1. 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

2. 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

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 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

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 Feasibility 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 Low
HBV DHBV Yes / DNA ~ 40 Medium
HCV BVDV Yes / RNA Medium
HAV HAV No / RNA High
CMV CMV/HSV Yes / DNA Low-Med
/PRV
B19 PPV No / DNA Very high

11. Viruses Used to Validate Product Derived from
Cell Lines
Virus Genome Size(nm) Enveloped Resistance
MVM ss-DNA No Very high
Reo-3 ds-RNA No High
MuLV ss-RNA Yes Low
PRV ds-DNA Yes Low-med

12. 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

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