Research paperA method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis
Introduction
Beyond their ability to neutralize pathogens, antibodies are able to mediate an array of effector functions through their interaction with Fc-receptors, complement molecules, and mammalian lectin-like molecules (Kapur et al., 2014). While the neutralizing activity of an antibody is mediated largely by its variable domain (Fab, antigen-binding fragment), its ability to perform extra-neutralizing functions is determined by the constant domain (Fc, crystallizable fragment) (Schroeder and Cavacini, 2010). Though the Fc is referred to as constant, it is in fact variable in two major aspects: a) protein sequence varies through subclass or isotype selection (Nimmerjahn and Ravetch, 2010) and b) glycosylation variation of N-linked glycans which change rapidly during an immunologic response (Xue et al., 2013). Together, these alterations in the antibody Fc significantly modify the effector function of antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) (Davies et al., 2001, Shields et al., 2002, Shinkawa et al., 2003, Shoji-Hosaka et al., 2006) and complement-dependent cytotoxicity (CDC) (Karsten and Köhl, 2012). While high-throughput methods are available to profile the isotype/subclass selection profile of polyclonal antibody pools (Brown et al., 2012), comparable methods for efficient analysis of glycan profiles are not available.
Studies of therapeutic monoclonal antibodies have clearly demonstrated the critical nature of the antibody glycan; ADCC activity is significantly increased in monoclonal therapeutic antibodies that lack fucose (Shields et al., 2002, Shinkawa et al., 2003, Shoji-Hosaka et al., 2006) or contain a bisecting N-acetylglucosamine (GlcNAc) (Davies et al., 2001). In addition to their role in determining effector function, inflammatory responses are dramatically modulated by Fc glycosylation. In particular, the addition of terminal sialic acids to the Fc glycan results in the induction of a potent anti-inflammatory response (Anthony and Ravetch, 2010, Böhm et al., 2012). Moreover, population level studies have shown that IgG glycosylation varies significantly with age, pregnancy, and during autoimmune-disease flares (Chen et al., 2012, Keusch et al., 1996, Parekh et al., 1988, Van De Geijn et al., 2009). More recent analyses point to antigen-specific antibody glycan alterations suggesting IgG-glycosylation is specifically programmed during immune responses (Ackerman et al., 2013, Collin and Ehlers, 2013, Selman et al., 2012, Yamada et al., 2013).
Studies of IgG-glycosylation in vivo have been historically limited by the low-throughput of existing analytical techniques, which generally require prohibitively expensive instrumentation and large quantities of sample, thus limiting the scope of research into natural regulation of IgG-glycosylation. Traditional approaches to analyze IgG N-glycosylation have relied primarily on high performance liquid chromatography (HPLC) or mass spectrometry (MS), both of which require relatively large quantities of material/antibody for accurate analysis as well as significant time and expertise to acquire and analyze data (Huhn et al., 2009). While MS offers remarkable structural resolution of N-glycans, it is poorly quantitative. On the other hand, while HPLC is highly quantitative, it is expensive, and both methods are distinctly low throughput. As studies of IgG glycosylation begin to focus on in vivo modifications, both in human populations and in animal models, the volume of samples often decreases as the number of samples increases. Thus a clear need exists for the development of a simple technique that combines sensitive quantitation with high-throughput capacity.
Capillary electrophoresis (CE) offers a unique high-throughput, quantitative analytical tool for the analysis of antibody glycosylation. Specifically, the use of common DNA sequencing equipment to perform glycan structure analysis by capillary electrophoresis is an excellent alternative to the established methods, with advantages in simplicity, throughput, structural resolution, and sensitivity (Callewaert et al., 2001, Huhn et al., 2012, Laroy et al., 2006, Reusch et al., 2014). Previously described CE techniques for antibody glycan analysis have focused on the analysis of whole IgG, as the large majority of monoclonal antibodies lack Fab glycan-sites (Ritamo et al., 2014). However, as many as 30% of serum-derived Fab fragments contain an N-glycosylation motif, and Fab glycans differ significantly from those typically found on the Fc-domain, in particular, Fab N-glycans contain higher proportions of sialic acid and fewer fucosylated structures (Anumula, 2012, Holland et al., 2006, Mimura et al., 2007). Thus, studies interrogating polyclonal antibody glycosylation aimed at understanding the functional significance of these regulated post-translational modifications will depend on the ability to resolve Fc and Fab glycans separately.
Here we describe a high-throughput, inexpensive, sensitive, and accurate approach for IgG N-glycan analysis of polyclonal antibodies. This methodology allows for separate analysis of the N-glycans from whole IgG, Fc, or Fab domains using capillary electrophoresis performed on a DNA-sequencer, providing a fast, accurate, quantitative, and relatively inexpensive and simple tool to probe IgG glycosylation, even when sample quantities are limited. This technique will be useful for the analysis of changes in antibody glycosylation following vaccination, in natural infection, as well as in non-infectious pathological conditions both in humans and in animal models, facilitating our understanding of the immunological impact of a B cell's ability to tune antibody activity through variations in glycosylation.
Section snippets
Samples
Optimization of digestion and separation conditions was performed on commercially available, pooled IgG from healthy donors (Sigma Aldrich, human IgG and mouse IgG) or a pool of IgGs purified from healthy rhesus monkeys (Non-Human Primate Reagent Resource). Healthy human subjects were recruited through Brigham and Women's Hospital PhenoGenetic Project. The Institutional Review Board of Partners Healthcare approved the study, and each subject gave written informed consent. Rhesus macaque plasma
Analysis of glycans on the DNA sequencer is highly sensitive and reproducible.
To test our glycan analysis technique, IgG glycans were enzymatically released, purified, fluorescently labeled and eletrophoretically separated on the DNA sequencer as described in the methods section. Individual peaks on the CE spectrum, as shown in Fig. 1A, were identified using standards and exoglycosidase digestion as described in Table 1. Individual glycan structures were broadly classified into categories as described in Table 2. These categories give a broad description of the
Discussion
With growing appreciation of the role of antibody glycosylation in tuning antibody effector function, an analytical technique capable of analyzing the glycosylation profiles of large numbers of serum-derived, polyclonal antibodies is needed. Ultimately, an analytical tool aimed at characterizing antibody glycosylation in vivo must (a) have a high-throughput capacity, (b) exhibit robust reliability and sensitivity for Fc-specific glycan analysis in multiple species, (c) be cost-effective, and
Acknowledgments
Thanks to PS for technical discussions and CS for sharing expertise in glycan structure analysis. This work was supported by R01 AI080289 and the Gates Foundation CAVD OPP 1032817.
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