All commercially-available therapeutic monoclonal antibodies (mAbs) contain a consensus N-linked ... more All commercially-available therapeutic monoclonal antibodies (mAbs) contain a consensus N-linked glycosylation site on the constant fragment (Fc) of their heavy chains. The composition of the carbohydrates (glycans) bound to these products determines their safety and therapeutic efficacy [1]. While production cell lines synthesize, glycosylate, and secrete the mAb, they also glycosylate their own components. Therefore, there is a partition of glycan biosynthetic precursors – nucleotide sugars (NSs) – that directly couples mAb glycosylation with cellular growth and metabolism. With this work, we present a novel metabolic flux analysis (MFA) model that establishes a mechanistic and quantitative representation of the partition of metabolic resources between cellular and mAb glycosylation. Experimentally, IgG-producing GS-CHO cells were cultured with three amino acid feeding strategies. Data for cell density, nutrient availability, metabolite accumulation, product titer, intracellular NS concentration [2], and mAb glycoprofiles [3] were collected. Computationally, a metabolic flux analysis (MFA) model that represents 101 metabolites connected by 143 reactions has been developed. In addition to central carbon, amino acid, nucleic acid and lipid metabolism, the MFA also includes the aspartate-malate shuttle, the urea cycle and detailed balances for ATP as well as the NAD(P)+/NAD(P)H redox pair. Demand of cellular resources towards cellular glycosylation has been represented by including stoichiometric coefficients for O-GalNAc, N-linked, glycosphingolipid and GPI anchor glycans in the biomass equation [4]. Product glycosylation has been included in the equation describing mAb composition. The underdetermined MFA (42 degrees of freedom) was constrained to account for reaction reversibility and was solved through multi-objective optimization, where the squared error between experimentally-determined and MFA-calculated fluxes is minimized and ATP synthesis per flux unit was simultaneously maximized [5]. The proposed MFA framework allows us to analyze how metabolic resources are partitioned between cellular and mAb glycosylation. Our results indicate that during exponential growth, cellular glycosylation consumes considerably higher amounts of NS biosynthetic precursors (ATP, glucose, and glutamine). As growth ceases, a larger fraction of metabolic resources is allocated to mAb glycosylation, but total NS consumption decreases. This suggests that cellular glycosylation is the larger metabolic ‘sink’ within our cell line, a result that is consistent with the intracellular accumulation of NSs observed towards latter stages of culture. With further refinement – in particular, with data for dynamic variations in cellular glycosylation – our MFA framework can serve as a computational tool to design optimal NS precursor feeding strategies that control mAb glycosylation and minimize negative impacts on cell growth and productivity. References 1. Jefferis R, 2009. Nat Rev Drug Discov, 8(3):226. 2. del Val IJ, et al., 2013. Anal Biochem, 443(2):172. 3. Stockmann H, et al., 2013. Anal Chem, 85(18):8841. 4. del Val IJ, et al., 2016. Sci Rep, 6:28547. 5. Schuetz R, et al., 2007. Mol Syst Biol, 3:119
ABSTRACT Several different chromatographic methods and a lectin-based assay have been compared fo... more ABSTRACT Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia lectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.
As in proteomics, the speed of the advances in glycomic discovery is dependent upon the developme... more As in proteomics, the speed of the advances in glycomic discovery is dependent upon the development of a specific bioinformatic knowledgebase that links various forms of glycoanalytical data to repositories of known glycan structures. The UniCarbKB project (www.unicarbkb.org) is a partnership of leading international research groups come together in an effort to develop and provide an informatic framework for the storage of high-quality structural glycan collections including informative meta-data and annotated experimental datasets. UniCarbKB seeks to advance the integration of data capture and management within the glycomics discipline.
<jats:p>Metabolic modelling has emerged as a key tool for the characterisation of biopharma... more <jats:p>Metabolic modelling has emerged as a key tool for the characterisation of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimise the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome scale metabolic models (GeMs) of CHO cells are very large (&gt;6000 reactions) and are, therefore, difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy multi-objective optimisation and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and, with further development, will serve as a platform to develop dynamic metabolic models for the control and optimisation of biopharmaceutical cell culture processes.</jats:p>
Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of variou... more Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC). Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis h...
Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly effica... more Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be "orchestrated" depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant's structural attribute and its impact on clinical performance.
