Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2.

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

Simple Routes to Stable Isotope-Coded Native Glycans.

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon or─following permethylation─on reversed phase.

Pseudomonas syringae DC3000 infection increases glucosylated N-glycans in Arabidopsis thaliana.

Studying the interaction between the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 and Arabidopsis thaliana has shed light onto the various forms of mechanisms plants use to defend themselves against pathogen attack. While a lot of emphasis has been put on investigating changes in protein expression in infected plants, only little information is available on the effect infection plays on the plants N-glycan composition. To close this gap in knowledge, total N-glycans were enriched from P. syringae DC3000-infected and mock treated Arabidopsis seedlings and analyzed via MALDI-TOF-MS. Additionally, fluorescently labelled N-glycans were quantified via HPLC-FLD. N-glycans from infected plants were overall less processed and displayed increased amounts of oligomannosidic N-glycans. As multiple peaks for certain oligomannosidic glycoforms were detected upon separation via liquid chromatography, a porous graphitic carbon (PGC)-analysis was conducted to separate individual N-glycan isomers. Indeed, multiple different N-glycan isomers with masses of two N-acetylhexosamine residues plus 8, 9 or 10 hexoses were detected in the infected plants which were absent in the mock controls. Treatment with jack bean α-mannosidase resulted in incomplete removal of hexoses from these N-glycans, indicating the presence of glucose residues. This hints at the accumulation of misfolded glycoproteins in the infected plants, likely because of endoplasmic reticulum (ER) stress. In addition, poly-hexose structures susceptible to α-amylase treatment were found in the DC3000-infected plants, indicating alterations in starch metabolism due to the infection process.

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy's success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI-MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris.

BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants.

BACKGROUND: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. RESULTS: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. CONCLUSIONS: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.

Bisecting Lewis X in Hybrid-Type N-Glycans of Human Brain Revealed by Deep Structural Glycomics.

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itself─in conjunction with collision induced dissociation─as a starting point for the overdue development of isomer-specific deep structural glycomics.

BGAL1 depletion boosts the level of ß-galactosylation of N- and O-glycans in N. benthamiana.

Glyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco-engineering facilitate customized N-glycosylation of plant-derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human-like ß1,4-galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of ß1,4-galactosyltransferase, many plant-derived glycoproteins still exhibit incomplete processed N-glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are ß-galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove ß1,4- and ß1,3-galactose residues on both N- and O-glycans. Transient BGAL1 down-regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce ß-galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N-glycans on several plant-produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N-glycans of plant-produced glycoproteins.

The Golgi Localization of GnTI Requires a Polar Amino Acid Residue within Its Transmembrane Domain.

The Golgi apparatus consists of stacked cisternae filled with enzymes that facilitate the sequential and highly controlled modification of glycans from proteins that transit through the organelle. Although the glycan processing pathways have been extensively studied, the underlying mechanisms that concentrate Golgi-resident glycosyltransferases and glycosidases in distinct Golgi compartments are poorly understood. The single-pass transmembrane domain (TMD) of n-acetylglucosaminyltransferaseI (GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the contribution of individual amino acid residues within the TMD of Arabidopsis (Arabidopsis thaliana) and Nicotiana tabacum GnTI toward Golgi localization and n-glycan processing. Conserved sequence motifs within the TMD were replaced with those from the established trans-Golgi enzyme α2,6-sialyltransferase and site-directed mutagenesis was used to exchange individual amino acid residues. Subsequent subcellular localization of fluorescent fusion proteins and n-glycan profiling revealed that a conserved Gln residue in the GnTI TMD is essential for its cis/medial-Golgi localization. Substitution of the crucial Gln residue with other amino acids resulted in mislocalization to the vacuole and impaired n-glycan processing in vivo. Our results suggest that sequence-specific features of the GnTI TMD are required for its interaction with a Golgi-resident adaptor protein or a specific lipid environment that likely promotes coat protein complexI-mediated retrograde transport, thus maintaining the steady-state distribution of GnTI in the cis/medial-Golgi of plants.

A subcellular proteome atlas of the yeast Komagataella phaffii.

The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

Oligomannosidic glycans at Asn-110 are essential for secretion of human diamine oxidase.

N-Glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for histamine catabolism, has multiple N-glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N-glycosylation sites Asn-168, Asn-538, and Asn-745 in recombinant hDAO (rhDAO) carry complex-type glycans, whereas Asn-110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn-110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occupied by oligomannosidic glycans. Glycans at Asn-168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn-538 and Asn-745 had similar complex-type glycans with some tissue- and cell line-specific variations. The related copper-containing amine oxidase human vascular adhesion protein-1 also exclusively displayed high-mannose glycosylation at Asn-137. X-ray structures revealed that the residues adjacent to Asn-110 and Asn-137 form a highly conserved hydrophobic cleft interacting with the core trisaccharide. Asn-110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic reticulum. Mutations of Asn-168, Asn-538, and Asn-745 reduced rhDAO secretion by 13, 71, and 32%, respectively. Asn-538/745 double and Asn-168/538/745 triple substitutions reduced rhDAO secretion by 85 and 94%. Because of their locations in the DAO structure, Asn-538 and Asn-745 glycosylations might be important for efficient DAO dimer formation. These functional results are reflected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney, and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues.

