TSE Clearance Studies for pdFVIII: Study Methods and Clearance Levels

1
TSE Clearance Studies for pdFVIII Study Methods
and Clearance Levels

• TSE Advisory Committee
• September 18, 2006
• Dorothy Scott, M.D.
• Office of Blood Research and Review/CBER

2
TSE Safety Concerns

• Theoretically, plasma derivatives might transmit
  vCJD or other TSE agents
• Any such risk is thought to be very low based on
  the fact that no cases of vCJD have been reported
  worldwide in any recipients of plasma
  derivatives, including in the UK, where vCJD risk
  is greatest
• FDA seeks to assure the safety of plasma
  derivatives, especially pdFVIII, against risk for
  transmission of TSE

3
Importance of TSE Clearance

• Clearance of TSE agents in manufacturing of
  pdFVIII and other plasma derivatives has a major
  impact on estimated risk
• FDA risk assessment for pdFXI, 2005
  (http//www.fda.gov/ohrms/dockets/ac/05/briefing/2
  005-4088b1.htm)
• However, standardized methods and assessment
  criteria for TSE clearance have not been defined.

4
Issue for the TSEAC

• FDA seeks the advice of the Committee whether
  standardized methods and assessment criteria are
  feasible and appropriate for determining TSE
  clearance in the manufacturing processes for
  plasma-derived FVIII (pdFVIII) products.

5
Items for Discussion

• Feasibility and scientific value of adopting
  standardized methods to assess TSE clearance in
  manufacturing of pdFVIII products
• Whether a minimum TSE agent reduction factor
  might reasonably serve as an appropriate standard
  for demonstrating vCJD safety of pdFVIII
  products and if so
• Actions FDA should consider if only lower levels
  of clearance can be demonstrated for a given
  pdFVIII product

6
FDAs Evaluation of Sponsors Voluntary Studies
of TSE Clearance

• FDA discussed TSE clearance with the TSEAC in
  2/03. FDA has engaged in case by case review of
  the following types of information on TSE
  clearance
• Rationale for animal model selected
• Rationale for selection of spiking preparation
• Characterization of the spiking agent
• Demonstration of accurately scaled-down processes
• Robust and reproducible experiments
• Well-characterized assay for TSE infectivity

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FDAs Evaluation of Sponsors Voluntary Studies
of TSE Clearance (continued)

• Estimated logs TSE clearance by processing steps
• Demonstration of mass balance (accounting for
  all input infectivity)
• Demonstration that mechanistically similar
  clearance steps are or are not additive
• Account for conditioning of infectivity where a
  prior step may affect physical state of TSE agent
  and in turn affect clearance step downstream

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TSE Clearance Labeling Approvals for
Plasma-derived Products

• Approved Steps RF
• Carimune NF precipitations 7.2
• nanofiltration 4.4
• Panglobulin NF precipitations 7.2
• nanofiltration 4.4
• Gamunex cloth filtration
• depth filtration 6.6
• Thrombate III precipitation 6.0

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pdFVIII manufacturing

• Cryoprecipitation is the first step in
  manufacturing pdFVIII
• Other steps can include precipitations, column
  purifications, some of which may result in
  further TSE clearance

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Starting material for pdFVIII (cryoprecipitate) is
precipitated early in plasma fractionation
schemes
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Experimental Clearance of PrPTSE and Infectivity
by Cryoprecipitation
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TSE Clearance Issues

• Exogenous (spiking) experiments
• Nature of spiking material and its relevance to
  blood-borne infection
• Endogenous experiments
• Relevance and feasibility
• TSE strain and animal model
• Output measure of infectivity reduction
• Bioassay
• In vitro assays

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TSE Clearance Evaluation Exogenous (Spiking
Experiment) Model
TSE Spike Plasma Cryoprecipitation Cryop
recipitate (FVIII) Cryopoor
Plasma Supernatant FIX, IGIV,
A1PI, Albumin, etc.

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Exogenous TSE clearance studies form of spiking
material

• Form infectious agent
• Brain homogenate, centrifuged
• Ultracentrifuged (microsomal)
• Caveolae-like domains
• Detergent-solubilized homogenate
• Membrane-free infectious material (e.g. fibrils)
• Very insoluble
• Probably NOT representative of blood infectivity

Membrane-associated
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Spike form impacts clearance by precipitationVey
et al. Biologicals 30 187-96, 2002
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Conditioning Detergent-treatment diminishes
clearance of scrapie agent by nanofiltration
Tateishi et al, Biologicals 29 17-35, 2001

• Detergent Log10 RF
• Feed solution - (8.13) ----
• (7.32) ----
• Filtrate
• 35 nM - 4.93
• 1.61
• 15 nM - gt 5.87
• gt 4.21
• 10 nM gt 3.80

Determined by bioassay Scrapie ME7
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Conditioning

• PrPTSE clearance by membrane filtration and depth
  filtration increases in presence of alcohol
  (evidence of aggregation)
• Van Holten et al, Vox Sang. 8520-24, 2003

