Q5A(R2) Viral Safety 
Evaluation of 
Biotechnology Products 
Derived From Cell Lines of 
Human or Animal Origin 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologic Evaluation and Research (CBER) 
 
January 2024 
ICH - Quality 
Revision 2 
   
Q5A(R2) Viral Safety 
Evaluation of 
Biotechnology Products 
Derived From Cell Lines of 
Human or Animal Origin 
Guidance for Industry 
 
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U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologic Evaluation and Research (CBER) 
 
January 2024 
ICH - Quality 
Revision 2 
  Contains Nonbinding Recommendations 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use (ICH) has the mission of achieving greater regulatory harmonization 
worldwide to ensure that safe, effective, and high-quality medicines are developed, 
registered, and maintained in the most resource-efficient manner.  By harmonizing the 
regulatory expectations in regions around the world, ICH guidelines have substantially 
reduced duplicative clinical studies, prevented unnecessary animal studies, standardized 
safety reporting and marketing application submissions, and contributed to many other 
improvements in the quality of global drug development and manufacturing and the products 
available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities 
and industry parties in detailed technical and science-based harmonization work that results 
in the development of ICH guidelines.  The commitment to consistent adoption of these 
consensus-based guidelines by regulators around the globe is critical to realizing the benefits 
of safe, effective, and high-quality medicines for patients as well as for industry.  As a 
Founding Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a 
major role in the development of each of the ICH guidelines, which FDA then adopts and 
issues as guidance to industry. 
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
I.  INTRODUCTION (1) .................................................................................................. 1 
II.  POTENTIAL SOURCES OF VIRUS CONTAMINATION (2) .............................. 3 
A.  Viruses That Could Occur in the Master Cell Bank (2.1) .................................................... 3 
B.  Adventitious Viruses That Could Be Introduced During Production (2.2) ........................ 3 
III.  CELL LINE QUALIFICATION: TESTING FOR VIRUSES (3) .......................... 4 
A.  Virus Tests for Master Cell Bank, Working Cell Bank, and Cells at the Limit of In Vitro 
Cell Age Used for Production (3.1) ......................................................................................... 4 
1. MCB (3.1.1) ............................................................................................................................... 4 
2. WCB (3.1.2) ............................................................................................................................... 4 
3. Cells at the LIVCA Used for Production (3.1.3) ........................................................................ 5 
B.  Recommended Virus Detection and Identification Assays (3.2)........................................... 5 
1. Tests for Retroviruses (3.2.1) ..................................................................................................... 5 
2. In Vitro Cell Culture Infectivity Assays (3.2.2) .......................................................................... 6 
3. In Vivo Assays (3.2.3) ................................................................................................................ 7 
4. Tests for Specific Viruses (3.2.4) ............................................................................................... 7 
5. Molecular Methods (3.2.5) ........................................................................................................ 8 
a.  NATs (3.2.5.1) .................................................................................................................... 8 
b.  NGS (3.2.5.2) ...................................................................................................................... 8 
C.  Acceptability of Cell Lines (3.3) ............................................................................................ 10 
IV.  TESTING FOR VIRUSES IN UNPROCESSED BULK (4) .................................. 10 
V.  RATIONALE AND ACTION PLAN FOR VIRAL CLEARANCE STUDIES 
AND VIRUS TESTS ON PURIFIED BULK (5) ..................................................... 11 
VI.  EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE 
PROCEDURES (6) .................................................................................................... 13 
A.  Choice of Viruses for Evaluation and Characterization of Viral Clearance (6.1) ........... 14 
1. Relevant Viruses and Model Viruses (6.1.1) ............................................................................ 14 
2. Other Considerations (6.1.2) ................................................................................................... 15 
B. Design and Implications of Viral Clearance Evaluation and Characterization  
Studies (6.2) ................................................................................................................................. 15 
1. Facility and Staff (6.2.1) .......................................................................................................... 15 
2. Scale-Down Model of the Production Process (6.2.2) ............................................................. 15 
3. Analysis of Stepwise Clearance of Virus (6.2.3) ...................................................................... 16 
4. Determining Physical Removal Versus Inactivation (6.2.4) .................................................... 16 
5. Inactivation Assessment (6.2.5) ............................................................................................... 17 
6. Function and Regeneration of Columns (6.2.6) ....................................................................... 17 
7. Specific Precautions (6.2.7) ..................................................................................................... 17 
C.  Interpretation of Viral Clearance Studies (6.3) ................................................................... 19 
D.  Limitations of Viral Clearance Studies (6.4) ....................................................................... 20 
E.  Statistics (6.5) ......................................................................................................................... 21 
F.  Application of Prior Knowledge for Evaluation of Viral Clearance (6.6) ........................ 21 
G.  Reevaluation of Viral Clearance (6.7) .................................................................................. 22 
i  Contains Nonbinding Recommendations 
 
VII.  POINTS TO CONSIDER FOR CM (7) ................................................................... 23 
A.  Viral Safety in CM (7.1) ........................................................................................................ 23 
2.  General Considerations for Viral Clearance in CM (7.2) .................................................. 23 
2.  Unique Considerations for Viral Clearance in CM (7.3).................................................... 24 
2. Potential Risk Related to Longer Periods in Production Culture (7.3.1) ................................ 24 
2. Approach to Viral Clearance Study (7.3.2) ............................................................................. 25 
VIII.  SUMMARY (8)........................................................................................................... 26 
GLOSSARY (9) ...................................................................................................................... 27 
REFERENCES (10) ............................................................................................................... 32 
TABLE 1. VIRUS TESTS RECOMMENDED FOR CHARACTERIZATION OF CELL 
SUBSTRATES ........................................................................................................................ 33 
TABLE 2. EXAMPLES OF THE USE AND LIMITATIONS OF ASSAYS THAT CAN 
BE USED TO TEST FOR VIRUSES ................................................................................... 34 
TABLE 3. VIRUSES DETECTED IN ANTIBODY PRODUCTION TESTSD ............... 36 
TABLE 4.  RECOMMENDED ACTION PLAN IN RESPONSE TO THE RESULTS 
OF VIRUS TESTS ON CELLS OR UNPROCESSED BULK .......................................... 37 
APPENDIX A: THE CHOICE OF VIRUSES FOR VIRAL CLEARANCE STUDIES 
(ANNEX 1) .............................................................................................................................. 39 
A.  Examples of Useful Model Viruses (Annex 1.1) ................................................................... 39 
B.  Examples of Viruses That Have Been Used in Viral Clearance Studies (Annex 1.2) ...... 39 
APPENDIX B: STATISTICAL CONSIDERATIONS FOR ASSESSING VIRUS AND 
VIRUS REDUCTION FACTORS (ANNEX 2) ................................................................... 41 
Probability of Detection of Viruses at Low Concentrations ....................................................... 41 
APPENDIX C: CALCULATION OF REDUCTION FACTORS IN STUDIES TO 
DETERMINE VIRAL CLEARANCE (ANNEX 3) ............................................................ 43 
APPENDIX D: CALCULATION OF ESTIMATED PARTICLES PER DOSE  
(ANNEX 4) .............................................................................................................................. 44 
APPENDIX E: EXAMPLES OF PRIOR KNOWLEDGE INCLUDING IN-HOUSE 
EXPERIENCE TO REDUCE PRODUCT-SPECIFIC VALIDATION EFFORT 
(ANNEX 5) .............................................................................................................................. 45 
A.  Introduction (Annex 5.1) ....................................................................................................... 45 
B.  Inactivation by Solvent/Detergent or Detergent Alone (Annex 5.2) .................................. 45 
C.  Incubation at Low pH (Annex 5.3) ....................................................................................... 48 
D.  Virus Filtration (Annex 5.4) .................................................................................................. 49 
APPENDIX F: GENETICALLY ENGINEERED VIRAL VECTORS AND VIRAL 
VECTOR-DERIVED PRODUCTS (ANNEX 6) ................................................................. 51 
A.  Introduction (Annex 6.1) ....................................................................................................... 51 
B.  Testing for Viruses (Annex 6.2) ............................................................................................ 51 
C.  Viral Clearance (Annex 6.3) .................................................................................................. 53 
  ii  Contains Nonbinding Recommendations 
Q5A(R2) Viral Safety Evaluation  
of Biotechnology Products Derived  
From Cell Lines of Human or Animal Origin 
Guidance for Industry1 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) 
on this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  
You can use an alternative approach if it satisfies the requirements of the applicable statutes and 
regulations.  To discuss an alternative approach, contact the FDA office responsible for this guidance 
as listed on the title page.   
 
 
 
I.  INTRODUCTION (1)2 
 
This guidance describes the evaluation of the viral safety of biotechnology products including 
viral clearance and testing, and it outlines what data should be submitted in marketing 
applications for those products. Biotechnology products include biotherapeutics and certain 
biological products derived from cell lines of human or animal origin (e.g., mammalian, 
avian, insect). In this guidance, the term virus excludes nonconventional transmissible agents 
like those associated with mammalian prions (e.g., bovine spongiform encephalopathy, 
scrapie). Applicants are encouraged to discuss transmissible spongiform encephalopathy 
associated issues with the appropriate regulatory authorities because they are not in scope of 
this guidance. 
 
This guidance includes products such as cytokines, monoclonal antibodies (mAbs), and 
subunit vaccines produced from in vitro cell culture using recombinant DNA technologies. 
This guidance also includes certain genetically engineered viral vectors and viral vector-
derived products (e.g., viral vaccines, gene therapy products), provided they are amenable to 
viral clearance, without a negative effect on the product. These products can include viral 
vectors, for example, adeno-associated virus (AAV), produced using transient or stable 
transfected cell lines, or by infection using a recombinant virus. It also includes viral vector-
derived products, for example, baculovirus-expressed virus-like particles (VLPs), protein 
subunits, and nanoparticle-based protein vaccines and therapeutics. AAV gene therapy 
vectors include those that depend on helper viruses such as herpes simplex virus or 
adenovirus for their production. Specific guidance on genetically engineered viral vectors and 
viral vector-derived products is provided in Appendix F (Annex 6). 
 
 
1 This guidance was developed within the Expert Working Group (Quality) of the International Council for 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has 
been subject to consultation by the regulatory parties, in accordance with the ICH process. This document has 
been endorsed by the ICH Assembly at Step 4 of the ICH process, November 2023. At Step 4 of the process, the 
final draft is recommended for adoption to the regulatory bodies of the ICH regions.  
2 The numbers in parentheses reflect the organizational breakdown of the document endorsed by the ICH 
Assembly at Step 4 of the ICH process, November 2023. 
1  Contains Nonbinding Recommendations 
 
Inactivated viral vaccines and live attenuated viral vaccines containing self-replicating agents 
are excluded from the scope of this guidance. Cell therapies are out of the scope of this 
guidance; however, the principles can be used as applicable (e.g., for biological starting or 
raw materials). 
 
It is no longer encouraged that materials be manufactured from hybridoma cells grown in 
vivo as ascites due to the contamination risk as well as to the ongoing global initiative to 
replace, reduce, and refine the use of animals. Where this situation exists, the principles of 
this guidance should be followed, including replacement of the in vivo assay by next 
generation sequencing (NGS).  
 
The risk of viral contamination is a concern for all biotechnology products derived from cell 
lines and needs to be reduced because such contamination could have serious clinical 
consequences. This risk can arise from the contamination of the source cell lines themselves 
(cell substrates) or from exogenous introduction of adventitious virus during production. 
However, biotechnology products derived from cell lines have not been historically 
implicated in the transmission of viruses. The viral safety of these products has been 
reasonably assured by applying a comprehensive virus testing program and assessing virus 
removal and inactivation achieved by the manufacturing process, as outlined below. Three 
principal, complementary approaches are applied to control the potential viral contamination 
of biotechnology products:  
 
  Selecting and testing cell lines and other raw materials, including media components, 
for the absence of undesirable infectious viruses 
 
  Assessing the capacity of the production processes to clear adventitious and 
endogenous viruses 
 
  Testing the product at appropriate steps of production for demonstrating the absence 
of contaminating infectious viruses 
 
Some viral clearance steps used during production of genetically engineered viral vectors and 
viral vector-derived products may not be as effective as those used for recombinant proteins. 
Therefore, considerations for further risk reduction (e.g., treatment of raw materials, 
extensive testing for broad virus detection) should be applied (see Appendix F (Annex 6)). 
 
Viral tests have inherent limitations. For example, the ability to detect low virus 
concentrations depends for statistical reasons on the size of the sample. Therefore, no single 
approach will necessarily establish the viral safety of a product. Confidence that infectious 
virus is absent from the final product will in many instances not be derived solely from direct 
testing for their presence, but also from a demonstration that the purification process is 
capable of removing and/or inactivating (clearing) viruses. 
 
The type and extent of viral tests and viral clearance studies performed at different steps of 
production will depend on various factors and should be considered on a case-by-case and 
step-by-step basis. The factors that should be considered include the origin of the cell line, 
the extent of cell bank characterization and testing; the nature of any viruses detected, culture 
medium constituents, culture methods; facility and equipment design; the results of viral tests 
after cell culture; the ability of the process to clear viruses; and the type of product and its 
intended clinical use. 
  2  Contains Nonbinding Recommendations 
 
 
The recommendations presented here should be adjusted to the specific product and its 
production process. Moreover, the approach used to assure viral safety should be explained 
and justified. In addition to the detailed data, an overall summary of the viral safety 
assessment should be provided. This summary should contain a brief description of all 
aspects of the viral safety studies and strategies used to prevent virus contamination. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys 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 Agency guidances means that something is suggested or 
recommended, but not required. 
 
 
II.  POTENTIAL SOURCES OF VIRUS CONTAMINATION (2) 
 
Virus contamination of biotechnology products may arise from the original source of the cell 
lines or from adventitious introduction of virus during generation of a production cell line 
and/or cell banking and during the production process. Use of well-characterized cell banks 
and virus seeds can reduce the risk of virus contamination. Introduction and control of 
potential adventitious viruses from a master virus seed or working virus seed (WVS) are 
discussed in Appendix F (Annex 6). Viruses used for production of the products (production 
viruses) described in Appendix F (Annex 6) are considered to be process-related impurities. 
 
A.  Viruses That Could Occur in the Master Cell Bank (2.1) 
 
Adventitious virus contaminants can be introduced in the master cell bank (MCB) by several 
routes such as (1) derivation of the cell line from an infected animal; (2) use of a virus to 
establish the cell line; (3) use of contaminated biological reagents (e.g., antibodies for 
selection) or raw materials for cell culturing (e.g., animal or human serum and porcine 
trypsin); or (4) contamination during cell handling and cell banking processes. 
 
Cells have endogenous retroviruses that are maintained from one cell generation to the next. 
The endogenous viral sequences may be constitutively expressed or may become activated to 
produce infectious or defective viral particles. Cells may also contain latent or persistent 
viruses (e.g., herpesvirus). 
 
B.  Adventitious Viruses That Could Be Introduced During Production (2.2) 
 
Adventitious viruses may contaminate the production process by several routes including, but 
not limited to: (1) the use of contaminated biological raw materials or reagents such as animal 
serum components during cell culture; (2) the use of a contaminated virus seed for production 
(see Appendix F (Annex 6)); (3) the use of a contaminated raw material or reagent used 
during downstream purification, such as a mammalian derived antibody-coupled affinity 
resin for product selection or purification; (4) the use of a contaminated inactive ingredient 
during formulation; and (5) contamination from the environment, including storage of 
nonbiological raw materials or during cell culture and medium handling by personnel. 
 
Monitoring cell culture parameters (e.g., cell growth and viability) may also be helpful in the 
early detection of potential adventitious viral contamination. Manufacturers should avoid 
  3  Contains Nonbinding Recommendations 
 
using human- and animal-derived raw materials (e.g., human serum, bovine serum, porcine 
trypsin) in their manufacturing processes when possible. When this is not possible, the use of 
human- and animal-derived raw materials should be supported by the relevant documentation 
or qualification of the material, commensurate with risk. Information such as the country of 
origin, tissue of origin, virus inactivation, or removal steps applied during the manufacturing 
process of the material, and the types of virus testing that have been performed on the raw 
material should be provided. This testing, if needed according to risk assessment, should be 
performed before inactivation, when possible. 
 
When possible, biological materials with a high risk of introducing adventitious viruses such 
as animal or human serum or porcine trypsin should be subjected to viral inactivation such as 
ionizing irradiation. Additional virus risk-mitigation strategies can include, for example, cell 
culture media or media supplement treatments, for example, virus filtration, high temperature 
short-time processing, or ultraviolet C irradiation, where appropriate. 
 
 
III.  CELL LINE QUALIFICATION: TESTING FOR VIRUSES (3) 
 
An important part of qualifying a cell line for use in the production of a biotechnology 
product is the appropriate testing for the presence of viruses. 
 
A.  Virus Tests for Master Cell Bank, Working Cell Bank, and Cells at the 
Limit of In Vitro Cell Age Used for Production (3.1) 
 
Table 1 shows the virus tests recommended to be performed once on the MCB, working cell 
bank (WCB) and cells at the limit of in vitro cell age (LIVCA) that are used for production. 
 
1.  MCB (3.1.1) 
 
Extensive screening for both endogenous viruses and adventitious viral contamination should 
be performed on the MCB. For hetero-hybrid cell lines, for example, in which one or more 
partners are human or nonhuman primate in origin, tests should be performed to detect 
viruses of human or nonhuman primate origin because viral contamination arising from these 
cells may pose a particular risk. Testing for adventitious viruses should include both broad 
and specific virus detection assays as described in Table 1. Introduction of new 
methodologies such as NGS for detecting a broad range of adventitious viruses is 
encouraged. To ensure detection of contaminating viruses, the testing approach should be 
based on a risk assessment, considering the origin and history of the parental cell line and the 
potential exposure to materials of human or animal origin during the generation of production 
cells and banking of the MCB. An alternative approach in which complete testing is carried 
out on the WCB rather than on the MCB could also be acceptable. 
 
2.  WCB (3.1.2) 
 
Each WCB should be tested for adventitious viruses as described in Table 1. When 
appropriate, if adventitious virus tests have been performed on the MCB, and cells cultured 
up to or beyond the LIVCA have been derived from the WCB and tested for the presence of 
adventitious viruses, then similar tests can be omitted from this WCB. 
 
  4  Contains Nonbinding Recommendations 
 
3.  Cells at the LIVCA Used for Production (3.1.3) 
 
The established LIVCA for production should be based on data derived from production cells 
expanded at small scale using a representative scale-down model and/or under pilot plant-
scale or commercial-scale conditions to the proposed in vitro cell age or beyond. LIVCA 
cells are obtained by expansion of the WCB or the MCB. Cells at the LIVCA should be 
evaluated once for viruses that may have been undetected in the MCB and/or WCB. The 
performance of suitable tests (as outlined in Table 1) once on cells at the LIVCA used for 
production will provide further assurance that the production process does not lead to 
induction of endogenous viruses, reactivation of latent viruses, or amplification of low-level 
or slow-growing viruses. If any adventitious viruses are detected at this stage, the process, 
including the MCB and WCB should be checked carefully to determine the source of the 
contamination. Cells at the LIVCA can also be referred to as end of production cells. 
 
B.  Recommended Virus Detection and Identification Assays (3.2) 
 
A number of assays are used to detect endogenous and adventitious viruses. Table 2 lists 
examples of such assays. These assays are recommended, but the list is not all-inclusive or 
definitive. The most appropriate techniques may change with scientific progress; proposals 
for alternative techniques should be accompanied by adequate supporting data. A 
comprehensive testing strategy should be developed following a thorough viral risk 
assessment that includes consideration of the cell line origin; the passage history; and the raw 
materials and reagents used for cell line generation, cell bank preparation, and production 
(e.g., steps that can inactivate or remove viruses). The strategy should include additional 
assays as appropriate based on risk assessment. For example, if there is a relatively high 
possibility of the presence of a particular virus, specific tests or other approaches for 
detection of that virus should be included unless otherwise justified. Appropriate controls 
should be included to demonstrate adequate assay sensitivity and specificity. Furthermore, 
potential matrix interference, where applicable, should be considered. 
 
The following is a brief description of a general framework that the manufacturer should use 
to develop a comprehensive virus testing scheme that is specific (or appropriate) to the 
product and manufacturing process. The testing strategy should be accompanied with 
appropriate justification for the approach. 
 
1.  Tests for Retroviruses (3.2.1) 
 
Cell lines should be characterized for the presence of retroviruses. Tests for retroviruses 
should be performed on the MCB and cells cultured up to or beyond the LIVCA used for 
production. These tests can include infectivity assays by direct inoculation of the cell 
supernatant or cocultivation of the cells, assays for reverse transcriptase (RT) activity using 
cell-free supernatant, and evaluation of the cells for particles by transmission electron 
microscopy (TEM). TEM is also able to detect other agents and, therefore, is generally 
recommended for characterization of cell banks. 
 
If a cell line is producing retroviral particles (as occurs in some cell lines derived from 
rodent, insect, and avian species), RT activity is expected and therefore a polymerase chain 
reaction (PCR)-based RT assay (e.g., product-enhanced reverse transcriptase (PERT) assay) 
may not be needed. TEM should be performed to examine the type of retroviral particles 
(e.g., type-A, type-C) present. To determine whether the endogenous retroviral particles are 
  5  Contains Nonbinding Recommendations 
 
infectious, infectivity assays should be performed using relevant permissive cells (e.g., Mus 
dunni for general murine retrovirus detection, SC-1 cells for ecotropic murine retrovirus 
detection) with sensitive readout assays for retrovirus detection (e.g., PERT assay, a sarcoma-
positive, leukemia-negative (S+L-) assay, or an XC plaque assay). 
 
If it is not known whether the cell line produces retroviral particles, TEM should be 
performed on cells, and an RT assay (e.g., PERT assay) should be carried out on clarified 
supernatant. The PCR-based RT assay is particularly useful because it can detect retrovirus 
RT activity with high sensitivity; however, the RT activity can be associated with an 
infectious or noninfectious retrovirus. Because some cellular DNA polymerases can lead to a 
positive RT result, confirmation of the RT activity (because of a retrovirus contamination) or 
a positive TEM result should be followed by testing to detect infectious retroviruses in 
permissible cells, including human cell lines and a sensitive readout assay for retrovirus 
detection. 
 
Retroviral testing results should be interpreted considering all available data. Cell lines 
expressing endogenous retrovirus particles are not precluded from use in manufacturing 
based on risk evaluation as discussed in sections III.C (3.3) and V (5). 
 
Chemical induction studies need not be performed for cell lines that have been well 
characterized for endogenous retroviruses (e.g., Chinese hamster ovary (CHO), NS0, Sp2/0, 
Vero, other cell lines based on prior knowledge). However, such studies may help to evaluate 
a new cell substrate for the presence of an unknown inducible endogenous retrovirus. 
Furthermore, induction studies for latent DNA viruses (e.g., herpesvirus in human cells) and 
latent RNA viruses (e.g., nodavirus in insect cells) may also be appropriate based on risk 
assessment. These studies may help inform the virus testing and clearance strategy for 
products derived from a new cell substrate. 
 
2.  In Vitro Cell Culture Infectivity Assays (3.2.2) 
 
In vitro assays are carried out by inoculating a test article (see Table 2) on to various 
indicator cell cultures capable of detecting a wide range of human and relevant animal 
viruses. The panel of indicator cell lines should include a cell line of the species of origin, 
human diploid cells (e.g., MRC-5), and a monkey kidney cell line (e.g., Vero). Additional 
cell lines can be included. The choice of cells used in the test should be based on a risk 
assessment considering the species of origin of the cell substrate to be tested. 
 
The nature of the infectivity assay and the sample to be tested are governed by the type of 
virus that may be present based on the origin or handling of the cells. The indicator cell 
cultures should be monitored for cytopathic viruses, hemadsorbing and hemagglutinating 
viruses, consistent with existing regional regulations and guidance. For qualification of the 
MCB, WCB, and LIVCA, 28-day testing on permissive cells should be performed with at 
least one subpassage at 2 weeks. 
 
NGS or other molecular methods can be used to supplement or replace the assay. This could 
address general limitations of the in vitro cell culture infectivity assay (e.g., susceptibility of 
cell lines to infection) and specific limitations of the production system (e.g., test article-
mediated interference or toxicity). 
 
  6  Contains Nonbinding Recommendations 
 
3.  In Vivo Assays (3.2.3) 
 
In vivo testing can be performed based on risk assessment considering the history and 
manufacturing of the cell bank and the testing strategy. However, in vivo testing is not 
necessary for extensively used, well-characterized cell lines such as CHO, NS0, and SP2/0, 
based on prior knowledge. Risk-based considerations include prior in vivo virus testing or 
NGS testing of the parental untransfected cell line and control of the derivation of the MCB 
from the parental cell bank. Prior knowledge of viral safety testing of other MCB derived 
from the same parental cell bank including the method used to establish the MCB also should 
be considered. The test is generally not necessary for the WCB if it is prepared under 
approved controlled conditions. For cells at the LIVCA, the test may not be necessary based 
on prior knowledge and other risk-based considerations. 
 
If residual risk remains, retention of the test or replacement with nontargeted NGS can be 
considered to detect viruses that may have been introduced during establishment of the MCB 
or during culture of the cells to the LIVCA stage. 
 
The use of the in vivo assay can include inoculation of the test article (see Table 2) into 
suckling mice, adult mice, and embryonated hens eggs. The health of the animals should be 
monitored, and any abnormality should be investigated to establish the cause. 
 
Nontargeted NGS is encouraged as a replacement for in vivo assays due to its breadth and 
sensitivity of virus detection and the limitations of the in vivo assays. Furthermore, this 
promotes the global initiative to replace, reduce, and refine the use of animal testing. 
 
4.  Tests for Specific Viruses (3.2.4) 
 
The list of specific viruses to be tested for is defined based on a viral contamination risk 
assessment and considers (but is not limited to) the origin of the cells and the potential 
sources of viral contamination (e.g., biological raw materials, especially those of human or 
animal origin). For cell lines with exposure to human- or animal-derived raw materials, (e.g., 
bovine serum, porcine trypsin), tests for human, bovine, and porcine viruses should be 
performed. For cell lines of rodent origin or exposure to rodent materials, nucleic acid 
amplification techniques (NATs) or antibody production tests in mice, rats, or hamsters are 
used to detect species-specific viruses. These tests should be performed when the potential 
exists for exposure to viruses of a specific animal species. For example, the presence of 
rodent viruses in cell lines of rodent origin, or cell lines generated by passage through rodents 
and the use of reagents that may have been derived from rodent materials, can be detected by 
inoculation of the test article (see Table 2) into specific pathogen-free animals, such as mice, 
rats, and hamsters, that are subsequently tested for antibodies to specific agents. 
 
Examples of such tests are the mouse antibody production (MAP) test, rat antibody 
production (RAP) test, and hamster antibody production (HAP) test. The viruses currently 
screened for in the antibody production assays are discussed in Table 3. 
 
NATs such as PCR assays or targeted or nontargeted NGS or other molecular methods can be 
used for replacing the animal assays described in Table 3, without head-to-head comparison. 
 
  7  Contains Nonbinding Recommendations 
 
5.  Molecular Methods (3.2.5) 
 
Molecular methods such as NAT and NGS are used for virus detection. NGS may be 
appropriate for broad (nontargeted) detection of known and novel viruses. NGS can also be 
used for targeted detection of viruses by the sequencing strategy or by bioinformatic analysis. 
Nontargeted NGS can be used to replace the in vivo assays and supplement or replace the in 
vitro cell culture assays, without a head-to-head comparison as long as the method is 
demonstrated to be suitable for its intended purpose. A head-to-head comparison is not 
recommended due to the different endpoints of the assay systems and limitations of the 
breadth of virus detection by the in vitro and in vivo methods compared to the enhanced 
capability of NGS for broad virus detection of known and unknown viruses. The results of 
the in vitro and in vivo assays rely on virus replication and biological properties for detection 
in the specific target system, which limits the breadth of detection. Replacement of in vivo 
assays by NGS also meets the intent of the global objective to replace, reduce, and refine the 
use of animals for testing. 
 
NATs such as PCR-based methods can be used for virus-specific detection. Targeted or 
nontargeted NGS can be used to replace the large number of PCR assays for virus-specific 
detection without a head-to-head comparison. This can also help overcome the limitation of 
detection of virus variants. Positive results should be investigated to determine whether 
detected nucleic acids are associated with an infectious virus. 
 
a.  NATs (3.2.5.1) 
 
NATs such as PCR-based methods are typically used singly or in a multiplex format to detect 
viral sequences from known viruses or known closely related virus families. These molecular 
methods can be used to supplement cell culture assays when there are limitations because of 
assay interference, and they are effective tools for specific virus detection when such viruses 
cannot be readily grown in cell culture for detection by infectivity assays. NATs also have the 
capacity to be adapted for broader range virus detection (e.g., degenerate PCR), but 
specificity may be reduced. Because of the assay specificity, multiple virus-specific PCR 
assays can be used to detect a broader range of viruses that would be detected by a single 
more general biological assay. NATs should be appropriately validated for their intended 
uses. This includes the method validation and matrix-specific verification, as applicable. 
 
b.  NGS (3.2.5.2) 
 
NGS (also known as high-throughput sequencing) is available with demonstrated capabilities 
for broad virus detection. NGS can provide defined sensitivity and breadth of virus detection 
and can reduce animal use and testing time. Nontargeted NGS can replace the in vivo tests 
with broad virus detection for unknown or unexpected virus species without a head-to-head 
comparison. 
 
Nontargeted NGS can also be used without a head-to-head comparison to supplement or 
replace the in vitro cell culture assays for detection of known and unknown or unexpected 
viruses. This could address general limitations of the in vitro cell culture infectivity assay 
(e.g., susceptibility of cell lines to infection) and specific limitations of the production system 
(e.g., test article-mediated interference or toxicity). 
 
  8  Contains Nonbinding Recommendations 
 
NGS can be used for targeted detection of known viruses based upon availability of their 
sequence information. Alternatively, the NGS bioinformatic analysis can be targeted for 
detection of specific viruses. 
 
NGS (targeted or nontargeted) can replace virus-specific PCR assays and rodent antibody 
production tests (see section III.B.4 (3.2.4)) without a head-to-head comparison. Replacement 
of in vivo assays by NGS also meets the intent of the global objective to replace, reduce, and 
refine the use of animals for testing. 
 
Use of NGS can be considered for characterization of the cell line or testing of the cell bank, 
virus seed, or unprocessed bulk harvest. This can be particularly useful in case of assay 
interference as a result of lack of effective neutralization of the viral vector (see Appendix F 
(Annex 6)) or toxicity caused by the product or media components. In such applications, 
NGS can be applied to analyze all genomic viral nucleic acids (genomics), viral mRNAs 
(transcriptomics), or encapsidated viral genomes (viromics). When analyzing cell culture-
derived materials, nucleic acids prepared from cells are used for genomics and 
transcriptomics, and cell culture supernatants or cell-free virus preparations are used for the 
viromics. The rationale for selecting these different strategies should be provided. 
 
When applying NGS for sensitive and broad detection of known and novel viruses, there are 
several critical steps in the NGS workflow for consideration: (1) sample pretreatment (when 
performed) and virus enrichment steps that may be needed to maximize virus detection based 
on the type of sample material; (2) efficiency of viral nucleic acid extraction (from enveloped 
and nonenveloped particles) and library preparation (DNA and RNA viruses); (3) selection of 
a suitable sequencing platform for sensitive virus detection; and (4) bioinformatics analysis 
against a database with diverse representation of viral sequences of different viral families 
using strategies for broad virus detection. A follow-up strategy may be needed to confirm the 
detection of a virus-specific signal to distinguish from nonviral sequences that could be 
present in the database. 
 
