Sp sepharose hp column

All four recombinant antibodies were expressed using the same proprietary Amgen modified Chinese hamster ovary (CHO) host cell line with expression titers ranging from 0.1 to 2.0 g/l. Antibodies were first purified using MabSelect Sure (GE Healthcare, Piscataway NJ) by directly loading the conditioned media and washing the column with Dulbecco’s phosphate buffered saline (PBS). Antibodies were eluted using 100 mM acetic acid pH 3.2 and the elution pools were brought to pH 5.0 using 1 M NaOH. The MabSelect Sure elution pools were then diluted with two volumes of water and loaded on to a SP-Sepharose HP column (GE Healthcare, Piscataway NJ) followed washing with SP-Buffer A (20 mM acetic acid pH 5.0). The column was then developed using a 20 column volume linear gradient to 100% SP-Buffer B (20 mM acetic acid, 600 mM NaCl, pH 5.0) and fractions were pooled based on Coomassie blue stained SDS-PAGE analysis (64–86% yield). The pooled antibodies were then concentrated and buffer exchanged into 10 mM acetic acid, 9% sucrose, pH 5.2, and aliquots were stored at -70°C until needed. Protein quality was assessed by Coomassie stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high performance liquid chromatography SE-HPLC (BioSep S3000 column, Phenomenex, Torrance, CA, USA) and liquid chromatography electrospray ionization mass-spectrometry.