Characterisation of Myeloma Paraprotein Glycosylation Reveals Functional Insights and Increased Sialylation of Residual Polyclonal Antibodies

Abstract

BackgroundA pathognomonic feature of multiple myeloma is the presence abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. All subclasses of Igs are post-translationally modified by the addition of N-glycans. The added N-glycosylation plays a key role in modulating the structure and function of the antibody. It has been recently demonstrated that high resolution mass spectrometry can facilitate the detection of myeloma associated monoclonal immunoglobulin. Herein, a simple method for the enrichment of monoclonal immunoglobulin from total polyclonal IgG enriched from patient sera is described, facilitating an insight into the structure functional relationship of the monoclonal immunoglobulin. Additionally, sensitivity of the platform was evaluated using longitudinal samples to determine the suitability of the method to determine disease emergence or recurrence.MethodsUsing Protein G affinity chromatography, polyclonal IgG was extracted from sera of patients with smouldering myeloma (SMM), newly-diagnosed, remission, and relapse-refractory myeloma. The quantity of extracted IgG was determined using the Bradford assay. Aliquots of polyclonal IgG were then injected on to a Thermo Scientific MabPac SCX-10 RS cation exchange column and separated using a linear pH elution gradient in the range of pH 5.6 to 10.2. Small aliquots of sample were initially injected to examine the resulting profile and to determine associated fraction collection parameters prior to operation in semi-prep mode for analyte collection. Dominant monoclonal IgG peaks and the general polyclonal background IgG were then buffer exchanged. N-glycans were subsequently liberated using overnight enzymatic digestion with PNGase F, purified, fluorescently derivatized and analysed using a combination of hydrophilic interaction ultra-performance liquid chromatography. Mass spectrometry and exoglycosidase digestions were performed for complete oligosaccharide structural elucidation. The deglycosylated IgG present in the dominant monoclonal IgG peak was additionally subjected to protease digestion with trypsin. Resulting tryptic peptides were analysed using nanoflow LC-MS on a Waters Xevo G2 QToF mass spectrometer. Data was searched against a database containing the FASTA sequences of human IgG 1-4 isotype constant regions and κ and λ light chain sequences, all extracted from the SwissProt Uniprot Human Proteome databaseResultsCation exchange chromatography operated using linear pH gradient elution proved to be an excellent, simple and non-destructive method for the detection of dominant monoclonal IgG populations present against the general polyclonal IgG background in the sera of patients at varying stages of multiple myeloma. The efficiency of the cation exchange method facilitated the determination of the isoform pattern of the dominant monoclonal IgG peaks which was unique from patient-to-patient. Increasing levels of the monoclonal IgG peaks were observed to be correlated with disease progression. N-glycan analysis of the enriched dominant monoclonal IgG population revealed a regular IgG associated glycosylation profile, namely biantennary glycans with variable levels of core fucosylation, galactosylation, sialylation and bisecting GlcNAc residues. When compared to the UPLC-fluorescence based profile for the total Protein G affinity enriched IgG N-glycan pool, it was noted that there was an excellent correlation in the levels of glycans present on the dominant IgG clones. However, the level of sialylation on the residual background polyclonal IgG population appeared to be higher, potentially suggesting a preferential modification of these molecules to lengthen their circulating half-life and anti-inflammatory properties following the emergence of the dominant clonal population with disease manifestation.ConclusionsGlycan analysis of both polyclonal IgG and the enriched monoclonal paraprotein extracted using Protein G affinity chromatography from the serum of patients with varying stages of myeloma progression is reported. Differences in glycosylation between the monoclonal paraprotein and the polyclonal IgG background were observed. DisclosuresNo relevant conflicts of interest to declare.