Patent GB-1345729-A (GB)
Title: Recovery and purification of antibiotics
IPC: C07D501/57
Filed: 1971-06-28
Abstract: 1345729 Purification and recovery of cephem antibiotics MERCK & CO Inc 28 June 1971 [30 June 1970] 30256/71 Addition to 1321412 Heading C2A Impure solutions of an antibiotic mixture comprising cephalosporin-like compounds of the general Formula I wherein R is -OH (compound Ia) or -OSO 3 H (compound Ib) are purified, and if required either isolated as a purified mixture of (Ia) and (Ib), or the purified solution separated into its components from which (Ia) and (Ib) may individually be isolated, by adsorbing the impure solution on an anion-exchange resin, a cationexchange resin or a cross-linked non-ionic adsorptive polymer, eluting the adsorbate in fractions with a solvent, collecting the eluted fractions and combining the antibiotically active fractions to obtain a purified solution of the mixed antibiotics from which the latter may be isolated as a mixture or which may be subjected to chromatography to separate a solution of (Ia) and a solution of (Ib) which solutions may then be treated to isolate the individual antibiotics. The impure solution may be a filtered fermentation broth from a Streptomyces griseus culture. Suitable anion-exchange resins are the chloride form of a cross-linked diethylaminoethyl dextran derivative, and a cross-linked acrylic copolymer containing weakly basic tertiary amine groups. Cation exchange resins used may be of the sulphonated styrene-divinylbenzene polymer type. The non-ionic adsorbent polymer may be a non-ionic macro-reticular cross-linked polystyrene. The impure solution is preferably added to the adsorbent at about pH 3-4, and the fractions may be eluted with an aqueous or aqueousalcoholic salt or acid. For elution from a non- ionic polymer, the eluant may be a 5%, 60% or 90% (v/v) solution of methanol in water or a 20% or 25% (v/v) solution of acetone in water. Elution from the ionic resins may be with: (1) dilute aqueous HCl, pH 0À95; (2) an aqueous solution of 1M. NaNO 3  and 0À1M. NaAc, pH 7À5; (3) an aqueous solution of 0À5M. NH 4 Br and 0À5M. acetic acid; and (4) a 1% (v/v) solution of formic acid in water. The antibiotically active eluate from this stage may be separated into solutions of (Ia) and of (Ib) by fractional chromatography using one of the following systems: (a) a strongly hydrophylic anion-exchanger which is a diethylaminoethyl dextran derivative in the chloride form, developing with a 0À5M. NH 4 Br-0À1M. formic acid aqueous solution; (b) a weakly basic anionexchanger (e.g. cross-linked acrylic copolymer having weakly basic tertiary amine groups) the material being charged on to the column at pH 3 to 3À5, and then developed first with an acid at pH about 2À0 and then with a mixture of NaCl and HCl at pH about 1; (c) a non-ionic crosslinked polystyrene adsorbent, eluting first with 10% v/v methanol in water followed by either 50% v/v methanol in water or 20% v/v acetone in water. The fractions obtained by these procedures are assayed by bioassay against a suitable sensitive micro-organism (e.g. Proteus vulgaris) to determine which fractions contained the highest levels of antibiotic. Generally, in the aforesaid systems, compound (Ia) eluted in the initial fractions and compound (Ib) was found in the later fractions. The individual product solutions thus obtained may be further purified by aqueous-alcoholic chromatography on the aforesaid non-ionic resin, the alcohol in the eluates then being removed by evaporation in vacuo, and the final aqueous solutions being freeze-dried to give the pure antibiotics (Ia) and (Ib).