Patent GB-786376-A (GB)
Title: Method of recovering proteins of bacterial origin
Inventor: TINT HOWARD
IPC: A61K39/08
Filed: 1955-04-22
Abstract: A concentrated and purified toxoid product, e.g. tetanus, diphtheria, scarlet fever and botulinus, is recovered from a toxoid-containing broth by adding to said broth a water soluble non-toxic polymeric metaphosphate which will form a complex with the toxoid, lowering the pH of the broth, e.g. using hydrochloric or phosphoric acid, to substantially completely precipitate the toxoid metaphosphate complex, e.g. pH 2 to 4, equilibrating the separated precipitated complex and undesired protein complexes with a non-toxic aqueous buffer solution at the least pH of solubility of substantially all of the desired complex and separating the solution of the desired complex from the mixture. In a variation of the procedure, the separated precipitated complex and undesired complexes are dissolved in a non-toxic aqueous buffer solution, e.g. phosphate, citrate-borate, bicarbonate, having a pH above that at which all of said complexes are soluble, e.g. pH 5.5 to 9, and the resulting solution is acidified to precipitate all the undesired protein complexes, e.g. pH 4.8 to 5.2, the precipitate being equilibrated with the solution in which it is formed and thereafter removed leaving the desired toxoid-metaphosphate complex in solution.  If required, metaphosphate ions can be removed from the solution by dialysis, precipitation as insoluble calcium or barium salts, or by treatment with an anion exchange resin, and any porphyrius remaining in the solution can be removed by treating the solution with activated carbon.  The term "polymeric metaphosphate" is defined as having a M2O:P2O5 mole. ratio of 1.0 to 1.66 (M is an alkali metalatom or ammonium radical), e.g. ammonium, potassium and sodium hexametaphosphate, and the metaphosphate is added in amount in excess of one metaphosphate polymer per protein amino group.ALSO:A concentrated and purified toxoid product, e.g. tetanus, diphtheria, scarlet fever and botulinus, is recovered from a toxoid-containing broth by adding to said broth a water-soluble non-toxic polymeric metaphosphate which will form a complex with the toxoid, lowering the pH of the broth, e.g. using hydrochloric or phosphoric acid, to substantially completely precipitate the toxoid metaphosphate complex, e.g. pH 2 to 4, equilibrating the separated precipitated complex and undesired protein complexes with a non-toxic aqueous buffet solution at the least pH of solubility of substantially all of the desired complex and separating the solution of the desired complex from the mixture. In a variation of the procedure, the separated precipitated complex and undesired complexes are dissolved in a non-toxic aqueous buffer solution, e.g. phosphate, citrate-borate, bicarbonate having a pH above that at which all of said complexes are soluble, e.g. pH 5.5 to 9, and the resulting solution is acidified to precipitate all the undesired protein complexes, e.g. pH 4.8 to 5.2, the precipitate being equilibrated with the solution in which it is formed and thereafter removed leaving the desired toxoid-metaphosphate complex in solution.  If required metaphosphate ions can be removed from the solution by dialysis, precipitation as insoluble calcium or barium salts, or by treatment with an anion exchange resin, and any porphyrius remaining in the solution can be removed by treating the solution with activated carbon.  The term "polymeric metaphosphate" is defined as having a M2O : P2O5 mol. ratio of 1.0 to 1.66 (M is an alkali metal atom or ammonium radical), e.g. ammonium, potassium and sodium hexametaphosphate, and the metaphosphate is added in amount in excess of one metaphosphate polymer per protein amino group.