July Dorion-Thibaudeau, Ph.D. student, Ecole Polytechnique
In the treatment of cancer or autoimmune diseases, monoclonal antibodies (Mabs) injected in the patient must interact with FcY receptors (FcYRs). It has been shown that the glycosylation of Mabs directly influence the antibody-receptor interactions. In order to get a functional drug for the patient, it is necessary to produce Mabs with an appropriate glycosylation pattern.
The project’s goal is to discriminate the antibodies according to their glycosylation pattern by their interactions with FcYRs. Characterization of antibody-receptor interactions will first be tested by surface plasmon resonance (SPR) and microcalorimetry. Later on, SPR, chromatography and ELISA techniques will be developed in order to get a pool of antibodies with a “good” glycosylation pattern.
Glycosylated and non-glycosylated herceptin will be used as model antibodies. Other Mabs will also be tested and will come from different labs within the network.
Keyvan Nowruzi, University of Guelph
Abdul Murayyan, University of Guelph
Carina Villacres, Ph.D. student, University of Manitoba
Mamalian cell culture bioprocesses have been used for the production of recombinant proteins, monoclonal antibodies (mAbs) and nucleic acid based products. Among the different cell lines, Chinese hamster ovary (CHO) cells are the most widely used for the production of biotherapeutics. Glycosylation is one of the post-translational modifications (PTM) to be considered when a biopharmaceutical is produced due to the influence that the protein content has not only an effect on the biological function of the recombinant protein, but also in the molecular stability, solubility, in vivo activity, serum half-life, and immunogenicity. Studies will be conducted to test the effects of glucose and glutamine deprivation in CHO-EG2 protein glycosylation.
Katrin Braasch, Ph.D. student, University of Manitoba
Monoclonal antibodies (Mabs) dominate the market of biopharmaceuticals accounting for 36% of the global value of biologics. Research is now focused on increasing the efficacy of Mabs by optimizing key features of the molecule, specifically the glycosylation, and also on the optimization of production to improve cost effectiveness. My research encompasses both of these key targets in the manufacturing process.While the factors influencing glycosylation are wide spread recent studies have indicated that the addition of carbohydrate precursors can shift the glycosylation profile of biopharmaceuticals in some cells. This offers the opportunity to shift the glycosylation of an antibody produced for therapeutic or diagnostic purpose towards the profile giving the optimal functional outcome. The question I am interested in is the correlation between the intracellular nucleotide / nucleotide sugar pool and the glycan structures observed. Since this pool contains the precursors for the glycosylation pathway as well as energy metabolites needed by the cell to function a direct correlation to the precursor feeding is expected. In a bioprocess it is also key to keep cells healthy (highly viable) for a prolonged time to increase volumetric productivity while maintaining product quality. This makes monitoring of a cell culture necessary to detect early sign of distress (e.g. nutrient limitations) to act appropriately to maintain cell health as well as antibody quality (mainly glycosylation). As part of my project we have shown that the use of an on-line capacitance probe or a novel dielectrophoretic (DEP) cytometer probe for viability monitoring is more sensitive than trypan blue exclusion to the physiological changes within a cell or cell population correlating very well with the flow cytometer assay for Annexin V detection (early sign of apoptosis). The use of either method gives us the opportunity to track changes in culture more precisely and intervene as needed to extend its life.
Natalie Krahn, M.Sc. student, University of Manitoba
The N-linked glycosylation pattern (Asn297) on monoclonal antibodies (mAbs) is known to have a significant effect on its structure and function. To understand how the glycan structure affects the function, structural studies will be done using small angle x-ray scattering (SAXS), dynamic light scattering (DLS), circular dichroism (CD) and crystallographic methods with the EG2-hFc chimeric mAb. In addition, the function of the antibody will be tested using isothermal titration calorimetry (ITC) as well as possible fluorescence experiments to analyze the kinetics (affinity) of how the EG2-hFc antibody interacts with the FcγRIIIa and FcγRI receptors. The antibodies will be subjected to various glycoprotein processing inhibitors including a fucosyltransferase inhibitor to produce varying amounts of fucosylated antibody and determine how the different glycosylation patterns affect the antibody structure as well as its function. Comparison studies will be done with an anti IL-8 IgG1 produced in CHO-DP12 cells to ensure that the camelid mAb has equivalent, if not more promising results, in terms of the function of the mAb.
Neha Mishra, M.Sc. student, University of Manitoba
Product consistency and quality are important factors in therapeutic antibody production. N-linked glycosylation contributes towards these factors by being responsible for efficacy/effector functions. My project focuses on investigating the effect of varying redox potential on glycosylation profiles. Currently, the aim is to apply a molecular biology approach towards studying redox status in cellular compartments of mammalian cells during monoclonal antibody production. In particular, the redox status and glycosylation profile of a CHO cell line producing a humanized camelid Mab, EG2-hFc, which contains only heavy chains. This will be compared to a CHO cell line producing full sized humanize anti‐IL8 Mab. Results from these studies will further add to our current knowledge of the link between redox potential of cells and glycosylation.
Ed Bodnar, Ph.D. student, University of Manitoba
Glyco-proteomics has evolved to the forefront of modern biosciences as it has been shown that the variance and structural diversity of oligosaccharides of glycoproteins can be coupled with particular disease stages that permit them to be viable biomarkers. Studying glycosylation at the glycopeptide level has several advantages, namely it allows for determination of site-specific heterogeneity, but in addition it circumvents the difficulty of identifying less abundant glycoforms that may be hindered in complex digests. My aim is to develop methods which simultaneous reduce handling time, and enhance the extraction and enrichment of glycoforms from digest mixtures by designing HILIC and glyco-like columns to efficiently isolate glycopeptides and improve ionization in mass spectrometric analysis.
Rahul Sadavarte, McMaster University
Calvin D’Eall – M.Sc. student, University of Ottawa/National Research Council Canada
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a critical component of protective immunity against both foreign pathogens and cancerous-self. Importantly, the reliance on immunoglobulin has made ADCC an attractive mechanism for immunotherapeutic exploitation, given the recent progress and success in the field of antibody engineering. However, these strategies have predominantly focused on improving a Mabs affinity and specificity for it’s respective tumor-associated antigen (TAA) through modification of the Fab domain, leaving the structurally distal Fc domain unmodified. Critically, the Fc fragment is required for binding FcgRIIIA (CD16); an Fc-receptor predominantly expressed on natural killer (NK) cells, critical for initiating ADCC. Thus, Fab-localized modifications that promote increased binding to TAAs may fail to augment Fc-FcR mediated ADCC, which ultimately facilitates tumor-cell killing. Fortunately, research has begun to delineate effective Fc-engineering strategies that give rise to improved ADCC through alteration of the glycosylation profile of a conserved oligosaccharide in the CH2 domain of the Fc fragment. Through implementation of the 51Chromium release assay, novel Fc-engineered EG2-hFc glycoforms will be characterized and ranked in-vitro for their ability to facilitate antitumor ADCC of EGFR-overexpressing breast cancer cells. Additionally, a polymorphism in CD16 imparts differential capacities of NK cells to facilitate ADCC, depending on the samples respective allotype. Thus, allotyping CD16 in each NK cell sample used may help account for relative differences in tumor cell lysis, as well as reductions in allotypic-specificity exhibited by novel EG2-hFc glycoforms.
Navid Ghaffari, M.Sc. student, University of British Columbia
Fed-batch culture is the most widely process for the production of monoclonal antibodies (MAb) from mammalian cells, due to its simplicity and high yield of products. However, large scale production of MAb by mammalian cells can be limited due to nutrient depletion and metabolite accumulation that result in decreased cell viability and productivity. Therefore, it is important to explore feeding strategies and technologies to avoid nutrient limitations while minimizing the formation of metabolic byproducts. Developing an effective feeding strategy is a multi-factorial challenge that requires in depth understanding of the factors influencing cell viability and antibody production. My study is focusing on Chinese Hamster Ovary (CHO) cells that are used extensively for the expression of mammalian proteins and provide a suitable model for studying the effect of the culture environment on recombinant protein production.
Celine Raymond, Ph.D. student NRC-Montreal/Universite de Montreal
Herceptin will be used as a model antibody (IgG1). Wild-type and mutant Herceptin will be co-expressed with sialyltransferase 6 (ST6Gal1) in HEK 293 and CHO DG44 cell lines under different culture conditions to determine the best expression platform for sialylated IgG1. The same approaches (mutations, overexpression of glycosyltransferases) will be used to produce other specific Mab glycoforms (core-fucosylation, presence of bisecting-GlcNAc, galactosylation). A flow cytometry method will be developed to assess the interactions between the different Mab glycoforms and Fc receptors.
Eric Blondeel, Ph.D. student, University of Waterloo
My proposed research project is to perform a metabolomic study on Chinese Hamster Ovary (CHO) cells producing the antibody EG2, using nuclear magnetic resonance (NMR) spectroscopy. NMR allows for a “global” quantification of intracelluar and extracellular metabolites, referred to collectively as the metabolome. Transient shifts in the metabolome and the resulting changes to the glycosylation profile of the antibodies will be measured to determine process conditions for manipulating glycosylation of antibodies.
