Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.
It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.
Research & Development, CSL Behring AG, Bern, Switzerland. fabian.kaesermann@cslbehring.com
Fabian
F
Käsermann
David J
DJ
Boerema
Monika
M
Rüegsegger
Andreas
A
Hofmann
Sandra
S
Wymann
Adrian W
AW
Zuercher
Sylvia
S
Miescher
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D016756:Immunoglobulins, Intravenous / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D054788:Ribosome Inactivating Proteins / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D016756:Immunoglobulins, Intravenous; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid