the tests needed to assure absence of contamination.   
 
Use of qualified raw materials can reduce the risk of introducing adventitious 
agents. For example, inactivation of viruses by irradiation of serum could provide 
additional assurance regarding the purity of the final product.   
 
3.	   Current Good Manufacturing Practices (cGMP) and Cell Substrate 
Development 
 
Concepts of quality design consistent with cGMP (see 21 CFR Part 211) are 
relevant to the selection of suitable cell substrates and viral seeds.  If your cell 
substrate or viral seed was not originally derived under concepts consistent with 
cGMP, 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 
 
We recommend the use of control cells when it is not feasible to directly test cells 
or product at various stages of manufacture.  For example, 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, when possible, 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. 
4   Contains Nonbinding Recommendations 

 
 
 
Control-cell cultures are also useful for testing in situations when your vaccine 
virus can interfere with the results of testing of the virus seed or vaccine product 
(e.g., when the virus cannot be adequately neutralized to permit testing for 
adventitious agents). 
 
Control cell cultures should be propagated under conditions similar to conditions 
of manufacture for a time appropriate to the reason for performing the cultures.  
This time period may be 14 days or more, when needed to allow for detection of 
potential adventitious agents that may be latent, endogenous, or poorly 
replicating. Control-cell cultures should be processed simultaneously with your 
production culture, but left uninfected. Control cells should be evaluated for the 
presence of adventitious agents using the same tests that would have been 
performed on the production cultures, if it were feasible.  Tests for adventitious 
agents should be performed on control cells at times at which you would perform  
similar tests on your manufactured product.   
 
Testing of control cells does not always eliminate the need for testing final 
product (see Section III.F.6). 
 
5.   Assay Validation 
 
You should demonstrate the reliability of assays or tests used to evaluate your cell 
substrate in the context of intended use. Assays related to assurance of safety 
should be scientifically valid (for example, by formal validation and/or inclusion 
of appropriate controls or standards) prior to initiation of clinical trials.  Guidance 
regarding validation of analytical assays may be obtained from the ICH Q2(R1) 
documents (Ref. 6).  Test methods should be validated according to the principles 
defined in these guidance documents.  Certain compendial tests (for example, 
those promulgated by the USP) might not require full validation, as long as you 
verify their suitability for their intended purpose under your actual conditions of 
use (Ref. 7). 
 
You should use appropriate statistical analyses to support 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 Q2(R1) document (Ref. 6). 
 
B.   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. 
 
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 Contains Nonbinding Recommendations 
 
 
1.   Properties Relevant to Cell Substrate Selection 
 
Assuring that the vaccine virus strain remains stable during passage in cell culture 
is an important component of cell substrate selection.  Different cell lines 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. 8).  If attenuating mutations or other genetic markers (including 
expression of antigens relevant to immune response) of the vaccine virus are 
known, data regarding 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 genetically engineered cells), any available 
information about those starting materials and their characterization (e.g., 
sequence of the plasmid) 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. 
 
2.   Source 
 
You should document the species of origin and the tissue type of your cell 
substrate. You should also document the medical history of the donor and the 
results of any screening and testing performed on the donor or on samples from  
the donor. When feasible, 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, and 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. 9).  (Issues 
specific to bovine- and porcine-derived materials are discussed in Sections 
IV.A.2.f and IV.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 
be more likely to 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. 
 
6   
 Contains Nonbinding Recommendations 
 
 
3. 	  History (including identifying characteristics) and other important 
characteristics 
 
The history of your vaccine cell substrate must be identified under 21 CFR 
610.18(c)(1)(i) and should include, to the extent available: sources and testing of 
the raw materials to which it was exposed in its passage history; listing of any 
other agents handled or grown in the same biosafety cabinet or incubators around 
the time of cell substrate passage; and the conditions under which it was passaged 
(including adherence to 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 from which it was obtained and its passage (or 
population doubling) level. All available documentation of all raw materials of 
human or animal origin you used for the entire passage history should be retained 
and considered in developing a testing program.  This is particularly important for 
passages using bovine materials acquired during and after 1980, when bovine 
spongiform encephalopathy (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 should provide the following information for any cell substrates 
used for the production of viral vaccines: 
 
 	  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 (including that required 
by 21 CFR 610.18(c)(1)(iv)); 
 	  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. 
 
For situations when the specified information is not available, data derived from  
testing of the cell substrate by other methods may prove supportive and may be 
sufficient. 
 
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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.   
 
A description of the tumorigenic property of cells is required for all diploid and 
non-diploid cells, including continuous cell lines (21 CFR 610.18(c)(1)(ii)).  
Because previous experience has consistently demonstrated the non-tumorigenic 
phenotype of the well-characterized diploid cell strains MRC-5, WI-38, and 
FRhL-2 (if they are not genetically or phenotypically modified), further 
tumorigenicity testing of banks consisting of these cell strains is not considered 
necessary to satisfy this requirement (please also see Section III.C.4).  A 
description of the tumorigenic property is not required for 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)). 
 
If there are specific markers that might be useful in characterizing your cell line 
(such as microscopic appearance, marker chromosomes, transfected genetic 
elements, specific surface markers, etc.), these should be analyzed for their 
stability through 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.  
 
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.  Selection of tests should be based on 
potential sources of contamination (See Section II.A.2).  Your production scheme  
and testing program should be designed to assure the absence of viruses that 
might replicate in your cell substrate. 
 
