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Evaluation of some selected vaccines and other biological products irradiated by gamma rays, electron beams and X-rays
Radiation Physics and Chemistry 63 (2002) 709–711
Evaluation of some selected vaccines and other biological products irradiated by gamma rays, electron beams and X-rays J.C. Maya,*, L. Reyb, Chi-Jen Leea a
Center for Biologics and Research, Food and Drug Administration, HMF-6 73, 1401 Rockville Pike, Rockville, MD 20852, USA b Professor des Universities, Conseiller Scientifique, CH-1010 Lausanne, Switzerland
Abstract Molecular sizing potency results are presented for irradiated samples of one lot of Haemophilus b conjugate vaccine, pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine. The samples were irradiated (25 kGy) by gamma rays, electron beams and X-rays. IgG and IgM antibody response in mice test results (ELISA) are given for the Hib conjugate vaccine irradiated at 01C or frozen in liquid nitrogen. Published by Elsevier Science Ltd. Keywords: Irradiated vaccines; Electron beams;
60
Co gamma irradiation; X-ray
1. Introduction In the area of licensed biological products, to date, dispettes (containers) and diluent for vaccines have been sterilized by 60Co gamma irradiation. A limited number of Food and Drug Administration (FDA) regulated foods and drugs and a large number of devices are currently approved for radiation sterilization. Transy and Fleurette (1976) characterized the effects of ionizing radiation on microorganisms and the application of radiosterilization. The effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin has been reported (Nedugova et al., 1984) with the sterilizing effect of gamma radiation achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. Vaccines have been gamma-irradiated for viral inactivation. Examples include gamma-irradiated influenza A virus vaccine (Pang et al., 1992). Gamma radiation has been used for the terminal sterilization and viral inactivation of blood products (Reid, 1998) and elimination of adventitious agents in continuous cell lines (Erickson et al., 1989). The effect of radiation sterilization on the *Corresponding author. Tel.: +1-301-496-4570; fax: +1301-435-4991. E-mail address: [email protected] (J.C. May). 0969-806X/02/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 5 6 5 - 5
immunobiological properties of the sorbed protective fraction of Bordetella pertussis has been reported (Bazhanova et al., 1986). Pneumococcal polysaccharide fragments have been produced (Pawlowski et al., 1999) by electron beam irradiation for the preparation of conjugate vaccines that retained their antigenic epitopes when compared to native, full-sized pneumococcal polysaccharides as determined by enzyme-linked immunoassay. Pneumococcal capsular polysaccharideprotein conjugate vaccines utilizing electron beam fragmentation technology have been produced which produce high IgG titers in both rabbits and mice (Pawlowski et al., 2000). The goal of this project is to study radiation sterilization dosing levels (McLaughlin et al., 1989) and their effect on potency for certain biological products regulated by FDA. In this project certain vaccines, namely, Haemophilus influenzae type b conjugate vaccine (Hib conjugate vaccine) (Booy et al., 1994), pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine (Acharya et al., 1987) have been irradiated using gamma (60Co), electron beam, and X-ray radiation at room temperature, 01C, and liquid nitrogen temperature. Potency testing in terms of molecular sizing (Bureau of Biologics, 1977; Plumb and Yost, 1996; Wong et al., 1977) and mouse IgG and IgM antibody response (Lu et al., 1994) have
J.C. May et al. / Radiation Physics and Chemistry 63 (2002) 709–711
710
then been performed to gather data to evaluate the impact of the radiation on the potency of the irradiated and control biological product samples.
2. Experimental Samples and control materials were from lots of vaccines licensed by the United States Food and Drug Administration. Irradiation facilities: gamma irradiation performed at CEA (Cadarache, France) and Studer AG Werk Hard (Daniken, Switzerland); electron beam irradiation performed at Studer AG Hard Werk; X-ray irradiation performed at the laboratory of Professor Louis Rey in Lausanne, Switzerland. Molecular sizing and mouse IgG and IgM antibody response ELISA potency assays were performed at the Center for Biologics Evaluation and Research of the US Food and Drug Administration (Rockville, MD, USA).
