Clinical pharmacokinetic studies of a human haemopoietic growth factor, GM‐CSF

Hovgaard, Doris; Mortensen, Børge Thing; Schifter, Søren; Nissen, Nis I.

doi:10.1111/j.1365-2362.1992.tb01934.x

Cited by 32 publications

(17 citation statements)

References 21 publications

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“…Moreover, some studies have suggested that hGM‐CSF lacking N‐linked carbohydrate has a significantly enhanced specific activity in vitro when compared to the native recombinant cytokine (2, 15). However, there are numerous evidences supporting the key role of carbohydrate chains in hGM‐CSF functions, such as pharmacokinetic, toxicity, and immunogenicity (16).…”

Section: Resultsmentioning

confidence: 99%

Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality

Bollati-Fogolín

Forno

Nimtz

et al. 2008

Biotechnol Progress

102

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We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.

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Section: Resultsmentioning

confidence: 99%

Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality

Bollati-Fogolín

Forno

Nimtz

et al. 2008

Biotechnol Progress

102

View full text Add to dashboard Cite

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“…In a previous clinical trial, we demonstrated that replication could result in detectable GM-CSF in blood when other inflammatory cytokines had returned to baseline (days 4-15)[7], despite the relatively short half-life of GM-CSF (< 2h) [10]. Therefore, detection of GM-CSF in blood is indicative of high-level GM-CSF expression, and may be a specific but insensitive marker of transgene expression.…”

Section: Pharmacokinetics and Sheddingmentioning

confidence: 94%

Phase 1b Trial of Biweekly Intravenous Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus in Colorectal Cancer

et al. 2015

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Fifteen patients with treatment-refractory colorectal cancer were enrolled on a phase 1b study of Pexa-Vec (pexastimogene devacirepvec; JX-594), an oncolytic and immunotherapeutic vaccinia designed to selectively replicate in cancer cells. Pexa-Vec was administered intravenously every 14 days, at dose levels of 1 × 10(6), 1 × 10(7), or 3 × 10(7) plaque-forming units (pfu)/kg. The primary endpoint was to determine the maximum tolerated dose. Secondary endpoints were pharmacokinetics and pharmacodynamics as well as antitumor activity. Patients were heavily pretreated (mean 4.5 lines of therapy). All patients received at least two Pexa-Vec doses (median = 4; range = 2-4). No dose-limiting toxicities were reported, and the maximum tolerated dose was not reached. The most common adverse events were grade 1/2 flu-like symptoms, generally lasting <24 hours. During the first and last cycles, genome pharmacokinetics were unchanged. Infectious pfu could be detected in plasma up to 2 hours after cycle 1 and up to 30 minutes after cycle 4 (when antivaccinia antibody titers are known to have peaked). Ten patients (67%) had radiographically stable disease. Given the acceptable safety profile of multiple intravenous Pexa-Vec infusions in patients with treatment-refractory colorectal cancer, further trials evaluating efficacy of intravenous Pexa-Vec, as monotherapy or in combination with chemotherapeutic agents, is warranted in this patient population.

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“…Since the action of PNGase F results in the liberation of N-glycans and the simultaneous conversion of the asparagine to aspartic acid, previously glycosylated peptides exhibit a mass increase of 1 Da which can be easily detected by mass spectrometry. The ratio of unglycosylated to glycosylated GP I [LLN(D)LSR (25)(26)(27)(28)(29)(30)] was found to be approximately 40 : 60. For GP II [DT AAEMN(D)ETVEVISEMFDLQEPTCLQTR(31-58)], a ratio of approximately 15 : 85 of unglycosylated and N-glycosylated peptide form was determined.…”

Section: Site Specific Glycosylation Of Gm-csfmentioning

confidence: 99%

N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster ovary cell line

Forno

Bollati-Fogolín

Oggero

et al. 2004

European Journal of Biochemistry

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GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Dm 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri-and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal a1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found a2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N-and C-termini integrity of the GM-CSF preparations.

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Clinical pharmacokinetic studies of a human haemopoietic growth factor, GM‐CSF

Cited by 32 publications

References 21 publications

Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality

Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality

Phase 1b Trial of Biweekly Intravenous Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus in Colorectal Cancer

N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster ovary cell line

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