Virus safety of human immunoglobulins: Efficient inactivation of hepatitis C and other human pathogenic viruses by the manufacturing procedure

Journal of Medical Virology 36209-216 (1992)

Virus Safety of Human Immunoglobulins: Efficient Inactivation of Hepatitis C and Other Human Pathogenic Viruses by the Manufacturing Procedure

Thomas Nowak, Jens-Peter Gregersen, Ulrich Klockmann, Larry Bill C u m i n s , and Joachim Hilfenhaus Research Laboratories of Behringwerke AG, Marburg, Germany ( T.N., J.-P.G., U.K., J.H.) and White Sands Research Center, Alamogordo, New Mexico (L.B.C.)

Human immunoglobulins are plasma derivatives with a low risk of transmitting viral infections. To the present, no proven case of human immunoglobulins transmitting human immunodeficiency viruses has been reported. However, there have been a few reports on the transmission of hepatitis C virus by these plasma proteins. To improve further the safety of both 5s iv human immunoglobulins and 7s im immunoglobulins, we introduced a 10-hour heat treatment of the aqueous solutions a t 60°C (i.e., pasteurization) into the manufacturing procedure. This treatment was not added to the manufacturing procedure of 7s iv immunoglobulin that already contained the S-sulfonation as a virus inactivating method. We now report on experimental data that show that the whole manufacturing procedures of the above immunoglobulins inactivate efficiently hepatitis C virus and that the specific virus inactivation methods alone, namely, pasteurization or S-sulfonation, also inactivate completely viruses of the flavivirus family, to which the hepatitis C virus belongs. The inactivation of the Flaviviridae bovine viral diarrhea virus, tick-borne encephalitis virus, and yellow fever virus by pasteurization or S-sulfonation was at least lo5. The clearance of HCV achieved by the entire manufacturing process of each of these immunoglobulins was also at least lo5. The experiments therefore show that pasteurization or S-sulfonation provides a high margin of safety to human immunoglobulins regard- ing the transmission of hepatitis C virus.

KEY WORDS: human immunoglobulins, pasteurization, flavivirus, S-sulfonation

INTRODUCTION Human plasma derivatives are important for prophy-

lactic or therapeutic treatment of human diseases. Sev- 0 1992 WILEY-LISS, INC.

era1 papers published during the last decade reported that human plasma and its derivatives could be con- taminated by viruses and that viral diseases could thus be transmitted to recipients [Prince et al., 1987; Cen- ters for Disease Control, 1988; Klein et al., 19901. The products that most frequently transmitted human pathogenic viruses were factor VIII concentrates. In the past, infections with hepatitis B virus (HBV), human immunodeficiency virus (HIV), and nonA/nonB hepatitis virus (now designated as hepatitis C virus, or HCV) frequently occurred in hemophiliacs treated with these concentrates [Gerety et al., 1982; Melief et al., 19861. In contrast, human albumin, and human immunoglobulins in general, were regarded as safe plasma derivatives [Centers for Disease Control, 19861. Only in the case of certain iv immunoglobulin preparations were a few reports published on transmission of nonA/nonB hepatitis [Lane, 1983; Weiland et al., 1986; Iwarson et al., 1987; Bjorkander et al., 1988; Williams et al., 19881.

It must be emphasized that the exceptionally high safety of human immunoglobulins does not simply depend on the exclusion of blood or plasma donations positive for HBsAg or anti-HIV. Nor does it depend on the introduction of a specific virus inactivation method into the manufacturing procedures of immunoglobulins, as had been demanded for factor VIII concentrates since 1985. There must, therefore, be other reasons for the safety of immunoglobulins, presumably: ( 1) the neu- tralization of infectious viruses by specific antibodies present in the plasma pools from which the immunoglobulins are isolated, and (2) the inactivation and/or elimination of viruses by certain steps of the manufacturing procedures already established before AIDS and the

Accepted for publication September 16,1991. Address reprint requests to Dr. Joachim Hilfenhaus, Behring-

werke AG, P.O. Box 1140,3550 Marburg, Germany. Trade names of the immunoglobulins studied Gamma Venin

HS = 5s iv human immunoglobulin; Beriglobin HS = 7s im human immunoglobulin; Venimmun = 7s iv human immunoglobulin.

210

safety of plasma proteins became such an important issue. In retrospect, the well-established ethanol fractionation method developed by Cohn et al. [1946] has subsequently been shown to be an efficient procedure for eliminating and/or inactivating HIV [Wells et al., 1986; Hilfenhaus et al., 19871.

Thus the only virus reported to have been transmitted by iv human immunoglobulins was nonA/nonB hepatitis. Whether this was due to the fact that the amounts of neutralizing antibodies to HCV were not high enough in certain plasma pools or that this virus was more resistant to the ethanol treatment of the Cohn procedure than other viruses is not known. Al- though no proven case of HCV transmission by our immunoglobulins had been reported, we introduced a specific virus inactivation step into the manufacturing procedure of these products, namely, heat treatment a t 60°C in aqueous solution of 10 hr (pasteurization), to improve their safety further. In the case of a 7s iv immunoglobulin, we did not add this inactivation method to the manufacturing process because we had found that S-sulfonation, a well-established step of this process, excellently inactivates viruses.

Here we report on the inactivation of viruses, partic- ularly those belonging to the same family as HCV [Choo et al., 19911, the Flaviviridae, by pasteurization or S-sulfonation. In addition, we investigated the ability of the entire manufacturing procedure of each of three different immunoglobulins to eliminate and/or inactivate HCV by subjecting human plasma that had been “spiked” with HCV to each of the different manufacturing procedures and then tested the resulting products for infectious HCV in chimpanzees.

Nowak et al.

MATERIALS AND METHODS Viruses

The following virudcell systems were used: bovine viral diarrhea virus (BVDV) (FlaviviridaeVbovine epithelial fetal lung cells; tick-borne encephalitis virus (TBEV) (FlaviviridaeYhuman carcinoma cell line (A549); yellow fever virus strain 27 D (,YFV) (Flaviviridae)/vero cells; human immunodeficiency virus type 1 or 2 (HIV-1, HIV-2) (RetroviridaeYhuman T4 lymphoblast cell line (Jurkat); herpes simplex virus type 1 (HSV-1) (Herpesviridae)/vero cells; poliovirus type 1 (Polio-3) (Picornaviridae)/human epithelial la- ryngeal tumor cell line (Hep2). With the exception of the Jurkat cells, all cells were grown in Eagle’s mini- mal essential medium supplemented with 5%-10% fetal calf serum. The Jurkat cells were grown in medium RPMI 1640 supplemented with 10% fetal calf serum. For the HCV studies, a sample of the HCV containing human plasma pool (H strain of HCV) was used [Ogata et al., 19911, which was kindly obtained from Dr. R. Purcell (NIAID, Bethesda, MD]. The infectivity titer of this HCV sample in chimpanzees had been determined as being lo6., CID,,/ml (CID,, = chimpanzee infec-

Virus Infectivity Assays Virus infectivity was titrated by standard microtitra-

tion assays using the cells noted above. With the exception of HIV-1, HIV-2, and HCV, the following procedure was used. Cell monolayers in flat-bottomed wells of standard 96-well microtiter plates were incubated with 0.1 ml of 10-fold serial dilutions in complete medium of the virus samples to be assayed. Eight wells per dilu- tion were used. The microtiter plates were incubated at 37°C for 7 days and evaluated microscopically for cytopathic effects. The infectivity titers (TCID,,/ml) were calculated according to the Reed and Muench method 119381. If no infectious virus was detected by microtitra- tion, four 25 cm2 or 100 cm2 cell culture vessels were incubated with 1 ml each of the original sample diluted in cell culture medium. These cell cultures were then also incubated at 37°C for 7 days and studied microscopically for cytopathic effects. If all four cultures re- mained negative, i.e., no infectious virus was detectable in four 1 ml aliquots of the original sample, the virus titer of this sample was given as < 10°TCID,,/ml.

Infectivity assays for HIV-1 or HIV-2 were started in microtiter plates or in larger culture vessels as men- tioned above. The entire incubation period, however, consisted of 4 weeks, and thus frequent feeding of the cell cultures with fresh medium was necessary. There- fore, every third day approximately two-thirds of the culture supernatants were replaced by fresh medium. After 2, 3, and 4 weeks, respectively, supernatants of the cell cultures were taken and concentrated 5-10-fold by PEG-precipitation (7.5% PEG 6000, 4”C, overnight incubation). Precipitates were resuspended in 0.85% NaCl + 0.6% Triton X-100 and tested for reverse transcriptase activity as described elsewhere [Gregersen, et al., 19881.

To determine infectious HCV, chimpanzees were inoculated intravenously with the samples to be tested and studied over a period of 9 months as described elsewhere [Mauler et al., 19871. During this period, blood was taken weekly to determine the activity of serum alanine aminotransferase (ALT), and monthly to determine the hepatitis B-markers: HBsAg, anti-HBs, anti- HBc, HBeAg, and anti-HBe. Liver biopsies were taken monthly for histopathological examinations. At the beginning and the end of the 9-month observation period, antibody titers to hepatitis A virus, cytomegalovirus, and Epstein-Barr virus were determined. If no infection with any of these viruses or hepatitis B virus was diagnosed and if the ALT levels in a chimpanzee increased more than twofold over the baseline, this particular chimpanzee was regarded as HCV infected, and conse- quently the inoculated sample as containing infectious HCV. When an antibody assay to HCV became available [Kuo et al., 19891, the chimpanzee sera were assayed retrospectively with the Ortho anti-HCV assay according to the manufacturer’s instructions.

Manufacturing of Immunoglobulins The manufacturing Drocedures of the immunodobu- u - -

tious dose 50%). lins studies are summarized schematically in Figure 1.

Virus Safety of Immunoglobulins

I 35 % Ethanol, -S°C Modified 7 s immunoglobulin Adsorptive filtration

25 % Ethanol,

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8 % Ethanol, -2°C

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sulfonation Pepsin treatment

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10 % Ethanol

stant 5s immunoglobulin I

20 % Ethanol, -Sac Adorptive Filtration

ant (redissolved)

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+ sucrose, glycine

Stabilized immunoglobulin

Adsorptive filtration

Sediment (redissolved)

Pasteurized, 5s iv imunoqlobulin 7s iv imunoqlobulin Pasteurization, 7s im immunoglobulin

Fig. 1. Scheme of the manufacturing procedures of a 7s im immunoglobulin, a 5s iv immunoglobulin, and a 7s iv immunoglobulin, respectively. The specific methods of these procedures that were investigated for their virus inactivating potency are boxed.

The crude immunoglobulin concentrate obtained as 25% ethanol precipitate according to the Cohn et al. [19461 procedure is the starting material for three different products, namely, a 7s im immunoglobulin, a 5s iv immunoglobulin, and a 7s iv immunoglobulin. The 7s im immunoglobulin is isolated from the crude concentrate by consecutive 10% ethanol and 25% ethanol fractionations followed by adsorptive filtration. High amounts of sucrose and glycine are then added as stabilizers, and this aqueous preparation is subjected to 10-hr heat treatment a t 60°C (i.e., pasteurization). Sub- sequently the stabilizers are removed by dialysis. The 5s iv immunoglobulin is developed from the crude immunoglobulin concentrate by pepsin treatment, which results in the F(ab’), fragment, followed by precipitation with 35% ethanol. Here, as described above for the 7s im immunoglobulin, the pasteurization is added as the final step to the manufacturing procedure. S-sulfonation was performed on the crude immunoglobulin preparation as described in detail elsewhere [Gronski et al., 19831, resulting in a modified 7s immunoglobulin, which is further purified by 25% ethanol precipitation and adsorptive filtration. Briefly, for the S-sulfonation of the immunoglobulins, CuSO, and Na2S20, are added to the protein solution while it is stirred. The pH is adjusted to 5.0, and this preparation is then incubated at 5°C for at least 10 hr.

Testing for Virus Inactivation

The ability of the pasteurization or S-sulfonation to inactivate viruses was tested separately by “spiking” intermediate product samples taken from a production lot with each virus immediately before pasteurization or S-sulfonation, respectively. For this purpose, one volume of the virus preparation was added to nine volumes of the product sample (for pasteurization studies samples already containing the stabilizers were used). The spiked samples were then treated according to the manufacturing protocol. Infectivity titers of each virus were determined for both the “spiked” starting material and the resulting sample after pasteurization or S-sulfonation.

In the studies with HCV, human plasma spiked with this virus was subjected to the entire manufacturing procedure of each of the immunoglobulins (see Fig. 1). Small samples of the spiked starting material as well as the total, resulting amounts of the final products were inoculated into chimpanzees, which were then moni- tored over a period of 9 months as described above.

The virus inactivation factor was calculated for each individual virus according to the CPMP guidelines [Commission of the European Communities, 19911 as the ratio of the virus load in the starting, spiked material and the virus load in the sample resulting from the

212 Nowak et al.

TABLE I. Virus Inactivation Achieved Under Conditions of the Manufacturing Procedure of Three Different Human Immunoglobulins by Either Pasturization or S-Sulfonation

Pasteurization of S-sulfonation of 7s im immunoglobulin 5s iv immunoglobulin 7s iv immunoglobulin

Virus Inact. factor (loglo) Inact. time (h) Inact. factor (loglo) Inact. time (h) Inact. factor (loglo) >5.0 4 >6.3 4 >5.0 >7.3 4 >7.5 4 >7.3 BVDV >5.9 6 >6.4 6 >6.3

TBEV >4.0

YFV >6.0 1.5 >6.0 2

>5.6 HIV-1

>7.8 0.5 >7.8 0.5 HSV-1 >6.3 2 >6.4 8 >4.6 HIV-2 Polio-3 >4.7 1 >4.6 4 >4.4

specific treatment to be tested. The formula used for this calculation is:

V a X a Vb X b

Inactivation factor = ___ .

Va = volume of the starting material, a = virus titer of

Vb = volume of the resulting material, b = virus titer the starting material.

of the resulting material.

All virus titers are given as log,,TCID,,/ml or log,,CID,,/ml, and all inactivation factors calculated are given as log,,.

RESULTS Virus Inactivation by Pasteurization or

S-Sulfonation Three different manufacturing procedures for human

immunoglobulins were studied. The specific methods for virus inactivation were pasteurization in the case of the 7s im immunoglobulin, and 5s iv immunoglobulin and S-sulfonation in the case of the 7s iv immunoglobulin, respectively (Fig. 1). Samples of the respective immunoglobulins were taken from the production lots and spiked separately with each of the viruses listed in Ta- ble I. The spiked samples were then subjected to either pasteurization or S-sulfonation and the amount of infectious virus before and after this treatment determined.

The resulting inactivation factors shown in Table I indicate that not only the viruses of the Flaviviridae family (BVDV, TBEV, and YFV) were completely inactivated (residual virus < 10°TCID,,/ml) by both methods but also some other human viruses tested, namely, HIV-1, HIV-2, HSV-1, and Polio-3. HCV, a member of the Flaviviridae, seems to be more closely related to the pestivirus group of this family than to the flavivirus group, but it does not clearly belong to one of these two groups. The proposal has therefore been made to regard HCV as an unclassified flavivirus [Choo et al., 19911. In our inactivation experiments, we used as test viruses related to HCV two representatives of the Flavivirus group, TBEV and YFV, and one representative of the pestivirus group, BVDV. When an attempt was made to determine the kinetics of virus inactivation by S-sul-

fonation using TBEV as a test virus, it was found that this virus was inactivated within a few minutes (data not shown 1. Inactivation of flaviviruses by pasteurization, however, occurred more slowly in the stabilized 5s iv immunoglobulin or 7s im immunoglobulin preparation (Fig. 2). As can be seen from the inactivation curves, all three viruses are comparably sensitive to heat treatment a t 60°C in aqueous solution. Whereas BVDV and TBEV were both completely inactivated after a 4-hr heat treatment, the vast majority of the infectious YF viruses was inactivated within 1 to 2 hr, but a very small amount of infectious YF viruses needed pro- longed heat treatment for complete inactivation, namely 6 hr.