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TSE Clearance Evaluation Endogenous
Infection model
Plasma from TSE-infected animal Cryoprecipitati
on Cryoprecipitate (FVIII) C
ryopoor Plasma Supernatant FIX, IGIV,
A1PI, Albumin, etc.
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Endogenous TSE studies Relevance to Blood
Infectivity

• Comparison of results from endogenous and
  exogenous infectivity studies suggest similar
  reductions for some precipitations
• Limited number of endogenous studies
• Endogenous infectivity characteristics in plasma
• Small size
• Difficult to sediment (in its native form)
• Poorly aggregated
• May be lipid/plasma-protein associated

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Endogenous TSE Clearance Studies

• Relevance to human blood highly likely
• Limited clearance can be demonstrated because
  starting infectivity is low (est. 2-30 ID/ml)
• Large numbers of donor and assay animals may
  compensate for low titers
• Recipients volume injectible i.c. for
  titration 0.02 ml mice 0.05 ml hamsters
• For 100 ml plasma 5000 mice or 2000 hamsters
• Large animal models (sheep Scrapie, BSE)
• Experimental logistics - herd management, limited
  locations, incubation time, availability
• Scale-down logistics dedicated pilot
  laboratories

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TSE Model Selection

• TSEs differ in resistance to inactivation
• To date, clearance in plasma products
  demonstrated by partitioning studies only
• Few direct strain comparisons
• EtOH precipitations clearance similar BSE,
  CJD, vCJD (Stenland et al)
• EtOH precipitations Nanofiltration could be
  influenced if strain-related differences exist in
  aggregation properties (theoretical) (Vey et al)
• Strain differences for partitioning clearance
  experiments not demonstrated

22
Assays for TSE Agents

• Bioassay limiting dilution titration into
  susceptible rodents
• PrPTSE proposed as surrogate marker for
  infectivity
• PrPTSE measured by Western Blot or
  Conformation-dependent immunoassay (based on
  binding of antibody to PrpTSE)

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Rationale for Bioassay Use

• Binding assays detect PrPTSE
• Examples of infectivity without detectable PrPTSE
• Examples of PrPTSE without infectivity
• Conditioning might differentially affect binding
  vs. infectivity
• Binding assays (currently) not as sensitive as
  bioassays (limit of detection typically 2-3 logs
  infectivity)

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Challenges in TSE Clearance interpretation how
much clearance is significant?

• Viral validation (clearance) studies typically
  demonstrate at least 2-3 logs greater clearance
  than maximum potential absolute amount of virus
  present
• Added clearance provides a margin of safety

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TSE clearance level and safety

• TSE infectivity if present, how much might be
  in a single plasma unit?
• 800 ml (plasma unit) x 2-30 ID/ml TSE
  infectivity 1600 24,000 IDs (total)
• 3.2 - 4.4 log10 total infectious units
  estimated possible infectivity in one unit of
  infected plasma
• Actual infectivity might be less due to
  blood-brain barrier (IC/IV ID50 1 to 1 to 1 to 10
  estimated TSEAC 10/2005), and host
  susceptibility

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Question 1A

• A. Please comment on the feasibility and
  scientific value of adopting standardized
  exogenous (spiking) study methods to assess TSE
  clearance in manufacturing of pdFVIII including
  the following
• Optimal spiking material and its preparation from
  the standpoint of relevance to blood infectivity
• Selection of TSE strain and animal model
• TSE immunoassays for PrPTSE and bioassays for
  infectivity
• Identification of manufacturing processes that
  might alter TSE agent properties

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Question 1B

• 1. B. Please comment on the feasibility and
  scientific value of adopting standardized
  endogenous study methods to assess TSE clearance
  in pdFVIII.

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Question 2

• 2. Based on the available scientific knowledge,
  please discuss whether a minimum TSE agent
  reduction factor, demonstrated using an exogenous
  spiking model in scaled-down manufacturing
  experiments, might reasonably serve as an
  appropriate standard for demonstrating TSE safety
  of the products.

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Question 3

• Considering the outcome of discussion on Question
  2, in cases where only lower levels of clearance
  can be demonstrated for a pdFVIII, should FDA
  consider the following
• Labeling that would differentiate the lower
  clearance products from other products with
  sufficient TSE clearance
• Recommending addition of TSE clearance steps to
  the manufacturing method
• Performance of TSE clearance experiments using
  endogenous infectivity models
• Any other actions?

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TSE Clearance Labeling

• Under DESCRIPTION Additionally, the
  manufacturing process was investigated for its
  capacity to decrease the infectivity of an
  experimental agent of transmissible spongiform
  encephalopathy (TSE), considered as a model for
  the vCJD and CJD agents.

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TSE Clearance Labeling

•    Under DESCRIPTION Several of the individual
  production steps in the product name
  manufacturing process have been shown to decrease
  TSE infectivity of an experimental model agent.
  TSE reduction steps include process logs,
  process logs, etc. These studies provide
  reasonable assurance that low levels of CJD/vCJD
  agent infectivity, if present in the starting
  material, would be removed.