O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurode... more O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurodegenerative conditions such as Parkinson's disease (PD) and incidental Lewy body disease (ILBD). This work outlines optimizations of a microwave-assisted nonreductive release to limit glycan degradation and employs this methodology to analyze O-glycosylation on the human striatum and substantia nigra tissue in PD, ILBD, and healthy controls, working alongside well-established reductive release approaches. A total of 70 Oglycans were identified, with ILBD presenting significantly decreased levels of mannose-core (p = 0.017) and glucuronylated structures (p = 0.039) in the striatum and PD presenting an increase in sialylation (p < 0.001) and a decrease in sulfation (p = 0.001). Significant increases in sialylation (p = 0.038) in PD were also observed in the substantia nigra. This is the first study to profile the whole nigrostriatal O-glycome in healthy, PD, and ILBD tissues, outlining disease biomarkers alongside benefits of employing orthogonal techniques for O-glycan analysis.
The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a p... more The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.
Background: Glycosylation is one of the most fundamental post-translational modifications. Import... more Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O 2) and compared to normoxia (21% O 2). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively. Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro.
The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughp... more The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughput generation of large amounts of glycomics data. This allows bioprocess engineers to identify critical process parameters that control the glycosylation critical quality attributes. The advances made in protocols for capillary electrophoresis-laser-induced fluorescence (CE-LIF) measurements of antibody N-glycans have increased the potential for generating large datasets of N-glycosylation values for assessment. With large cohorts of CE-LIF data, peak picking and peak area calculations still remain a problem for fast and accurate quantitation, despite the presence of internal and external standards to reduce misalignment for the qualitative analysis. The peak picking and area calculation problems are often due to fluctuations introduced by varying process conditions resulting in heterogeneous peak shapes. Additionally, peaks with co-eluting glycans can produce peaks of a non-Gaussian nat...
Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newbor... more Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity.In vivo, two B mAb-Ds with 77–81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fu...
expression was performed using SPSS. The Broad institute Morpheus tool was used for hierachical c... more expression was performed using SPSS. The Broad institute Morpheus tool was used for hierachical cluster analysis of the TCGA RNA-Seq data to identify associations of lncRNA expression with UC molecular subtypes. Results and discussions Consistent with the TCGA RNA-Seq data, qRT-PCR analysis of our large tissue set revealed both TINCR and DANCR to be upregulated in UC and in B-SCC compared to benign tissues. According to the qRT-PCR data, DANCR expression was significantly elevated in non-invasive over invasive tumours. Kaplan-Meier analysis did not reveal associations of patient outcome with upregulated lncRNA expression, except that high TINCR expression was associated with worse metastasis-free survival. Hierarchical cluster analysis indicated that the BASQ subtype, which is defined by low expression of luminal marker genes (FOXA1, GATA3) and high expression of basal and squamous marker genes (KRT5, KRT6, KRT14), is also characterised by intermediate TINCR expression. Conclusion Both TINCR and DANCR expression are frequently upregulated in UC, but are not strongly associated with clinical parameters. Instead, our data support the emerging consensus that specific lncRNA expression patterns are associated with and may contribute to the characteristics of UC molecular subtypes.
ABSTRACTGlycomics targets released glycans from proteins, lipids and proteoglycans. High throughp... more ABSTRACTGlycomics targets released glycans from proteins, lipids and proteoglycans. High throughput glycomics is based on mass spectrometry (MS) that increasingly depends on exchange of data with databases and the use of software. This requires an agreed format for accurately recording of experiments, developing consistent storage modules and granting public access to glycomic MS data. The introduction of the MIRAGE (Mimimum Requirement for A Glycomics Experiment) reporting standards for glycomics was the first step towards automating glycomic data recording. This report describes a glycomic e-infrastructure utilizing a well established glycomics recording format (GlycoWorkbench), and a dedicated web tool for submitting MIRAGE-compatible MS information into a public experimental repository, UniCarb-DR. The submission of data to UniCarb-DR should be a part of the submission process for publications with glycomics MSn that conform to the MIRAGE guidelines. The structure of this pipeli...
The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from prote... more The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.
Glycosylation, one of the most fundamental post-translational modifications, is altered in many c... more Glycosylation, one of the most fundamental post-translational modifications, is altered in many cancers. These alterations have been proven to impact on the progression of the tumor cell and promote survival. In literature published over the last half century, it is obvious there is a clear link between (a) chemo-resistance and hypoxia (b) hypoxia and epigenetics and more recently (c) glycosylation and epigenetics. We aim to bring these paradigms together with the remit of offering a more complete story and opening up new avenues of approach for the detection, diagnosis and treatment of breast and ovarian cancer. Ovarian and breast cancer cells were exposed to differential hypoxic conditions (0.5%, 1.0%, 2.0%) and normoxia. Firstly, we compared the methylation status of hypoxia exposed cells to the normoxic controls. A combination of hydrophilic interaction liquid chromatography (HILIC) and statistical analyses allowed comparisons of the secreted N-glycans from cells exposed to diff...
