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Inactivation of viruses in therapeutic products derived from human plasma
Inactivation of Viruses in Therapeutic Products Derived from Human Plasma
GAUTAM MITRA, Ph.D. MILTON B. DOBKIN, Ph.D. ROBERT E. LOUIE, Ph.D. MILTON M. MOZEN, Ph.D. Berkeley, California
Alpha-l-proteinase inhibitor concentrates have been prepared from pooled human plasma for therapeutic applications. Pasteurization (60°C, 10 hours) conditions have been defined to reduce risks of transmission of viral agents without significant loss of biologic activity of the purified product. Human clinical data collected to date support the model virus inactivation studies regarding viral safety. Transmission of infectious viruses through blood and blood products has led to various approaches towards their inactivation and removal. These include pasteurization [1-6], chemical inactivating agents [7], ultraviolet irradiation [8], solvent extraction of essential lipids [9,10], neutralization with specific antibodies [11,12], and removal by various fractionation techniques [13,14]. Any viable approach must selectively inactivate or remove viral agents without adversely affecting the safety or efficacy of the therapeutic product in question. It was demonstrated as early as 1948 [1] that human serum albumin in the presence of suitable stabilizers can be rendered free of hepatitis viruses when heated in solution at 60°C for 10 hours. Subsequent to that, it has become an industry-wide practice for therapeutic preparations of the so-called fraction V products, albumin, and plasma protein fractions to be pasteurized at 60°C for 10 hours. Immune globulin, when prepared from large plasma pools according to the Cohn Oncley procedures [15,16], has been historically free from the transmission of hepatitis B virus [17]. This is believed to be due to fractionation/inactivation during purification [18,19], and neutralization of residual virus by the antibodies present [20]. Proteinase inhibitor concentrates from pooled human plasma have recently been available for human clinical usage. Currently, antithrombin III concentrates are available from several European companies: Behringwerke (Federal Republic of Germany), KabiVitrum (Sweden), and Immuno (Austria). Comparable preparations of antithrombin III have also been tested clinically [21] in the United States. Pasteurization (60°C, 10 hours) of antithrombin III stabilized with 0.5 M citrate has been demonstrated to be hepatitis safe [2]. MATERIALS AND METHODS
From Cutter Biological of Miles Inc., Berkeley,California. Requestsfor reprints should be addressed to Dr. Gautam Mitra, Cutter Biological of Miles Inc., 4th and Parker Streets, P.O. Box 1986, Berkeley, California 94701.
The preparation and properties of an alpha-l-proteinase inhibitor (A1PI) concentrate from human plasma have previously been described [22]. The condilions for pasteurization (60°C, 10 hours) have also been optimized [23]. A1PI can be heated in liquid state at 60°C for 10 hours in the presence of 37 percent sucrose and 0.38 M sodium citrate without appreciable decrease in elastase-inhibiting activity. In order to assess the effectiveness of such heat treatment in eliminating potential infectious viral contaminants, a number of human and animal viruses, representing a broad spectrum of taxonomic
June 24, 1988 The American Journal of Medicine Volume 84 (suppl 6A)
'87
SYMPOSIUMON ALPHA-1-ANTITRYPSINDEFICIENCY--MITRAET AL
TABLE I
Indicator Viruses Used for Inactivation Studies Virus
Family
Poliovirus type II Herpes simplex virus type I Cytomegalovirusstrain AD169 Vaccinia virus lederle strain Vesicular stomatitis virus Indiana s t r a i n Porcine parvovirus Visna virus Human immonodeficiencyvirus
TABLE II
Picornavirus Herpesvirus Herpesvirus Poxvirus Rhabdovirus Parvovirus Lentivirus Lentivirus
Inactivation of Visna Virus in A I P I I-Jours at 61~'C
Titer*
0 0.5 1.0 2.0 5.0 10.0
6.0 <-1.5 -<1.5 <-1.5 <-1.5 <-1.5
"Loglo 50 percent tissue culture infective dose per milliliter.
