Coagulation controlling Glycoproteins

Modern medicine increasingly takes advantage of the biological effect of proteins and glycoproteins which are made available in concentrated and highly purified form as therapeutic preparations and are administered to patients. A top-importance in this context can be attributed to the various proteins involved in blood coagulation. These proteins are either attained from plasma sources via fractionation processes or gained by recombinant technology. Virus and pathogen deactivation is of eminent importance and a great task in both instances. Protein preparations used as therapeutics require a careful chemical and functional characterization in order to control the production process, to monitor the structural integrity of the proteins and their correct post-translational modifications and to guarantee the absence of adverse modifications. Modified carbohydrate structures, for instance, may be a source of adverse immunological responses in patients. In order to characterize the chemical structure of proteins and glyco-proteins, the combination of miniaturized high-performance separation methods with high sensitivity mass-spectrometry is the strategy of choice. In the present project the on-line hyphenation of capillary-high-performance-liquid-chromatography (C-HPLC) and capillary-electrophoresis (CE) with soft-ionization mass spectrometry (MS) is used. Tandem mass analyzers with collision induced fragmentation allow the control of the amino acid sequence, the detection of single modified amino acids, and give insight in the chemical structure of the carbohydrate moieties in glycoproteins. One of the project goals will be the improvement and further development of these mentioned analytical methods, particularly when dealing with highly glycosylated and highly modified proteins. As the second and equally important goal, these advanced analytical technology will be applied to the detailed analysis and characterization of the various isoforms of antithrombin III and coagulation factor IX, two plasmatic glycoproteins which carry an essential function in the regulation of blood coagulation and which are thus used as highly active agents in the therapy of bleeding disorders.

The research project dealt with the detailed analysis of the chemical structure of two glycoproteins which play a major role in the control of blood coagulation, i.e, coagulation factor IX (FIX) and antithrombin (AT). The analysis targeted the structure of the peptide chain and of the carbohydrate moieties (glycans) linked to the peptide backbone as well as the presence of further potential post- and co-translational modifications. Both glycoproteins are used as therapeutic drugs (Biopharmaka) for substitution in cases where in patients the respective glycoproteins are present in too low abundances or are dysfunctional due to congenital disorders or acquired diseases. Both glycoproteins can be gained either from human donor plasma after a series of separation-, enrichment-, and pathogen-deactivation steps, or, alternatively, via recombinant DNA technology from mammalian cell lines. Recombinant techniques play a very important role to ensure the availability of these products in sufficient amounts and to affordable costs. However, the glycosylation patterns in recombinant proteins differ from those found in human plasma in many instances with respect to certain structural features. One of the main goals of this project was thus, to determine the glycosylation patterns (i.e., structure of the protein-linked glycans, their position- specificity and their structural variability) of human plasmatic AT and FIX. These patterns of the plasmatic glycoproteins can then serve as standard, to which the pattern of the recombinant proteins have to refer to. The outcome of this project was published in six articles positioned in Peer-reviewed journals and were reported in six oral and three poster presentation at international scientific meetings. The major results are highlighted in the following. (i) Beyond the two already known glycoforms of AT five additional glycoforms could be identified for the first time and their relative abundances could be determined between 1 to 5 %. For the first time fucosylation was found to be present even in human plasmatic samples and the position of fucose was identified as core-linked as well as antenna-linked, forming the Lex antigen epitope. The Presence of fucosylation in human plasmatic AT was unknown up to now although it was frequently found in recombinant human AT out of mammalian cells. (ii) All modifications in the glycan structure were found position-specific with a prevalence for one single glycan out of four. AT has to be seen as being highly conserved with respect to its glycan structures. (This can be seen as indication for the important biological role glycosylation plays in the case of this glycoprotein.) (iii) The detailed glycosylation patterns reported here provide the standard to which the pattern of recombinant AT have to refer to. (iv) With FIX a higher number of glycosylation variant were found, all positioned in the so-called activation peptide of FIX. Here, N- and O-linked glycans contribute to this variability. A significant part of this research project was dedicated to the methodological development of the analytical techniques and strategies necessary for the in detail characterization of protein glycoforms in ranges down to g or pmole levels. For this purpose on-line coupling of high-performance separation methods (particularly capillary electrophoresis, capillary-liquid chromatography and affinity chromatography) and multi-stage mass spectrometry (MS) were used and refined with respect to sensitivity, reliability, robustness and speed as well as data interpretation from the multi-stage MS experiments. Particularly the analysis of glycoforms by CZE-ESI-MS was a rather newly applied strategy which was subsequently chosen by a number of scientific groups dealing with other pharmaceutically relevant glycoproteins. Practically and methodologically important findings were delivered by the investigations on the fragmentation patterns of glyco- and peptide-structures as attained by multi-stage MS in dependence of the type of ionization and the fragmentation conditions (ion-cooling, collision energies etc.). On the basis of these investigations important experience was gained with respect to the analysis of glycan structures by MS and improved strategies for the characterization of glycosylation in glycoproteins were developed.