Monoclonal Antibody Glycosylation Research Articles

Application of Analytical Quality by Design to the development and validation of reduced and non-reduced capillary electrophoresis analytical procedures for mAb purity determination

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a common analytical procedure used to quantitate critical quality attributes relating to the purity and glycosylation of monoclonal antibodies (mAbs). In this study, the application of an Analytical Quality by Design framework incorporating Design of Experiments was used to develop and validate both non-reduced (CE-NR) and reduced (CE-R) versions of this analytical procedure. Formal risk assessments were used to identify critical method attributes for optimization based on their potential impacts to performance criteria outlined in an analytical target profile. The resulting response surfaces connecting these critical factors to method performance were then utilized to generate a harmonized procedure to reduce execution risk across CE-R and CE-NR applications. Validation of these procedures according to regulatory guidelines support that they meet their required performance criteria, and a multivariate assessment of procedure robustness indicates that method parameters are in a sufficient state of control to ensure appropriate quantitation of mAb quality. Overall, this study demonstrates the utility of adopting an Analytical Quality by Design framework to leverage multidimensional knowledge from multiple critical method parameters to ensure an analytical procedure is fit-for-purpose.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Assessment of monoclonal antibody glycosylation: a comparative study using HRMS, NMR, and HILIC-FLD

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.Graphical

Impact of Glycosylation on Protein-Protein Self-Interactions of Monoclonal Antibodies.

Protein self-interactions measured via second osmotic virial coefficients (B22) and dynamic light scattering interaction parameter values (kD) are often used as metrics for assessing the favorability of protein candidates and different formulations during monoclonal antibody (MAb) product development. Model predictions of B22 or kD typically do not account for glycans, though glycosylation can potentially impact experimental MAb self-interactions. To the best of our knowledge, the impact of MAb glycosylation on the experimentally measured B22 and kD values has not yet been reported. B22 and kD values of two fully deglycosylated MAbs and their native (i.e., fully glycosylated) counterparts were measured by light scattering over a range of pH and ionic strength conditions. Significant differences between B22 and kD of the native and deglycosylated forms were observed at a range of low to high ionic strengths used to modulate the effect of electrostatic contributions. Differences were most pronounced at low ionic strength, indicating that electrostatic interactions are a contributing factor. Though B22 and kD values were statistically equivalent at high ionic strengths where electrostatics were fully screened, we observed protein-dependent qualitative differences, which indicate that steric interactions may also play a role in the observed B22 and kD differences. A domain-level coarse-grained molecular model accounting for charge differences was considered to potentially provide additional insight but was not fully predictive of the behavior across all of the solution conditions investigated. This highlights that both the level of modeling and lack of inclusion of glycans may limit existing models in making quantitatively accurate predictions of self-interactions.

A New Alternative Tool to Analyse Glycosylation in Monoclonal Antibodies Based on Drop-Coating Deposition Raman imaging: A Proof of Concept.

Glycosylation is considered a critical quality attribute of therapeutic proteins as it affects their stability, bioactivity, and safety. Hence, the development of analytical methods able to characterize the composition and structure of glycoproteins is crucial. Existing methods are time consuming, expensive, and require significant sample preparation, which can alter the robustness of the analyses. In this context, we developed a fast, direct, and simple drop-coating deposition Raman imaging (DCDR) method combined with multivariate curve resolution alternating least square (MCR-ALS) to analyze glycosylation in monoclonal antibodies (mAbs). A database of hyperspectral Raman imaging data of glycoproteins was built, and the glycoproteins were characterized by LC-FLR-MS as a reference method to determine the composition in glycans and monosaccharides. The DCDR method was used and allowed the separation of excipient and protein by forming a “coffee ring”. MCR-ALS analysis was performed to visualize the distribution of the compounds in the drop and to extract the pure spectral components. Further, the strategy of SVD-truncation was used to select the number of components to resolve by MCR-ALS. Raman spectra were processed by support vector regression (SVR). SVR models showed good predictive performance in terms of RMSECV, R2CV.

