Characterization of antithrombin isoforms and latent forms by ion exchange chromatography coupled to mass spectrometry.

Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.

C-terminal lysine clipping of IgG1: impact on binding to human FcγRIIIa and neonatal Fc receptors.

Monoclonal antibodies (mAbs) display numerous structural attributes, some of them may impact their safety and/or efficacy profiles. C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. If Fc-glycosylation has been by far the most documented critical quality attribute (CQA), the potential impacts of mAb C-terminal lysine content is far less reported, particularly on the ability of these basic variants to bind human Fc receptors. To address this question, three charge variant species having zero (K0), one (K1) and two (K2) C-terminal lysine(s) were isolated with high purity from an in-house human IgG1 by preparative strong-cation exchange (SCX) chromatography. A comprehensive biophysical characterization of these three fractions was undertaken, demonstrating their high similarity in terms of structural homogeneity, with a particular attention paid on their respective N-glycosylation profiles. The binding affinity of the fractions to human FcγRIIIa-Val176 was assessed both by affinity chromatography and surface plasmon resonance (SPR), and to human neonatal Fc receptor (FcRn) by affinity chromatography. Results demonstrate that the three charge variants did not show any significant binding difference for the two tested human Fc receptors, translating certainly to comparable biological properties. As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.

Human serum albumin presents isoform variants with altered neonatal Fc receptor interactions.

Human serum albumin (HSA) is the most abundant protein in plasma and presents the particularity, with IgG, to have an extraordinary long serum half-life conferred by its interaction with the neonatal Fc receptor (FcRn). If the impact of IgG post-translational modifications (PTMs) on FcRn binding is well documented, it is far less reported for HSA despite numerous PTMs occurring on the protein in plasma. HSA is susceptible to numerous degradation reactions in plasma, because of aging, oxidative stress or liver and pancreas related pathologies. In the present study, we combined FcRn affinity chromatography and mass spectrometry to investigate the impact of HSA PTMs upon FcRn binding. This methodology presents the advantage to distinguish the effect of a single modification from a plasma HSA preparation made of a mixture of different isoforms. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation, which are modifications related to several pathological conditions, were demonstrated to act negatively on HSA-FcRn interaction. The HSA-FcRn binding alteration generated by these modifications is consistent with their vicinity with the interaction interface of the two proteins. Results were discussed regarding altered half-life of HSA observed in several disease states and pave the way toward new understandings of the hypoalbuminemia pathogenesis. SIGNIFICANCE STATEMENT: In this study, we investigated the impact of several post-translational modifications of HSA toward its ability to bind to the neonatal Fc receptor using in vitro affinity chromatography, mass spectrometry, and surface plasmon resonance. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation were demonstrated to decrease HSA-FcRn binding. These modifications occurring in circulating HSA were discussed in relation to several pathologies as well as for the use of HSA as a therapeutic protein.

Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls.

Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities.

LC-MS analysis of polyclonal IgGs using IdeS enzymatic proteolysis for oxidation monitoring.

Susceptibility of IgGs to oxidation is a significant issue for intravenous immunoglobulin preparations (IVIG) in liquid solution and raises both safety and efficacy concerns. Here we present an optimized chromatography method coupled to mass spectrometry (MS) to determine the oxidation of Fc/2 fragments derived from polyclonal IgGs after IdeS treatment. Separation of the four IgG subclasses was achieved using a diphenyl column and UV/MS detections were used for quantification and characterization. Several oxidized Fc/2 fragments generated by stress conditions were resolved and oxidized methionines were identified. This procedure can be used to monitor the oxidative status of IVIG preparations during formulation or stability studies.