GlycopeptideGraphMS: Improved Glycopeptide Detection and Identification by Exploiting Graph Theoretical Patterns in Mass and Retention Time.

The leading proteomic method for identifying N-glycosylated peptides is liquid chromatography coupled with tandem fragmentation mass spectrometry (LCMS/MS) followed by spectral matching of MS/MS fragment masses to a database of possible glycan and peptide combinations. Such database-dependent approaches come with challenges such as needing high-quality informative MS/MS spectra, ignoring unexpected glycan or peptide sequences, and making incorrect assignments because some glycan combinations are equivalent in mass to amino acids. To address these challenges, we present GlycopeptideGraphMS, a graph theoretical bioinformatic approach complementary to the database-dependent method. Using the AXL receptor tyrosine kinase (AXL) as a model glycoprotein with multiple N-glycosylation sites, we show that those LCMS features that could be grouped into graph networks on the basis of glycan mass and retention time differences were actually N-glycopeptides with the same peptide backbone but different N-glycan compositions. Conversely, unglycosylated peptides did not exhibit this grouping behavior. Furthermore, MS/MS sequencing of the glycan and peptide composition of just one N-glycopeptide in the graph was sufficient to identify the rest of the N-glycopeptides in the graph. By validating the identifications with exoglycosidase cocktails and MS/MS fragmentation, we determined the experimental false discovery rate of identifications to be 2.21%. GlycopeptideGraphMS detected more than 500 unique N-glycopeptides from AXL, triple the number found by a database search with Byonic software, and detected incorrect assignments due to a nonspecific protease cleavage. This method overcomes some limitations of the database approach and is a step closer to comprehensive automated glycoproteomics.

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research.

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody's single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody's interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human ß-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

Thermal and pH stress dictate distinct mechanisms of monoclonal antibody aggregation.

Protein aggregation is a significant challenge in the development of monoclonal antibodies (mAbs), which can be exacerbated by stress conditions encountered along its production pipeline. In this study, we examine how thermal and pH stress conditions influence mAb aggregation mechanisms. We observe a complex interplay between these factors that significantly affects mAb stability, particularly under combined stress conditions. The mAb aggregates formed also varied distinctly in size and properties depending on the pH and thermal conditions, suggesting differences in their underlying mechanisms. Using a combination of experimental methods and kinetic modelling, we found that acidic pH conditions primarily promoted aggregation via the mAb unfolding step, while higher temperature conditions facilitated the formation of larger aggregates via monomer-independent cluster-cluster aggregation steps. These insights underscore the importance of extrinsic stress conditions in determining mAb aggregation propensity, and potentially provides a quantitative framework to holistically assess this across various accelerated stress conditions for the development of stable biologics.

An Integrative Glycomic Approach for Quantitative Meat Species Profiling.

It is estimated that food fraud, where meat from different species is deceitfully labelled or contaminated, has cost the global food industry around USD 6.2 to USD 40 billion annually. To overcome this problem, novel and robust quantitative methods are needed to accurately characterise and profile meat samples. In this study, we use a glycomic approach for the profiling of meat from different species. This involves an O-glycan analysis using LC-MS qTOF, and an N-glycan analysis using a high-resolution non-targeted ultra-performance liquid chromatography-fluorescence-mass spectrometry (UPLC-FLR-MS) on chicken, pork, and beef meat samples. Our integrated glycomic approach reveals the distinct glycan profile of chicken, pork, and beef samples; glycosylation attributes such as fucosylation, sialylation, galactosylation, high mannose, α-galactose, Neu5Gc, and Neu5Ac are significantly different between meat from different species. The multi-attribute data consisting of the abundance of each O-glycan and N-glycan structure allows a clear separation between meat from different species through principal component analysis. Altogether, we have successfully demonstrated the use of a glycomics-based workflow to extract multi-attribute data from O-glycan and N-glycan analysis for meat profiling. This established glycoanalytical methodology could be extended to other high-value biotechnology industries for product authentication.

