Validation of Viral Clearance

BY, Nirmal Patel M Pharm (Bio Tech)

S K Patel College Of Pharmaceutical Education & Research

Ganpat University

Viral clearance

The removal of viral contamination using specialized membranes or chromatography. In order to ensure that therapeutic drugs derived from certain sources are fully rid of any viral contamination, these protein solutions undergo viral clearance to inactivate or remove viral materials.

Biologics

• Monoclonal antibodies and recombinant products produced in cell culture

• Animal derived products

• Blood and blood products and other human derived products

The Aim of Viral Validation

• To provide evidence that the production process will effectively inactivate/remove viruses which could potentially be transmitted by the product

• To provide indirect evidence that the

production process has the capacity to inactivate/remove novel or yet undetermined virus contamination

Virus Clearance Methods

Virus inactivation:• Chemical: organic

solvents; pH extremes; solvent/detergent; alcohol

• Physical: Heat treatment (dry heat or pasteurization)

• Combined Methods: Photochemical

Virus removal:• Precipitation:

ammonium sulfate etc.• Chromatography: ion

exchange; gel filtration; affinity; reverse phase

• Membrane filtration: Omega, Planova, DV50

Validation of Virus Removal/inactivation

• Scaling down process steps• Spiking appropriate steps with high titer of

infectious virus (relevant or model)• Determining virus reduction factors for each

step• Summing reduction factors to give a total log10

reduction value (LRV)

Evaluation of Viral Clearance Steps

• Test viruses used• The design of the validation studies– Validity of scaled-down process– Kinetics of inactivation– Robustness– Assay sensitivity

• The log reduction

Virus Selection

• Viruses that can potentially be transmitted by the product (relevant or specific model viruses)

• Viruses with a wide range of physicochemical properties to evaluate robustness of the process (non-specific model viruses)

Virus Selection

• The nature of starting material– Cell lines– Human derived – Animal derived

• Feasibility– Availability of a suitable culture system– Availability of high-titer stocks– Reliable methods for quantification

Model viruses for human Blood-Derived Products

Virus Model Envelope/ Size Resistance Genome (nm)

HIV/HTLV HIV-1 Yes / RNA 80-130 Low

HBV DHBV Yes / DNA ~ 40 Medium

HCV BVDV Yes / RNA 40-50 Medium

HAV HAV No / RNA 28-30 High

CMV CMV/HSV Yes / DNA 150-200 Low-Med/PRV

B19 PPV No / DNA 18-26 Very high

Viruses Used to Validate Product Derived from Cell Lines

Virus Genome Size(nm) Enveloped Resistance

MVM ss-DNA 18-26 No Very high

Reo-3 ds-RNA 60-80 No High

MuLV ss-RNA 80-130 Yes Low

PRV ds-DNA 150-200 Yes Low-med

Virus Selection

• DNA and RNA genome (single and double-stranded)

• Lipid-enveloped and nonenveloped

• Large, intermediate, and small size

• From very highly resistant to inactivation to very easily inactivated

Scale-Down of Purification Steps

• Usually 1/10 to 1/100 scale• Must keep buffers, pH, protein

concentration, and product the same as full scale manufacturing

• Must keep operation parameters as close to full scale as possible

• Must show product is identical to production scale

Criteria for An Effective Virus Clearance Step

• Significant viral clearance• Reproducible and controllable at process scale

and model-able at the laboratory scale• Should have minimal impact on product yield

and activity• Not generate neo-antigens or leave toxic

residues

Common Features

• Involve "spiking" experiments, in which large amounts of a virus is added to the test article

• The reduction in the amount of added virus by the manufacturing step in question is then measured

• Appropriate controls are included to insure that the measurement of the amounts (titers) of the virus does not change the performance of the scaled-down manufacturing step

• Additional controls are included to confirm viability of the indicator cells and the infectivity of the virus

Common features

• In vitro analyses are most commonly used to quantify virus levels in the course of a validation study.

• These may take the form of "plaque assays" or assays that measure "cytopathic effect", both of which are performed in tissue culture.

• These assays measure infectivity of the virus used in the study. Biochemical assays may also be encountered, such as antigen-based or nucleic acid assays (e.g., PCR).

• These are acceptable when predictive of infectivity. Finally, some studies may use animal models such as primates or ducks, but these have become less frequent because they are expensive and adequate alternative methods are available.

Common features

• Most validation studies are performed with model viruses sharing characteristics of the relevant human viruses.

• The selection of appropriate models is of critical importance. • Among the human viruses of concern, only HIV and HAV have

appropriate in vitro systems with which their titers can be measured.

• The clearance of HBV and HCV, by some manufacturing processes, has been validated in primate models, but these studies are not common today.

Common features

• A validation study may be acceptable even if some detectable virus is found, as these studies should be designed to add many times more virus to the test article than would be encountered in actual practice.

• Large amounts of virus ("high titers") permit more precise quantification and provide safety margins to the manufacturing process.

Common Features

• When more than one manufacturing step in a process has been validated to clear a particular virus, the overall clearance of the process is the product of the two steps (often calculated by adding log reduction factors), provided that the two steps operate by independent principles.

• For instance, the results of a filtration step and a heating step may be combined because they operate by different mechanisms. On the other hand, merely repeating a step would not double the viral clearance because here would be no second mechanism involved; an additive effect cannot be presumed.

Common features

• Validation of the process or processes serves to establish that the perating parameters, used during normal production, are appropriate.

• Changes may be made based on the validation study. It is therefore important that the parameters established during the study are those used in actual manufacture.

Limitations of Viral Validation Studies

• Laboratory strains may behave differently than native viruses

• There may exist in any virus population a fraction that is resistant to inactivation

• Scale-down processes may be differ from full-scale

• Source plasma or Igs may have neutralizing antibodies

Limitations of Viral Validation Studies

• Total virus reduction may be overestimated because of repeated and similar process steps

• The ability of steps to remove virus after repeated use may vary.

How Much Clearance?

• The total viral reduction should be greater than the maximum possible virus titer that could potentially occurs in the source material

• A manufacturing process must be validated to remove/inactivate three to five orders of magnitude more virus than is estimated to be present in the starting materials

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