3. Results and discussion Table 1 lists the molecular sizing results for samples of lot A of freeze-dried Haemophilus b conjugate vaccine
Table 1 Molecular size determinations of irradiated (25 kGy) and control samples of lot A of freeze-dried Haemophilus influenzae type b conjugate vaccine (tetanus toxoid conjugate) Irradiation
Molecular sizing for Hib conjugate vaccine (Kd, sepharose CL-4B)
Control (no irradiation) Electron beam (25 kGy) Gamma ray (25 kGy) X-ray (25 kGy)
0.24 0.82 0.76 0.83
that had been irradiated (25 kGy) using gamma, electron beam and X-ray sources. In general, irradiation increased the value of the Kd, and therefore, decreased the molecular size of the polysaccharide. Table 2 lists the mouse antibody response for the gamma-irradiated samples, sample control and saline control. This data indicates that the IgG antibody response was not significantly changed in the irradiated vaccine compared to the vaccine control even though the irradiation has altered the molecular size of the irradiated vaccine. Similarly, Table 3 lists the IgM antibody response for the gamma-irradiated samples, sample control and saline control. This data also indicates that the IgM antibody response was not significantly changed in the irradiated vaccine compared to the vaccine control even though the irradiation has altered the molecular size of the irradiated vaccine. Table 4 lists the molecular sizing results for samples of a pneumococcal polysaccharide type 6B (powder) and typhoid vi polysaccharide vaccine (liquid) that had been irradiated (25 kGy) using gamma ray, and electron beam and unirradiated control samples. The values for the Kd increased for both irradiated products, therefore, indicating that the molecular size of both products decreased under these irradiation conditions. The Kd’s for the electron beam irradiated samples still met the product specifications for these two product types (0.20 and 0.25, respectively).
4. Conclusion In general, irradiation increased the value of the Kd and, therefore, decreased the molecular size of the Hib conjugate vaccine, pneumococcal polysaccharide type 6B and the typhoid vi polysaccharide vaccine. The data in Tables 2 and 3 indicated that although the molecular size has decreased under gamma irradiation at 25 kGy, the mouse IgG amd IgM antibody responses for the Hib conjugate vaccine have not significantly changed. The
Table 2 Mouse IgG antibody response (ELISA OD at 405 nm; 45 min at 251C) of gamma irradiated and non-irradiation samples of lot A of freeze-dried Haemophilus b conjugate vaccine (tetanus toxoid conjugate) and saline diluent As dilution
1/100 1/300 1/500 a
IgG antibody response (ELISA OD unit) Hib conjugate vaccine lot Aa 25 kGy in liq. N2
Hib conjugate vaccine lot Aa 25 kGy at 0oC
Hib conjugate vaccine lot Aa control Non-irradiated
Control
1.0870.06 0.8070.18 0.3770.03
0.7470.25 0.9070.16 0.3370.06
0.8470.19 0.7070.11 0.3870.02
0.2870.06 0.3270.02 0.3270.02
Mean and standard deviation of 4 samples.
Saline diluent
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Table 3 Mouse IgM antibody response (ELISA OD at 405 nm; 45 min at 251C) of gamma irradiated (at two temperatures) and non-irradiated samples of lot A of freeze-dried Haemophilus influenzae type b conjugate vaccine (tetanus toxoid conjugate) and saline diluent As dilution
1/100 1/300 1/500 a
IgM antibody response (ELISA OD unit) Saline diluenta
Hib conjugate vaccine lot Aa 25 kGy in liq. N2
Hib conjugate vaccine lot Aa 25 kGy at 01C
Hib conjugate vaccine lot A controla Non-irradiated
Control
0.9370.06 0.7570.18 0.4270.03
0.7570.25 0.8770.16 0.5270.06
0.4970.19 0.6670.11 0.4870.02
0.2470.06 0.3070.02 0.3470.02
Mean and standard deviation of 4 samples.
Table 4 Molecular size determinations for irradiated (25 kGy) and control pneumococcal polysaccharides type 6B and typhoid vi polysaccharide vaccine Radiation type
Molecular sizing (Kd, sepharose CL-4B) Pneumococcal polysaccharide type 6B (powder)
Control (no irradiation) 0.02 Electron beam 0.17 Gamma ray irradiated 0.30
Typhoid vi polysaccharide vaccine (liquid) 0.08 0.23 (in liq. N2) 0.76
Kd’s for the electron beam irradiated samples still met the product specifications for pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine.
References Acharya, I.L., et al., 1987. Prevention of typhoid fever in Nepal with the Vi capsular polysaccharide of Salmonella typhi. N. Engl. J. Med. 317, 1101–1104. Bazhanova, I.G., Tsevetkova, N.V., Bulk, V.F., Mashilova, G.M., Lazareva, E.S., 1986. Effect of radiation sterilization on the immunobiological properties of the sorbed protective fraction of Bordetella pertussis. Zh. Mikrobiol. Epidemiol. Immunobiol. 3, 78–82. Booy, R., Hodgson, S., Carpenter, L., et al., 1994. Efficacy of Haemophilus influenzae type b conjugate vaccine PRP-T. Lancet 344, 362–366. Bureau of Biologics, 1977. Molecular sizing of capsular polysaccharide antigens by Sepharose 4B column chromatography. BOB SOP, Food and Drug Administration, pp. 1–5.
