Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Patent
1995-03-01
2000-05-09
Housel, James C.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
4241841, 4242341, 4242361, 4242401, 4242411, 4242531, 4242541, 4242571, 4242611, 435243, 4352521, 436543, A61K 3900, A61K 3902, A61K 3910, A61K 39108
Patent
active
060600675
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a process for the preparation of tetanus toxin for use in tetanus vaccines.
The existing tetanus vaccine is produced from a 7-day bottle culture of Clostridium tetani which is inactivated ("toxoided") by the addition of formaldehyde. The formaldehyde is added as formalin. Toxoid is purified for use in a vaccine by salt fractionation to a specific activity of approximately 1200 flocculation units (Limes flocculationis, Lf)/mg protein nitrogen (PN), which is equivalent to 30% to 40% purity.
Attempts have been made to increase the specific activity of the vaccine by purifying the toxoid using immunoadsorbents (Hughes et al, J. Appl. Bact. 37, 603-621, 1974). This however only resulted in a limited increase, to 1600 Lf/mg PN. GB-A-969772 discloses a method for producing a toxoid from a purified bacterial toxin, especially purified diphtheria toxin, by treating the toxin in an aqueous medium with formaldehyde in the presence of an aliphatic diamine of molecular weight below 200 which contains a primary or secondary amino group. Preferred diamines are lysine and ethylenediamine.
We have now devised a new process for the preparation of tetanus toxoid. Instead of toxoiding the toxin and then purifying the toxoid, we purified the toxin and then toxoided the purified toxin. Surprisingly, however, many of these preparations were found to be unstable, showing varying degrees of reversion to toxicity when stored at 37.degree. C. in the absence of formalin.
We then looked at adding amino acids to the toxoiding reaction at different concentrations, also varying the formalin concentration, pH and incubation times. Reversion to toxicity was difficult to avoid, except at the price of low total combining power (TCP)/Lf ratios that resulted in preparations of poor immunogenicity. Only under specific conditions could stable and highly immunogenic preparations be obtained.
Accordingly, the present invention provides a process for the preparation of tetanus toxoid, which process comprises incubating purified tetanus toxin with 0.2 to 1% (v/v) formaldehyde in the presence of 0.005 to 0.25M lysine for from 24 to 32 days at a pH of from 6.0 to 8.0 and a temperature of from 30 to 45.degree. C.
The tetanus toxin is typically obtained from a culture of Clostridium tetani. Any appropriate strain of Cl. tetani can be employed. The toxin may be purified from a fermentation broth by first centrifuging the broth and then clarifying the culture supernatant, for example by a two stage filtration. The clarified supernatant may be concentrated. The supernatant may then be subjected to diafiltration to remove small charged molecular species and then to ion-exchange chromatography.
The purified toxin preferably has an Lf content of 250 Lf/ml or more, for example from 250 to 500 Lf/ml. If the Lf content is less than 250 Lf/ml, the toxin may be reprocessed by an additional concentration step to increase the toxin yield.
The specific activity of the toxin may be determined by estimating the protein nitrogen (PN) content of the purified material and by calculating the ratio of Lf to PN. Typically, the purified toxin has a ratio of Lf to PN of 2000 Lf/mg PN or more, for example from 2000 to 3000 Lf/mg PN or from 2000 to 2800 Lf/mg PN. If the specific activity of the toxin is less than 2000 Lf/mg PN, the purified material may be reprocessed through an ion-exchange column. A specific activity of 2000 Lf/mg PN is equivalent to a purity of about 70%. 100% pure toxin is reported to have a specific activity of 3000 to 3200 Lf/mg PN.
The purity of the toxin may alternatively or additionally be assessed by high pressure liquid chromatography (HPLC) analysis and/or by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Purity is preferably determined by HPLC whereby eluted peaks are integrated and the area of toxin-related peaks is expressed as a percentage of the total integrated peaks area. Analysis by SDS-PAGE under reducing conditions separates the toxin molecule into its constituent heavy (100 kD) and light (5
REFERENCES:
patent: 4996299 (1991-02-01), Ginnaga et al.
Relyveld et al. Methods in Enzymology 93:24-60, 1983.
Pillemer et al. J. Immunol. 54:213-224, 1946.
Physician's Desk Reference. Medical Economics Data, Oradell NJ. 1991 Edition, pp. 1206-1207.
Rene Germanier, Excerpt from Bacterial Vaccines, 1984, pp. 48-49, 60-61.
Chemical Abstracts, vol. 72, No. 3, Jan. 19, 1970, D.J. Dawson et al `Tetanus Toxin and Toxoid, IV. Interaction of Foraldehyde With Tetanus Toxin.` p. 172.
Biologicals (1993) 21, Anomalous Symptoms In Mice Injected With Reverte Tetanus Toxoid Knight et al, pp. 183-184.
Knight Peter Anthony
Sheppard Anthony James
Housel James C.
Medeva Pharma Limited
Ryan V.
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