Protein purification in the presence of nonionic organic polymers and electropositive surfaces

Patent metadata

Field	Value
Publication number	US-2016009759-A1
Application number	US-201414769101-A
Country	US
Kind code	A1
Filing date	Feb 26, 2014
Priority date	Feb 26, 2013
Publication date	Jan 14, 2016
Grant date	—

Abstract

A method of purifying a desired protein from a preparation includes (a) providing the preparation in a form having less than about 5% of chromatin residing in an original production medium, (b) contacting the preparation with a nonionic organic polymer and a salt, a concentration of nonionic organic polymer being sufficient to precipitate the desired protein or cause its accretion on a hydrophilic surface, or maintain it in a precipitated state or accreted on the hydrophilic surface, the salt concentration being sufficient to produce greater than physiological conductivity, and (c) contacting the preparation with at least one electropositive surface, optionally in the presence of a salt concentration sufficient to produce greater than physiological conductivity, the desired protein does not substantially adsorb to the at least one electropositive surface while not preventing adsorption of acidic contaminants to the at least one electropositive surface.

First claim

1 .- 21 . (canceled)

22 . A method of purifying a desired protein from a preparation comprising:
(a) providing the preparation in a form having less than about 5% of chromatin residing in an original production medium; (b) contacting the preparation with a nonionic organic polymer and a salt, wherein a concentration of the nonionic organic polymer is sufficient to precipitate the desired protein or cause its accretion on a hydrophilic surface, or maintain it in a precipitated state or accreted on the hydrophilic surface, the salt having a concentration being sufficient to produce greater than physiological conductivity; and (c) contacting the preparation with at least one electropositive surface, optionally in the presence of a salt concentration sufficient to produce greater than physiological conductivity, whereby the desired protein does not substantially adsorb to the at least one electropositive surface, wherein the contacting does not prevent adsorption of acidic contaminants to the at least one electropositive surface.

23 . The method of claim 22 , further comprising (d) contacting the preparation with at least one nonionic hydrophilic surface.

24 . The method of claim 22 , wherein the salt concentration of step (b) is the same as a concentration of the salt in the preparation of step (a).

25 . The method of claim 22 , further comprising retaining the precipitated desired protein on a porous membrane while soluble contaminants are eliminated by passage therethrough.

26 . The method of claim 22 , further comprising retaining the precipitated desired protein on a microporous membrane that is substantially inert while soluble contaminants are eliminated by passage there through, independently from the salt concentration.

27 . The method of claim 22 , further comprising retaining the precipitated desired protein on a microporous membrane that is electropositive while soluble contaminants pass there through when the conductivity is greater than physiological conductivity.

28 . The method of claim 22 , wherein the method is conducted in a single integrated apparatus.

29 . The method of claim 22 , wherein the nonionic organic polymer is polyethylene glycol (PEG).

30 . The method of claim 22 , wherein the average polymer size of the nonionic organic polymer is in the range of one from the group consisting of (a) from about 1,500 Daltons to about 15,000 Daltons, (b) from about 2,000 Daltons to about 12,000 Daltons (c) from about 3,000 Daltons to about 10,000 Daltons, (d) from about 4,000 Daltons to about 8,000 Daltons, and (e) from about 5,000 Daltons to about 6,000 Daltons.

31 . The method of claim 22 , wherein the conductivity is at least 1 mS/cm greater than physiological conductivity.

32 . The method of claim 22 , wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, sodium acetate, potassium acetate, sodium thiocyanate, potassium thiocyanate, guanidinium hydrochloride, and combinations thereof.

33 . The method of claim 32 , wherein the salt comprises sodium chloride at a concentration selected from the group consisting of (a) from about 0.5 M to about 1.5 M, (b) from about 2.0 M to about 3.0 M, and intermediate ranges thereof.

34 . The method of claim 22 , wherein the at least one electropositive surface comprises a membrane with pores of an average size selected from the group consisting of (a) about 100 nm, (b) about 220 nm, (c) about 450 nm, (d) about 1 micron, (e) about 2 microns, and intermediate values thereof.

35 . The method of claim 22 , wherein the at least one electropositive solid comprises a plurality of particles.

36 . The method of claim 22 , wherein the at least one electropositive surface is part of a chromatography device selected from the group comprising, a monolith-based chromatography device, a membrane-based chromatography device, a particle-based chromatography device, a macroreticulate skeleton supporting a hydrogel-based device, and combinations thereof.

37 . The method of claim 22 , wherein the salt concentration is selected from the group comprising a zero amount, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, and intermediate concentrations thereof.

38 . The method of claim 22 , wherein a pH during step (a) is in a range selected from the group consisting of (a) from about 8 to about 9, (b) from about 8 to about 8.5, (c) from about 7.5 to about 8.5, (d) from about 7.25 to about 8.25, (e) from about 7.0 to about 8.0, (f) from about 6 to about 7, and intermediate pH ranges thereof.

39 . The method of claim 22 , wherein a pH during step (b) is in a range selected from the group consisting of (a) from about 5 to about 9, (b) from about 6 to about 8, (c) from about 6.5 to about 7.5, (d) from about 7.5 to about 8.5, and intermediate pH ranges thereof.

40 . The method of claim 22 , wherein the at least one hydrophilic surface or the at least one electropositive surface comprise one selected from the group consisting of a membrane, a monolith, and a plurality of particles, wherein the plurality particles are optionally magnetic.

41 . The method of claim 22 , wherein the preparation comprises one selected from the group consisting of a cell culture medium, an extract from cultured organisms, and a bodily fluid.

Assignees

• Agency Science Tech & Res

Inventors

• Gagnon Peter Stanley

Classifications

• B01D15/3847

  Multimodal interactions · CPC title

• C07K1/30 Primary

  by precipitation · CPC title

• C07K1/303

  by salting out · CPC title

• B01D15/305

  Hydrophilic interaction chromatography [HILIC] · CPC title

• B01D15/363

  Anion-exchange · CPC title