Removal of Prions by Plasma Fractionation Processes

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Removal of Prions by Plasma Fractionation
Processes

• Henry Baron, M.D.
• Senior Director Medical and Scientific Affairs
• Aventis Behring

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CJD/vCJD and Human Blood Key Message

• Currently no scientific evidence to substantiate
  that persons with pre-clinical or clinical CJD,
  including vCJD, carry infectious prions in their
  blood or have transmitted them through blood or
  plasma products. Therefore, this risk is
  considered theoretical.
• Dozens of studies with classical CJD
• Experimental (animal)
• Epidemiological (human)
• Medical observation (human)
• Several decades of experience
• Less experience with variant CJD
• Oral, food-borne transmission of BSE prions

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Variant CJD and Human BloodCurrent State of
Knowledge

• Whole blood, red blood cells and platelets from
  UK donors have been and continue to be
  administered to UK recipients (estimated 30 to 40
  million transfusions in UK over past 10 years).
• Several patients with vCJD received transfusions
  but none could be linked to a donor who had CJD
  or vCJD.
• No cases of CJD or vCJD have been noted among
  identified recipients of blood or plasma products
  from known vCJD donors to date.
• To date, there is no evidence in the UK (where
  98 of all reported BSE and 95 of all
  reported vCJD worldwide have occurred) that vCJD
  has been transmitted through blood or plasma
  products.

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• Two Lancet publications report that prions were
  undetectable in blood, plasma and buffy coat of
  patients with vCJD, despite detection of prions
  in their lymphoid tissues.
• Bruce et al. Detection of variant
  Creutzfeldt-Jakob disease infectivity in
  extraneural tissues. Lancet, 2001 358208-209.
• Wadsworth et al. Tissue distribution of protease
  resistant prion protein in variant
  Creutzfeldt-Jakob disease using a highly
  sensitive immunoblotting assay. Lancet, 2001
  358171-180.

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A responsible approach to the manufacture of
plasma protein therapies is to treat this
theoretical risk as though it were real.

• RATIONAL, SCIENCE-BASED PRECAUTIONARY POLICIES
  TO MINIMIZE THE THEORETICAL RISK
• Individual donor deferral criteria.
• Geographic plasma rejection criteria.
• Withdrawal/notification policies.
• EXTREMELY RAPID AND HIGHLY SENSITIVE METHODS OF
  PRION DETECTION
• Research prion infectivity in bloods of CJD,
  including vCJD, cases.
• Assessment of prion partitioning in manufacturing
  processes.

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Prion Partitioning in the Manufacture of Human
Plasma Proteins

• Prions never detected in nor transmitted through
  human blood, plasma or plasma derivatives.
• Therefore, no knowledge as to the
  biophysicochemical nature of theoretical prion
  contaminant in plasma.
• Therefore, uncertainty as to appropriate,
  relevant prion spiking agent for study of prion
  partitioning in manufacturing processes.

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Key Issues In Prion Removal

• Validity of Scaledown Model
• Nature of the Spike
• Detection Methodology
• Immunoassay
• Infectivity bioassay
• Model (rodent) versus Human Prions
• Independent versus Coupled Process Step Removal

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Spiking Agents Used

• Brain Homogenates
• Microsomes
• Caveolae-Like Domains (CLDs)
• Purified PrPSc
• Prion Fibrils

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Prion Detection Methods

• PrPSc is Highly Correlated with Infectivity
• Immunoassays for PrPSc
• Western Blot
• Conformation Dependent Immunoassay (CDI)
• Infectivity Bioassays
• Rodents (mice, hamsters, transgenic mice)
• Removal Determined by Immunoassay Correlates with
  Removal Determined by Bioassay

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PrPSc Immunoassay vs Bioassay
PrP or infectivity clearance data for various
plasma protein and biotechnology processing steps
is plotted. For all cases presented, log
clearance of either PrPSc (Western blot) or
infectivity (bioassay) is similar. Lee et al,
Transfusion vol 41, April 2001
gt
gt
gt
gt
gt
gt
Kogenate
gt
Logs Cleared
3 PEG
CRYO
Fr IIIII
Fr III
Fr IV-1
Fr IV-4
11.5 PEG
DE3
DE2
Fractionation Step
gt Designates the limits of PrPSc detection for
the assay
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Clearance of Rodent versus Human Prions
Variant CJD and other human TSE agents partition
similarly to the rodent-adapted sheep scrapie
(263K strain)
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Clearance of Rodent versus Human Prions

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Clearance of Rodent versus Human Prions
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Clearance of Rodent versus Human Prions

• Conclusion
• Data showing removal of rodent prions can be
  considered predictive of removal of human CJD and
  vCJD prions.

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Prion Clearance Study - Cohn Coupled Series Steps
(5.2)
Effluent IV-1
Cryoeffluent
Effluent I
Effluent IIIII
Fr IIIII Paste
Fr I Paste Discard
Fr IV-1 Paste
Cryopaste

• Removal of PrPSC by a series of processing steps
  can be additive
• PrPSC that is not removed by an initial
  precipitation/centrifugation step is removed by a
  subsequent step
• No fraction of PrPSC resistant to
  precipitation/centrifugation was identified
• PrPSC removal is independent of the presence of
  brain homogenate
• Steps evaluated either independent or within a
  series demonstrated similar magnitudes of either
  infectivity or PrPSc clearance.

