A comparison of the removal of parvovirus B19 and model parvoviruses from plasma products by virus filtration

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1 A comparison of the removal of parvovirus B19 and model parvoviruses from plasma products by virus filtration Shirley Stagg, Samantha Fernando, Amy Shackell, Peter Roberts, Joan Dalton Bio Products Laboratory, Elstree UK

2 A comparison of the removal of parvovirus B19 and model parvoviruses from plasma products by virus filtration Shirley Stagg, Samantha Fernando, Amy Shackell, Peter Roberts, Joan Dalton Bio Products Laboratory, Elstree UK

3 Viruses of Concern Virus Envelope Nucleic acid Size (nm) HIV + RNA WNV + RNA HBV + DNA HCV + RNA HAV - RNA B19 - DNA 18-24

4 Parvovirus B19 High prevalence High levels of viraemia Can be asymptomatic Mini pool screening to reduce contamination (<10 4 IU/ml) <10 4 IU/ml may not be infective

5 Animal models Canine parvovirus (CPV) Porcine parvovirus (PPV) Minute virus of mice (MVM) Bovine parvovirus (BPV)

6 Virus inactivation / removal techniques Clearance technique B19 Susceptibility Animal model Solvent / detergent - - Pasteurisation Dry heat treatment Low ph +++ +/- Filtration??

7 Parvovirus filtration

8 Virus Product Filtration Protocol 0.1µm filtration (to remove aggregates) Spiked Product ~100 ml Spiked load sample Load VIRUS FILTRATION Collected in fractions Filtrate

9 B19 Measurement of B19 and CPV Qiagen QIAamp MinElute Virus Spin Extraction Kit Roche LightCycler Parvovirus B19 Quantification Kit CPV Plaque assay (A-72 cells) TCID 50

10 High-purity FIX Step Polio-1 HAV CPV B19 Planova 15N >5.6 > >7.2

11 IVIG Step EMC HAV B19 CPV Pall Ultipor DV20 >4.8 >

12 Coagulation factors A B C B19 in 11.8 log 10.6 log 10.4 log Planova 15N B19 out <4.6 log <4.7 log 5.0 log LRV >7.2 >

13 Coagulation factors A B C CPV in 6.3 log 6.3 log 6.8 log Planova 15N CPV out 3.1 log 2.2 log 3.1 log LRV

14 Robustness of parvovirus removal from IVIG Filter Pall Ultipor DV20 filter Parameters Pressure (Bar) Protein concentration (mg/ml) ph Load (L/m 2 )

15 Effect of pressure on DV20 filtration 7 6 Log Virus Removal B19 3 Bar B bar CPV 3 bar CPV 3.3 bar 0 Fr 1 Fr 2 Fr 3 Fr 4

16 Effect of ph on DV20 filtration 6 Log Virus Removal B19 ph 4.4 B19 ph 5.1 CPV ph 4.4 CPV ph Fr 1 Fr 2 Fr 3

17 Effect of protein concentration on DV20 removal 7 6 Log Virus Removal B19 0g/l B19 10 g/l B19 14 g/l CPV 0g/l CPV 10g/l CPV 14 g/l 0 Fr 1 Fr 2 Fr 3 Fr 4

18 B19 vs CPV Removal of B19 higher than CPV Removal is consistent Why?

19 Factors to be considered: Is it just CPV? Size of particles Presence of antibodies Aggregation B19 plasma sample Filter

20 Removal of parvovirus from buffer by DV20 filtration 6 Log Virus Removal B19 CPV PPV 0 Fr 1 Fr 2 Fr 3

21 PPV removal Filter BPL Study External Study DV Buffer 4.8 IgG

22 Size of virus particles? No obvious difference in size of particles nm in size B19 has smoother surface, may even be slightly smaller Difference in source PPV & CPV - tissue culture grown B19 from +plasma donation

23 Presence of B19 antibodies

24 Effect of protein concentration on DV20 removal 7 6 Log Virus Removal B19 0g/l B19 10 g/l B19 14 g/l CPV 0g/l CPV 10g/l CPV 14 g/l 0 Fr 1 Fr 2 Fr 3 Fr 4

25 12 10 B19 Log copies/ml Donation ID

26 IgM measurement Biotrin Parvovirus B19 IgM Enzyme Immunoassay Kit Cut off value: Donor 01 Donor 08 Donor 13 Donor 14 Donor 15 Donor

27 Aggregation

28 Aggregation of virus stocks No loss of B19 over 0.1µm pre-filtration of stock Aggregation also assessed by Planova 35N filtration

29 Planova 35N B19 in A 9.4 log B 8.3 log 35N B19 out 9.0 log 7.8 log LRV

30 B19 plasma

31 Influence of B19 spike Removal of B19 from buffer by DV20 filtration 6 Log Virus Removal Donor 01 Donor 14 Donor 15 0 Fr 1 Fr 2

32 Filter type

33 Preliminary data from alternative filters Filter Virus Solution A B Log Virus Removal B19 IgG 5.3 CPV PPV Buffer >5.8 IgG Buffer >4.6 >4.3 >4.3 >5.5 >7.3 B CPV Buffer 1.7 PPV 1.9

34 Regulatory Question the applicant has shown that this filter is less effective against canine parvovirus which is the same size and shape as human B 19. Does the applicant have any theories as to why the two viruses were not similarly cleared by the filter?

35 Conclusions/theories B19 is removed to a greater extent than CPV or PPV Viruses are the same size Virus stocks are not aggregated Could not demonstrate abs Different B19+ plasma samples gave similar results B19 may bind to some other factor in plasma e.g. globoside? The use of animal parvoviruses represents a true worst case challenge to filters Removal influenced by virus stocks, solution being filtered, filter type

36 Thankyou Biosafety group at BPL Anchal Johni

37 Company Filter Construction material Claims (LRV) Planova 15N >6.2 parvovirus >6.7 polio Asahi- Kasei Planova 20N Cuprammonium Regenerated Cellulose >4.3 parvovirus >5.4 EMC Planova 35N >5.9 BVDV >7.3 HIV Millipore Viresolve NFP Modified PVDF >4 φx-174 bacteriophage Viresolve NFR Polyether Sulphone >6 Retrovirus Viresolve Pro Polyether Sulphone [double layer] >4 MVM Pall Ultipor DV20 Modified PVDF [triple layer] >3 PP7 bacteriophage >6 PR772 bacteriophage Ultipor DV50 >6 PR772 bacteriophage Sartorius Virosart CPV Polyether Sulphone [double layer] >4 PR772 bacteriophage >6 Retrovirus Manufacturer s literature + Burnouf T & Radosevich, M., Haemophilia (2003), 9, 24-37

EMEA WORKSHOP ON VIRAL SAFETY OF PLASMA-DERIVED MEDICINAL PRODUCTS WITH PARTICULAR FOCUS ON NON-ENVELOPED VIRUSES. 13 September 2000 REPORT SUMMARY

The European Agency for the Evaluation of Medicinal Products Evaluation of Medicines for Human Use London, 28 March 2001 CPMP/BWP/BPWG/4080/00 EMEA WORKSHOP ON VIRAL SAFETY OF PLASMA-DERIVED MEDICINAL