Residual protein analysis in workup APIs using organic extraction.
As described in Materials and Methods, a workup procedure was developed to further remove residual proteins by using organic extraction and aqueous wash followed by a pH swing and aqueous back extraction (Figure 1b). The organic extraction efficiently extracts API to MeTHF using TGDE as a co-solvent and most residual proteins were removed in the rag layer and aqueous layer. The final API prepared from this procedure was analyzed using Cygnus kit ELISA, a silver-stained gel (Figure 9a), as well as a Coomassie blue stained gel (Figure 9b) with bands excising coupled with nano-LC-MS/MS (Table 4) again to analyze the residual protein level. No detectable amount of residual proteins was reactive to the Cygnus kit antibody and no proteins were detectable even at 20 mg/mL API load on a silver-stained SDS-PAGE gel (Figure 9a). Only five proteins were identified by LC-MS/MS from the bands excised from Coomassie blue-stained gel (Figure 9b), each with amount lower than 10 ng/mg. Similar results were obtained from three representative batches of APIs isolated by this new procedure (Table 4).