Fc-receptor based affinity chromatography
OwnerRoberto Falkenstein, Hubert Hertenberger +2inventors
InventorsRoberto Falkenstein, Hubert Hertenberger, Petra Rueger, Tilman Schlothauer
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Abstract
Herein is reported the use of an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand in general and, for example, for the determination of the in vivo half-live of an antibody by determining the ratio of the retention times of the antibody and a reference antibody.
Claims16
A method for screening a library of modified antibodies or modified fusion polypeptides of a parent antibody or a parent fusion polypeptide which comprise at least an FcRn binding portion of an Fe-region for those modified antibodies or modified fusion polypeptides that have an altered binding affinity for FcRn compared to the parent antibody or parent fusion polypeptide on an FcRn affinity column comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as ligand with a positive linear pH gradient, wherein the non-covalent complex is conjugated to biotin and immobilization to a solid support is performed via solid support immobilized avidin, and wherein the complex is mono-biotinylated, said method comprising the steps of:
- (a) applying the individual members of the library and the parent antibody or parent fusion polypeptide to the FcRn affinity chromatography column;
- (b) recovering the individual members of the library with a linear positive pH gradient and determining the individual retention times;
- and
- (c) selecting those antibodies or fusion polypeptides that have altered binding affinity for FcRn compared to the parent antibody or parent fusion polypeptide,
- wherein the neonatal Fc receptor has an amino acid sequence of SEQ ID NO:
- 07 with C-terminal His-Avi-tag of SEQ ID NO:
- 08.
A method for identifying antibodies or fusion polypeptides that comprise at least an FcRn-binding portion of an Fc-region which exhibit altered binding to the neonatal Fc receptor on an Ran affinity column comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as ligand with a positive linear pH gradient, said method comprising the steps of:
- (a) applying a variant antibody comprising an at least an FcRn-binding portion of an Fc-region, wherein one or more of the following amino acid residues according to the EU numbering of Kabat are altered F243, P244, P245 P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439, and S440, and the parent antibody to the FcRn affinity chromatography column;
- (b) recovering the variant and the parent antibody with a positive linear pH gradient and determining the individual retention times;
- and
- (c) identifying the variant antibody to have altered binding affinity for FcRn compared to the parent antibody if the variant antibody has a different retention time,
- wherein the neonatal Fc receptor has an amino acid sequence of SEQ ID NO:
- 07 with C-terminal His-Avi-tag of SEQ ID NO:
- 08.
Dependencies are inferred from the claim text.