Heterodimeric human IgG1 polypeptides with isoelectric point modifications
The invention relates generally to compositions and methods for purifying the desired species from a mixture of desired heterodimer and contaminating homodimer immunoglobulin variants by modifying the isoelectric point(s) of the individual chains.
1 . A method of separating heterodimeric antibodies from homodimeric antibodies, the method comprising:
(a) providing a mixture comprising heterodimeric antibodies and homodimeric antibodies,
wherein the heterodimeric antibodies comprise a first human IgG1 heavy chain constant region and a second human IgG1 heavy chain constant region, wherein the first human IgG1 heavy chain constant region comprises the amino acid modifications L351K and T366K, wherein numbering is according to the EU index, wherein the first human IgG1 constant region and the second human IgG1 constant region have at least 95% sequence identity to SEQ ID NO: 2,
wherein the homodimeric antibodies comprise a first homodimeric antibody comprising two of the first human IgG1 heavy chain constant regions and a second homodimeric antibody comprising two of the second human IgG1 heavy chain constant regions,
wherein the difference in the isoelectric point between each of the heterodimeric antibodies and the first homodimeric antibody and each of the heterodimeric antibodies and the second homodimeric antibody is at least 0.2 logs, and
(b) separating the heterodimeric antibodies from the first and second homodimeric antibodies by ion exchange chromatography based on the difference in the isoelectric points between the heterodimeric antibodies and the homodimeric antibodies.
2 . The method of claim 1 , wherein the difference in the isoelectric point is at least 0.3 logs.
3 . The method of claim 1 , wherein the difference in the isoelectric point is at least 0.4 logs.
4 . The method of claim 1 , wherein the difference in the isoelectric point is at least 0.5 logs.
5 . The method of claim 1 , wherein the pl of the heterodimeric antibodies is a pH at which the heterodimeric antibodies have an overall net charge of zero,
wherein the net charge is calculated by the following formula:
q
protein
(
pH
)
=
∑
i
=
H
,
K
,
R
,
N
termini
N
i
1
+
1
0
p
H
-
p
K
i
-
∑
i
=
D
,
E
,
C
,
Y
,
C
termini
N
i
1
+
1
0
pK
i
-
p
H
wherein q protein (pH) is the net charge of one of the heterodimeric antibodies at the pH, N i is the number of amino acids i, or N- or C-termini present in the heterodimeric antibody, and pK i is the pK of amino acid i, or N- or C-termini.
6 . The method of claim 1 , wherein the pI of the homodimeric antibodies is a pH at which the homodimeric antibodies have an overall net charge of zero,
wherein the net charge is calculated by the following formula:
q
protein
(
pH
)
=
∑
i
=
H
,
K
,
R
,
N
termini
N
i
1
+
1
0
p
H
-
p
K
i
-
∑
i
=
D
,
E
,
C
,
Y
,
C
termini
N
i
1
+
1
0
pK
i
-
p
H
wherein q protein (pH) is the net charge of one of the homodimeric antibodies at the pH, N i is the number of amino acids i, or N- or C-termini present in the homodimeric antibody, and pK i is the pK of amino acid i, or N- or C-termini.
7 . The method of claim 1 , wherein the method further comprises subjecting the mixture to protein A chromatography prior to step b).