Suitable reference materials should be used for method qualification and validation to 
evaluate performance of the different steps involved in the workflow and to demonstrate 
sensitivity, specificity, and breadth of virus detection. This can include using currently 
available reference virus reagents/panels, which contain viruses of distinct physical (size, 
enveloped and nonenveloped), chemical (low, medium, and high resistance), and genomic 
(DNA, RNA, double- and single-stranded, linear, circular) characteristics to evaluate the 
performance of the entire NGS workflow or specific steps. A comprehensive viral database 
should be used with diverse viral sequences for broad virus detection. Other types of 
reference materials can be used to evaluate the specific technical and bioinformatic steps. 
 
For any NGS method used, validation/qualification should be provided to support its intended 
use for the application. This includes method validation and matrix-specific verification when 
used as a replacement method. When used as a supplementary method, this includes method 
qualification and matrix-specific verification. Method validation requires predefined 
performance criteria while method qualification only evaluates the performance 
characteristics of the method. NGS is used as a limit test and, as such, the performance 
characteristics (specificity/breadth of detection and sensitivity) for method 
  9  Contains Nonbinding Recommendations 
 
validation/qualification should consider the principles of the ICH draft guidance for industry 
Q2(R2) Validation of Analytical Procedures (August 2022).3 
 
C.  Acceptability of Cell Lines (3.3) 
 
Some cell lines used to manufacture a product will contain endogenous retroviruses, other 
viruses, or viral sequences that may become reactivated as infectious viruses. In such 
circumstances, the action plan recommended for manufacture is described in section V (5). 
The acceptability of cell lines containing viruses other than endogenous retroviruses will be 
considered on an individual basis by the appropriate regulatory authorities, considering a 
benefit-risk analysis based on the benefit of the product and its intended clinical use, the 
nature of the contaminating viruses, their potential for infecting humans or for causing 
disease in humans, the purification process for the product (e.g., viral clearance evaluation 
data), and the extent of the virus tests conducted on the purified bulk. 
 
 
IV.  TESTING FOR VIRUSES IN UNPROCESSED BULK (4) 
 
It is recommended that manufacturers develop programs to routinely test for adventitious 
viruses in production batches. The extent of virus testing on the unprocessed bulk should 
consider several points including the nature of the cell lines used to produce the desired 
products, the results and extent of virus tests performed during the qualification of the cell 
lines, the cultivation method, the raw material and reagent sources, and the results of viral 
clearance studies. See Appendix F (Annex 6) for testing of unprocessed bulk for genetically 
engineered viral vectors and viral vector-derived products. 
 
For batch processing, the unprocessed bulk constitutes one or multiple pooled harvests of 
cells and culture media. For processes that involve a continuous product harvest from cell 
culture, there may be pooled intermediate or unprocessed bulk sample(s) collected from a 
flow stream. A representative sample of the unprocessed bulk, removed from the production 
reactor before further processing, represents one of the most suitable levels at which the 
possibility of adventitious virus contamination can be determined with a high probability of 
detection. Therefore, appropriate testing for viruses should be performed on the unprocessed 
bulk. The potential impact of cell separation technology on detecting cell-associated virus in 
the bioreactor should be considered. If unprocessed bulk is toxic or causes interference with 
virus detection in cell culture assays, minimal sample dilution or alternative testing (e.g., 
NGS) can be considered (see section III.B (3.2)). To detect cell-associated virus, a mixture of 
both intact cells and cell lysates (disrupted cells and cell culture supernatants) that are 
removed from the production reactor should be tested. For perfusion or continuous 
manufacturing (CM) processes, cells may not be readily accessible (e.g., because of the use 
of hollow fiber or similar microfiltration systems). In such cases, cell-free product harvest 
from cell culture can be used. For processes that involve continuous product harvest from cell 
culture, the sampling strategy (including periodicity and composition of the samples) should 
be justified because the level of adventitious viruses and endogenous virus particles may vary 
along the cell culture duration (see section VII (7)). 
 
 
3 When final, this guidance will represent the FDAs current thinking on this topic. For the most recent version 
of a guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-
guidance-documents.  
  10  Contains Nonbinding Recommendations 
 
Adventitious virus testing should be routinely applied to each unprocessed bulk. This testing 
can include in vitro assays using several indicator cell lines or nontargeted NGS (see section 
III.B (3.2)). The indicator cell cultures should be observed for 28 days including at least one 
subpassage at 2 weeks. Assay duration can be reduced with justification to 14 days for cell 
lines based on a risk assessment (considering the cell substrate, cultivation period for 
production, use of animal-derived raw materials or reagents, and level of viral clearance of 
the process). Additionally, methods, such as NAT or targeted NGS, may also be appropriate 
for detection of specific viruses (e.g., minute virus of mice) or virus families, based on risk 
assessment for potential introduction of contaminants. Such rapid test methods can facilitate 
real-time decision-making. 
 
If any adventitious viruses are detected at the unprocessed bulk stage, the product harvest 
from cell culture should not be used for product manufacture unless justified. (See section V 
(5) for guidance on the use of material in which an adventitious virus has been detected in the 
harvest material.) The process should be carefully checked to determine the root cause and 
extent of the contamination, and appropriate actions should be taken. For CM processes, final 
release of a batch should include documented absence of viral contamination for the period 
during which cultivation fluids were harvested for manufacture of that batch. If an 
adventitious virus is detected, a procedure to segregate potentially contaminated material 
should be established to mitigate a wider production effect. 
 
 
V.  RATIONALE AND ACTION PLAN FOR VIRAL CLEARANCE STUDIES 
AND VIRUS TESTS ON PURIFIED BULK (5) 
 
It is important to design the most relevant and rational protocol for virus tests from the MCB 
level, through the various steps of drug production, and to the final product including 
evaluation and characterization of viral clearance from unprocessed bulk. The evaluation and 
characterization of viral clearance play a critical role in this scheme. The goal should be to 
obtain assurance that the product is free of virus contamination. 
 
In selecting viruses to use for a clearance study, it is useful to distinguish between evaluating 
processes for their ability to clear viruses that are known to be present and estimating the 
robustness of the process by characterizing the clearance of nonspecific model viruses 
(described later). Definitions of relevant, specific, and nonspecific model viruses are given in 
the glossary. Viral risk evaluation should include knowledge of how much virus may be 
present in the process, such as in the unprocessed bulk, and how much can be cleared, to 
assess product safety. Knowledge of the time dependence for inactivation procedures is 
necessary in ensuring the effectiveness of the inactivation process. When evaluating viral 
clearance, in-depth time-dependent inactivation studies, demonstration of reproducibility of 
inactivation or removal, and evaluation of process parameters should be performed as 
appropriate. When a manufacturing process is characterized for robustness of clearance using 
nonspecific model viruses, particular attention should be paid to nonenveloped viruses in the 
study design (see section VI.A.1 (6.1.1) for explanation of model viruses). The extent of viral 
clearance studies may be influenced by the results of tests on cell lines and unprocessed bulk. 
These studies should be performed as described below (see section VI (6)). 
 
Table 4 presents an example of an action plan used in response to the results of virus tests on 
cells or unprocessed bulk. The plan includes the process evaluation and the characterization 
of viral clearance and virus tests on purified bulk. Various cases are presented in the table and 
  11  Contains Nonbinding Recommendations 
 
are described below. In all cases, characterization of clearance using nonspecific model 
viruses should be performed. The most common situations are Cases A, B, and F. Production 
systems contaminated with a virus other than a rodent retrovirus normally are not used. When 
there are well-justified reasons for drug production using a cell line from Cases C, D, or E, 
these should be discussed with the appropriate regulatory authorities. In Cases C, D, and E, it 
is important to have validated and effective steps to inactivate and/or remove the virus in 
question from the manufacturing process. 
 
Case A: When no virus, no VLP, or no retrovirus-like particle (RVLP) other than the drug 
substance (e.g., viral vector particles) has been demonstrated in the cells or the unprocessed 
bulk, virus removal and inactivation studies should be performed with nonspecific model 
viruses, as previously stated. If no RVLPs are detected and if the PERT assay is negative, no 
estimation of retroviral particles per dose is required. 
 
Case B: In rodent cell lines, if only a rodent retrovirus (such as rodent A-, C-, and R-type 
particle) is present, the process evaluation using a specific model virus (such as a murine 
leukemia virus (MLV)) should be performed in addition to nonspecific viral clearance 
evaluation as in Case A. For marketing authorization, typically RVLP quantification data 
from three lots or more of purified bulk at pilot plant scale or commercial scale should be 
provided. Well-characterized cell lines such as CHO, C127, BHK, and murine hybridoma cell 
lines (e.g., NS0 or SP2/0 cells) have frequently been used as substrates for drug production 
with no reported safety problems related to viral contamination of the products. For these cell 
lines in which the endogenous particles have been extensively characterized and clearance 
has been demonstrated, it is usually not recommended to test for the presence of the 
noninfectious particles in the purified bulk or drug substance. This also applies to other well-
characterized cell lines (e.g., Sf9 insect cell lines) that produce endogenous retroviral-like 
particles that have been extensively characterized. 
 
Case C: When the cells or unprocessed bulk are known to contain a virus (other than a rodent 
retrovirus) for which there is no evidence of infectivity to humans (e.g., Sf9 rhabdovirus), 
virus removal and inactivation evaluation studies should use the identified virus. If it is not 
possible to use the identified virus, relevant or specific model viruses should be used to 
demonstrate acceptable clearance. Time-dependent inactivation for identified (or relevant or 
specific model) viruses at the critical inactivation steps should be obtained as part of the 
process evaluation for these viruses. Purified bulk should be tested using suitable methods 
with high specificity and sensitivity for detecting the virus in question. For the purpose of 
marketing authorization, typically data from three batches or more of purified bulk 
manufactured at pilot plant scale or commercial scale should be provided. 
 
Case D: If a virus infectious to humans (such as those viruses indicated in Table 3, footnote 
a) is identified, the product should be considered acceptable only under exceptional 
circumstances. In such instances, the identified virus should be used for virus removal and 
inactivation evaluation studies and specific methods with high specificity, and sensitivity for 
the detection of the virus in question should be used. If it is not possible to use the identified 
virus, relevant and/or specific model viruses (described later) should be used. The process 
should be shown to remove and inactivate the selected viruses during downstream 
processing. Time-dependent inactivation data for the critical inactivation steps should be 
obtained as part of the process evaluation. Purified bulk should be tested using suitable 
methods with high specificity and sensitivity for the detection of the virus in question. For 
  12  Contains Nonbinding Recommendations 
 
marketing authorization, typically data from three batches or more of purified bulk 
manufactured at pilot plant scale or commercial scale should be provided. 
 
Case E: When a virus that cannot be classified by currently available methodologies is 
detected in the cells or unprocessed bulk, the product is usually considered unacceptable 
because the virus may be pathogenic. In the rare case in which there are well-justified reasons 
for drug production using such a cell line, this should be discussed with the appropriate 
regulatory authorities before proceeding further. 
 
Case F: When a production virus (helper virus or viral vector for expression of recombinant 
protein or VLP) is used in product manufacturing, clearance of the virus should be 
demonstrated using the production virus or a specific model virus (e.g., baculovirus, 
adenovirus, herpesvirus). Testing for the absence of the production virus should be performed 
for each purified bulk, unless justified by robust excess clearance (see section VI.C (6.3)). In 
this case, absence of residual production virus should be confirmed by testing of at least three 
batches of purified bulk. 
 
 
VI.  EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE 
PROCEDURES (6) 
 
Evaluation and characterization of virus removal or inactivation procedures are important for 
establishing the safety of biotechnology products. Past instances of contamination have 
occurred with agents whose presence was not known or even suspected. Though this 
happened due to various source materials other than fully characterized cell lines, it 
reinforces that assessment of viral clearance provides a measure of confidence that any 
unknown, unsuspected, and harmful viruses may be removed. Studies should be carried out in 
a well-documented and controlled manner. 
 
Process characterization/evaluation of viral clearance is performed to demonstrate the 
clearance of a virus known to be present endogenously in the cell substrate, or as a result of 
using production viruses as well as to ensure that adventitious viruses that could not be 
detected, or that might enter the production process, would be cleared. The objectives of viral 
clearance studies are (1) to assess process steps that effectively inactivate or remove viruses 
and (2) to estimate quantitatively the overall level of virus reduction obtained by the process. 
These should be achieved by the deliberate addition (i.e., spiking) of significant amounts of 
virus to the unprocessed bulk or to process intermediates obtained during the various process 
steps and demonstrating its removal or inactivation during the subsequent steps. It is not 
necessary to evaluate or characterize every step of a manufacturing process if adequate 
clearance is demonstrated by using fewer steps. It should be considered that other steps in the 
process may have an indirect effect on the viral inactivation or removal achieved. 
Manufacturers should explain and justify the approach used in studies to evaluate viral 
clearance. When appropriate, to determine the amount of virus particles that enter the 
purification process, quantification should be performed on three cell culture lots/batches. 
These data should be submitted as part of the marketing application. 
 
The reduction of virus infectivity can be achieved by removing virus particles or by 
inactivating viral infectivity. For each production step assessed, the possible mechanism of 
loss of viral infectivity should be described with regard to whether it results from inactivation 
  13  Contains Nonbinding Recommendations 
 
or removal. For inactivation steps, the study should be planned so that samples are taken at 
different times and an inactivation curve is constructed (see section VI.B.5 (6.2.5)). 
 
In addition to clearance studies for viruses known to be present, studies to characterize the 
ability to remove or inactivate other viruses should be conducted. The purpose of studies 
using viruses with diverse biochemical and biophysical properties is to characterize the 
robustness of the procedure to clear viruses that are not known or not expected to be present 
rather than to achieve a specific inactivation or removal goal. A demonstration of the capacity 
of the production process to inactivate or remove viruses is desirable (see section VI.C (6.3)). 
Such studies are not performed to evaluate a specific safety risk; therefore, achieving a 
specific clearance value is not needed. 
 
A.  Choice of Viruses for Evaluation and Characterization of Viral Clearance 
(6.1) 
 
Viruses for clearance evaluation and process characterization studies should be chosen to 
resemble viruses that may contaminate the product and to represent a wide range of 
physicochemical properties to test the ability of the system to eliminate viruses in general. 
The manufacturer should justify the choice of viruses according to the aims of the evaluation 
and characterization study provided in this guidance. 
 
1.  Relevant Viruses and Model Viruses (6.1.1) 
 
A major issue in performing a viral clearance study is to determine which viruses should be 
used. Such viruses fall into three categories: (1) relevant viruses, (2) specific model viruses, 
and (3) nonspecific model viruses. Relevant viruses are used in the viral clearance studies. 
Relevant viruses are the identified viruses or of the same species as the viruses that are 
identified or likely to contaminate the cell substrate or any other reagents or materials used in 
the production process. The downstream process should demonstrate the capability to remove 
and/or inactivate such viruses. When a relevant virus is not available or when it is not well 
adapted to the process evaluation of viral clearance studies (e.g., it cannot be grown in vitro 
to sufficiently high titers), a specific model virus should be used as a substitute. An 
appropriate specific model virus can be a virus that is closely related to the known or 
suspected virus (same genus or family), having similar physical and chemical properties to 
the observed or suspected virus. Cell lines derived from rodents usually contain endogenous 
retroviruses or RVLPs, which may be infectious (C-type particles) or noninfectious 
(cytoplasmic A- and R-type particles). The capacity of the manufacturing process to remove 
and/or inactivate rodent retroviruses from products obtained from such cells should be 
determined. This can be accomplished by using an MLV  a specific model virus in the case 
of cells of rodent origin. 
 
For CHO cell-derived products, CHO-derived endogenous virus particles can also be used for 
viral clearance experiments. There is no infectivity assay for these particles, and the detection 
assay (e.g., molecular, biochemical) should be qualified for its intended use. When human 
cell lines have been obtained by the immortalization of B lymphocytes by Epstein-Barr virus, 
the ability of the manufacturing process to remove and/or inactivate a herpesvirus should be 
determined. Pseudorabies virus can also be used as a specific model virus. 
 
