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

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

4. 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

5. 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

6. Common Virus Clearance Methods Virus inactivation: Chemical:organic solvents; pH extremes; solvent detergent; alcohol Physical: Heat treatment (dry heat or pasteurization) Virus removal Precipitation: ammonium sulfate etc. Chromatography: ion exchange; gel filtration; affinity; reverse phase Membrane filtration: Omega, Planova, DV50

7. Validation of Virus Removal/inactivation Include: 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 log 10 reduction value (LRV)

8. Evaluation of the Effectiveness of Viral Clearance Step Test viruses used The design of the validation studies –The validity of scaled-down process –The kinetics of inactivation –Susceptibility to small variations in process parameters (robustness) –The limits of assay sensitivity The log reduction achieved

9. 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)

10. 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

11. Model viruses for human Blood-Derived Products VirusModelEnvelope/Size Resistance Genome(nm) HIV/HTLVHIV-1Yes / RNA 80-130Low HBVDHBVYes / DNA ~ 40Medium HCVBVDV Yes / RNA 40-50Medium HAVHAVNo / RNA 28-30High CMVCMV/HSVYes / DNA 150-200Low-Med /PRV B19PPVNo / DNA 18-26Very high

12. Viruses Used to Validate Product Derived from Cell Lines VirusGenomeSize(nm)EnvelopedResistance MVMss-DNA18-26NoVery high Reo-3ds-RNA60-80NoHigh MuLVss-RNA80-130YesLow PRVds-DNA150-200YesLow-med

13. 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

14. 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 (e.g., bed height, flow rate) Must show product is identical to production scale

15. Criteria for An Effective Virus Clearance Step Significant viral kill 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. GENERAL CONSIDERATIONS Manufacturing processes for blood derived products should contain two effective steps for removal/inactivation of viruses At least one step should be effective against non-enveloped viruses

17. GENERAL CONSIDERATIONS (cont.) At least one stage in a production process must inactivate rather than remove viruses A single step having a large effect gives more assurance of viral safety than several steps having the same overall effect

18. Limitations of Viral Validation Studies Laboratory strains may behave differently than native viruses Source plasma or Igs may have neutralizing antibodies There may exist in any virus population a fraction that is resistant to inactivation Scale-down processes may be differ from full-scale

19. 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