Industrial production of high-titer tetanus toxin
Pettenella, G.; Sella, A.
Bollettino dell'Istituto Sieroterapico Milanese 37(9-10): 401-413
1958
ISSN/ISBN: 0021-2547 PMID: 13607653 Document Number: 3144
A method for the large-scale production of a highly potent tetanus toxin is detailed, based on the development of the toxigenic strain in simple, enriched isotonic saline solutions through a semipermeable cellophane membrane. After thermostatic incubation of the culture, the toxin and the bacteria producing it remain inside the cellophane walls, free of the non-dialyzable components usually present in the culture substrates used for the production of toxin by the classic methods. High commercial toxin yields are achieved by optimal regulation of various factors within and without the dialysis membrane. Best yields are achieved with a peptic hydrolysate of meat (100% yield), Difco proteose peptone (80%) and Witte peptone (80%). Of the various extracts tried, beef haunch muscle extract and beef heart extract give superior yields (100%). Addition of peptone after treatment at 45° C decreases the toxin yield to 35% (100% if added before treatment). Optimal NaCl content of the medium was found to be 0.517-0.550%. Lack of glucose in the medium reduced the 100% yield (with glucose) to less than 5%, and with medium containing 0.5 per thousand of Na thioglycolate, yield was only 15%. Optimal final pH was 7.8. Best adsorbent systems were 0.3, 0.5, or 1.0% Hyflo Supercel. The CN 761 strain of Clostridium tetani gave a yield of 100% toxin compared to 38% for Harvard-49205. Highest L + yields were obtained after 10, 12, or 14 days incubation. Tests of lethality in mice and guinea pigs showed that the technique used gives constant and reproducible data, offering a distinct advantage for continuous industrial production. The final medium is low in protein N and the final product is extremely pure.
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