When the purpose is to characterize the capacity of the manufacturing process to remove 
and/or inactivate viruses in general (i.e., to characterize the robustness of the clearance 
  14  Contains Nonbinding Recommendations 
 
process), viral clearance characterization studies should be performed with nonspecific model 
viruses with differing properties. Data obtained from studies with relevant and/or specific 
model viruses can also contribute to this assessment. It is not necessary to test all types of 
viruses. Preference should be given to viruses that display a significant resistance to physical 
and/or chemical treatments. The results obtained for such viruses provide useful information 
about the ability of the production process to remove and/or inactivate viruses in general. The 
choice and number of viruses used should be influenced by the quality and characterization of 
the cell lines and the production process. Generally, the process should be assessed for its 
ability to clear at least three different viruses with differing characteristics. 
 
Appendix 1 (Annex 1) and Table A-1 provide examples of useful model viruses representing 
a range of physicochemical structures and examples of viruses that have been used in viral 
clearance studies. 
 
2.  Other Considerations (6.1.2) 
 
Additional points to be considered include the following: 
 
  Viruses that can be grown to high titer are desirable, although this may not always be 
possible. 
 
  There should be an efficient and reliable assay for the detection of each virus used for each 
stage of manufacturing that is evaluated. 
 
  The health hazard that certain viruses may pose to the personnel performing the clearance 
studies should be considered. 
 
B.  Design and Implications of Viral Clearance Evaluation and 
Characterization Studies (6.2) 
 
1.  Facility and Staff (6.2.1) 
 
It is inappropriate to introduce any unintended virus into a production facility because of 
good manufacturing practice constraints. Therefore, viral clearance studies should be 
conducted in a separate laboratory equipped for virological work and performed by staff with 
virological expertise in collaboration with production personnel with experience in the 
specific process involved in designing and preparing a scale-down model of the purification 
process. 
 
2.  Scale-Down Model of the Production Process (6.2.2) 
 
The representativeness of the scale-down model should be demonstrated and should represent 
the production process as closely as possible. The scale-down model should also consider the 
robustness of viral clearance (i.e., ability of a process or process step to tolerate variability of 
materials and changes of the process without negative impact). In this context, it can be 
advisable to perform viral clearance studies under worst-case conditions for relevant 
parameters. It can also be acceptable that conditions may be outside of an operating range to 
demonstrate the robustness of viral clearance and to be prepared for potential manufacturing 
changes. The performance of the scale-down model should be demonstrated to represent the 
production process (e.g., comparable yield and purity). For chromatographic steps, for 
  15  Contains Nonbinding Recommendations 
 
example, column bed-height, linear flow rate, flow-rate-to-bed-volume ratio (i.e., contact 
time), buffer and resin types, pH, temperature, conductivity, and concentration of product 
should be shown to be representative of commercial-scale manufacturing. A similar 
chromatogram should result. For other procedures, similar considerations apply. Unavoidable 
deviations should be discussed with regard to their influence on the results. 
 
3.  Analysis of Stepwise Clearance of Virus (6.2.3) 
 
When viral clearance studies are performed, assessment of the contribution of more than one 
production step to virus elimination should be considered. Steps that are likely to clear virus 
should be individually assessed for their ability to remove or inactivate virus, and the exact 
definition of an individual step should be considered. Sufficient virus should be present in the 
material of each step to be tested so that an adequate assessment of the effectiveness of each 
step is obtained. Generally, virus should be added to process intermediate at each step to be 
tested. In some cases, adding high titer virus to an early process intermediate and testing its 
concentration between subsequent steps can be justified. When virus removal results from 
separation procedures, it is recommended that the distribution of the virus load in the 
different fractions be investigated, if appropriate and if possible. When buffers that inactivate 
viruses are used within the manufacturing process, alternative strategies such as parallel 
spiking in less virus-inactivating buffers (e.g., in pH adjusted buffer) can be carried out as 
part of the overall process assessment. Inactivation by the buffer itself might be tested in 
separate spiking experiments. Using quantitative assays not associated with infectivity may 
be applicable to determine partitioning of viral particles. The virus titer before and after each 
step being evaluated should be determined. Quantitative infectivity assays should have 
adequate sensitivity and reproducibility and should be performed with sufficient replicates to 
ensure adequate statistical validity of the result. Quantitative assays not associated with 
infectivity can be used if justified. Appropriate virus controls should be included in all 
infectivity assays to ensure the sensitivity of the method. Also, the statistics of sampling virus 
when at low concentrations should be considered (see Appendix B (Annex 2)). 
 
As a general principle, reproducible viral clearance should be confirmed in at least two 
independent experiments, unless a reduction of studies is justified by prior knowledge (see 
section VI.F (6.6)). Experiments can be performed at the same site, using the same 
experimental setting and with the same lot of in-process material although it is not expected 
that the two independent experiments are performed identically. 
 
4.  Determining Physical Removal Versus Inactivation (6.2.4) 
 
Reduction of infectious virus can be achieved by the removal or inactivation of virus. For 
each production step assessed, the possible mechanism of the loss of viral infectivity should 
be described as related to inactivation and/or removal. It may be necessary to distinguish 
between removal and inactivation for a particular step. As an example, when there is a 
possibility that a buffer used in more than one clearance step may contribute to inactivation 
during each step (i.e., the contribution to inactivation by a buffer shared by several 
chromatographic steps), the clearance achieved by each of these chromatographic steps 
should be distinguished. 
 
  16  Contains Nonbinding Recommendations 
 
5.  Inactivation Assessment (6.2.5) 
 
For the assessment of viral inactivation, process intermediate should be spiked with 
infectious virus, and the reduction factor calculated. It should be recognized that virus 
inactivation is not a simple first order reaction and is usually more complex with an initial 
fast phase and a subsequent slow phase. Therefore, the study should be planned in such a way 
that samples are taken at different times and an inactivation curve be constructed. It is 
recommended that studies for inactivation include at least one time point less than the 
minimum exposure time and greater than zero, in addition to the minimum exposure time. 
Additional data are particularly important if the virus is a relevant virus known to be a human 
pathogen, and an effective inactivation process is being designed. However, for inactivation 
studies in which nonspecific model viruses are used or when specific model viruses are used 
as surrogates for virus particles such as the CHO RVLPs, reproducible clearance should be 
demonstrated in typically two independent studies. Whenever possible, the initial virus load 
should be determined from the virus, which can be detected in the spiked starting material. If 
this is not possible, the initial virus load can be calculated from the titer of the spiking virus 
preparation. When inactivation is too rapid to plot an inactivation curve using process 
conditions, appropriate controls (e.g., samples with lower concentration of the inactivating 
agent) should be performed to demonstrate that infectivity is indeed lost by inactivation. 
 
6.  Function and Regeneration of Columns (6.2.6) 
 
Over time and after repeated use, the ability of chromatography resins and membranes used 
in the purification process to clear virus may vary. Chromatography media/resin lifetime use 
should be indicated, and parameters with potential impact on viral clearance should be 
defined. Viral clearance studies should be performed to support media/resin reuse. 
 
For Protein A affinity capture chromatography, prior knowledge indicates that virus removal 
is not affected or slightly increases for used (e.g., end of life) chromatography media/resin. 
Therefore product-specific studies with used resin are not expected. Prior knowledge might 
also apply to other chromatography types involved in viral clearance (e.g., anion exchange, 
cation exchange). Accordingly, in cases where product-specific studies with end-of-lifetime 
resin are not considered necessary to support repeated resin use for other chromatography 
types, equivalent prior knowledge including in-house experience and a detailed justification 
should be provided (see section VI.F (6.6)). 
 
Assurance should be provided so that any virus potentially retained by the production system 
would be adequately inactivated or removed before reusing the system. For example, 
evidence can be provided demonstrating that the cleaning and regeneration procedures 
inactivate or remove virus, for example, during viral clearance evaluation, and this can be 
supported by prior knowledge. 
 
7.  Specific Precautions (6.2.7) 
 
The following specific precautions should be considered: 
 
  Using highly pure and concentrated virus stocks for spiking can be helpful to 
minimize dilution of the product intermediate as well as to reduce introduction of 
nonrepresentative impurities that may impact process performance. However, care 
should be taken in preparing high titer virus to avoid aggregation, which may enhance 
  17  Contains Nonbinding Recommendations 
 
physical removal and decrease inactivation thus distorting the correlation with actual 
production. 
 
  Consideration should be given to the minimum quantity of virus that can be reliably 
assayed. 
 
  The study should include parallel control assays to assess the loss of infectivity of the 
virus because of such reasons as the dilution, concentration, filtration, or storage of 
samples before titration. 
 
  The virus spike should be added to the product in a small volume so as not to dilute or 
change the characteristics of the product. 
 
  Small differences in buffers, media, or reagents (for example) can affect viral clearance. 
 
  Virus inactivation is time-dependent; therefore, the amount of time a spiked product 
remains in a particular buffer solution or on a particular chromatography column 
should reflect the conditions of the commercial-scale process. 
 
  Buffers and product should be evaluated independently for toxicity and interference in 
assays used to determine the virus titer because these components may adversely 
affect the indicator cells. If the solutions are toxic to the indicator cells, dilution, 
adjustment of the pH or dialysis of the buffer containing spiked virus might be 
necessary. If the product itself has antiviral activity, the clearance study may need to 
be performed without the product in a mock run, or prior knowledge may be applicable 
as described in section VI.F (6.6). However, omitting the product or substituting a 
similar protein that does not have antiviral activity could affect the behavior of the virus 
in some production steps. Sufficient controls to demonstrate the effect of procedures 
used solely to prepare the sample for assay (e.g., dialysis, storage) on the 
removal/inactivation of the spiking virus should be included. 
 
  In case molecular biology assays are used for quantification of viral genomes, care 
should be taken to consider the effect from nonencapsulated viral nucleic acids, which 
may be nonrepresentative of intact viral particles removal in certain process steps. 
 
  Many purification processes use the same or similar buffers or columns in more than 
one purification step. When the overall virus reduction factor for a complete 
production process is based on the sum of the reduction factors of such purification 
processes, this approach should be justified; for example, the effectiveness of virus 
elimination by a particular process step may vary with the stage of manufacture at 
which it is used and the presence of accompanying proteins and other impurities 
clearly affecting the virus reduction capacity. 
 
  Overall reduction factors may be overestimated or underestimated when production 
conditions or buffers are too cytotoxic or virucidal and should be discussed on a case-
by-case basis. Overall reduction factors may also be overestimated because of the 
inherent limitations or inadequate design of viral clearance studies. 
 
  18  Contains Nonbinding Recommendations 
 
C.  Interpretation of Viral Clearance Studies (6.3) 
 
The objective of assessing virus inactivation/removal is to evaluate and characterize process 
steps considered effective in inactivating/removing viruses and to estimate quantitatively the 
overall level of virus reduction obtained by the manufacturing process. For viruses, as in 
Cases B through F, it is important to show that not only is the virus removed or inactivated, 
but that there is excess capacity for viral clearance built into the downstream process to 
ensure an appropriate level of viral safety for the final product. The amount of virus removed 
or inactivated by the manufacturing process should be compared to the amount of virus that 
may be entering the downstream process. 
 
To carry out this comparison, it is important to estimate the amount of virus in the 
unprocessed bulk. This estimate should be obtained using assays for infectivity or other 
methods such as TEM or a comparable quantitative NAT. The entire purification process 
should be able to eliminate substantially more virus than is estimated to be present in a single 
dose equivalent of unprocessed bulk. See Appendix C (Annex 3) for calculation of virus 
reduction factors and Appendix D (Annex 4) for calculation of estimated particles per dose. 
Manufacturers should recognize that clearance mechanisms may differ among virus classes. 
A combination of factors should be considered when judging the data supporting the 
effectiveness of virus inactivation/removal procedures. These include the following: 
 
  The appropriateness of the viruses evaluated 
 
  The design of the clearance studies 
 
  The virus reduction factor achieved 
 
  The time dependence of virus inactivation 
 
  The potential effects of variation in-process parameters on virus inactivation/removal 
 
  The assay sensitivity 
 
  The possible selectivity of inactivation/removal procedure(s) for certain classes of 
viruses 
 
It is recommended to design a downstream process that clears a wide range of potential 
viruses (potential adventitious virus contaminants, endogenous and/or production virus). In 
this context, whenever feasible and not adversely affecting the product, implementing two 
distinct viral clearance steps, one of which should provide effective clearance of 
nonenveloped virus, that complement each other in their mode of action is recommended. An 
effective viral clearance step generally gives reproducible reduction of virus load in the order 
of 4 log  or more shown by at least two independent studies. However, it is recognized that 
10
steps giving a reproducible reduction of 1 to 3 log  contribute toward viral safety and can be 
10
considered for assessment of overall virus reduction. Process steps dedicated to virus 
inactivation/removal such as solvent/detergent (SD) treatment, treatment with detergent 
alone, or incubation at low pH, have been very successful in clearing a wide range of 
enveloped viruses, and virus filtration removes viruses based on their sizes. Using virus filters 
designed for removal of small viruses is also an effective viral clearance step for the smaller 
viruses such as parvovirus or polyomaviruses. Finally, there is experience of efficient 
  19  Contains Nonbinding Recommendations 
 
inactivation of MLV and pseudorabies virus by incubation at low pH after the Protein A 
capture step for purification of mAbs. 
 
Acceptable overall clearance can be achieved by any of the following steps: multiple 
complementary inactivation steps, multiple complementary removal (separation) steps, or 
combinations of inactivation and removal steps. Virus removal methods may be dependent on 
the extremely specific physicochemical properties of viruses, which influence their 
interaction with stationary phases for chromatography (e.g., resins or chromatography 
membranes) and their precipitation properties. Therefore, model viruses can be separated in a 
different manner than a relevant virus. Manufacturing parameters influencing removal should 
be properly defined and controlled. Despite these potential variables, effective clearance can 
be obtained by a combination of complementary removal steps or combinations of inactivation 
and removal steps. Therefore, well-designed removal steps, such as chromatographic 
procedures, filtration steps, and extractions, can be effective virus removal steps if they are 
performed under appropriately controlled conditions. 
 
An overall reduction factor is generally expressed as the sum of the individual factors. 
However, reduction in virus load less than 1 log10 is considered negligible and should be 
ignored unless justified. 
 
If little reduction of infectivity is achieved by the production process, and the removal of 
virus is considered to be a major factor in the safety of the product, a specific, additional 
inactivation/removal step or steps should be introduced. For all viruses, manufacturers should 
justify the acceptability of the reduction factors obtained. The factors listed above will be 
considered in evaluating the results. 
 
D.  Limitations of Viral Clearance Studies (6.4) 
 
Viral clearance studies are useful for contributing to the assurance that an acceptable level of 
safety in the final product is achieved but do not by themselves establish safety. A number of 
factors in the design and execution of viral clearance studies may lead to an incorrect 
estimate of the ability of the process to remove virus infectivity. These factors include the 
following: 
 
  Virus preparations used in clearance studies for a production process are usually 
obtained from specific cell cultures. The behavior of such virus spike in a production 
step may be different from that of the native viral contaminant from a biological raw 
material in the cell culture medium or replicating in the manufacturing cells. For 
example, this might include if virus particles used for spiking and native virus from a 
respective production intermediate differ in purity or degree of aggregation. 
 
  Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid 
initial phase is followed by a slower phase. It is possible that virus escaping a first 
inactivation step may be more resistant to subsequent steps. For example, if the resistant 
fraction takes the form of virus aggregates, infectivity may be resistant to a range of 
different chemical treatments and to heating. 
 
  The ability of the overall process to remove or inactivate virus is expressed as the sum 
of the logarithm of the reductions at each step. The summation of the reduction factors 
from steps with nonsignificant reduction (e.g., below 1 log10), may overestimate the 
  20  Contains Nonbinding Recommendations 
 
true potential for virus elimination, and should not be performed. Addition of individual 
virus reduction factors resulting from similar inactivation mechanisms during the 
manufacturing process may also overestimate overall viral clearance. Furthermore, if 
reduction values achieved by repetition of identical or near identical procedures are 
included, they should be justified. 
 
