N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines.

Cells release vesicles to the surroundings, the extracellular vesicles (EVs), which may transmit biomolecules to other cells, and are found in bodily fluids, thus constituting emerging biomarker targets. Many studies on EV nucleic acid, lipid, and protein composition are available; however, detailed characterization of protein glycosylation has been less approached. Here, we describe a strategy for high-resolution quantitative profiling and structure elucidation of N-glycans from EV glycoproteins of three cell lines: human HEK-293, human glioma H4 and mouse glioma Tu-2449. EVs have been purified from cell supernatants by ultracentrifugation and compared with total cellular membranes (CMs). CMs and EVs have been characterized by immunoblotting using a panel of EV-specific antibodies, electron microscopy, and immunocytochemistry. N-Glycans were released from membrane-derived tryptic glycopeptides with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by normal phase-high-pressure liquid chromatography (NP-HPLC) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. For the three cell lines, enrichment in complex N-glycans was found in EVs concomitant to a small amount of high mannose glycans, whereas CMs were highly enriched in high mannose glycans. In HEK-293 and H4 EVs, the predominant N-glycan was tetraantennary proximally fucosylated with α2,3-linked N-acetylneuraminic acid; HEK-293 EVs also contained the LacdiNAc structure. Mouse Tu-2449 EV profiles were very heterogeneous, with di-, tri-, and tetraantennary proximally fucosylated glycans and the presence of peripheral Galα3Gal structure. The results opened novel perspectives to further investigate the roles of glycans in EVs biological properties and may contribute to the biomarker field in glioma.

Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis(x) levels and impaired neurite outgrowth.

BACKGROUND: Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. METHODS: The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. RESULTS AND CONCLUSIONS: One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis(x), as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. GENERAL SIGNIFICANCE: These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons.

N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin.

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.

Exploring Cerebrospinal Fluid IgG N-Glycosylation as Potential Biomarker for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease for which the existing candidate biomarkers (neurofilaments) have low specificity. Changes in blood IgG N-glycosylation have been observed in several diseases, including ALS, whereas cerebrospinal fluid (CSF) IgG has been less studied. Here, we characterized N-glycans of CSF IgG from ALS patients in comparison with a control group of other neurological diseases. Cerebrospinal fluid was collected from patients with ALS (n = 26) and other neurological diseases (n = 10). N-Glycans were released from CSF purified IgG with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by NP-HPLC chromatography in combination with exoglycosidase digestion and MALDI-TOF mass spectrometry. The N-glycosylation profile of ALS CSF IgG consisted of diantennary N-glycans predominantly with proximal fucose and some bisecting GlcNAc; agalacto-, mono-, and digalactosylated as well as α2,6-sialylated structures were detected. Differences between ALS and control patients were observed; most relevant was the increase in ALS CSF IgG of the level of galactosylated structures defined here as Gal-index (median 46.87 and 40.50% for ALS and controls, respectively; p = 0.006). The predictive value of the Gal-index (AUC = 0.792, p = 0.007) considering ROC analysis had potential utility as a diagnostic test for ALS and was comparable to that of phosphoneurofilament heavy chain (AUC = 0.777, p = 0.011), which was used as benchmark marker for our group of patients. The results provide the basis to further explore the potential of IgG N-glycan galactosylation as biomarker for ALS by using larger cohorts of patients and controls.

Characterization of the N-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS).

UNLABELLED: Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to alpha1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. CONCLUSION: Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.

Proteomic analysis of plasma from Portuguese patients with familial amyotrophic lateral sclerosis.

In ALS, the identification of abnormal proteins in biological fluids might be useful for the understanding of the ethiopathogenesis of the disease. Furthermore, it can provide biomarkers useful for diagnosis, to monitor disease progression and to study the effect of drugs. Plasma is a suitable fluid for screening such targets since blood collection is a relatively simple procedure. In this study, proteomic techniques consisting of two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) have been used for the analysis of plasma from a group of Portuguese familial ALS (FALS) patients not carrying SOD1 mutations, age-matched healthy controls, sporadic ALS patients and controls with other muscular disorders. Most relevant was the finding in the FALS patients of an isoform of vitamin D-binding protein (DBP) at pI 5.2, identified as GC2 by liquid chromatography electrospray ionization-TOF MS. GC2 was absent from the healthy controls. Concomitantly, decrease of more acidic isoforms of DBP was observed for the FALS patients. The results suggested that the GC2 polymorphism of DBP could constitute a risk factor for ALS.

LacdiNAc (GalNAcbeta1-4GlcNAc) is a major motif in N-glycan structures of the chicken eggshell protein ovocleidin-116.

The avian eggshell matrix protein ovocleidin-116 (OC-116) contains two N-glycosylation sites in its sequence. One of them, 293N-D-S, is modified only marginally while the second one, 62N-Q-T, is completely occupied by N-linked glycans. The glycopeptide bearing the modified site was isolated by size exclusion chromatography and reversed phase HPLC after cleavage of the protein with lysyl endopeptidase. The carbohydrate structures attached to Asn62 were determined by carbohydrate compositional analysis, methylation analysis and electrospray MS/MS. We identified 17 different oligosaccharide structures. Four of them were of the high-mannose type, eight were hybrid type and five were complex type structures. Both, hybrid and complex type glycans comprised core-fucosylated and peripherally fucosylated structures. Most of the antennae contained the relatively rare lacdiNAc (GalNAcbeta1-4GlcNAc) motif, which was fucosylated in 9 out of 15 structures. The lacNAc (Galbeta1-4GlcNAc) motif, which is the more frequent motif in mammals, only occurred in 3 of the 17 glycoforms. This is the first detailed study of N-glycan structures occurring in an avian shell-specific protein and, to our knowledge, the first description of fucosylated lacdiNAc structures present in avian glycoproteins.

Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.

We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. METHODS: We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. RESULTS: pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). CONCLUSIONS: Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker.

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS.

Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multianternary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3- and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B- and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. Alpha1-3fucosylation using GDP-fucose along with a soluble form of the recombinant human alpha1-3fucosyltransferase VI was carried out on tri- and tetraantennary precursors to test structural requirements for formation of Le(x) versus sLe(x) motifs.

Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells.

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

Changes in the hemagglutinin of H5N1 viruses during human infection--influence on receptor binding.

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.

Production and N-glycosylation of recombinant human cell adhesion molecule L1 from insect cells using the stable expression system. Effect of dimethyl sulfoxide.

L1 is a cell adhesion molecule that is heavily glycosylated and is essential for normal development of the central nervous system. In this work, we compare the N-glycosylation of the L1 mutant that consists of immunoglobulin domains 5 and 6 (L1/Ig5-6), expressed in insect Spodoptera frugiperda Sf9 and Trichoplusia ni Tn cells, using the stable expression system. L1/Ig5-6 levels of 30 and 8mgl(-1) were obtained from the two cell lines, respectively. The N-glycans were characterized by high-performance anion-exchange-chromatography with pulsed-amperometric-detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-glycans from Sf9 cells were more homogeneous and consisted predominantly of the paucimannose-type structure Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4(Fucalpha6)GlcNAc. On the other hand, the N-glycans from Tn cells were more heterogeneous and consisted of paucimannose-type structures with or without terminal N-acetylglucosamine. Allergenic proximal alpha3-linked fucose was only found in Tn cells. Dimethyl sulfoxide at 1.5% concentration has been found to increase the levels of L1/Ig5-6 and the L1 ectodomain in the Sf9 and Tn cells, without affecting cell viability nor protein integrity. Furthermore, the N-glycan composition of L1/Ig5-6 was not affected by dimethyl sulfoxide, with only a slight increase in the percentage of the minor high-mannose-type structures.

Highly glycosylated human alpha interferon: An insight into a new therapeutic candidate.

The type I human interferon alpha (hIFN-alpha) family consists of small proteins that exert a multiplicity of biological actions including antiviral, antiproliferative and immunomodulatory effects. However, though administration of recombinant hIFN-alpha2b is the current treatment for chronic hepatitis B and C and for some types of cancers, therapy outcomes have not been completely satisfactory. The short serum half-life and rapid clearance of the cytokine accounts for its low in vivo biological activity. Here we describe and characterize a long-acting rhIFN-alpha2b mutein, 4N-IFN, which has been created by introducing four N-glycosylation sites via site-directed mutagenesis. The hyperglycosylated protein was found to have a 25-fold longer plasma half-life than the non-glycosylated rhIFN-alpha2b, even greater than the commercial pegylated derivative Intron-A PEG. In addition, glycosylation increased the in vitro stability of the mutein against serum protease inactivation. Interestingly, despite its lower in vitro activity, 4N-IFN showed a markedly enhanced in vivo antitumor activity in human prostate carcinoma implanted in nude mice. MALDI-TOF MS and HPAEC-PAD carbohydrate analyses revealed the presence of high amounts of tetrasialylated (40%) and trisialylated (28%) N-glycan structures, which are consequently responsible for the improved characteristics of the cytokine, making 4N-IFN a new therapeutic candidate for viral and malignant diseases.

Generation of serum-stabilized retroviruses: reduction of alpha1,3gal-epitope synthesis in a murine NIH3T3-derived packaging cell line by expression of chimeric glycosyltransferases.

Retroviral vectors released from mouse-derived packaging cell lines are inactivated in human sera by naturally occurring antibodies due to the recognition of Galalpha1,3Galbeta1,4GlcNAc (alphagal-epitope) decorated surface proteins. In this study, an extensive analysis of the glycosylation potential of NIH3T3-derived PA317 packaging cells using combined MALDI/TOF-MS and HPAE-PAD reveals that 34% of the N-glycan moiety represents alphagal-epitope containing structures. Stable expression of glycosyltransferases and transport signal chimeras has been demonstrated to represent an efficient tool to alter cell- and species-specific glycosylation (Grabenhorst and Conradt, 1999. J. Biol. Chem. 274, 36107-36116). In order to reduce alphagal-epitope synthesis selected chimeric glycosyltransferases were constructed by fusing Golgi-signal sequences for compartment-specific localization with the catalytic domain of alpha2,3-sialyltransferase (ST3). Stable expression of these constructs in these cells resulted in a significant reduced alphagal-epitope synthesis, and moreover, a release of retroviral vectors showing an up to 3.5-fold increase in serum stability. Thus, our results suggest that the stably transfected cells stably transfected with chimeric glycosyltransferases compete efficiently with endogenous alpha1,3-galactosyltransferase. This approach allows favored glycodesign and we anticipate the applicability of such improved retroviral vectors produced by glycosylation engineered host cells for in vivo gene therapy and, furthermore, suggest the therapeutic benefit of this technology for xenotransplantation.

Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system.

We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.

Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding.

The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with alpha 2-->6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLe(x) motif. Proximal alpha 1-->6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively alpha 2-->3-linked N-acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin-Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans.

N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cell line.

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Deltam 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal alpha1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found alpha2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24.

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity.