ABSTRACT
This chapter shows that by changing the gradient slope one can optimize the reversed-phase chromatography (RPC) separation of closely related proteins. Several methods have been developed for correlating polypeptide structures with reversed-phase high-performance liquid chromatography retention in response to the growth of RPC as an efficient and popular method of separating polypeptides. One approach sums empirically derived retention coefficients representing the hydrophobic contribution of each amino acid residue. The assumption behind this approach is that each amino acid residue contacts the sorbent surface and that the total hydrophobicity of the solute determines reversed-phase retention. The absence of Peak A among some of the interleukin-2 (IL-2) muteins is consistent with their expected protein chemistry. The structural differences among this series of IL-2 muteins exemplify the utility of a gradient optimization scheme. Earlier studies on native IL-2 established that a single cystine disulfide is present in the biologically active native IL-2.
