Anionic exchange-hydrophobic mixed mode chromatography resins

Patent metadata

Field	Value
Publication number	US-2021069692-A1
Application number	US-202017012108-A
Country	US
Kind code	A1
Filing date	Sep 4, 2020
Priority date	Sep 5, 2019
Publication date	Mar 11, 2021
Grant date	—

Abstract

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands and methods of using such resins are provided.

First claim

1 . A chromatography matrix covalently linked to a ligand having the following formula:
Chromatography matrix-(X)—N(R 1 )—(R 2 -L) n -Ar
or an anionic salt thereof, wherein:
X is a spacer;
R 1 is hydrogen or C 1 to C 6 alkyl optionally substituted with an —OH;
R 2 is C 2 to C 6 alkyl or C 4 to C 6 cycloalkyl;
L is NR 4 , O, or S;
n=1 or 2; and
Ar is a 6-10 membered ring and:
if Ar is aryl, the aryl is optionally substituted with up to five C 1 to C 3 unsubstituted alkyl, C 3 to C 6 branched alkyl, unsubstituted aryl, or fluorine groups; or
if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four unsubstituted alkyl groups,
with the proviso that when R 1 is hydrogen, R 2 is C 2 alkyl, L is NR 4 or O, and n is 1, Ar is not phenyl.

2 . The chromatography matrix of claim 1 , wherein:
X is selected from the group consisting of —O—CH 2 —, —O—CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH(CH 2 —OH)—(O—CH 2 —CH(OH)—CH 2 ) 2 —, —O—CH 2 —CH 2 —CH(CH 2 —OH)—(O—CH 2 —CH 2 —CH(OH)—CH 2 ) 2 —, —O—CH 2 —CH(OH)—CH 2 —, —O—CH 2 —CH 2 —CH(OH)—CH 2 —CH 2 —, —O—CH 2 —CH(OH)—CH 2 —O—CH 2 —CH 2 —CH 2 —CH 2 —O—CH 2 —CH(OH)—CH 2 —, and —CO—NH—C(CH 3 ) 2 —CO—; R 1 is hydrogen or C 1 to C 3 alkyl; R 2 is C 2 to C 4 alkyl; L is O; n=1; and Ar is a 6 membered ring and:
if Ar is aryl, the aryl is optionally substituted with up to four C 1 to C 2 unsubstituted alkyl, C 3 or C 4 branched alkyl, or fluorine groups; or
if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups,
with the proviso that when R 1 is hydrogen, R 2 is C 2 alkyl, and n is 1, Ar is not phenyl.

3 . The chromatography matrix of claim 2 , wherein:
X is selected from the group consisting of —O—CH 2 —, —O—CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —CH 2 —, and —O—CH 2 —CH(OH)—CH 2 —; R 1 is hydrogen or C 1 to C 2 alkyl; R 2 is C 2 to C 3 alkyl; L is O; n=1; and Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C 1 to C 2 unsubstituted alkyl, with the proviso that when R 1 is hydrogen, R 2 is C 2 alkyl, and n is 1, Ar is not phenyl.

4 . The chromatography resin of claim 1 , wherein Ar is phenyl substituted with one or two C 1 to C 2 unsubstituted alkyl at the para or meta position relative to Chromatography matrix-(X)—N(R 1 )—(R 2 -L) n -.

5 . The chromatography resin of claim 1 , wherein —(X)—N(R 1 )—(R 2 -L) n -Ar is any one of the ligands of Table 1.

6 . A chromatography resin having the following formula:
Chromatography matrix-(X)—N—[(R 2 -L) n -Ar] 2
or an anionic salt thereof, wherein:
X is a spacer;
R 2 is C 2 to C 6 alkyl or C 4 to C 6 cycloalkyl;
L is NR 4 , O, or S;
n=1 or 2; and
Ar is a 6-10 membered ring and:
if Ar is aryl, the aryl is optionally substituted with up to five C 1 to C 3 unsubstituted alkyl, C 3 to C 6 branched alkyl, unsubstituted aryl, or fluorine groups; or
if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four unsubstituted alkyl groups.

7 . The chromatography matrix of claim 6 , wherein:
X is selected from the group consisting of —O—CH 2 —, —O—CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH(CH 2 —OH)—(O—CH 2 —CH(OH)—CH 2 ) 2 —, —O—CH 2 —CH 2 —CH(CH 2 —OH)—(O—CH 2 —CH 2 —CH(OH)—CH 2 ) 2 —, —O—CH 2 —CH(OH)—CH 2 —, —O—CH 2 —CH 2 —CH(OH)—CH 2 —CH 2 —, —O—CH 2 —CH(OH)—CH 2 —O—CH 2 —CH 2 —CH 2 —CH 2 —O—CH 2 —CH(OH)—CH 2 —, and —CO—NH—C(CH 3 ) 2 —CO—; R 2 is C 2 to C 4 alkyl; L is O; n=1; and Ar is a 6 membered ring and:
if Ar is aryl, the aryl is optionally substituted with—up to four C 1 to C 2 unsubstituted alkyl, C 3 or C 4 branched alkyl, or fluorine groups; or
if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups.

8 . The chromatography matrix of claim 7 , wherein:
X is selected from the group consisting of —O—CH 2 —, —O—CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —, —O—CH 2 —CH 2 —CH 2 —CH 2 —, and —O—CH 2 —CH(OH)—CH 2 —; R 1 is hydrogen, C 1 , or C 2 alkyl; R 2 is C 2 or C 3 alkyl; L is O; n=1; and Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C 1 to C 2 unsubstituted alkyl.

9 . The chromatography resin of claim 6 , wherein Ar is phenyl substituted with one or two C 1 to C 2 unsubstituted alkyl at the para or meta position relative to —(R 2 -L) n -.

10 . The chromatography resin of claim 6 , wherein —(X)—N—[(R 2 -L) n -Ar] 2 is any one of the ligands of Table 2.

11 . The chromatography resin of claim 1 , wherein Ar is heteroaryl and a heteroatom in the heteroaryl is N.

12 . The chromatography resin of claim 1 , wherein the anionic salt is a hydrochloride salt, a phosphate salt, or a sulfate salt.

13 . The chromatography resin of claim 1 , wherein X is attached to chromatography matrix via an amine, ether or amide bond.

14 . A chromatography resin prepared by reacting any one of the ligands of Table 1 with a chromatography matrix by any one of reductive amination, epoxide chemistry, or azalactone chemistry.

15 . The chromatography resin of claim 14 , wherein the chromatography matrix comprises an aldehyde group and any one of the ligands of Table 1 is reacted with the chromatography matrix by reductive amination.

16 . The chromatography resin of claim 14 , wherein the chromatography matrix comprises an epoxide group and any one of the ligands of Table 1 is reacted with the chromatography matrix by epoxide chemistry.

17 . The chromatography resin of claim 14 , wherein prior to reacting the chromatography matrix with the ligand, the chromatography matrix is reacted with allylglydicylether and bromine; 1,4-butanedioldiglycidyl; or epichlorohydrin.

18 . A chromatography resin prepared by reacting any one of the ligands of Table 2 with a chromatography matrix by epoxide chemistry.

19 . A method of purifying a biomolecule, the method comprising:
contacting a sample comprising the biomolecule to a chromatography resin of claim 1 , thereby separating the biomolecule from a contaminant; and collecting a purified biomolecule.

20 . The method of claim 19 , wherein the purified biomolecule is a protein.

21 . The method of claim 20 , wherein the contacting step comprises immobilizing the protein to the chromatography matrix and the collecting step comprises eluting the protein from the chromatography matrix.

22 . The method of claim 21 , wherein the protein is eluted by a step comprising reducing a pH of a solution in contact with the ligand from about 7-9 to about 4-6.

23 . The method of claim 20 , wherein the contacting step comprises flowing the protein through the chromatography matrix and the collecting step comprises collecting the protein in the flow through.

Assignees

• Bio Rad Laboratories

Inventors

• Belisle Christopher
• He Xuemei
• Chen Hong
• Xu Yueping
• Liao Jiali

Classifications

• B01D15/3847

  Multimodal interactions · CPC title

• B01J47/014

  in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds · CPC title

• B01J20/3293

  Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers · CPC title

• B01J20/3253

  comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures · CPC title

• B01J20/321

  consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds · CPC title