We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.