NOVEL MASS SPECTROMETRY BASED STRATEGIES TO STUDY HEPARIN-PROTEIN INTERACTIONS

Heparin and heparan sulfate (HS) are linear polyanions from the glycosaminoglycan (GAG) family. They are conceived to play critical roles in a variety of biophysiological processes by interacting with key proteins and regulating protein functions. Understanding their biological functions and exploit heparin’s unique versatility for therapeutic purposes critically depend on the characterization of their interactions with relevant proteins, which, however, is largely impeded by the structural heterogeneity of these polyanions. This work presented the development of novel mass spectrometry-based analytical strategies incorporated with liquid chromatography and ion mobility spectroscopy to investigate heparin-protein interactions and address their significance from different aspects

Ionic Charge Manipulation Using Solution and Gas-Phase Chemistry to Facilitate Analysis of Highly Heterogeneous Protein Complexes in Native Mass Spectrometry

Ionic signal in native MS typically populates high m/z regions of mass spectra, which frequently extend beyond the precursor ion isolation limits of most commercial mass spectrometers. An approach explored in this work relies on adding supercharging reagents to protein solutions as a means of increasing the extent of multiple charging of non-covalent complexes in ESI MS without compromising their integrity. This shifts the ionic signal down the m/z scale to the region where ion selection and isolation can be readily accomplished, followed by limited charge reduction of the isolated ionic population. The feasibility of the new approach is demonstrated using non-covalent complexes formed by hemoglobin with structurally heterogeneous haptoglobin

Mechanism of Reduction in IgG and IgE Binding of β‑Lactoglobulin Induced by Ultrasound Pretreatment Combined with Dry-State Glycation: A Study Using Conventional Spectrometry and High-Resolution Mass Spectrometry

Bovine β-lactoglobulin (β-Lg) is one of major allergens in cow’s milk. Previous study showed that ultrasound treatment induced the conformational changes of β-Lg and promoted the glycation in aqueous solutions, which is, however, less efficient compared with dry-state. In this work, the effect of ultrasound pretreatment combined with dry-state glycation on the IgG and IgE binding of β-Lg was studied. Dry-state glycation with mannose after ultrasound pretreatment at 0–600 W significantly reduced the IgG and IgE binding of β-Lg, with the lowest values observed at 400 W. The decrease in the IgG and IgE binding of β-Lg was attributed to the increase in glycation extent and the changes of secondary and tertiary structure, which reflected in the increase of UV absorbance, α-helix and β-sheet contents, as well as the decrease of intrinsic fluorescence intensity, surface hydrophobicity, β-turn, and random coil contents. Moreover, ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving glycation, reflecting in the increase of the glycation sites and the degree of substitution per peptide (DSP) value determined by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Ultrasound pretreatment at 400 W showed the most significantly enhanced glycation extent. Besides, the results suggested FTICR-MS could provide insights into the glycation at molecular level, which was conducive to the understanding of the mechanism of the reduction in the IgG and IgE binding of β-Lg. Therefore, ultrasound pretreatment combined with dry-state glycation may be a promising method for β-Lg hyposensitization

Mass Spectrometry Reveals a Multifaceted Role of Glycosaminoglycan Chains in Factor Xa Inactivation by Antithrombin

Factor Xa (fXa) inhibition by antithrombin (AT) enabled by heparin or heparan sulfate is critical for controlling blood coagulation. AT activation by heparin has been investigated extensively, while interaction of heparin with trapped AT/fXa intermediates has received relatively little attention. We use native electrospray ionization mass spectrometry to study the role of heparin chains of varying length [hexa-, octa-, deca-, and eicosasaccharides (dp6, dp8, dp10, and dp20, respectively)] in AT/fXa complex assembly. Despite being critical promoters of AT/Xa binding, shorter heparin chains are excluded from the final products (trapped intermediates). However, replacement of short heparin segments with dp20 gives rise to a prominent ionic signal of ternary complexes. These species are also observed when the trapped intermediate is initially prepared in the presence of a short oligoheparin (dp6), followed by addition of a longer heparin chain (dp20), indicating that binding of heparin to AT/fXa complexes takes place after the inhibition event. The importance of the heparin chain length for its ability to associate with the trapped intermediate suggests that the binding likely occurs in a bidentate fashion (where two distinct segments of oligoheparin make contacts with the protein components, while the part of the chain separating these two segments is extended into solution to minimize electrostatic repulsion). This model is corroborated by both molecular dynamics simulations with an explicit solvent and ion mobility measurements in the gas phase. The observed post-inhibition binding of heparin to the trapped AT/fXa intermediates hints at the likely role played by heparan sulfate in their catabolism

Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the CH1-Cλ interface

ABSTRACTBispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics