Guidance for Industry

Q5A Viral Safety Evaluation

of Biotechnology Products

Derived From Cell Lines of

Human or Animal Origin

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

September 1998

ICH
Guidance for Industry

Q5A Viral Safety Evaluation

of Biotechnology Products

Derived From Cell Lines of

Human or Animal Origin

Additional copies are available from:

Office of Training and Communications

Division of Drug Information (HFD-240)

Center for Drug Evaluation and Research (CDER)

Food and Drug Administration

5600 Fishers Lane, Rockville, MD 20857

(Tel) 301-827-4573

http://www.fda.gov/cder/guidance/index.htm

or

Office of Communication, Training, and Manufacturers Assistance (HFM-40)

Center for Biologics Evaluation and Research (CBER)

1401 Rockville Pike, Rockville, MD 20852-1448

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

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

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

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

September 1998

ICH
i

TABLE OF CONTENTS

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

II. POTENTIAL SOURCES OF VIRUS CONTAMINATION ....................................................................................2

A. VIRUSES THAT COULD OCCUR IN THE MASTER CELL BANK (MCB)............................................................................2

B. ADVENTITIOUS VIRUSES THAT COULD BE INTRODUCED DURING PRODUCTION...........................................................2

III. CELL LINE QUALIFICATION: TESTING FOR VIRUSES.................................................................................3

A. SUGGESTED VIRUS TESTS FOR MCB, WORKING CELL BANK (WCB), AND CELLS AT THE LIMIT OF IN VITRO CELL AGE

USED FOR PRODUCTION.............................................................................................................................................3

B. RECOMMENDED VIRAL DETECTION AND IDENTIFICATION ASSAYS..............................................................................4

C. ACCEPTABILITY OF CELL LINES..................................................................................................................................5

IV. TESTING FOR VIRUSES IN UNPROCESSED BULK..........................................................................................5

V. RATIONALE AND ACTION PLAN FOR VIRAL CLEARANCE STUDIES AND VIRUS TESTS ON

PURIFIED BULK .................................................................................................................................................................6

VI. EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE PROCEDURES...............................8

A. THE CHOICE OF VIRUSES FOR THE EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE ..............................9

B. DESIGN AND IMPLICATIONS OF VIRAL CLEARANCE EVALUATION AND CHARACTERIZATION STUDIES........................10

C. INTERPRETATION OF VIRAL CLEARANCE STUDIES; ACCEPTABILITY..........................................................................14

D. LIMITATIONS OF VIRAL CLEARANCE STUDIES ..........................................................................................................15

E. STATISTICS..............................................................................................................................................................16

F. REEVALUATION OF VIRAL CLEARANCE ....................................................................................................................16

VII. SUMMARY..........................................................................................................................................................16

GLOSSARY.........................................................................................................................................................................18

APPENDIX 1.......................................................................................................................................................................24

APPENDIX 2.......................................................................................................................................................................25

APPENDIX 3.......................................................................................................................................................................27

APPENDIX 4.......................................................................................................................................................................29

APPENDIX 5.......................................................................................................................................................................30
1

GUIDANCE FOR INDUSTRY1

Q5A Viral Safety Evaluation of Biotechnology Products

Derived From Cell Lines of Human or Animal Origin

I. INTRODUCTION

This document is concerned with testing and evaluation of the viral safety of biotechnology

products derived from characterized cell lines of human or animal origin (i.e., mammalian, avian,

insect), and outlines data that should be submitted in the marketing application/registration

package. For the purposes of this document, the term virus excludes nonconventional

transmissible agents like those associated with bovine spongiform encephalopathy (BSE) and

scrapie. Applicants are encouraged to discuss issues associated with BSE with the regulatory

authorities.

The scope of the document covers products derived from cell cultures initiated from characterized

cell banks. It covers products derived from in vitro cell culture, such as interferons, monoclonal

antibodies, and recombinant deoxyribonucleic acid (DNA)-derived products including

recombinant subunit vaccines, and also includes products derived from hybridoma cells grown in

vivo as ascites. In this latter case, special considerations apply and additional information on

testing cells propagated in vivo is contained in Appendix 1. Inactivated vaccines, all live

vaccines containing self-replicating agents, and genetically engineered live vectors are excluded

from the scope of this document.

The risk of viral contamination is a feature common to all biotechnology products derived from

cell lines. Such contamination could have serious clinical consequences and can arise from the

contamination of the source cell lines themselves (cell substrates) or from adventitious

 

1

 This guidance was developed within the Expert Working Group (Quality) of the International Conference on

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

Steering Committee at Step 4 of the ICH process, March 1997. At Step 4 of the process, the final draft is recommended for

adoption to the regulatory bodies of the European Union, Japan, and the United States. This guidance was published in the

Federal Register on September 24, 1998 (63 FR 51074), and is applicable to drug and biological products. 

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

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

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

requirements of the applicable statutes and regulations.
2

introduction of virus during production. To date, however, biotechnology products derived from

cell lines have not been implicated in the transmission of viruses. Nevertheless, it is expected that

the safety of these products with regard to viral contamination can be reasonably assured only by

the application of a virus testing program and assessment of virus removal and inactivation

achieved by the manufacturing process, as outlined below.

Three principal, complementary approaches have evolved 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 viruses which may be infectious and/or pathogenic for humans;

Assessing the capacity of the production processes to clear infectious viruses;

Testing the product at appropriate steps of production for absence of contaminating infectious

viruses.

II. POTENTIAL SOURCES OF VIRUS CONTAMINATION

Viral contamination of biotechnology products may arise from the original source of the cell lines

or from adventitious introduction of virus during production processes.

A. Viruses That Could Occur in the Master Cell Bank (MCB)

Cells may have latent or persistent virus infection (e.g., herpesvirus) or endogenous

retrovirus which may be transmitted vertically from one cell generation to the next, since

the viral genome persists within the cell. Such viruses may be constitutively expressed or

may unexpectedly become expressed as an infectious virus.

Viruses can be introduced into the MCB by several routes such as: (1) derivation of cell

lines from infected animals; (2) use of virus to establish the cell line; (3) use of

contaminated biological reagents such as animal serum components; (4) contamination

during cell handling.

B. Adventitious Viruses That Could Be Introduced During Production

Adventitious viruses can be introduced into the final product by several routes including,

but not limited to, the following: (1) use of contaminated biological reagents such as

animal serum components; (2) use of a virus for the induction of expression of specific

genes encoding a desired protein; (3) use of a contaminated reagent, such as a monoclonal

antibody affinity column; (4) use of a contaminated excipient during formulation; and (5)

contamination during cell and medium handling. Monitoring of cell culture parameters can

be helpful in the early detection of potential adventitious viral contamination.
3

III. CELL LINE QUALIFICATION: TESTING FOR VIRUSES

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 virus. 

A. Suggested Virus Tests for MCB, Working Cell Bank (WCB), and Cells at the

Limit of In Vitro Cell Age Used for Production

Table 1 shows examples of virus tests to be performed once only at various cell levels,

including MCB, WCB, and cells at the limit of in vitro cell age used for production.

1. Master Cell Bank

Extensive screening for both endogenous and nonendogenous viral contamination

should be performed on the MCB. For heterohybrid cell lines in which one or

more partners are human or nonhuman primate in origin, tests should be performed

in order to detect viruses of human or nonhuman primate origin because viral

contamination arising from these cells may pose a particular hazard.

Testing for nonendogenous viruses should include in vitro and in vivo inoculation

tests and any other specific tests, including species-specific tests such as the mouse

antibody production (MAP) test, that are appropriate, based on the passage history

of the cell line, to detect possible contaminating viruses.

2. Working Cell Bank

Each WCB as a starting cell substrate for drug production should be tested for

adventitious virus either by direct testing or by analysis of cells at the limit of in

vitro cell age, initiated from the WCB. When appropriate nonendogenous virus

tests have been performed on the MCB and cells cultured up to or beyond the limit

of in vitro cell age have been derived from the WCB and used for testing for the

presence of adventitious viruses, similar tests need not be performed on the initial

WCB. Antibody production tests are usually not necessary for the WCB. An

alternative approach in which full tests are carried out on the WCB rather than on

the MCB would also be considered acceptable.

3. Cells at the Limit of In Vitro Cell Age Used for Production

The limit of in vitro cell age used for production should be based on data derived

from production cells expanded under pilot-plant scale or commercial-scale

conditions to the proposed in vitro cell age or beyond. Generally, the production

cells are obtained by expansion of the WCB; the MCB could also be used to
4

prepare the production cells. Cells at the limit of in vitro cell age should be

evaluated once for those endogenous viruses that may have been undetected in the

MCB and WCB. The performance of suitable tests (e.g., in vitro and in vivo ) at

least once on cells at the limit of in vitro cell age used for production would

provide further assurance that the production process is not prone to contamination

by adventitious virus. If any adventitious viruses are detected at this level, the

process should be carefully checked in order to determine the cause of the

contamination, and should be completely redesigned if necessary

.

B. Recommended Viral Detection and Identification Assays

Numerous assays can be used for the detection of endogenous and adventitious viruses.

Table 2 outlines examples for these assays. They should be regarded as assay protocols

recommended for the present, but the list is not all-inclusive or definitive. Since the most

appropriate techniques may change with scientific progress, proposals for alternative

techniques, when accompanied by adequate supporting data, may be acceptable. 

Manufacturers are encouraged to discuss these alternatives with the regulatory authorities.

Other tests may be necessary depending on the individual case. Assays should include

appropriate controls to ensure adequate sensitivity and specificity. Wherever a relatively

high possibility of the presence of a specific virus can be predicted from the species of

origin of the cell substrate, specific tests and/or approaches may be necessary. If the cell

line used for production is of human or nonhuman primate origin, additional tests for human

viruses, such as those causing immunodeficiency diseases and hepatitis, should be

performed unless otherwise justified. The polymerase chain reaction (PCR) may be

appropriate for detection of sequences of thioe human viruses as well as for other specific

viruses. The following is a brief description of a general framework and philosophical

background within which the manufacturer should justify what was done.

1. Tests for Retroviruses

For the MCB and for cells cultured up to or beyond the limit of in vitro cell age

used for production, tests for retroviruses, including infectivity assays in sensitive

cell cultures and electron microscopy (EM) studies, should be carried out. If

infectivity is not detected and no retrovirus or retrovirus-like particles have been

observed by EM, reverse transcriptase (RT) or other appropriate assays should be

performed to detect retroviruses that may be noninfectious. Induction studies have

not been found to be useful.

2. In Vitro Assays

In vitro tests are carried out by the inoculation of a test article (see Table 2) into

various susceptible indicator cell cultures capable of detecting a wide range of

human and relevant animal viruses. The choice of cells used in the test is governed

by the species of origin of the cell bank to be tested, but should include a human
5

and/or a nonhuman primate cell line susceptible to human viruses. The nature of the

assay and the sample to be tested are governed by the type of virus which may

possibly be present based on the origin or handling of the cells. Both cytopathic

and hemadsorbing viruses should be sought.

3. In Vivo Assays

A test article (see Table 2) should be inoculated into animals, including suckling

and adult mice, and in embryonated eggs to reveal viruses that cannot grow in cell

cultures. Additional animal species may be used, depending on the nature and

source of the cell lines being tested. The health of the animals should be monitored

and any abnormality should be investigated to establish the cause of the illness.

