Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression

Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells.     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1?-?6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2?-?3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2?-?6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.     

Expression of anti-apoptotic 30Kc6 gene inhibiting hyperosmotic pressure-induced apoptosis in antibody-producing Chinese hamster ovary cells

In mammalian cell culture, elevating osmotic pressure can improve recombinant protein production by increasing the specific productivity. However, this operation also induces cell apoptosis. Thus, its beneficial effect is compromised. Previously, the expression of the 30Kc6 gene was found to inhibit apoptosis in Chinese hamster ovary (CHO) cells, resulting in an increase in recombinant protein production. In this study, the 30Kc6 gene was introduced into an antibody-producing CHO cell line, and its effect on hyperosmotic pressure-induced apoptosis was investigated. In the standard medium, the expression of 30Kc6 increased cell viability by 34.1% and productivity to 2.3 folds. After the osmotic pressure shift to 410 mOsm/kg, it was found that the viability of the 30Kc6-expressing cell decreased only by 8.5% as compared with that of the standard culture, while it decreased by 27.1% for the control cell. Consequently, the maximum production of the 30Kc6-expressing cell increased to 3.8 folds relative to that of the control cell in the standard condition. However the production rate did not increase for the control cell under the same conditions. 30Kc6 expression inhibited the hyperosmotic pressure-induced apoptosis at least partially because it repressed the mitochondrial membrane potential (MMP) reduction.     

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019     

A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines

Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.     

Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Enhancement of therapeutic monoclonal antibody production in CHO cells using 30Kc6 gene

Over-expression of anti-apoptotic cloned-genes is a widely used strategy for inhibiting apoptosis in mammalian cell culture. In our previous study, we reported Bombyx mori 30K gene improved the production of recombinant proteins in Chinese hamster ovary (CHO) cells. In this study, we reengineered the CHO cells with the 30Kc6 gene and 30Kc19 gene for the production of a therapeutic monoclonal antibody (mAb) directed against the glycoprotein receptor of human platelets. After the medium was changed from serum containing one to serum-free one, expression of 30Kc6 in CHO cells increased the cell viability by 40.8% in 4 days and mAb production by 2.3-fold in 5 days. However, no significant changes in cell viability and mAb production were observed for the cells expressing 30Kc19. In the case of the cells expressing 30Kc6, the specific production rate was also improved. The expression of the 30Kc6 gene increased the cell viability and productivity because it maintained the mitochondrial membrane potential (MMP) and reduced the downstream cascade responses for apoptosis. These results indicate that 30Kc6 outperformed 30Kc19 in terms of cell death-protective capability and the production of monoclonal antibodies in CHO cells.     

Site- and branch-specific sialylation of recombinant human interferon-gamma in Chinese hamster ovary cell culture

Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn(25) and Asn(97)). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn(25) were more heavily sialylated than those of Asn(97). Furthermore, Man(alpha1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.     

Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation

The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

Enhancement of cell growth and viability of CHO cells in serum-free media by 30Kc6 gene expression

Many attempts have been made to develop a serum-free medium on account of the problems caused by serum in mammalian cell culture. However, serum deprivation inhibits cell growth and induces apoptosis. Moreover, adapting host cells to the serum-free medium is difficult and time-consuming. In a previous study, the anti-apoptotic 30K proteins were identified from silkworm hemolymph, which suggests that the 30K genes coding for the anti-apoptotic compound can be used for the anti-apoptosis engineering of mammalian cells. In this study, the 30K genes (30Kc6, 30Kc19, and 30Kc123) were introduced to DG44 CHO cells, which are the mammalian cell line most commonly used by industry for the production of biopharmaceuticals, in order to make them resistant to the apoptosis induced by serum deprivation. Among the 30K genes, the 30Kc6 gene exhibited the highest apoptosis-inhibition activity. When the 30Kc6-expressing cells cultivated in the serum-containing medium were transferred directly to commercially available serum-free media, 30Kc6 expression increased the viable cell density by four-fold through inhibiting serum deprivation-induced apoptosis.     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

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.     

Oligosaccharides modulate the apoptotic activity of glycodelin

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.