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).