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Mobile Phase Optimization for the Analysis of an Antibody-Based Fusion Protein and Aggregates using a TSKgel Super SW3000 Size-Exclusion Column

ning buffer consisted of 0.1M NaH2PO4 (pH 6.0) to which was added 15% acetonitrile and 0.2M NaCl. In the redesigned buffer system, NaCl was replaced by NaClO4 and the phosphate buffer concentration was reduced to 0.05M. Initially, the concentration of sodium perchlorate investigated was 0.2M, however, best results were obtained at 0.4M. The flow rate of 0.35mL/min was left unchanged. Note that recent work has shown that acetonitrile can be omitted from the mobile phase without affecting separation quality or method robustness.


Results:
As shown in Figure 1 , excessive peak tailing of "fusion protein 1" is evident with the use of 0.2M NaCl. Additionally, the expected protein dimer and trimer aggregates are not visible in the chromatogram. By switching from 0.2M sodium chloride to 0.2M of the more chaotropic sodium perchlorate salt, together with a two-fold reduction in the buffer concentration, less peak tailing and distinct peaks for the dimer and trimer species of MAb1 resulted. Doubling the perchlorate concentration to 0.4M provided further improvement in the peak shape of fusion protein 1 and associated aggregate species. Additionally, the re-development increased the methods' robustness allowing for a substantial decrease in column "break-in" time from two weeks to 4 days as well as better peak area reproducibility and linearity of both monomer and aggregate species.


As a precaution, a more rigorous cleaning protocol consisting of flushing the system with DI water was implemented during periods of instrument inactivity. Although perchlorate is slightly less corrosive to stainless steel components relative
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