Process for purifying insulin

Claim 2

A PROCESS FOR REDUCING THE ANTIGENICITY OF THE INSULIN THAT HAS BEEN RECOVERED FROM PANCREAS GLANDS OF DOMESTIC MAMMALS AND WHICH CONTAINS INSULIN-LIKE IMPURITIES, WHICH COMPRISES DISSOLVING THE INSULIN IN A WATER-CONTAINING MEDIUM, ADJUSTING THE INSULIN SOLUTION TO A PH-VALUE WITHIN THE RANGE OF 6 TO 9, APPLYING THEINSULIN SOLUTION ON A CHROMATOGRAPHIC COLUMN OF AN ANION EXCHANGE TO FIX THEINSULIN TO THE COLUMN, FRACTIONALLY ELUTING THE COLUMN WITH AN ELUENT CONTAINING WATER TOGETHER WITH A WATER-MISCIBLE MONOHYDRIC ALIPHATIC ALCOHOL IN A CONCENTRATION OF 40 TO 80 PER CENT (V/), THEREBY SEPARATING THE INSULIN FROM ITS IMPURITIES, COLLECTING THE INSULIN-CONTAINING FRACTIONS SHOWING ESSENTIALLY A SINGLE COMPONENT WHEN ANALYSED BY DISCONTINUOUS POLYACRYLAMIDE GET ELECTROPHORESIS (DISC PAGE), AND RECOVERING THE INSULIN CONTAINED IN THE COLLECTED FRACTIONS.

Claim 3

A process as defined in claim 1, in which the insulin solution is adjusted to a pH-value within the range of 6.5 to 8.5.

Claim 4

A process as defined in claim 1, in which the alcohol concentration of the eluent is 50 to 70 per cent (v/v).

Claim 5

A process as defined in claim 1, in which the eluent has almost the same pH-value as that of the insulin solution applied to the column.

Claim 6

A process as defined in claim 1, in which the eluent has almost the same pH-value as that of the insulin solution applied to the column and the elution is effected without changing the ion strength of the eluent applied to the loaded column.

Claim 7

A process as defined in claim 1, in which the insulin has been recovered from pork pancreas glands.

Claim 8

A process according to claim 1, in which the insulin has been recovered from bovine pancreas glands.

Claim 9

A process as defined in claim 1, in which the anion exchanger is a strongly basic anion exchanger.

Claim 10

A process according to claim 1, in which the eluent has almost the same pH-value as that of the insulin solution applied to the column and the elution is effected without changing the pH-value of the eluent.

Claim 11

A process according to claim 1, in which the column is equilibrated with a buffer solution having a similar composition as that of the eluent before the insulin solution is applied to the column.

Claim 12

A process for reducing the antigenicity of commercial or crude insulin recovered from pancreas glands and containing insulin-like impurities having a molecular weight of about 6,000, which comprises preparing a buffer solution containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v) and having a pH-value within the range of 6 to 9, applying said buffer solution of a chromatographic column of an anion exchanger and equilibrating the column with the buffer, dissolving the crude or commerical insulin in a water-containing medium, adjusting the insulin solution to a pH-value within the range of 6 to 9, applyiNg said insulin solution to the column, eluting the column in fractions with an eluent containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v) and having a pH-value within the same range as that of the equilibrating solution, collecting the fractions showing essentially a single component when analysed by discontinuous polyacrylamide gel electrophoresis (DISC PAGE) and recovering the insulin contained in the collected fractions.

Claim 13

A process for reducing the antigenicity of commercial or crude insulin recovered from pancreas glands and containing insulin-like impurities having a molecular weight of about 6,000, which comprises preparing a buffer solution containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v) and having a pH-value within the range of 6 to 9, applying said buffer solution of a chromatographic column of an anion exchanger and equilibrating the column with the buffer, dissolving the crude or commerical insulin in a water-containing medium, adjusting the insulin solution to a pH-value within the range of 6 to 9, applyiNg said insulin solution to the column, eluting the column in fractions with an eluent containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v) and having a pH-value within the same range as that of the equilibrating solution, collecting the fractions showing essentially a single component when analysed by discontinuous polyacrylamide gel electrophoresis (DISC PAGE) and recovering the insulin contained in the collected fractions.

Claim 14

A process as defined in claim 11, in which the alcohol in the eluent is in a concentration of 50 to 70 per cent (v/v).

Claim 15

A process for reducing the antigenicity of insulin recovered from pancreas glands of domestic mammals and containing antigenic impurities, which comprises fractionally eluting a chromatographic column of an anion exchanger having the insulin fixed thereto with an eluent containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v), collecting the eluate fractions containing insulin essentially free of the antigenic impurities, and recovering the insulin from the collected fractions.

Claim 16

A process for reducing the antigenicity of insulin recovered from pancreas glands of domestic mammals and containing antigenic impurities, which comprises fractionally eluting a chromatographic column of an anion exchanger having the insulin fixed thereto with an eluent containing water together with a water-miscible monohydric aliphatic alcohol in a concentration of 40 to 80 per cent (v/v), collecting the eluate fractions containing insulin essentially free of the antigenic impurities, and recovering the insulin from the collected fractions.