Steven A. Berkowitz

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

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.

Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals.

In developing and manufacturing protein biopharmaceuticals, aggregation is a parameter that needs careful monitoring to ensure the quality and consistency of the final biopharmaceutical drug product. The analytical method of choice used to perform this task is size-exclusion chromatography (SEC). However, it is becoming more and more apparent that considerable care is required in assessing the accuracy of SEC data. One old analytical tool that is now reappearing to help in this assessment is analytical ultracentrifugation (AUC). Developments in AUC hardware and, more importantly, recent developments in AUC data analysis computer programs have converged to provide this old biophysical tool with the ability to extract very high resolution size information about the molecules in a given sample from a simple sedimentation velocity experiment. In addition, AUC allows sample testing to be conducted in the exact or nearly exact liquid formulation or reconstituted liquid formulation of the biopharmaceutical in the vial, with minimal surface area contact with extraneous materials. As a result, AUC analysis can provide detailed information on the aggregation of a biopharmaceutical, while avoiding many of the major problems that can plague SEC, thus allowing AUC to be used as an orthogonal method to verity SEC aggregation information and the associating properties of biopharmaceuticals.

Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site.

The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complexs catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.

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.

Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis.

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti‐LINGO‐1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

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.