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.  
8   
 Contains Nonbinding Recommendations 
 
 
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 
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 derivative 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. 
 
C.   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 contaminant.   
 
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), sometimes called a manufacturers 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 MCB, 
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 your cell substrate characterization, including at 
end-of-production level (See Section III.C.2). 
 
When using a two-tiered banking system, we generally recommend that you 
perform an extensive characterization of the MCB.  Because all WCBs would be 
9   Contains Nonbinding Recommendations 

 
 
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 
manufacturing from the WCB.  Alternatively, some manufacturers might choose to 
extensively characterize each WCB in lieu of a one-time thorough characterization 
of the MCB. The testing strategy chosen will depend on your own circumstances, 
but you should characterize at least one cell bank (MCB or WCB) extensively.  
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, with a 
justification for the choice of the passage level tested.  In either case, the derivation 
and cell bank designations should be thoroughly described and explained.   
 
The requirements set out in 21 CFR 610.18 relate to cultures, including cell banks.  
Cell banks should be stored under conditions shown to be suitable for long-term  
stability (e.g., liquid nitrogen, ultra-low temperature freezer) (21 CFR 610.18(a)).  
Storage of cell banks in the vapor phase (as compared to the liquid phase) of liquid 
nitrogen might reduce the potential for cross-contamination.  Cell stability under 
the freezing and storage conditions should be validated using cell recovery or 
viability data.  You should store the MCB and WCB in two or more separate 
locations within the facility or at a distant site in order to avoid loss of the cell 
substrate due to local disaster or equipment malfunction.  Access to these cell 
banks by your staff should be limited and controlled. Under 21 CFR 610.18(d), 
appropriate records must be maintained.  You should maintain a record of the 
location, identity, and inventory of individual ampoules of cells.   
 
2.   Qualification of Cell Banks and Primary Cell Cultures 
 
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. 
 
The same basic principles apply to the qualification of cell banks and to the 
qualification of primary cell cultures.  Characterization of a cell bank is dependent 
on its use, and your particular use should guide your determination of which 
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 
indications. 
 
10   Contains Nonbinding Recommendations 

 
 
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 
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.  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 products produced in avian cells.  If you are using primary monkey kidney 
cell cultures, for example, you should test for species-specific simian agents, such 
as SV40 and herpes B virus, and other simian agents, such as simian 
polyomaviruses and simian cytomegalovirus. 
 
Testing either the MCB or the reagents to which it was exposed may be done to 
provide assurance that these reagents were not a source of adventitious agents, an 
issue of particular concern when reagents are animal-derived.  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 (Section IV.A.2.g) agents.  
Additional considerations are described for serum in Section III.E.1 and for trypsin 
in Section III.E.2. In the case of bovine derived materials, the issue of TSE agents 
should be considered (Ref. 33). 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.  Methods that may be used for identity 
testing of the MCB are discussed in Section IV.B.3.  
 
You should characterize 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, if applicable, growth characteristics, 
tumorigenic phenotype, expression of endogenous viruses, stability of expression 
of the inserted or engineered genes, and genetic stability of the cells (such as 
karyotyping or DNA fingerprinting). 
 
3.   Special Considerations for Primary Cell Cultures 
 
Primary cells are obtained directly from the tissues of healthy animals.  Primary 
cells are more likely to contain adventitious agents than banked, well-characterized 
cells. This concern with primary cells is mitigated by rigorous qualification of  
source animals and primary cell substrates.  Animals from which primary cultures 
11   
 Contains Nonbinding Recommendations 

 
 
are established should be from specific-pathogen-free (SPF) closed 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 
leukosis virus, reticuloendotheliosis virus, and other adventitious agents pathogenic 
for chickens. Appropriate records must be retained for documentation (21 CFR 
610.18(d)). If eggs are procured from non-SPF flocks, assurance that the vaccine is 
free of such agents may be provided by testing or by validating the process for their 
removal.  
 
Control cells are often used with primary cell substrates and are described in more 
detail in Section III.A.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 non-
diploid. 
 
You should determine the karyotype of new diploid cell strains 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.  
We recommend that you monitor the genetic stability of the diploid cell strain 
throughout production. Other tests of identity (see Section IV.B.3) may also be 
appropriate. 
 
Animal tumorigenicity testing is not needed if you are using genetically or 
phenotypically unmodified previously characterized diploid cell strains, such as 
MRC-5, WI-38, and FRhL-2 (see Section III.B.4).  However, tumorigenicity 
testing must be performed 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.  
 
12   
 Contains Nonbinding Recommendations 
 
 
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 cell lines compared with diploid cells, including the potential  
that transformation was caused by an adventitious agent and potential risks from 
residual DNA.       
 
Products prepared in cell lines (including viral vaccines) should be purified to be 
free (see Section V. 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 express endogenous viruses (e.g., retroviruses), tests capable 
of detecting such agents should be completed on cells grown under production 
conditions (See Section IV.A. Testing for Adventitious Agents).  With the exception 
of chicken cells (see Section IV.A.2.c), if specific contaminants have been identified 
as endogenous agents in the MCBs and WCBs you are using, you should 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). 
 
The testing recommended for qualification of cell substrates and cell banks should 
also be applied to rodent cell lines.  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). 
 
Under 21 CFR 610.18(c)(1)(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 
13   
 Contains Nonbinding Recommendations 
 
 
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-of
production level.   
 