  The expression of reduction factors as logarithmic reductions in titer implies that, 
although residual virus infectivity may be greatly reduced, it will never be reduced to 
zero. For example, a reduction in the infectivity of a preparation containing 8 log10 
infectious units per milliliter (mL) by a factor of 8 log10 leaves 0 log10 per mL or one 
infectious unit per mL, taking into consideration the limit of detection of the assay. 
 
  Processing during validation studies for viral clearance performed at small scale may 
differ from processing at commercial scale despite care taken to design the scale-
down process. 
 
E.  Statistics (6.5) 
 
The viral clearance studies should include the use of statistical analysis of the data to evaluate 
the results. The study results should be statistically valid to support the conclusions reached 
(refer to Appendix B (Annex 2)). 
 
F.  Application of Prior Knowledge for Evaluation of Viral Clearance (6.6) 
 
As a general principle, viral clearance is evaluated by experiments when the virus is added to 
the product-specific process intermediate of each step to be investigated. When a 
manufacturer is developing similar products by established and well-characterized processes 
(i.e., using the same platform technology), viral clearance data generated for other products 
might be applicable to the new product for the same processing step. However, to make use 
of data from such a step, the process step should be well understood. The representativeness 
of the prior knowledge for the specific process step should be clearly justified. The prior 
knowledge including external and in-house experience should cover the aspects outlined 
below: 
 
  There should be an understanding of the mechanism underlying viral clearance. 
 
  There should be comprehensive understanding of the process parameters that may 
affect viral clearance. 
 
  It should be clear that interactions between virus and product do not affect viral 
clearance. If there is a potential risk that the virus-product interaction may affect viral 
clearance, applying prior knowledge from manufacturing of other products should be 
justified. If data for more than one product is available for the specific step, the 
effectiveness of virus reduction should be comparable in each case. 
 
  The composition of a specific process intermediate may affect viral clearance. For 
some process steps, even small differences in buffers, media, reagents, and profile of 
impurities (for example) may affect viral clearance. Therefore, the representativeness 
of the composition of the process intermediate(s) from other products should be 
justified. Processing before the specific step for the new and the established product(s) 
  21  Contains Nonbinding Recommendations 
 
should follow a similar strategy unless prior knowledge indicates robustness of viral 
clearance with respect to composition of the process intermediate. 
 
  The general limitations of viral clearance studies as outlined in section VI.D (6.4) 
should be considered when applying prior knowledge to a specific product. 
 
External prior knowledge (including published data) can be supportive in indicating the 
potential of a step to inactivate/remove viruses and can provide insight to the mechanisms 
involved. Such data can also be used to define the process parameters critical for viral 
clearance and in setting worst-case limits for testing in a specific viral clearance step. 
Performing viral clearance studies at worst-case conditions can help reduce the number of 
product-specific experiments. However, the application of published reduction factors to a 
specific product should be supported by demonstration of similarity of the processes across 
manufacture of different products involved, similarity of the product intermediates, and an 
assurance that product-specific attributes do not affect virus reduction. Therefore, published 
data should be carefully assessed and supplemented with in-house experience (internal prior 
knowledge) for a given platform. 
 
The decision on the acceptability of viral clearance data without product-specific experiments 
is made on a case-by-case basis while considering the whole viral safety strategy for a 
medicinal product, including the nature and characterization of the cell substrate and raw 
materials, and the overall viral clearance strategy. If the data package does not sufficiently 
support the use of prior knowledge, product-specific viral clearance studies should be 
performed. 
 
When deriving a reduction factor claim using prior knowledge, the claim should be justified 
considering all reduction factors from the relevant platform data. A conservative reduction 
factor claim is advised to avoid the risk of overestimating the reduction capacity of the 
process step. 
 
Appendix E (Annex 5) provides cases when, according to current understanding, prior 
knowledge including in-house experience with viral reduction data from other products could 
be used to claim a reduction factor for a new product from the same manufacturing platform. 
 
G.  Reevaluation of Viral Clearance (6.7) 
 
Whenever significant changes in the upstream or downstream manufacturing processes are 
made, the effect of that change, both direct and indirect, on viral clearance should be 
considered and the system reevaluated, as needed. For example, changes in upstream 
manufacturing processes may cause significant changes in the amount of virus produced by 
the cell line; changes in process steps may change the extent of viral clearance. 
 
Changes in the manufacturing process during life-cycle management that may affect viral 
clearance efficacy could be evaluated using internal knowledge and the platform concept. If 
the internal knowledge (in-house experience) with other products cannot be extrapolated to 
specific products and/or the platform concept can no longer be applied, product-specific viral 
clearance studies should be performed. 
 
 
  22  Contains Nonbinding Recommendations 
 
VII.  POINTS TO CONSIDER FOR CM (7) 
 
CM can involve the continuous feeding of input materials into a manufacturing process 
comprised of a series of linked unit operations that transform the feed and provide a 
continuous stream of an output material (i.e., product). CM can be applied to some or all unit 
operations of a manufacturing process. An understanding of the integrated process and its 
dynamics, in addition to each unit operation, is essential to identify and mitigate the risk to 
viral safety. A description of the modes of CM processes for the manufacture of therapeutic 
proteins can be found in the ICH guidance for industry Q13 Continuous Manufacturing of 
Drug Substances and Drug Products (March 2023), (at Annex III). 
 
In terms of viral safety, technical aspects for CM may differ from those encountered in batch 
processes, including approaches of detection and removal of viruses, material traceability, 
process dynamics, and monitoring frequency start-up/shutdown. 
 
However, basic principles and expectations (such as science- and risk-based approaches and 
their implementation to control virus contamination risk) that are based on process 
understanding are the same as for batch manufacturing. This also includes contamination 
prevention strategies (see section II.B (2.2)). For example, the physical and chemical 
conditions to inactivate or remove viruses derived from experience or prior knowledge of 
batch production may also be applicable to CM processes (see section VI.F (6.6)). 
 
A.  Viral Safety in CM (7.1) 
 
The viral safety strategy in CM processes should be based on a risk assessment of potential 
sources of contamination (e.g., the starting and raw materials and extended cell culture 
duration), the ability of the process to remove viruses, and the testing capability to assure 
absence of viruses. Guidance on testing provided in sections III (3) and IV (4) is also 
considered applicable to CM. Based on this assessment, a strategy should be developed to 
include the type and frequency of adventitious virus testing undertaken to demonstrate that 
the23rocesss is free of contamination during manufacturing. 
 
2.  General Considerations for Viral Clearance in CM (7.2) 
 
To design the manufacturing process and the viral clearance study, the following should be 
considered: 
 
  The manufacturing process can be partially run in an integrated (connected) or 
continuous mode of operation, and it is possible to use the scientific understanding of 
viral clearance performance based on batch processes for the evaluation of unit 
operations if suitable (see section VII.C.2 (7.3.2)). 
 
  The potential risk of each unit operation and the connection between equipment (e.g., 
use of a surge or mixing tank between unit operations to mitigate differences in mass 
flow rate or nonhomogenous input materials) should be assessed to address any effects 
on the virus reduction capabilities. 
 
 
  23  Contains Nonbinding Recommendations 
 
  There should be appropriate process monitoring in place to detect (1) process 
disturbance (e.g., pressure/flow interruption that impacts viral clearance) and (2) 
adventitious virus contamination. The final impact of process disturbance may differ 
based on the type of event and should be addressed on a case-by-case basis. 
 
 
  If conducting real-time decision-making, this should include a procedure to determine 
the effects of (1) the process disturbance on viral clearance or (2) the contamination 
on the output material quality and product. Based on these considerations, the diversion 
of the potential nonconforming material from the product stream or the disposition of 
the material produced should be considered (see section IV (4)). 
 
 
  The viral clearance study designs should consider potential effects of the following, if 
applicable: 
 
 
 Variability  of  input  material  attributes  (e.g.,  concentration  and 
homogeneity of protein or impurities, level of aggregation) 
 
 Residence time 
 
 
 Planned  events  (e.g.,  process  start-up,  shutdown,  and  pause)  and unplanned 
transient events (e.g., disturbances) 
 
 
 Operational loading capacity 
 
 
 Alternative loading strategies (e.g., multicolumn cycling, serial loading) 
 
 
 Virus control strategy 
 
 
2.  Unique Considerations for Viral Clearance in CM (7.3) 
 
CM also presents unique aspects to consider for viral safety as described below. 
 
2.  Potential Risk Related to Longer Periods in Production Culture (7.3.1) 
 
Fluctuations in the levels of endogenous virus may occur over time in the production culture, 
so an assessment should be made of the appropriate sampling strategy so as not to affect the 
dose risk factor calculation for the drug product (see section IV (4) and considerations in 
section III (3) for cell line qualification). 
  24  Contains Nonbinding Recommendations 
 
 
2.  Approach to Viral Clearance Study (7.3.2) 
 
Although CM is expected to maintain a state of control, the manufacturing process can 
include periods when the process output can vary during start-up, termination, and process 
disturbance (e.g., potentially high virus load for a short period of time in case of a virus 
contamination). The risks for such periods are addressed elsewhere in this guidance (see 
section VI.B (6.2)). 
 
Furthermore, simultaneous validation of two or more connected unit operations could be an 
option according to the equipment design and system integration. 
 
Considerations specific to CM include the following: 
 
  Chromatography 
 
 In the case of repeating cycles (e.g., multicolumn processing), a batch process 
could serve as a scale-down model with well-justified target process conditions 
(e.g., flow rate, resin load versus column overload, resin cleanability); see also 
guidance for scaling down in section VI.B.2 (6.2.2). 
 
 Validation of two or more connected chromatography steps can be considered 
(e.g., bind and elute mode of cation exchange chromatography and flow through 
mode of anion exchange chromatography). For connected unit operations, it is an 
option to evaluate viral clearance using a conventional batch scale-down model if 
the operating conditions during manufacturing are adequately reflected, with 
respect to the load material. 
 
  Viral inactivation 
 
 Validation as a batch process could be appropriate with well-justified target 
process conditions. 
 
 For virus inactivation (e.g., pH, SD), the control of relevant dynamic process 
parameters should be ensured (e.g., pH, SD concentration, homogeneity and 
mixing, temperature, residence time distribution). 
 
 Care should be taken in evaluating/justifying the effects of scale on the process 
dynamics (e.g., residence time distribution) and control strategy when a scale-
down model is applied for inactivation in a dynamic process. 
 
  Virus filtration 
 
 Validation as a batch process could be appropriate if settings of parameters, which 
affect viral clearance, do not vary beyond ranges tested in the viral clearance study 
(e.g., worst-case set point). 
 
 Process controls should be defined to allow for filter changes and post use 
integrity testing while maintaining viral clearance capacity. 
  25  Contains Nonbinding Recommendations 
 
 
 
VIII.  SUMMARY (8) 
 
This guidance recommends approaches for evaluating the risk of viral contamination of the 
product, controlling potential sources of virus, and qualifying the manufacturing process for 
viral clearance, thus contributing to the production of safe biotechnology products derived 
from animal or human cell lines. These control and clearance approaches include the 
following: 
 
  Thorough characterization/testing of cell substrate starting material to identify which, if 
any, virus contaminants are present 
 
  Assessment of potential risk from virus contaminants by determination of the human cell 
tropism or knowledge of human infections 
 
  Establishment of an appropriate program of testing for adventitious viruses in 
unprocessed bulk 
 
  Establishment of a program for appropriate viral clearance 
 
  Careful design and performance of viral clearance studies for different methods of 
virus inactivation or removal in the same production process. 
  26  Contains Nonbinding Recommendations 
GLOSSARY (9) 
 
Adventitious virus 
 
Unintentionally introduced contaminant viruses. See virus. 
 
Cell substrate 
 
Cells used to manufacture product. 
 
Control cells 
 
Cells cultured in parallel with the production of the virus or viral vector without inoculation 
of the virus/viral vector seed. Control cells are maintained in conditions that are essentially 
equivalent to those used for the production cell cultures, including use of the same batches of 
media and media changes. 
 
End of production cells (EOPCs) 
 
Cells harvested (under conditions comparable to those used in production) from the master 
cell bank (MCB) or working cell bank (WCB) cultured to a passage level or population 
doubling level comparable to or beyond the highest level reached in production. In certain 
situations, the chronological time in culture can be measured. The EOPCs are also referred to 
as extended cell banks (ECB), and these terms can be used interchangeably with limit of in 
vitro cell age (LIVCA) cells. 
 
Endogenous virus 
 
Retrovirus whose genome is part of the germ line of the species of origin of the cell line and 
is stably integrated into the genome of the host species from which the parental cell line was 
derived. In this guidance, this also includes intentionally introduced, nonintegrated viruses 
such as Epstein-Barr virus used to immortalize cell substrates or bovine papilloma virus. 
 
Extended cell bank (ECB) 
 
Cells cultured from the MCB or WCB and propagated to the proposed in vitro cell age used 
for production or beyond. May also be referred to as EOPC. 
 
Inactivation 
 
Reduction of virus infectivity caused by chemical or physical treatment. 
 
In vitro cell age 
 
A measure of the period between thawing the MCB vial(s) and harvesting the production 
vessel that is measured by elapsed chronological time in culture, population doubling level of 
the cells, or passage level of the cells when subcultivated by a defined procedure for dilution 
of the culture. 
 
27  Contains Nonbinding Recommendations 
 
Limit of in vitro cell age (LIVCA) cells 
 
LIVCA cells are derived from production cells at or beyond in vitro cell age by expansion of 
the MCB or WCB. LIVCA cells may be also referred to as EOPC or ECB, and these terms 
can be used interchangeably. 
 
Master cell bank (MCB) 
 
An aliquot of a single pool of cells, which generally has been prepared from the cell substrate 
or selected cell clone under defined conditions, dispensed into multiple containers and stored 
under defined conditions. 
 
Master virus seed (MVS) 
 
An MVS (stock, lot, or bank) is a preparation of a vector virus, a viral vector or production 
virus (i.e., helper virus or viral protein expression vector), from which all future production 
will be derived, either directly or via a working virus seed (WVS). It is 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. 
 
Minimum exposure time 
 
The shortest period for which a treatment step will be maintained. 
 
Next generation sequencing (NGS) 
 
Also referred to as high-throughput sequencing or massive parallel sequencing or deep 
sequencing, multistep nucleic acid-based technology with broad capabilities for nontargeted 
(agnostic) detection of known and unknown adventitious viruses. In some cases, NGS can be 
used for targeted detection of known viruses by the sequencing strategy or by bioinformatic 
analysis. 
 
Platform manufacturing (see the International Council for Harmonisation guidance for 
industry Q11 Development and Manufacture of Drug Substances (November 2012))1 
 
The approach of developing a production strategy for a new drug starting from manufacturing 
processes similar to those used by the same applicant to manufacture other drugs of the same 
type (e.g., as in the production of monoclonal antibodies (mAbs) using predefined host cell, 
cell culture, and purification processes for which considerable experience already exists). 
 
 
1 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the 
FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.  
  28  Contains Nonbinding Recommendations 
 
Platform validation2 
 
Throughout this guidance, this term exclusively refers to validation of the process platform 
regarding viral clearance. In this context, platform validation is defined as the use of prior 
knowledge including in-house experience with viral reduction data from other products, to 
claim a reduction factor for a new similar product, according to current understanding. 
 
Prior knowledge 
 
Prior knowledge refers to existing knowledge and includes internal knowledge (e.g., 
development and manufacturing experience), external knowledge (e.g., scientific and 
technical publications, including vendors data, literature, and peer-reviewed publications), or 
the application of established scientific principles (e.g., chemistry, physics, and engineering 
principles). 
 
Process characterization of viral clearance 
 
Viral clearance studies in which nonspecific model viruses are used to assess the robustness 
of the manufacturing process to remove and/or inactivate viruses. 
 
