Damian Houde

Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.

The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies

The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confidant comparability assessments of protein higher-order structures than are currently available within the biopharmaceutical industry.

Characterization of IgG1 Conformation and Conformational Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

Protein function is dictated by protein conformation. For the protein biopharmaceutical industry, therefore, it is important to have analytical tools that can detect changes in protein conformation rapidly, accurately, and with high sensitivity. In this paper we show that hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can play an important role in fulfilling this need within the industry. H/DX-MS was used to assess both global and local conformational behavior of a recombinant monoclonal IgG1 antibody, a major class of biopharmaceuticals. Analysis of exchange into the intact, glycosylated IgG1 (and the Fab and Fc regions thereof) showed that the molecule was folded, highly stable, and highly amenable to analysis by this method using less than a nanomole of material. With improved chromatographic methods, peptide identification algorithms and data-processing steps, the analysis of deuterium levels in peptic peptides produced after labeling was accomplished in 1-2 days. On the basis of peptic peptide data, exchange was localized to specific regions of the antibody. Changes to IgG1 conformation as a result of deglycosylation were determined by comparing exchange into the glycosylated and deglycosylated forms of the antibody. Two regions of the IgG1 (residues 236-253 and 292-308) were found to have altered exchange properties upon deglycosylation. These results are consistent with previous findings concerning the role of glycosylation in the interaction of IgG1 with Fc receptors. Moreover, the data clearly illustrate how H/DX-MS can provide important characterization information on the higher order structure of antibodies and conformational changes that these molecules may experience upon modification.

Conformation and dynamics of biopharmaceuticals: Transition of mass spectrometry-based tools from academe to industry

Mass spectrometry plays a very visible role in biopharmaceutical industry, although its use in development, characterization, and quality control of protein drugs is mostly limited to the analysis of covalent structure (amino acid sequence and post-translational modifications). Despite the centrality of protein conformation to biological activity, stability, and safety of biopharmaceutical products, the expanding arsenal of mass spectrometry-based methods that are currently available to probe higher order structure and conformational dynamics of biopolymers did not, until recently, enjoy much attention in the industry. This is beginning to change as a result of recent work demonstrating the utility of these experimental tools for various aspects of biopharmaceutical product development and manufacturing. In this work, we use a paradigmatic protein drug interferon β-1a as an example to illustrate the utility of mass spectrometry as a powerful tool not only to assess the integrity of higher order structure of a protein drug, but also to predict consequences of its degradation at a variety of levels.

Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity.

In this work several aspects of imaging capillary IEF (icIEF) application for charge heterogeneity analysis of recombinant proteins and monoclonal antibodies have been discussed. Advantages of the method as compared with traditional approaches for determination of biomolecule charge heterogeneity, such as gel and IEC, have been demonstrated. Correlation of icIEF‐detected protein isoforms with the charge heterogeneity determined by IEC has been shown for a representative recombinant monoclonal antibody. Identification of charged variants collected from IEC has been performed by ESI‐MS. Qualification of an icIEF method for use in quality control environment for quantitative analysis of recombinant protein charge heterogeneity and monitoring protein stability has also been discussed. The intermediate precision for determination of pI of main or main acidic species was ≤0.2% RSD. Relative % peak areas for acidic, main and basic species were reproducible within 1.9, 0.9 and 16.6% RSD, respectively. Based on the assay performance evaluation, icIEF assay has been shown to allow for fast method development, short analysis time and high sample throughput. Some aspects of the method specificity for use as an identity test in biopharmaceutical development have been discussed.

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX‐MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.

Conformational analysis of recombinant monoclonal antibodies with hydrogen/deuterium exchange mass spectrometry.

Understanding the conformation of antibodies, especially those of therapeutic value, is of great interest. Many of the current analytical methods used to probe protein conformation face issues in the analysis of antibodies, either due to the nature of the antibody itself or due to the limitations of the method. One method that has recently been utilized for conformational analysis of antibodies is hydrogen/deuterium exchange mass spectrometry (H/DX MS). H/DX MS can be used to probe the conformation and dynamics of proteins in solution, requires small sample quantities, is compatible with many buffer systems, and provides peptide-level resolution. The application of H/DX MS to immunoglobulin gamma 1 (IgG1) recombinant monoclonal antibodies can provide information about IgG1 conformation, dynamics, and changes to conformation as a result of protein modification(s), changes in storage conditions, purification procedures, formulation, and many other parameters. In this article we provide a comprehensive H/DX MS protocol for the analysis of an antibody.

Fine Details of IGF-1R Activation, Inhibition, and Asymmetry Determined by Associated Hydrogen /Deuterium-Exchange and Peptide Mass Mapping

The structural features of the asymmetric activated states of the insulin receptor family are still poorly understood. We investigated hydrogen/deuterium (H/D)-exchange within the extracellular domain of the type-I insulin-like growth factor receptor (IGF-1R) in the absence and presence of IGF-1 (active state) and in the presence of antibody inhibitors (inactive state). Near complete coverage of the 210 kDa receptor sequence was obtained by mass mapping of proteolytically derived peptides at all H/D-exchange time points. The data provide details regarding solvent exposure and dynamics across the extracellular region as well as conformational changes induced by activation or inactivation. Multiple peptides, distant in structure, individually demonstrated two distinct H/D-exchange rates, suggesting that each of these peptides exists in two separate environments in IGF-1R. The dual-exchange behavior of these peptides was enhanced on ligand binding and eliminated on inhibitor binding, clearly associating these regions with active state asymmetry and enabling them to serve as reporters of receptor activity.

Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography.

Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG(1) monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG(1) mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG(1) mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG(1) molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture.