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TSE Agent Clearance Issues. TSE Advisory Committee February 20, 2003 Dorothy Scott, M.D. DH/OBRR/CBER/FDA. Paradigm: Validation of Virus Removal/inactivation Includes:. Scaling down process steps Spiking appropriate steps with high titer of infectious agent (actual or model)

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tse agent clearance issues

TSE Agent Clearance Issues

TSE Advisory Committee

February 20, 2003

Dorothy Scott, M.D.


paradigm validation of virus removal inactivation includes
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
scale down of purification steps
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
criteria for acceptable pathogen detection assays
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

TSE Clearance Evaluation: Example

TSE Spike Plasma

Cryoprecipitation Cryoprecipitate


Cryopoor Plasma Supernatant

Albumin, IGIV,

1PI, ATIII, etc.

published tse clearance studies for plasma fractionation
Published TSE Clearance Studies for Plasma Fractionation
  • Brown, P et al, Transfusion 1998 38:810-6
  • Brown, P et al, Transfusion 1999 39: 1169-78
  • Lee, DC et al, J. Virol. Meth. 2000 84: 77-89
  • Foster, PR et al, Transfusion Science 2000 22:53-56
  • Foster, PR et al, Vox Sanguinis 2000 78:86-95
  • Lee, DC et al, Transfusion 2001 41: 449-55
  • Cai, K et al, Biochem Biophys. Acta 2002 1597: 28-35
  • Stenland, JS et al, Transfusion 2002 42:1497-1500
  • Vey, M et al, Biologicals 2002 30:187-96
  • Reichl, HE et al, Vox Sanguinis 2002 83:137-45
challenges in studies of clearance of tse agents
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 TSE’s (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
challenges in studies of clearance of tse agents9
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
challenges in evaluating clearance of tse agents
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
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
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
parameters influencing prp sc partitioning
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

example 2 cryoprecipitation variable clearance among studies with different conditions
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)
    • <1 – 1.7 logs clearance in precipitate
    • (Foster et al., Vox Sang 78:86- 95, 2000)
Example (3) Clearance PrPsc (microsomal spike) by Depth Filtration – Influence of Starting Materials and Filter

Starting MaterialDepth FilterReduction Factor (log10)

Fr V (albumin) Seitz KS80 > 4.9

Fr V (albumin) CUNO Delipid 1 2.3

S I + III (IGIV) Millipore AP20 < 1

Fr II (IGIV) Seitz K200 > 2.8

Foster et. al., Vox Sang 78: 86-95, 2000

Fr I supernatant (IGIV, albumin) Supra P80 < 1

Fr V supernatant (albumin) Supra P80 > 1.1

Fr V supernatant (albumin) –

Prp-sc spike Supra P80 > 2.4

Vey et al, Biologicals 30:187-96, 2002

tse clearance and the manufacturing process
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
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
evaluation of submissions to support labeling claims
Evaluation of Submissions to Support Labeling Claims
  • Clearance “beltline” to support labeling
    • At least 2 orthogonal steps with > 4 logs clearance (total 8 logs)
    • At least 2 steps demonstrated to be additive with > 4 logs clearance/step (total 8 logs)
    • ? At least 2 steps (orthogonal or demonstrated to be additive) with > 3 logs/step (total 6 logs)
    • Is a single clearance step of > 4 logs sufficient if robust and reproducible?
    • Are clearance steps of > 2 logs reliable if they are robust and reproducible?
  • Cumulative clearance/risk analysis
labeling for tse risk
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 manufacturer’s] 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