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Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

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Washed cells from 72-h cultures of Streptomyces fradiae GS14 were used to examine the distribution of radiolabel from 14C-amino acids and related compounds into tylactone, C02, and cells. Test compounds were categorized according to products of their oxidative degradation. Those compounds known to produce propionyl-coenzyme A by direct catabolic oxidation were designated as group I. Group II included those compounds oxidized to methylmalonyl-coenzyme A via succinyl-coenzyme A and the tricarboxylic acid cycle. Group III contained compounds known to be oxidized to acetoacetyl-coenzyme A. The total amount of label recovered after 60 min ranged from 3 to 65%. Although label from all test compounds except proline (group II) and lysine (group III) was incorporated into tylactone after 60 min, label from group I and group III compounds was incorporated at levels five times greater than label from group II compounds. From 55 to 75% of the recovered label from propionate (I), asparagine (II), glutamine (II), glutamate (II), aketoglutarate (II), and succinate (II) was recovered as 14 C02. From 75 to 95% of the recovered label from the remaining compounds tested was located in the cells. Based on the data, a pathway for the role of amino acids in the biosynthesis of tylactone is proposed.Tylactone (Fig. 1) is the initial lactone precursor to tylosin, an antibiotic produced commercially by Streptomyces fradiae (1-3). Omura et al. (6), using 13C enrichment, showed that tylactone was formed from five propionate molecules, two acetate molecules, and one butyrate molecule. Subsequent inhibition studies with cerulenin, an inhibitor of fatty acid synthetase, showed that tylactone and related macrolide aglycone molecules were assembled in a manner similar to fatty acid biosynthesis (5, 7-9). The data of suggested that the precursors for tylactone biosynthesis were incorporated in the following manner: propionate as one propionyl-coenzyme A (CoA) (primer) and four methylmalonyl-CoA molecules, acetate as two malonyl-CoA molecules, and butyrate as one ethylmalonylCoA molecule.In this study, the role of amino acids as sources for the tylactone precursors was examined by using a tylA mutant of S. fradiae (1). This strain was blocked in the production of the tylosin sugars and therefore produced tylactone as its product (1, were grown in a complex medium as described previously (1).Preparation of washed cell suspensions. After 72 h of incubation, the cells from 300 ml of broth were removed by centrifugation at 16,000 x g for 5 min in a Sorvall RC-5 centrifuge. Cell pellets were washed twice with 300 ml of 0.1 M potassium phosphate buffer, pH 7.0. Approximately 6.5 g of wet cells were suspended in the buffer to a volume of 50 ml, and portions were diluted 10-fold with buffer to yield final cell suspensions containing 1.2 to 1.7 mg of dry cells per ml.Uptake and distribution reactions. Reactions were carried out in 35-ml flint glass bottles (Kimble, Toledo, Ohio) that were closed with gray butyl rubber stoppers (West Rubber Co., Phoenix...