A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation

Assessment of the interactions between bioprocess conditions and protein glycosylation in antibody- producing mammalian cell cultures

The pharmaceutical industry is going through a rather turbulent period. Many blockbuster drugs have fallen off patent over the past two years and many more are expected to do so in the near future. In response, pharmaceutical companies have continued searching for products that will replace those that have lost patent protection. However, drug development and approval is extremely time-consuming and costly. So that this critical issue is addressed, industry experts and regulatory agencies have jointly proposed the implementation of Quality by Design (QbD) principles in the development and manufacture of all new drugs. Adoption of QbD is expected to reduce drug development cost and approval time. It is also expected to encourage innovation by developing drugs, and the processes used to manufacture them, around the mechanisms that relate process inputs with end product quality. Within this context, monoclonal antibodies (mAbs) are currently the highest-selling products of the biopharmaceutical industry and are projected to account for nearly half of the world’s top-selling drugs by 2018. All currently commercialized mAbs contain N-linked glycans (complex carbohydrates) bound to their protein backbone. These carbohydrates, in turn, have been widely reported to impact the safety and efficacy of mAbs. Furthermore, it has widely been reported that bioprocess conditions heavily impact the composition and distribution of these glycans. For these reasons, mAb glycosylation is considered a critical quality attribute (CQA) of these therapeutic proteins under the QbD scope. Based on QbD principles, the objective of this thesis was to generate a mathematical model that mechanistically relates the effect of nutrient availability throughout cell culture with the glycan profile of a mAb. The model was constructed from three individual ones. The first model describes the N-linked glycosylation process which occurs in the Golgi apparatus. The second model is unstructured and describes cell culture dynamics. The third and final model describes the biosynthetic pathway for nucleotide sugars. All three models were developed independently, but were adapted with features so that they could be interconnected. The glycosylation model approximates the Golgi apparatus to a single plug flow reactor where resident proteins (glycosylation enzymes and transport proteins) are recycled from distal portions of the Golgi space to proximal ones. Optimisation-based methods were developed to estimate unknown parameters of the model. The cell culture dynamics model was developed to represent cell growth, nutrient consumption and mAb synthesis. It was originally based on Monod kinetics, but was adapted to include experimentally-encountered complexity. The model for nucleotide metabolism was heuristically reduced from 35 constituting reactions to 7. Additional mechanistic features were adapted or included to ensure model fidelity. Experimentally, batch cultures were performed with hybridoma (CRL-1606 from ATCC). Data for viable cell density, glucose, glutamine, lactate, ammonia and mAb titre were collected. Intracellular samples were produced by perchloric acid extraction. These samples were then analysed for nucleotide sugar content using a high performance anion exchange chromatographic method which was optimized to quantify eight nucleotide sugars and four nucleotides in 30min. mAb bound glycans were analysed by MALDI mass spectrometry. The experimental data was used to estimate the unknown parameters of the models. The models – along with their associated parameters – were then combined to produce a coupled model that mechanistically relates nutrient availability with mAb glycosylation-associated quality. With further validation, such a model could be used for bioprocess design, control and optimization

Seeing cannibals : European colonial discourses on the Latin American other

The figure of the cannibal has been central in the development of European colonial discourses on Latin America. It has functioned as a locus for coming to grips with otherness and as a crucial marker for differentiating between the "civilised" and the "savage" in European discourses. While there is an extensive academic body of work on the figure of the Latin American cannibal in written texts, a study dedicated exclusively to the images of Latin American cannibals is lacking. The present dissertation addresses this gap by looking at the role that printed images of cannibalism played in the construction of European discourses on Latin American otherness during the colonial period of the region (1500-ca. 1750). It focuses on a corpus consisting mainly of woodcuts and copperplates that illustrated the main European travel narratives, New World compendiums, maps and atlases of the period. Centrally, this work proposes that visual representations of the cannibal functioned as discursive sites for the deployment of strategic othering at the service of European colonialism in the Americas. The theoretical framework for this study is based on Foucault's work on discourse and the impact that particular systems of power/knowledge had on the representational regimes of the period. Further theoretical references include postcolonial theory through figures such as Said, Bhabha and Mignolo, as well as current debates on visual culture and visuality. In terms of methodology, the thesis locates the shifts in European forms of discursive othering over time and space by following a Foucauldian method of discourse analysis based on archaeological and genealogical analyses of the corpus. It also addresses the intertextual and interdiscursive threads that connect these printed images of Latin American cannibals to their accompanying texts and surrounding discourses

H-2Ld class I molecule protects an HIV N-extended epitope from in vitro trimming by endoplasmic reticulum aminopeptidase associated with antigen processing.

In the classical MHC class I Ag presentation pathway, antigenic peptides derived from viral proteins by multiple proteolytic cleavages are transported to the endoplasmic reticulum lumen and are then exposed to ami-nopeptidase activity. In the current study, a long MHC class I natural ligand recognized by cytotoxic T lymphocytes was used to study the kinetics of degradation by aminopeptidase. The in vitro data indicate that this N-extended peptide is efficiently trimmed to a 9-mer, unless its binding to the MHC molecules protects the full-length peptide.We thank Dr. A. K. Stout of the NIH Tetramer Facility for providing the peptide/MHC complex reagent. This work was suppor ted by grants provi ded by Programa Ramón y Cajal, and Fundación FIPSE to D. L.; by grants provided by Comunidad de Madrid and Ministerio de Educación y Ciencia to M. D. V.; and by a joint grant provided by Instituto de Salud Carlos III to D. L., and M. D. V.S

Lectin-aided flow cytometry reveals a close correlation between cell surface and mAb glycosylation- aided flow cytometry reveals a close correlation between cell surface and mAb glycosylation

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Next-gen glycoengineering: Combining cellular and metabolic engineering to fine-tune β1,4-Galactosylation

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