Rapid antibody conformational screening by matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry

Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.     

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.     

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

A synthetic camel anti-lysozyme peptide antibody (peptibody) with flexible loop structure identified by high-resolution affinity mass spectrometry

We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.