6. 	  Additional Considerations for Cell Lines that are Tumorigenic or Tumor-
derived 
 
Use of tumorigenic and tumor-derived cells for vaccine production is associated 
with additional issues. This document does not provide guidance on a pathway for 
licensure of live-attenuated or minimally purified vaccine produced in these cells. 
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 tumorigenicity testing and section IV.B.2 for a 
discussion of oncogenicity testing.) 
 
In cases where you know the transforming event (e.g., 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. 
 
D.   VIRAL SEEDS 
 
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 cold-adaptation, development of 
temperature-sensitivity, or attenuation of virulence, should be well documented.  Any 
genetic manipulations, such as reassortment or recombination, should also be well 
documented, including determining the nucleic acid sequences and sourcing of each 
biological starting material (e.g., plasmids, parental viruses). 
 
Vaccine virus banks are commonly referred to as master viral seed (MVS) and working 
viral seed (WVS). Viral seeds should be stored under conditions that maintain their 
14   
 Contains Nonbinding Recommendations 
 
 
stability (i.e., in liquid nitrogen or ultra-low temperature freezers) and in more than one 
location within a 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 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 it 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 
 
We recommend that you extensively characterize your MVS, as this 
characterization also provides assurance regarding the characteristics of subsequent 
passages, including the WVS. In addition, you should demonstrate the stability of 
the genotype and phenotype following viral passages beyond the level used in your 
production. Genotypic characterization of a viral seed includes its sequence, and 
may include analysis of viral subpopulations and its genetic stability, including 
susceptibility to reversion. Phenotypic characterization of a viral seed may include 
assessment of tissue tropism, attenuation properties, and temperature sensitivity, if  
applicable.  
 
Under 21 CFR Part 610, you must perform tests for safety, purity, and potency of a 
product, as required. This includes tests for identity, bacterial and fungal sterility, 
the presence of mycoplasmas, Mycobacterium tuberculosis (if appropriate), and 
adventitious viruses (in vitro and in vivo tests).  You should also consider specific 
tests for agents that might be present in the seed due to its passage history. 
 
In some cases, it may be necessary to neutralize the vaccine virus in order to 
adequately test for adventitious agents in vitro and in vivo.  Preferably, neutralizing 
antibodies should be monoclonal to reduce the likelihood of also neutralizing 
adventitious viruses that could be present in the seed.  In addition, due to the 
potential for cross-neutralization 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, introduction of additional tests (e.g., polymerase 
chain reaction (PCR), Antibody Production assays (Ref. 10) or testing of control 
cells (Section III.A.4). 
 
Assessment of neurovirulence might be appropriate for MVSs derived from wild 
type viruses known or suspected to possess neurotropic or neurovirulent properties, 
and we recommend that you consult with CBER on appropriate animal models, 
methods, and scoring systems for this assessment before you initiate such studies.  
15   
 Contains Nonbinding Recommendations 

 
 
Animal models other than non-human primates may be acceptable. For live-
attenuated 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 
 
The level of characterization of a WVS should be based on the extent of 
characterization of the MVS from which it was 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, you 
only need to show that your WVS is free of adventitious agents that could have 
been introduced in generating the WVS (e.g., from production cells and raw 
materials used in propagation and processing).  Alternatively, more extensive 
testing could be performed on WVS material if less extensive testing has been 
performed on the MVS in an effort to conserve the supply of MVS. 
  
E.   BIOLOGICAL RAW MATERIALS AND ANCILLARY REAGENTS 
 
You should test your raw materials and ancillary reagents to assure that they are 
appropriate for use in all stages of vaccine production, including the production of your 
cell banks and viral seeds.  Raw materials derived from animals or humans should be 
shown to be free from adventitious agents of the species from which they are derived. 
Appropriate records must be retained for all reagents and biological raw materials used for 
vaccine production (21 CFR 610.18(d)).  
 
If your documentation for the raw materials used in the passage of your cell substrate is 
insufficient to assure that the raw materials did not introduce adventitious agents, you 
should test the cell substrate itself for adventitious agents of the relevant species.  For 
human-derived raw materials and reagents, you should be able to document 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.  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 
steps that you rely upon to remove or inactivate potential infectious contaminants from 
biological raw materials, you should validate those steps.  
 
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 licensed for human use in the U.S. or  derived  from  donors  
16   
 Contains Nonbinding Recommendations 
 
 
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). 
 
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, Sourcing  and  
Testing 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. 
 
4.   Other Biological Reagents 
 
You should assess (including testing, where appropriate) the potential risk for 
introduction of adventitious agents via other biological reagents used during 
manufacturing, including transferrin, insulin, or other growth factors used in 
growth medium, as appropriate for their species of origin.  If derived from humans, 
the ancillary reagent 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 
viral vaccines. Also, other beta-lactam antibiotics should not be present in 
production cell cultures. 
 
F.   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.C. 
 
2.   Pre-Production Cells 
 
Visual inspection or an identity test may be performed on cells directly prior to 
production. 
 
17   Contains Nonbinding Recommendations 
 
 
3.   Pre-Filtered Harvest or Post-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 the viral or vaccine harvest for cultivatable mycoplasmas, as 
required under 21 CFR 610.30, you should also test the viral or vaccine harvest for 
non-cultivatable mycoplasmas (and spiroplasmas, if appropriate), and adventitious 
viruses by in vitro methods.  Adventitious virus tests should also include  in vivo 
methods if the cells and vaccine seeds have not already been tested.  This might 
include a test for hemadsorbing viruses.  If the production process is capable of 
inducing expression of retroviruses, PCR-based reverse transcriptase (RT) testing 
or infectivity studies might also be recommended at this stage.  If appropriate, a test 
for Mycobacterium tuberculosis should be performed.   
 
As discussed for the MVS (Section III.D.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.D.1 might be applicable. 
 
If cells survive the production process (e.g.,  if the vaccine virus does not result in a 
lytic infection), post-production cells may also be tested for adventitious agents.    
If multiple harvests are performed for  a single vaccine lot, testing should be  
performed on each individual harvest (rather than the pooled 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 III.A. 4.    
 
5.   Post-Filtered Harvest (Final Bulk) 
 
The post-filtered harvest (generally referred to as final bulk) should be tested for 
bacterial and fungal sterility. Depending on the product, other testing might be 
appropriate to assure the safety, identity, purity, potency, and quality of the final 
bulk. These may 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 
18   
 Contains Nonbinding Recommendations 
 
 
for removal of residual cells, a test to demonstrate the absence of residual cells is 
recommended at this stage.  
 
6.   Final Filled Product 
 
The regulations for General Biological Products Standards under 21 CFR Part 610 
provide that each lot must be tested for potency, general safety, sterility, purity and 
identity. 
 
Sterility must be demonstrated as required under 21 CFR 610.12.  Products 
excepted from these requirements (e.g., under 21 CFR 610.12(g)(4)(ii)) should still 
be assessed by the microbial limits test for bioburden (Ref. 11).  Under 21 CFR 
610.11a, general safety testing for each lot of inactivated influenza vaccine includes 
a demonstration that it contains less endotoxin than 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. 
 
 
IV.   DESCRIPTION OF TEST METHODS 
 
The tests described in this Section may be used to address the considerations discussed in 
Section III. Advances in science and technology are likely to yield additional information that 
could lead to modification or replacement of some of these tests, and except where prohibited by 
regulation, manufacturers may use alternative test methods, where scientifically justified.  
Manufacturers should consider how they can replace, refine, or reduce their use of in vivo tests. 
 
A.   TESTING FOR ADVENTITIOUS AGENTS  
 
Assurance that products are free of adventitious agents is a critical component of meeting 
the 21 CFR 610.13 requirement for purity.  Cultures must be tested for the presence of 
detectable microbial agents and tests necessary to assure the safety and purity of the 
product may be required (21 CFR 610.18).  Your biological starting materials should be 
characterized sufficiently to ensure that they do not contaminate the final product with 
extraneous infectious organisms, such as bacteria, fungi, cultivatable and non-cultivatable 
mycoplasmas and spiroplasma, mycobacteria, viruses, and the agent(s) responsible for 
transmissible spongiform encephalopathies (TSEs).  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 
19   Contains Nonbinding Recommendations 
 
 
production process is sufficiently robust to eliminate or inactivate the agent with 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 (such as TSEs).   
You should have appropriate and sufficient controls in place to assure that such 
exposures do not occur. 
 
This section applies to testing of cell substrates, control cell cultures, viral seeds, and 
vaccine harvests.  When cell substrates are tested, generally, a preparation at 107 cells/mL  
(or when appropriate, cell lysate from an equivalent number of cells in culture medium) 
is used, with the indicated volumes.  When viral seeds or vaccine harvests are tested, the 
tests are performed either at the indicated volumes or dose-equivalents.  When viral seeds 
and vaccine harvests are tested, it is often necessary to neutralize the virus prior to 
inoculation. 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. 
 
The most appropriate tests for adventitious agents in a given vaccine 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 in selecting the appropriate tests.  See Section 
III for additional information on factors to take into account in selecting appropriate tests. 
 
For each of the tests for adventitious agent described in this section, alternatives such as 
those recommended by the World Health Organization (WHO) or the European 
Pharmacopoeia (EP) or different inoculation strategies for testing cell bank lysates and 
viral harvests will be considered when justified in the context of the entire testing 
program. 
 
1.   In Vivo Tests 
 
In vivo adventitious agent testing for vaccines 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 (e.g., through exposure to rodents or rodent cells).  These tests also 
may be performed in other species to detect species-specific viruses.  For 
example, inoculation of rabbits or guinea pigs may also be used to detect 
additional agents, when appropriate. The described animal studies must be 
performed in accordance with Good Laboratory Practices (GLP) regulations (21 
CFR Part 58). 
 
a.   Adult Mice 
 
This test detects adventitious viruses including lymphocytic choriomeningitis 
virus (LCMV), coxsackieviruses, flaviviruses, and rabies virus. 
 
20   Contains Nonbinding Recommendations 
 
 
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 by intraperitoneal and intracerebral inoculation of appropriate 
homogenized tissue into at least five additional mice.  Each mouse inoculated 
with the homogenized tissue should then 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) (Ref. 12). 
 
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 (e.g., polioviruses and echoviruses), alphaviruses, 
bunyaviruses (e.g., phleboviruses and nairoviruses), arenaviruses, flaviviruses, 
rabies, and herpesviruses (e.g., 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 
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) (Ref. 12).  
 
c.   Guinea Pigs  
 
This test detects Mycobacterium tuberculosis and adventitious viruses 
including paramyxoviruses (including Sendai virus), reoviruses, and 
filoviruses. 
21   Contains Nonbinding Recommendations 

 
 
 
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) (Ref. 12). 
   
