Process for the recovery of human β-interferon-like polypeptides

Claim 2

In a process for the recovery of a lipophilic protein from a microorganism transformed to produce said protein wherein the steps comprise: solubilizing said protein in an aqueous medium with an appropriate solubilizing agent; extracting said solubilized protein from the aqueous medium with an aliphatic alcohol selected from the group consisting of 2-butanol, 2-methyl-2-butanol and mixtures thereof; precipitating said protein from the alcohol phase; the improvement which comprises purifying said precipitated protein by diafiltration at a pH of about 10.5 to 12.5.

Claim 3

A process according to claim 1, wherein said lipophilic protein is β-HIFN.

Claim 4

A process according to claim 2, wherein said solubilizing agent is an anionic surfactant.

Claim 5

A process according to claim 3, wherein said surfactant is sodium dodecyl sulfate or sodium laurate.

Claim 6

A process according to claim 4, wherein the weight ratio of said surfactant to said β-HIFN is in the ratio of about 1:1 to about 5:1.

Claim 7

A process according to claim 5, wherein said ratio is about 3:1.

Claim 8

In a process for the recovery of unglycosylated β-HIFN from a microorganism transformed to produce said β-HIFN wherein the steps comprise: a. disrupting the cell wall of said microorganism; b. solubilizing said β-HIFN in the disruptate into an aqueous medium with solubilizing agent; c. extracting said solubilized β-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 β-HIFN; the improvement which comprises the additional steps of: e. adjusting the pH of a solution containing the purified β-HIFN to about 10.5 to 12.5; and f. diafiltering said β-HIFN solution at a pH of about 10.5 to 12.5.

Claim 9

In a process for the recovery of unglycosylated β-HIFN from a microorganism transformed to produce said β-HIFN wherein the steps comprise: a. disrupting the cell wall of said microorganism; b. solubilizing said β-HIFN in the disruptate into an aqueous medium with solubilizing agent; c. extracting said solubilized β-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 β-HIFN; the improvement which comprises the additional steps of: e. adjusting the pH of a solution containing the purified β-HIFN to about 10.5 to 12.5; and f. diafiltering said β-HIFN solution at a pH of about 10.5 to 12.5.

Claim 10

A process according to claim 7, including reducing said β-HIFN prior to step (e).

Claim 11

A process according to claim 8, wherein said β-HIFN is reduced with dithiothreitol or mercaptoethanol or glutathione or cysteine.

Claim 12

A process according to claim 7, wherein said β-HIFN solution in step (f) is diafiltered against distilled water adjusted to a pH of about 10.5 to 12.5.

Claim 13

A process according to claim 7, wherein said β-HIFN solution in step (f) is diafiltered against a 10% aqueous solution of ethanol adjusted to a pH of about 10.5 to 12.5.

Claim 14

A process according to claim 7, wherein said β-HIFN solution in step (f) is diafiltered against a 10% aqueous solution of a polyhydric alconol selected from a group consisting of a glycerol, mannitol, sorbitol and dextrose, adjusted to a pH of about 12.

Claim 15

A process according to claim 7, wherein said microorganism is a bacterium.

Claim 16

A process according to claim 13, wherein said bacterium is E. coli.

Claim 17

A process according to claims 7 or 14, wherein said microorganism is contained in a fermentation medium and is concentrated prior to step (a).

Claim 18

A process according to claims 7 or 15, wherein said solubilizing agent is an anionic surfactant.

Claim 19

A process according to claim 7 or 16, wherein said solubilizing agent is an alkali metal, alkyl sulfate or an alkali metal salt of a fatty acd of 10 to 14 carbon chain length.

Claim 20

A process according to claim 17, wherein said solubilizing agent is sodium dodecyl sulfate or sodium laurate.

Claim 21

A process according to claim 18, wherein said solubilizing agent is sodium dodecyl sulfate and the weight ratio of said sodium dodecyl sulfate to β-HIFN is in the range of about 1:1 to about 5:1.

Claim 22

A process according to claim 19, wherein said weight ratio is about 3:1.

Claim 23

A process according to claim 7, wherein said alcohol is 2-butanol.

