Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

Biosimilar drugs must closely resemble the pharmacological attributes of innovator products to ensure safety and efficacy to obtain regulatory approval. Glycosylation is one critical quality attribute that must be matched, but it is inherently difficult to control due to the complexity of its biogenesis. This usually implies that costly and time-consuming experimentation is required for clone identification and optimization of biosimilar glycosylation. Here, we describe a computational method that utilizes a Markov model of glycosylation to predict optimal glycoengineering strategies to obtain a specific glycosylation profile with desired properties. The approach uses a genetic algorithm to find the required quantities to perturb glycosylation reaction rates that lead to the best possible match with a given glycosylation profile. Furthermore, the approach can be used to identify cell lines and clones that will require minimal intervention while achieving a glycoprofile that is most similar to the desired profile. Thus, this approach can facilitate biosimilar design by providing computational glycoengineering guidelines that can be generated with a minimal time and cost

Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic acid as determined by constraint based metabolic network analysis.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.The metabolism of gluconate is well characterized in prokaryotes where it is known to be degraded following phosphorylation by gluconokinase. Less is known of gluconate metabolism in humans. Human gluconokinase activity was recently identified proposing questions about the metabolic role of gluconate in humans. Here we report the recombinant expression, purification and biochemical characterization of isoform I of human gluconokinase alongside substrate specificity and kinetic assays of the enzyme catalyzed reaction. The enzyme, shown to be a dimer, had ATP dependent phosphorylation activity and strict specificity towards gluconate out of 122 substrates tested. In order to evaluate the metabolic impact of gluconate in humans we modeled gluconate metabolism using steady state metabolic network analysis. The results indicate that significant metabolic flux changes in anabolic pathways linked to the hexose monophosphate shunt (HMS) are induced through a small increase in gluconate concentration. We argue that the enzyme takes part in a context specific carbon flux route into the HMS that, in humans, remains incompletely explored. Apart from the biochemical description of human gluconokinase, the results highlight that little is known of the mechanism of gluconate metabolism in humans despite its widespread use in medicine and consumer products.info:eu-repo/grantAgreement/EC/FP7/23281

Subcellular fractionation coupled to shotgun proteomics allows the identification of novel targets of the classical secretion pathway associated with increased productivity in recombinant CHO cells

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Engineering CHO cells for the production of Hard-To-Produce proteins

Over the past decades, the CHO cell has become increasingly popular as the favorite host cell line for the production of protein based therapeutic drugs. In comparison with the popularity of the CHO cells and the frequent use of these cells to produce a large part of the bestselling blockbuster drugs, less intensive efforts have been done to understand the machinery used by the CHO cells during growth and production. The main approach has (broadly speaking) been to approach the CHO cell as a “black box” where one could insert the gene of interest, perform a number of amplifying steps, like gene amplification, selection for stable clones, intense screening for stably expressing high producers, and massive efforts to optimize a specific bioprocess for the selected cell line(s). Since 2013, the Novo Nordisk Foundation Center for Biosustainablity at the Technical University of Denmark has embarked on a large CHO program to open up the “black box”, to get a deeper understanding of the available machinery inside the protein producing “cell factory” that is CHO cells. We are using this understanding to engineer new CHO cell lines having significantly improved features for the production of therapeutic proteins. We are not only doing this by improving the titer, quality, downstream processing and speed of development for already well-known proteins (e.g. Ab), but also for the production of therapeutic proteins that cannot be produced in CHO cells today, due low titer, wrong post translational modifications, and/or low activity. By combining the competences embedded in the CHO program, we are able to exploit the combination of genome scale modelling, high throughput protein expression, deep understanding of both the glycosylation machinery as well as the secretory and metabolic pathways involved in the expression of secreted proteins. This knowledge is being used as input to a high throughput CHO cell line engineering pipeline, able to engineer up to 10 cell lines and 25 gene targets in parallel. This has resulted in a large number of new CHO cell lines enabling the production of proteins with specific tailor-made glycoprofiles, higher quality, less degradation, improved bioprocess, higher viable cell density and better cell viability. We have made a cell lines where we have removed a number of naturally expressed host cell proteins (HCP) from CHO, which has resulted in higher titer and higher VCD, cell lines showing increased resistance to viral infections, cell lines displaying homogenous glycoprofiles, reduced degradation, and drastically changed cell lines that does not produce lactate. These features are currently being combined to engineer CHO cells able to produce proteins that have not been possible to produce with adequate product quality and titer using CHO cells to date

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies

Discovery of a human monoclonal antibody that cross-neutralizes venom phospholipase A2s from three different snake genera

Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects (using a mouse myoblast cell line that may demonstrate the impact of venoms on skeletal muscle) of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to potentially compose more effective, safer, and globally accessible polyvalent antivenoms that hopefully can be widely applicable for snakebite envenomings

Combating viral contaminants in CHO cells by engineering innate immunity

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited activation of cellular immune responses and increased resistance to the RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications

Variants of alpha-1-antitrypsin

Provided are variants of alpha-1-antitrypsin comprising mutations which render the variants oxidation- as well as protease-resistant, polynucleotides encoding said variants, methods of producing the variants and the variants for use in the treatment of alpha-1-antitrypsin deficiency, cystic fibrosis and chronic obstructive pulmonary disease.</p