Patent ID: 9163227
Filing Date: 2015-10-20
Classification: A61K,A61P,C07K,C12N

Abstract:
1. A process for purifying a sample of h38C2 antibody or variant thereof, wherein both antigen binding sites are fully available for antigen binding in at least about 85% of the antibodies in the sample, comprising (a) Equilibrating a hydrophobic interaction chromatography (HIC) column with a pre-loading equilibration wash comprising a base buffer that comprises between about 15 mM and about 100 mM sodium phosphate, potassium phosphate or ammonium phosphate HEPES, Tris and bis-Tris, at between about pH6.5 to about 7.5, and further comprises a salt selected from the group consisting of NaCl, KCl, and monosodium citrate, at a first concentration of between about 0.5 M and 1.5 M; wherein the HIC column comprises phenyl conjugated resin beads below about 50 μm in diameter and comprising pores of at least about 500 Å; (b) Loading the column with a sample of h38C2 at between about 4 and about 80 g/L in loading buffer comprising the base buffer and further comprising the salt at the first concentration; (c) Washing the column with post-loading equilibration wash comprising the base buffer and the salt at the first concentration; (d) Washing the column with a salt gradient, comprising the base buffer and further comprising a linear concentration gradient from about 1.5 M to about 0.25 M of the salt, characterised in that the salt concentration decreases by between about -90 mM and 100 mM per 1column volume CV; (e) Washing the column with a salt plateau wash, comprising between about 4CV and about -8CV of the salt at between about 0.25 M and about 0.4M in the base buffer; (f) Washing the column with a buffer wash comprising the base buffer; (g) Eluting the h38C2 with an elution buffer, comprising the base buffer and a linear concentration gradient of 1,6 hexanediol beginning at a concentration of between about 0 to about 1% of 1,6 hexanediol and ending at an upper limit selected from the group consisting of about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, and 22% about 0% to about 22% of 1,6 hexanediol for between about 0.5CV to about 3CV or until the elution pool is collected, (h) Optionally running a further elution step comprising the base buffer and 1,6 hexanediol at a concentration selected from the group consisting of about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, and 22% about 0% to about 22%, for up to 5CV or until the elution pool is collected; wherein the h38C2 antibody or variant thereof is an IgG1 aldolase antibody comprising a light chain variable region (V L ) as set forth in SEQ ID NO:27; and a heavy chain variable region (V H ) as set forth in SEQ ID NO:28.