Evaluation of Viral Clearance Studies Mahmood Farshid, Ph.D. Div. Of Hematology OBRR/ CBER/FDA AS we know FDA and European regulatory agencies require that the manufacturing process for bio;pgoc products be validated for its capacity to inactivate and remove viruses

Biologics Monoclonal antibodies and recombinant products produced in cell culture Animal derived products Blood and blood products and other human derived products Biopharmaceuticals or biologics fall loosely into three categories: these include

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 Feasibility 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 Lipid-enveloped and nonenveloped 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