TSE Agent Clearance Issues

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TSE Agent Clearance Issues

• TSE Advisory Committee
• February 20, 2003
• Dorothy Scott, M.D.
• DH/OBRR/CBER/FDA

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Paradigm Validation of Virus Removal/inactivation
Includes

• Scaling down process steps
• Spiking appropriate steps with high titer of
  infectious agent (actual or model)
• Determination reduction factors for each step
• Summing reduction factors from orthogonal
  processes to give a total log10 reduction value

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Scale-Down of Purification Steps

• Usually 1/10 to 1/100 scale no set guidelines
• 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 Acceptable Pathogen Detection Assays

• Accuracy
• Assay repeatability and reproducibility
• Linearity
• The limit of detection (LOD)
• The limit of quantitation (LOQ)
• Assay robustness and reproducibility

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TSE Clearance Evaluation Example
TSE Spike Plasma Cryoprecipitation Cryop
recipitate (FVIII) Cryopoor
Plasma Supernatant Albumin, IGIV,
?1PI, ATIII, etc.
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Published TSE Clearance Studies for Plasma
Fractionation

1. Brown, P et al, Transfusion 1998 38810-6
2. Brown, P et al, Transfusion 1999 39 1169-78
3. Lee, DC et al, J. Virol. Meth. 2000 84 77-89
4. Foster, PR et al, Transfusion Science 2000
   2253-56
5. Foster, PR et al, Vox Sanguinis 2000 7886-95
6. Lee, DC et al, Transfusion 2001 41 449-55
7. Cai, K et al, Biochem Biophys. Acta 2002 1597
   28-35
8. Stenland, JS et al, Transfusion 2002 421497-1500
9. Vey, M et al, Biologicals 2002 30187-96
10. Reichl, HE et al, Vox Sanguinis 2002 83137-45

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Challenges in Studies of Clearance of TSE Agents

• What source of infectivity to use
• Brains preparations from experimentally infected
  animals most easily available
• Hamsters (scrapie)
• Mice (GSS, BSE)
• BSL-3 facility needed to study vCJD, BSE
• PrpSc partitioning similar when source is human
  (CJD, vCJD), or animal TSEs (Stenland et al,
  Transfusion 42 1497-1500, 2002 single study)
• What form of infectious agent most relevant to
  blood?
• Brain homogenate
• Subcellular membrane fractions
• Membrane-free infectious material

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Challenges in Studies of Clearance of TSE Agents

• The lower limits of assay sensitivity (2-3 logs),
  and upper limits of titers available for spiking
• Range of infectivity removal detectable 4-5 logs
• Throughput experiments to assess additiveness
  of clearance steps therefore have limitations
• What assays are best to measure outcomes
• In vivo infectivity (time, expense)
• In vitro surrogates measurements of PrpSc
• Bridging in vivo to in vitro results (Transfusion
  2001 41 449-55)
• Mass balance retention TSE agents by columns
  loss of mass balance

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Challenges in Evaluating Clearance of TSE Agents

• How much reduction is enough? (risk assessment)
• How many disparate clearance steps should there
  be?
• What steps can be summed, which cannot?
• Summed reduction factors for similar steps, e.g.
  EtOH precipitation

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TSE Clearance depends upon specific
characteristics of starting material and process
conditions Examples

• Partitioning of infectivity depends upon pH,
  ionic strength, and alcohol concentration
• Cryoprecipitation methods may influence degree of
  clearance
• Depth filtration effectiveness depends upon
  filter used and/or properties of starting material

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Example (1) PrpSc Partitioning is
condition-dependent Cai, A. et. al. Biochem.
Biophys. Acta 597 28-35, 2002

• Scrapie brain homogenate spiked into buffers with
  varied
• EtOH concentrations
• Salt concentrations
• pH
• Incubation
• Centrifugation
• Measurement PrpSc in supernatant

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Parameters Influencing Prpsc Partitioning

• Precipitation best at
• Mildly acidic pH
• With EtOH
• At higher pH, with salt and EtOH

Cai, K. et. al. Biochem Biophys Acta 1597(1)
28-35, 2002
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Example (2) Cryoprecipitation variable clearance
among studies with different conditions

• FVIII partitions with cryoprecipitate
• 2. Clearance of PrpSc in cryoprecipitation
• - 1 log clearance in effluent (Lee et al.,
• Transfusion 41 449-55, 2001)
• 1 log clearance in effluent (Brown et al.,
• Transfusion 38 810-16, 1998)
• lt1 1.7 logs clearance in precipitate
• (Foster et al., Vox Sang 7886- 95, 2000)

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Example (3) Clearance PrPsc (microsomal spike) by
Depth Filtration Influence of Starting
Materials and Filter
Starting Material Depth Filter Reduction Factor
(log10) Fr V (albumin) Seitz KS80 gt
4.9 Fr V (albumin) CUNO Delipid 1 2.3 S I
III (IGIV) Millipore AP20 lt 1 Fr II
(IGIV) Seitz K200 gt 2.8 Foster et. al., Vox
Sang 78 86-95, 2000 Fr I supernatant (IGIV,
albumin) Supra P80 lt 1 Fr V supernatant
(albumin) Supra P80 gt 1.1 Fr V supernatant
(albumin) Prp-sc spike Supra P80 gt 2.4 Vey
et al, Biologicals 30187-96, 2002
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TSE Clearance and the Manufacturing Process

• Manufacturing processes are highly individual
• Cohn-Oncley process variations
• Other fractionation methods
• Variations in downstream processing/purification
  of products (e.g. column chromatography)
• Rigorous demonstrations of TSE clearance
  therefore need to be based upon the specific
  manufacturing process
• Published studies may prove useful to identify
  steps with potential for TSE clearance

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Evaluation of TSE clearance studies from
industry, to support labeling claims of lowering
possible TSE risk

• Characterization of spiking agent
• Accurately scaled-down processes
• Robust and reproducible experiments
• Well-characterized assay for TSE infectivity
• Bridging binding assays to bioassays
• Estimated logs clearance of TSE by processing
  steps (reduction factor and clearance factor)
• Demonstration of mass balance
• Demonstration, where relevant, that non-
  orthogonal (similar) clearance steps are/are not
  additive

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Evaluation of Submissions to Support Labeling
Claims

• Clearance beltline to support labeling
• At least 2 orthogonal steps with gt 4 logs
  clearance (total 8 logs)
• At least 2 steps demonstrated to be additive with
  gt 4 logs clearance/step (total 8 logs)
• ? At least 2 steps (orthogonal or demonstrated to
  be additive) with gt 3 logs/step (total 6 logs)
• Is a single clearance step of gt 4 logs sufficient
  if robust and reproducible?
• Are clearance steps of gt 2 logs reliable if they
  are robust and reproducible?
• Cumulative clearance/risk analysis

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Labeling for TSE Risk

• Current proposal Because this product is made
  from human plasma, it carries a risk of
  transmitting infectious agents, e.g. viruses,
  and, theoretically the vCJD agent. It has been
  demonstrated that the manufacturers
  manufacturing process provides substantial
  clearance of agents similar to those causing CJD
  and vCJD. Thus the theoretical risk of
  transmission of CJD or vCJD is extremely remote.
• Future improvements in risk assessment,
  understanding of plasma infectivity, and study
  methods could provide a basis for additional
  labeling content