Note for guidance

Biologiculs

(1991)19,247-251

EEC REGULATORY DOCUMENT NOTE FOR GWQANCE Validation Committee

of Virus Removal and hwtiva~n

for Proprietary

Medicinal Products: Ad Hoc Working Party on Biotechnology/Pharmacy and Working Party on Safety Medicines

Introduction A feature common to all biologicals of which production has involved the use of any material of animal or human origin is the risk of viral contamination. Potential viral contamination of a biological may arise from the source material, e.g. cell banks of animal origin, human blood, human or animal tissues or as adventitious agents introduced by the production process, e.g. the use of animal sera in cell culture. In the past, a number of biologicals administered to humans have been contaminated with viruses, and in several instances the contaminant virus was identified many years after the product had been introduced into the market. Yellow fever vaccine was contaminated by avian leukosis virus by virtue of its production in infected hens eggs and also by hepatitis B virus contained in human sera added to the vaccine as stabilizer. Other examples are SV40 contamination of poliovirus and adenovirus vaccines prepared in the 1950s on primary cultures of kidney cells obtained from rhesus monkeys which naturally harboured a clinically inapparent infection with SV40. Also, HIV has contaminated blood products, and human growth hormone extracted from the pituitaries of cadavers was implicated in the transmission of the aetiological agent responsible for CreutzfeldtJakob disease. Three principal complementary approaches can be adopted to control potential viral contamination of biologicals: selecting and testing source material for the absence of viruses, testing the capacity of the Requests for reprints should be made to: Permanent Office Biostandards, Case Postale 456, CH-1211, GenBve, Switzerland This paper is covered by EC copyright. It is published with the agreement of the Office of EC Publications. 1045-1056/91/030247+05$03.00/0

Protadures

production processes to remove or inactivate viruses and testing the product at appropriate stages of production for freedom from contaminating viruses. All testing suffers from the inherent limitation of quantitative virus assays, of which the ability to detect low virus concentrations depends for statistical reasons (see Appendix 1) on the size of the sample. Therefore, no single approach will necessarily establish the safety of a product and, due to the hazardous nature of many potential viral contaminants, establishing the freedom of a biological from infectious virus will in many instances not derive solely from direct testing for their presence, but also from a demonstration that the purification regime is capable of removing or inactivating them. Validation of the purification and/or inactivation procedures can play an essential and important role in establishing the safety of biological products especially when there is a high potential for the source material to be contaminated with a virus known to be pathogenic for man. Also, since many instances of contamination in the past have occurred with agents of which the presence was not known or even suspected, a validation will provide a measure of confidence that any unknown, unsuspected and harmful agent may be removed. The need for extensive validation studies is greatest for products derived from variable source materials, such as blood, tissues and organs of human or animal origin, or where, for technical or practical reasons, it is not possible to employ a fully validated and tested cell bank. In contrast, the justification for extensive validation studies is less where the product derives from well characterized source material, such as a fully characterized cell bank. In general, some validation of the ability of the process to remove or inactivate viruses will be

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expected, but the extent of the studies required will depend on the product and will be reviewed on a case-by-case basis. The objective of validation is to estimate quantitatively (see Appendix 2) the overall level of virus reduction obtained along the various stages of purification and/or any viral inactivation stages. This will be achieved by the deliberate addition (‘spiking’) of significant amounts of a virus to the crude material to be purified and to different fractions obtained during the various purification stages, and its removal or inactivation during the subsequent stage of purification and/or inactivation determined. The validation of virus elimination from biologicals is a complex issue because of the variety of source material and the diversity of procedures used for preparation. The intention of this note for guidance is to provide only a general framework for validation studies and the virological approach which should be used in the design of virus validation studies. The manufacturers should adjust the recommendations presented here to their specific product, taking into consideration the nature of the source material, the procedures used for production and purification and any other factors which can have consequences on this safety issue. The approach used by producers in validation studies for virus elimination should be explained and justified. This note for guidance concerns the validation of virus removal and/or inactivation procedures for the following categories of biologicals: 1. products derived from cell lines of human or animal origin; 2. products derived from organs or tissues of human or animal origin; 3. products biological Sources

derived fluids.

from blood or urine

or other

of viral contamination

Viral contamination of biologicals can arise in the following ways Source material may be contaminated with a virus indigenous to the species of origin. Blood can harbour many viruses and the use of products derived from human blood or plasma has caused diseases including hepatitis B, C and other non-A, non-B hepatitis and AIDS. Human blood can carry other viruses like HTLV-1, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), herpesviruses 6 and 7 (HHVG or 7) or parvovirus B19, which are

document

responsible for various clinical syndromes. Murine viruses, some of which are pathogenic for man, may contaminate monoclonal antibodies derived from murine hybridomas. Cell lines, which are intended to be used for genetic manipulations, may be contaminated by viruses and, therefore, they should be chosen carefully and tested for freedom of adventitious agents even before genetic manipulation, in order to start with a clean material. Cells may have a latent or persistent infection which may be transmitted vertically from one cell generation to the next, since the viral genome persists within the cell, e.g. a herpesvirus or a retrovirus, and which may be expressed unexpectedly as infectious virus. The process of construction of a production cell line may introduce a contaminant virus indigenous to another species, e.g. human lymphoblastoid cell lines secreting monoclonal antibodies can be infected with a murine retrovirus when murine feeder cells have be used. Adventitious viruses may be introduced by the use of contaminated animal products in the production process, e.g. cell cultures may be contaminated with bovine viruses through the use of bovine sera or a murine monoclonal antibody used in affinity chromatography may contaminate a product with a murine virus. The possibility of contamination by an adventitious virus because of a failure in. GMP should also be considered. The choice of viruses for validation A major issue in performing a validation is the determination of which viruses should be used. Such viruses fall into two categories: relevant viruses and model viruses. (a) Relevant viruses. Relevant viruses are viruses which are known to, or likely to, contaminate the source material or other materials used in the production process. The purification and/or inactivation process must be shown to remove or inactivate those viruses or appropriately similar viruses. Examples of situations in which a relevant virus is appropriate are: l Cell lines

derived from rodents usually contain endogenous retroviral particles, which may be infectious (C-type particles) or non-infectious (intracisternal A-type particles). It is therefore necessary to validate the capacity of the purification process to remove murine retro-

Virus removal/Inactivation

viruses where cells of rodent origin are used in production. l Human lymphoblastoid cells secreting monoclonal antibodies are usually obtained by deliberate addition of Epstein-Barr virus (EBV) to B lymphocytes in order to immortalize them. EBV establishes a latent or a persistent infection in lymphoblastoid cells. It is therefore necessary to check the ability of the purification process to remove EBV or another suitable herpes virus during the purification of a human monoclonal antibody. l Human clotting factor concentrates have been contaminated by HIV. Thus, HIV must be taken as a relevant virus for products derived from human blood.

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249

of a thoroughly characterized non-primate cell bank will differ considerably from that of product derived from human blood plasma. Examples of useful model viruses representing a range of physicochemical structures could be: SV40 or Sabin type 1 poliovirus or some other small non-enveloped virus l A parainfluenza virus or influenza virus, a murine retrovirus or a lentivirus such as Visna, or some other medium to large enveloped RNA l

Vii-US.

l Vaccinia

or a herpes virus, e.g., HSV-1, other medium to large DNA virus.

or some

These viruses are examples only and their use is not mandatory. Viruses which can be grown to high titre will be desirable, although this may not always be possible. There should be an efficient and reliable assay for the detection of the viruses used, before and after processing through a stage. Consideration should be given to the health hazard which certain viruses may pose to the personnel performing the validation. Examples of viruses which have been used in the past in validation studies are given in Table 1. However, since these and the viruses mentioned above 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.

(b) Model viruses. If the use of relevant viruses does not encompass viruses with a wide range of physico-chemical properties, then, where required, validation should be performed with ‘model’ viruses in order to do so. A preference should be given to viruses which display a significant resistance to physical and/or chemical agents. Although the source material may not necessarily be a host for specific model viruses, reduction values obtained for such viruses provide useful information on the ability of the production process to remove and/or inactivate viruses in general. The choice and number of viruses used will also be influenced by the quality and characterization of the source material and the production process, e.g. the validation requirements

Table 1. Examples of viruses which have been used in virus validation studies. This table gives an incomplete list of viruses which have been used in validation studies either as relevant viruses, i.e. viruses considered to be potential contaminants of the source material, or as model viruses. Consequently, 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

Virus

Family

Natural host

Poliovirus, Sabin type 1 Reovirus 3 sv 40 Murine leukemia virus (MuLV) Human immunodeficiency virus (HIV) Vesicular stomatitis virus Parainfluenza virus Pseudorabies virus

Picorna Reo Papova

Man Various Monkey

RNA RNA DNA

No No No

25-30 60-80 45

Retro

Mouse

RNA

Yes

80-l 10

Retro Rhabdo Paramyxo Herpes

Man Bovine Various Swine

RNA RNA RNA DNA

Yes Yes Yes Yes

Genome

Enveloped

Size (nm)

80-100 80-90 150-300 120-200

Shape

Resistance to physico-chemical reagents

Icosahedral Spherical Icosahedral

Med. High High

Spherical

Low

Spherical Bullet shaped Pleo-Spher Spherical

Low Low Low Med.