In vitro methods, such as culture and PCR, are also acceptable for identifying 
Mycobacterium tuberculosis when validated. 
 
d. 	  Rabbits 
 
This test detects simian herpes B virus, and should be considered when 
primary monkey cells are used. 
 
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) (Ref. 12). 
 
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 
22   
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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 35C 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 
passes the test if at least 80% of the embryos appear normal and there is no 
evidence of viral or 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 35C 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 35C for at least 9 days.  The material passes the test if at 
least 80% of the embryos in both the initial test and the passage appear normal 
and there is no evidence of viral agents (Ref. 12).  
 
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 viruses of a specific species. 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 virus (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 might 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 non
virulent strains of LCMV will survive this challenge.  If you use alternative 
23   
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methods, such as PCR, you should demonstrate sensitivity comparable to that 
of the described test. 
 
Antibody tests such as Test for Extraneous Agents Using Chicks (Ref. 13), 
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 Tests 
 
Cell culture safety tests can detect a variety of adventitious viruses that 
include cytopathic viruses, hemadsorbing 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.  For 
example, in vitro adventitious agent testing of insect cells should be capable 
of detecting different families of insect viruses that could infect the host from  
which the cell substrate was derived. This should also include a cell line 
broadly susceptible to arboviruses, such as BHK21. 
   Human Diploid Cells 
 
Human viruses:  adenoviruses, alphaviruses,  coronaviruses, 
enteroviruses (including poliovirus, echovirus, and coxsackie B 
virus), flaviviruses, certain strains of hepatitis A virus, 
herpesviruses (including herpes simplex virus, varicella zoster 
virus, and cytomegalovirus), orthomyxoviruses, paramyxoviruses, 
rabies virus, reoviruses, some rhinoviruses, and rubella virus.  
 
Simian viruses:  SCMV (simian cytomegalovirus). 
   Monkey Kidney Cell Line (usually Vero cells) 
 
Human viruses: adenoviruses (some strains), alphaviruses, 
arenaviruses, bunyaviruses (including phleboviruses and 
nairoviruses), enteroviruses (including polioviruses, coxsackie B 
viruses, echoviruses) , filoviruses, flaviviruses, herpes simplex 
24   Contains Nonbinding Recommendations 
 
 
virus, influenza viruses, orthomyxoviruses, paramyxoviruses, 
polyomaviruses, poxviruses, rabies viruses, reoviruses, 
rhinoviruses, rotaviruses, rubella virus, and vesiculoviruses. 
 
Simian viruses:  herpes B virus.                        
 
An appropriate volume should be inoculated onto monolayer cultures of at 
least 3 cell types. The sample to be tested should be diluted as little as 
possible. The cell cultures should be observed for at least 2 weeks.  After 2 
weeks of observation, the original cultures of supernatants or lysates from cell 
banks are subcultured onto fresh cells and observed for at least an additional 2 
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 virus-induced 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 4 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 cell lysate equivalent to 107 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 (Ref. 12).  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 or monkey RBCs, which are added independently 
to different portions of the cell cultures or to aliquots of the supernatants at 2
8C for 30 minutes, and observed for hemadsorption and hemagglutination, 
respectively. This is followed by incubation with RBCs for 30 minutes at 
room temperature, followed by observation for 
25   Contains Nonbinding Recommendations 
 
 
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 (see Section III.A.4).  Historically, one quarter to one  
third of the control vessels were tested for hemadsorbing 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. 14). While TEM is fairly insensitive, it is a 
test 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. 15). 
 
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 and insect cell substrates (or that use or include mammalian-
derived materials) using a highly sensitive PCR-based RT (PBRT) assay.  In 
certain situations, it may be appropriate to pre-treat the cells with chemical 
inducers of endogenous retroviruses.  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 assay 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 (see, for example, Refs. 16-19).  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 (Ref. 20). 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, which are less amenable to 
characterization than banked cells, might need to be assessed lot-by-lot.  In 
26   
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cases when control cell cultures are set up in parallel with production cells 
(Section III.A.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 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 exogenous retroviruses, such as avian leukosis 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.    
 
d.   Infectivity Tests for Retroviruses 
 
For non-murine retroviruses, infectivity testing on indicator cells selected for 
their susceptibility to different retrovirus types might be appropriate.  Such 
testing strategies should be determined on a case-by-case basis 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. 21). 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. 20) and other avian 
retroviruses might be appropriate for cell substrates of avian origin.  Often, 
infectivity tests may be augmented with sensitive PBRT 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 
(Ref. 14). 
 
e.   PCR or Other Specific In Vitro Tests 
 
In cases where viruses cannot be readily grown in culture, PCR is an 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 be able 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 determining what testing 
is appropriate. Specific PCR tests for human viruses that you should consider 
27   
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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 potentially infect humans, including simian 
polyomaviruses (e.g., SV40), simian foamy virus (SFV), simian  
immunodeficiency virus (SIV), simian retrovirus (SRV), and simian T-cell 
lymphotropic virus (STLV), should also be considered.  You also should 
consider using PCR assays that detect several agents using degenerate or 
consensus primers provided that the sensitivity of these assays is sufficient to 
improve assurance of product safety.  PCR testing also can play an important 
role in qualification of insect cell substrates. 
 
Although less sensitive, you also 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 BSE (Ref. 33).  We recommend that you consult 
with CBER for more information.  In some cases, a BSE risk assessment can 
address BSE-related issues. 
   
