1. Removal of Prions by Plasma Fractionation Processes
Henry Baron, M.D.
Senior Director Medical and Scientific Affairs
Aventis Behring

2. 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

3. Variant CJD and Human Blood: Current 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.

4. 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; 358:
Wadsworth et al. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet, 2001; 358:

5. 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.

6. 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.

7. 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

8. Scaledown of Experimental Process
Experimental Process is Equivalent to Manufacturing Process.
Effluent
Precipitate
Separate
Input Solution
Sample
Spike
Experimental Process Step
Manufacturing Process Step
scaled down
Prove
Clearance = Prove - Effluent

9. Spiking Agents Used Brain Homogenates Microsomes
Caveolae-Like Domains (CLDs)
Purified PrPSc
Prion Fibrils

10. 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

11. 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
>
>
>
>
>
>
Kogenate
>
Logs Cleared
CRYO
3% PEG
Fr II+III
Fr III
Fr IV-1
11.5% PEG
Fr IV-4
DE2
DE3
Fractionation Step
> Designates the limits of PrPSc detection for the assay

12. Clearance of Rodent versus Human Prions
Variant CJD and other human TSE agents partition similarly
to the rodent-adapted sheep scrapie (263K strain)

13. Clearance of Rodent versus Human Prions

14. Clearance of Rodent versus Human Prions

15. 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.

16. Prion Clearance Study - Cohn Coupled Series Steps
SPIKE
Fr IV-1
Separation
Cryo
Fr I
Fr II+III
Fr II+IIIw
Fr III
(5.2)
Cryoeffluent
Effluent I
Effluent II+III
Effluent IV-1
Cryopaste
Fr I Paste
Discard
Fr II+III
Paste
Fr IV-1
Paste
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 II+IIIw
Fr II+IIIw
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.

17. Prion Removal Factors Major product categories F VIII Immunoglobulins
Albumin
Protease inhibitors

18. Prion Removal Factors F VIII
Cryoprec.: 1.0 (1), <1.0 (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), (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), (5)
1 Foster et al., Vox. Sang. [2000] 78: Lee et al., J Virol Meth [2000] 84: 77
3 Lee et al., Transfusion [2001] 41: Rohwer / Baxter & ARC, internal report
5 Vey et al., Biologicals [2002] 30: Brown et al., Transfusion [1999] 39: 1169
7 Rohwer / Baxter & ARC, preliminary results 8 Bayer, internal report.
9 Biotest, internal report.

19. Prion Removal Factors Immunoglobulins Immunoglobulins
Cryoprecipitation: <1 (1), 1.0 (2), < (5)
Precipitation of fraction I: 1.1 (2), <1 – 3.1 (5)
Precipitation of fraction (I+)III: > (9), 3.5(6), >3.7 (1), >4.0 (2), >4.3 (3), 5.3 (3)
PEG precipitation: >3.0(9)
Depth filtration: >2.8 (1), 2.8(6), 4.4(6), 6.4 (2,3), 6.0 (4)
Nanofiltration: 4.4(6)
Ig:  3.0(7),  (8), 6.5 (1),  (9), 6.4 (2&3), 7.9 (4)
Immunoglobulins
Cryoprecipitation: <1 (1), 1.0 (2), < (5)
Precipitation of fraction I: 1.1 (2), <1 – 3.1 (5)
Precipitation of fraction (I+)III: >3.7 (1), >4.0 (2), >4.3 (3), 5.3 (3)
Depth filtration: >2.8 (1), 6.4 (2,3), 6.0 (4)
Ig safety margin: 6.5 (1), 6.4 (2&3), 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: Lee et al., J Virol Meth [2000] 84: 77
3 Lee et al., Transfusion [2001] 41: Rohwer / Baxter & ARC, preliminary results
5 Vey et al., Biologicals [2002] 30: ZLB, internal report.
7 Biotest, internal report Aventis Behring, internal report.
9 Baxter, internal report.

20. Prion Removal Factors Albumin
Cryoprecipitation: <1 (1), 1.0 (2), <1 – 2.4 (5)
Precipitation of fraction I: 1.1 (2) , <1 – 3.1 (5)
Ppt. of fr. (I+II+)III: 1.3 (1), 1.9 (7), 2.2(6), 4.0 (2), 6.0 (3), 4.7 (3), (4) , (5)
Precipitation of fraction IV: 3.0 (1),3.0(6), 3.9 (7), 4.6 (3), 4.1 (3),  (5)
Depth filtration: 4.9 (1)
Albumin: 5.8 (7), 11.5 (1), 16.0 (3),  (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

21. Prion Removal Factors Proteinase inhibitor
Cryoprecipitation: 1.0 (1,2), < (3)
Precipitation of fraction I: 1.1 (1,2), < (3)
Ppt. of fr. (I+II+)III:  (2), (3)
PEG precipitation: 5.4 (2)
Proteinase inhibitor: 12.2 (2), (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

22. 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 !