Secondary metabolite scale‐up to minimize homolog impurity levels

Abstract

A mutant strain of Streptomyces hygroscopicus was found to produce up to 9.0 units/L of an immunoregulant precursor, immunomycin, with up to 3.5% of a lower homolog impurity under either dual fed-batch or batch conditions. Glycerol and valine were key nutrients influencing productivity and impurity levels. Soybean oil was successfully substituted for glycerol as a carbon source to minimize shot additions to batch culture. The remainder of the production medium was composed largely of defined components with the exception of yeast extract.Valine limitation increased lower homolog formation while decreasing higher homolog formation; excess valine decreased lower homolog formation below 2-3% while increasing higher homolog formation. Higher homolog formation in the presence of valine seemed to be slower than lower homolog formation in the absence of valine. Valine was believed to be the major butyrate precursor; consequently its availability influenced the impurity profile. A preliminary cost analysis suggests that elimination of added valine from the cultivation and replacement of glycerol with soybean oil can result in a 6.6-fold reduction in media costs relative to the original fed-batch process.

Use of soybean oil and ammonium sulfate additions to optimize secondary metabolite production

“…Thus, the evaluation of broth ammonia levels appeared critical to maximizing titer and productivity in addition to carbon utilization. This behavior sharply contrasted both the behavior of the same culture in Pharmamedia-based GYG5 medium as well as the behavior of the earlier MA6678 culture (Junker et al, 1998) in which there were no adverse affects of ammonium hydroxide additions.…”

“…Production medium was prepared using deionized water, adjusted to pH 7.3, after which calcium carbonate was added. Methods for seed-flask preparation have been described previously (Junker et al, 1998). Two production media were evaluated.…”

“…The cultures utilized in the previous work, MA6678 (Junker et al, 1998) and the current work, MA7040, were obtained from Streptomyces hygroscopicus var ascomyceticus (ATCC141891, MA6475) via a classical mutation and selection program. Lower homologs were composed of a methyl group instead of an ethyl group at a distinct location on the carbon skeleton, resulting from the substitution of a propionate for a butyrate during polyketide synthesis.…”

“…Lower homologs were composed of a methyl group instead of an ethyl group at a distinct location on the carbon skeleton, resulting from the substitution of a propionate for a butyrate during polyketide synthesis. In contrast, higher homologs were composed of an ethyl group substituted for a methyl group, specifically a butyrate in lieu of a propionate (Junker et al, 1998). Based on purity profiles of material used in safety assessment studies, the specification for lower homologs was 2%, while that for higher homologs was 0.5%.…”

“…Earlier work (Junker et al, 1998) utilized culture MA6678, which required a dual-fed (glycerol and valine) batch process to reach a titer of 0.9 g/L L-683,590 with 2-3% of the L-683,795 lower homolog after about 150 h. Valine was converted to isobutyryl CoA and then butyryl CoA. Due to the high cost of valine and the tendency of high broth valine concentrations to raise levels of difficult-toremove higher homolog impurities up to 1-1.5%, a valineover producing mutant, MA7040, was identified.…”