Removal of hepatitis B virus from a concentrate of coagulation factors II, VII, IX and X by hydrophobic interaction chromatography

Journalof

VirologicalMethods,

Elsevier/North-Holland

3 (1981)

Biomedical

213-228

213

Press

REMOVAL OF HEPATITIS B VIRUS FROM A CONCENTRATE FACTORS II, VII, IX AND X BY HYDROPHOBIC

OF COAGULATION

INTERACTION

CHROMATOGRAPHY MONICA ‘Research

EINARSSON',LENNART Department,

Medical Microbiology, (Accepted

17 June

Deliberately removed II, VII,

by

ularly

hepatitis

useful

same stability

and 2Department

of

University of Lund, Lund, Sweden

B surface

interaction

X. Chromatography 4B, resulted

in removing

in high

activities

(HBsAg) from

concentrations

and hepatitis

B virus

a concentrate of salt,

reduction

preferably

of HBsAg.

factor as before

of virus-related concentrate

material

was about

(HBV)

could

of coagulation

from

properties

method protein

be

factors

on octanoic

The binding

gels were similar and the chromatographic

low concentrations

from the coagulation

and biological

antigen

chromatography

in a lo4 -lo’-fold

and HBV to the hydrophobic

yield of protein

and EVA MILLER2

1981)

added

IX and

ERIK NORDENFELT’

S-I 12 87, KabiVitrum AB, Stockholm;

Virology Section,

hydrophobic

hydrazide-Sepharose HBsAg

Biochemistry,

KAPLAN’,

acid of

seems particsolutions.

85% and the material

showed

The the

the chromatography.

INTRODUCTION

The discovery of hepatitis B surface antigen (HBsAg) in 1964 (Blumberg et al., 1965, 1967) and its role as an immunological marker of type B viral hepatitis (Prince, 1968; Blumberg et al., 1970; Gocke et al., 1970; Krugman and Giles, 1970; Gocke, 1972) provided the knowledge and the means for screening donor blood for HBsAg. In contaminated plasma, the most abundant forms of HBsAg are 22 nm spherical particles, which have been shown to be non-infectious (Kim and Tilles, 1973; Robinson and Lutwick, 1976). It is generally agreed that the 42 nm Dane particle is the complete hepatitis B virus (HBV), since DNA polymerase activity (Kaplan et al., 1973; Melnick et al., 1976; Robinson and Lutwick, 1976) and double-stranded DNA, which could act as the template for the polymerase, have been demonstrated (Kaplan et al., 1973; Summers et al., 1975). There is no absolute correlation between the antigen titre and the infective potential in any given sample. In early experiments in man, 95% of the patients receiving 1 ml of a lo-’ dilution of a contaminated plasma pool showed clinical signs of disease (Murray, 1955; Barker et al., 1970; Melnick et al., 1976). From these test results, the minimal number of Dane particles associated with infection has been estimated to be less than lo2 particles/ml (Robinson and Lutwick, 1976). In titration experiments on chimpanzees (Barker et al., 1975) using human sera containing varying dilutions of hepatitis B 0166-0934/81/0000-0000/$02.50

@Elsevier/North-Holland

Biomedical

Press

214

infectious

material,

it has been demonstrated

that the material was infectious

10-r to lo-’ below the level of sensitivity of the assay. Practical procedures for large-scale fractionation of human

plasma

in d~utions

to isolate

and

purify many clinically valuable plasma proteins often start with large pools of plasma for maximum efficiency. Despite sensitive test methods for HBsAg, there is a considerable risk of contamination of the plasma pool, and hepatitis caused by the administration to patients of plasma fractions that cannot be heat-treated is well documented (Grady and Bennett, 19’72; Sandier et al., 1973; Roberts and Blatt, 1975). Only ~bu~n pasteurized at 6O*C for 10 h and gammaglobulin isolated by the Cohn fraction II method are generally non-infectious (Hoofnagle et al., 1976). Efforts have been made to develop techniques to reduce the infectivity of clinically valuable plasma fractions. Procedures employing polye~ylene glycol (PEG) have been shown to decrease HBsAg deliberately added to plasma fractions (Johnson et al., 1976). However, inoculation experiments in chimpanzees showed that the material was still infectious (Johnson et al., 1976). The aim of this investigation has been to develop methods for remo~ng HBV from contaminated plasma fractions by the use of adsorption chromatographic methods. HBsAg has been shown to have a high affinity for matrix-bound linear and cyclic hydrocarbons (Andersson et al., 1976; Einarsson et al., 1976; Einarsson, 1977; Neurath et al., 1978). In our work, special attention has therefore been devoted to finding the best and most practical procedures for maximal reduction of the number of virus particles from plasma fractions, especially concentrate of coagulation factors II, VII, IX and X, with a minimal loss of protein activity. MATERIALS

AND METHODS

Reagents Berol WASC (alkylphenol-ethyleneoxide adduct) was purchased from Berol Kemi AB, Stenungssund, Sweden. Cholic acid was obtained from Koch-Light Laboratories Ltd., England. Cyanogen bromide, dicyclohexyl~arbodi-i~de and caprylic acid ethylester were obtained

from Fluka AG, Buchs, Switzerland.

Octylamine,

decylamine

and hydrazine

hydroxide (100%) were purchased from Kebo-Grave, Stockholm, Sweden. Sepharose products were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The agarose used for electro~unoassays was from ~Industrie Bioiogique Fran@se, Issy La Molineux, France. H-D-Phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide dihydrochloride (S-2238), a synthetic chromogenic substrate for thrombin determination, was obtained from Kabi Diagnostica, Stockholm, Sweden. The concentrate of coagulation factors II, VII, IX and X used was clinical-grade material (Preconativ) prepared by KabiVitrum AB, Stockholm, Sweden. In the following account this material will be referred to as factor IX concentrate.

215

ANALYTICAL

TECHNIQUES

HBsAg determinations HBsAg was identified by immunoelectroosmophoresis 1971)

and by solid-phase

radioimmunoassay

(RIA)

(IEOP) (Hansson and Johansson, (Ausria

Chicago, IL). Quantitative HBsAg determinations were by making consecutive 2-fold dilutions of the sample (Gibco Bio-Cult, Glasgow, U.K.) to the point of antigen of the sample which was positive by RIA was considered HBeAg determination Hepatitis B e antigen (HBeAg) was determined (Abbott HBe, Abbott Laboratories).

II, Abbott

Laboratories,

performed by the RIA method with 10% newborn calf serum negativity. The highest dilution to be the titre of the sample.

by solid-phase

radioimmunoassay

DNA polymerase assay Assay conditions for the DNA polymerase were essentially as described earlier (Nordenfelt et al., 1980). The DNA polymerase activity was measured in the acidprecipitable material after incorporation of [“2P] dTTP. A ratio between 3 and 0 h incorporation of the radioactivity was calculated; when this ratio was below 2.0 the sample was considered negative (Cappel et al., 1975; Nordenfelt and AndrCn-Sandberg, 1976). In titration experiments a linear relationship between the titre and the measured c.pm. was regularly observed. For quantitative presentation of the data obtained, the DNA polymerase activity has been converted to HBV titres. A HBV titre of a sample was defined as the highest dilution of the sample considered to be DNA polymerase positive (ratio above 2.0). Assays for coagulation factors The assays for coagulation factors II, VII, IX and X were performed by previously established methods (Owren, 1949; Denson, 1961; Quick, 1966; Veltkamp et al., 1968). Factor IX-deficient plasma was obtained from a subject with severe haemophilia B and was used in the factor IX assay. Congenital factor VII-deficient plasma was used as a substrate in the factor VII assay. Factor IX concentrates were examined for in vitro thrombogenicity by the nonactivated partial thromboplastin time (NAPTT) (Kingdon et al., 1975) and the thrombin generation time (TG,50) (Sas et al., 1975) tests. Traces of thrombin in factor IX concentrates were screened for by incubating 200 ~1 of the sample (30 mg protein/ml buffer of 0.05 mol/l citrate and 0.09 mol/l sodium chloride) with 400 4 of 0.25% human fibrinogen at 37°C. The test requirement was that no clot should be observed within at least 4 11. Protein determination Protein was determined

according to Lowry et al. (195 1).