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Products derived from ovine or bovine tissues raise the problem of contamination by scrapie or scrapie-like agents which accumulate in central nervous system and lymphoid tissue. Validation studies can be performed in rodents using rodent-adapted scrapie agent. So far, the most satisfactory approach, however, has been to ensure that the source materials are free of these agents, for example by obtaining them from a geographical region or from a closed herd free of these diseases. Design and implications

of validation

studies

It will be inappropriate to introduce any virus into the production facilities because of GMP constraints. Therefore, the validation should be conducted in a separate laboratory equipped for virological work and performed by staff with virological expertise in conjunction with production bio-engineers who should be involved in designing and preparing a scaled-down version of the purification process. The validity of the scaling down should be demonstrated. The level of purification of the product achieved with stages of the scaled-down version which contribute to purification of the product should mimic as closely as possible the production process. Reduction in virus infectivity may be achieved by removal of virus particles or by inactivation of the infectivity. In both cases, validation is performed following a similar approach. 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 or an additional inactivation/removal step should be introduced. Where virus removal is demonstrated, the distribution of the removed virus should be investigated. Assurance should be provided that any virus potentially retained by the production system would be adequately destroyed prior to reuse of the system, e.g. by sanitization of columns etc. Essential stages of the purification process should be individually assessed for their ability to remove or inactivate virus and careful consideration should be given to the exact definition of an individual stage. The overall reduction factor should be determined from the sum of the individual reduction factors. The overall reduction factor for relevant viruses for a production process should be substantially greater than the maximum possible virus titre which could potentially occur in the source material. For all viruses, manufacturers will be expected to demonstrate large reduction factors or to justify the

acceptability of the reduction factors obtained. Results will be considered on a case-by-case basis. Whilst consideration should be given to performing the validation in the presence of the product, some products, e.g. interferon, may interfere with the virus assay. Attention should be paid to the dilution effect of adding the viral suspension to the product, which might alter the protein concentration so that that test sample is no longer identical to the product obtained in full-scale. Consideration should be given to the minimum quantity of virus which can be assayed reliably. Care should be taken that the products, column buffers, etc., are not toxic for the cell line used for the virus assay. The validation exercise should include parallel control assays to assess loss of infectivity of the virus due to such reasons as dilution or storage of samples before titration. For validation of viral inactivation, the source or intermediate material should be spiked with infectious virus and the reduction factor calculated. Whenever possible, it is desirable to obtain a kinetics of viral inactivation in order to measure the slope of the curve and to determine the theoretical time necessary to inactivate the total virus population. 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 validation should, therefore, be planned in such a way that samples are taken at different times and an inactivation curve constructed. Limitations

of validation

studies

Validation studies are useful in contributing to the assurance that an acceptable level of safety in the final product is established and do not by themselves establish safety. However, a number of factors in the design and execution of virus validation experiments may lead to an incorrect estimate of the ability of the process to remove virus infectivity. These factors include the following. Virus preparations used to validate a production process are likely to be produced in tissue culture. The behaviour of 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. The ability of the overall process to remove infectivity is often expressed as the sum of the logarithm of the reductions at each step. This is a useful way of calculating the, overall reduction factor, though

Virus removal/inactivation

there are some situations in which it may not be valid to add logarithmic reductions, e.g. where reduction depends on virus adsorption to a matrix such as column bed material. Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid initial phase is followed by a slower phase (see above). It is possible that virus escaping a first inactivation step may be more resistant to subsequent steps. As a consequence, the overall reduction factor is not necessarily the sum of reduction factors calculated from each individual step, using each time a fresh virus spike-suspension. 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 expression of reduction factors as logarithmic reductions in titre 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 eight log,, infectious units per ml by a factor of eight log,, leaves zero log,, or one infectious unit per ml. Pilot-scale processing may differ from full-scale processing despite care taken to design the scaleddown process. Changes to the production process may necessitate a new validation study.

Appendix 1. Probability at low concentrations

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251

the Poisson distribution: p = e-Cu where c is the concentration of infectious particles per litre. If a sample volume of 1 ml is tested, the probabilities P at virus concentrations ranging from 10 to 1000 infectious particles per litre are: c 10 100 P o-99 0.90

1000 0.37

This indicates that for a concentration of 1000 viruses per litre, in 37% of sampling, 1 ml will not contain a virus particle.

Appendix

2. Calculation

of reduction

factors

The virus reduction factor of an individual purification or inactivation step is defined as the log,, of the ratio of the virus load in the pre-purification material and the virus load in the post-purification material which is ready for use in the next step of the process. If the following abbreviations are used: Starting u’ lo”.

material:

Final material: the individual according to

of virus&

vol u’; titer

lo”‘;

virus

load:

vol u”; titer lo”“; virus load: ~“10”“. reduction

factors Ri are calculated

1oR’ = u’10”‘/u”10””

At low virus concentrations (e.g. in the range of 10 to 1000 infectious particles per litre) it is evident that a sample of a few millilitres may or may not contain infectious particles. The probability P that this sample does not contain infectious viruses is: P = ((V-u)/V) where V (litre) is the overall volume of the material to be tested, u (litre) the volume of the sample and n the absolute number of infectious particles statistically distributed in V. With V >> u this equation

procedures

can be approximated

by

This formula takes into account both the titres 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 has normally to be greater than 1. The overall reduction factor for a complete production process is the sum of the reduction factors of the individual steps. It represents the logarithm of the ratio of the virus load at the beginning and at the end of the process.