On the cause and consequences of IgE to galactose-α-1,3-galactose: A report from the National Institute of Allergy and Infectious Diseases Workshop on Understanding IgE-Mediated Mammalian Meat Allergy

The mammalian meat allergy known as the "α-Gal syndrome" relates to IgE specific for galactose-α-1,3-galactose (α-Gal), an oligosaccharide that is present in cells and tissues of nonprimate mammals. The recognition of delayed reactions to food derived from mammals in patients with IgE to α-Gal and also the association with tick bites have been increasing worldwide. In 2018, the National Institute of Allergy and Infectious Diseases, Division of Allergy, Immunology and Transplantation, sponsored a workshop on this emerging tick-related disease. International experts from the fields of tick biology, allergy, immunology, infectious disease, and dermatology discussed the current state of our understanding of this emerging medical condition. The participants provided suggestions for specific research priorities and for the development of resources to advance our knowledge of the mechanisms, diagnosis, management, and prevention of this allergic disease. This publication is a summary of the workshop and the panel's recommendations are presented herein.

Development and validation of a platform reduced intact mass method for process monitoring of monoclonal antibody glycosylation during routine manufacturing

ABSTRACT N-linked glycosylation is a primary source of heterogeneity associated with recombinant monoclonal antibodies and plays a key role in a myriad of drug properties associated with biological function. The glycosylation profile of recombinant monoclonal antibodies is influenced by an array of cell culture inputs which must be carefully controlled in order to engineer the desired glycan distribution. A platform reduced intact mass method applied to monoclonal antibodies has been validated as a quantitative method to monitor the relative mannose-5 level as a surrogate for overall high mannose content in cell culture as a control strategy to ensure product quality and process consistency. The method was shown to be linear, accurate, specific, and precise for an IgG1 and IgG4 mAb allowing relative quantitation of mannose-5 in the range 0.8–11.0% and 1.0–6.2%, respectively. The method can be applied at several stages of the production process from cell culture harvest to drug substance/drug product and is amenable to routine GMP batch testing in a quality control laboratory. Testing upstream during cell culture rather than for product release allows for an earlier assessment of product quality as the glycosylation profile remains unchanged during downstream purification.

Support Vector and Locally Weighted regressions to monitor monoclonal antibody glycosylation during CHO cell culture processes, an enhanced alternative to Partial Least Squares regression

Since monoclonal antibodies (mAb) are sensitive to the manufacturing process, several mAb variants can be the result of a single batch production. The most critical source of heterogeneity is glycosylation which has a profound impact on safety and efficacy of the final product. Implementation of monitoring and control of the process using the Quality by Design (QbD) approach may help to ensure mAb specifications, although its implementation is limited by the availability of real-time specific measurements. All current approaches to elucidate mAb glycoforms require sampling and labour-intensive efforts. Thus, glycosylation analysis is often performed with the objective of detecting quality defects at the end of the culture process. In this work, the capability of Near Infrared spectroscopy and chemometric treatment to accurately monitor mAb glycosylation during CHO cells cultures using in situ probes is shown for the first time. Real-time monitoring of glycosylation, in terms of high mannose isoforms, fucosylated, sialylated and galactosylated isoforms as well as non-glycosylated mAb, has been successfully performed by the novel use of Locally Weighted Regression (LWR) and Support Vector Regression (SVR). These encouraging results open the way for the implementation of control systems on the impact of cell culture operating parameters on mAb heterogeneity, particularly glycosylation, during CHO cell culture processes through the QbD approach.

Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and β-1,4-galactosyltransferase (β4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and β4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.

Chemical Structure and Composition of Major Glycans Covalently Linked to Therapeutic Monoclonal Antibodies by Middle-Down Nuclear Magnetic Resonance.