216

Electron microscopy Electron 1981). METHODS

microscopic

FOR REMOVAL

studies were performed

as described previously (Einarsson

OF HBsAg AND HBV FROM

PLASMA

et al.,

FRACTIONS

Source of HBsAg HBsAg was purified from serum from chronically infected individuals by chromatography on dextran sulphatesepharose 4B, as described by Einarsson et al. (1978). Part of the lipoproteins was removed from the HBsAg material by flotation in an MSE superspeed 65 ultracentrifuge with a titanium angle rotor, 8 X 25 ml, at 45,000 r.p.m. (150,000 g,) for 4 h at 4°C. Titres of the HBsAg obtained are shown in Table 1. Source of HB V Sera were taken from two patients (01s and A.W.) who were chronic carriers of HBsAg and undergoing haemodialysis. Part of the serum of Ols was used without treatment, and its HBV and HBsAg titres are shown in Table 1. Fifty and 120 ml, respectively, of the serum from the two patients (Ols and A.W.) were enriched by a routinely used pelleting procedure in an MSE superspeed 65 ultracentrifuge at 30,000 r.p.m. (6O,OOOg,) for 3 h at 4’C in a titanium angle rotor, 8 X 25 ml. The pelleted material was washed once in physiological phosphate buffer (PBS), pelleted again in the ultracentrifuge, and then suspended in 1 ml of PBS. Titre of the HBV concentrates obtained (Ols pellet and A.W. pellet) are shown in Table 1. HBsAg and HBV were also prepared from an infected human liver (B.S.). The liver was cut into small pieces, mixed with 1 volume of 0.45% NaCl and then frozen at -90°C. After thawing, the tissue was homogenized for 5 min in an MSE homogenizer (Tillquist AB, Stockholm, Sweden). The material was centrifuged twice at 2500 r.p.m. (16OOg,) for 30 min in a 4 X 750 ml aluminium angle rotor in an MSE centrifuge, model Mistral 4L. Then HBV in the supernatant was pelleted and resuspended three times by the routinely used pelleting procedure. The final pellet was dissolved in 10 ml of PBS (B.S. liver) and showed the titres indicated in Table 1. TABLE

1

Titres of the HBsAg and HBV preparations Analytical

technique

HBsAg

used in the experiments Source

of HBV

Serum 01s RIA

I :

IEOP

1 : 32 (64)

HBV by DNA polymerase assay

200,000

1

:

100,000

Ols, pellet 1

:

1,000,000

1 : 256

1 : 2,048

1 : 15

1

:

100

A.W., pellet

B.S., liver

:

1 : 500,000

1

1 : 512 (1,024)

1 : 64

100,000

1 : 10

1 : 300

217

Preparation of agarose derivatives Alkylamines

and octanoic

acid hydrazide

were coupled to CNBr-activated

as described by Jost et al. (1974). Cross-linking

Sepharose

of octanoic acid hydrazideBepharose

4B

was kindly performed by Pharmacia Fine Chemicals, Uppsala, Sweden. The procedure for preparation of cholic acid-AH-Sepharose was the same as described by Wichman (1979). ‘The degree of ligand substitution (Rosengren et al., 1975).

on the gels was determined

by proton

NMR

Studies on the extent and rate of adsorption of HBsAg to cross-linked octanoic acid hydrazide_Sepharose 4B in batchwise experiments In order to study the effect of different concentrations of HBsAg on the binding to cross-linked octanoic acid hydrazide-Sepharose 4B, the following method was used. A series of test tubes containing 0.5 ml of the gel derivative equilibrated in 0.75 mol/l ammonium bicarbonate (NH4HCOJ) was prepared. The degree of ligand substitution on the gel was 4.8% (w/w), and thus 0.5 ml of gel contained about 4 pmol of octanoic acid hydrazide. Various concentrations of HBsAg in 1.5 ml of 0.75 mol/l NH4HC03 were added. The titres by RIA of the HBsAg were between 1 : 40,000 and 1 : 40. The gel suspensions were gently mixed at 4°C for 1 h. After centrifugation the supernatants were collected for analysis of HBsAg. Experiments were also performed to determine the rate at which the HBsAg was bound to the cross-linked octanoic acid hydrazide-Sepharose 4B. In three beakers a slurry of 5 ml of sedimented gel and 5 ml of dilutions of HBsAg with titres of 1 : 20,000, 1 : 2000 and 1 : 200, respectively, were stirred at 4°C. While stirring, 1.0 ml aliquots of the gel slurries were taken out at various intervals between 0.5 and 60 min. The gel in the respective aliquots was immediately separated from the protein solution by filtering on a glass filter under suction. The solutions obtained were analysed with respect to HBsAg. Chromatography of factor IX concentrate Various gel derivatives were tested for the removal of HBV and HBsAg from factor IX concentrate. Among those investigated were Sepharose 4B substituted with cholic acid, octylamine, decylamine and octanoic acid hydrazide. Phenyl-Sepharose CL4B, octyl-Sepharose CL4B and cross-linked octanoic acid hydrazide-Sepharose 4B were also used. Columns filled with hydrophobic gel derivatives (1.6 X 5 cm) were equilibrated at 4’C in 0.75 mol/l NH4HC03. Various amounts of HBsAg or HBV were added to factor IX concentrates (10 mg/ml) in the same buffer. After application of varying sample volumes, tilution was performed with the buffer at 4°C. The flow rate was about 10 ml/h. Fractions of about 2 ml were collected and analysed for the presence of protein, DNA polymerase activity, HBV and related particles. The fractions containing protein were pooled, and, when found to be HBsAg-negative by RIA, concentrated in a Minicon B 15 macrosolute concentrator (Amicon Corp, Lexington, MA). The concentrate was then analysed for HBsAg. HBV was routinely concentrated by pelleting and then suspended in PBS before analysis.

218

A number

of experiments

with factor IX concentrate

above in order to investigate

its properties

fractions

protein

obtained

containing

when active, lyophilized. mol/l sodium chloride,

as described

procedure.

The

were pooled, analysed for factor IX activity and,

After redissolving the solution

were performed

after the chromatographic (30 mg/ml)

in 0.05 mol/l citrate and 0.09

was analysed for factors II, VII, IX and X and the

absence of thrombin. In vitro tests to trace potentially thrombogenic material, i.e. thrombin generation time (TG$O) and non-activated partial thromboplastin time (NAPTT), were also performed. If no factor IX activity was found in the original protein peak, attempts were made to elute it by decreasing the concentration of NH4HC03 to 0.05 mol/l. Attempts were made to regenerate the gel matrix after use by careful washing of the column with the following solutions: 1% (v/v) Berol in water, increasing concentrations of ethanol in water, n-butanol, decreasing concentrations of ethanol in water, and, finally, with the buffer to be used in the experiment. Fractions of about 2 ml were collected and analysed for protein, DNA polymerase activity, HBV and related particles. The same technique as described above was used to concentrate HBsAg and HBV. RESULTS

Adsorption properties of gel derivatives Various hydrophobic ligands linked covalently to Sepharose gels were investigated with respect to yield of factor IX activity after passage of solutions of factor IX concentrates in 0.75 mol/l NH4HC0a through columns filled with the gel derivatives. It was found that all, or nearly all, factor IX activity was lost, along with 40-50% of the protein, during passage through the columns, with one exception, viz matrix-bound octanoic acid hydrazide. When phenylSepharose CL4B was used, about 60% of the applied amount of factor IX activity could be eluted simply by decreasing the ionic strength of the buffer. However, under these conditions part of the bound HBsAg-positive material was also eluted along with the factor IX activity. In chromatography

on octanoic

acid hydrazidesepharose 4B the yield of factor IX activity and protein was SS%, and the composition of the factor IX concentrate, i.e. factors II, VII, IX and X, was the same before and after passage through the column. The protein eluate was found to be stable and could be lyophilized without any loss of activity. In vitro assays. TG,50 and NAPTT, to trace potentially thrombogenic material in the factor IX concentrate showed that no change in clotting times was caused by the chromatographic procedure. Furthermore, no clot was observed within 4 h in the thrombin test. Extent and rate of adsorption of HBsAg to cross-linked octanoic acid hydrazideSepharose 4B in batch-wise procedures Data from batch-wise adsorption

of various amounts of HBsAg to cross-linked octanoic

219

acid hydrazide-Sepharose are presented

4B using a high concentration

of salt (0.75 mol/l NH4HC03)

in Table 2 and Fig. 1. HBsAg was detected

by RIA in the supernatant

of

the adsorbed sample when the titre of the HBsAg had originally been about 1 : 1600 or more. By repeating the adsorption with fresh gel, the rest of the detectable HBsAg could be removed from the supernatant (not shown in the table). The amount of free HBsAg as a function of added material is shown in Fig. 1. The curve is biphasic, indicating at least two modes of binding to the gel, one with high affinity and low capacity and one with lower affinity and high capacity. The gel was never saturated with HBsAg. By varying the time for the binding of HBsAg to cross-linked octanoic acid hydrazideSepharose 4B in a batch-wise procedure, it was found (Fig. 2) that more than 10 min were necessary to reduce the amount of HBsAg and HBV below 1% of the added amount.

1:2OOxl 1:lOOOo

1:5000

pmoles

. .

i._ 1:loo

,

1. HBsAg

Varying

adsorption

amounts

Sepharose bound

l

050

IO

Free HEsAg (pmoledml)

1

Fig.

