Aaron T. Wecksler

Effect of ambient light on IgG1 monoclonal antibodies during drug product processing and development

Photostability studies are standard stress testing conducted during drug product development of various pharmaceutical compounds, including small molecules and proteins. These studies as recommended by ICH Q1B are carried out using no less than 1.2× 10(6)lux-hours in the visible region and no less than 200Wh/m(2) in UV light. However, normal drug product processing is carried out under fluorescent lamps that emit white light almost exclusively in the >400nm region with a small UV quotient. We term these as ambient or mild light conditions. We tested several IgG1 monoclonal antibodies (mAbs 1-5) under these ambient light conditions and compared them to the ICH light conditions. All the mAbs were significantly degraded under the ICH light but several mAbs (mAbs 3-5) were processed without impacting any product quality attributes under ambient or mild light conditions. Interestingly we observed site-specific Trp oxidation in mAb1, while higher aggregation and color change were observed for mAb2 under mild light conditions. The recommended ICH light conditions have a high UV component and hence may not help to rank order photosensitivity under normal protein DP processing conditions.

Characterizing monoclonal antibody structure by carboxyl group footprinting

Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.

Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

AbstractWe previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies. Graphical abstractᅟ

Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

AbstractA proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes. Graphical Abstractᅟ

Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal

Significance The outer membrane of Gram-negative bacteria presents a formidable barrier to the discovery of new antibiotics needed to combat infections by multidrug-resistant bacteria. Targeting essential proteins or processes directly exposed to the environment could bypass this obstacle. Here, we describe a monoclonal antibody that selectively and potently antagonizes BamA, which folds and inserts integral outer membrane β-barrel proteins, by binding to a surface-exposed BamA epitope and, as a result, inhibits bacterial cell growth. Mechanisms of resistance to the antibody reveal that membrane fluidity affects BamA activity. This antibody validates the potential therapeutic strategy of targeting essential, exposed functions and provides a powerful tool for dissecting the fundamental process of folding integral membrane β-barrel proteins in vivo. The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction.

AbstractNative mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)–VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis. Graphical Abstractᅟ

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting.

Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs.

Photo-Disruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O18-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

AbstractWe describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody–antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody–antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. Graphical Abstractᅟ