Synthetic Phosphodiester-Linked 4-Amino-4-deoxy-l-arabinose Derivatives Demonstrate that ArnT is an Inverting Aminoarabinosyl Transferase.

4-Amino-4-deoxy-l-arabinopyranose (Ara4N) residues have been linked to antibiotic resistance due to reduction of the negative charge in the lipid A and core regions of the bacterial lipopolysaccharide (LPS). To study the enzymatic transfer of Ara4N onto lipid A, which is catalysed by the ArnT transferase, we chemically synthesised a series of anomeric phosphodiester-linked lipid Ara4N derivatives containing linear aliphatic chains as well as E- and Z-configured monoterpene units. Coupling reactions were based on sugar-derived H-phosphonates, followed by oxidation and global deprotection. The enzymatic Ara4N transfer was performed in vitro with crude membranes from a deep-rough mutant from Escherichia coli as acceptor. Product formation was detected by TLC and LC-ESI-QTOF mass spectrometry. Out of seven analogues tested, only the α-neryl derivative was accepted by the Burkholderia cenocepacia ArnT protein, leading to substitution of the Kdo2 -lipid A acceptor and thus affording evidence that ArnT is an inverting glycosyl transferase that requires the Z-configured double bond next to the anomeric phosphate moiety. This approach provides an easily accessible donor substrate for biochemical studies relating to modifications of bacterial LPS that modulate antibiotic resistance and immune recognition.

Engineering of complex protein sialylation in plants.

Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

An oligosaccharyltransferase from Leishmania major increases the N-glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana.

N-glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central-protein complex facilitating the N-glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N-glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single-subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well-established production platform for recombinant proteins. A fluorescent protein-tagged LmSTT3D variant was predominately found in the ER and co-located with plant oligosaccharyltransferase subunits. Co-expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N-glycosylation site occupancy on all N-glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N-glycosylation efficiency in plants.

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison.

BACKGROUND: The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce. RESULTS: A Man5GlcNAc2 glycosylating and a Man8-10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8-10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C. CONCLUSION: This study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

Reduced paucimannosidic N-glycan formation by suppression of a specific ß-hexosaminidase from Nicotiana benthamiana.

Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco-engineering facilitate the elimination of nonmammalian-type glycosylation and introduction of missing pathways for customized N-glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N-glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N-glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, ß-hexosaminidases (HEXOs) that account for the formation of paucimannosidic N-glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO-deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core α1,3-fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N-glycans on plant-produced human α1-antitrypsin.

Glycan profile of CHO derived IgM purified by highly efficient single step affinity chromatography.

Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity. Here we show that IgMs are sensitive to aggregation at very acidic conditions (pH ≤ 3.0) despite often being used for affinity chromatography. We combined pH 3.5 with a high salt concentration to prevent aggregation during elution. The elution strategy presented in this paper will improve IgM processes for further applications. The herein used IgMs were produced in Chinese hamster ovary (CHO) cells. We present the first detailed glycan analysis of IgM produced in CHO cells with predominantly complex type structures at Asn171, Asn332 and Asn395 and oligomannosidic structures at Asn402 and Asn563 similar to human serum-IgM.

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry.

An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar "glyco tune mix". When subjected to liquid chromatography-ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment. Graphical Abstract Discrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS.

Effects of N-glycans on the structure of human IgA2.

The transition of IgA antibodies into clinical development is crucial because they have the potential to create a new class of therapeutics with superior pathogen neutralization, cancer cell killing, and immunomodulation capacity compared to IgG. However, the biological role of IgA glycans in these processes needs to be better understood. This study provides a detailed biochemical, biophysical, and structural characterization of recombinant monomeric human IgA2, which varies in the amount/locations of attached glycans. Monomeric IgA2 antibodies were produced by removing the N-linked glycans in the CH1 and CH2 domains. The impact of glycans on oligomer formation, thermal stability, and receptor binding was evaluated. In addition, we performed a structural analysis of recombinant IgA2 in solution using Small Angle X-Ray Scattering (SAXS) to examine the effect of glycans on protein structure and flexibility. Our results indicate that the absence of glycans in the Fc tail region leads to higher-order aggregates. SAXS, combined with atomistic modeling, showed that the lack of glycans in the CH2 domain results in increased flexibility between the Fab and Fc domains and a different distribution of open and closed conformations in solution. When binding with the Fcα-receptor, the dissociation constant remains unaltered in the absence of glycans in the CH1 or CH2 domain, compared to the fully glycosylated protein. These results provide insights into N-glycans' function on IgA2, which could have important implications for developing more effective IgA-based therapeutics in the future.