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There are numerous reports of phosphonate analogues of biologically interesting compounds.1 A related class of analogues

There are numerous reports of phosphonate analogues of biologically interesting compounds.1 A related class of analogues

PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the A-mating factor expression system. To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200-l scale and the PIXY321 molecule isolated by four successive steps of ion-exchange chromatography. Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry. Four different N-terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions. Molecules lacking 1Ϫ6 residues at the C-terminus were identified and their relative frequencies quantified. Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level. Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM-CSF portion and three in the IL-3 portion of the molecule. The sites were shown to be differentially occupied and to carry 0Ϫ 10 mannose residues according to their location in the sequence. Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots.