The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc,... Show moreThe conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes.To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness.This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs. Show less
Gstöttner, C.; Hutanu, A.; Boon, S.; Raducanu, A.; Richter, K.; Haindl, M.; ... ; Domínguez-Vega, E. 2023
After decades ofresearch, gene therapy products havereached marketmaturity in recent years. Recombinant adeno-associated viruses (rAAVs)are one of the most promising gene delivery vehicles and are... Show moreAfter decades ofresearch, gene therapy products havereached marketmaturity in recent years. Recombinant adeno-associated viruses (rAAVs)are one of the most promising gene delivery vehicles and are currentlyunder intense scientific investigation. These next-generation medicinesremain very challenging when it comes to designing appropriate analyticaltechniques for quality control. One critical quality attribute isthe integrity of ssDNA incorporated in these vectors. The genome isthe active compound driving rAAV therapy and therefore requires properassessment and quality control. Current techniques for rAAV genomecharacterization include next-generation sequencing, quantitativepolymerase chain reaction, analytical ultracentrifugation (AUC), andcapillary gel electrophoresis (CGE), yet each of them presents theirlimitations or lack of user-friendliness. In this work, we demonstratefor the first time the potential of ion pairing-reverse phase-liquidchromatography (IP-RP-LC) to characterize the integrity of rAAV genomes.The obtained results were supported by two orthogonal techniques,AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures,avoiding the detection of secondary DNA isoforms, and does not requirethe use of dyes due to UV detection. We demonstrate that this techniqueis suitable for batch comparability, different rAAV serotypes (AAV2and AAV8), internal vs external (inside vs outside the capsid) DNAanalysis, and contaminated samples. Overall, it is exceptionally user-friendly,needs limited sample preparation, has high reproducibility, and permitsfractionation for further peak characterization. All of these factorsadd significant value of IP-RP-LC to the analytical toolbox of rAAVgenome assessment. Show less
