Elucidating the Mechanism of Multispecific Antibody Aggregation through Subunit Analysis.

Multispecific antibody constructs are quickly becoming more common constructs in biopharmaceuticals to improve specificity and efficacy. While the advent of this technology has led to improved therapeutics, its development has challenged the analytical tools through which these therapeutics are characterized. Moreover, new critical quality attributes, such as aggregation, have challenged the approaches to characterization even further. Herein, we describe a novel native subunit analysis using IdeS and IgdE analyzed by native size exclusion chromatography coupled with mass spectrometry to interrogate the mechanism of aggregation in a multispecific antibody. Digestion by IdeS and IdgE allows for the retention and detection of noncovalent interactions thereafter. Aggregation was localized to single-chain fragment variables (scFvs) wherein a domain swapping mechanism between VH1/VL2 and VH2/VL1 occurs.

"Lab of the Future"─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics.

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system. Subsequently, the purified protein samples are prepared for MS, including deglycosylation and reduction steps for intact and reduced mass analysis, and proteolytic digestions, desalting, and buffer exchange via centrifugation for peptide map analysis. The prepared samples are then loaded into the LC-MS instrumentation for data acquisition. The acquired raw data are initially stored on a local area network storage system that is monitored by watcher scripts that then upload the raw MS data to a network of cloud-based servers. The raw MS data are processed with the appropriately configured analysis workflows such as database search for peptide mapping or charge deconvolution for undigested proteins. The results are verified and formatted for expert curation directly in the cloud. Finally, the curated results are appended to sample metadata in the corporate data aggregation system to accompany the biotherapeutic cell lines in subsequent processes.

Functional Relevance of Interleukin-1 Receptor Inter-domain Flexibility for Cytokine Binding and Signaling.

The interleukin 1 (IL-1) receptor family, whose members contain three immunoglobulin-like domains (D1-D3) in the extracellular region, is responsible for transmitting pleiotropic signals of IL-1 cytokines. The inter-domain flexibility of IL-1 receptors and its functional roles have not been fully elucidated. In this study, we used small-angle X-ray scattering to show that ligand-binding primary receptors and co-receptors in the family all have inherent inter-domain flexibility due to the D2/D3 linker. Variants of the IL-1RAcP and IL-18Rß co-receptors with mutated D2/D3 linkers cannot form a cytokine-receptor complex and mediate signaling. Our analysis further revealed that these mutated co-receptors exhibited a changed conformational ensemble, suggesting that loss of function is due to the alteration of receptor dynamics. Taken together, our results demonstrate that the D2/D3 linker is a critical functional determinant of IL-1 receptor and underscore the important roles of the inter-domain flexibility in cytokine/receptor binding and signaling.

Conformational Plasticity of the Immunoglobulin Fc Domain in Solution.

Fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibody binds to specific Fc receptors (FcγRs) to control antibody effector functions. Currently, engineered specific Fc-FcγR interactions are validated with a static conformation derived from the crystal structure. However, computational evidence suggests that the conformational variability of Fcs plays an important role in receptor recognition. Here we elucidate Fc flexibility of IgG1, IgG2, and IgG1 Fc with mutations (M255Y/S257T/T259E) in solution by small-angle X-ray scattering (SAXS). Measured SAXS profiles and experimental parameters show variations in flexibility between Fc isotypes. We develop and apply a modeling tool for an accurate conformational sampling of Fcs followed by SAXS fitting. Revealed conformational variability of the CH2 domain as low as 10 Å in displacement, illustrates the power of the atomistic modeling combined with SAXS. This inexpensive SAXS-based approach offers to improve the engineering of antibodies for tailoring Fc receptor interactions through altering and measuring Fc flexibility.

A novel method for determination of drug distribution in rat brain tissue sections by LC/MS/MS: functional tissue microanalysis.

Determination of drug distribution in brain and other tissues is important in pharmaceutical research. Tissue drug levels need to be determined routinely as they are usually diagnostic for both efficacy and toxicity. Determination of tissue levels in small organ subregions is frequently performed due to important functional considerations. These measurements have traditionally been very tedious requiring extensive dissection and specimen pooling to achieve detection of analytes of interest. Direct and indirect methods utilizing mass spectrometry have been reported for detection of analytes in tissue specimens. Typically, these require very specialized MS or sampling equipment and are only partially successful due to analyte response. We have developed a novel approach for quantitation of tissue sections called Functional Tissue Microanalysis (FTM) in which small circular samples are removed from subregions of interest, extracted and analyzed by conventional LC/MS/MS utilizing electrospray ionization. This allows direct measurement of regional concentrations without dissection and homogenization of tissue specimens as many subregions can be sampled from a single mounted section. Utilization of the FTM approach for analysis of both sagittal and coronal rat brain sections is shown for quantitation of raclopride and rimonabant. Reproducibility of this approach and comparison to conventional methods is reported.

Structural basis for elastolytic substrate specificity in rodent alpha-chymases.

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.