.

I

0.25

4B in a total

HBsAg has been

were

volume plotted

20

30

40

50

60

Incubation time (minutes)

to cross-linked

of HBsAg

. Lb_

octanoic

incubated

acid hydrazideSepharose

with

0.5 ml of cross-linked

of 2.0 ml of 0.75 against

mol/l

the amount

NH,HCO,

in batch octanoic

experiments.

acid hydrazide-

at 4°C for 1 h. The amount

of free HBsAg in the system.

For calculations

of see

legend to Table 2. Fig. 2. Time dependence 4B. Equal

volumes

of the binding

of sedimented

of HBsAg

in 0.75 mol/l

incubated

at 4°C for various

been plotted

against

NH,HCO, periods

the adsorption

of HBsAg

cross-linked with titres

to cross-linked

octanoic of 1

of time between time.

octanoic

acid hydrazidaSepharose

acid hydrazidsSepharose

: 20,000

(O), 1

4B and

dilutions

: 2000 (+) and 1 : 200 (A) were

0.5 and 60 min. The amount

of free HBsAg has

220

TABLE

2

Batch-wise Sepharose

adsorption

of HBsAg

in various

concentrations

to cross-linked

octanoic

acid hydrazide-

4B

Titre of HBsAg

Total amounta

Titre of HBsAg

Total amount

Detectable

in sample

of HBsAg

in supernatant

of unbound

HBsAg

added

after gel adsorption

HBsAg

bound

(nmol X 10m5)

(%)

added

(nmol

X 10F5)

RIA

RIA

1

: 40,000 : 24,000 : 12,000 : 4,000 : 3,200 : 2,400 : 1,600 : 800 : 400 : 320 : 160

1:

80

1:

40

1 1 1 1 1 1 1 1 1 1

a

3,240

1

1,950

: 800,l : 900 1 : 400

912 324

1:

80

98

8.7

99 99

40

3.8

1:

6

0.6

99

196

1:

5

0.5

99

64 32

1

30,l:

91

43

260 132

1:

92

: 10,l

:

4

0.7

Neg. ,,

99

GO.05 ,,

%I00 I,

26

,,

,,

I,

13

I,

,I

#I

6.4

,,

,,

,,

3.2

I,

4,

I,

A limit for the detection

of 2.4 X lo6 are parameters

of HBsAg by RIA of 1.3 ng HBsAg/ml used for the calculations

(Harris

and a molecular

weight

for HBsAg

et al., 1977).

On the basis of these results, it was assumed that in order to remove HBsAg efficiently from plasma fractions, to levels not detectable by the most sensitive assay methods available, it was preferable to perform the adsorption by a column chromatographic procedure. The linear flow rate was chosen so that the eluate was retained on the column for 1 h. Removal of HBsAg and HBVfrom factor IX concentrates Various concentrations of HBsAg and HBV were added to factor IX concentrates in 0.75 mol/l NH4HC0a. Experiments were then performed (Tables 3 and 4) to find out at what concentration HBsAg- or HBV-positive material leaked through octanoic acid hydrazide-Sepharose when the column procedure was used. Octanoic acid hydrazideSepharose 4B was examined before and after cross-linking of the gel matrix in order to investigate the effect of cross-linking on the binding of HBsAg and HBV. The data in Tables 3 and 4 show that the cross-linking caused a decrease in the binding capacity of HBsAg including HBV by about a power of 10 in titre. When a sample of HBsAg with a titre of 1 : 20,000 and a volume equal to the bed volume of the column was applied on

3

IX concentrates

10

10

10:

4B

4B cross-linked

4B cross-linked

experiments

IX concentrate

The rest of the protein

Factor

were performed.

: 20,000 : 20,000

: 200,000d

the gel was used again after regeneration

1

1

: 10

10

1

: 50

: 20,000

eluate was positive.

with Berol.

after

Pos. titre 1

Neg.

was negative

:8

< 36 ml of the eluatee

:4

10X, ~0s.’

titres ?, 1

cont.

Neg.

RIA

RIA 1

gel adsorption

IX

concentrate

factor

IX concentrate

on octanoic

HBsAga in the factor

chromatography

original

interaction

Titre of HBsAg in the

by hydrophobic

: 10

: ml)

was not present.

In two of the experiments

Four identical

IEOP was negative.

10

4Bb

(ml

Sample volume

factor

Gel volume

from

Sepharose

of HBsAg

gel type

NH., HCO,

Separation

TABLE in 0.75 mol/l

Cont.

