Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.     

O-Glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins: Attachment of glycosaminoglycans and other intermediates with phosphorylation at the xylose sugar subunit

A xylose-based glycosaminoglycan (GAG) core was recently identified at a Ser residue in the linker sequence of a recombinant Fc fusion protein. The linker sequence, G-S-G-G-G-G, and an upstream acidic residue were serving as a substrate for O-xylosyltransferase, resulting in a major glycan composed of Xyl-Gal-Gal-GlcA and other minor intermediates. In this paper, a portion of an unrelated protein was fused to the C-terminus of an IgG Fc domain using the common (G4S)₄ linker repeat. This linker resulted in a heterogenous population of xylose-based glycans all containing at least a core Xyl. Commonly observed glycan structures include GAG-related di-, tri-, tetra-, and penta-saccharides (e.g., Xyl-Gal, Xyl-Gal-Gal, Xyl-Gal-Gal-GlcA, and Xyl-Gal-Gal-GlcA-HexNAc), as well as Xyl-Gal-Neu5Ac. Following alkaline phosphatase or sialidase treatment combined with CID fragmentation, low-level glycans with a mass addition of 79.9 Da were confirmed to be a result of phosphorylated xylose. A minute quantity of phosphorylated GAG pentasaccharides may also be sulfated (also 79.9 Da), possibly at the HexNAc moiety due to non-reactivity to alkaline phosphatase. The xylose moiety may be randomly incorporated in one of the three G-S-G sequence motifs; and the linker peptide shows evidence for multiple additions of xylose at very low levels.     

O-Glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins

A xylose-based glycosaminoglycan (GAG) core was recently identified at a Ser residue in the linker sequence of a recombinant Fc fusion protein. The linker sequence, G-S-G-G-G-G, and an upstream acidic residue were serving as a substrate for O-xylosyltransferase, resulting in a major glycan composed of Xyl-Gal-Gal-GlcA and other minor intermediates. In this paper, a portion of an unrelated protein was fused to the C-terminus of an IgG Fc domain using the common (G4S)₄ linker repeat. This linker resulted in a heterogenous population of xylose-based glycans all containing at least a core Xyl. Commonly observed glycan structures include GAG-related di-, tri-, tetra-, and penta-saccharides (e.g., Xyl-Gal, Xyl-Gal-Gal, Xyl-Gal-Gal-GlcA, and Xyl-Gal-Gal-GlcA-HexNAc), as well as Xyl-Gal-Neu5Ac. Following alkaline phosphatase or sialidase treatment combined with CID fragmentation, low-level glycans with a mass addition of 79.9 Da were confirmed to be a result of phosphorylated xylose. A minute quantity of phosphorylated GAG pentasaccharides may also be sulfated (also 79.9 Da), possibly at the HexNAc moiety due to non-reactivity to alkaline phosphatase. The xylose moiety may be randomly incorporated in one of the three G-S-G sequence motifs; and the linker peptide shows evidence for multiple additions of xylose at very low levels.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Human Dickkopf-1 (huDKK1) protein: characterization of glycosylation and determination of disulfide linkages in the two cysteine-rich domains

Human Dickkopf‐1 (huDKK1), an inhibitor of the canonical Wnt‐signaling pathway that has been implicated in bone metabolism and other diseases, was expressed in engineered Chinese hamster ovary cells and purified. HuDKK1 is biologically active in a TCF/lef‐luciferase reporter gene assay and is able to bind LRP6 coreceptor. In SDS‐PAGE, huDKK1 exhibits molecular weights of 27–28 K and 30 K at ～ 1:9 ratio. By MALDI‐MS analysis, the observed molecular weights of 27.4K and 29.5K indicate that the low molecular weight form may contain O‐linked glycans while the high molecular weight form contains both N‐ and O‐linked glycans. LC‐MS/MS peptide mapping indicates that ～ 92% of huDKK1 is glycosylated at Asn²²⁵ with three N‐linked glycans composed of two biantennary forms with 1 and 2 sialic acid (23% and 60%, respectively), and one triantennary structure with 2 sialic acids (9%). HuDKK1 contains two O‐linked glycans, GalNAc (sialic acid)‐Gal‐sialic acid (65%) and GalNAc‐Gal[sialic acid] (30%), attached at Ser 30 as confirmed by β‐elimination and targeted LC‐MS/MS. The 10 intramolecular disulfide bonds at the N‐ and C‐terminal cysteine‐rich domains were elucidated by analyses including multiple proteolytic digestions, isolation and characterization of disulfide‐containing peptides, and secondary digestion and characterization of selected disulfide‐containing peptides. The five disulfide bonds within the huDKK1 N‐terminal domain are unique to the DKK family proteins; there are no exact matches in disulfide positioning when compared to other known disulfide clusters. The five disulfide bonds assigned in the C‐terminal domain show the expected homology with those found in colipase and other reported disulfide clusters.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

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     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.     

Comparison of VHH‐Fc antibody production in Arabidopsis thaliana,Nicotiana benthamiana and Pichia pastoris

VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH‐Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH‐Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH‐Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N‐glycans were expected for KDEL‐tagged VHH‐Fcs, several VHH‐Fcs with an intact KDEL‐tag carried complex‐type N‐glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH‐Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.     

Isotype‐specific glycosylation analysis of mouse IgG by LC‐MS

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono‐ and disialylated N‐glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N‐glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3‐galactosylated glycans were found.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N‐glycans in protein functionality. Endo‐β‐N‐acetylglucosaminidase (EndoBI‐1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat‐stable enzyme that cleaves the N‐N′‐diacetyl chitobiose moiety found in the N‐glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI‐1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI‐1 is able to cleave a high diversity of N‐glycan structures. Nano‐LC‐Chip–Q‐TOF MS data also revealed that different reaction conditions resulted in different N‐glycan compositions released, thus modifying the relative abundance of N‐glycan types. In general, more sialylated N‐glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI‐1 is able to release a wide variety of N‐glycans, whose compositions can be selectively manipulated using different processing conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1323–1330, 2015     

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Immunoglobin G with α‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies with α‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusively α‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α‐2,6 sialyltransferase produced IgG with significant levels of both α‐2,6 and α‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α‐2,6 sialylation level relative to α‐2,3 sialylation for the α‐2,3 sialyltransferase knockouts when combined with α‐2,6 sialyltransferase overexpression. Indeed, α‐2,3 linked sialic acids were not detected on IgG produced from the α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

Controlling the time evolution of mAb N‐linked glycosylation,Part I: Microbioreactor experiments

N‐linked glycosylation is of key importance for the efficacy of many biotherapeutic proteins such as monoclonal antibodies (mAbs). Media components and cell culture conditions have been shown to significantly affect N‐linked glycosylation during the production of glycoproteins using mammalian cell fed‐batch cultures. These parameters inevitably change in modern industrial processes with concentrated feed additions and cell densities beyond 2 × 10⁷ cells/mL. In order to control the time‐dependent changes of protein glycosylation, an automated microbioreactor system was used to investigate the effects of culture pH, ammonia, galactose, and manganese chloride supplementation on nucleotide sugars as well as mAb N‐linked glycosylation in a time‐dependent way. Two different strategies comprising of a single shift of culture conditions as well as multiple media supplementations along the culture duration were applied to obtain changing and constant glycosylation profiles. The different feeding approaches enabled constant glycosylation patterns throughout the entire culture duration at different levels. By modulating the time evolution of the mAb glycan pattern, not only the endpoint but also the ratios between different glycosylation structures could be modified. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1123–1134, 2016     

BGAL1 depletion boosts the level of β‐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 β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐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 β‐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 β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐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.