Glycosylation of monoclonal antibodies (mAbs) is a critical quality attribute that can impact mAb drug efficacy and safety. The mAb glycans are inherently heterogeneous in chemical structure and composition of monosaccharides. The established fluorescence or mass-spectrometry (MS) detection methods for glycosylation evaluation may require multiple steps of glycan cleavage or extensive digestion of the mAb, chemical labeling of the glycans, column separation and report the chemical identity of glycans indirectly through retention time and molecular weight values. In demonstrating chemical structure similarity and comparability among mAb drugs, orthogonal analytical methods for measuring glycan chemistry are needed to ensure the quality of drug products. Here, a "middle-down" NMR method is developed as a proof-of-concept approach to measure the domain-specific glycosylation of marketed mAb drugs without cleavage of the glycan moieties. Complete glycan 1H/13C chemical shift assignments were obtained at 13C natural abundance from commercial standard glycans that allowed unambiguous determination of the chemical structure, glycosidic linkage position, and anomeric configuration of each monosaccharide in the major N-glycan scaffolds found in mAb molecules. The analysis of glycan anomeric peaks in two-dimensional (2D) 1H-13C NMR spectra yielded metrics for clinically important mAb quality attributes (i.e., galactosylation (Gal%) and fucosylation (Fuc%)), consistent with literature results using a standard glycan-mapping method. Therefore, the middle-down NMR method provided a facile orthogonal measurement for mAb glycosylation characterization with improved chemical information content on glycan structure determination and quantification, compared to standard approaches.

Fast, robust and high-resolution glycosylation profiling of intact monoclonal IgG antibodies using nanoLC-chip-QTOF

Optimal glycosylation of immunoglobulins is essential in the generation of therapeutic biologicals with respect to efficacy, pharmacokinetics and immunogenic properties. This challenge in the field of biopharmaceuticals requires technologies for fast, robust and quantitative analysis of glycosylation. Current analyses of monoclonal antibody glycosylation are proteolysis-based mass spectrometry methods, which provide detailed structural information, but suffer a number of drawbacks such as lengthy sample preparation with the possibility to introduce artifacts.Here, we describe a fast, robust and high-resolution nanoLC-chip-QTOF method for quantitative analysis of intact monoclonal IgG glycosylation profiling. The method is able to detect hypoglycosylation, i.e. the lack of whole glycans, which is an important advantage over the well-established methods for free N-glycan or glycopeptide analysis. Moreover, the method is highly amenable to automation and because no digestion steps are involved, it provides direct relative quantitative information of both glycans on each IgG attachment site. We demonstrate that the ease and robustness make this technique ideally suited for quality control of the production process of mAb biopharmaceuticals, and provides new opportunities to study the clinical impact of mAb-glycosylation in patients with monoclonal gammopathies.

Designing an Artificial Golgi reactor to achieve targeted glycosylation of monoclonal antibodies

The therapeutic efficacy of monoclonal antibodies (mAbs) is dependent on their glycosylation patterns. As the largest group of currently approved biopharmaceuticals, the microheterogeneity in mAb oligosaccharide profiles deriving from mammalian cell production is a challenge to the biopharmaceutical industry. Disengaging the glycosylation process from the cell may offer significant enhancement of product quality and allow better control and reproducibility in line with the Quality‐by‐Design paradigm. Three potential designs of an Artificial Golgi reactor implementing targeted sequential glycosylation of mAbs are proposed including a (1) microcapillary film reactor, (2) packed bed reactor with nonporous pellets, and (3) packed bed reactor with porous pellets. Detailed mathematical models are developed to predict their performance for a range of design and operational parameters. While all three reactor designs can achieve desired conversion levels, the choice of a particular one depends on the required throughput and the associated cost of enzymes and co‐substrates. © 2016 The Authors AIChE Journal published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers AIChE J, 62: 2959–2973, 2016

Understanding the impact of different bioprocess conditions on monoclonal antibody glycosylation in CHO cell cultures through experimental and computational analyses

Understanding the impact of different bioprocess conditions on monoclonal antibody glycosylation in CHO cell cultures through experimental and computational analyses

In vitro glycoengineering of IgG1 and its effect on Fc receptor binding and ADCC activity.

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.

How does mild hypothermia affect monoclonal antibody glycosylation?

The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution.