Cont.

Pos.

Pos.

IEOP

10X, neg.

8X, neg.

desorbate

Pos.

Pos.

Pos.

Pos.

RIA

HBsAg in the 1% Berol

acid hydrazide-Sepharose

of HBV (and

e

were performed.

B.S. livers were used as a source of HBsAg and HBV.

experiments

Serum 01s was used as a source of HBsAg and HBV.

Three identical

’ d

3,000 1

1

: 9e titre ?, 1

Pos.

: 40

1

: 17

IEOP was negative.

9.6

4B cross-linkedd

cont.

cont.

20,000

1

10X, neg.

Neg.

Neg.

: 3c

1

: 50

25,000

01s pellets were used as a source of HBsAg and HBV.

10

4B

Neg.

: 3b

1

: 17

a

9.6

4B

1

: 10

24X,

15X, pos.

:2

cont. titre 1

Neg.

20X, neg.

IEOP pos.

RIA pos.

IEOP pos.

RIA pos.

titre 1

Cont.

ND

:2

15X,

experiments

titre

Cont.

cont.

ND

polymerase

HBV by DNA

The gel was used for other 40X, neg.

47X, neg.

RIA, IEOP

HBsAg by

desorbate

Neg.

cont.

Neg.

Neg.

1

: 3b

25,000

1

polymerase

acid hydrazide-Sepharose

Titres in the 1% Berol

on octanoic

HBV by DNA

: 10

HBsAg by RIA

after gel adsorption

IX

chromatography

in the factor

concentrate

Titre?

interaction

polymerase

HBV by DNA

factor

by hydrophobic

: ml)

b

10

4B

(ml

volume

HBsAg by RIA

IX concentrate

Sample

Titres in the original

IX concentrates

volume

factor

Gel

from

Sepharose

HBsAg)

gel type

0.75 mol/l NH,, HCO,

Separation

TABLE 4 in

K W

223

non-cross-linked

gel, no HBsAg could be detected

in the eluate by IEOP or by RIA

(Table 3). However, when a sample of HBsAg with a titre as high as 1 : 200,000 and a volume five times the bed volume was applied, HBsAg material, which was positive by RIA but not by IEOP, broke through the column after the passage of about 70% of the sample, i.e. after 3.5 times the bed volume. Table 4 displays the data from experiments with HBsAg-positive samples containing high DNA polymerase activity. A concentrate of Dane particles (Ols, pellet) was added to factor IX concentrate in 0.75 mol/l NH4HC03. The final titre of HBsAg in this protein solution was 1 : 25,000 and of HBV 1 : 3. In separate experiments, volumes of this sample equal to one and two times the bed volume were passed through a column of the octanoic acid hydrazide-Sepharose gel. No HBsAg, and no HBeAg, were found in the eluate in the radioimmunoassays and no HBV could be detected as indicated by either electron microscopy or DNA polymerase activity. Even when the eluate was concentrated 40-fold, no HBsAg-related material or DNA polymerase activity could be detected. On desorption of bound proteins with 1% Berol in water (Fig. 3) HBVrelated proteins, i.e. HBeAg and DNA polymerase, could be detected in the desorbate. When a serum (01s) containing high DNA polymerase activity was used as a source of HBV in the factor IX concentrate in a dilution of 1 : 5, the result obtained showed that no hepatitis-related material was found in the eluate, with the exception of HBeAg. 1.30

15

10

? m E 5

10

30

20

40

ElUtlO""Ol"me,ml)

Fig. 3. Chromatography has been

added

tographed

at 4°C on a column

the same buffer. of protein HBeAg

on octanoic

HBV. Factor Desorption

determined

determined

determined

in samples

radioactivity

obtained

of factor

1X concentrate

containing

(0) were

concentrated

material

method

NH, HCO,

acid hydrazide-Sepharose

was effected

against

the elution

volume.

to a HBeAg-negative

in HBsAg by RIA.

control

to which

was chroma-

4B equilibrated

with 1% Berol in water. The amount

20 times. The HBeAg is expressed

with the sample relative was negative

IX concentrate

(o), the titre of HBV by DNA polymerase

plotted about

4B of factor HBV in 0.75’ mol/l

(1.6 x 5 cm) of octanoic (D) of bound

by the Lowry by RlA

acid hydrazidssepharose

IX concentrate

in

The amount assay (0) and of HBV was

as the ratio between

the

in the RIA test. The eluate

224

This experiment

also demonstrated

that HBsAg and HBV bind very effectively

despite the co-existence

in the serum of high concentrations

well-documented

for hydrophobic

affinity

of albumin,

to the gel,

which has a

gels.