4. Antibody Production Tests

Species-specific viruses present in rodent cell lines may be detected by inoculating

test article (see Table 2) into virus-free animals and examining the serum antibody

level or enzyme activity after a specified period. 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.

C. Acceptability of Cell Lines

It is recognized that some cell lines used for the manufacture of product will contain

endogenous retroviruses, other viruses, or viral sequences. In such circumstances, the

action plan recommended for manufacture is described in section V of this document. The

acceptability of cell lines containing viruses other than endogenous retroviruses will be

considered on an individual basis by the regulatory authorities, by taking into account a

risk/benefit 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

The unprocessed bulk constitutes one or multiple pooled harvests of cells and culture media. 

When cells are not readily accessible (e.g., hollow fiber or similar systems), the unprocessed bulk

would constitute fluids harvested from the fermenter. A representative sample of the unprocessed

bulk, removed from the production reactor prior to 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. Appropriate testing for viruses should be performed at the

unprocessed bulk level unless virus testing is made more sensitive by initial partial processing
6

(e.g., unprocessed bulk may be toxic in test cell cultures, whereas partially processed bulk may not

be toxic).

In certain instances, it may be more appropriate to test a mixture consisting of both intact and

disrupted cells and their cell culture supernatants removed from the production reactor prior to

further processing. Data from at least three lots of unprocessed bulk at pilot-plant scale or

commercial scale should be submitted as part of the marketing application/registration package.

It is recommended that manufacturers develop programs for the ongoing assessment of adventitious

viruses in production batches. The scope, extent, and frequency of virus testing on the unprocessed

bulk should be determined by taking several points into consideration, 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, raw material sources, and results

of viral clearance studies. In vitro screening tests, using one or several cell lines, are generally

employed to test unprocessed bulk. If appropriate, a PCR test or other suitable methods may be

used.

Generally, harvest material in which adventitious virus has been detected should not be used to

manufacture the product. If any adventitious viruses are detected at this level, the process should

be carefully checked to determine the cause of the contamination, and appropriate actions taken.

V. RATIONALE AND ACTION PLAN FOR VIRAL CLEARANCE STUDIES AND

VIRUS TESTS ON PURIFIED BULK

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, to the final product including evaluation and

characterization of viral clearance from unprocessed bulk. The evaluation and characterization of

viral clearance plays a critical role in this scheme. The goal should be to obtain the best

reasonable assurance that the product is free of virus contamination.

In selecting viruses to use for a clearance study, it is useful to distinguish between the need to

evaluate processes for their ability to clear viruses that are known to be present and the desire to

estimate 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. Process evaluation requires knowledge of how much virus may be present in the

process, such as the unprocessed bulk, and how much can be cleared in order to assess product

safety. Knowledge of the time dependence for inactivation procedures is helpful in assuring the

effectiveness of the inactivation process. When evaluating clearance of known contaminants,

indepth, time-dependent inactivation studies, demonstration of reproducibility of

inactivation/removal, and evaluation of process parameters should be provided. 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. The extent
7

of viral clearance characterization studies may be influenced by the results of tests on cell lines

and unprocessed bulk. These studies should be performed as described in section VI below.

Table 4 presents an example of an action plan in terms of process evaluation and characterization

of viral clearance as well as virus tests on purified bulk, in response to the results of virus tests on

cells and/or the unprocessed bulk. Various cases are considered. In all cases, characterization of

clearance using nonspecific model viruses should be performed. The most common situations are

Cases A and B. Production systems contaminated with a virus other than a rodent retrovirus are

normally not used. Where there are convincing and well justified reasons for drug production

using a cell line from Cases C, D, or E, these should be discussed with the regulatory authorities. 

With Cases C, D, and E, it is important to have validated effective steps to inactivate/remove the

virus in question from the manufacturing process.

Case A: Where no virus, virus-like particle, or retrovirus-like particle has been demonstrated in

the cells or in the unprocessed bulk, virus removal and inactivation studies should be performed

with nonspecific model viruses as previously stated.

Case B: Where only a rodent retrovirus (or a retrovirus-like particle that is believed to be

nonpathogenic, such as rodent A- and R-type particles) is present, process evaluation using a

specific model virus, such as a murine leukemia virus, should be performed. Purified bulk should

be tested using suitable methods having high specificity and sensitivity for the detection of the

virus in question. For marketing authorization, data from at least three lots of purified bulk at

pilot-plant scale or commercial scale should be provided. Cell lines such as Chinese hamster

ovary (CHO), C127, baby hamster kidney (BHK), and murine hybridoma cell lines 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 not usually necessary to assay

for the presence of the noninfectious particles in purified bulk. Studies with nonspecific model

viruses, as in Case A, are appropriate.

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 capacity for infecting humans (such as those identified

by footnote 2 in Table 3, except rodent retroviruses (Case B)), 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. Timedependent inactivation for identified (or relevant or specific model) viruses at the critical

inactivation step(s) should be obtained as part of process evaluation for these viruses. Purified

bulk should be tested using suitable methods having high specificity and sensitivity for the

detection of the virus in question. For the purpose of marketing authorization, data from at least

three lots of purified bulk manufactured at pilot-plant scale or commercial scale should be

provided.

Case D: Where a known human pathogen, such as those indicated by footnote 1 in Table 3, is

identified, the product may be acceptable only under exceptional circumstances. In this instance, it
8

is recommended that the identified virus 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 be employed. 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 achieve the

removal and inactivation of the selected viruses during the purification and inactivation processes.

