Analysis of human parvovirus B19 components

and strategies of non-enveloped virus removal

from factor VIII concentrates

Japanese Red Cross Plasma Fractionation Center

K. Furuya, T. Yokoyama, H. Maeno, T. Murozuka

M. Tanifuji, A. Wakisaka and T. Tomono

Elution profile for B19 components on Q-Sepharose

0

1

2

3

4

5

0 5 10 15 200

200

400

600

Pass through

38 x 10n IU / mL

Fraction number

NaCl [mM]

pH 6.4

pH 5.5

Peak 1 Peak 3Peak 2

 

Detection of B19 capsid proteins, VP-1 and VP-2, in each eluted fraction by immuno-blot analysis

　 1 　　　 2 　　　　 3 　　　　 4 　　　　　 5 　　　　 6

　 7 　　　 8 　　　 9 　　　 10 　　 11 　　　 12 　　 13 　　　 14 　　　　 15　　　　　　　　　　　　　　　　　　　　　　　　　 peak 2

　 16 　　 17 　　 18 　　 19 　　　 20 　　　 21 　 positive 　　　　　　　　　　　　　 peak 3 　　　　　 　 control

B19 spiked solution

Pass through peak 1

Characteristics and expected shape of B19 separated by Q-Sepharose

peak １ peak 2 peak 3

Capsid protein 　　　 ○ 　　　　　　○ 　　　　　　 ×

DNase treatment resistant 　　 sensitivesensitive

　Charge 　 not negative negative negative

Shape 　　　

　 Elution profile for B19 on Q-Sepharose after nanofiltration

▲

00.51

1.52

2.53

3.54

4.5

0 5 10 15 200

200

400

600

Pass through

38 x 10n IU / mL NaCl [mM]

pH 6.4

pH 5.5

Fraction number

Not filtrated ◆20 nm filtration ■35 nm filtration ▲◆

▲

■

B19 DNA fragment and factor VIII with different elution conditions

Spiked solution 　　　 1.9 　　 100%

Pass through 　　　　 ＜ 0.0* 　　　　　 Wash ＜ 0.0*

Eluted at

450 mM NaCl pH 5.8 　 ＜ 0.0* 　 97.6

%

600 mM NaCl pH 5.5 1.7 4.2%

B19 DNA fragment Amount of FVIII

（ 38 x 10n IU / mL ） ( U )

* Not detected

Conclusions

1. At least three forms of B19 were found ,i.e. , intact

virus virion, disrupted virion and DNA fragment

2. B19 virus virion and disrupted virion can be remo

ved by implementing a 20-nm-pore-size filter

3. B19 DNA fragment can be separated from factor

VIII on Q-Sepharose with adequate elution conditi

ons