Erickson, G.A., Landgraf, J.G., Wessman, S.J., Koski, T.A., Moss, L.M., 1989. Detection and elimination of adventitious agents in continuous cell lines. Dev. Biol. Stand. 70, 59–66. Lu, C.S., Lee, C.J., Kind, P., 1994. Immune responses of young mice to pneumococcal type 9 V polysaccharide-tetanus toxoid conjugate. Infect. Immun. 62, 2754–2760. McLaughlin, W.L., Boyd, A.W., Chadwick, K.H., McDonald, J.C., Miller, A., 1989. Dosimetry for Radiation Processing. Taylor & Francis, New York. Nedugova, G.I., Rubtsov, I.V., Samoilenko, I.I., 1984. Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. I. Change in the biological activity of non purified cholera exotoxin as affected by ionizing radiation. Zh. Mikrobiol. Epidemiol. Immunobiol. 2, 47–51. Pang, G.T., Clancy, R.L., O’Reilly, S.E., Cripps, A.W., 1992. A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization. J. Virol. 66, 1162–1170. Pawlowski, A., Svenson, S.B., 1999. Electron beam fragmentation of bacterial polysaccharides as a method of producing oligosaccharides for the preparation of conjugate vaccines. FEMS Microbiol. Lett. 174, 255–263. Pawlowski, A., Kallenius, G., Svenson, S.B., 2000. Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing fragmentation and conjugation technologies. Vaccine 18, 1873–1885. Plumb, J.J., Yost, S.E., 1996. Molecular size characterization of Haemophilus influenzae type b polysaccharide-protein conjugates vaccines. Vaccine 14, 399–404. Reid, B.D., 1998. The sterways process: a new approach to inactivating viruses using gamma radiation. Biologicals 26, 125–129. Transy, M.J., Fleurette, J., 1976. Sterilization by ionizing radiations. Rev Epidemiol Sante Publique 24, 165–184. Wong, K.H., et al., 1977. Standardization and control of meningococcal vaccines, group A and group C polysaccharides. J. Biol. Stand. 8, 197–214.
Evaluation of some selected vaccines and other biological products irradiated by gamma rays, electron beams and X-rays J.C. Maya,*, L. Reyb, Chi-Jen Leea a
Center for Biologics and Research, Food and Drug Administration, HMF-6 73, 1401 Rockville Pike, Rockville, MD 20852, USA b Professor des Universities, Conseiller Scientifique, CH-1010 Lausanne, Switzerland
Abstract Molecular sizing potency results are presented for irradiated samples of one lot of Haemophilus b conjugate vaccine, pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine. The samples were irradiated (25 kGy) by gamma rays, electron beams and X-rays. IgG and IgM antibody response in mice test results (ELISA) are given for the Hib conjugate vaccine irradiated at 01C or frozen in liquid nitrogen. Published by Elsevier Science Ltd. Keywords: Irradiated vaccines; Electron beams;
60
Co gamma irradiation; X-ray
1. Introduction In the area of licensed biological products, to date, dispettes (containers) and diluent for vaccines have been sterilized by 60Co gamma irradiation. A limited number of Food and Drug Administration (FDA) regulated foods and drugs and a large number of devices are currently approved for radiation sterilization. Transy and Fleurette (1976) characterized the effects of ionizing radiation on microorganisms and the application of radiosterilization. The effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin has been reported (Nedugova et al., 1984) with the sterilizing effect of gamma radiation achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. Vaccines have been gamma-irradiated for viral inactivation. Examples include gamma-irradiated influenza A virus vaccine (Pang et al., 1992). Gamma radiation has been used for the terminal sterilization and viral inactivation of blood products (Reid, 1998) and elimination of adventitious agents in continuous cell lines (Erickson et al., 1989). The effect of radiation sterilization on the *Corresponding author. Tel.: +1-301-496-4570; fax: +1301-435-4991. E-mail address: [email protected] (J.C. May). 0969-806X/02/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 5 6 5 - 5
immunobiological properties of the sorbed protective fraction of Bordetella pertussis has been reported (Bazhanova et al., 1986). Pneumococcal polysaccharide fragments have been produced (Pawlowski et al., 1999) by electron beam irradiation for the preparation of conjugate vaccines that retained their antigenic epitopes when compared to native, full-sized pneumococcal polysaccharides as determined by enzyme-linked immunoassay. Pneumococcal capsular polysaccharideprotein conjugate vaccines utilizing electron beam fragmentation technology have been produced which produce high IgG titers in both rabbits and mice (Pawlowski et al., 2000). The goal of this project is to study radiation sterilization dosing levels (McLaughlin et al., 1989) and their effect on potency for certain biological products regulated by FDA. In this project certain vaccines, namely, Haemophilus influenzae type b conjugate vaccine (Hib conjugate vaccine) (Booy et al., 1994), pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine (Acharya et al., 1987) have been irradiated using gamma (60Co), electron beam, and X-ray radiation at room temperature, 01C, and liquid nitrogen temperature. Potency testing in terms of molecular sizing (Bureau of Biologics, 1977; Plumb and Yost, 1996; Wong et al., 1977) and mouse IgG and IgM antibody response (Lu et al., 1994) have
J.C. May et al. / Radiation Physics and Chemistry 63 (2002) 709–711
710
then been performed to gather data to evaluate the impact of the radiation on the potency of the irradiated and control biological product samples.
2. Experimental Samples and control materials were from lots of vaccines licensed by the United States Food and Drug Administration. Irradiation facilities: gamma irradiation performed at CEA (Cadarache, France) and Studer AG Werk Hard (Daniken, Switzerland); electron beam irradiation performed at Studer AG Hard Werk; X-ray irradiation performed at the laboratory of Professor Louis Rey in Lausanne, Switzerland. Molecular sizing and mouse IgG and IgM antibody response ELISA potency assays were performed at the Center for Biologics Evaluation and Research of the US Food and Drug Administration (Rockville, MD, USA).
3. Results and discussion Table 1 lists the molecular sizing results for samples of lot A of freeze-dried Haemophilus b conjugate vaccine
Table 1 Molecular size determinations of irradiated (25 kGy) and control samples of lot A of freeze-dried Haemophilus influenzae type b conjugate vaccine (tetanus toxoid conjugate) Irradiation
Molecular sizing for Hib conjugate vaccine (Kd, sepharose CL-4B)
Control (no irradiation) Electron beam (25 kGy) Gamma ray (25 kGy) X-ray (25 kGy)
0.24 0.82 0.76 0.83
that had been irradiated (25 kGy) using gamma, electron beam and X-ray sources. In general, irradiation increased the value of the Kd, and therefore, decreased the molecular size of the polysaccharide. Table 2 lists the mouse antibody response for the gamma-irradiated samples, sample control and saline control. This data indicates that the IgG antibody response was not significantly changed in the irradiated vaccine compared to the vaccine control even though the irradiation has altered the molecular size of the irradiated vaccine. Similarly, Table 3 lists the IgM antibody response for the gamma-irradiated samples, sample control and saline control. This data also indicates that the IgM antibody response was not significantly changed in the irradiated vaccine compared to the vaccine control even though the irradiation has altered the molecular size of the irradiated vaccine. Table 4 lists the molecular sizing results for samples of a pneumococcal polysaccharide type 6B (powder) and typhoid vi polysaccharide vaccine (liquid) that had been irradiated (25 kGy) using gamma ray, and electron beam and unirradiated control samples. The values for the Kd increased for both irradiated products, therefore, indicating that the molecular size of both products decreased under these irradiation conditions. The Kd’s for the electron beam irradiated samples still met the product specifications for these two product types (0.20 and 0.25, respectively).
4. Conclusion In general, irradiation increased the value of the Kd and, therefore, decreased the molecular size of the Hib conjugate vaccine, pneumococcal polysaccharide type 6B and the typhoid vi polysaccharide vaccine. The data in Tables 2 and 3 indicated that although the molecular size has decreased under gamma irradiation at 25 kGy, the mouse IgG amd IgM antibody responses for the Hib conjugate vaccine have not significantly changed. The
Table 2 Mouse IgG antibody response (ELISA OD at 405 nm; 45 min at 251C) of gamma irradiated and non-irradiation samples of lot A of freeze-dried Haemophilus b conjugate vaccine (tetanus toxoid conjugate) and saline diluent As dilution
1/100 1/300 1/500 a
IgG antibody response (ELISA OD unit) Hib conjugate vaccine lot Aa 25 kGy in liq. N2
Hib conjugate vaccine lot Aa 25 kGy at 0oC
Hib conjugate vaccine lot Aa control Non-irradiated
Control
1.0870.06 0.8070.18 0.3770.03
0.7470.25 0.9070.16 0.3370.06
0.8470.19 0.7070.11 0.3870.02
0.2870.06 0.3270.02 0.3270.02
Mean and standard deviation of 4 samples.
