Anionic exchange-hydrophobic mixed mode chromatography resin
Chromatography resins having anionic exchange-hydrophobic mixed mode ligands, that are useful for purifying target biomolecules using anionic exchange (i.e., where the ligand is positively charged) and hydrophobic mixed mode chromatography. The chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody-drug conjugates, or antibody derivatives including, but not limited to, antibody fragments and antibody fusions) from a sample, and have been found to be useful in purifying monomeric target biomolecules from aggregate target biomolecules. In an embodiment, the chromatography resins are useful for separating antibodies from one or more components (e.g., contaminants) in the sample.
1. A chromatography resin having the following formula:
Chromatography matrix-(X)—N(R 1 )(R 2 )—(R 3 -L) n -Ar
or an anionic salt thereof,
wherein:
X is a spacer 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 and R 2 are each independently C 1 to C 6 alkyl optionally substituted with —OH;
R 3 is C 2 to C 6 alkyl or C 4 to C 6 cycloalkyl;
L is O;
n is 1 or 2; and
Ar is a phenyl or naphthyl group optionally substituted with up to 5: C 1 to C 3 unsubstituted alkyl, C 3 to C 6 branched alkyl, unsubstituted aryl, or flourine groups; or
a pyridal group optionally substituted with up to four alkyl groups.
2. The chromatography matrix of claim 1 , wherein:
R 1 and R 2 are each independently C 1 to C 3 alkyl;
R 3 is C 2 to C 4 alkyl;
L is O;
n is 1; and
Ar is a phenyl or naphthyl group optionally substituted with up to four C 1 to C 2 unsubstituted alkyl, C 3 to C 4 branched alkyl, or flourine groups; or
a pyridal group optionally substituted with up to three alkyl groups.
3. The chromatography resin 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 —, and —O—CH 2 —CH(CH 2 —OH)—(O—CH 2 —CH(OH)—CH 2 ) 2 —;
R 1 and R 2 are each independently C 1 or C 2 alkyl;
R 3 is C 2 or C 3 alkyl;
L is O;
n is 1; and
Ar is phenyl, naphthyl, or pyridyl optionally substituted with up to three C 1 to C 2 unsubstituted alkyl.
4. The chromatography resin of claim 1 , wherein Ar is phenyl optionally substituted with one or two C 1 to C 2 unsubstituted alkyl.
5. The chromatography resin of claim 1 , wherein the chromatography resin has one of the following structures, the sphere representing the chromatography matrix and the spacer X:
Resin
Number
13
94
34-1
34-2
86-1
86-2
97-1
97-2
98-1
32-1
91-1
91-2
91-3
91-4
91-5
91-6
32-2
CB216
50-1
71-2
71-4
71-3
71-1
6. The chromatography resin of claim 1 , wherein Ar is unsubstituted.
7. The chromatography resin of claim 1 , wherein Ar is a pyridyl group optionally substituted with up to four alkyl groups.
8. The chromatography resin of claim 1 , wherein the anionic salt is a hydrochloride salt or a sulfate salt.
9. The chromatography resin of claim 1 , wherein X is attached to chromatography matrix via an amine, ether or amide bond.
10. A chromatography resin having the following formula:
Chromatography matrix-(X)—N(R 1 )—[(R 3 -L) n -Ar] 2
or an anionic salt thereof,
wherein:
X is a spacer 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 C 1 to C 6 alkyl optionally substituted with —OH;
R 3 is C 2 to C 6 alkyl or C 4 to C 6 cycloalkyl;
L is O;
n is 1; and
Ar is a phenyl or naphthyl group optionally substituted with up to 5: C 1 to C 3 unsubstituted alkyl, C 3 to C 6 branched alkyl, unsubstituted aryl, or flourine groups; or
a pyridal group optionally substituted with up to four alkyl groups.
11. The chromatography resin of claim 10 , 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 —, —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 C 1 to C 3 alkyl;
R 3 is C 2 to C 4 alkyl;
L is O;
n is 1; and
L is O;
n is 1; and
Ar is a phenyl or naphthyl group optionally substituted with up to four C 1 to C 2 unsubstituted alkyl, C 3 to C 4 branched alkyl, or flourine groups; or
a pyridal group optionally substituted with up to three alkyl groups.
12. The chromatography resin of claim 11 , 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(CH 2 —OH)—(O—CH 2 —CH(OH)—CH 2 ) 2 —;
R 1 is C 1 or C 2 alkyl;
R 3 is C 2 or C 3 alkyl; and
L is O;
n is 1; and
Ar is phenyl, naphthyl, or pyridyl optionally substituted with up to three C 1 to C 2 unsubstituted alkyl.
13. The chromatography resin of claim 10 , wherein Ar is phenyl optionally substituted with one or two C 1 to C 2 unsubstituted alkyl.
14. The chromatography resin of claim 10 , wherein the chromatography resin has one of the following structures, the sphere representing the chromatography matrix and the spacer X: