Purpose: Treatment of BRAF V600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi.Experimental Design: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre-and ontreatment tumor specimens.Results: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARa-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAF V600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo.Conclusions: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAF V600E -mutant melanoma.

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.

To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

N-linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N-linked glycosylation patterns in a CHO-S cell fed-batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High-throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N-linked glycosylation patterns during a fed-batch process as a function of time as well as the manipulated variables. A constant and varying mAb N-linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model-based evaluation of feeding regimes using high-throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135-1148, 2016.

N-linked glycosylation plays a fundamental role in determining the thermodynamic stability of proteins and is involved in multiple key biological processes. The mechanistic understanding of the intracellular machinery responsible for the stepwise biosynthesis of N-glycans is still incomplete due to limited understanding of in vivo kinetics of N-glycan processing along the secretory pathway. We present a glycoproteomics approach to monitor the processing of site-specific N-glycans in CHO cells. On the basis of a model-based analysis of structure-specific turnover rates, we provide a kinetic description of intracellular N-glycan processing along the entire secretory pathway. This approach refines and further extends the current knowledge on N-glycans biosynthesis and provides a basis to quantify alterations in the glycoprotein processing machinery.

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Synthetic substrates able to prevent the adhesion of cells, commonly referred to as nonfouling surfaces, are useful in a wide range of applications, including the culture of multicellular spheroids and the engineering of complex cell sheets. Nonfouling properties are commonly conferred to surfaces through superhydrophilic polymer-based coatings. At the same time, this type of treatment requires difficult, expensive, and time-consuming procedures, leading to high prices of the final product. In this perspective, the development of quick single-step methods to coat surfaces with nonfouling polymers would dramatically reduce the costs and increase the flexibility of such products. Moreover, this strategy would enable the process to be carried out directly by the end user. In this work, we report a fast and easy-to-use coating method to prevent the nonspecific adhesion of cells on tissue culture polystyrene (TCPS) surfaces. Our approach is based on the adsorption of poly(styrene-co-3-sulfopropyl methacrylate) copolymers comprising nonfouling hydrophilic moieties as well as functionalities that enhance the polymer adhesion to the substrate. The polymer adsorption is obtained from an aqueous mixture with a procedure that can be conveniently performed in short time. The kinetic of polymer adsorption as well as the effect of the polymer concentration was studied in order to reduce the time and cost of the entire procedure. Additionally, the polymer composition and the polymer density were optimized to completely avoid the adhesion of three different adherent cell lines, that is, CHO, A375-P, and HFF-1 cells.

The efficacy of several cell therapy products is directly impacted by trypsinization, which can diminish the engrafting capacity of transplanted cells by cleaving cell surface receptors. Thermoresponsive surfaces can alleviate this drawback, enabling temperature-driven and enzyme-free cell harvesting. However, the production of thermoresponsive surfaces relies on dedicated and complex equipment, often involving protocols dependent on high surface activation energies that prevent the development of scalable and universal platforms. In this work, we developed thermoresponsive copolymers incorporating styrene units that enable the copolymer adsorption on tissue culture polystyrene surfaces from an alcoholic solution in a short time, regardless of the vessel size and geometry, and without any particular equipment. In this way, the procedure can be performed with minimal effort by the end user on any surface. The thermoresponsive copolymers were synthesized via reversible addition–fragmentation chain transfer polymerization, providing high control over the polymer microstructure, a key parameter for tuning its cloud point and architecture. Block copolymers comprising a thermoresponsive segment and a polystyrene block exhibited optimal adhesion on conventional cell culture surfaces and permitted a more efficient temperature-mediated harvesting of adipose-derived stromal cells and Chinese hamster ovary cells compared to their statistical counterparts. To expand the application of this polymer deposition protocol to serum-free cell culture, we also considered the polymer modification with the tripeptide arginine-glycine-aspartic acid, known to promote the cell adhesion to synthetic substrates. The incorporation of this peptide enabled the collection in serum-free conditions of intact cell sheets from surfaces prepared shortly before their usage.