The scene of the protein micro-heterogeneity of recombinant hirudin-II (HV2) expressed in Pichia pastoris was investigated. It was shown that three derivatives of HV2 were present in the fermentation broth of P. pastoris, which were intact HV2 and its two derivatives truncated the C-terminal amino acid residue Gln and Leu-Gln, respectively. To purify the minor degradation derivatives of HV2, a simple, biocompatible and scale-up-feasible purification process with two-step ion-exchange chromatography was established instead of usual reverse phase chromatography. The purities of end products were over 96% and the residual endotoxin less than 0.5 EU/ml. (c) 2006 Elsevier Ltd. All rights reserved.