Fucose Content Research Articles

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14%, respectively), while the control cells produced predominantly bi-antennary branching (70%). About 53% of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77%, whereas only 61% of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6% after the addition of 0.2mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

ESIMS analysis of fucoidan preparations from Costaria costata, extracted from alga at different life-stages

Four fucoidan fractions from brown alga Costaria costata, collected at different life-stages: vegetative, May (5F2 and 5F3) and generative, July (7F1 and 7F2) collections were characterized. It was found that seaweed synthesizes different set of fucoidans – one with high fucose content and substantial percentage of hexoses and uronic acid and lower sulfate content (7F1, 5F2 and 5F3) and other – highly sulfated galactofucan (7F2). Structural features of fractions 7F2 and 5F3 were predominantly determined by mass spectrometric analysis of low-molecular-weight (LMW) oligosaccharide fragments, obtained by autohydrolysis of 7F2 and mild acid hydrolysis of 5F3 fucoidans. It was found that oligosaccharides from 7F2 fractions were mainly built up of sulfated at C-2 and/or at C-2/C-4 (1→3)-linked α-l-fucopyranose residues. d-Galactose residues, sulfated either at C-2 or C-6, were found as parts of mixed di- and trisaccharides at both termini and, probably, internal. Fucose residues in 5F3 fucoidan fragments were sulfated at C-2 and sometimes at C-4. Galactose residues were sulfated at C-4 and less frequently at C-2. Resistant to hydrolysis fraction was probably a core, built up with fucose, mannose and glucuronic acid. Presumably, oligosaccharide fragments were branches at C-4 of GlcA. They were sulfated at C-2 and sometimes at C-4 (1→3)- and/or (1→4)-linked fucooligosaccharides (sometimes terminated with (1→3)-linked galactose) and sulfated at C-4 or C-2 (1→4)- or, probably, (1→6)-linked galactooligosaccharides, probably, with own branches, formed by (1→2)-linked galactose residues. Unsulfated xylose residues were probably terminal in chains built up of fucose. It was confirmed, that monosaccharide content and structure of fucoidans of vegetative algae changed following its life stage. Generative alga in general produced highly sulfated galactofucan having lower MW along with less sulfated mannoglucuronofucan with higher MW, which was extensively synthesized by vegetative algae.

Mogamulizumab

Mogamulizumab (Poteligeo®) is a defucosylated, humanized monoclonal antibody targeting CC chemokine receptor 4 (CCR4). Development is being carried out by its owner Kyowa Hakko Kirin for various haematological malignancies, and by licensee Amgen for asthma. Mogamulizumab was conceived through Kyowa Hakko Kirin's Potelligent® technology, which produces antibodies with enhanced antibody-dependent cellular cytotoxicity. This is achieved largely by reducing fucose content in the oligosaccharide structure of the Fc region. Mogamulizumab has been approved in Japan for the treatment of relapsed or refractory adult T-cell leukaemia-lymphoma (ATL) and is the first Potelligent® antibody to receive marketing approval anywhere in the world. Phase II development is underway for adult T-cell leukaemia-lymphoma (ATL) and cutaneous T-cell lymphoma in the US, and for peripheral T-cell lymphoma in the US and Europe. Amgen is conducting a phase I US-based study in patients with asthma. This article summarizes the milestones in the development of intravenous mogamulizumab leading to this first approval.

Study of the interactive effect of temperature and pH on exopolysaccharide production by Enterobacter A47 using multivariate statistical analysis

Enterobacter A47 synthesizes fucose-containing exopolysaccharides (EPS). Maximum EPS production (>7.00gL−1) was obtained for temperature and pH within 25–35°C and 6.0–8.0, respectively. Under these conditions, the polymers contained over 30% fucose. Glucose, galactose, and glucuronic acid contents were about 28%, 25%, and 10%, respectively, and the total acyl groups content was about 20wt.%. The average molecular weight (Mw) was around 4.0×106. Outside the optimal temperature and pH ranges, fucose, galactose and glucuronic acid, and the total acyl group contents were reduced, while the glucose content increased, new monomers (rhamnose and glucosamine) were detected, and the Mw increased to ⩾1.10×107.This study revealed the ability of Enterobacter A47 to synthesize different heteropolysaccharides as a function of pH and temperature, a feature that can be exploited to obtain tailored polymer composition. Moreover, the production of high fucose content EPS was stable for wide pH and temperature ranges, which is important for the envisaged industrial development of the bioprocess.