A quantitative and mechanistic model for monoclonal antibody glycosylation as a function of nutrient availability during cell culture

Monoclonal antibodies (mAbs) are currently the highest-selling products of the biopharmaceutical industry, having had global sales of over $45 billion in 2012 [1]. All commercially-available mAbs contain a consensus N-linked glycosylation site on each of the Cγ2 domains of their constant fragment (Fc). The monosaccharide composition and distribution of these N-linked carbohydrates (glycans) has been widely reported to directly impact the safety and efficacy of mAbs when administered to patients. Many studies have also shown that manufacturing bioprocess conditions (e.g. nutrient availability, metabolite accumulation, dissolved oxygen, pH, temperature and stirring speed) directly influence the composition and distribution of N-linked glycans bound to mAbs and other recombinant proteins. Given this tight interconnection between manufacturing process conditions, product quality and ensuing safety and therapeutic efficacy, mAbs and their glycosylation present a clear opportunity where process development can be guided by quality by design (QbD) principles. QbD is a conceptual framework that aims to build quality into drug products at every stage of process development. Specifically, implementation of QbD to pharmaceutical process development requires identifying critical quality attributes (CQAs) that define the drug's safety and therapeutic efficacy. QbD then uses all available information on the mechanisms that quantitatively relate process inputs with product quality to control the manufacturing process so that product CQAs are maintained and end-product quality is ensured. Within the QbD context, composition and distribution of the glycans present on the Fc of mAbs is defined as a CQA, and thus, the processes employed in their manufacture must be controlled so that their glycan distribution ensures the required safety and efficacy profiles. Under this perspective, we have defined a mathematical model that mechanistically and quantitatively describes mAb Fc glycosylation as a function of nutrient availability during cell culture. Such a model aims to be used for bioprocess design, control and optimisation, thus facilitating the manufacture of mAbs with built-in glycosylation-associated quality under the QbD scope.

Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection.

The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis.

Industry and regulatory experience of the glycosylation of monoclonal antibodies

We surveyed 23 antibody-related marketing applications for glycoform analytical and functional information. Our database analysis shows a clear trend of increasing sophistication of analytical methods used to identify and quantify glycans. These have revealed a high degree of complexity and heterogeneity of glycans attached to antibody products. The nature of the complexity is influenced by product type and expression system, and may be associated with functional consequences in some but not all cases.

Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals

Therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. The use of mass spectrometry (MS) for the evaluation of recombinant protein sequence and structure provides detailed information regarding amino acid modifications and sequence alterations that have the potential to affect the safety and activity of therapeutic protein products. General MS approaches for the characterization of recombinant therapeutic protein products will be reviewed with particular attention given to the standard MS tools available in most biotechnology laboratories. A number of recent examples will be used to illustrate the utility of MS strategies for evaluation of recombinant protein heterogeneity resulting from post-translational modifications (PTMs), sequence variations generated from proteolysis or transcriptional/translational errors, and degradation products which are formed during processing or final product storage. Specific attention will be given to the MS characterization of monoclonal antibodies as a model system for large, glycosylated, recombinant proteins. Detailed examples highlighting the use of MS for the analysis of monoclonal antibody glycosylation, deamidation, and disulfide mapping will be used to illustrate the application of these techniques to a wide variety of heterogeneous therapeutic protein products. The potential use of MS to support the selection of cell line/clone selection and formulation development for therapeutic antibody products will also be discussed.

Development of a dynamic model of monoclonal antibody production and glycosylation for product quality monitoring

A comprehensive dynamic model to describe monoclonal antibody (MAb)-producing mammalian cell cultures is presented featuring: (i) cell growth kinetics, (ii) cell metabolism, (iii) MAb synthesis and secretion and (iv) MAb glycosylation. Its simulation results are in good agreement with experimental data for macroscopic culture variables and qualitative observations for glycosylation. The combined model allows us to examine in silico and a priori the impact of feeding schedules on the extent of MAb glycosylation and productivity. Optimisation results for different feeding strategies and their effect on glycosylation patterns are also presented, based on which product quality monitoring strategies can be developed.