Glycoform Distribution Research Articles

Concomitant reduction of lactate and ammonia accumulation in fed‐batch cultures: Impact on glycoprotein production and quality

Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed-batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine-free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 and 1 mM, respectively. In flask, this resulted in more than a 2.5-fold increase in IFNα2b titer in comparison to the control glutamine-supplied fed-batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC-MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high-quality therapeutic glycoproteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:494-504, 2018.

Identification of methionine oxidation in human recombinant erythropoietin by mass spectrometry: Comparative isoform distribution and biological activity analysis

ABSTRACTBackground: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO. Methods: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins. Results: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2–4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93 mg/mL for the samples of A, B, and C, respectively. Conclusion: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms.

Abstract 1808: Vascular endothelial growth factor receptor-2 (VEGFR-2) N-glycosylation modulates angiogenic signaling

Abstract Angiogenesis, the formation of new blood vessels from pre-existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR-2 is essential for tumor angiogenesis. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like (Ig) domains, each with multiple potential N-glycosylation sites. N-glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the occupancy and glycoform distributions at each of the potential N-glycosylation sites and their putative role(s) in VEGFR-2 function remain largely unknown. The objective of this study is to investigate the functional importance of VEGFR-2 N-glycosylation in VEGFR-2 angiogenic signaling. Porcine aortic endothelial (PAE) cells with ectopic expression of VEGFR-2 were treated with PNGase F to remove N-linked glycans or heat-denatured PNGase F as a control. Following PNGase F treatment for 4 hours, cells were treated with VEGF-A ligand for 0, 5, 10 or 30 minutes. VEGFR-2 phosphorylation (activation) was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody. In addition, a polyclonal anti-VEGFR2 antibody was used to immunoprecipitate untreated and PNGase F-treated VEGFR-2 from PAE cell lysates. Evaluation of the N-glycosylation sites targeted by PNGase F was carried out by gel electrophoresis, followed by protease digestion and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site-specific glycosylation information we performed proteolysis of VEGFR-2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation. We detected a dramatic increase in ligand-mediated activation of VEGFR-2 after treatment with PNGase F, suggesting that certain N-linked glycans may hinder ligand access to the VEGF binding site, or that removal of N-linked glycans results in conformational changes that lead to increased activation of the receptor by VEGF-A. To explore this observation in greater detail, we have created a series of VEGFR-2 N-glycosylation site mutants, and we are now using the mutants to determine which glycosylation sites are involved in the observed modulation of VEGFR-2 signaling. Citation Format: Kevin B. Chandler, Deborah R. Leon, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. Vascular endothelial growth factor receptor-2 (VEGFR-2) N-glycosylation modulates angiogenic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1808. doi:10.1158/1538-7445.AM2017-1808

Model-based analysis of N-glycosylation in Chinese hamster ovary cells.

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products.

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.

Analysis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR‐2) Glycosylation and its Role in Angiogenesis

Angiogenesis, the formation of new blood vessels from pre‐existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR‐2 is essential for tumor angiogenesis. The extracellular domain of VEGFR‐2 contains seven immunoglobulin‐like (Ig) domains, each with multiple potential N‐glycosylation sites, and N‐glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the N‐glycosylation sites and their putative role(s) in VEGFR‐2 function remain largely unknown. The objective of this study is to characterize VEGFR‐2 N‐glycosylation sites using tandem mass spectrometry (MS/MS) and investigate the functional importance of these modifications on VEGFR‐2 angiogenic signaling.VEGFR‐2 isolated from porcine aortic endothelial cell (PAEC) lysate was immunoprecipitated with a polyclonal anti‐VEGFR2 antibody. Evaluation of the purity and site occupancy of VEGFR‐2 were carried out by gel electrophoresis, followed by protease digestion, PNGaseF/H218O treatment, and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site‐specific glycosylation information we performed proteolysis of VEGFR‐2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides via an Agilent 6550 Quadrupole Time‐of‐Flight (Q‐TOF) MS using collision‐induced dissociation.MS/MS analysis of the VEGFR‐2 tryptic digest achieved 56% sequence coverage. We detected candidate N‐glycosylated peptides in the extracellular domain of VEGFR‐2 after treatment with PNGaseF/H218O, including N98, N160, N247, N297, N521, N578, N702 and N719. MS/MS spectra were used to define the locations of the glycosylation sites. These results are the first evidence for which potential VEGFR‐2 N‐glycosylation sites are occupied by N‐linked glycans. In addition, VEGFR‐2 glycopeptide MS/MS data provide information on the glycoform distributions at each occupied site. All seven VEGFR‐2 immunoglobulin domains contain at least one occupied N‐glycosylation site. Observed glycosylation on Ig domain 2, involved in VEGFR‐2 ligand binding, and on Ig domain 7, involved in receptor dimerization and activation, could have important implications for protein folding, ligand binding and receptor dimerization. These analyses are essential for determination of the role of glycosylation in VEGFR‐2 function and trafficking.Support or Funding InformationThis research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.

MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

A new tool for monoclonal antibody analysis

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.

Kidney-synthesized erythropoietin is the main source for the hypoxia-induced increase in plasma erythropoietin in adult humans

Erythropoietin (EPO) is mainly synthesized within renal peritubular fibroblasts, and also other tissues such as the liver possess the ability. However, to what extent non-kidney produced EPO contributes to the hypoxia-induced increase in circulating EPO in adult humans remains unclear. We aimed to quantify this by assessing the distribution of EPO glycoforms which are characterized by posttranslational glycosylation patterns specific to the synthesizing cell. The analysis was performed on samples obtained in seven healthy volunteers before, during and after 1 month of sojourn at 3,454 m altitude. Umbilical cord (UC) plasma served as control. As expected a peak (p < 0.05) in urine (2.3 ± 0.5-fold) and plasma (3.3 ± 0.5-fold) EPO was observed on day 1 of high-altitude exposure, and thereafter the concentration decreased for the urine sample obtained after 26 days at altitude, but remained elevated (p < 0.05) by 1.5 ± 0.2-fold above the initial sea level value for the plasma sample. The EPO glycoform heterogeneity, in the urine samples collected at altitude, did not differ from values at sea level, but were markedly lower (p < 0.05) than the mean percent migrated isoform (PMI) for the umbilical cord samples. Our studies demonstrate (1) UC samples express a different glycoform distribution as compared to adult humans and hence illustrates the ability to synthesis EPO in non-kidney cells during fetal development (2) as expected hypoxia augments circulating EPO in adults and the predominant source here for remains being kidney derived.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.

Comparison of the glycosylation of in vitro generated polyclonal human IgG and therapeutic immunoglobulins

We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography–mass spectrometry, we analyzed IgGs’ glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro.

Applying quality by design to glycoprotein therapeutics: experimental and computational efforts of process control

Therapeutic glycoproteins represent one of the most important classes of products in the pharmaceutical industry, accounting for 77 high-value drugs out of 642 pharmaceuticals approved by the European Medicines Agency. Their therapeutic efficacy, serum half-life and immunogenicity depend on glycosylation, a complex and prominent post-translational event, which in turn is influenced by manufacturing process conditions. For this reason, protein glycosylation is a critical quality attribute for these drugs. Herein, we review the impact of glycosylation on product function, and the role of manufacturing conditions on the resulting glycoform distribution. We further present promising developments in terms of alternative, genetically engineered hosts, as well as advances in process operation that influence the glycan profile of the recombinant product. Finally, we review work on dynamic mathematical modeling for protein glycosylation that allows researchers to evaluate genetic engineering and process operation ...

Applying high‐throughput methods to develop a purification process for a highly glycosylated protein

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns.

Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

High performance liquid chromatography evaluation of serum carbohydrate-deficient transferrin and more sialylated transferrin glycoforms in children

Objectives. The evaluation of the age-specific distribution of transferrin glycoforms in paediatric patients may help in defining reference intervals which are critical for an improved and earlier diagnosis. Design and methods. Serum samples from 224 children (age: 2 months–14 years) were analyzed by HPLC (Bio-Rad CDT/HPLC kit) and glycoforms expressed as percentage of the total area of transferrin (Tf). Results. Asialo- and Monosialo-Tf were not detectable in any patient. Medians (IQR) were respectively 0.92% (0.80–1.04%) for Disialo-Tf; 3.47% (2.69–4.18%) for Trisialo-Tf; 82.54% (81.32–83.53%) for Tetrasialo-Tf; 12.73% (11.91–14.09%) for Pentasialo-Tf. Statistically significant differences in Trisialo-Tf (p < 0.0005), Tetrasialo-Tf (p = 0.001), Pentasialo-Tf (p < 0.0005), but not in Disialo-Tf, were observed between the age groups. Conclusions. Age-specific Disialo-Tf cut-offs are not necessary. In children 1.3% and 6.4% may be suggested as upper limits of normal range to detect increases of Disialo- and Trisialo-Tf. The presence of Asialo- and Monosialo-Tf should be considered an abnormal finding and prompt further investigations.

Immuno-magnetic beads-based extraction-capillary zone electrophoresis-deep UV laser-induced fluorescence analysis of erythropoietin

Erythropoietin (EPO) is an important glycoprotein hormone. Recombinant human EPO (rhEPO) is an important therapeutic drug and can be also used as doping reagent in sports. The analysis of EPO glycoforms in pharmaceutical and sports areas greatly challenges analytical scientists from several aspects, among which sensitive detection and effective and facile sample preparation are two essential issues. Herein, we investigated new possibilities for these two aspects. Deep UV laser-induced fluorescence detection (deep UV-LIF) was established to detect the intrinsic fluorescence of EPO while an immuno-magnetic beads-based extraction (IMBE) was developed to specifically extract EPO glycoforms. Combined with capillary zone electrophoresis (CZE), CZE-deep UV-LIF allows high resolution glycoform profiling with improved sensitivity. The detection sensitivity was improved by one order of magnitude as compared with UV absorbance detection. An additional advantage is that the original glycoform distribution can be completely preserved because no fluorescent labeling is needed. By combining IMBE with CZE-deep UV-LIF, the overall detection sensitivity was 1.5×10−8mol/L, which was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection. It is applicable to the analysis of pharmaceutical preparations of EPO, but the sensitivity is insufficient for the anti-doping analysis of EPO in blood and urine. IMBE can be straightforward and effective approach for sample preparation. However, antibodies with high specificity were the key for application to urine samples because some urinary proteins can severely interfere the immuno-extraction.

Immunochemical Analysis of Mutant CFTR in Lung Explants

Background/Aims: Knowledge about the abundance and distribution of CFTR protein glycoforms in native lung tissue is scarce. For upcoming studies with correctors and potentiators for CFTR it is important to get more information about mutant CFTR protein biochemistry. Target for novel treatment is the most afflicted organ in cystic fibrosis (CF), the lung. Methods: Lung tissue sampled from patients with CF and non-CF donors prior to lung transplantation was examined for CFTR-immunoreactive signals by immunoblot. Quantitation of the immunoreactive signals was carried out by densitometry. Results: The complexglycosylated and mannose-rich CFTR isoforms were present in all non-CF specimens, whereas no or only the immature CFTR isoform was visible in CF samples. Whereas some complexglycosylated CFTR was often present in rectal biopsies of F508del homozygous subjects, no mature CFTR was detectable in CF lungs at the stage of terminal respiratory insufficiency. Conclusion: Immunoblot analysis of CFTR in lung tissue is feasible, but in context of the upcoming studies of CFTR correctors and potentiators rectal biopsies seem to be a more appropriate choice because of their safe and repeatable excision.

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)–TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006–2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.

Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.

IgG Fc N-Glycosylation Changes in Lambert-Eaton Myasthenic Syndrome and Myasthenia Gravis

N-glycosylation of the immunoglobulin Fc moiety influences its biological activity by, for example, modulating the interaction with Fc receptors. Changes in IgG glycosylation have been found to be associated with various inflammatory diseases. Here we evaluated for the first time IgG Fc N-glycosylation changes in well-defined antibody-mediated autoimmune diseases, that is, the neurological disorders Lambert-Eaton myasthenic syndrome and myasthenia gravis, with antibodies to muscle nicotinic acetylcholine receptors or muscle-specific kinase. IgGs were purified from serum or plasma by protein A affinity chromatography and digested with trypsin. Glycopeptides were purified and analyzed by MALDI-FTICR-MS. Glycoform distributions of both IgG1 and IgG2 were determined for 229 patients and 56 controls. We observed an overall age and sex dependency of IgG Fc N-glycosylation, which was in accordance with literature. All three disease groups showed lower levels of IgG2 galactosylation compared to controls. In addition, LEMS patients showed lower IgG1 galactosylation. Notably, the galactosylation differences were not paralleled by a difference in IgG sialylation. Moreover, the level of IgG core-fucosylation and bisecting N-acetylglucosamine were evaluated. The control and disease groups revealed similar levels of IgG Fc core-fucosylation. Interestingly, LEMS patients below 50 years showed elevated levels of bisecting N-acetylglucosamine on IgG1 and IgG2, demonstrating for the first time the link of changes in the level of bisecting N-acetylglucosamine with disease.