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 regarding the transmission of hepatitis C virus.

era1 papers published during the last decade reported that human plasma and its derivatives could be contaminated by viruses and that viral diseases could thus be transmitted to recipients [Prince et al., 1987; Centers 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 neutralization 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

KEY WORDS: human immunoglobulins, pasteurization, fonation

flavivirus,

S-sul-

INTRODUCTION Human plasma derivatives are important for prophylactic or therapeutic treatment of human diseases. Sev0 1992 WILEY-LISS, INC.

Accepted for publication September 16,1991. Address reprint requests to Dr. Joachim Hilfenhaus, Behringwerke AG, P.O. Box 1140,3550Marburg, 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.

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Virus Infectivity Assays Virus infectivity was titrated by standard microtitration 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 dilution 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 microtitration, four 25 cm2 or 100 cm2 cell culture vessels were incubated with 1ml 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 remained 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 mentioned above. The entire incubation period, however, consisted of 4 weeks, and thus frequent feeding of the cell cultures with fresh medium was necessary. Therefore, 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 MATERIALS AND METHODS and studied over a period of 9 months as described elseViruses where [Mauler et al., 19871. During this period, blood The following virudcell systems were used: bovine was taken weekly to determine the activity of serum viral diarrhea virus (BVDV)(FlaviviridaeVbovine epi- alanine aminotransferase (ALT),and monthly t o deterthelial fetal lung cells; tick-borne encephalitis virus mine the hepatitis B-markers: HBsAg, anti-HBs, anti(TBEV) (FlaviviridaeYhuman carcinoma cell line HBc, HBeAg, and anti-HBe. Liver biopsies were taken (A549); yellow fever virus strain 27 D (,YFV) monthly for histopathological examinations. At the be(Flaviviridae)/vero cells; human immunodeficiency vi- ginning and the end of the 9-month observation period, rus type 1or 2 (HIV-1, HIV-2) (RetroviridaeYhumanT4 antibody titers to hepatitis A virus, cytomegalovirus, lymphoblast cell line (Jurkat); herpes simplex virus and Epstein-Barr virus were determined. If no infection type 1 (HSV-1) (Herpesviridae)/vero cells; poliovirus with any of these viruses or hepatitis B virus was diagtype 1 (Polio-3) (Picornaviridae)/human epithelial la- nosed and if the ALT levels in a chimpanzee increased ryngeal tumor cell line (Hep2). With the exception of more than twofold over the baseline, this particular the Jurkat cells, all cells were grown in Eagle’s mini- chimpanzee was regarded as HCV infected, and consemal essential medium supplemented with 5%-10% fe- quently the inoculated sample as containing infectious tal calf serum. The Jurkat cells were grown in medium HCV. When an antibody assay to HCV became availRPMI 1640 supplemented with 10% fetal calf serum. able [Kuo et al., 19891, the chimpanzee sera were asFor the HCV studies, a sample of the HCV containing sayed retrospectively with the Ortho anti-HCV assay human plasma pool (H strain of HCV) was used [Ogata according to the manufacturer’s instructions. et al., 19911, which was kindly obtained from Dr. R. Purcell (NIAID, Bethesda, MD]. The infectivity titer of Manufacturing of Immunoglobulins this HCV sample in chimpanzees had been determined as being lo6., CID,,/ml (CID,, = chimpanzee infecThe manufacturing Drocedures of the immunodobutious dose 50%). lins studies are summarized schematically in Figure 1. 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. Although 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, particularly 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.

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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 Testing for Virus Inactivation 25% ethanol precipitate according to the Cohn et al. [19461 procedure is the starting material for three difThe ability of the pasteurization or S-sulfonation to ferent products, namely, a 7s im immunoglobulin, a 5s inactivate viruses was tested separately by “spiking” iv immunoglobulin, and a 7s iv immunoglobulin. The intermediate product samples taken from a production 7s im immunoglobulin is isolated from the crude con- lot with each virus immediately before pasteurization centrate by consecutive 10% ethanol and 25% ethanol or S-sulfonation, respectively. For this purpose, one volfractionations followed by adsorptive filtration. High ume of the virus preparation was added to nine volumes amounts of sucrose and glycine are then added as stabi- of the product sample (for pasteurization studies samlizers, and this aqueous preparation is subjected to ples already containing the stabilizers were used). The 10-hr heat treatment a t 60°C (i.e., pasteurization). Sub- spiked samples were then treated according to the mansequently the stabilizers are removed by dialysis. The ufacturing protocol. Infectivity titers of each virus were 5s iv immunoglobulin is developed from the crude im- determined for both the “spiked” starting material and munoglobulin concentrate by pepsin treatment, which the resulting sample after pasteurization or S-sulfonaresults in the F(ab’), fragment, followed by precipita- tion. tion with 35%ethanol. Here, as described above for the In the studies with HCV, human plasma spiked with 7s im immunoglobulin, the pasteurization is added as this virus was subjected to the entire manufacturing the final step to the manufacturing procedure. S-sul- procedure of each of the immunoglobulins (see Fig. 1). fonation was performed on the crude immunoglobulin Small samples of the spiked starting material as well as preparation as described in detail elsewhere [Gronski the total, resulting amounts of the final products were et al., 19831, resulting in a modified 7s immunoglobu- inoculated into chimpanzees, which were then monilin, which is further purified by 25% ethanol precipita- tored over a period of 9 months as described above. The virus inactivation factor was calculated for each tion and adsorptive filtration. Briefly, for the S-sulfonation of the immunoglobulins, CuSO, and Na2S20, are individual virus according to the CPMP guidelines added to the protein solution while it is stirred. The pH [Commission of the European Communities, 19911 as is adjusted to 5.0, and this preparation is then incu- the ratio of the virus load in the starting, spiked material and the virus load in the sample resulting from the bated at 5°C for at least 10 hr.

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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 BVDV >7.3 4 >7.5 4 >7.3 TBEV >5.9 6 >6.4 6 >6.3 YFV >6.0 1.5 >6.0 2 >4.0 HIV-1 >7.8 0.5 >7.8 0.5 >5.6 HIV-2 HSV-1 >6.3 2 >6.4 8 >4.6 Polio-3 >4.7 1 >4.6 4 >4.4

specific treatment to be tested. The formula used for this calculation is: VaXa Inactivation factor = ___ . Vb X b Va

volume of the starting material, a = virus titer of the starting material. Vb = volume of the resulting material, b = virus titer 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 Table 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 t o the flavivirus group, but it does not clearly belong to one of these two groups. The proposal has therefore been made to regard HCV a s a n 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 a n 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 1to 2 hr, but a very small amount of infectious YF viruses needed prolonged 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 increase 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 Epstein-Barr virus infection diagnosed nor did any hepatitis B marker became positive. Histopathological alterations observed in liver biopsies of weeks 8,12,16, and 20 postinoculation showed a n 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 example 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

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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 5.4log,,.

DISCUSSION HCV is the only virus reported to have been transmitted 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 efficiency 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 result 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 theoretically, 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. Furthermore, 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 t o 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

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to the significant elevation of ALT, the only commercially 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 [Commission 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