Experiments were also performed to demonstrate the ability of the gels to concentrate HBsAg and HBV. The aim was to show that the removal of virus-related material is effective even at concentrations not detectable by the most sensitive methods for analysis. 400 ~1 of a Dane particle concentrate (A.W. pellet) with a HBV titre of 1 : 10 was added to 50 ml of factor IX concentrate in 0.75 mol/l NH4HC0a. This protein sample, which was negative with respect to DNA polymerase activity, was applied to a 10 ml column of cross-linked octanoic acid hydrazide-Sepharose 4B. The eluate obtained was concentrated 127X by pelleting in an ultracentrifuge and was shown to be negative with respect to DNA polymerase activity. By desorption of bound material with 1% Berol in water and then concentration six times by pelleting, DNA polymerase activity could be demonstrated in a titre of 1 : 2. Since the volume of this concentrate was 2.1 ml, the results indicate that HBV could be concentrated efficiently from a dilute solution and then eluted almost quantitatively by Berol. In an analogous experiment with a sample of factor IX concentrate containing HBsAg, but negative by RIA, at least 75% of the applied HBsAg could be recovered in the 1% Berol desorbate. Studies were performed on the capacity of octanoic acid hydrazide-Sepharose 4B to bind HBsAg after regeneration with 1% Berol. By repeated adsorption of HBsAg followed by desorption in 1% Berol, careful washing in various alcohol-water mixtures and equilibration in salt, it was demonstrated that the procedure used for regeneration of the gel matrix somewhat reduced the capacity to bind HBsAg (Table 5). DISCUSSION

In recent years, frequent use has been made of hydrophobic interaction chromatography for protein purification, since proteins often contain hydrophobic domains or pockets. By varying the hydrophobicity of the ligand and/or by varying the degree of ligand substitution to the gel matrix, the affinity of proteins for the gel can be controlled. Sometimes the binding is so strong that the proteins denature on the gel surface and are thus very difficult to elute in a native form. The binding properties of HBsAg to matrixbound linear and cyclic hydrocarbons have been investigated previously (Andersson et al., 1976; Einarsson et al., 1976; Einarsson, 1977) and it was found that a straight carbon chain of at least eight carbon atoms was necessary for binding. Branched molecules showed very little affinity for HBsAg (Andersson et al., 1976) unless one chain was at least eight carbon atoms long. The earlier experiments indicate that there are two types of binding of HBsAg to hydrophobic ligands. First there are specific binding pockets for linear hydrocarbons and, secondly, there are more general hydrophobic surfaces where hydrophobic molecules can bind and the configuration of the ligand is not restricted to the same extent. Our present experiments described in Fig. 1 indicate that the octanoic acid hydrazide-

225

TABLE

5

Studies

on the

regeneration Number

capacity

of octanoic

acid

hydrazide-Sepharose

4B

to bind

HBsAg

after

use and

with 1% Berol Gel volume

Titre of HBsAg

of regeneration

of times

Sample

in the original

of the gel

(ml

volume

HBsAg in the sample

eluate

: ml) RIA

: 10 : 10 10 : 10

: : 1 : 1 :

RIA

10

1

18,000a

Neg.

10

1

18,000a

Neg.

18,000a

Neg.

10 : 50

200,000b

d 36 mlc of the eluate was negative

10 : 50

1

: 200,000b

< 22 mP of the eluate was negative

10

: 40

1

: 200,000b

<13mlcof the eluate was negative

9.6

: 40

1

: 200,000b

d 11 mlcof the eluate was negative

a

The HBsAg material

was added

b To the HBsAg material ’

The rest of the eluate

to factor

solid NH,HCO, was positive

IX concentrate was added

in 0.75 mol/l NH,HCO,.

to a final concentration

by RIA but negative

of 0.75 mol/l.

by IEOP.

Sepharose gel can bind HBsAg by two modes, one with low capacity but high affinity and one with high capacity but lower affinity. The higher affinity binding seems particularly useful in removing HBsAg and HBV from solutions of low concentration with a high degree of efficiency.