All commercially-available therapeutic monoclonal antibodies (mAbs) contain a consensus N-linked ... more All commercially-available therapeutic monoclonal antibodies (mAbs) contain a consensus N-linked glycosylation site on the constant fragment (Fc) of their heavy chains. The composition of the carbohydrates (glycans) bound to these products determines their safety and therapeutic efficacy [1]. While production cell lines synthesize, glycosylate, and secrete the mAb, they also glycosylate their own components. Therefore, there is a partition of glycan biosynthetic precursors – nucleotide sugars (NSs) – that directly couples mAb glycosylation with cellular growth and metabolism. With this work, we present a novel metabolic flux analysis (MFA) model that establishes a mechanistic and quantitative representation of the partition of metabolic resources between cellular and mAb glycosylation. Experimentally, IgG-producing GS-CHO cells were cultured with three amino acid feeding strategies. Data for cell density, nutrient availability, metabolite accumulation, product titer, intracellular NS concentration [2], and mAb glycoprofiles [3] were collected. Computationally, a metabolic flux analysis (MFA) model that represents 101 metabolites connected by 143 reactions has been developed. In addition to central carbon, amino acid, nucleic acid and lipid metabolism, the MFA also includes the aspartate-malate shuttle, the urea cycle and detailed balances for ATP as well as the NAD(P)+/NAD(P)H redox pair. Demand of cellular resources towards cellular glycosylation has been represented by including stoichiometric coefficients for O-GalNAc, N-linked, glycosphingolipid and GPI anchor glycans in the biomass equation [4]. Product glycosylation has been included in the equation describing mAb composition. The underdetermined MFA (42 degrees of freedom) was constrained to account for reaction reversibility and was solved through multi-objective optimization, where the squared error between experimentally-determined and MFA-calculated fluxes is minimized and ATP synthesis per flux unit was simultaneously maximized [5]. The proposed MFA framework allows us to analyze how metabolic resources are partitioned between cellular and mAb glycosylation. Our results indicate that during exponential growth, cellular glycosylation consumes considerably higher amounts of NS biosynthetic precursors (ATP, glucose, and glutamine). As growth ceases, a larger fraction of metabolic resources is allocated to mAb glycosylation, but total NS consumption decreases. This suggests that cellular glycosylation is the larger metabolic ‘sink’ within our cell line, a result that is consistent with the intracellular accumulation of NSs observed towards latter stages of culture. With further refinement – in particular, with data for dynamic variations in cellular glycosylation – our MFA framework can serve as a computational tool to design optimal NS precursor feeding strategies that control mAb glycosylation and minimize negative impacts on cell growth and productivity. References 1. Jefferis R, 2009. Nat Rev Drug Discov, 8(3):226. 2. del Val IJ, et al., 2013. Anal Biochem, 443(2):172. 3. Stockmann H, et al., 2013. Anal Chem, 85(18):8841. 4. del Val IJ, et al., 2016. Sci Rep, 6:28547. 5. Schuetz R, et al., 2007. Mol Syst Biol, 3:119
ABSTRACT Several different chromatographic methods and a lectin-based assay have been compared fo... more ABSTRACT Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia lectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.
As in proteomics, the speed of the advances in glycomic discovery is dependent upon the developme... more As in proteomics, the speed of the advances in glycomic discovery is dependent upon the development of a specific bioinformatic knowledgebase that links various forms of glycoanalytical data to repositories of known glycan structures. The UniCarbKB project (www.unicarbkb.org) is a partnership of leading international research groups come together in an effort to develop and provide an informatic framework for the storage of high-quality structural glycan collections including informative meta-data and annotated experimental datasets. UniCarbKB seeks to advance the integration of data capture and management within the glycomics discipline.
<jats:p>Metabolic modelling has emerged as a key tool for the characterisation of biopharma... more <jats:p>Metabolic modelling has emerged as a key tool for the characterisation of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimise the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome scale metabolic models (GeMs) of CHO cells are very large (&gt;6000 reactions) and are, therefore, difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy multi-objective optimisation and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and, with further development, will serve as a platform to develop dynamic metabolic models for the control and optimisation of biopharmaceutical cell culture processes.</jats:p>
Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of variou... more Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC). Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis h...
Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly effica... more Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be "orchestrated" depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant's structural attribute and its impact on clinical performance.
