Evaluation of Viral Clearance Studies

1. Evaluation of Viral Clearance Studies Mahmood Farshid, Ph.D. Div. Of Hematology OBRR/ CBER/FDA

2. Biologics • Monoclonal antibodies and recombinant products produced in cell culture • Animal derived products • Blood and blood products and other human derived products

3. 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

4. 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

5. 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 Virus Clearance Methods

6. 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)

7. 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

8. 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)

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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