Investigating protein-excipient interactions of a multivalent VHH therapeutic protein using NMR spectroscopy.

ALPHABETICAL LIST OF ABBREVIATIONS: Fab Fragment antigen-binding; Fc Fragment crystallizable; HMW High molecular weight; ∆HMW Difference between HMW species at stress temperature and 5°C controls; IgG Immunoglobulin G; mAbs Monoclonal antibodies; MV-VHH Multivalent VHH molecule with the format aC-L1-aC-L1-aD; NMR Nuclear magnetic resonance; scFv Single-chain fragment variable; SEC Size-exclusion chromatography; VHH Variable domain of Heavy chain of Heavy chain-only antibody.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein.

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118-152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φeq value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φeq value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding.

Immunogenicity of Therapeutic Protein Aggregates.

Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation. In this review, we provide an overview of the basic immune mechanisms in the context of interactions with protein aggregates. We then critically examine the literature with emphasis on the underlying immune mechanisms as they relate to aggregate properties. Finally, we highlight the gaps in our current understanding of this issue and offer recommendations for future research.

Analyzing subvisible particles in protein drug products: a comparison of dynamic light scattering (DLS) and resonant mass measurement (RMM).

Aggregation is common in protein drug manufacture, and while the effects of protein particulates are under investigation, many techniques applicable for their characterization have been recently developed. Among the methods available to characterize and quantify protein aggregates, none is applicable over the full size range and different methods often give conflicting results. The studies presented here compare two such methods: dynamic light scattering (DLS) and resonant mass measurement (RMM). The performance of each method was first characterized using polystyrene particle size standards (20, 60, 100, 200, 400, and 1,000 nm) over a range of concentrations. Standard particles were measured both singly and in binary mixtures containing 20 nm particles at a fixed concentration (10(14) particles/mL) and various concentrations of one of the other particle sizes (i.e., 60, 100, 200, 400, or 1,000 nm). DLS and RMM were then used to detect unknown aggregate content in stressed samples of IgG. Both instruments were shown to have a working range that depends on particle size and concentration. In binary mixtures and polydisperse solutions, DLS was able to resolve two species in a manner dependent on both concentration and particle size. RMM was able to resolve particles above 200 nm (150 nm for protein) at concentrations below 10(9) particles/mL. In addition, dilution was evaluated as a technique to confirm and quantify the number of particles in solution.

Microarrays and microneedle arrays for delivery of peptides, proteins, vaccines and other applications.

INTRODUCTION: Peptide and protein microarray and microneedle array technology provides direct information on protein function and potential drug targets in drug discovery and delivery. Because of this unique ability, these arrays are well suited for protein profiling, drug target identification/validation and studies of protein interaction, biochemical activity, immune responses, clinical prognosis and diagnosis and for gene, protein and drug delivery. AREAS COVERED: The aim of this review is to describe and summarize past and recent developments of microarrays in their construction, characterization and production and applications of microneedles in drug delivery. The scope and limitations of various technologies in this respect are discussed. EXPERT OPINION: This article offers a review of microarray/microneedle technologies and possible future directions in targeting and in the delivery of pharmacologically active compounds for unmet needs in biopharmaceutical research. A better understanding of the production and use of microarrays and microneedles for delivery of peptides, proteins and vaccines is needed.