Rapid characterization of protein productivity and production stability of CHO cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The biopharmaceutical industry has been in pursuit of strategies which can isolate stable and high-producing cell lines. The whole cell mass spectrometry method by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a rapid and simple method for cell characterization based on the differences in the fingerprints of the mass spectra. This work describes how the method was evaluated for the application of screening for stable and high-producing clones from a panel of recombinant Chinese hamster ovary (CHO) cell lines. Detectable m/z values and their relative intensities were collected and processed by partial least squares (PLS). To reduce the errors introduced by the preparation method and spectra noise, high intensity preliminary data was selected and the number of variables introduced was validated by leave-one-out cross-validation. The differences in recombinant protein productivity and titer were revealed by PLS regression with promising results. Partial least-squares discriminant analysis (PLS-DA) was applied to differentiate stable and unstable cell lines as traditional stability testing would require several months involving numerous continuous passages. Results confirmed that the whole cell MALDI-TOF method can be a powerful method for routine monitoring of bioprocesses and study can be further developed by extending the number of the cell lines tested to establish a recombinant cell line database.

Semi-Automated Glycoproteomic Data Analysis of LC-MS Data Using GlycopeptideGraphMS in Process Development of Monoclonal Antibody Biologics.

The glycosylation of antibody-based proteins is vital in translating the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. Simplified mass spectrometers offer ease of operation as well as the portability of method development across various operations. Furthermore, data analysis would need to have a degree of automation to relay information back to the manufacturing line. We set out to investigate the applicability of using a semiautomated data analysis workflow to investigate glycosylation in different biologic development test cases. The workflow involves data acquisition using a BioAccord LC-MS system with a data-analytical tool called GlycopeptideGraphMS along with Progenesis QI to semi-automate glycoproteomic characterisation and quantitation with a LC-MS1 dataset of a glycopeptides and peptides. Data analysis which involved identifying glycopeptides and their quantitative glycosylation was performed in 30 min with minimal user intervention. To demonstrate the effectiveness of the antibody and biologic glycopeptide assignment in various scenarios akin to biologic development activities, we demonstrate the effectiveness in the filtering of IgG1 and IgG2 subclasses from human serum IgG as well as innovator drugs trastuzumab and adalimumab and glycoforms by virtue of their glycosylation pattern. We demonstrate a high correlation between conventional released glycan analysis with fluorescent tagging and glycopeptide assignment derived from GraphMS. GraphMS workflow was then used to monitor the glycoform of our in-house trastuzumab biosimilar produced in fed-batch cultures. The demonstrated utility of GraphMS to semi-automate quantitation and qualitative identification of glycopeptides proves to be an easy data analysis method that can complement emerging multi-attribute monitoring (MAM) analytical toolsets in bioprocess environments.

Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum.

Although Phaeodactylum tricornutum is gaining importance in plant molecular farming for the production of high-value molecules such as monoclonal antibodies, little is currently known about key cell metabolism occurring in this diatom such as protein glycosylation. For example, incorporation of fucose residues in the glycans N-linked to protein in P. tricornutum is questionable. Indeed, such epitope has previously been found on N-glycans of endogenous glycoproteins in P. tricornutum. Meanwhile, the potential immunogenicity of the α(1,3)-fucose epitope present on plant-derived biopharmaceuticals is still a matter of debate. In this paper, we have studied molecular actors potentially involved in the fucosylation of the glycoproteins in P. tricornutum. Based on sequence similarities, we have identified a putative P. tricornutum GDP-L-fucose transporter and three fucosyltransferase (FuT) candidates. The putative P. tricornutum GDP-L-fucose transporter coding sequence was expressed in the Chinese Hamster Ovary (CHO)-gmt5 mutant lacking its endogenous GDP-L-fucose transporter activity. We show that the P. tricornutum transporter is able to rescue the fucosylation of proteins in this CHO-gmt5 mutant cell line, thus demonstrating the functional activity of the diatom transporter and its appropriate Golgi localization. In addition, we overexpressed one of the three FuT candidates, namely the FuT54599, in P. tricornutum and investigated its localization within Golgi stacks of the diatom. Our findings show that overexpression of the FuT54599 leads to a significant increase of the α(1,3)-fucosylation of the diatom endogenous glycoproteins.

LC/MS-based Intact IgG and Released Glycan Analysis for Bioprocessing Applications.

Robust plate based antibody glycan analysis platforms are urgently needed for biopharmaceutical development and manufacturing as well as for clinical biomarker research. A 96-well plate based workflow has been developed to analyze both intact IgG antibodies and released N-glycans using an Orbitrap Fusion Mass Spectrometer and an LC/MS method on the Waters UNIFI platform. Here, such a workflow including protein A purification, PNGaseF digestion, 2-AB labeling, and SPE clean-up is described. The measured IgG glycan profile is consistent with that obtained from non-plate based method and commercial kit and has the advantage of less hands-on time. Also the application of the workflow in cell culture monitoring and clonal selection work is demonstrated. Apart from checking the major glycan structure changes among clones, post translational modifications (PTMs) such as C-terminal lysine residue clipping and N-terminal pyroglutamic acid formation can also be deduced from the workflow.

Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line.

Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner.

Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies.

Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with restored GnT I function.

Therapeutic glycoprotein drugs require a high degree of sialylation of their N-glycans for a better circulatory half-life that results in greater efficacy. It has been demonstrated that Chinese hamster ovary (CHO) glycosylation mutants lacking N-acetylglucosaminyltransferase I (GnT I), when restored by introduction of a functional GnT I, produced highly sialylated erythropoietin (EPO). We have now further engineered one of such mutants, JW152, by inactivating the dihydrofolate reductase (DHFR) gene to allow for the amplification of the EPO gene with methotrexate (MTX). Several MTX-amplified clones maintained the ability to produce highly sialylated EPO and one was selected for culture in a perfusion bioreactor that is used in an existing industrial EPO-production bioprocess. Extensive characterization of the EPO produced was performed using total sialic quantification, HPAEC-PAD and MALDI-TOF MS analyses. Our results demonstrated that the EPO produced by the mutant line exhibits superior sialylation compared to the commercially used EPO-producing CHO clone cultured under the same conditions. Therefore, this mutant has the industrial potential for producing highly sialylated recombinant EPO and potentially other recombinant glycoprotein therapeutics.

Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells.

Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.

Immunoglobulins M survive low-pH conditions used for virus inactivation and for elution from bioaffinity columns.

Tolerance of low pH is crucial for recombinant proteins to survive conditions that might be experienced during manufacturing such as virus inactivation and elution from bioaffinity columns. In this study, we exposed three different purified immunoglobulins M (IgMs) to pH 3.5 for 60 min at room temperature. Treated samples showed no significant aggregation or fragmentation and retained full immunoreactivity, an intact glycosylation profile, and unchanged thermal stability. Because IgMs are serious candidates for next-generation therapeutics, it is essential to know that some of them are stable at low pH.

Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability.

Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by disulfide bridges. Due to the kinetics of mAb assembly, it is suggested that expression of LC and HC in equal amounts is not optimal for IgG production. We designed a set of vectors using internal ribosome entry site (IRES) elements to control LC and HC expression. The intracellular LC:HC ratio of stable IgG expressing Chinese hamster ovary (CHO) cell pools can be controlled effectively at four different ratios of 3.43, 1.24, 1.12, and 0.32. The stable pools were used to study the impact of LC:HC ratio on mAb expression and quality. Gene amplification was most effective for pools with excess LC and generated the highest mAb titers among the transfected pools. When LC:HC ratio was greater than one, more than 97% of the secreted products were IgG monomers. The products also have similar N-glycosylation profiles and conformational stabilities at those ratios. For pools presented a lower LC:HC ratio of 0.32, monomers only constituted half of the product with the other half being aggregates and mAb fragments. High mannose-type N-glycans increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC:HC ratio. Product stability was also adversely affected. The results obtained provide insights to the impact of different LC:HC ratios on stable mAb production and useful information for vector design during generation of mAb producing cell lines.