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 (Requirements for ingredients of animal origin used for 
production of biologics) and 113.47 (Detection of extraneous viruses 
by the fluorescent antibody technique). 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. 
 
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g.   Testing of Porcine-Derived Reagents 
 
Products that will be administered to humans should not be contaminated with 
animal adventitious agents.  Products should thus be free of the agents listed 
in the requirements for porcine adventitious virus testing of products intended 
for use in animals (9 CFR 113.47).  Testing of porcine-derived reagents for 
these agents is an important component of providing this assurance. Under 
certain circumstances, additional tests might be needed.  Agents of concern  
from porcine cells listed in 9 CFR 113.47(b)(1) and (6) include:  porcine 
parvovirus, bovine viral diarrhea virus (BVDV), reovirus, rabies virus, 
porcine adenovirus, transmissible gastroenteritis virus (TGE), and porcine 
hemagglutinating encephalitis virus.  At a minimum, you should test porcine 
trypsin for porcine parvovirus (PPV).  
 
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. 22), Nipah virus and porcine 
retroviruses.  
 
3.   In Vitro Tests for Non-Viral Agents  
 
a.   Mycoplasma/Spiroplasma  
 
All licensed viral vaccines produced in cell substrates (and similar products) 
should be tested to ensure the absence of mycoplasma contamination.  Testing 
is usually performed on the virus seed and/or MCBs, 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 sampled for 
testing 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 and 
control fluid pool (when control cells are used) 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: 1) between 2 and 
8 C for no longer than 24 hours, or 2) 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 21 CFR 610.30 specifies agar and broth media culture procedures, 
recent data suggest that some fastidious strains of mycoplasma, especially 
Mycoplasma hyorhinis, might not be detected by these agar and broth media 
29   
 Contains Nonbinding Recommendations 
 
 
procedures. 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 specified in 21 CFR 610.30.  However, 
you must seek and obtain FDA approval for these modifications in accordance 
with 21 CFR 610.9(b). Additional information regarding mycoplasma testing 
is available (Ref. 23). 
 
For Spiroplasma testing by the culture method and by DNA staining, you 
should use media that supports the growth of Spiroplasma, Entomoplasma, 
and Mesoplasma. 
 
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 
 1C 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 
  30   Contains Nonbinding Recommendations 

 
 
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 colony-
forming 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 

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  1C 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. 
 
31   
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   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. 
 
(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.  Such assays may combine culture 
and PCR. 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 
 
A 2.0 mL sample of the filtrate in its most concentrated form should be 
inoculated onto Lowenstein  Jensens egg medium or other media 
32   Contains Nonbinding Recommendations 
 
 
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 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 (Ref. 12).  
 
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.  
 
B.   TESTING OF CELL PROPERTIES  
 
Tests for tumorigenicity and oncogenicity as outlined in the section below are required to 
follow good laboratory practices for conducting nonclinical laboratory studies as 
described in the GLP regulations (21 CFR Part 58). 
 
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 V. Glossary).  Tumorigenicity 
is a characteristic of the immortalized cells themselves, rather than of the agents or 
components present in them.  We recommend you contact CBER for advice as to 
the need for tumorigenicity testing. 
 
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 TPD  (tumor
50
producing 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.  
33   Contains Nonbinding Recommendations 

 
 
 
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. 
24), 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.   
 
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. 24), and these also 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.  Intercurrent infections, 
including subclinical infections, have the potential to alter the incidence of tumors, 
affecting the quality of the data and interfering with the purpose of the study (Ref. 
25). 
 
You should perform a necropsy on each animal when it dies, when it 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 all major 
organ systems such as lymph nodes, lungs, brain, spleen, kidneys, and liver.  All 
lesions, detectable regional lymph nodes, the site of inoculation, and the lungs are 
to be examined histologically.  In some cases (for example, tumors distant from the 
site of inoculation), 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 test cells or positive control tumor 
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 animals 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. 
 
34   
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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 testing (see Section V. Glossary) is designed to assure that agents 
that could immortalize cells and endow them with the capacity to form tumors are 
not present in a cell substrate.  If your vaccine is manufactured in a cell substrate 
that was derived from a tumor, or that has 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, we recommend that you perform oncogenicity 
testing in animals using lysates of the cell substrate.  For cell substrates with a 
tumorigenic phenotype, we recommend that you perform oncogenicity testing in 
animals using DNA from the cell substrate in order to provide assurance that 
residual DNA is non-oncogenic (also see Section III.C.6).  Oncogenicity testing 
might also be appropriate for some 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. 
 
All tumors that develop in oncogenicity assays should be examined for the 
presence of DNA from the species that the cell substrate was derived from, and of 
the animal in which oncogenicity testing was performed.  We recommend 
retention of samples from any tumors observed in oncogenicity assays to permit 
further investigation when necessary.  
 
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 
manufacturers label.  Identity tests also provide extra assurance in facilities or 
laboratories that are propagating multiple cell lines or viruses.   
 
Biochemical tests such as polyacrylamide gel electrophoresis (PAGE) analysis 
may be used to determine the species from which the cell substrate was derived.  
Comparison of the migration patterns of the cellular enzymes malate 
35   Contains Nonbinding Recommendations 

 
 
dehydrogenase, glucose-6-phosphate dehydrogenase, nucleoside phosphorylase 
and lactate dehydrogenase is another method (Ref. 26). 
 
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 than 200 such cells, 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. 27).  Specific methods for this purpose can be found in Stacey, et al. 
(Ref. 28). 
 
