Variants of intact polypeptides/proteins ranging in mass from 6,500 to 70,000 Da were easily separated using reversed-phaseHPLC (rpHPLC) or affinity chromatography. A variant of rhlGF-I, where the racemization of a serine residue was detected in the intact molecule, was resolved from rhlGF-I within 25 minutes by rpHPLC. Other variants of rhlGF-I separated by this method include methionine sulphoxide at position 59, des Gly1, des Gly1Pro2, Glu for Asp substitution at position 20 and incorrectly folded IGF-I. For rhDNase (approximately 40 kDa), affinity chromatography was able to clearly resolve three different amino acids (Asn, Asp and iso-Asp) at position 74 of the intact glycoprotein. The presence or absence of O-linked sugars on Thr -37 of recombinant human thrombopoietin was rapidly demonstrated by rpHPLC. While the separation of these types of variants is essential, the demonstration of biological activity is critical for designing specifications that allow the administration of these proteins into humans. Once a correlation exists between the variant and its biological activity, control of the manufacturing process can be better achieved with analytical methodology.