TABLE III
Inactivation of Human Immunodeficiency Virus in A I P I Dilution at Which Virus Is below Detection
Sample
10.3 10-3 Undiluted Undiluted
Virus concentrate AIPI plus virus concentrate 60 -+ 0.5°C, 1 hour 60 -+ 0.5°C, 10 hours
TABLE IV
Inactivation of Model Viruses under Pasteurization Conditions for A I P I Virus
Initial Tiler (Ioglo)
Hours*
Poliovirus Herpes simplex virus Cytomegalovirus Vaccinia virus Vesicular stomatitis virus Porcine parvovirus Visna virus Human immunodeficiencyvirus
5.0 8.0 4.0 4.0 5.0 4.75 6.0 3.0
5.0 1.0 1.0 5.0-10.0 1.0 >10.0 0.5 <1.0
"Time required for virus titer to decrease below detectability.
88
June 24, 1988
The American Journal of Medicine
Nucleic Acid
Cell Indicator
RNA DNA DNA DNA RNA DNA RNA RNA
Human fibroblast (MRC5) Human fibroblast (MRC5) Human tibroblast (MRC5) Human fibroblast (MRC5) Human fibroblast (MRC5) Embryonic porcine kidney Sheep fibroblast (ShNF) Human peripheral mononuclear cells
groups, were selected as model systems for this study (Table I). Viral safety in patients receiving A1PI concentrates was also demonstrated by six months of post-infusion follow-up. Tissue Culture. Vesicular stomatitis virus, vaccinia virus, poliovirus, herpes simplex virus, and cytomegalovirus samples were titrated on monolayers of MRC5 cells grown in 25cm 2 flasks, using one flask culture specimen per dilution. After removing the growth medium by suction, one ml of viral inoculum was added to each culture specimen. The culture specimens were incubated for one to 1.5 hours at 37 -+ 0.5°C with intermittent bathing of the fluid over the monolayers to permit adsorption of residual infectious virus. Inoculum fluids were removed by suction, and 7 to 8 ml of maintenance medium was added to each flask. All culture specimens were incubated at 37 -+ 0.5°C. Culture specimens were examined microscopically, and final readings were made after seven days (14 days for cytomegalovirus) of incubation. Any culture specimen showing characteristic cytopathogenic effects was considered positive. The titers are expressed as the log of that virus dilution that produces cytopathic effect on 100 percent of the virusinoculated culture specimens. Porcine parvovirus titrations were carried out on three-day culture specimens of primary embryonic porcine kidney cells (first through tenth passages) grown on Lab-Tek tissue culture chamber slides; 0.2 ml of virus was added to each well, using four wells per dilution. Following incubation for 60 minutes to permit adsorption, the inocula were removed and replaced with fresh tissue culture medium. The infectious culture specimens were incubated at 37 +- 0.5°C in the presence of 5 percent carbon dioxide for 72 hours. The chamber slides were washed, fixed with acetone, and stained with porcine parvovirus fluorescent antibody conjugate. The slides were read by means of an ultraviolet microscope, and any well containing one or more cells showing fluorescing nuclei was considered positive. Titers are expressed as 50 percent fluorescent antibody infectious doses per ml. Visna virus samples were tested on monolayers of ShNF cells grown in 24-well plates (Coming), using four wells per dilution. Growth medium was removed by suction, and 0.1 ml of virus dilution was added to each well. Following adsorption for two hours at 37 - 0.5°C, inoculum fluids were removed by suction, and each well was re-fed with 2 ml of maintenance medium. Culture specimens were incubated at 37 -+ 0.5°C under 5 percent carbon dioxide for seven days and examined
Volume 84 (suppl 6A)
SYMPOSIUM ON ALPHA-1-ANTITRYPSIN DEFICIENCY--MITRA ET AL
TABLE V
Viral Safety Follow-up of Patients Receiving A I P I Center
National Institutes of Health" Completed study Ongoing study University of California, San Diego Ongoing study
Number of Patients
Numberof Infusions
Durationof Therapy (weeks)
23 7
517 133
1-26 (median = 26) 1-33 (median = 22)
6
6
1
HBsAg/NANI] HIV Follow-up Period for Surveillance Seroconversion HIV Testing (months)
Negative Negative
Negative Pending
Negative
Not available
3-17 (median = 9)
HBsAg = hepatitis B surface antigen; NANB = non-A non-B; HIV = human immunodeficiency virus. *Received hepatitis B vaccination.
