This is the institutional Repository of the Helmholtz Centre for Infection Research in Braunschweig/Germany (HZI), the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken/Germany, the TWINCORE Zentrum für Exprerimentelle und Klinische Infektionsforschung, Hannover/Germany,Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI), Würzburg/Germany, Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) the Study Centre Hannover, Hannover/Germany and the Centre for Individualised Infection Medicine (CiiM).



    Nimtz, Manfred; Conradt, Harald S.; Department of Genetics and Cell Biology, GBF- Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg1, D-3300 Braunschweig, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Our group has elucidated the carbohydrate structures of a number of pharmaceutically relevant recombinant glycoproteins (IFN-8, IL-2, t-PA, AT II, EPO as well as mutant proteins thereof) expressed in several mammalian host cell lines (CHO, BHK, C127, Ltk) . After enzymatic liberation of the N-linked oligosaccharide chains by action of polypeptide:N-glycanase from the intact glycoprotein or tryptic fragments thereof, the individual oligosaccharides were separated by a combination of ion exchange chromatography and HPLC on NH,-phase. Oligosaccharide structures were elucidated using several analytical techniques: GC/MS (SIM-mode) for compositional and methylation analyses, FAB-MS for the determination of their molecular weight and the terminal substitution pattern as well as 600 MHz 7H-NMR spectrometry for the determination of anomeric configuration and linkage pattern of the monosaccharide building blocks. The recently introduced high-pH-anion-exchange-chromatography with pulsed amperometric detection (HPAE-PAD) was applied for comparison of differently charged oligosaccharide fractions after enzymatic desialylation, determination of oligosaccharide structures at individual glycosylation sites and for control of batch-to-batch consistency of biotechnologically produced glycoproteins.

    Stanley, Pamela; Albert Einstein College of Medicine, Bronx, New York USA 10461 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Most glycoproteins require their carbohydrates in order to be synthesized efficiently and in a biologically active, stable form. However it is clear that the actual structures of these carbohydrates can vary widely; severly truncated forms will often confer the desired characteristics. Minimizing carbohydrate heterogeneity may be advantageous for many reasons, especially in the production of recombinant glycocoproteins. To obtain glycoproteins with a limited set of carbohydrate structures glycosylation mutants with mutations in carbohydrate biosynthesis can be used. Chinese hamster ovary (CHO) glycosylation mutants that are missing an enzyme activity (e.g. a transferase or translocase) synthesize truncated carbohydrates with predictable structures. For purification or tissue-targeting purposes, it may be desirable to embellish carbohydrates with particular sugar residues. Dominant CHO mutants or CHO cells transfected with a cloned glycosyltransferase can be used for this purpose. Most of the CHO mutants that synthesize altered carbohydrates grow well in culture showing that a wide range of carbohydrate structures are compatible with viability. These lines can readily be used to engineer the carbohyrates of recombinant glycoproteins for a multitude of purposes.
  • Remodeling of the Carbohydrate Chains of hCG by Use of Sialyltransferases: Effects on the Biological Activity

    Nemansky, Martin; Eijnden, Dirk H. van den; Dedem, Gijs W. K. van; Mannaerts, Bernadette M. J. L.; Department of Medical Chemistry, Vrije Universiteit, Amsterdam and °Diosynth B.V./Organon B.V., Oss, The Netherlands (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Human chorionic gonadotropin (hCG) is a glycoprotein hormone which contains both N- and O-linked carbohydrate chains. It consists of two subunits, a and 8. The a-subunit contains two N-linked carbohydrate chains: a mono-antenna and a non-fucosylated bi-antenna. The $-subunit contains both (two) N and (four) O-linked carbohydrate structures. Both of the N-linked carbohydrate chainsare biantennary, one of which is fucosylated.

    Zettlmeißl, G.; Conradt, Harald S.; Nimtz, Manfred; Haigwood, N.; Paques, E. P.; 1) Behringwerke AG, Postfach 1140, 3550 Marburg, F.R.G. 2) GBF mbH, Mascheroder Weg 1, 3300 Braunschweig, F.R.G. 3) Chiron Corporation, 456 Horton St. Emeryville, CA 94608, USA (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Comparative analysis of the carbohydrate structure of plasma antithrombin III and recombinant antithrombin III synthesized in Chinese Hamster Ovary cells revealed differences in the linkage of NeuAc, the presence of higher than biantennary structures and the presence of proximal fucose. Treatment of the carbohydrate part of antithrombin III from both sources with glycopeptidase F or sialidase had a strong negative effect on the serum half-life. In order to analyze the effects of elimination of individual carbohydrate side chains on the pharmacokinetic and functional properties of AT III the four N-linked glycosylation sites of the recombinant molecule were altered individually or in combination by site directed mutagenesis of Asn to Gln. All mutants showed a shorter serum half-life compared to natural antithrombin II]. However molecules modified at residues Asn 135, Asn 155 and Asn 192 showed higher heparin affinity and/or maximal stimulation at lower heparin concentrations. As in the case of antithrombin III the three N-glycosylation sites of tissue plasminogen activator mutated individually or in combination. Whereas the specific activities of single glycosylation mutants were unaltered, simultaneous mutation of two (Asn 117 and Asn 184) or three Asn residues to Gln resulted in molecules with 2-3 fold higher specific activities.

    Parekh, R. B.; Oxford GlycoSystems Limited, Unit 4 Hitching Court, Blacklands Way, Abingdon, Oxon. OX14 1RG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    It is being increasingly recognised that many polypeptides of therapeutic interest, which in their native form are glycoproteins, need to be glycosylated in order to be of benefit in vivo..A consideration of polypeptide glycosylation therefore becomes relevant throughoutthe development and production of recombinant glycoproteins, principally for the following reasons. First, cell lines differ in their glycosylation characteristics, and the same polypeptide expressed in two different cell lines will generally be glycosylated differently. As a consequence, a recombinant glycoprotein is usually glycosylated differently to the native form, and such 'non-physiological' glycosylation can have profoundeffects on functional activity, physicochemical properties, and pharmacokinetic behaviour in vivo . A limited set of oligosaccharide determinants has been identified, the members of which influence the pharmacokinetic and immunogenic properties of a glycoprotein. It can therefore prove valuable to screen anycell line chosen for the production of a recombinant polypeptide, for expression of such determinants. Second, to ensurethat any changes in culture method (for example, during scale-up) are not associated with alterations in glycosylation, and that batch-to-batch uniformity is maintained during production,it is necessary to follow the glycosylation pattern of the secreted protein. Third, individual glycoformsof a polypeptide can differ with respect to functional properties. Identification of an improved product may, in some cases, involve nothing morethan isolation of a particular glycosylation variant. These and other aspects of the glycosylation of recombinant glycoproteins are discussed ir this paper.

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