HCV Inactivation in the Whole Manufacturing Procedures

Human plasma that did not contain any antibodies to HCV was spiked with infectious HCV and subjected to the manufacturing procedure of the 5s iv, 7s iv, or 7s im immunoglobulin, respectively. Table I1 gives the volumes of human plasma spiked with HCV, the amounts of infectious HCV used, the volumes of the resulting final products, and the volumes inoculated into chimpanzees of the “spiked” starting material and the final products. Figure 3 shows the follow-up of ALT activities during the 9-month observation period after inoculation of a chimpanzee with either 5 ml of HCV spiked plasma (Fig. 3A) or with 45 ml of the resulting 7s iv immunoglobulin (Fig. 3B). Furthermore, the levels of anti-HCV antibodies are shown. The significant in- crease of ALT activity beginning 12 weeks after inoculation of the chimpanzee with the spiked starting material indicated that this sample contained infectious HCV because during this observation period there was neither a hepatitis A virus, cytomegalovirus, or Ep- stein-Barr virus infection diagnosed nor did any hepatitis B marker became positive. Histopathological alter- ations observed in liver biopsies of weeks 8,12,16, and 20 postinoculation showed an inflammatory process of the liver. Retrospectively, this indirect diagnosis was confirmed by the detection of HCV specific antibodies. In contrast to the animal inoculated with the HCV spiked plasma, the other one, shown as a negative ex- ample here, did not develop a HCV infection after inoculation of 45 ml of the 7s iv immunoglobulin preparation derived from this HCV spiked plasma (Fig. 3B). In

Virus Safety of Immunoglobulins

8 -

7 t T 213

A

- 1 C I ! ! I ! I I I ! I I I I I I I ! I --c--cI 0 1 2 3 4 5 6 7 8 9 10

time of heat treatment (h)

Fig. 2. Kinetics of inactivation of the pestivirus BVDV (V ) and the flaviviruses TBEV 1*) and YFV (0) by pasteurization in a stabilized 5s iv immunoglobulin preparation (A) or in a stabilized 7s im immunoglobulin preparation (B). If four 1 ml samples of the virus spiked, but heat-treated immunoglobulin preparation was free of any infectious virus assayed, this is indicated as “no virus detectable.”

Table 11, the results of the chimpanzee experiments performed with the spiked plasma samples and the resulting immunoglobulin preparations are summarized. The inactivation factors calculated from these experimental data (last line of Table 11) resulted in each case in an inactivation factor greater than 5.4 log,,.

DISCUSSION HCV is the only virus reported to have been trans-

mitted by human immunoglobulins [Lane, 1983; Lever

et al., 1984; Ochs et al., 1986; Williams et al., 19881. We therefore paid particular attention to viruses that are closely related to HCV while investigating the effi- ciency of the virus inactivation methods, pasteurization and S-sulfonation, included in the manufacturing procedures of three different human immunoglobulin products. Furthermore, we studied whether the entire manufacturing process of these immunoglobulins is able to eliminatehnactivate HCV. Both kinds of experiments were performed to find out whether the manufac-

Nowak et al.

turing procedures of these immunoglobulins could re- sult in HCV-free final products.

