US 11,814,409 B2
Fc-receptor based affinity chromatography
Roberto Falkenstein, Munich (DE); Hubert Hertenberger, Weilheim (DE); Petra Rueger, Penzberg (DE); and Tilman Schlothauer, Penzberg (DE)
Filed by Hoffmann-La Roche Inc., Nutley, NJ (US)
Filed on Jan. 25, 2019, as Appl. No. 16/258,294.
Application 16/258,294 is a continuation of application No. 14/378,808, abandoned, previously published as PCT/EP2013/052932, filed on Feb. 14, 2013.
Claims priority of application No. 12155630 (EP), filed on Feb. 15, 2012.
Prior Publication US 2019/0276492 A1, Sep. 12, 2019
Int. Cl. C07K 1/22 (2006.01); B01D 15/16 (2006.01); B01D 15/38 (2006.01); B01J 20/289 (2006.01); B01J 20/32 (2006.01); C07K 16/06 (2006.01); C07K 16/28 (2006.01); B01J 39/26 (2006.01); G01N 33/68 (2006.01)
CPC C07K 1/22 (2013.01) [B01D 15/168 (2013.01); B01D 15/3809 (2013.01); B01J 20/289 (2013.01); B01J 20/3204 (2013.01); B01J 20/3206 (2013.01); B01J 20/3219 (2013.01); B01J 20/3274 (2013.01); B01J 39/26 (2013.01); C07K 16/065 (2013.01); C07K 16/2866 (2013.01); G01N 33/6854 (2013.01)] 16 Claims
 
1. 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.