Fc-receptor based affinity chromatography

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.

1 . Method of using an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as an affinity chromatography ligand in an affinity chromatography with a positive linear pH gradient. 
     
     
         2 . The method according to  claim 1 , wherein the affinity chromatography with a positive linear pH gradient for separating antibodies or fusion polypeptides comprises at least an Fc-region. 
     
     
         3 . The method according to  claim 1 , characterized in that the neonatal Fc receptor and the beta-2-microglobulin are independently of each other of human origin, of mouse origin, of cynomolgus origin, of rat origin, or of rabbit origin. 
     
     
         4 - 37 . (canceled) 
     
     
         38 . The method according to  claim 3 , wherein the neonatal Fc receptor and the beta-2-microglobulin are independent of each other of human origin, of mouse origin, of cynomolgus origin, of rat origin, or of rabbit origin. 
     
     
         39 . The method according to  claim 38 , wherein the beta-2-microglobulin is from the same species as the neonatal Fc receptor. 
     
     
         40 . The method according to  claim 1 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each independently of each other with 0 to 10 amino acid residue modifications. 
     
     
         41 . The method according to  claim 2 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each independently of each other with 0 to 10 amino acid residue modifications. 
     
     
         42 . The method according to  claim 3 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each independently of each other with 0 to 10 amino acid residue modifications. 
     
     
         43 . The method according to  claim 40 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each is independent of each other with 0 to 10 amino acid residue modifications. 
     
     
         44 . The method according to  claim 41 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each is independent of each other with 0 to 10 amino acid residue modifications. 
     
     
         45 . The method according to  claim 42 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each is independent of each other with 0 to 10 amino acid residue modifications. 
     
     
         46 . The method according to  claim 1 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         47 . The method according to  claim 2 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         48 . The method according to  claim 3 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         49 . The method according to  claim 40 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         50 . The method according to  claim 41 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         51 . The method according to  claim 42 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         52 . The method according to  claim 43 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         53 . The method according to  claim 44 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         54 . The use according to  claim 45 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 
     
     
         55 . The method according to  claim 47 , wherein the solid phase is a chromatography material. 
     
     
         56 . The method according to  claim 48 , wherein the solid phase is a chromatography material. 
     
     
         57 . The method according to  claim 49 , wherein the solid phase is a chromatography material. 
     
     
         58 . The method according to  claim 50 , wherein the solid phase is a chromatography material. 
     
     
         59 . The method according to  claim 51 , wherein the solid phase is a chromatography material. 
     
     
         60 . The method according to  claim 52 , wherein the solid phase is a chromatography material. 
     
     
         61 . The method according to  claim 53 , wherein the solid phase is a chromatography material. 
     
     
         62 . The method according to  claim 54 , wherein the solid phase is a chromatography material. 
     
     
         63 . The method according to  claim 1 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         64 . The method according to  claim 2 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         65 . The method according to  claim 3 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         66 . The method according to  claim 40 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         67 . The method according to  claim 41 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         68 . The method according to  claim 42 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         69 . The method according to  claim 43 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         70 . The method according to  claim 44 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         71 . The method according to  claim 45 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         72 . The method according to  claim 46 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         73 . The method according to  claim 47 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         74 . The method according to  claim 48 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         75 . The method according to  claim 49 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         76 . The method according to  claim 50 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         77 . The method according to  claim 51 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         78 . The method according to  claim 52 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         79 . The method according to  claim 1 , wherein the pH gradient is from a first pH value to a second pH value whereby the first pH value is from about pH 3.5 to about pH 7.5 and the second pH value is from about pH 6.0 to about pH 9.5, preferably wherein the first pH value is about pH 5.5 and the second pH value is about pH 8.8. 
     
     
         80 . The method according to  claim 1 , wherein the method is selected from 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; is for determining methionine oxidation of an antibody; is for determining the oligomerization level of an antibody; or is 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 Fc-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. 
     
     
         81 . The method according to  claim 1 , wherein the method is selected from the use 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; the use is for the removal of half antibodies from IgG preparations; the use is for the removal of antibody aggregates and antibody oligomers from IgG preparations; the antibody is a monospecific antibody or antibody fragment of fusion polypeptide, or a bispecific antibody or antibody fragment of fusion polypeptide, or a trispecific antibody or antibody fragment of fusion polypeptide, or a tetraspecific antibody or antibody fragment of fusion polypeptide. 
     
     
         82 . Method of using an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand in an affinity chromatography with a negative linear pH gradient. 
     
     
         83 . The method according to  claim 82 , wherein an affinity chromatography with a negative linear pH gradient for separating antibodies or fusion polypeptides comprises at least an Fc-region. 
     
     
         84 . The method according to  claim 82 , wherein the neonatal Fc receptor and the beta-2-microglobulin are independently of each other of human origin, or of mouse origin, or of cynomolgus origin, or of rat origin, or of rabbit origin. 
     
     
         85 . The method according to  claim 84 , wherein the beta-2-microglobulin is from the same species as the neonatal Fc receptor. 
     
     
         86 . The method according to  claim 85 , wherein the neonatal Fc receptor and the beta-2-microglobulin are the human wild-type neonatal Fc receptor and the human wild-type beta-2-microglobulin each independently of each other with 0 to 10 amino acid residue modifications. 
     
     
         87 . The method according to  claim 82 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase that is chromatography material. 
     
     
         88 . The method according to  claim 82 , wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 
     
     
         89 . The method according to  claim 82 , wherein the pH gradient is from a first pH value to a second pH value whereby the first pH value is from about pH 7.0 to about pH 8.5 and the second pH value is from about pH 5.5 to about pH 6.9; preferably wherein the first pH value is about pH 7.4 and the second pH value is about pH 6.0. 
     
     
         90 . The method according to  claim 82 , wherein the use is selected from the use 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; the use is for determining methionine oxidation of an antibody; the use is for determining the oligomerization level of an antibody; the use is 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 Fc-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; the use is 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; the use is for the removal of half antibodies from IgG preparations; or the use is for the removal of antibody aggregates and antibody oligomers from IgG preparations. 
     
     
         91 . The method according to  claim 90 , wherein the antibody is a monospecific antibody or antibody fragment of fusion polypeptide, or a bispecific antibody or antibody fragment of fusion polypeptide, or a trispecific antibody or antibody fragment of fusion polypeptide, or a tetraspecific antibody or antibody fragment of fusion polypeptide. 
     
     
         92 . An Fc-region variant of human IgG1 isotype in which the amino acid at position 252 is changed from methionine to histidine and the amino acid at position 428 is changed from methionine to glutamic acid.