Saline diluent
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Table 3 Mouse IgM antibody response (ELISA OD at 405 nm; 45 min at 251C) of gamma irradiated (at two temperatures) and non-irradiated samples of lot A of freeze-dried Haemophilus influenzae type b conjugate vaccine (tetanus toxoid conjugate) and saline diluent As dilution
1/100 1/300 1/500 a
IgM antibody response (ELISA OD unit) Saline diluenta
Hib conjugate vaccine lot Aa 25 kGy in liq. N2
Hib conjugate vaccine lot Aa 25 kGy at 01C
Hib conjugate vaccine lot A controla Non-irradiated
Control
0.9370.06 0.7570.18 0.4270.03
0.7570.25 0.8770.16 0.5270.06
0.4970.19 0.6670.11 0.4870.02
0.2470.06 0.3070.02 0.3470.02
Mean and standard deviation of 4 samples.
Table 4 Molecular size determinations for irradiated (25 kGy) and control pneumococcal polysaccharides type 6B and typhoid vi polysaccharide vaccine Radiation type
Molecular sizing (Kd, sepharose CL-4B) Pneumococcal polysaccharide type 6B (powder)
Control (no irradiation) 0.02 Electron beam 0.17 Gamma ray irradiated 0.30
Typhoid vi polysaccharide vaccine (liquid) 0.08 0.23 (in liq. N2) 0.76
Kd’s for the electron beam irradiated samples still met the product specifications for pneumococcal polysaccharide type 6B and typhoid vi polysaccharide vaccine.
References Acharya, I.L., et al., 1987. Prevention of typhoid fever in Nepal with the Vi capsular polysaccharide of Salmonella typhi. N. Engl. J. Med. 317, 1101–1104. Bazhanova, I.G., Tsevetkova, N.V., Bulk, V.F., Mashilova, G.M., Lazareva, E.S., 1986. Effect of radiation sterilization on the immunobiological properties of the sorbed protective fraction of Bordetella pertussis. Zh. Mikrobiol. Epidemiol. Immunobiol. 3, 78–82. Booy, R., Hodgson, S., Carpenter, L., et al., 1994. Efficacy of Haemophilus influenzae type b conjugate vaccine PRP-T. Lancet 344, 362–366. Bureau of Biologics, 1977. Molecular sizing of capsular polysaccharide antigens by Sepharose 4B column chromatography. BOB SOP, Food and Drug Administration, pp. 1–5.
Erickson, G.A., Landgraf, J.G., Wessman, S.J., Koski, T.A., Moss, L.M., 1989. Detection and elimination of adventitious agents in continuous cell lines. Dev. Biol. Stand. 70, 59–66. Lu, C.S., Lee, C.J., Kind, P., 1994. Immune responses of young mice to pneumococcal type 9 V polysaccharide-tetanus toxoid conjugate. Infect. Immun. 62, 2754–2760. McLaughlin, W.L., Boyd, A.W., Chadwick, K.H., McDonald, J.C., Miller, A., 1989. Dosimetry for Radiation Processing. Taylor & Francis, New York. Nedugova, G.I., Rubtsov, I.V., Samoilenko, I.I., 1984. Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. I. Change in the biological activity of non purified cholera exotoxin as affected by ionizing radiation. Zh. Mikrobiol. Epidemiol. Immunobiol. 2, 47–51. Pang, G.T., Clancy, R.L., O’Reilly, S.E., Cripps, A.W., 1992. A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization. J. Virol. 66, 1162–1170. Pawlowski, A., Svenson, S.B., 1999. Electron beam fragmentation of bacterial polysaccharides as a method of producing oligosaccharides for the preparation of conjugate vaccines. FEMS Microbiol. Lett. 174, 255–263. Pawlowski, A., Kallenius, G., Svenson, S.B., 2000. Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing fragmentation and conjugation technologies. Vaccine 18, 1873–1885. Plumb, J.J., Yost, S.E., 1996. Molecular size characterization of Haemophilus influenzae type b polysaccharide-protein conjugates vaccines. Vaccine 14, 399–404. Reid, B.D., 1998. The sterways process: a new approach to inactivating viruses using gamma radiation. Biologicals 26, 125–129. Transy, M.J., Fleurette, J., 1976. Sterilization by ionizing radiations. Rev Epidemiol Sante Publique 24, 165–184. Wong, K.H., et al., 1977. Standardization and control of meningococcal vaccines, group A and group C polysaccharides. J. Biol. Stand. 8, 197–214.