Hengameh Aghamoseni, Ph.D. student, University of Waterloo
The pathway of protein glycosylation is known to involve several enzymatic reactions for adding a polysaccharide chains to the appropriate glycosylation sites followed by a stepwise removal of one or more individual monosaccharide. The pathway, however, can be affected by various environmental factors such as cell culture conditions.
The aim of this project is to develop a dynamic mathematical model that relates Mab glycan distribution patterns in the Golgi apparatus to CHO cell culture conditions. In order to find the optimum glycosylation pattern, the metabolic profile of CHO-DG44 through major processing modes such as batch and fed-batch or perfusion culture conditions will be monitored.
In the experimental phase of this research, a comprehensive experimental design with different levels of medium supplements will be applied to investigate the major effects of key nutrient consumption and byproducts accumulation on the therapeutic properties and glycan profile of the final Mab product. In the modeling phase, the experimental data of metabolite and biomass production will be applied to develop a metabolic flux based methodology and define the conditions (retention time, for example) in the Golgi apparatus for optimum glycoprotein profile synthesis.
Raymond Ho, Ph.D. student, University of Waterloo
The therapeutic potential and clinical success of monoclonal antibodies (mAbs) continue to drive the development with the focus of optimizing antibody production. A frequent approach is through genetic manipulation of mammalian cell lines to improve antibody production levels. The effects of such genetic changes on the biochemistry of the cell may provide valuable information for the optimization of mAb production. Therefore, we take a proteomics approach to compare the protein expression patterns of a recombinant monoclonal antibody (EG2-hFc)-producing CHO-DG44 strain to that of its non-producing counterpart. Key differentially expressed proteins are identified by method of Two-dimensional Differential In-Gel Electrophoresis (2D-DIGE) in combination with tandem mass spectrometry. The set of identified proteins provides an insight on protein expression changes associated with our antibody-producing CHO strain. Additionally, this data provides potential protein targets for optimization of antibody production.
Steffen Schulze, University of Waterloo
My research project is the development and optmization of a cultivation process for the expression of a chimeric heavy chain antibody (EG2). To accomplish this, I used a metabolomics approach using Nuclear Magnetic Resonance (NMR) spectroscopy as an analytical tool for the optimization of the growth media. The majority of ingredients in the media are detectable metabolites. The analysis of the consumption or production of metabolites can reveal growth limiting factors and the accumulation of toxic side-products. The aim is to find limiting nutrients for the cell culture. Furthermore, these compounds and different mixtures of these compounds will be tested concerning their ability to improve cell growth and mAb production.
Jann Cathrine Ang, M.Sc. student, University of Waterloo
Glycosylation structure significantly impacts the bioactivity of therapeutic proteins. My proposed research project involves the in vitro modification of N-glycans using N-acetylglucosaminyltransferase III (GnT3). The proposed glycan, EG2, is to be modified in vitro and are produced from the Chinese Hamster Ovary (CHO) cells. For monoclonal antibodies, N-glycosylation is essential for Fc-receptor mediated effector functions. The degree of glycosylation of EG2 that is found in CHO cells are mostly sialic acid containing glycans and glycans with glucose moieties attached to mannose-1,3 and 1,6 alpha branch. In particular, the glycans are modified using GnT3 for the addition of bisecting GlcNAc which shows an increase of ADCC that would be beneficial for cancer and immune therapy. The main goal of the project is to express a functional GnT3 enzyme to successfully modify the glycans in vitro. This would result for the creation of a robust in vitro process that will lead for a uniform population of glycans with N-acetyl glucosamine in the antibody pool.
Katharina Hassel, Ph.D. student, University of Waterloo
My topic is the purification of monoclonal antibodies (mAbs) with ion exchange membranes. The target mAbs are EG2 and Herceptin. Improved mAbs purification processes are needed because purification operations represent 50-80% of the total production cost. The most common purification process has relied on a series of resin chromatography steps with Protein-A affinity chromatography as the initial capture step. Protein-A resins display an excellent specificity for mAbs but are extremely costly, limited by pore diffusion mass transfer and may lead to leaching of the protein A ligand in the eluted product stream. As a result of these limitations, alternative strategies for the purification of mAbs are being investigated. The aim of this project is to develop and evaluate the potential of ion exchange membrane chromatography as an alternative to Protein-A affinity chromatography for the initial capture of mAbs. A weak cation exchange membrane material developed by Natrix is considered. We are interested in the characterization of this weak cation exchange membrane chromatography material as well as surface characteristic, binding and elution capacity of this material in dead-end and cross-flow systems. We collaborate closely with Natrix Separations (http://www.natrixseparations.com/) providing membrane material and BRI (http://www.nrc-cnrc.gc.ca/eng/ibp/bri.html) providing culture samples of the two different mAbs.
Ali Nikdel, University of Waterloo
Kaveh Ohadi, University of Waterloo