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globulins permitting intravenous application. I. Physico-chemical and binding properties of S-sulfonated and reconstituted IgG. Vox Sanguinis 45:14&154. Hilfenhaus J, Geiger H, Lemp J, Hung CL (1987): A strategy for testing established human plasma protein manufacturing proceACKNOWLEDGMENTS dures for their ability to inactivate or eliminate human immunodeficiency virus. Journal of Biological Standardization 15:251-263. We are very grateful to Mr. Heinrich Eife, Mrs. Sab- Hilfenhaus J , Mauler R (1987):Effzienz der Erhitzung humaner Plasine Mehdi, Mrs. Eva Weber, Mr. Klaus Weber, and Mrs. mapraparationen bei 60°C in Losung im Hinblick auf die Virusinaktivierung: In Mammen EF (ed): “Intensivmedizin Aktuell: Ramona Volk for their excellent technical assistance. Sicherheit in Diagnose und Therapie von Gerinnungs storungen.” The careful preparation of this manuscript by Mrs. AnMarburg: Medizinische Verlagsgesellschaft, pp 134-142. drea Kirch is highly acknowledged. We much appreci- Iwarson St, Wejstal R, Ruttimann E, Bjorkander J, Set0 B, Snoy P, Gerety RJ (1987):Non-A, Non-B hepatitis associated with the adate the skillful work of Dr. Cummin’s staff when mainministration of intravenous immunoglobulin-transmission studtaining and treating the chimpanzees during the ies in chimpanzees. Serodiagnosis and Immunotherapy 1:261-266. hepatitis C studies. Experimental studies involving Klein RS, Friedland GH (1990):Transmission of human immunodeficiency virus type 1(HIV-1)by exposure to blood: Defining the risk. chimpanzees were conducted in accordance with the Annals of Internal Medicine 113:729-730. Ethical Practices Committee of the laboratories. Kuo G, Choo QL, Alter H, Gitnick GL, Redeker AG, Prucell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegmeier GE, REFERENCES Bonino F, Colombo LM, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M (1989):An assay for circuBjorkander J , Cumminghamrundless C, Lundin P, Olsson R, Soderlating antibodies to a major etiologic virus of human non-A, non-B strom R, Hanson LA (1988):Intravenous immunoglobulin prophyhepatitis. Science 244:362-364. laxis causing liver damage in 16 of 17 patients with hypogammaglobulinemia or IgG subclass deficiencies. American Journal of Lane RS (1983):Non-A, non-B hepatitis from intravenous immunogloMedicine 84:107-111. bulin. Lancet ii:97&975. Centers for Disease Control, Atlanta (1986): Safety of therapeutic Lever AML, Webster ADB, Brown D, Thomas HC (1984): Non-A, immune globulin preparations with respect to transmission of hunon-B hepatitis occurring in agammaglobulinaemic patients after man T-lymphotropic virus type IIUlymph-adenopathy-associated intravenous immunoglobulin. Lancet ii:1062-1064. virus infection. Morbidity and Mortality Weekly Report 35:231Mauler R, Merkle W, Hilfenhaus J (1987):Inactivation of HTLV-HI/ 233. LAV, Hepatitis B and Non-AiNon-B viruses by pasteurization in Centers for Disease Control, Atlanta (1988): Safety of therapeutic human plasma protein preparations. Development of Biological products used for hemophilia patients. Journal of the American Standardization 67:337351. Medical Association 260:901-903. MeliefCJM, Goudsmit J (1986):Transmission of lymphotropic retroviChoo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, Gallegos C, ruses (HTLV-I and LAVIHTLV-111)by blood transfusion and blood Coit D, Medina-Selby A, Barr PJ, Weiner AJ, Bradley DW, Kuo G, products. Vox Sanguinis 50:l-11. Houghton M (1991):Genetic organization and diversity ofthe hepOchs HD, Fischer SH, Virant FS, Lee ML, Mankarios S, Kingdon HS, atitis C virus. Proceedings of the National Academy of Sciences Wedgwood RJ (1986): Non-A, non-B hepatitis after intravenous 88:2451-2455. immunoglobulin. Lancet i:322-323. Cohn EJ, Strong LE, Hughes WL J r , Nulford DJ, Ashworth J N , Melin Ogata N, Alter HJ, Miller RH, Purcell R (1991):Nucleotide sequence N, Taylor HL (1946): Preparation and properties of serum and and mutation rate of the H strain of hepatitis C virus. Proceedings plasma proteins. IV: A system for the separation into fractions of of the National Academy of Sciences 88:3392-3396. protein and lipoprotein components of biological tissues and fluids. Prince AM, Horowitz B, Horowitz MS, Zang E (1987): The developJournal of the American Chemical Society 68:459-475. ment of virus-free labile blood d e r i v a t i v e s a review. European Commission of the European Communities (1991): Note for guidance Journal of Epidemiology 3:103-118. of the CPMP: “Validation of virus removal and inactivation proceReed U,Muench H (1938): A simple method of estimating fifty per dures.” cent endpoints. American Journal of Hygiene 27:493497. Feinstone SM, Alter HK, Diemes HP, Shimuzu YK, Popper H, BlackWeiland 0, Mattsow L, Glaumann H (1986):Non-A, Non-B hepatitis more D, Sly D, London WT, Purcell RH (1981): NonNNonB hepaafter intravenous gammaglobulin. Lancet I:976-977. titis in chimpanzees and marmosets. Journal of Infectious Diseases Wells MA, Wittek AE, Epstein JS, Marcus-Sekura C, Daniel S,Tan144:588-598. kersley DL, Preston MS, Quinnan GV (1986): Inactivation and Gerety R J , Aronson DL (1982):Plasma derivatives and viral hepatitis. partition of human T-cell lymphotrophic virus, type 111, during Transfusion 22:347-351. ethanol fractionation of plasma. Transfusion 26:210-213. Gregersen JP, Wege H, Preiss L, Jentsch KD (1988): Detection of human immunodeficiency virus and other retroviruses in cell cul- Williams PE, Yap PL, Gillon J, Crawford RJ, Galea G, Cuthbertson B (1988): Non-A, Non-B hepatitis transmission by intravenous imture supernatants by a reverse transcriptase microassay. Journal munoglobulin. Lancet II:501. of Virological Methods 19:161-168. Gronski P, Hofstaetter T, Kanzy E J , Luben G, Seiler FR (1983):S-sulfonation: a reversible, chemical modification of human immuno-

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

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

Human immunoglobulins are plasma derivatives with a low risk of transmitting viral infections. To the present, no proven case of human immunoglobulins...
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