Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte‐macrophage colony‐stimulating factor/interleukin‐3 fusion protein expressed in yeast

Abstract

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.

Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture

“…This cytokine evidences clinical applicability in treatments for neutropenia and aplastic anemia, and has also significantly reduced the infection risk associated with bone marrow transplantation, as the result of its ability to accelerate the recovery of neutrophil counts (Dale et al 1995). GM-CSF has been expressed previously in a variety of different systems, including Escherichia coli (Libby et al 1987), yeast (Balland et al 1998), Aspergillus niger (Kim et al 2000), mammalian cells (Chiou and Wu 1990) and plant cells , and is currently being generated for clinical use (Metcalf 1991).…”

Prophylactic Treatment of Endotoxic Shock with Monophosphoryl Lipid A in Newborn Rats

“…Therefore, IL-10 may not have been responsible for the MPL-induced endotoxin tolerance. GM-CSF stimulates the function and proliferation of neutrophils and macrophages, resulting in a decreased host response to LPS [22]. As GM-CSF may cross the rat placenta, it attenuates the effects of LPS on newborn rats.…”

“…As GM-CSF may cross the rat placenta, it attenuates the effects of LPS on newborn rats. However, the half-life of GM-CSF is between 2 and 20 h [21,22].…”

Cell cycle‐dependent protein secretion by Saccharomyces cerevisiae

“…The recombinant strain (XV2181:321) was transformed with a 2-m plasmid (pIXY321) encoding a 35-kDa fusion cytokine, GMCSF/IL3 (human granulocyte-macrophage colony stimulating factor and human interleukin 3), which was also provided by Immunex (Price et al, 1990;Balland et al, 1998). Transcription of the GMCSF/IL3 is controlled by an alcohol dehydrogenase II (ADHII) promoter, which is repressed in the presence of glucose, but permits transcription when ethanol is the sole carbon source.…”

“…The diploid yeast cells used in this study express and secrete an engineered recombinant human cytokine, GMCSF/IL3 (Balland et al, 1998). Expression is controlled by the homologous alcohol dehydrogenase II (ADHII) promoter.…”