Patent ID: 11932704
Assignee: SANOFI
Field: Pharmaceuticals (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 0:
1. A method of purifying a trispecific binding protein produced by a host cell, comprising:
(a) producing in a host cell a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL  [I], and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3  [II], and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3  [III], and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

VL3-CL  [IV], wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and
L1, L2, L3 and L4 are each independently amino acid linkers or zero amino acids in length;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair of the binding protein, and wherein VH1 and VL1 form a binding pair and a first antigen binding site, VH2 and VL2 form a binding pair and a second antigen binding site, and VH3 and VL3 form a binding pair and a third antigen binding site,
wherein only one of the CH3 domain of the second polypeptide chain and the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F;
(b) contacting the binding protein produced in (a) with Protein A;
(c) eluting the binding protein from Protein A under conditions suitable for isolating the binding protein away from binding proteins comprising either 0 or 2 CH3 domains comprising the amino acid substitutions H435R and Y436F and detecting the isolated binding protein using hydrophobic interaction chromatography (HIC).