Investigation of Antiangiogenic Tumor Therapy Potential of Microencapsulated HEK293 VEGF165b Producing Cells

To investigate the antiangiogenic potential of encapsulated VEGF165b producing HEK293 cells, Human Embryonic Kidney 293 (HEK293) cells were stably transfected to produce VEGF165b. Then they were encapsulated in alginate - polylysine -alginate (APA) microcapsules. VEGF165b productivity and viability of encapsulated cells were analyzed and compared with the non-encapsulated cells. Results showed that encapsulated cells proliferated and remained viable within the microcapsules throughout the 28-day period of the experiment. The quantity of VEGF165b increased from 6.5 ± 1.2 μg/ml at day 13 to 13 ± 0.96 μg/ml at day 16. Then it gradually dropped to 5 ± 1.2 μg/ml for the last 3 days period as measured at day 28. Production of VEGF165b from encapsulated and non-encapsulated cells was similar. The effect of VEGF165b harvested from encapsulated cells on Human Umbilical Vein Endothelial cells (HUVECs) proliferation were also examined.The same inhibitory effects on HUVECs proliferation was seen when the cells were incubated with a mixture of VEGF165b and a 2-fold VEGF165b or with VEGF165b and 2-fold excess VEGF165b released from encapsulated cells. Subcutaneous injection of microencapsulated VEGF165b producing cells in tumor site of nude mice resulted in the reduction of the number of vessels around the tumors

From meticulous professionals to superheroes of the business world : a historical portrait of a cultural change in the field of accountancy

Purpose The purpose of this paper is to examine the relative cultural shift from professionalism to commercialism in the accounting profession, based on an analysis of the promotional brochures used by the Ordre des comptables agréés du Québec (Institute of Chartered Accountants of Québec), over the last 40 years, to attract new members. Design/methodology/approach The study's specific objectives are: to examine accountancy's cultural representations depicted in promotional brochures; to evaluate the extent to which these representations are indicative of the commercialist shift as documented in the literature; and to establish whether the representations under study provide further insight into the nature of the cultural shift. Drawing on the semiotic approach developed by Roland Barthes, the authors' analysis is predicated on the idea that promotional brochures and advertisements, though often simple in appearance, constitute complex representations that convey meaningful information about influential values and cultural change. Findings The authors found that commercial values are increasingly apparent through the celebration of multidisciplinary services and the emphasis on generous compensation and high dynamism. Originality/value Barthes' framework was especially useful to analyze the interplay between images and text to gain insight into the historical emergence of what has become the accountant's representation of today. As such, this study points to promotional representations participating to the inculcation of a cosmopolitan culture, where the internationalization of business is supposedly natural, inevitable, and beneficial to everyone. The authors' research also highlights the increasingly significant role played by marketing experts in designing professional institutes' brochures, consistent with the broader view of marketization as a key trend within the accounting industry

From meticulous professionals to superheroes of the business world : a historical portrait of a cultural change in the field of accountancy

Purpose – The purpose of this paper is to examine the relative cultural shift from professionalism to commercialism in the accounting profession, based on an analysis of the promotional brochures used by the Ordre des comptables agréés du Québec (Institute of Chartered Accountants of Québec), over the last forty years, to attract new members. Design/methodology/approach – The study’s specific objectives are: (1) to examine accountancy’s cultural representations depicted in promotional brochures; (2) to evaluate the extent to which these representations are indicative of the commercialist shift as documented in the literature; and (3) to establish whether the representations under study provide further insight into the nature of the cultural shift. Drawing on the semiotic approach developed by Roland Barthes, our analysis is predicated on the idea that promotional brochures and advertisements, though often simple in appearance, constitute complex representations that convey meaningful information about influential values and cultural change. Findings – We found that commercial values are increasingly apparent through the celebration of multidisciplinary services and the emphasis on generous compensation and high dynamism. Originality/value – Barthes’ framework was especially useful to analyze the interplay between images and text to gain insight into the historical emergence of what has become the accountant’s representation of today. As such, our study points to promotional representations participating to the inculcation of a cosmopolitan culture, where the internationalization of business is supposedly natural, inevitable, and beneficial to everyone. Our research also highlights the increasingly significant role played by marketing experts in designing professional institutes’ brochures, consistent with the broader view of marketization as a key trend within the accounting industry

Improving the metabolic efficiency of mammalian cells and its impact on glycoproteins quality