microscopically for the presence of characteristic cytopathogenic effects (megakaryocytes) after fixation and staining with Giemsa. Titers are expressed as Ioglo 50 percent tissue culture infective dose per milliliter. Human immune deficiency virus strain ARV-19 was detected by testing for reverse transcriptase activity in the supernatant culture medium of human peripheral mononuclear cells maintained for one month after virus inoculation. Positive reverse transcriptase activity was judged as counts above background. Experimental Design. One part virus was added to nine parts non-heated A1 PI. After thorough mixing, a sample was removed (time 0) and frozen. The remaining material was placed in a water bath at 60 _ 0.5°C. Samples were removed at various time intervals and frozen for subsequent titrations of residual infectious virus. Sera from patients receiving A1PI concentrates were drawn at regular intervals for up to six months post-infusion and assayed for the presence of hepatitis B virus (surface antigen assay, third-generation test kit), human immunodeficiency virus (anti-human immunodeficiency virus by enzymelinked immunosorbent assay), and non-A non-B hepatitis virus (serum alanine aminotransferase assay). RESULTS A N D C O M M E N T S
The inactivation kinetics of the two retroviruses studied, visna and human immunodeficiency virus, at 60°C in liquid A1PI are shown in Tables II and III, respectively. The data obtained with poliovirus type II, herpes simplex virus-l, cytomegalovirus, vaccinia virus, and porcine parvovirus are summarized in Table IV. The presence of sucrose as stabilizer prevented testing of undiluted samples because
of toxicity for MRC5, ShNF, and peripheral mononuclear cells. Vesicular stomatitis virus, herpes simplex virus-l, visna, and human immunodeficiency virus were the most thermolabile viruses tested. These were inactivated within one hour of heat treatment. Poliovirus appeared to be inactivated within five hours, and vaccinia virus reached the lower limit of detectability at between five and 10 hours. Porcine parvovirus, however, although reduced by 3 logs, still demonstrated 1.7 logs of infectivity after 10 hours at 60°C. Porcine parvovirus is one of the most thermo-stable viruses known, and a previous report [24] had indicated that, starting with 4.4 logs of this virus in 5 percent human serum albumin with stabilizers, 3.2 logs of infectivity remained after 10 hours at 60°C. These are the same conditions under which hepatitis B is inactivated in human serum albumin preparations. More than 30 years of human administration experience exists attesting to the safety of this product. The presence of 37 percent sucrose as protein stabilizer is not believed to offer significant protection for viruses. Effects of comparable levels of sucrose have shown minimal protective effect for human immunodeficiency virus [25] and porcine parvovirus [24]. Table V shows clinical follow-up data up to six months post-infusion of A1PI concentrates. All indications were negative for hepatitis B (including the non-vaccinated group), non-A non-B hepatitis, and human immunodeficiency virus. To date, the data strongly support the heat treatment of stabilized liquid A1 PI for the preparation of a therapeutically effective and virus-safe product.
REFERENCES
1. Gellis SS, Neefe JR, Stokes J, Strong L, Janeway C, Scatchard G: Chemical, clinical and immunological studies on the products of human plasma fractionation. XXXVI. Inactivation of the virus of homologous serum hepatitis in solutions of normal human serum albumin by means of heat. J Clin Invest 1948; 27: 239-244. 2. Tabor E, Murano G, Snoy P, Gerety RJ: Inactivation of hepatitis B virus by heat in antithrombin III stabilized with citrates.
June 24, 1988
Thromb Res 1981; 22: 233-238. 3. Heimburger VN, Schwinn H, Gratz P: Factor VllI-Konzentrat, hochgereinigt und in LSsung erhitzt. Arzneimittelforschung 1981; 31: 619-622, 4. Ng PK, Eguizabal HC, Mitra G: Preparation of high purity factor VIII concentrates. Thromb Res 1986; 42: 825-834. 5. Mozen MM, Louie RE, Mitra G: Heat inactivation of viruses in antihemophilic factor concentrates (abstr). Proceedings of the
The American Journal of Medicine
Volume 84 (suppl 6A)
89
SYMPOSIUM ON ALPHA-1-ANTITRYPSIN DEFICIENCY--MITRA ET AL
6. 7. 8. 9. 1Q.