 Time-dependent inactivation data for the critical inactivation step(s) should be obtained as part of

process evaluation. Purified bulk should be tested using suitable methods having high specificity

and sensitivity for the detection of the virus in question. For the purpose of marketing

authorization, data from at least three lots 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 since the virus may

prove to be pathogenic. In the very rare case where there are convincing and well justified

reasons for drug production using such a cell line, this should be discussed with the regulatory

authorities before proceeding further.

VI. EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE

PROCEDURES

Evaluation and characterization of due virus removal and/or inactivation procedures play an

important role in establishing the safety of biotechnology products. Many instances of

contamination in the past have occurred with agents whose presence was not known or even

suspected, and though this happened to biological products derived from various source materials

other than fully characterized cell lines, assessment of viral clearance will provide a measure of

confidence that any unknown, unsuspected, and harmful viruses may be removed. Studies should

be carried out in a manner that is well documented and controlled.

The objective of viral clearance studies is to assess process step(s) that can be considered to be

effective in inactivating/removing viruses and to estimate quantitatively the overall level of virus

reduction obtained by the process. This should be achieved by the deliberate addition (spiking) of

significant amounts of a virus to the crude material and/or to different fractions obtained during the

various process steps and demonstrating its removal or inactivation during the subsequent steps. It

is not considered necessary to evaluate or characterize every step of a manufacturing process if

adequate clearance is demonstrated by the use of fewer steps. It should be borne in mind that other

steps in the process may have an indirect effect on the viral inactivation/removal achieved. 

Manufacturers should explain and justify the approach used in studies for evaluating virus

clearance.

The reduction of virus infectivity may be achieved by removal of virus particles or by inactivation

of viral infectivity. For each production step assessed, the possible mechanism of loss of viral

infectivity should be described with regard to whether it is due to inactivation or removal. For
9

inactivation steps, the study should be planned in such a way that samples are taken at different

times and an inactivation curve constructed (see section VI.B.5.).

Viral clearance evaluation studies are performed to demonstrate the clearance of a virus known to

be present in the MCB and/or to provide some level of assurance that adventitious viruses which

could not be detected, or might gain access to the production process, would be cleared. 

Reduction factors are normally expressed on a logarithmic scale, which implies that, while

residual virus infectivity will never be reduced to zero, it may be greatly reduced mathematically.

In addition to clearance studies for viruses known to be present, studies to characterize the ability

to remove and/or inactivate other viruses should be conducted. The purpose of studies with

viruses exhibiting a range of biochemical and biophysical properties that are not known or

expected to be present is to characterize the robustness of the procedure 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.). Such studies are not performed to

evaluate a specific safety risk. Therefore, a specific clearance value need not be achieved.

A. The Choice of Viruses for the Evaluation and Characterization of Viral

Clearance

Viruses for clearance evaluation and process characterization studies should be chosen to

resemble viruses which may contaminate the product and to represent a wide range of

physico-chemical properties in order to test the ability of the system to eliminate viruses in

general. The manufacturer should justify the choice of viruses in accordance with the aims

of the evaluation and characterization study and the guidance provided in this document.

1. Relevant Viruses and Model Viruses

A major issue in performing a viral clearance study is to determine which viruses

should be used. Such viruses fall into three categories: Relevant viruses, specific

model viruses, and nonspecific model viruses.

Relevant viruses are viruses used in process evaluation of viral clearance studies

which are either the identified viruses, or of the same species as the viruses that are

known, or likely to contaminate the cell substrate or any other reagents or materials

used in the production process. The purification and/or inactivation 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 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 may be a virus which 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.
10

Cell lines derived from rodents usually contain endogenous retrovirus particles or

retrovirus-like particles, 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 may be

accomplished by using a murine leukemia virus, a specific model virus in the case

of cells of murine origin. When human cell lines secreting monoclonal antibodies

have been obtained by the immortalization of B lymphocytes by Epstein-Barr Virus

(EBV), the ability of the manufacturing process to remove and/or inactivate a

herpes virus should be determined. Pseudorabies virus may 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 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 may 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 will be influenced by the quality and

characterization of the cell lines and the production process.

Examples of useful model viruses representing a range of physico-chemical

structures and examples of viruses which have been used in viral clearance studies

are given in Appendix 2 and Table A-1.

2. Other Considerations

Additional points to be considered are as follows:

a. Viruses which can be grown to high titer are desirable, although this

may not always be possible.

b. There should be an efficient and reliable assay for the detection of

each virus used, for every stage of manufacturing that is tested.

c. Consideration should be given to the health hazard which certain

viruses may pose to the personnel performing the clearance studies.

B. Design and Implications of Viral Clearance Evaluation and Characterization

Studies
11

1. Facility and Staff

It is inappropriate to introduce any virus into a production facility because of good

manufacturing practice (GMP) constraints. Therefore, viral clearance studies

should be conducted in a separate laboratory equipped for virological work and

performed by staff with virological expertise in conjunction with production

personnel involved in designing and preparing a scaled-down version of the

purification process.

2. Scaled-Down Production System

The validity of the scaling down should be demonstrated. The level of purification

of the scaled-down version should represent as closely as possible the production

procedure. For chromatographic equipment, column bed-height, linear flow-rate,

flow-rate-to-bed-volume ratio (i.e., contact time), buffer and gel types, pH,

temperature, and concentration of protein, salt, and product should all be shown to

be representative of commercial-scale manufacturing. A similar elution profile

should result. For other procedures, similar considerations apply. Deviations that

cannot be avoided should be discussed with regard to their influence on the results.

3. Analysis of Step-Wise Elimination of Virus

When viral clearance studies are being performed, it is desirable to assess the

contribution of more than one production step to virus elimination. Steps that are

likely to clear virus should be individually assessed for their ability to remove and

inactivate virus and careful consideration should be given to the exact definition of

an individual step. 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 in-process material of each step to

be tested. In some cases, simply adding high titer virus to unpurified bulk and

testing its concentration between steps will be sufficient. Where virus removal

results from separation procedures, it is recommended that, if appropriate and if

possible, the distribution of the virus load in the different fractions be investigated.

