Evaluation of Viral Clearance Studies

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Evaluation of Viral Clearance Studies

• Mahmood Farshid, Ph.D.
• Div. Of Hematology
• OBRR/ CBER/FDA

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Biologics

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

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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

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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

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Common Virus Clearance Methods

• Virus inactivation
• Chemicalorganic 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

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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 log10
  reduction value (LRV)

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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

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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)

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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

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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
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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
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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

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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

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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

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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

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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

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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

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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