11. 12. 13. 14.
15.
90
XVl International Congress of the World Federation of Hemophilia, Rio de Janeiro, 1984. Hollinger FB, Dolana G, Thomas W, Gyorkey F: Reduction in dsk of hepatitis transmission by heat treatment of a human factor VIII concentrate. J Infect Dis 1984; 150 (2): 250-262. Logrippo GA, Wolfram BR, Rupe CF: Human plasma treated with ultraviolet and propiolactone, a six year clinical evaluation. JAMA 1964; 178: 722-726. Openheimer F, Benesi E, Taylor AR: The ultraviolet irradiation of biological fluids in their flowing films. Am J Public Health 1959; 49: 903-923. Feinstone SM, Mihalik KB, Kamimura T, Alter HJ, London WT, Purcell RH: Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform. Infect Immun 1983; 41: 816-821. Prince AM, Horowitz B, Brotman B, Huima T, Richardson L, van der Ende MC: Inactivation of hepatitis B and Hutchinson strain non-A, non-B hepatitis viruses by exposure to Tween 80 and ether. Vox Sang 1984; 46: 36-43. Tabor E, Aronson DL, Gerety RJ: Removal of hepatitis B virus infectivity from Factor IX complex. Lancet 1980; I1: 68-70. Brummelhuis HGJ, Over J, Duivis-Vorst CC, et al: Elimination of hepatitis B transmission by (potentially) infectious plasma derivatives. Vox Sang 1983; 45: 205-216. Johnson AJ, Semar M, Newman J, et al: Removal of hepatitis B surface antigen (HBsAg) from plasma fractions. J Lab Clin Med 1976; 88: 91-101. Galpin SS, Karayiannis P, Middleton SM, Thomas HC: The removal of hepatitis B virus from factor VIII concentrates by fractionation on ethylene maleic anhydride polyelectrolyte. J Med Virol 1984; 14: 229-233. Cohn EJ, Strong LE, Hughes WL Jr, et al: Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946;
June 24, 1988
The American Journal of Medicine
68: 459-475. 16. Oncley JL, Melin M, Richert DA, Cameron JW, Gross PM Jr: The separation of the antibodies, isoagglutinins, prothrombin, plasminogen, and/3-1-1ipoprotein into subtractions of human plasma. J Am Chem Soc 1949; 71: 541-550. 17. Murray R, Ratner F: Safety of immune serum globulin with respect to homologous serum hepatitis. Proc Soc Exp Biol Med 1953; 83: 554-555. 18. Wells MA, Wittek AE, Epstein JS, et al: Inactivation and partition of human T-cell lymphotropic virus, type III, during ethanol fractionation of plasma. Transfusion 1986; 26: 210-213. 19. Mitra G, Wong MF, Mozen MM, McDougal JS, Levy JA: Elimination of infectious retroviruses during preparation of immunoglobulins. Transfusion 1986; 26: 394-396. 20. Hoofnagle JH, Gerety RJ, Barker LF: Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-413. 21. Bauer KA, Goodman TL, Kass BL, Rosenberg RD: Elevated factor Xa activity in the blood of asymptomatic patients with cogenital antithrombin deficiency. J Clin Invest 1985; 76: 826836. 22. Coan MH, Brochway WJ, Eguizabal H, Krieg T, Fournel M: Preparation and properties of a'l proteinase inhibitor concentrate from human plasma. Vox Sang 1985; 48: 333-342. 23. Coan MH, Mitra G: Stabilization of o~1 proteinase inhibitor by citrate. Vox Sang 1984; 46: 142-148. 24. Ng PK, Dobkin MB: Pasteurization of antihemophilic factor and model virus inactivation studies. Thromb Res 1985; 39: 439447. 25. McDougal JS, Martin LS, Cort SP, Mozen M, Heldebrant CM, Evatt BL: Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-Ill/ lymphadenopathy-associated virus, with special reference to antihemophilic factor. J Clin Invest 1985; 76: 875-877.