 When virucidal buffers are used in multiple steps within the manufacturing

process, alternative strategies such as parallel spiking in less virucidal buffers may

be carried out as part of the overall process assessment. The virus titer before and

after each step being tested 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 may 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 (Appendix 3).
12

4. Determination of Physical Removal Versus Inactivation

Reduction in virus infectivity may be achieved by the removal or inactivation of

virus. For each production step assessed, the possible mechanism of loss of viral

infectivity should be described with regard to whether it is due to inactivation or

removal. If little clearance of infectivity is achieved by the production process,

and the clearance of virus is considered to be a major factor in the safety of the

product, specific or additional inactivation/removal steps should be introduced. It

may be necessary to distinguish between removal and inactivation for a particular

step. For example, when there is a possibility that a buffer used in more than one

clearance step may contribute to inactivation during each step, the contribution to

inactivation by a buffer shared by several chromatographic steps and the removal

achieved by each of these chromatographic steps should be distinguished.

5. Inactivation Assessment

For assessment of viral inactivation, unprocessed crude material or intermediate

material 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 a fast phase 1 and a slow phase 2. The study

should, therefore, be planned in such a way that samples are taken at different times

and an inactivation curve 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 where 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

intracytoplasmic retrovirus-like particles, reproducible clearance should be

demonstrated in at least two independent studies. Whenever possible, the initial

virus load should be determined from the virus that can be detected in the spiked

starting material. If this is not possible, the initial virus load may be calculated

from the titer of the spiking virus preparation. Where inactivation is too rapid to

plot an inactivation curve using process conditions, appropriate controls should be

performed to demonstrate that infectivity is indeed lost by inactivation.

6. Function and Regeneration of Columns

Over time and after repeated use, the ability of chromatography columns and other

devices used in the purification scheme to clear virus may vary. Some estimate of

the stability of the viral clearance after several uses may provide support for

repeated use of such columns. Assurance should be provided that any virus

potentially retained by the production system would be adequately destroyed or

removed prior to reuse of the system. For example, such evidence may be
13

provided by demonstrating that the cleaning and regeneration procedures do

inactivate or remove virus.

7. Specific Precautions

a. Care should be taken in preparing the high-titer virus to avoid

aggregation which may enhance physical removal and decrease

inactivation, thus distorting the correlation with actual production.

b. Consideration should be given to the minimum quantity of virus

which can be reliably assayed.

c. The study should include parallel control assays to assess the loss

of infectivity of the virus due to such reasons as the dilution,

concentration, filtration or storage of samples before titration.

d. 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. Diluted,

test-protein sample is no longer identical to the product obtained at

commercial scale.

e. Small differences in, for example, buffers, media, or reagents can

substantially affect viral clearance.

f. 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.

g. Buffers and product should be evaluated independently for toxicity

or interference in assays used to determine the virus titer, as 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 anti-viral activity, the clearance study may

need to be performed without the product in a mock run, although

omitting the product or substituting a similar protein that does not

have anti-viral 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.
14

h. Many purification schemes use the same or similar buffers or

columns repetitively. The effects of this approach should be taken

into account when analyzing the data. The effectiveness of virus

elimination by a particular process may vary with the manufacturing

stage at which it is used.

i. Overall reduction factors may be underestimated where 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 due to inherent limitations or inadequate

design of viral clearance studies.

C. Interpretation of Viral Clearance Studies; Acceptability

The object of assessing virus inactivation/removal is to evaluate and characterize process

steps that can be considered to be effective in inactivating/removing viruses and to

estimate quantitatively the overall level of virus reduction obtained by the manufacturing

process. For virus contaminants, as in Cases B through E, it is important to show that not

only is the virus eliminated or inactivated, but that there is excess capacity for viral

clearance built into the purification process to assure an appropriate level of safety for the

final product. The amount of virus eliminated or inactivated by the production process

should be compared to the amount of virus that may be present in unprocessed bulk.

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 transmission electron microscopy (TEM). 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 4 for calculation of virus

reduction factors and Appendix 5 for calculation of estimated particles per dose.

Manufacturers should recognize that clearance mechanisms may differ between virus

classes. A combination of factors should be considered when judging the data supporting

the effectiveness of virus inactivation/removal procedures. These include:

• The appropriateness of the test viruses used;

• The design of the clearance studies;

• The log reduction achieved;

• The time dependence of inactivation;

• The potential effects of variation in process parameters on virus

inactivation/removal;

• The limits of assay sensitivities;

• The possible selectivity of inactivation/removal procedure(s) for certain

classes of viruses.
15

Effective clearance may be achieved by any of the following: Multiple inactivation steps,

multiple complementary separation steps, or combinations of inactivation and separation

steps. Since separation methods may be dependent on the extremely specific physicochemical properties of a virus which influence its interaction with gel matrices and

precipitation properties, model viruses may be separated in a different manner than a target

virus. Manufacturing parameters influencing separation should be properly defined and

controlled. Differences may originate from changes in surface properties such as

glycosylation. However, despite these potential variables, effective removal can be

obtained by a combination of complementary separation steps or combinations of

inactivation and separation steps. Therefore, well-designed separation steps, such as

chromatographic procedures, filtration steps, and extractions, can be effective virus

removal steps provided that they are performed under appropriately controlled

conditions. An effective virus removal step should give reproducible reduction of virus

load shown by at least two independent studies.

An overall reduction factor is generally expressed as the sum of the individual factors. 