O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurode... more O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurodegenerative conditions such as Parkinson's disease (PD) and incidental Lewy body disease (ILBD). This work outlines optimizations of a microwave-assisted nonreductive release to limit glycan degradation and employs this methodology to analyze O-glycosylation on the human striatum and substantia nigra tissue in PD, ILBD, and healthy controls, working alongside well-established reductive release approaches. A total of 70 Oglycans were identified, with ILBD presenting significantly decreased levels of mannose-core (p = 0.017) and glucuronylated structures (p = 0.039) in the striatum and PD presenting an increase in sialylation (p < 0.001) and a decrease in sulfation (p = 0.001). Significant increases in sialylation (p = 0.038) in PD were also observed in the substantia nigra. This is the first study to profile the whole nigrostriatal O-glycome in healthy, PD, and ILBD tissues, outlining disease biomarkers alongside benefits of employing orthogonal techniques for O-glycan analysis.
The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a p... more The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.
Background: Glycosylation is one of the most fundamental post-translational modifications. Import... more Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O 2) and compared to normoxia (21% O 2). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively. Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro.
The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughp... more The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughput generation of large amounts of glycomics data. This allows bioprocess engineers to identify critical process parameters that control the glycosylation critical quality attributes. The advances made in protocols for capillary electrophoresis-laser-induced fluorescence (CE-LIF) measurements of antibody N-glycans have increased the potential for generating large datasets of N-glycosylation values for assessment. With large cohorts of CE-LIF data, peak picking and peak area calculations still remain a problem for fast and accurate quantitation, despite the presence of internal and external standards to reduce misalignment for the qualitative analysis. The peak picking and area calculation problems are often due to fluctuations introduced by varying process conditions resulting in heterogeneous peak shapes. Additionally, peaks with co-eluting glycans can produce peaks of a non-Gaussian nat...
Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newbor... more Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity.In vivo, two B mAb-Ds with 77–81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fu...
expression was performed using SPSS. The Broad institute Morpheus tool was used for hierachical c... more expression was performed using SPSS. The Broad institute Morpheus tool was used for hierachical cluster analysis of the TCGA RNA-Seq data to identify associations of lncRNA expression with UC molecular subtypes. Results and discussions Consistent with the TCGA RNA-Seq data, qRT-PCR analysis of our large tissue set revealed both TINCR and DANCR to be upregulated in UC and in B-SCC compared to benign tissues. According to the qRT-PCR data, DANCR expression was significantly elevated in non-invasive over invasive tumours. Kaplan-Meier analysis did not reveal associations of patient outcome with upregulated lncRNA expression, except that high TINCR expression was associated with worse metastasis-free survival. Hierarchical cluster analysis indicated that the BASQ subtype, which is defined by low expression of luminal marker genes (FOXA1, GATA3) and high expression of basal and squamous marker genes (KRT5, KRT6, KRT14), is also characterised by intermediate TINCR expression. Conclusion Both TINCR and DANCR expression are frequently upregulated in UC, but are not strongly associated with clinical parameters. Instead, our data support the emerging consensus that specific lncRNA expression patterns are associated with and may contribute to the characteristics of UC molecular subtypes.
ABSTRACTGlycomics targets released glycans from proteins, lipids and proteoglycans. High throughp... more ABSTRACTGlycomics targets released glycans from proteins, lipids and proteoglycans. High throughput glycomics is based on mass spectrometry (MS) that increasingly depends on exchange of data with databases and the use of software. This requires an agreed format for accurately recording of experiments, developing consistent storage modules and granting public access to glycomic MS data. The introduction of the MIRAGE (Mimimum Requirement for A Glycomics Experiment) reporting standards for glycomics was the first step towards automating glycomic data recording. This report describes a glycomic e-infrastructure utilizing a well established glycomics recording format (GlycoWorkbench), and a dedicated web tool for submitting MIRAGE-compatible MS information into a public experimental repository, UniCarb-DR. The submission of data to UniCarb-DR should be a part of the submission process for publications with glycomics MSn that conform to the MIRAGE guidelines. The structure of this pipeli...
The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from prote... more The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.
Glycosylation, one of the most fundamental post-translational modifications, is altered in many c... more Glycosylation, one of the most fundamental post-translational modifications, is altered in many cancers. These alterations have been proven to impact on the progression of the tumor cell and promote survival. In literature published over the last half century, it is obvious there is a clear link between (a) chemo-resistance and hypoxia (b) hypoxia and epigenetics and more recently (c) glycosylation and epigenetics. We aim to bring these paradigms together with the remit of offering a more complete story and opening up new avenues of approach for the detection, diagnosis and treatment of breast and ovarian cancer. Ovarian and breast cancer cells were exposed to differential hypoxic conditions (0.5%, 1.0%, 2.0%) and normoxia. Firstly, we compared the methylation status of hypoxia exposed cells to the normoxic controls. A combination of hydrophilic interaction liquid chromatography (HILIC) and statistical analyses allowed comparisons of the secreted N-glycans from cells exposed to diff...
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Papers by Pauline Rudd