Volume 84 (suppl 6A)
GAUTAM MITRA, Ph.D. MILTON B. DOBKIN, Ph.D. ROBERT E. LOUIE, Ph.D. MILTON M. MOZEN, Ph.D. Berkeley, California
Alpha-l-proteinase inhibitor concentrates have been prepared from pooled human plasma for therapeutic applications. Pasteurization (60°C, 10 hours) conditions have been defined to reduce risks of transmission of viral agents without significant loss of biologic activity of the purified product. Human clinical data collected to date support the model virus inactivation studies regarding viral safety. Transmission of infectious viruses through blood and blood products has led to various approaches towards their inactivation and removal. These include pasteurization [1-6], chemical inactivating agents [7], ultraviolet irradiation [8], solvent extraction of essential lipids [9,10], neutralization with specific antibodies [11,12], and removal by various fractionation techniques [13,14]. Any viable approach must selectively inactivate or remove viral agents without adversely affecting the safety or efficacy of the therapeutic product in question. It was demonstrated as early as 1948 [1] that human serum albumin in the presence of suitable stabilizers can be rendered free of hepatitis viruses when heated in solution at 60°C for 10 hours. Subsequent to that, it has become an industry-wide practice for therapeutic preparations of the so-called fraction V products, albumin, and plasma protein fractions to be pasteurized at 60°C for 10 hours. Immune globulin, when prepared from large plasma pools according to the Cohn Oncley procedures [15,16], has been historically free from the transmission of hepatitis B virus [17]. This is believed to be due to fractionation/inactivation during purification [18,19], and neutralization of residual virus by the antibodies present [20]. Proteinase inhibitor concentrates from pooled human plasma have recently been available for human clinical usage. Currently, antithrombin III concentrates are available from several European companies: Behringwerke (Federal Republic of Germany), KabiVitrum (Sweden), and Immuno (Austria). Comparable preparations of antithrombin III have also been tested clinically [21] in the United States. Pasteurization (60°C, 10 hours) of antithrombin III stabilized with 0.5 M citrate has been demonstrated to be hepatitis safe [2]. MATERIALS AND METHODS
From Cutter Biological of Miles Inc., Berkeley,California. Requestsfor reprints should be addressed to Dr. Gautam Mitra, Cutter Biological of Miles Inc., 4th and Parker Streets, P.O. Box 1986, Berkeley, California 94701.
The preparation and properties of an alpha-l-proteinase inhibitor (A1PI) concentrate from human plasma have previously been described [22]. The condilions for pasteurization (60°C, 10 hours) have also been optimized [23]. A1PI can be heated in liquid state at 60°C for 10 hours in the presence of 37 percent sucrose and 0.38 M sodium citrate without appreciable decrease in elastase-inhibiting activity. In order to assess the effectiveness of such heat treatment in eliminating potential infectious viral contaminants, a number of human and animal viruses, representing a broad spectrum of taxonomic
June 24, 1988 The American Journal of Medicine Volume 84 (suppl 6A)
'87
SYMPOSIUMON ALPHA-1-ANTITRYPSINDEFICIENCY--MITRAET AL
TABLE I
Indicator Viruses Used for Inactivation Studies Virus
Family
Poliovirus type II Herpes simplex virus type I Cytomegalovirusstrain AD169 Vaccinia virus lederle strain Vesicular stomatitis virus Indiana s t r a i n Porcine parvovirus Visna virus Human immonodeficiencyvirus
TABLE II
Picornavirus Herpesvirus Herpesvirus Poxvirus Rhabdovirus Parvovirus Lentivirus Lentivirus
Inactivation of Visna Virus in A I P I I-Jours at 61~'C
Titer*
0 0.5 1.0 2.0 5.0 10.0
6.0 <-1.5 -<1.5 <-1.5 <-1.5 <-1.5
"Loglo 50 percent tissue culture infective dose per milliliter.