Glycosylation is a critical quality attribute for recombinant therapeutic proteins, which can be impacted by a number of process conditions, including waste metabolite accumulation. While fed-batch strategies that consist in substituting or controlling the main substrates at low concentrations have proven generally effective at improving protein titer, they can also adversely affect product glycosylation. Metabolic engineering strategies aiming at reducing by-product formation may thus be beneficial for ensuring product quality consistency. In this work, we have specifically investigated the impact of PYC2 overexpression on the quality of a recombinant glycoprotein of therapeutic interest, the interferon α2b (IFNα2b) that has one O-glycosylation site. To this end, batch and fed-batch cultures were performed and product characteristics were measured for both the PYC expressing HEK293 clone and the parental cells. SDS-PAGE and Western Blot analysis of batch culture harvests revealed two distinct bands corresponding to glycosylated and non-glycosylated fractions of IFNα2b, as subsequently confirmed via SDS-PAGE analysis of purified samples loaded along with a non-glycosylated commercial standard produced in E.coli. As inferred from densitometry analysis of the gels, the cultures with PYC-expressing cells were shown to sustain a significantly higher percentage of glycosylated IFNα2b at the late stage of the culture, which was correlated with the prolonged viability and reduced accumulation of waste metabolites. Differences between the two cell lines in terms of cell viability and protein quality were even more pronounced when performing fed-batch cultures during which glucose was maintained at high levels. To investigate the potential impact of ammonia, batch cultures with various glutamine substitutes were also performed. Among the different substitutes tested, pyruvate led to the lowest ammonia production with no significant impact on protein titer. Of salient interest, the results suggest that substituting glutamine by α-ketoglutarate, glutamate or pyruvate may allow to maintain a higher fraction of glycosylated proteins during late-stage batch cultures

CHO stable pool fed-batch process development of SARS-CoV-2 spike protein production

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Improving transient gene expression in CHO-EBNA1 cells

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly generating recombinant proteins that are highly similar to those produced in stable CHO clones used for biomanufacturing. The higher cost of reagents necessary for TGE, specifically the requirement for large amounts of purified DNA and transfection agent for each production, means that improving the performance of CHO TGE could substantially augment the method’s utility. In the current study, we have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1). Transfection of EBNA-1-expressing cells with plasmid vectors encoding the Epstein-Barr virus OriP sequence boosted TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture in shake flasks, we optimized transfection parameters (plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added at 24 h post-transfection, followed by periodic glucose supplementation. This method gave a maximum yield of 900 mg/L (for the chimeric IgG4 B72-3 mAb), with an average of 570 mg/L (standard deviation: 250 mg/L) for a panel of six mAbs and 320 mg/L (standard deviation: 140 mg/L) for five His-tagged recombinant proteins at 14 days post-transfection. Compared to our current low-density TGE process using CHO-3E7 cells and different culture medium, the new procedure gave on average 3-fold higher yields; purified mAbs produced using the two methods had distinct glycosylation profiles (by HILIC analysis) but showed identical target binding kinetics by SPR. Key advantages of the improved CHO-3E7-based protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change

Overexpression of G6PDH does not affect the behavior of HEK-293 clones stably expressing interferon-α2b

HEK293 cells are gaining in interest for the production of recombinant proteins. However, further understanding and engineering of cell metabolism are still needed to improve protein titers. The importance of G6PDH has already been studied regarding redox balance, and a correlation has recently been established between the oxidative pentose phosphate pathway (PPP) and antibody peak production. In this work, HEK293 cells stably expressing interferon-α2b, a parental clone, and a further engineered clone expressing the cytosolic yeast pyruvate-carboxylase (PYC) were transiently transfected to overexpress glucose-6-phosphate dehydrogenase (G6PDH) and increase fluxes through the PPP. The aim of the study was thus to evaluate the effect of overexpressing G6PDH on the “pull” effect brought by the PYC phenotype. Results indicate that the cell metabolism is, however, highly robust, showing a highly regulated PPP damping the potential effects of overexpressing G6PDH. A metabolomic study also clearly demonstrates, by metabolites profiling, that the PYC clone has a highly robust and more efficient metabolic efficiency, compared to its parental clone

Determination of the composition of heterogeneous binder solutions by surface plasmon resonance biosensing

Surface plasmon resonance-based biosensors have been extensively applied to the characterization of the binding kinetics between purified (bio)molecules, thanks to robust data analysis techniques. However, data analysis for solutions containing multiple interactants is still at its infancy. We here present two algorithms for (1) the reliable and accurate determination of the kinetic parameters of N interactants present at different ratios in N mixtures and (2) the estimation of the ratios of each interactant in a given mixture, assuming that their kinetic parameters are known. Both algorithms assume that the interactants compete to bind to an immobilized ligand in a 1:1 fashion and necessitate prior knowledge of the total concentration of all interactants combined. The effectiveness of these two algorithms was experimentally validated with a model system corresponding to mixtures of four small molecular weight drugs binding to an immobilized protein. This approach enables the in-depth characterization of mixtures using SPR, which may be of considerable interest for many drug discovery or development applications, notably for protein glycovariant analysis

Process for generation of high-producing CHO cell lines for biomanufacturing of biologics using our CHOrcTA platform

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