Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others. Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.     

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics

Protein glycosylation is an important post‐translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N‐glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody‐dependent cell‐mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1419–1431, 2014     

Glycan analysis of therapeutic glycoproteins

Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content.     

Glycosylation control technologies for recombinant therapeutic proteins

Protein glycosylation is a very important quality attribute of any biopharmaceutical product as it affects the efficacy, serum half-life, and antigenicity of a molecule. The present expression hosts commercially utilized for a recombinant glycoprotein production generally cannot produce a desired and uniform glycan composition and generally exhibit non-human glycans that can lead to unwanted side effects. The authors provide a comprehensive review of various approaches which can be implemented to minimize the glycan heterogeneity for the production of the desired protein with improved glycoforms. The authors also describe that the industry standard expression systems such as mammalian, insect, and yeast are glycoengineered to produce human-like glycan composition of a recombinant product. This review summarizes the recent technologies used for the improvement of the glycan composition of the biotherapeutics, focusing largely on the selection of an appropriate expression host, glycoengineering, and upstream process optimization to control protein glycosylation and thus enhanced biological activity with fewer side effects. Here, we also suggest various approaches such as host and clone selection to achieve expected glycosylation in a recombinant protein. The cell culture, biochemical, and physical process parameters play a key role in the manufacturing of the desired glycoform of a therapeutic protein. Hence, these components are to be considered very carefully while developing such glycoproteins. Also, glycoengineering of production host to modulate the protein glycosylation is also recommended in the present review.

     

Modulation of mAb quality attributes using microliter scale fed‐batch cultures

A high‐throughput DoE approach performed in a 96‐deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO‐S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed‐batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N‐glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines ‐ some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small‐scale bioreactors. Being fast and resource effective, this integrated high‐throughput approach can provide information which is particularly useful during early stage cell culture development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:571–583, 2014     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015     

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell‐ and antibody class‐dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel‐purified MAbs produced in NS0 and CHO cells, in which small‐scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI‐ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi‐well formats. Biotechnol. Bioeng. 2010;107: 902–908. © 2010 Wiley Periodicals, Inc.     

新型冠状病毒的糖基化、糖受体识别及糖链抑制剂的发现北大核心CSCD

新型冠状病毒疫情(COVID-19)是21世纪截至目前人类面对的最为严重的公共卫生事件。疫苗、中和抗体以及小分子化合药物的出现有效预防和阻止了COVID-19的快速传播,而不断出现的病毒突变体却使这些疫苗及药物的效价降低,这对COVID-19的预防及治疗提出了新的挑战。新型冠状病毒(SARS-CoV-2)通常会先黏附于呼吸道表面的大分子糖链——硫酸乙酰肝素,进而与特异性受体人血管紧张素转化酶2(human angiotensin-converting enzyme 2,hACE2)结合,从而实现对人体的侵入。SARS-CoV-2的刺突(spike,S)蛋白是高度糖基化的,而糖基化对于hACE2与S蛋白的结合也有着重要影响,S蛋白在宿主体内还会被一系列凝集素受体所结合,这意味着糖链在SARS-CoV-2的入侵及感染过程中有着重要的作用。基于SARS-CoV-2的糖基化及糖受体识别机制开发糖链抑制剂可能是预防或治疗新型冠状病毒感染的有效手段,相关研究发现海洋来源的硫酸化多糖、肝素分子及其他的一些糖类具有抗SARS-CoV-2的活性。本文系统阐述了新型冠状病毒的糖基化及其糖链在入侵、感染中的作用,并对抗SARS-CoV-2糖链抑制剂的发现和机制研究现状进行了总结,在此基础上还对糖类抗病毒药物的机遇与挑战进行了展望。     

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety,functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as nextgeneration therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.     

Harnessing glycosylation to improve cellulase activity

Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.     

Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers

Glycosylation is among the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we established a binary analysis of cell binding and hierarchical organization of 24 mammalian cell lines. The array was also used to document changes in cell surface glycosylation during cell development and differentiation of primary murine immune system cells. To establish the biological and clinical importance of glycan signatures, the lectin microarray was applied in two systems. First, we analyzed the cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen. Second, we used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification.     

Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcγRIIIa receptor binding activity of an IgG1 antibody

N‐linked Fc glycosylation of IgG1 monoclonal antibody therapeutics can directly influence their mechanism of action by impacting IgG effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). Therefore, identification and detailed characterization of Fc glycan critical quality attributes (CQAs) provides important information for process design and control. A two‐step approach was used to identify and characterize the Fc glycan CQAs for an IgG1 Mab with effector function. First, single factor experiments were performed to identify glycan critical quality attributes that influence ADCC and CDC activities. Next, a full‐factorial design of experiment (DOE) to characterize the possible interactions and relative effect of these three glycan species on ADCC, CDC, and FcγRIIIa binding was employed. Additionally, the DOE data were used to develop models to predict ADCC, CDC, and FcγRIIIa binding of a given configuration of the three glycan species for this IgG1 molecule. The results demonstrate that for ADCC, afuco mono/bi has the largest effect, followed by HM and β‐gal, while FcγRIIIa binding is affected by afuco mono/bi and β‐gal. CDC, in contrast, is affected by β‐gal only. This type of glycan characterization and modeling can provide valuable information for development, manufacturing support and process improvements for IgG products that require effector function for efficacy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1181–1192, 2016     

Modeling the Effect of Amino Acids and Copper on Monoclonal Antibody Productivity and Glycosylation: A Modular Approach

In manufacturing monoclonal antibodies (mAbs), it is crucial to be able to predict how process conditions and supplements affect productivity and quality attributes, especially glycosylation. Supplemental inputs, such as amino acids and trace metals in the media, are reported to affect cell metabolism and glycosylation; quantifying their effects is essential for effective process development. We aim to present and validate, through a commercially relevant cell culture process, a technique for modeling such effects efficiently. While existing models can predict mAb production or glycosylation dynamics under specific process configurations, adapting them to new processes remains challenging, because it involves modifying the model structure and often requires some mechanistic understanding. Here, a modular modeling technique for adapting an existing model for a fed-batch Chinese hamster ovary (CHO) cell culture process without structural modifications or mechanistic insight is presented. Instead, data is used, obtained from designed experimental perturbations in media supplementation, to train and validate a supplemental input effect model, which is used to “patch” the existing model. The combined model can be used for model-based process development to improve productivity and to meet product quality targets more efficiently. The methodology and analysis are generally applicable to other CHO cell lines and cell types.     

Glycosylation and glycan interactions can serve as extracellular machinery facilitating clathrin‐independent endocytosis

In contrast to clathrin‐mediated endocytosis (CME) which is well characterized and understood, little is known about the regulation and machinery underlying clathrin‐independent endocytosis (CIE). There is also a wide variation in the requirements each individual CIE cargo has for its internalization. Recent studies have shown that CIE is affected by glycosylation and glycan interactions. We briefly review these studies and explore how these studies mesh with one another. We then discuss what this sensitivity to glycan interactions could indicate for the regulation of CIE. We address the spectrum of responses CIE has been shown to have with respect to changes in glycan interactions and attempt to reconcile disparate observations onto a shared conceptual landscape. We focus on the mechanisms by which cells can alter the glycan interactions at the plasma membrane and propose that glycosylation and glycan interactions could provide cells with a tool box with which cells can manipulate CIE. Altered glycosylation is often associated with a number of diseases and we discuss how under different disease settings, glycosylation‐based modulation of CIE could play a role in disease progression.      

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.     

Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches

For therapeutic monoclonal antibodies (mAbs), detailed analysis of the structural integrity and heterogeneity, which results from multiple types of post-translational modifications (PTMs), is relevant to various processes, including product characterization, storage stability and quality control. Despite the recent rapid development of new bioanalytical techniques, it is still challenging to completely characterize the proteoform profile of a mAb. As a nearly indispensable tool in mAb analysis, mass spectrometry (MS) provides unique structural information at multiple levels. Here, we tested a hybrid strategy for the comprehensive characterization of micro-heterogeneity by integrating 2 state-of-the-art MS-based approaches, high-resolution native MS and targeted glycan profiling, to perform complementary analysis at the intact protein level and released glycan level, respectively. We compared the performance of these methods using samples of engineered half-body IgG4s and a panel of mAbs approved for human use. The glycosylation characterization data derived from these approaches were found to be mutually consistent in composition profiling, and complementary in identification and relative-quantitation of low-abundant uncommon glycoforms. In addition, multiple other sources of micro-heterogeneity, such as glycation, lack of glycosylation, and loss of light chains, could be detected by this approach, and the contribution of multiple types of modifications to the overall micro-heterogeneity could be assessed using our superposition algorithm. Our data demonstrate that the hybrid strategy allows reliable and thorough characterization of mAbs, revealing product characteristics that would easily be missed if only a single approach were used.     

N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

We studied the variations in N‐linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO‐K1SV cells. The glycans detected on the Fc fragment were mainly of the core‐fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non‐core‐fucosylation between the 105 different cell lines, suggesting clone‐to‐clone variation. These differences may change the Fc‐mediated effector functions by such antibodies. Large variation was also observed in the oligomannose‐5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed‐batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone‐to‐clone glycosylation variation but batch‐to‐batch consistency provides a rationale for selection of optimal production cell lines for large‐scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009