We have demonstrated that temperature reduction from 37 to 33 °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 °C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 °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 °C. Furthermore, the total rhGM‐CSF production increased 6 times in the culture shifted to 33 °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.

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 (Δm 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 α1–6‐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 α2–3 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.

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

International audienceInterferons (IFNs) are potent biologically active proteins synthesized and secreted by somatic cells of all mammalian species. They play an important role in the immune response and defence against viruses because they have antiproliferative, antiviral and immunomodulatory activities [1]. They are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells. Antiviral assays may be subject to high inter- and intraassay variations having the drawbacks of using viruses [2]. Therefore, a set a new human cell lines derived from different tissues were developed using the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter. In addition, having reporter gene assays derived from cells of different tissue origins would allow us to design studies aiming to evaluate how IFNs induce their actions through the human body

Activated mPEG carbonates are important reagents that have been widely used for the PEGylation of several peptides and proteins by means of stable urethane linkages. In fact, mPEG-N-hydroxysuccinimidyl carbonate and mPEG-p-nitrophenyl carbonate are among the most used reagents in PEGylation technology. However, the synthesis and storage of these reagents are not always easy to resolve. With the aim of surpassing some of the drawbacks associated with the use of activated mPEG-carbonates we have prepared and evaluated a new mPEG-carbonylimidazolium iodide, which can be used for the conjugation of the NH₂ group by means of urethane linkages, as an interesting alternative to the known reagents. It is noteworthy that the novel reagent is prepared by a simple two-step procedure under mild experimental conditions. Moreover, we performed a detailed study of the conjugation reaction of interferon-α2b with the carbonylimidazolium derivative, and evaluated the conjugate in in vitro and in vivo studies.