However, reduction in virus titer of the order of 1 log10 or less would be considered

negligible and would 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. Results would be

evaluated on the basis of the factors listed above.

D. Limitations of Viral Clearance Studies

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. 

However, 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

likely to be produced in tissue culture. The behavior of a tissue culture virus

in a production step may be different from that of the native virus, for

example, if native and cultured viruses 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,
16

infectivity may be resistant to a range of different chemical treatments and to

heating.

• The ability of the overall process to remove infectivity is expressed as the

sum of the logarithm of the reductions at each step. The summation of the

reduction factors of multiple steps, particularly of steps with little reduction

(e.g., below 1 log10), may overestimate the true potential for virus

elimination. Furthermore, reduction values achieved by repetition of

identical or near identical procedures should not be included unless justified.

• The expression of reduction factors as logarithmic reductions in titer implies

that, while 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 zero log10 per mL or one infectious unit per mL, taking into

consideration the limit of detection of the assay.

• Pilot-plant scale processing may differ from commercial-scale processing

despite care taken to design the scaled-down process.

• Addition of individual virus reduction factors resulting from similar

inactivation mechanisms along the manufacturing process may overestimate

overall viral clearance.

E. Statistics

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 (see Appendix 3).

F. Reevaluation of Viral Clearance

Whenever significant changes in the production or purification process are made, the effect

of that change, both direct and indirect, on viral clearance should be considered and the

system re-evaluated as needed. For example, changes in production 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.

VII. SUMMARY

This document suggests approaches for the evaluation of the risk of viral contamination and for the

removal of virus from product, thus contributing to the production of safe biotechnology products

derived from animal or human cell lines, and emphasizes the value of many strategies, including:
17

• Thorough characterization/screening of cell substrate starting material in order to

identify which, if any, viral contaminants are present

• Assessment of risk by determination of the human tropism of the contaminants

• Establishment of an appropriate program of testing for adventitious viruses in

unprocessed bulk

• Careful design of viral clearance studies using different methods of virus

inactivation or removal in the same production process in order to achieve

maximum viral clearance

• Performance of studies which assess virus inactivation and removal
18

GLOSSARY

Adventitious Virus: See virus.

Cell Substrate: Cells used to manufacture product.

Endogenous Virus: See virus.

Inactivation: Reduction of virus infectivity caused by chemical or physical modification.

In Vitro Cell Age: A measure of the period between thawing of the MCB vial(s) and harvest of

the production vessel 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.

Master Cell Bank (MCB): An aliquot of a single pool of cells which generally has been

prepared from the selected cell clone under defined conditions, dispensed into multiple containers,

and stored under defined conditions. The MCB is used to derive all working cell banks. The

testing performed on a new MCB (from a previous initial cell clone, MCB, or WCB) should be the

same as for the original MCB, unless justified.

Minimum Exposure Time: The shortest period for which a treatment step will be maintained.

Nonendogenous Virus: See virus.

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.

Production Cells: Cell substrate used to manufacture product.

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 fermenter.

Virus: Intracellularly replicating infectious agents that are potentially pathogenic, possess only a

single type of nucleic acid (either ribonucleic acid (RNA) or DNA), are unable to grow and

undergo binary fission, and multiply in the form of their genetic material.

Adventitious Virus: Unintentionally introduced contaminant virus.
19

Endogenous Virus: Viral entity whose genome is part of the germ line of the species of origin of

the cell line and is covalently integrated into the genome of animal from which the parental cell

line was derived. For the purposes of this document, intentionally introduced, nonintegrated

viruses such as EBV used to immortalize cell substrates or Bovine Papilloma Virus fit in this

category.

Nonendogenous Virus: Virus from external sources present in the MCB.

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 purification process.

Relevant Virus: Virus used in process evaluation studies which 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.

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.

Viral Clearance: Elimination of target virus by removal of viral particles or inactivation of viral

infectivity.

Virus-like Particles: Structures visible by electron microscopy which morphologically appear to

be related to known viruses.

Virus Removal: Physical separation of virus particles from the intended product.

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.
20

Table 1. – Examples of Virus Tests To Be Performed Once at Various Cell Levels

MCB WCB1 Cells at the limit2

Tests for Retroviruses and Other Endogenous Viruses

Infectivity + - +

Electron microscopy3+

3

- +

3

Reverse transcriptase4+

4

- +

4

Other virus-specific tests5as appropriate5- as appropriate5

Tests for Nonendogenous or Adventitious Viruses

In vitro Assays + -

6

+

In vivo Assays + -

6

+

Antibody production tests7+

7

- -

Other virus-specific tests8+

8

- -

 

1

 See text — section III.A.2.

 

2

 Cells at the limit: Cells at the limit of in vitro cell age used for production. (See text — section III.A.3.)

 

3

 May also detect other agents.

 

4

 Not necessary if positive by retrovirus infectivity test.

 

5

 As appropriate for cell lines which are known to have been infected by such agents.

 

6

 For the first WCB, this test should be performed on cells at the limit of in vitro cell age, generated from that WCB; for WCB's

subsequent to the first WCB, a single in vitro and in vivo test can be done either directly on the WCB or on cells at the limit of in

vitro cell age.

 

7

 E.g., MAP, RAP, HAP — usually applicable for rodent cell lines.

