| Ansamitocin is a benzoquinone ansamycin antibiotic well known for its extraordinary antitumor activity. Ansamitocin is an important constituent in many antibody-drug conjugates (ADCs). A shortage of commercial ansamitocin supply exists in China because of the very low production level of ansamitocin, which hinders the research progress of Chinese ADCs development. Establishing an efficient ansamitocin fermentation and purification process and making a stable and economical supply of ansamitocin should relieve the present urgent demand for ansamitocin in ADCs development.In particular, as a major institute of microbial drug development in China that shows also very high interests in ADCs development, the Institute of Medicinal Biotechnology, PUMC & CAMS, is also seeking for a bulk supply of ansamitocin for its ADCs programs.The present study aims at establishing a laboratory level (grams) of a comprehensive ansamitocin production process, from ansamitocin fermentation, extraction, separation and purification. And Solid state fermentation of Actinosynnema pretiosum ATCC 31565 is chosen for the primary ansamitocin fermentation mode.First, an agar medium, which consists of glucose 8 g 1-, malt extract 5 g 1-, yeast extract 1 g 1-1, NaCl 5 g 1-1, agar 15 g 1-1, was successfully designed for production of stable and abundant spores of Actinosynnema pretiosum ATCC 31565 used as seed culture for the following solid state fermentation scale-up culture. Several fermentation media were tested for solid state fermentation of Actinosynnema pretiosum ATCC 31565, and their ansamticocin production levels were compared. Among them, the fermentation medium, which consisted of glucose 20 g 1-1, corn starch 30 g 1-1, cottonseed meal 30 g 1-1, CaCl2 10g 1-1, agar 20 g 1-1, gave an ansamitocins production level of about 100 mg/L after purification (purity ≥ 85% by HPLC). This was about three times more than the ansamitocins production level with the ISP medium.Second, a purification process which contains ethyl acetate extraction and silica gel column fractionation was established for making ansamitocins preparation, a mixture of ansamitocins P-2, P-3 and P-4.Third, many efforts were made by us to improve ansamitocins production by Actinosynnema pretiosum ATCC 31565. Isobutanol, valine, or scandium trichloride were supplemented into the fermentation media, to observe their effects on ansamitocins production. Isobutanol improved significantly the ansamitocin P-3 proportion in ansamitocins (containing ansamitocin P-2,3 and 4), but showed no stimulation effect for the overall ansamitocins production level. Valine or scandium trichloride displayed no effect on improving the overall ansamitocins production levels. Liquid state fermentation in shake flasks was also conducted for ansamitocin fermentation. It produced similar levels of ansamitocins compared to solid state fermentation. But liquid state fermentation consumes more energy than solid state fermentation, and the following ansamitocins extraction process is more difficult than that of the solid state fermentation.Finally, our ansamitocins preparation was used to generate ansamitocin P-0 by chemical de-acylation. The yield of ansamitocin P-0 (purity≥ 99% by HPLC) was about 82% from ansamitocins. A total amount of 1.5 grams ansamitocins were provided for the ADCs development program of the Institute of Medicinal Biotechnology, PUMC & CAMS, which guaranteed the initial urgent demand for ansamitocins in DM-1 preparation in ADCs development. |
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