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 DaltonBio Products Laboratory, Elstree UK

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 DaltonBio Products Laboratory, Elstree UK

Virus Envelope Nucleic acid Size (nm)

HIV + RNA 80-100

WNV + RNA 80-100

HBV + DNA 40-50

HCV + RNA 40-50

HAV - RNA 25-30

B19 - DNA 18-24

Viruses of Concern

Parvovirus B19

• High prevalence• High levels of viraemia• Can be asymptomatic• Mini pool screening to reduce

contamination (<104 IU/ml)• <104 IU/ml may not be infective

Animal models

• Canine parvovirus (CPV)• Porcine parvovirus (PPV)• Minute virus of mice (MVM)• Bovine parvovirus (BPV)

SusceptibilityClearance technique B19 Animal model

Solvent / detergent - -

Pasteurisation +++ ++

Dry heat treatment +++ ++

Low pH +++ +/-

Filtration ? ?

Virus inactivation / removal techniques

Parvovirus filtration

Filtration ProtocolVirus Spiked Product

~100 mL

Spiked load

sample

0.1µm filtration(to remove aggregates)

Load

VIRUS FILTRATION

Filtrate

Product

Collected in fractions

Measurement of B19 and CPV• B19

• Qiagen QIAamp MinElute Virus Spin Extraction Kit • Roche LightCycler Parvovirus B19 Quantification

Kit• CPV

• Plaque assay (A-72 –cells)• TCID50

High-purity FIX

Step Polio-1 HAV CPV B19

Planova15N >5.6 >6.0 3.2 >7.2

IVIG

Step EMC HAV B19 CPV

Pall UltiporDV20 >4.8 >4.8 6.0 3.2

11.8 log

A B C

LRV

B19 in

B19 out

>7.2 >5.9 5.4

<4.6 log

10.6 log 10.4 log

<4.7 log 5.0 log

Coagulation factors

Planova15N

6.3 log

A B C

LRV

CPV in

CPV out

3.2 4.1 3.7

3.1 log

6.3 log 6.8 log

2.2 log 3.1 log

Coagulation factors

Planova15N

Robustness of parvovirus removal from IVIG

• Filter – Pall Ultipor DV20 filter• Parameters

• Pressure (Bar)• Protein concentration (mg/ml)• pH• Load (L/m2)

Effect of pressure on DV20 filtration

0

1

2

3

4

5

6

7

Log

Viru

s R

emov

al

Fr 1 Fr 2 Fr 3 Fr 4

B19 3 BarB19 3.3 barCPV 3 barCPV 3.3 bar

Effect of pH on DV20 filtration

0

1

2

3

4

5

6

Log

Viru

s R

emov

al

Fr 1 Fr 2 Fr 3

B19 pH 4.4B19 pH 5.1CPV pH 4.4CPV pH 5.1

Effect of protein concentration on DV20 removal

0

1

2

3

4

5

6

7

Log

Viru

s R

emov

al

Fr 1 Fr 2 Fr 3 Fr 4

B19 0g/l B19 10 g/lB19 14 g/lCPV 0g/lCPV 10g/lCPV 14 g/l

• Removal of B19 higher than CPV• Removal is consistent • Why?

B19 vs CPV

• Is it just CPV?• Size of particles• Presence of antibodies• Aggregation• B19 plasma sample• Filter

Factors to be considered:

Removal of parvovirus from buffer by DV20 filtration

0

1

2

3

4

5

6

Log

Viru

s R

emov

al

Fr 1 Fr 2 Fr 3

B19CPVPPV

PPV removal

Filter BPL Study External Study

DV203.1

Buffer4.8IgG

Size of virus particles?

• No obvious difference in size of particles• 18 – 24 nm in size• B19 has “smoother” surface, may even be

slightly smaller• Difference in source

• PPV & CPV - tissue culture grown• B19 – from +plasma donation

Presence of B19 antibodies

Effect of protein concentration on DV20 removal

0

1

2

3

4

5

6

7

Log

Viru

s R

emov

al

Fr 1 Fr 2 Fr 3 Fr 4

B19 0g/l B19 10 g/lB19 14 g/lCPV 0g/lCPV 10g/lCPV 14 g/l

0

2

4

6

8

10

12B

19 L

og c

opie

s/m

l

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Donation ID

IgM measurement

• Biotrin Parvovirus B19 IgM Enzyme Immunoassay Kit

• Cut off value: 0.344

Donor 01

Donor 08

Donor 13

Donor 14

Donor 15

Donor 16

0.090 0.072 0.058 0.062 0.060 0.905

Aggregation

Aggregation of virus stocks

• No loss of B19 over 0.1µm pre-filtration of stock

• Aggregation also assessed by Planova35N filtration

9.4 log

A B

LRV

B19 in

B19 out

0.4 0.5

9.0 log

8.3 log

7.8 log

35N

Planova 35N

B19 plasma

Influence of B19 spike

0

1

2

3

4

5

6

Log

Viru

s R

emov

al

Fr 1 Fr 2

Donor 01Donor 14Donor 15

• Removal of B19 from buffer by DV20 filtration

Filter type

Filter Virus Solution Log Virus Removal

B19 IgG 5.3

A

B19 3.1

PPV 1.9

BufferCPV 1.7B

Buffer >5.8

>4.6

>4.3

>4.3

>5.5PPV Buffer

>7.3

IgGCPV

Preliminary data from alternative filters

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?

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

Thankyou

• Biosafety group at BPL • Anchal Johni

Company Filter Construction material Claims (LRV)

Planova 15N>6.2 parvovirus

>6.7 polio

Planova 20N>4.3 parvovirus

>5.4 EMC

Planova 35N>5.9 BVDV>7.3 HIV

Viresolve NFP Modified PVDF >4 φX-174 bacteriophage

Ultipor DV20>3 PP7 bacteriophage

>6 PR772 bacteriophageModified PVDF

[triple layer]Pall

Ultipor DV50 >6 PR772 bacteriophage

Viresolve NFR Polyether Sulphone >6 RetrovirusMillipore

Viresolve Pro Polyether Sulphone[double layer] >4 MVM

Sartorius Virosart CPVPolyether Sulphone

[double layer]>4 PR772 bacteriophage

>6 Retrovirus

CuprammoniumRegenerated Cellulose

Asahi-Kasei

Manufacturer’s literature + Burnouf T & Radosevich, M., Haemophilia (2003), 9, 24-37