High Levels Of Mannose Research Articles

Evaluation of Effects of Fc Domain High-Mannose Glycan on Antibody Stability

Elevated levels of CH2 domain N-linked high-mannose (HM) glycans are commonly observed in therapeutic monoclonal antibodies at various stages of the development. The effect of HM glycans on antibody stability was evaluated by using two approaches. In the first approach, immunoglobulin G (IgG) 1 material containing 21% HM was incubated at 29°C for 6 weeks and fractionated into monomeric and aggregate species by using size-exclusion chromatography (SEC). These fractions were analyzed for the levels of HM. No significant difference was observed in the amount of HM in aggregate and monomer fractions indicating that the HM-containing fractions did not have a greater tendency to form aggregates. In the second approach, both IgG1 material and IgG2 material were separated by Concanavalin-A affinity chromatography into a HM-enriched fraction and a HM-depleted fraction, respectively. Real-time and accelerated stability studies were carried out with these fractions together with untreated samples under standard formulation conditions. The stability of these fractions over time was monitored using SEC and cation-exchange chromatography. No significant difference was observed in rates of aggregation or charge variant formation. These data indicate that HM glycans had no effect on the IgG1 and IgG2 product's stability under the formulation conditions studied.

Sustained high plasma mannose less sensitive to fluctuating blood glucose in glycogen storage disease type Ia children

Plasma mannose is suggested to be largely generated from liver glycogen-oriented glucose-6-phosphate. This study examined plasma mannose in glycogen storage disease type Ia (GSD Ia) lacking conversion of glucose-6-phosphate to glucose in the liver. We initially examined fasting--and postprandial 2 h--plasma mannose and other blood carbohydrates and lipids for seven GSD Ia children receiving dietary interventions using cornstarch and six healthy age-matched children. Next, one-day successive intra-individual parameter changes were examined for six affected and two control children. Although there were no significant differences in fasting--and postprandial 2 h--glucose and insulin levels, the mannose level of the affected group was invariably much higher than that of the control group (p < 0.001): the fasting level of the affected group was about two-fold that of the control group; the postprandial-2 h level remained almost unchanged in the affected group, although it was one-half of the fasting level in the control group. Inter-individual analyses revealed that the GSD Ia group mannose level was significantly and positively correlated with lactate and triglycerides levels at both time points (p < 0.01). In each control, mannose levels fluctuated greatly, maintaining strong and significant negative correlations with glucose and insulin levels (p < 0.001). Correlations were lower or nonexistent in GSD Ia children. In individuals with high lactate and triglycerides levels, strikingly high mannose levels never changed against glucose and insulin fluctuations. Plasma mannose is less sensitive to blood glucose and insulin in GSD Ia children. Its basal level and the fluctuation pattern differ by their metabolic activity.

A high‐throughput microchip‐based glycan screening assay for antibody cell culture samples

A high-throughput screening assay was developed to quantify major glycan species in the crude mammalian cell culture samples for monoclonal antibodies (mAbs). This method utilizes high-speed microchip electrophoresis separation following a fast sample preparation procedure. Using a 96-well ultra-filtration membrane, interfering species in the cell culture media were efficiently removed as the samples were concentrated. A commercial microchip electrophoresis instrument was used for high-speed separation, allowing each sample to be analyzed in less than 1 min. This method is well suited for the purpose of high-throughput antibody glycan profiling during cell culture expression, including clone selection and cell culture process optimization. The relative levels of high mannose (HM), fucosylated and galactosylated glycan species in the Fc domain can be determined for hundreds of crude cell culture samples in a few hours.

High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans

Glycan structures attached to the C(H)2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species.

The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose. The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis. However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis. The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results. RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

Demonstration of a keratan sulfate proteoglycan and a mannose-rich glycoconjugate in corpora amylacea of the brain by immunocytochemical and lectin-binding methods

The aim of the present report is to call attention to the fact that corpora amylacea (CA) of the brain have similar biochemical and immunological characteristics as the connective tissue matrix. We have demonstrated that CA reacted positively toward a monoclonal antibody that specifically recognized the polysaccharide determinant on cartilage proteoglycans. Material strongly bound to concanavalin A was demonstrated in CA, blood vessel wall and cartilage and suggested high levels of mannose in these structures. These findings are in agreement with the known fact that both brain and cartilage contain keratan sulfate and high mannose glycoproteins. We propose that CA may result from accumulation of glycoconjugates normally present in the brain tissue matrix as the result of aging.

Carbohydrate-binding component of amphibian embryo cell surfaces: restriction to surface regions capable of cell adhesion.

Superficial cells from early amphibian embryos display regional specializations of their cell surfaces. That portion of the plasma membrane facing the perivitelline space (apical surface) is nonadhesive, whereas, in the same cell, the lateral and basal portions of the plasma membrane will adhere to other cells. These adhesive differences are maintained on single cells that have been dissociated from the intact embryo. Extracts of cleavage-stage Rana pipiens embryos are capable of agglutinating formalinized sheep erythrocytes. The hemagglutination activity can be blocked by a yeast mannan and a family of glycoproteins containing high levels of mannose, indicating the presence of a lectin with oligomannosyl specificity. The cell surface location of this carbohydrate-binding component can be demonstrated by the ability of the formalinized sheep erythrocytes to form rosettes with living dissociated embryonic superficial cells. Rosette formation is blocked by the same inhibitors that are effective in blocking the activity of the crude extracts. The formalinized sheep erythrocytes form rosettes only to those cell surface regions of the superficial cells that are capable of adhering to other amphibian embryo cells. Receptors for concanavalin A, a lectin that binds D-mannose and D-glucose residues, have also been shown to be present exclusively over the adhesive regions of the superficial cells. The involvement of a carbohydrate-binding component with oligomannosyl specificity in the adhesive mechanisms of these cells is suggested by this restriction of both the embryonic amphibian lectin and its possible receptors (concanavalin A receptors) to adhesive regions of the cell surface.

Gargoylism: hydrolysis of beta-galactosides and tissure accumulation of galactose- and mannose-containing compounds.

The sugars present in hydrolyzed extracts of human liver and brain were analyzed by gasliquid chromatography after conversion to their alditol acetates. The samples analyzed were obtained from control subjects, patients with gargoylism, and patients with a few other kinds of storage disorders. Accumulation of galactose was demonstrated in the liver and the brain of two patients with gargoylism, and in the liver samples, high levels of mannose were found too. We also studied the hydrolysis of a number of galactosides by homogenates from different tissues in the control subjects and in the patients. Separation methods and kinetic studies demonstrated the presence in normal human tissues of two different beta-galactosidases, which we call enzyme A and enzyme B, respectively. Enzyme A hydrolyzed all the beta-galactosides tested. Enzyme B hydrolyzed the synthetic substrates tested (4-methylumbelliferyl-, p-nitrophenyl-, o-nitrophenyl-, and phenyl-beta-galactoside) but not the natural substrates tested (ceramide-beta-galactoside, ceramide lactoside, transferrin glycopeptide, and keratan sulfate). Enzyme B also exerted beta-glucosidase activity. In various tissues from patients with gargoylism, deficiency of beta-galactosidase A could be demonstrated. It is suggested that the high level of galactose found in the hydrolyzed extracts of tissues from gargoylism patients is due to storage of galactose-rich glycosaminoglycans and glycopeptides, and that this storage is a result of the deficiency of beta-galactosidase A. The high level of mannose in the liver from gargoylism patients seems to indicate storage of glycopeptide, adding a new group of substances to those known to be stored in gargoylism.