TABLE III
Inactivation of Human Immunodeficiency Virus in A I P I Dilution at Which Virus Is below Detection
Sample
10.3 10-3 Undiluted Undiluted
Virus concentrate AIPI plus virus concentrate 60 -+ 0.5°C, 1 hour 60 -+ 0.5°C, 10 hours
TABLE IV
Inactivation of Model Viruses under Pasteurization Conditions for A I P I Virus
Initial Tiler (Ioglo)
Hours*
Poliovirus Herpes simplex virus Cytomegalovirus Vaccinia virus Vesicular stomatitis virus Porcine parvovirus Visna virus Human immunodeficiencyvirus
5.0 8.0 4.0 4.0 5.0 4.75 6.0 3.0
5.0 1.0 1.0 5.0-10.0 1.0 >10.0 0.5 <1.0
"Time required for virus titer to decrease below detectability.
88
June 24, 1988
The American Journal of Medicine
Nucleic Acid
Cell Indicator
RNA DNA DNA DNA RNA DNA RNA RNA
Human fibroblast (MRC5) Human fibroblast (MRC5) Human tibroblast (MRC5) Human fibroblast (MRC5) Human fibroblast (MRC5) Embryonic porcine kidney Sheep fibroblast (ShNF) Human peripheral mononuclear cells
groups, were selected as model systems for this study (Table I). Viral safety in patients receiving A1PI concentrates was also demonstrated by six months of post-infusion follow-up. Tissue Culture. Vesicular stomatitis virus, vaccinia virus, poliovirus, herpes simplex virus, and cytomegalovirus samples were titrated on monolayers of MRC5 cells grown in 25cm 2 flasks, using one flask culture specimen per dilution. After removing the growth medium by suction, one ml of viral inoculum was added to each culture specimen. The culture specimens were incubated for one to 1.5 hours at 37 -+ 0.5°C with intermittent bathing of the fluid over the monolayers to permit adsorption of residual infectious virus. Inoculum fluids were removed by suction, and 7 to 8 ml of maintenance medium was added to each flask. All culture specimens were incubated at 37 -+ 0.5°C. Culture specimens were examined microscopically, and final readings were made after seven days (14 days for cytomegalovirus) of incubation. Any culture specimen showing characteristic cytopathogenic effects was considered positive. The titers are expressed as the log of that virus dilution that produces cytopathic effect on 100 percent of the virusinoculated culture specimens. Porcine parvovirus titrations were carried out on three-day culture specimens of primary embryonic porcine kidney cells (first through tenth passages) grown on Lab-Tek tissue culture chamber slides; 0.2 ml of virus was added to each well, using four wells per dilution. Following incubation for 60 minutes to permit adsorption, the inocula were removed and replaced with fresh tissue culture medium. The infectious culture specimens were incubated at 37 +- 0.5°C in the presence of 5 percent carbon dioxide for 72 hours. The chamber slides were washed, fixed with acetone, and stained with porcine parvovirus fluorescent antibody conjugate. The slides were read by means of an ultraviolet microscope, and any well containing one or more cells showing fluorescing nuclei was considered positive. Titers are expressed as 50 percent fluorescent antibody infectious doses per ml. Visna virus samples were tested on monolayers of ShNF cells grown in 24-well plates (Coming), using four wells per dilution. Growth medium was removed by suction, and 0.1 ml of virus dilution was added to each well. Following adsorption for two hours at 37 - 0.5°C, inoculum fluids were removed by suction, and each well was re-fed with 2 ml of maintenance medium. Culture specimens were incubated at 37 -+ 0.5°C under 5 percent carbon dioxide for seven days and examined
Volume 84 (suppl 6A)
SYMPOSIUM ON ALPHA-1-ANTITRYPSIN DEFICIENCY--MITRA ET AL
TABLE V
Viral Safety Follow-up of Patients Receiving A I P I Center
National Institutes of Health" Completed study Ongoing study University of California, San Diego Ongoing study
Number of Patients
Numberof Infusions
Durationof Therapy (weeks)
23 7
517 133
1-26 (median = 26) 1-33 (median = 22)
6
6
1
HBsAg/NANI] HIV Follow-up Period for Surveillance Seroconversion HIV Testing (months)
Negative Negative
Negative Pending
Negative
Not available
3-17 (median = 9)
HBsAg = hepatitis B surface antigen; NANB = non-A non-B; HIV = human immunodeficiency virus. *Received hepatitis B vaccination.