Seven different viruses, including three flaviviruses and both known serotypes of HIV, were completely inactivated in the respective immunoglobulin preparations by pasteurization or by S-sulfonation. The kinetics of the inactivation of the three Flaviviridae by pasteurization show that the flavivirus TBEV and the pestivirus BVDV in either the 5s iv immunoglobulin preparation or the 7s im immunoglobulin preparation stabilized with high amounts of sucrose and glycine were completely inactivated after a 4-hr heat treatment (duration of heat treatment during manufacturing: 10 hr). The YFV preparations seemed to consist of two different fractions, the majority being rather heat labile (inactivation after 1 to 2 hr) and a very small fraction needing as long as 6 hr for complete inactivation. The relative stability of HSV-1 to heat treatment in the two immunoglobulins, namely, in the 7s im immunoglobulin (complete inactivation after 2 hr) and the 5s iv immunoglobulin (complete inactivation after 8 hr) differed considerably. This observation concurs well with other examples, in which the heat inactivation of a certain virus varies with the plasma protein preparation used [Hilfenhaus and Mauler, 19871. The reasons for such differences can be discussed theoreti- cally, but more importantly such different results cannot be predicted. It is therefore necessary to test the inactivation of each specific virus separately by the same method, e.g., heat inactivation, case by case for each human plasma derivative. In contrast, these data again demonstrate that results obtained with test viruses provide some solid information on a virus inactivation procedure but may not be identical for the virus of real interest, i.e., the inactivation factors elaborated for BVDV, TBEV, or YFV need not to be the same as for HCV.

The only system available in which infectious HCV can be determined are chimpanzees. Plasma samples obtained from infected chimpanzees or from patients suffering from nonAinonB hepatitis are the only source of infectious HCV. Since the amount of infectious HCV available for spiking experiments is very limited and since no in vitro assays exist for infectious HCV, it is not possible to conduct an extensive step-by-step investigation of HCV eliminationlinactivation by the manufacturing procedures of human plasma proteins. Fur- thermore, individual steps that may efficiently inactivate HCV cannot be studied in detail because a large number of chimpanzees would be needed. We therefore decided to investigate the elimination1 inactivation of HCV by the entire manufacturing procedures of the immunoglobulins discussed here, instead of doing step-by-step studies. We were able to confirm the HCV infectivity of the “spiked” starting material as well as the lack of any infectious HCV in the final immunoglobulin preparation. We were also able to demonstrate that anti-HCV antibodies were developed in the chimpanzees that showed nonAInonB hepatitis after HCV infection. Here as in other serum samples of HCV infected chimpanzees, it was found that compared

Virus Safety of Immunoglobulins 215

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E 0 .- - $ 150 3

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Fig. 3. Follow-up of the alanine aminotransferase activity 1ALT) (V ) and of the anti-HCV antibody (0) response in chimpanzees inoculated with either 5 ml of the spiked plasma (A) or 45 ml of the 7s iv immunoglobulin produced from this spiked plasma (B).

to the significant elevation of ALT, the only commer- cially available anti-HCV assay a t the time detected seroconversion rather late.

The new European guidelines for the validation of virus eliminatiodinactivation of the manufacturing procedure of plasma proteins require the step-by-step investigation of a certain manufacturing procedure and the elaboration of a cumulative clearance factor [Com- mission of the European Communities, 19911. Of course, such cumulative clearance factors will in due course be elaborated for test viruses such as HIV, HSV, and poliovirus, and possibly a close relative of HCV as

test virus, in the immunoglobulin products. The purpose of the experiments described here, however, was to study the inactivation not only of viruses closely related to HCV but also HCV itself. The results demonstrate that pasteurization or S-sulfonation efficiently and completely inactivated the testviruses of the flaviviridae family and that HCV itself was completely inactivated by the end of the whole manufacturing procedures. It is concluded that immunoglobulins, namely, a 5s iv immunoglobulin, 7s iv immunoglobulin, and a 7s im immunoglobulin, respectively, manufactured by these procedures will provide a high margin of safety

216 Nowak et al.

and that transmission of infectious HCV or other human pathogenic viruses by these immunoglobulins is highly unlikely.

ACKNOWLEDGMENTS We are very grateful to Mr. Heinrich Eife, Mrs. Sab-

ine Mehdi, Mrs. Eva Weber, Mr. Klaus Weber, and Mrs. Ramona Volk for their excellent technical assistance. The careful preparation of this manuscript by Mrs. An- drea Kirch is highly acknowledged. We much appreci- ate the skillful work of Dr. Cummin’s staff when main- taining and treating the chimpanzees during the hepatitis C studies. Experimental studies involving chimpanzees were conducted in accordance with the Ethical Practices Committee of the laboratories.

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Virus safety of human immunoglobulins: Efficient inactivation of hepatitis C and other human pathogenic viruses by the manufacturing procedure