AbSorber AB | Date: 2011-02-28
The present invention provides compositions and methods for treating or preventing antibody mediated graft rejection and blood typing.
Kenny D.T.,National University of Ireland |
Gaunitz S.,Karolinska Institutet |
Hayes C.A.,Gothenburg University |
Gustafsson A.,Recopharma AB |
And 4 more authors.
Methods in Molecular Biology | Year: 2013
Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS2 with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exempli fied by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O -glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS n experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence. © Springer Science+Business Media New York 2013. Source
Gustafsson A.,Recopharma AB |
Sjoblom M.,Lule University of Technology |
Strindelius L.,Recopharma AB |
Strindelius L.,Uppsala University |
And 8 more authors.
Glycobiology | Year: 2011
Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG2b) carrying mostly O-glycans and, as a control, the 1-acid glycoprotein (AGP/mIgG2b) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG2b was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by-mannosidases including an 1,2-but not an 1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta-and hexasaccharides being the predominant ones. 1,2-and 1,3-linked, but not 1,6-linked, mannose residues were detected by 1H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG2b to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG2b expressed in P. pastoris carried O-glycans mainly comprised of-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo. © 2011 The Author. Source
Alheim M.,Karolinska University Hospital |
Johansson S.M.,Karolinska University Hospital |
Hauzenberger D.,Karolinska University Hospital |
Grufman P.,AbSorber AB |
Holgersson J.,Karolinska University Hospital
Tissue Antigens | Year: 2010
Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE ®) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8 + and CD 4 + T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3 + lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19 + lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R 2 = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R 2 = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE ® assay. © 2010 John Wiley & Sons A/S. Source
Lindberg L.,AbSorber AB |
Liu J.,AbSorber AB |
Gaunitz S.,Karolinska University Hospital |
Nilsson A.,Recopharma AB |
And 3 more authors.
Glycobiology | Year: 2013
Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIgG2b) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG2b carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored. © 2012 The Author. Source