microscopically for the presence of characteristic cytopathogenic effects (megakaryocytes) after fixation and staining with Giemsa. Titers are expressed as Ioglo 50 percent tissue culture infective dose per milliliter. Human immune deficiency virus strain ARV-19 was detected by testing for reverse transcriptase activity in the supernatant culture medium of human peripheral mononuclear cells maintained for one month after virus inoculation. Positive reverse transcriptase activity was judged as counts above background. Experimental Design. One part virus was added to nine parts non-heated A1 PI. After thorough mixing, a sample was removed (time 0) and frozen. The remaining material was placed in a water bath at 60 _ 0.5°C. Samples were removed at various time intervals and frozen for subsequent titrations of residual infectious virus. Sera from patients receiving A1PI concentrates were drawn at regular intervals for up to six months post-infusion and assayed for the presence of hepatitis B virus (surface antigen assay, third-generation test kit), human immunodeficiency virus (anti-human immunodeficiency virus by enzymelinked immunosorbent assay), and non-A non-B hepatitis virus (serum alanine aminotransferase assay). RESULTS A N D C O M M E N T S
The inactivation kinetics of the two retroviruses studied, visna and human immunodeficiency virus, at 60°C in liquid A1PI are shown in Tables II and III, respectively. The data obtained with poliovirus type II, herpes simplex virus-l, cytomegalovirus, vaccinia virus, and porcine parvovirus are summarized in Table IV. The presence of sucrose as stabilizer prevented testing of undiluted samples because
of toxicity for MRC5, ShNF, and peripheral mononuclear cells. Vesicular stomatitis virus, herpes simplex virus-l, visna, and human immunodeficiency virus were the most thermolabile viruses tested. These were inactivated within one hour of heat treatment. Poliovirus appeared to be inactivated within five hours, and vaccinia virus reached the lower limit of detectability at between five and 10 hours. Porcine parvovirus, however, although reduced by 3 logs, still demonstrated 1.7 logs of infectivity after 10 hours at 60°C. Porcine parvovirus is one of the most thermo-stable viruses known, and a previous report [24] had indicated that, starting with 4.4 logs of this virus in 5 percent human serum albumin with stabilizers, 3.2 logs of infectivity remained after 10 hours at 60°C. These are the same conditions under which hepatitis B is inactivated in human serum albumin preparations. More than 30 years of human administration experience exists attesting to the safety of this product. The presence of 37 percent sucrose as protein stabilizer is not believed to offer significant protection for viruses. Effects of comparable levels of sucrose have shown minimal protective effect for human immunodeficiency virus [25] and porcine parvovirus [24]. Table V shows clinical follow-up data up to six months post-infusion of A1PI concentrates. All indications were negative for hepatitis B (including the non-vaccinated group), non-A non-B hepatitis, and human immunodeficiency virus. To date, the data strongly support the heat treatment of stabilized liquid A1 PI for the preparation of a therapeutically effective and virus-safe product.
REFERENCES
1. Gellis SS, Neefe JR, Stokes J, Strong L, Janeway C, Scatchard G: Chemical, clinical and immunological studies on the products of human plasma fractionation. XXXVI. Inactivation of the virus of homologous serum hepatitis in solutions of normal human serum albumin by means of heat. J Clin Invest 1948; 27: 239-244. 2. Tabor E, Murano G, Snoy P, Gerety RJ: Inactivation of hepatitis B virus by heat in antithrombin III stabilized with citrates.
June 24, 1988
Thromb Res 1981; 22: 233-238. 3. Heimburger VN, Schwinn H, Gratz P: Factor VllI-Konzentrat, hochgereinigt und in LSsung erhitzt. Arzneimittelforschung 1981; 31: 619-622, 4. Ng PK, Eguizabal HC, Mitra G: Preparation of high purity factor VIII concentrates. Thromb Res 1986; 42: 825-834. 5. Mozen MM, Louie RE, Mitra G: Heat inactivation of viruses in antihemophilic factor concentrates (abstr). Proceedings of the
The American Journal of Medicine
Volume 84 (suppl 6A)
89
SYMPOSIUM ON ALPHA-1-ANTITRYPSIN DEFICIENCY--MITRA ET AL
6. 7. 8. 9. 1Q.