 

8

 E.g., tests for cell lines derived from human, nonhuman primate, or other cell lines as appropriate.
21

Table 2. – Examples of the Use and Limitations of Assays Which May Be Used to Test for Virus

Test Test Article Detection Capability Detection Limitation

Antibody production Lysate of cells and their

culture medium

Specific viral antigens Antigens not infectious for animal

test system

In vivo virus screen Lysate of cells and their

culture medium

Broad range of viruses

pathogenic for humans

Agents failing to replicate or produce

diseases in the test system

In vitro virus screen for:

 1. Cell bank 

characterization

2. Production screen

1. Lysate of cells and

their culture medium (for

cocultivation, intact cells

should be in the test

article)

2. Unprocessed bulk

harvest or lysate of cells

and their cell culture

medium from the

production reactor

Broad range of viruses

pathogenic for humans

Agents failing to replicate or produce

diseases in the test system

TEM on:

 1. Cell substrate

 2. Cell culture supernatant

 1. Viable cells

 2. Cell-free culture

supernatant

Virus and virus-like

particles

Qualitative assay with assessment of

identity

Reverse transcriptase

(RT)

Cell-free culture

supernatant

Retroviruses and

expressed

retroviral RT

Only detects enzymes with optimal

activity under preferred conditions. 

Interpretation may be difficult due to

presence of cellular enzymes; background with some concentrated

samples

Retrovirus (RV) infectivity Cell-free culture

supernatant

Infectious

retroviruses

RV failing to replicate or form

discrete foci or plaques in the chosen

test system

Cocultivation

 1. Infectivity endpoint

 2. TEM endpoint

 3. RT endpoint

Viable cells Infectious

retroviruses

RV failing to replicate

 1. See above under RV 

infectivity

 2. See above under TEM1

 3. See above under RT

PCR (Polymerase chain

reaction)

Cells, culture fluid and

other materials

Specific virus

sequences

Primer sequences must be present. 

Does not indicate whether virus is

infectious.

 

1

 In addition, difficult to distinguish test article from indicator cells.
22

Table 3. – Virus Detected in Antibody Production Tests

MAP HAP RAP

Ectromelia Virus2,3 Lymphocytic

 Choriomeningitis

 Virus (LCM)1,3

Hantaan Virus1,3

Hantaan Virus1,3 Pneumonia Virus of Mice

 (PVM)2,3

Kilham Rat Virus (KRV)2,3

K Virus2 Reovirus Type 3 (Reo3)1,3 Mouse Encephalomyelitis

 Virus (Theilers, GDVII)2

Lactic Dehydrogenase

 Virus (LDM)1,3

Sendai Virus1,3 Pneumonia Virus of Mice

 (PVM)2,3

Lymphocytic

 Choriomeningitis Virus

 (LCM)1,3

SV5 Rat Coronavirus (RCV) 2

Minute Virus of Mice2,3 Reovirus Type 3 (Reo3)1,3

Mouse Adenovirus (MAV) 2,3 Sendai Virus1,3

Mouse Cytomegalovirus

 (MCMV)2,3

Sialoacryoadenitis Virus

 (SDAV)2

Mouse Encephalomyelitis

 Virus (Theilers, GDVII)2

Toolan Virus (HI)2,3

Mouse Hepatitis Virus

 (MHV)2

Mouse Rotavirus (EDIM)2,3

Pneumonia Virus of Mice

 (PVM)2,3

Polyoma Virus2

Reovirus Type 3 (Reo3)1,3

Sendai Virus1,3

Thymic Virus2

 1

 Viruses for which there is evidence of capacity for infecting humans or primates.

 

2

 Viruses for which there is no evidence of capacity for infecting humans.

 

3

 Virus capable of replicating in vitro in cells of human or primate origin.
23

Table 4. – Action Plan for Process Assessment of Viral Clearance and Virus Tests on 

 Purified Bulk

Case A Case B Case C2 Case D2 Case E2

Status

Presence of virus1- - + + (+)3

Virus-like particles1- - - - (+)3

Retrovirus-like particles1- + - - (+)3

Virus identified not applicable + + + -

Virus pathogenic for humans not applicable -

4

-

4

+ unknown

Action

Process characterization of

 viral clearance using

 nonspecific model viruses

yes5yes5

yes5yes5yes7

Process evaluation of viral

 clearance using relevant or

 specific model viruses

no yes6yes6yes6yes7

Test for virus in purified bulk not applicable yes8yes8yes8yes8

 

1

 Results of virus tests for the cell substrate and/or at the unprocessed bulk level. Cell cultures used for production which are

contaminated with viruses will generally not be acceptable. Endogenous viruses (e.g., retroviruses), or viruses that are an integral

part of the MCB, may be acceptable if appropriate viral clearance evaluation procedures are followed.

 

2

 The use of source material, which is contaminated with viruses, whether or not they are known to be infectious and/or

pathogenic in humans, will only be acceptable under very exceptional circumstances.

 

3

 Virus has been observed by either direct or indirect methods.

 

4

 Believed to be nonpathogenic.

 

5

 Characterization of clearance using nonspecific model viruses should be performed.

 

6

 Process evaluation for relevant viruses or specific model viruses should be performed.

 

7

 See text under Case E.

 

8

 The absence of detectable virus should be confirmed for purified bulk by means of suitable methods having high specificity and

sensitivity for the detection of the virus in question. For the purpose of marketing authorization, data from at least 3 lots of purified

bulk manufactured at pilot-plant or commercial scale should be provided. However, for cell lines such as CHO cells for which the

endogenous particles have been extensively characterized and adequate clearance has been demonstrated, it is not usually

necessary to assay for the presence of the noninfectious particles in purified bulk.
24

APPENDIX 1

Products Derived from Characterized Cell Banks Which Were

Subsequently Grown In Vivo

For products manufactured from fluids harvested from animals inoculated with cells from

characterized banks, additional information regarding the animals should be provided.

Whenever possible, animals used in the manufacture of biotechnological/biological products

should be obtained from well defined, specific pathogen-free colonies. Adequate testing for

appropriate viruses, such as those listed in Table 3, should be performed. Quarantine procedures

for newly arrived as well as diseased animals should be described, and assurance provided that

all containment, cleaning, and decontamination methodologies employed within the facility are

adequate to contain the spread of adventitious agents. This may be accomplished through the use

of a sentinel program. A listing of agents for which testing is performed should also be included. 

