Virus Removalinactivation Methods

A complementary approach to screening and testing for viruses is to treat the product in order to remove physically or inactivate any viruses that might be present (Roberts 1994, 1996). This approach (hereafter termed 'virus reduction' for convenience) has the potential advantage that it can be effective against a wide spectrum of viruses. A virus-reduction step may be part of the standard purification process for the product that may fortuitously also remove viruses. However, it may not be possible to modify such a step to maximise virus reduction. Alternatively, a specific or dedicated step may be used which has no other purpose.

The ideal method would bring about a high level of virus reduction, be effective against all types of virus, and be robust, i.e. effective over a wide range of process conditions. In practice such a goal is not easily accomplished. For instance, most non-enveloped viruses are relatively resistant to inactivation and are not easy to remove by virus filtration due to their small size. In 'spiking' studies (see Section 19.4.1) the level of virus titre reduction that is considered substantial for a single virus reduction step is about 104-fold (commonly referred to as 4 logs). The titre of the virus stock and the toxicity of the samples to be assayed may mean that this is difficult to demonstrate in practice. The reduction values from several process steps can be combined to give an estimate for the total purification process, although this is only strictly applicable where different reduction mechanisms are involved. This can give figures in the order of, for example, around 10 logs for small resistant viruses and 20 logs or more for viruses of low to medium resistance. Quantitative risk assessment, taking into account the level of virus that is likely to be present in the cell supernatant harvest, is used to determine if the level of virus reduction is sufficient. The target is to ensure that the concentration of infectious virus in a therapeutic dose of product reaches a level several orders of magnitude below that which could result in an infection. Some further guidance on the residual levels that may be considered acceptable is given in the European Pharmacopoeia, which requires that containers of final sterile products contain less than 10~6 viable microorganisms per container. In addition, the application for which the product is to be used is also important. For instance, where the product is to be used for treating a life-threatening condition for which no current treatment exists, e.g. some cancers, a higher degree of risk may be acceptable. Thus the acceptability of any virus reduction procedure for a specific product or application must be judged on a case-by-case basis. Because of the limitation of any particular removal or inactiva-tion method with regard to the range of viruses affected, there has been pressure to include two or more specific virus-reduction steps in a purification process. In addition, the methods used should act by different mechanisms. The inclusion of several virus reduction steps also has the advantage that, in the unlikely case of one step failing, there should still remain an adequate margin of virus safety. The use of specific virus reduction steps, rather than just the use of standard process steps, is advocated. This is because these are generally more readily controlled, can be specifically optimized for virus reduction and can be more readily validated. An example of a protein purification process, incorporating several virus reduction steps is shown in Figure 19.1. The virus reduction values obtained for various viruses in virus spiking studies is given in Table 19.3.

Figure 19.1 Monoclonal antibody purification process with virus reduction steps. Virus reduction steps are shown in bold boxes. These include dedicated virus reduction steps as well as purification steps shown, in validation studies (Table 19.3), also to make a significant contribution to virus reduction.

VIRUS REMOVAL/INACTIVATION METHODS Table 19.3 Example of virus reduction during the purification of a monoclonal antibody.

Virus reduction (log10)

VIRUS REMOVAL/INACTIVATION METHODS Table 19.3 Example of virus reduction during the purification of a monoclonal antibody.

Virus reduction (log10)

Stepa

HSV-16

Sindbis

SFV6

Vaccinia

MMV6

Polio-1

Solvent/detergentc

>5.7

>5.7

>6.7

nd

>3.6

0d

Protein-G affinity chromatography8

7.1

6.0

nd

nd

nd

3.1

Ion-exchange chromatography

5.5

nd

nd

nd

nd

2.4

Size-exclusion chromatography

2.7

nd

nd

nd

nd

1.2

Virus filtration (50 nm)f

>7.6

6.7

6.7

6.8

nd

1.0

Total reduction

>28.6

>18.4

>13.4

6.8

>3.6

7.7

aPurification of human anti-D monoclonal antibody BRAD-3

bHSV-1, herpes simplex virus-1; SFV, Semliki Forest virus; MMV, minute virus of mice c0.3 % tri-n-butyl phosphate/1 % Triton X-100 for 1 hr dNon-enveloped virus, thus not susceptible to solvent/detergent treatment eStep includes elution at acidpH which contributes to virus reduction by causing inactivation fUltipore DV-50 (Pall) or Viresolve-180 (Millipore) or equivalent, e.g. Planova 35 (Asahi)

nd = Not Done aPurification of human anti-D monoclonal antibody BRAD-3

bHSV-1, herpes simplex virus-1; SFV, Semliki Forest virus; MMV, minute virus of mice c0.3 % tri-n-butyl phosphate/1 % Triton X-100 for 1 hr dNon-enveloped virus, thus not susceptible to solvent/detergent treatment eStep includes elution at acidpH which contributes to virus reduction by causing inactivation fUltipore DV-50 (Pall) or Viresolve-180 (Millipore) or equivalent, e.g. Planova 35 (Asahi)

In addition to the inclusion of effective virus reduction steps, it is essential to ensure that virus safety is not subsequently compromised by recontaminating the product during the manufacturing process. The various precautions taken to prevent recontamination are part of good manufacturing practice (GMP) and include the use of closed processing systems and/or physical separation in different rooms or areas (see Chapters 12 and 34). Effective cleaning and sterilization procedures for equipment are also used (see Chapter 14). One approach commonly employed is to process product intermediate, after a major virus reduction step, in a segregated area commonly known as a 'virus-secure area'. One advantage of using a terminal virus inactivation step on the product in the final container is that there is no possibility of recontamination.

A major factor that must be considered with any virus removal/inactivation method is the possibility that the step may have adversely affected the product. Most effective virus inactivation methods are relatively severe, and a compromise between maximum virus inactivation and maximum product yield must be reached. The activity, structure, immunogenicity or thrombogenicity of the product can also be affected. For instance, the heat-treatment of coagulation factors has, in some cases, led to the production of neoantigens and thus the induction of inhibitors, i.e. antibodies directed to the protein, in recipients (Rosentaal et al. 1993). This has been a problem with some heat-treated factor VIII products. In the case of antibodies, the product can be altered in such a way that anti-complement activity can result. While virus filtration is a very gentle procedure, product losses on the filter itself can be high if the size of the protein is too near to that of the filter pores.

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