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. 2012;7(6):e37243.
doi: 10.1371/journal.pone.0037243. Epub 2012 Jun 4.

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation

Fabian Käsermann  1 , David J Boerema, Monika Rüegsegger, Andreas Hofmann, Sandra Wymann, Adrian W Zuercher, Sylvia Miescher
Affiliations

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation

Fabian Käsermann et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors are employees of CSL Behring as indicated in the affiliations. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Lectin affinity chromatography.
A: Typical fractionation chromatogram of 1 gram IVIG on a 90 mL SNA-agarose column, showing the SNA non-binding (-SNA IVIG) flowthrough fraction (FT) and the SNA binding fractions (+SNA IVIG) eluate 1 (E1) and eluate 2 (E2). B: SDS PAGE and C: Lectin Blot (SNA) analysis of the SNA-purified IVIG fractions. IVIG: source IVIG before fractionation, FT: SNA non-binding flowthrough, E1: SNA binding eluate 1, E2: SNA binding eluate 2, NAase IVIG: Neuraminidase treated (desialylated) IVIG, M: molecular marker, red.: gel run under reducing conditions, non red.: gel run under non-reducing conditions.
Figure 2
Figure 2. Overview of SNA affinity chromatography with IgG, Fc and F(ab’)2 fragments.
Schematic presentation of purification strategy showing various starting materials for SNA-chromatography and resulting fractions.
Figure 3
Figure 3. Analysis of total and Fc sialylation by HPLC and LC-MS.
Upper row: sialylation in lectin purified IVIG. Lower row: sialylation in IgG fragments. A: Total sialic acid in IgG of the indicated fractions by HPLC. B: Percentage of sialylated IgG1 Fc-glycopeptides in the indicated IgG fractions (LC-MS). C: Percentage of sialylated glycans in F(ab’)2 fragments produced from the indicated IgG fractions (LC-MS). D: Total sialic acid content in fractions from Fc run over the lectin column (HPLC). E: Percentage of sialylated IgG1 Fc-glycopeptides in the indicated Fc fractions (LC-MS). F: Total sialic acid content in F(ab’)2 fragments purified over the lectin column (HPLC). Mean values and standard deviations measured from three independently produced batches are shown. NAase: Neuraminidase treated, S+ Fc: SNA binding fraction of Fc fragments, S+ F(ab’)2: SNA binding fraction of F(ab’)2 fragments. FT: flow through (SNA non-binding), E1/E2 IVIG: SNA binding fractions of IVIG.
Figure 4
Figure 4. Specific antibody concentrations and human erythrocyte binding.
A: Concentrations of antibodies against a panel of antigens. Results are expressed as % of specific concentrations measured in IVIG. RV: rubella virus, MV: measles virus, TetTox: tetanus toxoid, EBV (EBNA): Epstein-Bar virus (nuclear antigen), B19V: human parvovirus B19, Hib: Haemophilius influencae, VZV: varizella zoster virus, CMV: cytomegalovirus, EBV (VCA): Epstein-Barr virus (capsid antigen). Concentrations are normalized to IVIG and shown in linear scale (left axes). ANA: anti-nuclear antibodies, absolute titres are shown in a log2 scale (right axis). Mean values and standard deviations measured from three independently produced batches are shown. B: Binding of different IVIG fractions to erythrocytes (RBC) from donors with blood group A, B or AB have been analysed by flow cytometry. MFI: mean fluorescence intensity.
Figure 5
Figure 5. Whole blood stimulation assay.
A: Whole blood was stimulated with LPS (left panels) or PHA (right panel). Upper panels: concentration dependent inhibition of CD54 (ICAM-1) expression on monocytes by the indicated IVIG fractions was monitored by flow cytometry. MFI: mean fluorescence intensity. Lower panels: concentration dependent inhibition of MCP-1 (CCL2) release by IVIG was quantified by ELISA in the supernatant of stimulated blood cultures. Mean values from representative experiments using blood from single donors are shown. neg: no stimulation, pos: stimulation without IVIG, FT: flow through, E1/E2 SNA binding IVIG fractions, NAase: neuraminidase treated. B: Inhibition of PHA mediated MCP-1 release by Fc and F(ab')2 fragments. Left panel: Mean MCP-1 concentration of stimulated minus non stimulated samples from a representative experiment is shown. Right panel: PHA stimulated whole blood was treated with 40 µM IgG, Fc, F(ab’)2 or highly sialylated Fc (S+Fc). Normalized values from experiments with blood from two donors are shown. S+ Fc: SNA binding Fc fraction (highly sialylated Fc).
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