Example 2
Purification or enrichment for different romosozumab species from a composition comprising wild-type romosozumab and the romosozumab PARG LSEQ ID NO: 8) C-terminal variant is achieved by Cation Exchange Chromatography (CEX) fractionation. CEX separates proteins based on differences in their surface charges. At a set pH, positively charged variants of wild-type romosozumab are separated on a cation-exchange column (e.g., Dionex Pro Pac WCX-10 analytical column, 2.0 mm×250 mm) and eluted using a salt gradient (e.g., Mobile Phase A: 10:90 (v/v) ACN, 19 mM MES pH 6.2; Mobile Phase B: 10:90 (v/v) ACN, 19 mM MES, 250 mM NaCl, pH 6.2). The different C-terminal variants of romosozumab are charged differently and the more positively charged variant elutes later in CEX. Thus, the elution order is: PG (wild-type), P-amide (amidated proline of wild-type), PARG (SEQ ID NO: 8) variant, and PAR-amide. The fraction collector can be programmed to collect CEX eluents containing different variants at different elution times.
