Process for the recovery of human .beta.-interferon-like polypeptides

In a process for the recovery of unglycosylated .beta.-HIFN from a microorganism transformed to produce said .beta.-HIFN wherein the steps comprise:

• a. disrupting the cell wall of said microorganism;
• b. solubilizing said .beta.-HIFN in the disruptate into an aqueous medium with solubilizing agent;
• c. extracting said solubilized .beta.-HIFN from the aqueous medium with an aliphatic alcohol selected from the group consisting of 2-butanol, 2-methyl-2-butanol and mixtures thereof;
• and
• d. isolating and purifying said .beta.-HIFN;
• the improvement which comprises the additional steps of:
• e. adjusting the pH of a solution containing the purified .beta.-HIFN to about 10.5 to 12.5;
• and
• f. diafiltering said .beta.-HIFN solution at a pH of about 10.5 to 12.5.

In a process for the recovery of .beta.-HIFN from transformed E. coli bacteria comprising concentrating said bacteria, disrupting the cell wall of said bacteria, solubilizing said .beta.-HIFN with SDS, extracting said .beta.-HIFN with 2-butanol and precipitating said .beta.-HIFN by mixing said butanol phase with an aqueous mixture, the improvement comprising:

• dissolving the precipitated .beta.-HIFN in an appropriate buffer at a pH of about 9;
• reducing said .beta.-HIFN with dithiothreitol;
• diafiltering the .beta.-HIFN solution against water at a pH of about 10.5 to 12.5 or against a 10% (v/v) ethanol at a pH of about 10.5 to 12.5;
• adding to said .beta.-HIFN a solution of human serum albumin and dextrose adjusted to a pH of about 10.5 to 12.5;
• and
• lowering the pH of the resulting .beta.-HIFN solution to about 7 to 7.5.

In a process for the production of an unglycosylated .beta.-HIFN from E. coli genetically transformed to produce said .beta.-HIFN and grown in an appropriate fermentation medium wherein the steps comprise:

• (a) concentrating the bacteria in the fermentation medium by cross-flow filtration;
• (b) mechanically homogenizing the bacteria to disrupt the cell walls of the bacteria;
• (c) separating solid cellular material from the remainder of the homogenate by centrifugation;
• (d) solubilizing the solid cellular material by suspending it in an aqueous solution of sodium sulfate at a SDS to protein ratio of about 3:1;
• (e) extracting the .beta.-HIFN from the aqueous phase with 2-butanol by continuous cocurrent extraction;
• (f) aging the 2-butanol phase overnight;
• (g) heating the 2-butanol phase to about 60.degree. C. for about 10 to 20 minutes;
• (h) contacting the 2-butanol phase with an aqueous buffer and adjusting the pH of the mixture to aobut 5.5 to precipitate the .beta.-HIFN;
• (i) collecting the precipitated .beta.-HIFN by centrifugation;
• (j) solubilizing the .beta.-HIFN with an aqueous solution of sodium dodecyl sulfate at a SDS to protein ratio of about 3:1;
• (k) reducing said solubilized .beta.-HIFN with dithiothreitol;
• (l) purifying the reduced .beta.-HIFN by chromatography;
• (m) collecting the eluted fraction of the purified .beta.-HIFN;
• (n) concentrating the purified .beta.-HIFN to a concentration of about 20 mg protein/ml;
• (o) further purifying the .beta.-HIFN by gel chromatography;
• and
• (p) collecting and dissolving the fraction eluate containing the purified .beta.-HIFN in an aqueous buffer;
• the improvement which comprises the steps of:
• (q) adjusting the pH of the .beta.-HIFN solution to about 12;
• (r) diafiltering the solution containing the .beta.-HIFN at a pH of about 12 against pure water or a H.sub.2 O-ethanol mixture adjusted to a pH of about 12;
• (s) stabilizing the .beta.-HIFN by the addition of a 0.5-10% by weight solution of dextrose and human serum albumin adjusted to a pH of about 12;
• and
• (t) lowering the pH of the resulting solution to physiological pH.