11. 12. 13. 14.
15.
90
XVl International Congress of the World Federation of Hemophilia, Rio de Janeiro, 1984. Hollinger FB, Dolana G, Thomas W, Gyorkey F: Reduction in dsk of hepatitis transmission by heat treatment of a human factor VIII concentrate. J Infect Dis 1984; 150 (2): 250-262. Logrippo GA, Wolfram BR, Rupe CF: Human plasma treated with ultraviolet and propiolactone, a six year clinical evaluation. JAMA 1964; 178: 722-726. Openheimer F, Benesi E, Taylor AR: The ultraviolet irradiation of biological fluids in their flowing films. Am J Public Health 1959; 49: 903-923. Feinstone SM, Mihalik KB, Kamimura T, Alter HJ, London WT, Purcell RH: Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform. Infect Immun 1983; 41: 816-821. Prince AM, Horowitz B, Brotman B, Huima T, Richardson L, van der Ende MC: Inactivation of hepatitis B and Hutchinson strain non-A, non-B hepatitis viruses by exposure to Tween 80 and ether. Vox Sang 1984; 46: 36-43. Tabor E, Aronson DL, Gerety RJ: Removal of hepatitis B virus infectivity from Factor IX complex. Lancet 1980; I1: 68-70. Brummelhuis HGJ, Over J, Duivis-Vorst CC, et al: Elimination of hepatitis B transmission by (potentially) infectious plasma derivatives. Vox Sang 1983; 45: 205-216. Johnson AJ, Semar M, Newman J, et al: Removal of hepatitis B surface antigen (HBsAg) from plasma fractions. J Lab Clin Med 1976; 88: 91-101. Galpin SS, Karayiannis P, Middleton SM, Thomas HC: The removal of hepatitis B virus from factor VIII concentrates by fractionation on ethylene maleic anhydride polyelectrolyte. J Med Virol 1984; 14: 229-233. Cohn EJ, Strong LE, Hughes WL Jr, et al: Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946;
June 24, 1988
The American Journal of Medicine
68: 459-475. 16. Oncley JL, Melin M, Richert DA, Cameron JW, Gross PM Jr: The separation of the antibodies, isoagglutinins, prothrombin, plasminogen, and/3-1-1ipoprotein into subtractions of human plasma. J Am Chem Soc 1949; 71: 541-550. 17. Murray R, Ratner F: Safety of immune serum globulin with respect to homologous serum hepatitis. Proc Soc Exp Biol Med 1953; 83: 554-555. 18. Wells MA, Wittek AE, Epstein JS, et al: Inactivation and partition of human T-cell lymphotropic virus, type III, during ethanol fractionation of plasma. Transfusion 1986; 26: 210-213. 19. Mitra G, Wong MF, Mozen MM, McDougal JS, Levy JA: Elimination of infectious retroviruses during preparation of immunoglobulins. Transfusion 1986; 26: 394-396. 20. Hoofnagle JH, Gerety RJ, Barker LF: Antibody to the hepatitis B surface antigen in immune serum globulin. Transfusion 1975; 15: 408-413. 21. Bauer KA, Goodman TL, Kass BL, Rosenberg RD: Elevated factor Xa activity in the blood of asymptomatic patients with cogenital antithrombin deficiency. J Clin Invest 1985; 76: 826836. 22. Coan MH, Brochway WJ, Eguizabal H, Krieg T, Fournel M: Preparation and properties of a'l proteinase inhibitor concentrate from human plasma. Vox Sang 1985; 48: 333-342. 23. Coan MH, Mitra G: Stabilization of o~1 proteinase inhibitor by citrate. Vox Sang 1984; 46: 142-148. 24. Ng PK, Dobkin MB: Pasteurization of antihemophilic factor and model virus inactivation studies. Thromb Res 1985; 39: 439447. 25. McDougal JS, Martin LS, Cort SP, Mozen M, Heldebrant CM, Evatt BL: Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-Ill/ lymphadenopathy-associated virus, with special reference to antihemophilic factor. J Clin Invest 1985; 76: 875-877.
Volume 84 (suppl 6A)