Purification Process for Anitbody Fragments Using Derivatized Triazines as Affinity Ligands
A process for the separation of a fragment antibody from a medium is provided. The process comprises contacting the medium comprising the fragment antibody with a synthetic affinity ligand attached to a support matrix under conditions whereby the fragment antibody binds to the synthetic affinity ligand. The synthetic affinity ligand has the formula (I): wherein Q represent an attachment to a solid support matrix, optionally via a spacer group; A and B are each independently —Y-phenyl or —Y-naphthyl groups substituted with one or more substituents capable of hydrogen bonding, preferably one or more of —OH, —SH or —CO 2 H groups; each Y independently represents —NR—, —O— or —S—; and each R independently represents H or a C 1-4 alkyl group.
1 - 13 . (canceled)
14 . A process for the separation of a fragment antibody from a medium which comprises contacting the medium comprising the fragment antibody with a synthetic affinity ligand attached to a support matrix under conditions whereby the fragment antibody binds to the synthetic affinity ligand, wherein the synthetic affinity ligand has the formula:
wherein
Q represents an attachment to a solid support matrix, optionally via a spacer group;
A and B are each independently —Y-phenyl or —Y-naphthyl groups substituted with one or more substituents capable of hydrogen bonding;
each Y independently represents —NR—, —O— or —S—; and
each R independently represents H or a C 1-4 alkyl group.
15 . A process according to claim 14 , wherein the substituents capable of hydrogen bonding are independently selected from —OH, —SH or —CO 2 H groups.
16 . A process according to claim 15 , wherein the synthetic affinity ligand attached to a support matrix is selected from compounds of formula:
wherein Q represents an attachment to a solid support matrix, optionally via a spacer group.
17 . A process according to claim 16 , wherein Q represents a spacer group selected from the group consisting of groups of formula —NH—(CH 2 ) n NH-G;
—NH—(CH 2 ) n O-G; —O—(CH 2 ) n O-G; —O—(CH 2 CH 2 ) n O-G; —NH—(CH 2 ) n O-G; and
—NH—(CH 2 ) n NH—(CH 2 ) x O-G
wherein n is a positive integer up to 12;
x is from 1 to 6; and
G is a solid support matrix.
18 . A process according to claim 17 , wherein n is from 2 to 6
19 . A process according to claim 14 , wherein the fragment antibody is bound to the ligand at a pH of from 6 to 8.
20 . A process according to claim 19 , wherein the fragment antibody is bound to the ligand at a pH of from 6.5 to 7.5.
21 . A process according to claim 14 , wherein the fragment antibody is bound to the ligand using a solution having an ionic strength of less than 50 mS/cm.
22 . A process according to claim 21 , wherein the fragment antibody is bound to the ligand using a solution having an ionic strength of between 10 and 40 mS/cm.
23 . A process according to claim 14 , wherein the fragment antibody is a Fab; an scFv; a single domain, or a fragment thereof; a bis scFv, Fab 2 , Fab 3 , minibody, diabody, triabody, tetrabody or tandab.
24 . A process according to claim 23 , wherein the fragment antibody is a single domain from a variable heavy chain or a fragment thereof, or a singe domain from a variable light chain or a fragment thereof.
25 . A process for the preparation of a fragment antibody comprising:
(a) preparing a fragment antibody by recombinant technology to produce a medium comprising fragment antibody;
(b) separation of the fragment antibody from the medium by a process comprising contacting the medium comprising the fragment antibody with a synthetic affinity ligand attached to a support matrix under conditions whereby the fragment antibody binds to the synthetic affinity ligand, wherein the synthetic affinity ligand has the formula:
wherein
Q represents an attachment to a solid support matrix, optionally via a spacer group;
A and B are each independently —Y-phenyl or —Y-naphthyl groups substituted with one or more substituents capable of hydrogen bonding;
each Y independently represents —NR—, —O— or —S—; and
each R independently represents H or a C 1-4 alkyl group ; and
(c) releasing the fragment antibody from the affinity ligand.
26 . A process according to claim 25 , where the fragment antibody is produced by expression of an E. coli or Pichia pastoris host cell.
27 . A process according to claim 25 , wherein the substituents capable of hydrogen bonding are independently selected from —OH, —SH or —CO 2 H groups.
28 . A process according to claim 27 , wherein the synthetic affinity ligand attached to a support matrix is selected from compounds of formula:
wherein Q represents an attachment to a solid support matrix, optionally via a spacer group.
29 . A process according to claim 28 wherein Q represents a spacer group selected from the group consisting of groups of formula —NH—(CH 2 ) n NH-G;
—NH—(CH 2 ) n O-G; —O—(CH 2 ) n O-G; —O—(CH 2 CH 2 ) n O-G; —NH—(CH 2 ) n O-G; and
—NH—(CH 2 ) n NH—(CH 2 ) x O-G
wherein n is a positive integer up to 12;
x is from 1 to 6; and
G is a solid support matrix.
30 . A process according to claim 29 , where n is from 2 to 6
31 . A process according to claim 25 , wherein the fragment antibody is bound to the ligand at a pH of from 6 to 8.
32 . A process according to claim 31 , wherein the fragment antibody is bound to the ligand at a pH of from 6.5 to 7.5.
33 . A process according to claim 25 , wherein the fragment antibody is bound to the ligand using a solution having an ionic strength of less than 50 mS/cm.
34 . A process according to claim 33 , wherein the fragment antibody is bound to the ligand using a solution having an ionic strength of between 10 and 40 mS/cm.
35 . A process according to claim 25 , wherein the fragment antibody is a Fab; an scFv; a single domain, or a fragment thereof; a bis scFv, Fab 2 , Fab 3 , minibody, diabody, triabody, tetrabody or tandab.
36 . A process according to claim 35 , wherein the fragment antibody is a single domain from a variable heavy chain or a fragment thereof, or a singe domain from a variable light chain or a fragment thereof.
37 . A process for the preparation of a fragment antibody comprising:
(a) expression of the fragment antibody in an E. coli or Pichia pastoris host cell to produce a medium comprising fragment antibody;
(b) separation of the fragment antibody from the medium by a process comprising contacting the medium comprising the fragment antibody with a synthetic affinity ligand attached to a support matrix at a pH of from 6.5 to 7.5 in a solution having an ionic strength of between 10 and 40 mS/cm whereby the fragment antibody binds to the synthetic affinity ligand, wherein the synthetic affinity ligand has the formula:
wherein Q represents a spacer group selected from the group consisting of groups of formula —NH—(CH 2 ) n NH-G;
—NH—(CH 2 ) n O-G; —O—(CH 2 ) n O-G; —O—(CH 2 CH 2 ) n O-G; —NH—(CH 2 ) n O-G; and
—NH—(CH 2 ) n NH—(CH 2 ) x O-G
wherein n is from 2 to 6;
x is from 1 to 6; and
G is a solid support matrix; and
(c) releasing the fragment antibody from the affinity ligand.
38 . A process according to claim 37 , wherein the fragment antibody is a Fab; an scFv; a single domain, or a fragment thereof; a bis scFv, Fab 2 , Fab 3 , minibody, diabody, triabody, tetrabody or tandab.