Process evaluation studies of viral clearance 
 
Viral clearance studies in which relevant and/or specific model viruses are used to determine 
the ability of the manufacturing process to remove and/or inactivate these viruses. 
 
Process robustness of viral clearance 
 
The term robustness is used to describe one or both of the different characteristics. One 
characteristic is the ability of a process or process step to tolerate variability of materials and 
changes of the process without negative effect on clearing a virus. The other characteristic is 
the ability to clear a wide range of specific and nonspecific model viruses. 
 
Production cells 
 
Cell substrate used to manufacture product. 
 
Production virus  
 
A production virus is a process-related virus and may include a helper virus or a viral vector 
for protein expression. 
 
 
2 The Food and Drug Administration (FDA) acknowledges that the terms platform and platform technology have 
been used by both industry and FDA to describe technologies in ways differing from the term designated 
platform technology described in section 506K of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 356k). 
Use of the terms platform or platform technology within this guidance does not constitute a determination that 
the technology would meet that statutory definition and eligibility criteria for purposes of the platform 
technology designation program.   
  29  Contains Nonbinding Recommendations 
 
Helper virus 
 
A virus that provides helper functions allowing an otherwise replication-deficient 
coinfecting virus to replicate. 
 
Viral vector for protein expression 
 
A recombinant virus, such as baculovirus, that can be used to express a recombinant 
protein or a virus-like particle or to produce a viral vector. 
 
Purified bulk 
 
The term purified bulk refers to material at the end of the purification process. While in most 
cases this represents drug substance, the purified material without inactive ingredients can be 
used instead to avoid interference with testing assays. 
 
Supplementary test method 
 
A test method used to provide additional data to support the conventional testing. 
 
Unprocessed bulk 
 
One or multiple pooled harvests of cells and culture media. When cells are not readily 
accessible, the unprocessed bulk would constitute fluid harvested from the bioreactor. 
 
Viral clearance 
 
Removal of viral particles or inactivation of viral infectivity. 
 
Viral vector 
 
A recombinant virus that may be applied in vivo as a medicinal product or applied ex vivo for 
other advanced therapeutic applications. 
 
Viral vector-derived product 
 
A product encoded and expressed by a viral vector, where the recombinant virus is referred to 
as a viral vector for production, such as a baculovirus. 
 
Virus 
 
Intracellularly replicating infectious agents that are potentially pathogenic, that possess only a 
single type of nucleic acid (either RNA or DNA), that are unable to grow and undergo binary 
fission, and that multiply in the form of their genetic material. 
 
Nonspecific model virus 
 
A virus used for characterization of viral clearance of the process when the purpose is 
to characterize the capacity of the manufacturing process to remove and/or inactivate 
viruses in general (i.e., to characterize the robustness of the downstream process). 
  30  Contains Nonbinding Recommendations 
 
 
Relevant virus 
 
Virus used in the process evaluation studies that is either the identified virus, or of the 
same species as the virus that is known, or likely to contaminate the cell substrate or 
any other reagents or materials used in the production process. 
 
Replication competent virus (RCV) 
 
Recombination of the viral vector with transcomplementing viral sequences generating 
an RCV. 
 
Specific model virus 
 
Virus, which is closely related to the known or suspected virus (same genus or 
family), having similar physical and chemical properties to those of the observed or 
suspected virus. 
 
Virus-like particles 
 
Structures that morphologically appear to be related to known viruses. May or may not contain 
the viral genome. 
 
Working cell bank (WCB) 
 
The WCB is prepared from aliquots of a homogeneous suspension of cells obtained from 
culturing the MCB under defined culture conditions. 
 
Working virus seed (WVS) 
 
A WVS (stock, lot, or bank) is produced from the MVS. 
  31  Contains Nonbinding Recommendations 
REFERENCES (10) 
 
International Council for Harmonisation draft guidances for industry Q2(R2) Validation of 
Analytical Procedures (August 2022) and Q14 Analytical Procedure Development (August 
2022)1 
 
International Council for Harmonisation guidance for industry Q11 Development and 
Manufacture of Drug Substances (November 2012) 
 
International Council for Harmonisation guidance for industry Q13 Continuous 
Manufacturing of Drug Substances and Drug Products (March 2023) 
 
 
1 When final, these guidances will represent the FDAs current thinking on these topics. For the most recent 
version of a guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-
fda-guidance-documents.  
32  Contains Nonbinding Recommendations 
TABLE 1. VIRUS TESTS RECOMMENDED FOR CHARACTERIZATION OF CELL 
SUBSTRATES 
 
 
  MCB  WCB  Cells at the LIVCA 
Tests for Retroviruses and Other Endogenous Viruses 
Retrovirus testsa  +  -  + 
Other endogenous virus testsb  as appropriateb  -  as appropriateb 
     
Tests for Adventitious Viruses 
In vitro assays or NGSe  +  +c  +c 
In vivo assays or NGSe  +d  -  +d 
Tests for specific virusesf  as appropriatef  -  - 
 
MCB = master cell bank; WCB = working cell bank; LIVCA = limit of in vitro cell age; NGS = next generation 
sequencing. 
a.  Refer to section III.B.1 (3.2.1) for additional details. 
b.  As appropriate for cell lines that are known to contain such agents. 
c.  The in vitro virus assay is performed directly on the WCB or on LIVCA cells directly derived from this WCB. 
d.  The in vivo assay can be performed based on risk assessment. If residual risk remains, retention of the test or 
replacement with nontargeted NGS can be considered to detect viruses that may have been introduced during 
establishment of the MCB or during culture of the cells at the LIVCA stage. Refer to section III.B.3 (3.2.3) for 
details. 
e.  Nontargeted NGS can replace the in vivo assay (see section III.B.3 (3.2.3)) and supplement or replace the in 
vitro assay (see section III.B.2 (3.2.2)). 
f.  Testing is based on risk assessment including the origin and history of the cell substrate, and potential exposure 
to human- or animal-derived raw materials. Methods such as cell culture-based infectivity assays, antibody 
production tests (MAP, HAP, RAP), virus-specific NAT or other molecular methods (e.g., NGS) can be used. 
Refer to section III.B.4 (3.2.4) for details. This can include testing for species-specific viruses, for example, 
arboviruses in insect cells, and bovine or porcine viruses if serum or trypsin are used, respectively. Refer to Table 
4 (Cases B, C, and E) for action steps to be taken for virus detection in cell substrates used for production. 
33  Contains Nonbinding Recommendations 
TABLE 2. EXAMPLES OF THE USE AND LIMITATIONS OF ASSAYS THAT CAN 
BE USED TO TEST FOR VIRUSES 
 
Test  Test Article  Detection Capability  Assay Limitation 
Antibody Production  Lysate of cells and their  Specific viral antigens  Viruses failing to replicate and/or 
Test  culture medium  produce antibodies in animals. 
Not all virus infections result in a 
measurable antibody response. 
In vivo virus assay  Lysate of cells and their  Limited range of viruses  Viruses failing to replicate or 
culture medium  produce diseases in the test 
system. Assay interference. 
In vitro virus assay    Broad range of viruses  Viruses failing to replicate or 
  produce signs of infection in the 
test system. Assay interference. 
1. Cell bank testing   Lysate of cells and their     
culture medium (for 
cocultivation, intact cells 
should be in the test article) 
2. Production testing  Unprocessed bulk harvest 
or lysate of cells and their 
cell culture medium from 
the production reactor 
       
TEM:  Low sensitivity. Does not indicate 
whether virus is infectious. 
  Cell bank testing    Viable cells  Virus and virus-like 
Qualitative assessment of viral 
    particles including 
particles. 
  Production testing    Cell-free material  endogenous retroviruses 
      Quantitative assay (for viral 
clearance assessment). 
Reverse transcriptase  Cell-free culture  Retroviral particles and RT  May need to distinguish 
(RT) assay (e.g.,  supernatant  activity  retrovirus RT from cellular 
PERT assay)  polymerases. 
  Cell bank testing       
  Production testing 
       
Retrovirus (RV)  Cell-free culture  Infectious retroviruses  RV failing to replicate or form 
infectivity  supernatant  discrete foci or plaques in the 
chosen test system. 
Cocultivation  Viable cells  Infectious retroviruses  RV failing to replicate 
  Infectivity endpoint    See above under RV infectivity. 
2.  TEM endpoint      2.  See above under TEMa 
3.  RT endpoint      3.  See above under RT 
34  Contains Nonbinding Recommendations 
 
Test  Test Article  Detection Capability  Assay Limitation 
      PCR  ls, culture fluid, and other  Specific viral sequences  Primer sequences are present. 
  Cell bank testing  materials  Does not indicate whether virus is 
  Production testing  infectious. 
ls, culture fluid, and other  Broad range of viral sequences Positive result does not indicate 
NGS 
materials  whether virus is infectious and 
should be investigated. 
   
   
a. In addition, may be difficult to distinguish test article from indicator cells. 
TEM = transmission electron microscopy; PERT = product-enhanced reverse transcriptase; NGS = next 
generation sequencing. 
  35  Contains Nonbinding Recommendations 
TABLE 3. VIRUSES DETECTED IN ANTIBODY PRODUCTION TESTSD 
 
MAP  HAP  RAP 
Ectromelia Virusb,c  Lymphocytic Choriomeningitis Virus  Hantaan Virusa,c 
(LCM)a,c 
Hantaan Virusa,c  Pneumonia Virus of Mice (PVM)b,c  Kilham Rat Virus (KRV)b,c 
K Virusb  Reovirus Type 3 (Reo3)a,c  Mouse Encephalomyelitis Virus 
(Theilers, GDVII)b 
Lactic Dehydrogenase Virus  Sendai Virus (SV)a,c SV5a,c  Pneumonia Virus of Mice (PVM)b,c 
(LDM)a,c 
Rat Coronavirusb 
Lymphocytic Choriomeningitis 
 
Virus (LCM)a,c 
Reovirus Type 3 (Reo3)a,c Sendai 
Minute Virus of Miceb,c Mouse 
Virusa,c 
Adenovirus (MAV)b,c 
Sialodacryoadenitis Virus (SDAV)b 
Mouse Cytomegalovirus 
Toolans H-1 Virusb,c 
(MCMV)b,c 
   
Mouse Encephalomyelitis Virus 
(Theilers, GDVII)b 
   
Mouse Hepatitis Virus (MHV)b 
Mouse Rotavirus (EDIM)b,c 
   
Pneumonia Virus of Mice (PVM)b,c 
   
Polyoma Virusb 
   
Reovirus Type 3 (Reo3)a,c Sendai 
Virusa,c 
   
Thymic Virus b,e 
 
MAP = mouse antibody production; HAP = hamster antibody production; RAP = rat antibody production. 
a.  Viruses for which there is evidence of capacity for infecting humans or primates. 
b.  Viruses for which there is no evidence of capacity for infecting humans. 
c.  Virus capable of replicating in vitro in cells of human or primate origin. 
d.  NAT such as PCR assays or targeted or non-targeted NGS or other molecular methods can be used instead. 
e.  Also called murid herpesvirus 3. 
36  Contains Nonbinding Recommendations 
TABLE 4.  RECOMMENDED ACTION PLAN IN RESPONSE TO THE RESULTS 
OF VIRUS TESTS ON CELLS OR UNPROCESSED BULK 
 
  Case A  Case B  Case Cb  Case Db  Case Eb  Case F 
           
STATUS 
Presence of adventitious virusa  -  -  +  +  +c  - 
Virus-like particlesa  -  -  -  -  +c  - 
Retrovirus-like particlesa  -  +  -  -  +c  - 
Virus identified  not  +  +  +  -  + 
applicable 
Virus infectious for humans  not  -d  -d  +  unknown  +i 
applicable 
Presence of production virus  -  -  -  -  -  + 
           
ACTION 
Process characterization of  yese  yese  yese  yese  yese  yese 
viral clearance using 
nonspecific model viruses 
Process evaluation of viral  no  yesf  yesf  yesf  yesg  yesi 
clearance using relevant or 
specific model viruses 
Test for virus in purified bulk  not  noj  yesh  yesh  yesh  yesi 
applicable 
 
a.  Results of virus tests for the cell substrate and/or at the unprocessed bulk level. Cell cultures used for production 
that are contaminated with viruses generally should not be used unless justified by specific viral clearance and 
risk assessment. Endogenous viruses (such as retroviruses) or viruses that are an integral part of the MCB can be 
acceptable if appropriate viral clearance evaluation procedures are followed. 
b.  Source material that is contaminated with viruses, where they are known to be infectious and/or pathogenic in 
humans, should only be used under exceptional circumstances by demonstration of specific viral clearance and 
risk assessment. 
c.  Virus has been observed by either direct or indirect methods. 
d.  Believed to be nonpathogenic. 
e.  Characterization of clearance using nonspecific model viruses should be performed. 
f.  Process evaluation for relevant viruses or specific model viruses should be performed. 
g.  See text under Case E. 
h.  The absence of detectable virus should be confirmed for purified bulk by means of suitable methods having high 
specificity and sensitivity for the detection of the virus in question. For marketing authorization, data from at 
least three lots/batches of purified bulk manufactured at pilot plant scale or commercial scale should be 
provided. 
i.  Virus may or may not be infectious for humans. Process evaluation for the production virus should be performed. 
If this is not possible, then a specific model virus should be used. When utilized in production, the production 
virus is quantified in the unprocessed bulk stage using at least three cell culture lots/batches to determine the 
target for viral clearance. In purified bulk, absence of detectable production virus is determined using an 
infectivity assay with relevant permissive cell lines for sensitive virus detection. Alternatively, molecular 
37  Contains Nonbinding Recommendations 
 
methods can be used. Testing for the absence of residual production virus should be performed for each purified 
bulk, unless justified by robust clearance (see section VI.C (6.3)). 
j.  Please refer to section V (5), description of Case B.  
  38  Contains Nonbinding Recommendations 
APPENDIX A: THE CHOICE OF VIRUSES FOR VIRAL CLEARANCE STUDIES 
(ANNEX 1) 
 
A.  Examples of Useful Model Viruses (Annex 1.1) 
 
1.  Nonspecific model viruses representing a range of physicochemical structures: 
 
  A  polyomavirus  (e.g.,  SV40),  an  animal  parvovirus,  or  some  other  small  and 
physicochemically resistant, nonenveloped virus. 
 
  A parainfluenza virus or influenza virus, Sindbis virus, or some other medium-to- 
large, enveloped, RNA virus. Alternatively, another nonenveloped virus such as 
reovirus,  SV40,  or  a  picornavirus  can  be  used  to  extend  the  spectrum  of 
nonenveloped virus. 
 
  A herpesvirus (e.g., herpes simplex virus-1, a pseudorabies virus), or some other 
medium-to-large DNA virus. 
 
These viruses are examples only, and their use is not mandatory. 
 
2.  For cell substrates producing retroviral-like particles, murine retroviruses are commonly 
used as specific model viruses. It may be also possible to use endogenous murine or 
other rodent retrovirus particles. 
 
B.  Examples of Viruses That Have Been Used in Viral Clearance Studies 
(Annex 1.2) 
 
Several viruses that have been used in viral clearance studies are listed in Table A-1. 
However, as these are merely examples, the use of any of the viruses in the table is not 
mandatory, and manufacturers are invited to consider other viruses, especially those which 
may be more appropriate for their individual production processes. Generally, the process 
should be assessed for its ability to clear three or more different viruses with differing 
characteristics. 
39  Contains Nonbinding Recommendations 
 
 
Table A-1: Examples of Viruses That Have Been Used in Viral Clearance Studies 
 
Virus  Family  Genus  Natural host Genome Env  Size   Shape  Resistance a 
(nanometer) 
Vesicular Stomatitis  Rhabdo  Vesiculovirus  Equine  RNA  yes  70x150  Bullet  Low 
Virus (VSV)b  Bovine 
Parainfluenza Virus  Paramyxo  Respirovirus or  Various  RNA  yes  100-200+  Pleo/Sphere  Low 
Orthorubulavirus 
Murine Leukemia  Retro  Gammaretrovirus  Mouse  RNA  yes  80-110  Spherical  Low 
Virus (MLV) 
Sindbis Virus  Toga  Alphavirus  Human  RNA  yes  60-70  Spherical  Low 
Bovine Viral Diarrhea Pesti  Pestivirus  Bovine  RNA  yes  50-70  Pleo/Sphere  Low 
Virus (BVDV) 
Pseudorabies Virusc  Herpes  Varicellovirus  Swine  DNA  yes  120-200  Spherical  Med 
                 
                 
Autographa  Baculo  Alphabaculovirus  Insect  DNA  yes  250-300  Polyhedral  Med 
Californica Multiple 
Nucleopolyhedrovirusc 
                 
Adenovirus Type 2 or Adeno  Mastadenovirus  Human  DNA  no  70-90  Icosahedral  Med 
Type 5c 
Vesivirus 2117  Calici  Vesivirus  Unknown  RNA  no  27-40  Icosahedral  Med 
Encephalomyocarditis Picorna  Cardiovirus  Mouse  RNA  no  25-30  Icosahedral  Med 
Virus (EMCV)                 
               
Bovine Enterovirus 
Picorna  Enterovirus  Bovine  RNA  no  25-30  Icosahedral  Med 
(BEV) 
                 
Reovirus 3  Reo  Orthoreovirus  Various  RNA  no  60-80  Spherical  Med 
SV40  Polyoma  Betapolyomavirus  Monkey  DNA  no  40-50  Icosahedral  Very high 
Parvoviruses (canine, Parvo  Protoparvovirus  Canine  DNA  no  18-24  Icosahedral  Very high 
murine, porcine)d  Mouse 
Porcine 
a.  Resistance to physicochemical treatments based on studies of production processes. Resistance is relative to the 
specific treatment, and it is used in the context of the understanding of the biology of the virus and the nature of 
the manufacturing process. Actual results will vary according to the treatment. 
b.  Specific model virus for rhabdovirus found in insect cells. 
c.  Specific model virus or relevant virus for helper virus or viral protein expression vector used for viral vector 
production. 
d.  Can be used as single worst-case model virus for larger spherical/icosahedral viruses and enveloped viruses for 
validation of virus filters. 
These viruses are examples only, and their use is not mandatory. 
  40  Contains Nonbinding Recommendations 
APPENDIX B: STATISTICAL CONSIDERATIONS FOR ASSESSING VIRUS AND 
VIRUS REDUCTION FACTORS (ANNEX 2) 
 
Virus titrations suffer the problems of variation common to all biological assay systems. 
Assessment of the accuracy of the virus titrations and reduction factors derived from them, 
and the validity of the assays should be performed to define the reliability of a study. The 
objective of statistical evaluation is to establish that the study has been carried out to an 
acceptable level of virological competence. 
 
1.  Assay methods can be either quantal or quantitative. Quantal methods include infectivity 
assays in animals or in tissue-culture-infectious-dose assays, in which the animal or cell 
culture is scored as either infected or not. Infectivity titers are then measured by the 
proportion of animals or culture infected. In quantitative methods, the infectivity 
measured varies continuously with the virus input. Quantitative methods include 
molecular-based methods or plaque assays in which each plaque counted corresponds to 
a single infectious unit. Both quantal and quantitative assays are amenable to statistical 
evaluation. 
 
2.  Variation can arise within an assay as a result of dilution errors, statistical effects, and 
differences within the assay system that are either unknown or difficult to control. 
These effects are likely to be greater when different assay runs are compared 
(between-assay variation) than when results within a single assay run are compared 
(within-assay variation). 
 
3.  The 95 percent confidence limits for results of within-assay variation normally should be 
on the order of +0.5 log  of the mean. Within-assay variation can be assessed by 
10
standard textbook methods. Between-assay variation can be monitored by the 
inclusion of a reference preparation, the estimate of whose potency should be within 
approximately 0.5 log  of the mean estimate established in the laboratory for the 
10
assay to be acceptable. Assays with lower precision can be acceptable with 
appropriate justification. 
 
4.  The 95 percent confidence limits for the reduction factor observed should be calculated 
wherever possible in studies of clearance of relevant and specific model viruses. If the 
95 percent confidence limits for the viral assays of the starting material are +s, and for the 
viral assays of the material after the step are +a, the 95 percent confidence limits for the 
reduction factor are: 
 
                            
 
Probability of Detection of Viruses at Low Concentrations 
 
At low virus concentrations (e.g., in the range of 10 to 1,000 infectious particles per liter (L)), it 
is evident that a sample of a few milliliters (mL) may or may not contain infectious particles. 
The probability, p, that this sample does not contain infectious viruses is: 
 
p = ((V-v)/V)n 
 
When V (L) is the overall volume of the material to be tested, v (L) is the volume of the 
sample and n is the absolute number of infectious particles statistically distributed in V. 
41  Contains Nonbinding Recommendations 
 
 
If V >> v, this equation can be approximated by the Poisson distribution: 
 
p = e-cv 
 
When c is the concentration of infectious particles per liter. 
 
Or, c = ln p /-v 
 
As an example, if a sample volume of 1 mL is tested, the probabilities p at virus 
concentrations ranging from 10 to 1,000 infectious particles per liter are: 
 
c  10  100  1000 
p  0.99  0.90  0.37 
 
This indicates that for a concentration of 1,000 viruses per liter, in 37 percent of sampling, 1 
mL will not contain a virus particle. 
 
If only a portion of a sample is tested for virus, and the test is negative, the amount of virus 
that would be present in the total sample to achieve a positive result should be calculated and 
this value taken into account when calculating a reduction factor. Confidence limits at 95 
percent are desirable. However, in some instances, this may not be practical because of 
material limitations. 
  42  Contains Nonbinding Recommendations 
 
APPENDIX C: CALCULATION OF REDUCTION FACTORS IN STUDIES TO 
DETERMINE VIRAL CLEARANCE (ANNEX 3) 
 
The virus reduction factor of an individual removal or inactivation step is defined as the log  
10
of the ratio of the virus load in the preprocessed material and the virus load in the 
postprocessed material that is ready for use in the next step of the process. If the following 
abbreviations are used: 
 
Starting material: 
 
Vol v; titer 10a; Virus load: (v)(10a), Final material: 
 
Vol v; titer 10a; Virus load: (v)(10a), 
 
The individual reduction factors Ri are calculated according to 10Ri = (v)(10a) / (v)(10a) 
 
This formula takes into account both the titers and volumes of the materials before and after 
the process step. 
 
Because of the inherent imprecision of some virus titrations, an individual reduction factor 
used for the calculation of an overall reduction factor should be greater than one. 
 
The overall reduction factor for a complete production process is the sum logarithm of the 
reduction factors of the individual steps. It represents the logarithm of the ratio of the virus 
load at the beginning of the first process clearance step and at the end of the last process 
clearance step. Reduction factors are normally expressed on a logarithmic scale, which 
implies that, although residual virus infectivity will never be reduced to zero, it may be 
greatly reduced mathematically. 
43  Contains Nonbinding Recommendations 
APPENDIX D: CALCULATION OF ESTIMATED PARTICLES PER DOSE  
(ANNEX 4) 
 
This appendix is applicable to those viruses, such as endogenous retroviruses, for which an 
estimate of starting numbers can be made. 
 
Example: 
 
1)  Assumptions 
 
Measured or estimated concentration of virus entering the purification process (e.g., virus 
concentration in cell culture harvest) = 106/ (milliliter) mL 
 
Calculated viral clearance factor = >1015 
 
Volume of culture harvest used to make a dose of product = 1 liter (103mL) 
 
2)  Calculation of Estimated Particles/Dose  
 
(106 virus units/mL) x (103mL/dose)  
Clearance factor >1015 
=  109 particles/dose 
 
Clearance factor >1015 
 
= <10-6 particles/dose 
 
Therefore, less than one particle per million doses would be expected. 
 
The case above is typical for the reduction of endogenous retroviruses during the 
manufacture of monoclonal antibodies (mAbs) or other recombinant proteins from rodent 
cells (Case B). In a comprehensive risk assessment for a specific virus, additional factors 
should be considered, such as the host range of the virus, the infectivity and pathogenicity of 
the virus, measures to avoid contamination, testing, the route of administration, and the 
human infectious dose. 
 
In the Case B scenario for Chinese hamster ovary (CHO) cells, since endogenous retroviruses 
have been extensively characterized as noninfectious, a value of less than or equal to 10-4 
particles/dose is considered acceptable for retroviral-like particles for mAbs or other 
recombinant proteins if testing fails to identify the presence of infectious retroviruses. 
44  Contains Nonbinding Recommendations 
APPENDIX E: EXAMPLES OF PRIOR KNOWLEDGE INCLUDING IN-HOUSE 
EXPERIENCE TO REDUCE PRODUCT-SPECIFIC VALIDATION EFFORT 
(ANNEX 5) 
 
A.  Introduction (Annex 5.1)  
 
According to the general principles for a platform validation approach, robust viral clearance 
should be demonstrated across products from the same platform, and the procedure for viral 
clearance should follow established and well-characterized conditions. In addition, it should 
be shown that the composition of the process intermediate is comparable to the intermediates 
used in viral clearance studies unless prior knowledge indicates robustness of viral clearance 
with respect to process intermediate composition. 
 
In this context, as opposed to product-specific process validation, platform validation is 
defined as the use of prior knowledge including in-house (applicant-owned data) experience 
with viral clearance from other products, to claim a reduction factor for a new similar 
product. In general, viral clearance claims for a new product based on prior knowledge 
including in-house experience should include a discussion of all relevant platform data 
available and the rationale to support the platform validation approach (see section VI.F (6.6) 
of the main guidance). Part of the prior knowledge and in-house data used to reduce product-
specific validation could be provided as a comparison of the new product and its 
manufacturing process with other in-house products, related process conditions, and process 
intermediates. 
 
Process steps dedicated to viral clearance (e.g., inactivation by detergent, low pH and 
removal by virus filtration) are suitable for a platform validation approach. 
 
Therefore, examples for application of prior knowledge to xenotropic murine leukemia virus 
(XMLV) inactivation by detergent and incubation at low pH, as well as virus removal by 
virus filtration are given below. 
 
These examples are provided for illustrative purposes, only suggesting how the platform 
validation approach could be applied, and should not be used as a template or the sole basis 
for a regulatory submission. 
 
Tables A-2 to A-4 summarize process parameters and their potential criticality for the 
individual process step according to the current understanding of a wide range of process 
conditions applied across industry. The actual effect of process parameters and intermediates 
on viral clearance should be assessed by prior knowledge and in-house experience. 
 
Based on evolving process understanding, further process steps can be considered for 
platform validation in the future. 
 
B.  Inactivation by Solvent/Detergent or Detergent Alone (Annex 5.2) 
 
Based on the mechanism of action, detergent concentration of solvent/detergent (SD) reagents 
or detergent alone is an important process parameter. 
 
45  Contains Nonbinding Recommendations 
 
In addition, hydrophobic impurities such as lipids, cell debris, or components of cell culture 
media such as antifoaming agents can affect virus inactivation by challenging the detergent or 
SD mixture in solubilizing the virus lipid envelope and, therefore, should be assessed. 
 
There is, so far, no indication that the interaction between virus and a specific therapeutic 
protein affects inactivation by detergent. Aggregates (e.g., cell debris, aggregated virus 
particles) can potentially entrap and protect viral particles from detergent access. Therefore, 
at manufacture, the product intermediate (e.g., harvested cell culture fluid (HCCF)) should be 
clarified from cells/cellular debris including a filtration step of less than or equal to 0.2 
micrometer (m) nominal pore size before detergent inactivation. 
 
The following paragraph describes how to apply a platform validation approach to XMLV 
inactivation using SD or Triton X-100 as an example. The approach may also be applicable to 
alternate detergents shown to provide robust and efficient XMLV inactivation. 
 
Triton X-100 is a nonionic detergent commonly used in membrane research to solubilize lipid 
bilayers. It inactivates enveloped viruses by solubilizing the virus lipid envelope thus 
rendering the virus noninfectious. Triton X-100 has been widely used for viral inactivation in 
manufacturing processes of plasma derived products for many years as well as in platform 
purification processes for monoclonal antibodies (mAbs) by addition to HCCF. 
 
The European Chemicals Agency included Triton X-100 in the Authorisation List (at Annex 
XIV) because of the hormone-like activity of degradation compounds in the environment. 
Therefore, though widely used, the pharmaceutical industry is looking into alternate 
detergents. Other detergents with similar physicochemical properties are commercially 
available and achieve efficient XMLV inactivation. Inactivation by Triton X-100 is included 
as an example in this guidance because it is well characterized and a critical mass of 
knowledge regarding its effectiveness exists. 
 
Because of the nonionic nature of Triton X-100, its effectiveness should not be sensitive to pH, 
to ionic strength, or to the nature of the counter ions in HCCF. Prior experience indicates 
effective XMLV inactivation in HCCF at 0.2 percent Triton X100 concentration, at 15C, and 
at 60-minute incubation across multiple products from platform processes covering a range of 
typical lipid and total protein content in HCCF. However, as indicated below, applying a 
Triton X-100 concentration of 0.5 percent is recommended to ensure effective and reliable 
inactivation when omitting product-specific experiments. 
 
Table A-2 summarizes process parameters and their potential criticality for detergent-based 
inactivation of lipid-enveloped virus using Triton X-100 or SD reagents and murine leukemia 
virus (MLV) as an example. 
 
  46  Contains Nonbinding Recommendations 
 
Table A-2: Summary of Process Parameters or Factors and Their Potential Effect for 
Detergent Inactivation or SD Treatment 
 
Process Parameter or Factor  Potential Effect  Rationale 
SD or Triton X-100  high  Inactivating agent 
concentration 
Incubation time  high  Mechanism of inactivation is 
time-dependent 
Temperature  high  Effect on inactivation kinetics 
Pretreatment by  0.2 m  high  Removal from the starting 
filtration  intermediate of aggregates 
potentially entrapping and 
protecting viral particles from 
detergent access 
Total lipid content or  low  Low effect observed with worst-
surrogate parameter in  case HCCF 
HCCF 
     
Type of product  low  No effect on inactivation 
observed for mAbs, half 
antibody, fusion protein or 
recombinant protein 
Total protein content  low  Low effect observed 
pH  low  Triton X-100 is a nonionic 
detergent 
Ionic strength  low  Triton X-100 is a nonionic 
detergent 
Buffer salt in HCCF  low  Triton X-100 is a nonionic 
detergent 
Potential interaction between  low  No effect on inactivation 
virus particle and product  observed and disruption of lipid 
envelope lowers 
probability of interaction with 
product 
SD = solvent/detergent; m = micrometer; HCCF = harvested cell culture fluid; mAbs = monoclonal antibodies. 
 
Thus, consistent with current process understanding for using Triton X-100 with clarified 
HCCF, at a concentration of greater than or equal to 0.5 percent Triton X-100 for greater than 
or equal to 60 minutes at greater than or equal to 15C effectively inactivates XMLV for 
multiple cell culture-derived products. When using SD reagents, treatment with 1 percent 
Triton X-100 and 0.3 percent Tri-N-Butylphosphate for greater than or equal to 30 minutes or 
treatment with 1 percent polysorbate 80 and 0.3 percent Tri-N-Butylphosphate for greater 
than or equal to 6 hours at greater than or equal to 23C effectively inactivates retroviruses. 
According to current process understanding, a platform validation approach can be applied 
for XMLV inactivation by SD treatment or treatment with Triton X-100 alone. 
  47  Contains Nonbinding Recommendations 
 
 
C.  Incubation at Low pH (Annex 5.3) 
 
Low pH treatment inactivates enveloped viruses by denaturing proteins located in the viral 
envelope, thus preventing the adsorption to and infection of cells. Low pH treatment of the 
capture chromatography product pool has been widely used for retrovirus inactivation in 
manufacturing processes of cell culture-derived products such as mAbs. 
 
