Rigorous Analysis of Multimodal HDX-MS Spectra.

An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations.

Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS).

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy.

Optimization of a Hydrogen Exchange-Mass Spectrometry Robotic Liquid Handler Using Tracers.

Hydrogen exchange-mass spectrometry (HX-MS) is a valuable analytical technique that can provide insight into protein interactions and structure. The deuterium labeling necessary to gain this insight is affected by many physical and chemical factors, making it challenging to achieve high reproducibility. Poor precision during dispensing, transfer, and mixing of solutions during the experiment contributes substantially to the overall variability.

Interaction between preservatives and a monoclonal antibody in support of multidose formulation development.

Multidose formulations have patient-centric advantages over single-dose formats. A major challenge in developing multidose formulations is the prevention of microbial growth that can potentially be introduced during multiple drawings. The incorporation of antimicrobial preservatives (APs) is a common approach to inhibit this microbial growth.

Structure of a transmission blocking antibody in complex with Outer surface protein A from the Lyme disease spirochete, Borreliella burgdorferi.

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.

Structural Elucidation of a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease Spirochete, Borreliella burgdorferi.

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B.

High-Throughput Epitope Mapping by Hydrogen Exchange-Mass Spectrometry.

In this paper, we introduce a screening protocol for epitope mapping by hydrogen exchange mass spectrometry (HX-MS) that has higher throughput than a traditional HX-MS epitope mapping. In the screening protocol, three HX labeling times (20, 1000, and 86400 s) are each measured without replicates. The experimental protocol is anchored on a single epitope mapping experiment conducted using the traditional complete protocol (five HX times measured in triplicate) that is used to define HX times and define significance limits.

Identification of Agitation-Induced Unfolding Events Causing Aggregation of Monoclonal Antibodies Using Hydrogen Exchange-Mass Spectrometry.

Due to significant safety tolerances on maximum levels of visible and sub-visible particles in parenterally dosed drug products like monoclonal antibodies (mAbs), particle formation rates must be determined during development and minimized. Agitation stress, encountered during transportation and manufacturing, increases particle formation rates in a protein and formulation dependent fashion in a phenomenon thought to be partially mediated by mAb adsorption to the continuously regenerating air-water interface that results from agitation. The goal of this study was to explore the structural dynamics of three mAbs with variable sensitivity to agitation to develop a mechanistic understanding of exactly what occurs at the air-water interface that leads to aggregation and particle formation.

Interaction of Aluminum-adjuvanted Recombinant P[4] Protein Antigen With Preservatives: Storage Stability and Backbone Flexibility Studies.

Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m-cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4 °C, 25 °C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.

Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS).

Hydrogen exchange-mass spectrometry (HX-MS) is widely recognized for its potential utility for establishing the equivalence of the higher-order structures of proteins, particularly in comparability and similarity contexts. However, recent progress in the statistical analysis of HX-MS data has instead placed an emphasis on significance testing to identify regions of proteins where there are significant differences in HX between two or more protein states. In the cases involving assessment of similarity or equivalence of the higher-order structure of different protein samples (e.

Recommendations for the Propagation of Uncertainty in Hydrogen Exchange-Mass Spectrometric Measurements.

Hydrogen exchange-mass spectrometry (HX-MS) is used widely to characterize higher-order protein structure and to locate changes in protein structure and dynamics that accompany, for example, ligand binding and protein-protein interactions. Quantitative differences in the amount of hydrogen exchange between two states (i.e.

Protein A does not induce allosteric structural changes in an IgG1 antibody during binding.

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix.

In Vivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by in vitro studies to probe its effect on antibody function and stability, their in vivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. In vivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests.

Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine.

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017).

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 1.

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 2.

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, NaSO), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations.

Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach.

Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures of proteins. Typically, the data sets are large with many differential HX values corresponding to many peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant differences in HX-MS measurements, a statistical analysis approach is needed.

A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements.

Hydrogen exchange-mass spectrometry (HX-MS) is widely promoted for its ability to detect subtle perturbations in protein structure, but such perturbations will result in small differences in HX. However, the detection limit of HX-MS has not been widely investigated, nor is there a useful approach for defining the detection limit of HX-MS measurements. In this work, we designed a well-characterized structural variant spiking model to investigate the detection limit of conventional peptide-based HX-MS.

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical.