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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. TSE Safety Concerns. Theoretically, plasma derivatives might transmit vCJD or other TSE agents - PowerPoint PPT Presentation
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TSE Clearance Studies for pdFVIII: Study Methods and
Clearance Levels
TSE Advisory CommitteeSeptember 18, 2006Dorothy Scott, M.D.
Office of Blood Research and Review/CBER
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
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/2005-4088b1.htm)
• However, standardized methods and assessment criteria for TSE clearance have not been defined.
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.
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
FDA’s 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
FDA’s 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
TSE Clearance Labeling Approvals for Plasma-derived Products
Approved Steps RF
Carimune NF precipitations 7.2nanofiltration 4.4
Panglobulin NF precipitations 7.2nanofiltration 4.4
Gamunex cloth filtration +depth filtration 6.6
Thrombate III precipitation 6.0
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
ANTITHROMBIN III
ANION EXCHANGE CHROMATOGRAPHYHEPARIN AFFINITY CHROMATOGRAPHY
cohnfrac.org by DJFrazier 12/96
Pasteurization
(Diafiltration)(Chromatography)(Viral inactivation)(Limited proteolysis)(Lyophilization)
The Cohn-Oncley Blood Plasma Fractionation Process
Fraction IV-1 Supernate
Cryoprecipitate Supernate
Fraction I Supernate
Fraction II+III Supernate
Fraction II+IIIw Supernate
Fraction II Supernate
Fraction III Supernate
Fraction V Supernate
Fraction IV-4 Supernate
freeze, thaw
IMMUNE GLOBULINS
pH 7.425% ETOH-5 oC
pH 5.217% ETOH-6 oC
pH 7.620% ETOH
-5 oC
ALBUMIN
pH 4.610% ETOH
-2 oCFilterpH 5.1540% ETOH
-5 oCResuspend
pH 4.840% ETOH-5 oC
pH 5.840% ETOH
-5 oC
pH 5.318% ETOH
-5 oC
pH 6.8520% ETOH-5 oC
pH 7.28% ETOH-2oC
FACTOR IX COMPLEXANTIHEMOPHILIC FACTOR
Plasma
Starting material for pdFVIII (cryoprecipitate)is precipitated early in plasma fractionation schemes
Experimental Clearance of PrPTSE and Infectivity by Cryoprecipitation
Reference
Log10 Reduction Assay
Brown et al., Transfusion 38: 810-16, 1998
< 1.0 Bioassay
Foster et al., Vox Sang 78:86- 95, 2000
1.0 PrPTSE
Lee et al., Transfusion 41: 449-55, 2001
< 1.0 Bioassay
Vey et al., Biologicals 30: 187-96, 2002
< 1.0 PrPTSE
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
TSE Spike Plasma
Cryoprecipitation Cryoprecipitate(FVIII)
Cryopoor Plasma Supernatant
FIX, IGIV, A1PI,Albumin, etc.
TSE Clearance Evaluation: Exogenous(“Spiking Experiment”) Model
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
Spike form impacts clearance by precipitation
Vey et al. Biologicals 30: 187-96, 2002
Manufacturing
Process
Log10 PrPTSE Reduction (with respect to supernatant)
Microsomal Spike
Purified PrP-Scrapie Spike
Cryoppt. 0.2 2.4
8% EtOH ppt. 0.9 3.1
25% EtOH ppt. 3.1 4.0
Glycine ppt. 1.7 3.3
“Conditioning”: Detergent-treatment diminishes clearance of scrapie agent by nanofiltration
Detergent Log10 RF*
Feed solution - (8.13) ----+ (7.32) ----
Filtrate35 nM - 4.93
+ 1.61
15 nM - > 5.87+ > 4.21
10 nM + > 3.80
Tateishi et al, Biologicals 29: 17-35, 2001
* Determined by bioassay [Scrapie ME7]
“Conditioning”
• PrPTSE clearance by membrane filtration and depth filtration increases in presence of alcohol (evidence of aggregation)
Van Holten et al, Vox Sang. 85:20-24, 2003
Plasma from TSE-infected animal
Cryoprecipitation Cryoprecipitate(FVIII)
Cryopoor Plasma Supernatant
FIX, IGIV, A1PI,Albumin, etc.
TSE Clearance Evaluation: Endogenous Infection model
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
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
TSE Model Selection
• TSE’s 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
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)
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)
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
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 ID’s (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
Question 1A
1. 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
I) Optimal spiking material and its preparation from the standpoint of relevance to blood infectivity
II) Selection of TSE strain and animal modelIII) TSE immunoassays for PrPTSE and bioassays for
infectivityIV) Identification of manufacturing processes that might
alter TSE agent properties
Question 1B
1. B. Please comment on the feasibility and scientific value of adopting standardized endogenous study methods to assess TSE clearance in pdFVIII.
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.
Question 3
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:
A. Labeling that would differentiate the lower clearance products from other products with sufficient TSE clearance;
B. Recommending addition of TSE clearance steps to the manufacturing method;
C. Performance of TSE clearance experiments using endogenous infectivity models;
D. Any other actions?
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.”
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.