This is further supported

by the experiments

in which HBsAg

and HBV were concentrated from dilute solutions by chromatography. The particles were recovered from the column almost quantitatively. In this study it was also shown that HBsAg and HBV could be removed from plasma fractions deliberately contaminated with hepatitis B-positive material to final titres by RIA of at least 1 : 10,000. The protein eluates obtained after gel adsorption were concentrated lo-fold and shown to be negative or, occasionally, slightly positive for HBsAg, with RIA titres of about 1 : 4. Some of these positive eluates were obtained, however, after use of regenerated gel. Thus the reduction of HBsAg-positive material was between 104- and 10’ -fold. Data also show that the binding properties to the hydrophobic gels of both HBsAg and HBV are about the same. Since HBV cannot yet be cultured in vitro, experiments to demonstrate inactivation of HBV are difficult to perform. A few studies have been made with chimpanzees as experimental animals (Shikata et al., 1978). The investigators found that when serum

226

with a titre of 10’ times the chimpanzee-infectious dose had been heated at 60°C for 10 h, it was still infectious. This is supported by in vitro experiments, since we have found that lo-25% of the DNA polymerase activity remained after 10 h of inactivation at 60°C (unpublished results). Furthermore, thermal inactivation of HBsAg (De Flora, 1978) has been shown to be dependent on the experimental conditions. Treatment of hepatitis B virus-infected plasma with a combination of /!I-propiolactone and UV light resulted in a 103-fold reduction of infectivity (Stephan et al., 1980) but the yield of factor IX concentrate was poor. The use of UV light on proteins must be considered hazardous, since this treatment might cause modification of the proteins, with adverse immunological treatment.

reactions

as a possible side effect in patients,

especially during repeated

Immunoadsorption has also been used for the removal of HBsAg from blood plasma (Wong and Charm, 1977). A rather low affinity of HBsAg for the anti-HBs-Sepharose was obtained, however, and the time required for adsorption was long, especially at low temperatures. Experiments with the addition of relatively high concentrations of human anti-HBs to factor IX concentrates have also been performed (Tabor et al., 1980). Since there is a limited supply of human anti-HBs, such use seems to be of limited practical value. Procedures employing precipitation in PEG have been used to reduce the amount of HBsAg from plasma fractions about 104-fold. The method is influenced by pH, ionic strength, protein concentration and specific salts. In most instances it is difficult to adjust these parameters to allow separation of HBsAg and HBV from the plasma proteins without simultaneous loss of the latter. The removal of HBsAg by PEG is not absolute, because the principle is based on exclusion and a residue will always remain in solution (Laurent, 1963). Cross-linked octanoic acid hydrazide-Sepharose 4B as adsorbent afforded an effective agent for the removal of HBsAg, as demonstrated in Table 2. The particles were adsorbed nearly quantitatively in the batch experiments. Furthermore, as indicated in Fig. 1, this procedure is especially effective at low particle concentrations, which contrasts with the PEG procedure which utilizes the exclusion phenomenon. The efficiency of the adsorption can be further increased by using a chromatographic procedure instead of the batch type adsorption, especially on non-cross-linked gel, as can be seen from Tables 3 and 4. The method has also been applied with success to the removal of HBsAg and HBV from solutions of antithrombin III (unpublished). By selecting the ligand, the type of salt and the salt concentration, conditions could be created in which the HBsAg were adsorbed but the plasma proteins could be recovered with a yield of 85% and with intact biological activity and stability. The major problems concerning the clinical use of factor IX concentrates are their potential for transmitting hepatitis (Kingdon et al., 1975; Rossiter et al., 1979) and the possibility of thrombogenic complications in the recipient (Aronson, 1978). Infusion of the factor IX concentrate Preconativ has not resulted in any thrombogenic complications. Passage of this protein concentrate through a column of octanoic acid

227

hydrazide-Sepharose, could possibly

in order to reduce the amount

result in the formation

of any HBsAg and HBV present,

of thrombogenic

material.

However, the results

obtained by the in vitro assays (NAPTT, TG,50, thrombin test) to trace potentially thrombogenic material indicate that no thrombogenic material is formed. The procedure used to remove hepatitis B virus might also be effective in adsorbing the non-A, non-B hepatitis virus(es), especially if it (they) turns out to be a lipidcontaining virus(es) or a HBV-like virus(es) as indicated by some research groups. ACKNOWLEDGEMENTS

We wish to thank Professor T. Laurent, Biomedical Centre, Uppsala, for valuable discussion and criticism of the manuscript. Parts of the investigation have been supported by the Swedish Medical Research Council (project No. B79-16X-02865-10A and No. 13X-4). REFERENCES

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