Effluent IIIIIw
Fr IIIIIw Paste
(4.0)
Effluent III
(0)
Fr III Paste Discard
(4.2)
Partitioning determined for independent steps is
consistent with partitioning determined
for coupled processes.
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Prion Removal Factors

• Major product categories
• F VIII
• Immunoglobulins
• Albumin
• Protease inhibitors

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Prion Removal Factors

• F VIII
• Cryoprec. 1.0 (1), lt1.0 (2,3), 1.5 (7), 1.0 (6)
• Aluminium-hydroxide adsorption 1.7 (1), 1.3 (5)
• PEG or glycine precipitation 2.2 (3), 3.0 (3),
  1.7-3.3 (5)
• Ion exchange or size exclusion chromatography
  3.1 (1), 1.0 (8), 3.5 (4)
• Monoclonal antibody purification 4.1 (4)
• 0.45 µm / 0.2 µm filtration 1.0 (1), 1.0 (5)
• F VIII 6.8 (1), 3.2 (2,3), 3.2(9), 8.0 (4),
  4.8-5.5 (5)

1 Foster et al., Vox. Sang. 2000 78 86 2 Lee
et al., J Virol Meth 2000 84 77 3 Lee et al.,
Transfusion 2001 41 449 4 Rohwer / Baxter
ARC, internal report 5 Vey et al., Biologicals
2002 30 187 6 Brown et al., Transfusion
1999 39 1169 7 Rohwer / Baxter ARC,
preliminary results 8 Bayer, internal report. 9
Biotest, internal report.
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Prion Removal Factors

• Immunoglobulins
• Cryoprecipitation lt1 (1), 1.0 (2), lt1 - 2.4 (5)
• Precipitation of fraction I 1.1 (2), lt1 3.1
  (5)
• Precipitation of fraction (I)III gt3.3-3.8(9),
  3.5(6), gt3.7 (1), gt4.0 (2), gt4.3 (3), 5.3
  (3)
• PEG precipitation gt3.0(9)
• Depth filtration gt2.8 (1), 2.8(6), 4.4(6), 6.4
  (2,3), 6.0 (4)
• Nanofiltration 4.4(6)
• Ig ? 3.0(7), ? 5.0-9.4(8), ?6.5 (1), ?
  6.3-6.8(9), ?6.4 (23), 7.9 (4)

• Immunoglobulins
• Cryoprecipitation lt1 (1), 1.0 (2), lt1 - 2.4 (5)
• Precipitation of fraction I 1.1 (2), lt1 3.1
  (5)
• Precipitation of fraction (I)III gt3.7 (1),
  gt4.0 (2), gt4.3 (3), 5.3 (3)
• Depth filtration gt2.8 (1), 6.4 (2,3), 6.0 (4)
• Ig safety margin ?6.5 (1), ?6.4 (23), 7.7 (4)
• 1 Foster et al., Vox. Sang. 2000 78 86
• 2 Lee et al., J Virol Meth 2000 84 77
• 3 Lee et al., Transfusion 2001 41 449
• 4 Rohwer / Baxter ARC, preliminary results
• 5 Aventis, submitted

1 Foster et al., Vox. Sang. 2000 78 86 2 Lee
et al., J Virol Meth 2000 84 77 3 Lee et al.,
Transfusion 2001 41 449 4 Rohwer / Baxter
ARC, preliminary results 5 Vey et al.,
Biologicals 2002 30 187 6 ZLB, internal
report. 7 Biotest, internal report. 8 Aventis
Behring, internal report. 9 Baxter, internal
report.
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Prion Removal Factors

• Albumin
• Cryoprecipitation lt1 (1), 1.0 (2), lt1 2.4 (5)
• Precipitation of fraction I 1.1 (2) , lt1 3.1
  (5)
• Ppt. of fr. (III)III 1.3 (1), 1.9 (7),
  2.2(6), ?4.0 (2), 6.0 (3), ?4.7 (3), 2.4 (4) ,
  3.1-4.0 (5)
• Precipitation of fraction IV ?3.0 (1),3.0(6),
  3.9 (7), 4.6 (3), ?4.1 (3), ?4.5-4.6 (5)
• Depth filtration ?4.9 (1)
• Albumin 5.8 (7), ?11.5 (1), ?16.0 (3),
  ?7.7-14.1 (5)

1 Foster et al., Vox. Sang. 2000 78 86 2 Lee
et al., J Virol Meth 2000 84 77 3 Lee et al.,
Transfusion 2001 41 449 4 Rohwer / Baxter
ARC, preliminary results 5 Vey et al.,
Biologicals 2002 30 187 6 ZLB, internal
report 7 Baxter, internal report
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Prion Removal Factors

• Proteinase inhibitor
• Cryoprecipitation 1.0 (1,2), lt1 - 2.4 (3)
• Precipitation of fraction I 1.1 (1,2), lt1 - 3.1
  (3)
• Ppt. of fr. (III)III ?4.7-6.0 (2), 3.1 - 4.0
  (3)
• PEG precipitation ?5.4 (2)
• Proteinase inhibitor ?12.2 (2), 3.1-9.5 (3)

1 Lee et al., J Virol Meth 2000 84 77 2 Lee et
al., Transfusion 2001 41 449 3 Vey et al.,
Biologicals 2002 30 187
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Conclusions

• Removal of prions by plasma manufacturing
  processes
• is very significant, and further minimizes the
  theoretical risk addressed by donor deferral
• donor deferral - 1 log10 reduction of exposure
  risk
• is process specific and demonstrated across all
  prion spike materials, different prion assay
  systems, and manufacturing step specifics
• is very substantial as compared to the still
  theoretical level of risk !