Abstract 2945: Antitumor activities of 2-fluorofucose, an orally active agent that inhibits cell-surface fucosylation

Abstract Fucose is a component of many carbohydrates that are found on the outer membranes of cancer cells. There is evidence that loss of this sugar from cell-surface glycoproteins can change cancer cell behavior, including growth and metastasis. We have previously demonstrated that 2-fluorofucose (SGD-2083) blocks the fucosylation of antibodies in vitro and in vivo due to the depletion of GDP-activated fucose inside cells. These findings provide the basis for evaluating the antitumor activities of SGD-2083, since alterations of the fucose content on cell surfaces could potentially impact tumor growth and adhesion. To explore this, SGD-2083 was given orally to mice with carcinoma and hematologic tumor xenografts. Significant antitumor activities were obtained in prostate, renal cell and colorectal carcinomas, but not in the two hematologic cancer models tested. The doses used to achieve efficacy were well-tolerated. FACS analysis was used to detect alterations in tumor cell surface fucosylation in animals that responded to treatment with SGD-2083. Signal reduction was observed on E-selectin ligand and several other cell surface fucose linkages, while little or no response to the treatment was observed in the fucosylation of cell surface P-selectin ligand. These results show that oral administration of SGD-2083 leads to the alteration of tumor cell surface fucosylation in a well-defined manner, and this may account for the observed activities of this novel antitumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2945. doi:1538-7445.AM2012-2945

A Method to Produce and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression.

α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis. Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE Correspondence: Paul.Martin@nationwidechildrens.org

Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages

Clearance of apoptotic cells, so-called efferocytosis, by alveolar macrophages (AMs) is important for lung homeostasis and is impaired in pulmonary inflammatory diseases, such as chronic obstructive pulmonary disease and asthma. Carbocisteine, a mucoregulatory drug, corrects the contents of fucose in airway mucus and has anti-inflammatory properties in airway inflammation. Thus, we conducted the present study to better understand the anti-inflammatory properties of carbocisteine. First, we induced airway inflammation in mice with lipopolysaccharide intratracheally. Carbocisteine significantly decreased neutrophil numbers in bronchoalveolar lavage fluid at the resolution phase of inflammation, implying the promotion of neutrophil clearance. Then, we investigated whether carbocisteine would enhance the efferocytosis by AMs isolated from mice and found that this drug promoted not only the phagocytosis but also the binding of apoptotic cells to AMs in vitro. Furthermore, carbocisteine decreased the fucose residues stained with fluorescent fucose-binding lectin, Lens culinaris agglutinin, on the cell surface of AMs. We found here that removing fucose residues from cell surfaces of AMs by fucosidase markedly enhanced both the binding and phagocytosis of apoptotic cells. Finally, AMs from mice orally given carbocisteine also promoted both the binding and phagocytosis ex vivo similarly to in vitro. These results suggest that carbocisteine could promote the clearance of apoptotic cells by AMs in airway. In addition, the present findings suggest that the binding and phagocytosis of apoptotic cells may be modulated by fucose residues on the cell surface of AMs.

Structural and Functional Characterization of Fucoidan and Its Procoagulant Effect in An Ex Vivo Model of Factor VIII-Inhibited Guinea Pigs

Abstract 2262Fucoidans are sulfated polysaccharides extracted and purified from brown seaweeds. Described as non-anticoagulant polysaccharides (NASPs), they have been shown to improve clotting in FVIII- and FIX-deficient plasma (Liu et al. Thromb Haemost 2006;95:68), making them good candidates for hemophilia treatment. NASP orally administered to hemophilia A dogs over several weeks resulted in correction to normal ranges in thromboelastography (TEG) parameters and improvement of the cuticle bleeding time (Prasad et al. Blood 2008;111:672). We screened extracts of eleven brown algae species to select the most active, high-quality, homogeneous fucoidan. In-depth structural analysis and assessment of the pro-/anticoagulant and TFPI-inhibiting activities of a selected set of fucoidan preparations was performed. The effect of the best fucoidan candidate was studied in a novel guinea pig model by whole blood TEG monitoring supported by calibrated automated thrombographic (CAT) analysis.Comparative structural studies of fucoidans included monosaccharide composition and elemental analysis, molecular weight and overall structural determination by NMR. 13C-NMR spectroscopy gave a comprehensive picture of fucoidan structure including fucose and alginate content and the relative heterogeneity of the preparation distinguishing between fucoidans of different algae species. Heterogeneity of the materials was highest in U. pinnatifida and L. japonica and lowest in F. vesiculosus (F.v.) samples. Procoagualant activity was shown by CAT and rotation thromboelastometry. Clotting parameters of FVIII-inhibited human blood or plasma were corrected to normal levels at 1 and 3 μg/mL depending on the fucoidan. In addition, the fucoidans were analyzed by BiaCore and a modified dilute prothrombin time assay to show binding to TFPI and inhibition of full-length TFPI activity. The screening process supported the identification and optimization of new fucoidan-specific analytical methods.Furthermore, a CAT protocol optimized for guinea pig plasma was used to show the procoagulant effect of the overall best fucoidan candidate. We spiked F.v. fucoidan into FVIII-inhibited guinea pig plasma between 1 and 100μg/mL and triggered thrombin generation with low amounts of tissue factor. The FVIII-inhibited guinea pig plasma was corrected to normal CAT parameters at ∼30μg/mL NASP. Based on our findings in the CAT assay, we designed a dosage pattern for studying the procoagulant effect using TEG in FVIII-inhibited Dunkin Hartley guinea pigs (n=10) after intravenous administration of 0.1 to 1.6mg/kg NASP. The primary endpoint assessed was R-time (time to clotting). FVIII-inhibited guinea pigs showed a median R-time of ≥120min (no clotting), while R-time was reduced in a non-linear dose-dependent manner after administration of NASP down to 69min (0.4mg/kg NASP). Thus, the results generated in in vitro tests, were confirmed in vivo. This new animal model of induced hemophilia will support the development of alternative hemophilia therapeutics. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Zhang:Baxter Healthcare Corporation: Employment. Till:Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Szabo:Baxter Healthcare Corporation: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Physicochemical properties and bioactivity of brown seaweed fucoidan prepared by ultra high pressure-assisted enzyme treatment

The effects of ultra-high pressure (UHP, 100 MPa, 40, 24 h) processing and enzyme treatment on the physicochemical properties and anticoagulant activity of fucoidan obtained from Undaria pinnatifida sporophyll (UPS) were investigated. Crude UPS fucoidan (FUPS) with an average MW of 877 kDa was slightly depolymerized by UHPassisted Tunicase treatment, yielding fucoidan with a lower MW (600–800 kDa). UHP-enzyme treatment decreased the sulfate, fucose and galactose contents of FUPS but increased its glucose content. From FT-IR spectrum, UHP-enzyme treatment was found not to cause a structural change on S=O and C-O-S. After UHP-enzyme treatment, the sulfate content and average MW of FUPS decreased with the increase of Tunicase concentration. It was found that among the UHP-enzyme-treated fucoidans, FUPS-T0.3-U (Tunicase 0.3% treatment+UHP treatment) possessed the highest anticoagulant activity. FUPS-T0.3-U appeared to inhibit blood coagulation via intrinsic pathway. With the increase of sulfate content in FUPS, activated partial thromboplastin time (APTT) and thrombin time (TT) showed a tendency of increase. APTT and TT had the highest values in FUPS-T0.3-U in which the sulfate concentration was 24%, but on the contrary decreased at a sulfate concentration of above 24%. This result indicates that there is an optimum sulfate concentration for the anticoagulant activity of fucoidan. Consequently, UHP-assisted enzymatic treatment was found to be helpful for the improvement of anticoagulant activities of fucoidan.

Influence of viscous Rhodella grisea (Rhodophyceae) proteoglycan on chemically induced cough reflex

An algal extracellular biopolymer (over 8.5×105Da) composed of carbohydrates (52%) and protein (∼13%) has been isolated from a red alga Rhodella grisea growing in natural conditions by concentration of water medium, alcohol precipitation, dialysis and freeze-drying. This mucilagineous biopolymer contained xylose and its 3-O- and 4-O-methyl derivatives (∼63%), galactose (∼12%), glucuronic acid (11–12%), glucose (∼5%), rhamnose (∼4%), fucose (∼3–4%) and low content of others accompaning sugars. When tested on the citric acid-induced cough and reactivity of airways smooth muscle in vivo in the test system guinea pigs, this biopolymer assigned a significant cough suppressing effect. The reactivity of airways smooth muscle was not affected indicating that expectoration effect was not suppressed by biopolymer application, which is important from the pharmacological point of view.

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.

Designed optimization of a single-step extraction of fucose-containing sulfated polysaccharides from Sargassum sp.

Fucose-containing sulfated polysaccharides can be extracted from the brown seaweed, Sargassum sp. It has been reported that fucose-rich sulfated polysaccharides from brown seaweeds exert different beneficial biological activities including anti-inflammatory, anticoagulant, and anti-viral effects. Classical extraction of fucose-containing sulfated polysaccharides from brown seaweed species typically involves extended, multiple-step, hot acid, or CaCl2 treatments, each step lasting several hours. In this work, we systematically examined the influence of acid concentration (HCl), time, and temperature on the yield of fucose-containing sulfated polysaccharides (FCSPs) in statistically designed two-step and single-step multifactorial extraction experiments. All extraction factors had significant effects on the fucose-containing sulfated polysaccharides yield, with the temperature and time exerting positive effects, and the acid concentration having a negative effect. The model defined an optimized single-step FCSPs extraction procedure for Sargassum sp. (a brown seaweed). A maximal fucose-containing sulfated polysaccharides yield of ∼7% of the Sargassum sp. dry matter was achieved by the optimal extraction procedure of: 0.03 M HCl, 90°C, 4 h. HPAEC-PAD analysis confirmed that fucose, galactose, and glucuronic acid were the major constituents of the polysaccharides obtained by the optimized method. Lower polysaccharide yield, but relatively higher fucose content was obtained with shorter extraction time. The data also revealed that classical multi-step extraction with acid ≥0.2 M HCl at elevated temperature and extended time had a detrimental effect on the FCSPs yield as this treatment apparently disrupted the structural integrity of the polymer and evidently caused degradation of the carbohydrate chains built up of fucose residues.

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive grown characteristics such as a very short population doubling time of 12 to 14 hours, a capacity to reach very high cell density (> 30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.

Carbocisteine normalizes the viscous property of mucus through regulation of fucosylated and sialylated sugar chain on airway mucins

Almost all of fucose and sialic acid in mucus are found on the mucus glycoproteins (mucins), and these sugar components on mucins are known to be associated with the viscous property of mucus. We have reported some aspects of carbocisteine, a mucoregulatory drug, correcting fucose and sialic acid contents in mucus. At present, carbocisteine's expectorant action of airway mucus is postulated to involve — the regulation of fucose and sialic acid contents on mucins. However little information is available about the relationship between the viscosity and sugar contents on mucins when treated with carbocisteine. To investigate further the mechanism behind the action of carbocisteine, the present study prepared MUC5AC fusion protein which has tandem repeat regions associated with MUC5AC, and evaluated the effects of carbocisteine on tumor necrosis factor (TNF)-α-induced increases of mucus viscosity and sialyl-Lewis x-epitopes antigen, an antigen which consists of fucosylated and sialylated sugar chains on the MUC5AC fusion proteins. Carbocisteine inhibited the TNF-α-induced increases of the viscosity and sialyl-Lewis x-epitopes on MUC5AC fusion protein. These findings suggest that carbocisteine may normalize the viscosity of mucus through “balancing” of fucose and sialic acid contents on airway mucins.

Les anticorps EMABling®

The Laboratoire français du fractionnement et des biotechnologies (LFB), the leading manufacturer of plasma-derived medicinal products in France and 6th worldwide, is strongly involved in the development of therapeutic monoclonal antibodies (mAb). For more than 15 years, LFB has been focusing its research effort on the study of structure-function relationship of antibodies. Its studies on the molecular basis of IgG interaction with the receptors for the Fc portion of IgG (FcgRs) has made it possible to develop antibodies with high antibody-dependent cellular cytotoxicity (ADCC) activity and enhanced affinity to FcgRIII (CD16), both correlated to a glycosylation pattern characterized by a low fucose content. Based on these studies, LFB has developed EMABling, a technological platform for the production of antibodies with enhanced cytotoxicity ability. Two EMABling antibodies recently entered clinical development: LFB-R593, a fully human anti-rhesus D (RhD) antibody, for the prevention of feto-maternal allo-immunization in RhD- women, as a substitute for human polyclonal anti-RhD immunoglobulins, and LFB-R603, a monoclonal antibody directed against CD20, for the treatment of B cell malignancies. LFB investment in bioproduction through the recent acquisition of MAbgène company, a fully integrated French contract biopharmaceutical manufacturing company, allows the production of antibodies to a large GMP scale. As a whole, LFB owns a portfolio of several EMABling antibodies with high therapeutic interest, in line with its public health mission.

Effects of cold stress on alterations of physiochemical and chemical properties of rice polysaccharides

The purpose of this research was to physiochemically characterize the temporal expression profiles of polysaccharides from cold-stressed rice. Rice seedlings (at the three-leaf stage) were subjected to cold treatment, and polysaccharides were characterized by size-exclusion chromatography (SEC) and high-performance anion-exchange chromatography (HPAEC). In general, the molecular weight distribution of polysaccharides was similar during the 6-day exposure to cold stress. We observed that very-low-molecular-weight (<1 kDa) polysaccharides increased during extended cold stress. We determined correlations between sugar components in the polysaccharides and prolonged exposure to cold stress. Arabinose was the main sugar component among polysaccharides found in this study. A positive correlation was observed for the contents of glucose and fructose with prolonged cold stress. Negative correlations existed for the contents of fucose and arabinose with cold-stress duration. The present results indicate that changes in the chemical profile of rice polysaccharides were correlated with extended exposure to cold stress.

Comparative Study of the Physicochemical Properties of Rice Endosperm Components Expressing Sugary-2 Mutant in Different Genetic Backgrounds

The physicochemical properties of the endosperm components of sugary-2 mutant rice were examined, characterized and compared to that of the normal type. The protein and amylose contents as well as the palatability score were all higher in sugary-2. For fatty acids, palmitic, linoleic and linolenic acid were all present in significantly higher proportion in the sugary-2 mutant. Analysis of the minor sugars in the endosperm showed that inducing the sugary-2 gene did not increase the amount of all sugar types, higher amounts were only measured in rhamnose and fucose content. Regarding the characteristics of starch granules, the ratio of longer glucose chain length to the distribution of endosperm starches decreased with the insertion of the sugary-2 mutant gene. And it was also found that the gelatinization properties of sugary-2 mutant rice showed a general trend of lower gelatinization temperature while the gelatinization enthalphy was considerably lower.

Evaluation of fruit authenticity and determination of the fruit content of fruit products using FT-NIR spectroscopy of cell wall components

An analytical procedure using Fourier transform near infrared (FT-NIR) spectroscopy and chemometrics with multivariate techniques for the rapid determination of the fruit authenticity and for the quantification of the fruit content was developed, based on the cell wall constituents (alcohol-insoluble residue, AIR, and hemicellulose, HC). The contents of rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose in the hemicellulose fraction of apricots, peaches, and pumpkins determined by gas chromatography were used as references. Furthermore, spectral information was correlated with the fruit content and the gravimetric data obtained from sequential fractionation of the alcohol-insoluble residue. Samples of self-made and commercial apricot and peach fruit preparations, jams, and spreads were included in the investigations. Hemicellulose from 109 samples and AIR from 92 samples was recorded, and principal component regression was used to create calibration models relating chemical and gravimetrical reference values to spectral data. The calibration models provided a good predictability in comparison with the results obtained by reference methods. Good agreement was also obtained for the prediction of the neutral sugar composition of the HC and the fruit content from the AIR. FT-NIR spectroscopy allowed a rapid, accurate and non-destructive assignment of specified fruit from spectral data of the HC fraction and the AIR. Thus, FT-NIR could be applied for investigations on quality control complementing, or even replacing, gas chromatography as the most widespread method for the determination of neutral sugars. Furthermore, preliminary investigation on classification of fruit blends was performed. The probability of belonging to a specific fruit was significantly lowered or precluded for samples with fraudulent admixture like peach or pumpkin to apricot, or pumpkin to peach.

L - Fucosegehalt im Humansperma*

Human semen contains beside the major carbohydrate fructose: galactose, glucose, sorbitol and inositol. By means of gas-liquid chromatography it is possible to identify also L-fucose. Our method requires the removal of hexosamines, amines and amino acids after hydrolysis. The gas-chromatographic separation of the carbohydrates is achieved in form of their trimethylsilyl derivatives. By this method the fucose content of several freeze dried human semen samples of different diagnosis could be determined.