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

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

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

Biochemical Characterization of Human Gluconokinase and the Proposed Metabolic Impact of Gluconic Acid as Determined by Constraint Based Metabolic Network Analysis

<div><p>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.</p></div

Modelling the contribution of gluconate to core metabolism.

<p><b>A</b>) Metabolic flux through glycolysis and the HMS is affected upon gluconate (yellow) consumption via GntK. The two added reactions (grey) feed gluconate into the HMS as described in the text. Reactions in the HMS and glycolysis with changed flux ranges upon gluconate uptake are indicated by arrow thickness as defined in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098760#pone.0098760.s001" target="_blank">File S1</a></b>. Metabolite abbreviations are consistent with the BiGG database. <b>B</b>) Flux variability analysis showed changes in the flux ranges of 329 out of the 416 reactions in the RBC model upon incorporation of gluconate. 56 miscellaneous reactions, which were not defined properly in the model, have been removed from this graph to simplify the graph. Gluconate degradation also activated reactions that otherwise did not carry flux due to constraints imposed by flux into the HMS through glucose-6-phosphate dehydrogenase which was floated at physiological 5–10% of glucose uptake.</p

Structural comparison of human GntK to <i>E.coli</i> GntK.

<p><b>A</b>) An iTasser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098760#pone.0098760-Roy1" target="_blank">[44]</a> structural model of human GntK (cyan) superimposed on E.coli GntK (magenta) to which it shows 34% sequence homology. The 18 a.a. insert observed in human GntK is predicted to form a protruding α-helix. <b>B</b>) SDS-PAGE of purified human GntK vs. <i>E.coli</i> GntK. <b>C</b>) Human GntK was observed to oligomerize as multiples of protein dimers. <b>D</b>) The ionization pattern (inset) and deconvoluted mass spectrum of purified human GntK <b>E</b>) Circular dichroism spectra of the two proteins are indicative of a similar tertiary structure.</p