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 
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. 29). 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 demonstrate the genetic stability of your cell substrate from the 
establishment of the MCB 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 comparable levels throughout production.  
Also, a diploid cell strain should remain diploid throughout.  If such characteristics 
are not stable, then you should demonstrate that the instability does not adversely 
impact manufacturing or product consistency.  For methods to assess a cell 
substrates genetic stability, reference is made to the ICH Q5B and Q5D documents 
(Refs. 30 and 3, respectively). 
 
C.   OTHER TESTS 
 
1.   Testing for the Presence of Residual Cells 
 
You should assure that your final vaccine product does not contain 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 (or other materials known to be smaller than intact 
cells) are cleared by these processes is an important part of this validation.   
 
36   
 Contains Nonbinding Recommendations 
 
 
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 
(based on current evidence, approximately 200 base pairs).  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. 31).  Because orally 
administered DNA is taken up approximately 10,000-fold less efficiently than 
parenterally administered DNA, we recommend limiting DNA to less than 100 
g/dose for oral vaccines (Ref. 32).  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 for, and the exceptions and criteria for exception from, the GST 
are set forth in 21 CFR 610.11. 
 
 
37   
 Contains Nonbinding Recommendations 

 
 
V.   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 REAGENT: Reagents 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, and 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 passed 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 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 an intact 
or infectious virus. 
 
10.  END-OF-PRODUCTION CELLS (EOPC):  	Cells cultured (under conditions comparable 
to those used in production) from the MCB or WCB  to a passage level or population 
doubling level comparable to or beyond the highest level reached in production. 
 
38   Contains Nonbinding Recommendations 

 
 
11.  FINAL BULK: 	The stage of vaccine production directly prior to filling of individual 
vials. The final bulk often represents a processed harvest. 
 
12.  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 is 
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. 
 
13.  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. 
 
14.  IMMORTALIZATION:	  The process by which cells with finite lifespan (e.g., primary 
cells, diploid cell strains) are converted to those with infinite lifespan.  
 
15.  LATENT VIRUS: 	 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. 
 
16.  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. 
 
17.  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. 
 
18.  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.   
 
19.  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). 
 
20.  PARENTAL VIRUS: 	A virus that has been  manipulated in some manner to generate a 
viral seed with characteristics needed for production. 
 
21.  PARENT CELL BANK: 	A few vials consisti ng of cells from which the Master Cell 
Bank was derived. Parental Cells may be manipulated to derive a cell substrate with 
desired characteristics. 
 
22.  PASSAGE LEVEL: 	 The number of times, since establishment from a primary cell 
culture, a culture has been split or re-seeded. 
39   Contains Nonbinding Recommendations 

 
 
 
23.  POPULATION DOUBLING LEVEL:  T	 he number of times, since establishment from a 
defined point in the history of a cell substrate (often the primary cell culture), a culture 
has doubled in number of cells. 
 
24.  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. 
 
25.  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)) 
 
26.  QUALIFICATION:  	Determination of the suitability of a cell substrate for manufacturing 
based on its characterization. 
 
27.  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.  
 
28.  NON-TUMORIGENIC:	  A cell type is non-tumorigenic if it is shown not to form tumors 
in appropriate animal models. 
 
29.  TUMORIGENICITY: 	Tumorigenicity is the  process by which  immortalized cells form  
tumors when inoculated into animals (see Tumorigenic).  Tumorigenicity is distinct from  
Oncogenicity (See Oncogenicity). 
 
30.  TUMORIGENICITY TESTING: 	An assay/test that determines whether or not 
immortalized cells are tumorigenic when injected into animals. 
 
31.  VALIDATION OF ANALYTICAL PROCEDURES:  V	 alidation defines the 
performance characteristics of an analytical procedure, based on the demonstration that 
the procedure is suitable for its intended purpose or use.  Validation is generally 
performed in accordance with the relevant ICH guidelines. 
 
32.  VALIDATION OF PROCESSES: Process validation is establishing documented 
evidence that provides a high degree of assurance that a specific process will consistently 
produce a product meeting its pre-determined specifications and quality characteristics. 
 
33.  VIRAL CLEARANCE:	  The combination of the physical removal of viral particles and 
the reduction of viral infectivity through inactivation. 
 
34.  VIRUS PRE-SEED: 	A few vials consisting of  vaccine virus from which the MVS was 
derived. 
 
40   
 Contains Nonbinding Recommendations 

 
 
35.  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. 
 
36.  WORKING CELL BANK (WCB):  See Manufacturers Working Cell Bank (MWCB). 
 
37.  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. 
 
 
  
41   
 Contains Nonbinding Recommendations 

 
 
 
VI.   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. 	  The International Conference on Harmonisation (ICH) Guideline, Q2(R1):  Validation of 
Analytical Procedures: Text and Methodology, (60 FR 11260, March 1, 1995) and (62 FR 
27463, May 19, 1997). 
 
7. 	  U.S. Pharmacopeia (USP) Chapter 1225: Validation of Compendial Methods. 
 
8. 	  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. 
 
9. 	  Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning 
the Use of Xenotransplantation Products in Humans, April 2003.  (see also 21 CFR 
610.18(c)(1)(iii)). 
 
10.  European Pharmacopoeia, 4th Edition, Section 2.6.6. 
 
11.  U.S. Pharmacopeia, Chapter 61: Microbial Examination of Non Sterile Products: Microbial 
Enumeration Tests. 
 
12.  Office of the Federal Register, Code of Federal Regulations, 21 CFR part 630 (Additional 
Standards for Viral Vaccines). 1996 and earlier versions. 
 
13.  European Pharmacopoeia, 4th Edition, Test for Extraneous Agents Using Chicks, Section 
2.6.6. 
 
14.  Khan, A.S., Muller, J., and Sears, J.F., Early detection of endogenous retroviruses in 
chemically induced mouse cells, Virus Research 2001; 79:39-45. 
42   Contains Nonbinding Recommendations 

 
 
 
15.  Bierley,S.T. et al., A comparison of methods for the estimation of retroviral burden., Dev. 
Biol. Stand. 1996; 88:163-5. 
 
16.  Arnold et al., One-step fluorescent probe product-enhanced reverse transcriptase assay, 
Biotechniques 1998; 25: 98-106. 
 
17.  Maudru and Peden, Adaptation of the fluorogenic 5'-nuclease chemistry to a PCR-based 
reverse transcriptase assay, Biotechniques 1998; 25: 972-975. 
 
18.  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.  
 
19.  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. 
 
20.  Khan AS, et al., The reverse transcriptase activity in cell-free medium of chicken embryo 
fibroblast cultures is not associated with a replication competent retrovirus, J Clin Virol 1998; 
11:7-18. 
 
21.  European Pharmacopoeia, 4th Edition, Section 2.6.4. 
 
22.  Leman, A.D., et al., Diseases of the Swine, 1992, 7th Edition. 
 
23.  Barile, M.F. and Rottem, S., Mycoplasmas in Cell Culture, Rapid Diagnosis of Mycoplasmas, 
I. Kahane and A. Adoni (ed.) Plenum Press, New York, 1993; p.155-193. 
 
24.  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 1977; p. 313-326. 
 
25.  National Research Council (Commission on Life Sciences, Institute of Laboratory Animal 
Resources), Surveillance, Diagnosis, Treatment and Control of Disease in Guide for the Care 
and Use of Laboratory Animals, National Academy Press, Washington DC, 1996, pp. 59-60. 
 
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. 
 
43   Contains Nonbinding Recommendations 

 
 
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 
Construct in Cells Used for Production of r-DNA Derived Protein Products, (61 FR 7006, 
February 23, 1996). 
 
31.  World Health Organization, Requirements for the use of animal cells as in vitro substrates for 
the production of biologicals. World Health Organization (WHO) Technical Report.  Series 
878, 1998, Annex 1 
 
32.  World Health Organization, Guidelines To Assure The Quality, Safety And Efficacy Of Live 
Attenuated Rotavirus Vaccines (Oral), World Health Organization (WHO) Technical Report 
Series 941, 2007, Annex 3 
 
33. Proposed Rule, Use of Materials Derived from Cattle in Medical Products Intended for Use in 
Humans and Drugs Intended for Use in Ruminants, (72 FR 2007 1582; Jan. 12, 2007). 
44   
 Contains Nonbinding Recommendations 

 
 
Appendix 1 
 
 
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 table shows a typical testing scheme; actual selected tests 
will depend upon the actual cell substrate, the product, and its manufacture.  Additional tests may 
be included on a case-by-case basis depending upon the cell substrate and other biological 
materials as well as production conditions for vaccine manufacture. 
45   
 Contains Nonbinding Recommendations 
 
Table 1: Example of a Biosafety Testing Scheme for Manufacture of a Viral Vaccine. 
 
  Cell Testing  Testing of Intermediates and Final 
MCB  WCB  Control  EOP  Virus  Virus  Vaccine  Vaccine  Final 
Cells  Seed  Seed  Harvest  Bulk  Filled 
Harvest  Final 
IDENTITY & 
+  (+)    (+)  (+)  +    (+)  + 
PURITY1  
STABILITY 
(genotype/  (+)      (+)    (+)       
phenotype/serotype) 
STERILITY                   
Bacteria and 
+  +  +  +  +  +  +  +  + 
Fungal 
Mycoplasma or 
+  +  +  +  +  +  +     
Spiroplasma 
Mycobacteria  (+)  (+)  (+)  (+)  (+)2     (+)     
ADVENTITIOUS 
                 
AGENT TESTING 
Cytopathic and 
Hemadsorbing      +             
viruses 
   In vitro cell 
+  +  (+)  +  +    (+)     
cultures 
   In vivo assays  +    (+)  +  +    (+)     
TEM4   +      +           
Antibody 
(+)        (+)         
Production assay 
Bovine viruses  (+)  (+)  (+)  (+)  (+)    (+)     
   Porcine viruses  (+)  (+)  (+)  (+)  (+)    (+)     
   Retroviruses4   +    (+)  +  +    (+)     
   Tests for specific  
(+)  (+)  (+)3     (+)    (+)     
viruses4  
TUMORIGENICITY        +           
ONCOGENICITY        (+)           
OTHER AGENTS                   
Residual cell DNA                +   
Residual cellular 
              +   
protein 
Residual serum,               
antibiotics, 
+   
benzonase, 
ancillary reagents2  
GENERAL SAFETY               
  + 
TEST 
ENDOTOXIN                  + 
46   
 Contains Nonbinding Recommendations 

 
 
1 tests may be different for cells versus intermediate and final products    
2 if applicable 
3 including ALV test in case of chicken cells
4 may be combined with virus induction assays, as applicable
+ required stage for testing
(+) alternative stage for testing  
47