Veterinary support services should be available on-site or within easy access. The degree to

which the vivarium is segregated from other areas of the manufacturing facility should be

described. Personnel practices should be adequate to ensure safety.

Procedures for the maintenance of the animals should be fully described. These would include

diet, cleaning and feeding schedules, provisions for periodic veterinary care if applicable, and

details of special handling that the animals may require once inoculated. A description of the

priming regimen(s) for the animals, the preparation of the inoculum, and the site and route of

inoculation should also be included.

The primary harvest material from animals may be considered an equivalent stage of manufacture

to unprocessed bulk harvest from a bioreactor. Therefore, all testing considerations previously

outlined in section IV. of this document should apply. In addition, the manufacturer should assess

the bioburden of the unprocessed bulk, determine whether the material is free of mycoplasma, and

perform species-specific assay(s) as well as in vivo testing in adult and suckling mice.
25

APPENDIX 2

The Choice of Viruses for Viral Clearance Studies

A. Examples of Useful Model Viruses:

1. Nonspecific model viruses representing a range of physico-chemical structures:

• SV40 (Polyomavirus maccacae 1), human polio virus 1 (Sabin), animal

parvovirus or some other small, nonenveloped viruses;

• a parainfluenza virus or influenza virus, Sindbis virus or some other

medium-to-large, enveloped, RNA viruses;

• a herpes virus (e.g., HSV-1 or a pseudorabies virus), or some other

medium-to-large, DNA viruses.

These viruses are examples only and their use is not mandatory.

2. For rodent cell substrates murine retroviruses are commonly used as specific

model viruses.

B. Examples of Viruses That Have Been Used in Viral Clearance Studies.

Several viruses that have been used in viral clearance studies are listed in Table A-1. However,

since these are merely examples, the use of any of the viruses in the table is not considered

mandatory and manufacturers are invited to consider other viruses, especially those that may be

more appropriate for their individual production processes. Generally, the process should be

assessed for its ability to clear at least three different viruses with differing characteristics.
26

TABLE A-1 – Examples of Viruses Which Have Been Used in Viral Clearance Studies

Virus Family Genus Natural

Host

Genome Env Size

(nm)

Shape Resistance1

Vesicular

Stomatitis Virus

Rhabdo VesiculovirusEquine

Bovine

RNA yes 70 x 150 Bullet Low

Parainfluenza

Virus

ParamyxoParamyxovirusVarious RNA yes 100-200+ Pleo/

Spher

Low

MuLV Retro Type C

oncovirus

Mouse RNA yes 80-110 Spherical Low

Sindbis Virus Toga Alphavirus Human RNA yes 60-70 Spherical Low

BVDV Flavi Pestivirus Bovine RNA yes 50-70 Pleo/

Spher

Low

Pseudo-rabies

Virus

Herpes Swine DNA yes 120-200 Spherical Med

Poliovirus Sabin

Type 1

Picorna Enterovirus Human RNA no 25-30 IcosahedralMed

Encephalomyocarditis Virus

(EMC)

Picorna CardiovirusMouse RNA no 25-30 IcosahedralMed

Reovirus 3 Roe OrthoreovirusVarious RNA no 60-80 Spherical Med

SV40 Papova PolyomavirusMonkey DNA no 40-50 IcosahedralVery high

Parvoviruses

(canine, porcine)

Parvo Parvovirus Canine

Porcine

DNA no 18-24 IcosahedralVery high

1

 Resistance to physico-chemical 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. These viruses are examples only and their use is not considered mandatory.
27

APPENDIX 3

Statistical Considerations for Assessing Virus Assays

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 may be either quantal or quantitative. Quantal methods include infectivity

assays in animals or in tissue-culture-infectious-dose (TCID) 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 plaque assays

where 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 which 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 log10 of the mean. Within-assay variation can be assessed by

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

log10 of the mean estimate established in the laboratory for the assay to be acceptable. 

Assays with lower precision may 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

+ S +a

2 2

.
28

Appendix 3 (continued)

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) it is

evident that a sample of a few milliliters may or may not contain infectious particles. The

probability, p, that this sample does not contain infectious viruses is:

p = ((V-v)/V) n

where V (liter) is the overall volume of the material to be tested, v (liter) is the volume of the

sample and n is the absolute number of infectious particles statistically distributed in V.

If V >> v, this equation can be approximated by the Poisson distribution:

p = e-cv

where 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 1,000

 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 which

would have to be present in the total sample in order 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 due to material

limitations.
29

APPENDIX 4

Calculation of Reduction Factors in Studies to Determine Viral Clearance

The virus reduction factor of an individual purification or inactivation step is defined as the log10

of the ratio of the virus load in the pre-purification material and the virus load in the postpurification material which 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

purification 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 1.

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, while

residual virus infectivity will never be reduced to zero, it may be greatly reduced mathematically.
30

APPENDIX 5

Calculation of Estimated Particles Per Dose

This is applicable to those viruses for which an estimate of starting numbers can be made, such as

endogenous retroviruses.

Example:

1. Assumptions

Measured or estimated concentration of virus in cell culture harvest = 106/mL

Calculated viral clearance factor = >1015

Volume of culture harvest needed to make a dose of product = 1 liter (l03mL)

2. Calculation of Estimated Particles/Dose

Clearance factor >10

( 10 virus units/mL)x ( 10 mL/dose)

15

6 3

 = 

Clearance factor >10

10 particles/dose

15

9

 = <10-6 particles/dose

Therefore, less than one particle per million doses would be expected.