Inactivation efficiency depends on the concentration of hydrogen ions as the inactivating 
agent, measured as pH, incubation time and temperature, and buffer matrix. Extremely high 
ionic strength may affect inactivation efficiency as well. 
 
Table A-3 summarizes process parameters and their potential effect on low pH inactivation of 
XMLV. 
 
Table A-3: Summary of Process Parameters or Factors and Their Potential Effect for 
Low pH Inactivation and Effect on XMLV 
 
Process Parameter or Factor  Potential Effect  Rationale 
pH  high  Inactivating agent 
Incubation time  high  Mechanism of inactivation is time-
dependent 
Temperature  high  Effect on inactivation kinetics 
Buffer matrix  high  Available data show that 
inactivation robustness depends on 
buffer matrix 
Product concentration  low  No effect observed under the 
conditions outlined below 
Formation of protein aggregates  low  No effect observed under the 
conditions outlined below 
Type of product  low  No effect on inactivation observed 
for mAbs, 
half antibody, bispecific antibody, 
fusion 
protein or recombinant protein 
NaCl concentrationa  low  No effect if  500 mmol/L sodium 
chloride 
Potential interaction between  low  No effect on inactivation 
virus particle and product  observed 
a. To date, data on influence of ionic strength of other buffers is limited. 
mAbs = monoclonal antibodies; NaCl = sodium chloride; mmol = millimole; L = liter. 
 
Consistent with current process understanding, low pH treatment at less than or equal to pH 
3.6, greater than or equal to 15C for greater than or equal to 30 minutes at less than or equal 
to 500 millimoles/liter sodium chloride concentration is effectively inactivating XMLV. 
Acetate and citrate buffer are most commonly used and allow for robust XMLV inactivation. 
 
According to the current process understanding, a platform validation approach can be 
applied for XMLV inactivation by low pH treatment. 
 
  48  Contains Nonbinding Recommendations 
 
D.  Virus Filtration (Annex 5.4) 
 
The primary mechanism of action of virus filtration is size-based particle removal. In general, 
volumetric throughput of the product intermediate as well as the volumetric throughput of the 
buffer used for flushing filters and pressure including pressure/flow interruptions are 
potentially critical parameters in virus filtration. 
 
A potential interaction of virus particles with the product is not critical when the virus particle 
size is much larger than the distribution of filter pore size. However, when the virus particle 
size and pore size are similar, the influence of the potential interaction on virus retention is 
not fully understood. 
 
The rest of this section focuses on using prior knowledge and in-house experience in virus 
filtration of other products to claim large virus removal by small and large virus-retentive 
filters. Prior knowledge can be used for both large and small virus-retentive filters, but this 
section focuses on small virus-retentive filters because they are more commonly used. 
 
Factors that affect efficient retrovirus removal by small virus-retentive filters are well 
understood with respect to variation of process parameters such as filter type (model and 
characteristics), flow- or pressure-controlled filtration mode, tangential or dead-end flow 
filtration, and pressure interruptions. Based on predictability and robustness of virus removal, 
this process step is considered suitable for a platform validation approach. 
 
For virus removal using small virus-retentive filters, one option is to apply parvovirus 
reduction factors for larger spherical/icosahedral viruses and enveloped viruses thus avoiding 
the need to perform validation studies with larger viruses. However, sometimes this could 
result in underestimating virus removal capacity (e.g., large virus removal capacity) as a 
result of parvovirus passage. Given the size-based mechanism of action, and industrys 
experience of robust complete retrovirus removal with small virus-retentive filters, companies 
could use their in-house data from parvovirus and large virus removal to build a platform 
large viral clearance claim for commonly used small virus-retentive filters. 
 
According to the size-based removal mechanism, the theoretical risk of virus passage through 
a small virus-retentive filter is higher for small viruses than for large viruses. 
 
A thorough understanding of the effect of pressure/flow interruptions, as well as volume 
throughput and postfiltration flush volume reflecting manufacturing conditions should be in 
place. It should be considered that there may be a limited negative impact of low 
pressure/flow or pressure/flow interruptions on specific virus filter types. 
 
If using prior knowledge and in-house experience from other products to claim parvovirus 
removal, at least one confirmatory product-specific validation run using a parvovirus should 
be performed considering worst-case conditions (see Table A-4). The brand/model of virus-
retentive filter is important for virus reduction and its robustness with respect to the effect of 
process parameters and should be considered when designing platform data. 
 
Table A-4 summarizes process parameters and their potential effect on parvovirus retention 
using small virus-retentive filters.  
 
  49  Contains Nonbinding Recommendations 
 
Table A-4. Summary of Process Parameters or Factors and Their Potential Effect for 
Parvoviral Clearance by Small Virus-Retentive Filters 
 
Process Parameter or Factor  Potential Effect  Rationale 
Volumetric throughput of  high  High volumetric/protein 
product intermediate loaded  throughput is considered worst- 
onto the virus filter  case with effects varying for 
specific membrane types. 
Volumetric throughput of the  high  High throughput is considered 
buffer used for flushing  worst-case with effects varying 
filters  for specific membrane types. 
Pressure/flow  high  Pressure/flow should not exceed 
the upper limit for filter operation. 
Low pressure/flow can be worse 
case for a specific membrane type. 
Pressure/flow interruption (if 
occurring during filtration or at 
switching from filtration of 
product 
intermediate to filter flush) should 
be considered. 
Type of product  low  No effects on viral clearance 
observed for mAbs, half antibody, 
bispecific antibody, fusion protein, 
or recombinant 
protein 
Product concentration  low  No negative effects on viral 
clearance observed. 
pH  low  No negative effects on viral 
clearance caused by size-based 
removal. May have limited 
negative effects at lower pH 
values (< pH 5) with varying 
effects for specific membrane 
types. 
Ionic strength  low  Limited effects on viral clearance 
have been observed. 
Buffer matrix  low  Limited effects on viral clearance 
have been observed. 
Potential interaction between  low  Specific interaction between 
virus particle and product  virus and antibody can enhance 
virus retention. 
  50  Contains Nonbinding Recommendations 
APPENDIX F: GENETICALLY ENGINEERED VIRAL VECTORS AND VIRAL 
VECTOR-DERIVED PRODUCTS (ANNEX 6) 
 
A.  Introduction (Annex 6.1) 
 
Advances in biotechnology have led to the emergence of new and advanced production 
platforms expressing new product types manufactured using characterized cell banks of 
human or animal origin (i.e., avian, mammalian, or insect). The scope of Appendix F (Annex 
6) includes genetically engineered viral vectors and viral vector-derived products that can be 
produced using helper virus and viral vectors for protein expression (collectively referred to 
as production viruses) or from stable or transient transfected cell lines. The products included 
here are those amenable to viral clearance based on the physicochemical properties of the 
product. These products include protein subunits and virus-like particles (VLPs) that are 
produced using baculovirus/insect cells, nanoparticle-based protein vaccines, and viral vector 
products such as adeno-associated virus (AAV). These vectors can be applied in vivo or ex 
vivo. 
 
Helper virus independent products can be manufactured using stable or transient transfected 
cell lines or by infection with a viral vector for expression of recombinant protein or VLP 
(e.g., recombinant baculovirus). Helper virus dependent products require a helper virus to 
enable expression of the product or replication of the viral vector (e.g., AAVs that require a 
virus such as herpes simplex virus or adenovirus). 
 
The potential sources for virus contamination for a biopharmaceutical product are described 
in section II (2) of the main guidance. Additional viral contamination risks such as those 
introduced by the expression system and the potential for contamination with replication 
competent virus should be considered. The susceptibility of the cell substrate to adventitious 
viruses should be carefully considered when assessing the potential for extrinsic 
contamination during product manufacture. The use of well-characterized cell banks and 
virus seeds can reduce the risk of virus contamination. Production viruses are considered 
process-related impurities. 
 
Viral safety and contamination controls of new product types should be assured through the 
application of a comprehensive program of material sourcing, virus testing at appropriate 
steps of the manufacturing process, and removal and/or inactivation of adventitious viruses 
and production viruses by the manufacturing process. If viral clearance is limited, viral safety 
should focus on the testing and control of the raw materials and reagents and the 
manufacturing process. 
 
Accordingly, a risk-based approach should be applied for demonstrating viral safety of the 
product. 
 
B.  Testing for Viruses (Annex 6.2) 
 
Extensive testing and characterization for both endogenous viruses and adventitious viruses 
should be performed at suitable stages of manufacturing to support the overall product safety. 
Based on the product type and its associated risk factors, the testing scheme should apply 
across the product life cycle. Table A-5 below provides an outline of the tests to be performed 
at various stages during production. The tests applied for virus seeds, unprocessed bulk 
(harvest), and purified bulk/drug substance are described. The testing and characterization 
51  Contains Nonbinding Recommendations 
 
schemes for the cell substrates used for viral vector production are referred to in Table 1 in 
the main guidance; additional considerations that may apply for these product types are also 
indicated in Table A-5. In cases where the producer cells cannot survive the production 
process, limit of in vitro cell age (LIVCA) applies to untransfected cells. 
 
The type and extent of testing depends on a risk assessment considering the specific risk 
factors associated with the cell substrate and the manufacturing process. Factors that should 
be considered include the origin, passage history and characteristics of the cell substrate and 
viral seeds/vector, the source and production of the raw materials and reagents, and cell 
culture process used, the use of production viruses, and the capacity of the manufacturing 
process to inactivate and/or remove viruses. 
 
Table A-5: Virus Tests That Should Be Performed at Applicable Stages 
 
Test  MCB, WCB,  Virus Seedsi        Unprocessed Bulk (harvest)  Drug Substance 
Cells at the  (purified bulk) 
LIVCA 
 
Test for Adventitious Viruses 
In vitro assays or  See Table 1 of 
+g  +g  - 
NGS a,b  main guidance 
In vivo assays or 
+g  -g, j  - 
NGSb 
Tests for specific       
virusc  +h  +  - 
 
Test for Retrovirus and Endogenous Virus, Production Virus and Replication Competent Virus, As Applicable 
Retrovirus and  See Table 1 of       
endogenous virus  main guidance  +  -d, j  - 
Production virus  -  -  +e  +e 
Replication 
-  +f  +f  +f 
competent virus 
MCB = master cell bank; WCB = working cell bank; LIVCA = limit of in vitro cell age; NGS = next generation 
sequencing. 
a.  A 28-day testing on permissive cells for MCB, WCB, LIVCA, MVS, and WVS should be performed with at 
least one subpassage at 2 weeks. For the unprocessed bulk, this can be reduced with justification to 14 days for 
products based on a risk assessment (considering the cell substrate, cultivation period for production, use of 
animal-derived raw materials or reagents, and level of viral clearance of the process). The indicator cell cultures 
should be monitored for cytopathic viruses, hemadsorbing and hemagglutinating viruses, consistent with existing 
regional regulations and guidance. For products produced in insect cell lines, the testing should include a 
permissive cell line for arboviruses (e.g., BHK cells). NGS can be used as an alternative assay and can be 
particularly useful if the viral vector or viral vector-derived product cannot be neutralized. Testing should be 
performed on the virus seed and the unprocessed bulk harvest. In some cases, the unprocessed bulk harvest may 
be the same as the drug substance. 
b.  Nontargeted NGS can replace the in vivo assay (see section III.B.3 (3.2.3) of the main guidance) and supplement 
or replace the in vitro assay (see section III.B.2 (3.2.2) of the main guidance). 
c.  Testing is based on risk assessment including the origin and history of the cell substrate, derivation of the virus 
seeds, and potential exposure to human- or animal-derived raw materials. Methods such as cell culture-based 
infectivity assays, antibody production tests (MAP, HAP, RAP), virus specific NAT or other molecular methods 
(e.g., NGS) can be used. Refer to section III.B.4 (3.2.4) of the main guidance for details. This can include testing 
for species-specific viruses, e.g., arboviruses in insect cells, and bovine or porcine viruses if serum or trypsin are 
used, respectively. 
d.  If either the MCB or virus seeds are positive for retrovirus, follow-up should include quantification of potential 
retroviral particles in the unprocessed bulk harvest from at least three lots/batches to determine the target level for 
viral clearance. For the MVS, if there is remaining residual risk, then testing of the unprocessed bulk harvest can 
be considered. 
  52  Contains Nonbinding Recommendations 
 
e.  Quantification of the production virus should be performed at the unprocessed bulk stage using at least three cell 
culture lots/batches to determine the target for viral clearance. Absence of production virus should be 
demonstrated in the drug substance (purified bulk) using an infectivity assay with relevant permissive cell lines 
for sensitive virus detection. Alternatively molecular methods can be used for testing. Testing for the absence of 
residual production virus should be performed for each purified bulk, unless justified by robust excess clearance 
(see Case F, Table 4, in the main guidance). 
f.  Replication competent virus (RCV) may develop at any step during manufacturing. Recommendations include 
testing for RCV at multiple stages of manufacture. RCV testing is performed on the virus seed/bank and on each 
unprocessed bulk harvest or on each drug substance or drug product as applicable for maximum detection. 
g.  When interference may occur with the in vitro or in vivo assays, control cells cultured in parallel are tested at the 
virus seed and/or unprocessed bulk harvest stages. When NGS is used for testing the virus seeds and 
unprocessed bulk, testing of the control cell cultures is not needed. 
h.  Testing should be performed on the virus seeds if the cell bank has not been tested. 
i.  Testing should be applied on the virus seeds. Depending on the product type, the virus seed can be used to 
manufacture a vaccine virus, viral vector, or helper virus. The virus seeds are generated from an established cell 
line. Consistent with a risk-based approach, the virus testing should consider the origin of the cell line and raw 
materials and reagents used for preparation of the virus seeds, to ensure the absence of adventitious virus and the 
absence of replication competent virus. Because the WVS is derived directly from the MVS, a subset of 
adventitious virus testing applies based on a risk assessment. An alternative approach in which complete testing is 
carried out on each WVS rather than on the MVS could also be acceptable. 
j.  Testing based on risk assessment. 
 
C.  Viral Clearance (Annex 6.3) 
 
The risk of contamination with adventitious viruses, endogenous viruses, and residual 
production viruses should be mitigated following the general principles of this guidance to 
the extent possible. Some viral vector products such as AAVs are amenable to viral clearance 
steps, assuring adventitious and production viral clearance (inactivation or removal). 
 
The viral clearance should be evaluated using representative and qualified scale-down 
models. The physicochemical characteristics of the viral vector and the viral vector-derived 
product will determine how viral clearance will apply within the downstream process. Virus-
clearance studies should include the production virus itself or a specific model virus (e.g., 
baculovirus, adenovirus, herpesvirus) and model viruses representative of adventitious and 
endogenous viruses (see Table A-1 in Appendix A (Annex 1)). Refer to sections V (5) and (6) 
of the main guidance for an action plan for the selection of specific and nonspecific model 
viruses described in Table 4 of the main guidance. The manufacturing processes should have 
robust clearance of the viral vector for protein expression or helper virus. Common virus 
inactivation steps such as treatment with detergent or solvent/detergent (SD) may be suitable, 
when the product is compatible, such as for nonenveloped viral vectors. Alternatively, virus 
filtration may be more suitable for small viral vectors such as AAV or nanoparticle-based 
vaccines when virus removal can be based on the size exclusion. Chromatography steps can 
provide viral clearance for viruses with different surface properties from the viral vector. 
When appropriate, viral clearance studies should be performed to determine virus reduction 
factors for the relevant step(s) of the production process. Examples include manufacture of 
subunit proteins and VLPs that are produced using baculovirus/insect cells, and which can be 
purified, and for which viral clearance can be achieved through the manufacturing process. 
 
The viral safety of these products may also rely on closed processing, testing, and other 
preventative controls (see sections II.B.3 (2.2, 3) and IV (4) of the main guidance). Viral 
clearance steps during production may not achieve the same robustness as for recombinant 
proteins; therefore, viral safety of the products should be supported by a risk assessment. 
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