Claim 24

A process according to claim 7, wherein said isolation in step (d) includes the steps of: contacting the alcohol extract with an aqueous buffer; precipitating the β-HIFN from the mixture by lowering the pH; and separating the precipitated β-HIFN from the supernatant.

Claim 25

A process according to claim 22, wherein the pH of the solution is lowered to about 5 to 7.

Claim 26

A process according to claims 7, 8 or 22, including adding a β-HIFN stabilizer adjusted to a pH of about 12.

Claim 27

A process according to claim 24, wherein said stabilizer is human serum albumin.

Claim 28

A process according to claim 24, wherein said stabilizer is dextrose.

Claim 29

A process according to claim 24, wherein said stabilizer is a mixture of human serum albumin and dextrose.

Claim 30

A process according to claim 25, wherein said human serum albumin is in the concentration range of about 0.5 to 10 (w/v) percent.

Claim 31

In a process for the recovery of β-HIFN from transformed E. coli bacteria comprising concentrating said bacteria, disrupting the cell wall of said bacteria, solubilizing said β-HIFN with SDS, extracting said β-HIFN with 2-butanol and precipitating said β-HIFN by mixing said butanol phase with an aqueous mixture, the improvement comprising: dissolving the precipitated β-HIFN in an appropriate buffer at a pH of about 9; reducing said β-HIFN with dithiothreitol; diafiltering the β-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 β-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 β-HIFN solution to about 7 to 7.5.

Claim 32

In a process for the recovery of β-HIFN from transformed E. coli bacteria comprising concentrating said bacteria, disrupting the cell wall of said bacteria, solubilizing said β-HIFN with SDS, extracting said β-HIFN with 2-butanol and precipitating said β-HIFN by mixing said butanol phase with an aqueous mixture, the improvement comprising: dissolving the precipitated β-HIFN in an appropriate buffer at a pH of about 9; reducing said β-HIFN with dithiothreitol; diafiltering the β-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 β-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 β-HIFN solution to about 7 to 7.5.

Claim 33

The improvement according to claim 29, wherein said diafiltration is carried out at a pH of about 12.

Claim 34

The improvement according to claim 29 or including lyophilizing said β-HIFN.

Claim 35

In a process for the production of an unglycosylated β-HIFN from E. coli genetically transformed to produce said β-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 β-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° 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 β-HIFN; (i) collecting the precipitated β-HIFN by centrifugation; (j) solubilizing the β-HIFN with an aqueous solution of sodium dodecyl sulfate at a SDS to protein ratio of about 3:1; (k) reducing said solubilized β-HIFN with dithiothreitol; (l) purifying the reduced β-HIFN by chromatography; (m) collecting the eluted fraction of the purified β-HIFN; (n) concentrating the purified β-HIFN to a concentration of about 20 mg protein/ml; (o) further purifying the β-HIFN by gel chromatography; and (p) collecting and dissolving the fraction eluate containing the purified β-HIFN in an aqueous buffer; the improvement which comprises the steps of: (q) adjusting the pH of the β-HIFN solution to about 12; (r) diafiltering the solution containing the β-HIFN at a pH of about 12 against pure water or a H 2 O-ethanol mixture adjusted to a pH of about 12; (s) stabilizing the β-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.

Claim 36

In a process for the production of an unglycosylated β-HIFN from E. coli genetically transformed to produce said β-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 β-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° 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 β-HIFN; (i) collecting the precipitated β-HIFN by centrifugation; (j) solubilizing the β-HIFN with an aqueous solution of sodium dodecyl sulfate at a SDS to protein ratio of about 3:1; (k) reducing said solubilized β-HIFN with dithiothreitol; (l) purifying the reduced β-HIFN by chromatography; (m) collecting the eluted fraction of the purified β-HIFN; (n) concentrating the purified β-HIFN to a concentration of about 20 mg protein/ml; (o) further purifying the β-HIFN by gel chromatography; and (p) collecting and dissolving the fraction eluate containing the purified β-HIFN in an aqueous buffer; the improvement which comprises the steps of: (q) adjusting the pH of the β-HIFN solution to about 12; (r) diafiltering the solution containing the β-HIFN at a pH of about 12 against pure water or a H 2 O-ethanol mixture adjusted to a pH of about 12; (s) stabilizing the β-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.