Evaluation of Viral Clearance Studies

Mahmood Farshid, Ph.D.

Div. Of Hematology

OBRR/ CBER/FDA

Biologics

• Monoclonal antibodies and recombinant products produced in cell culture

• Animal derived products

• Blood and blood products and other human derived products

Risk Reduction Strategies

• Donor Screening: – donor history assessment,– written and oral questionnaire

• Donors Testing:– Anti- HIV-1/2, HIV-1 p24 Ag ,anti-HCV, HBsAg , anti HBc,

anti-HTLV-1/2, syphilis – (NAT for HCV and HIV)

• Pharmacovigilance/ look back studies• Inactivation/Removal

– Validating the manufacturing processes for removal / inactivation of viruses

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

Important Factors for Validation of Photochemical Processes

• Concentration of the chemical with changes in donor plasma/cell volume

• Lipemia and other impurities in the donor unit• The degree of impurity removal prior to treatment• The total quantity (fluence) of light as well as its

intensity and wavelength • Plastic bag transparency• Sample depth• Mixing efficiency • Residual level of chemical and its breakdown

products

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

Other Considerations

• Manufacturing processes for blood derived products must contain two effective steps for removal/inactivation of viruses

• At least one step should be effective against non-enveloped viruses

• At least one stage in a production process must inactivate rather than remove viruses

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

Factors influencing TSE clearance

• Selection of TSE agent strain– CJD, vCJD or GSS

• Infectivity assay– Animal species– Genotype– Period observed

• Spiking preparation– Crude brain homogenate – Microsomal preparation– Bolton preparation