Supervised and unsupervised machine learning approaches for monitoring subvisible particles within an aluminum-salt adjuvanted vaccine formulation.

Suspensions of protein antigens adsorbed to aluminum-salt adjuvants are used in many vaccines and require mixing during vial filling operations to prevent sedimentation. However, the mixing of vaccine formulations may generate undesirable particles that are difficult to detect against the background of suspended adjuvant particles. We simulated the mixing of a suspension containing a protein antigen adsorbed to an aluminum-salt adjuvant using a recirculating peristaltic pump and used flow imaging microscopy to record images of particles within the pumped suspensions. Supervised convolutional neural networks (CNNs) were used to analyze the images and create "fingerprints" of particle morphology distributions, allowing detection of new particles generated during pumping. These results were compared to those obtained from an unsupervised machine learning algorithm relying on variational autoencoders (VAEs) that were also used to detect new particles generated during pumping. Analyses of images conducted by applying both supervised CNNs and VAEs found that rates of generation of new particles were higher in aluminum-salt adjuvant suspensions containing protein antigen than placebo suspensions containing only adjuvant. Finally, front-face fluorescence measurements of the vaccine suspensions indicated changes in solvent exposure of tryptophan residues in the protein that occurred concomitantly with new particle generation during pumping.

Antibodies Adsorbed to the Air-Water Interface Form Soft Glasses.

When monoclonal antibodies are exposed to an air-water interface, they form aggregates, which negatively impacts their performance. Until now, the detection and characterization of interfacial aggregation have been difficult. Here, we exploit the mechanical response imparted by interfacial adsorption by measuring the interfacial shear rheology of a model antibody, anti-streptavidin immunoglobulin-1 (AS-IgG1), at the air-water interface. Strong viscoelastic layers of AS-IgG1 form when the protein is adsorbed from the bulk solution. Creep experiments correlate the compliance of the interfacial protein layer with the subphase solution pH and bulk concentration. These, along with oscillatory strain amplitude and frequency sweeps, show that the viscoelastic behavior of the adsorbed layers is that of a soft glass with interfacial shear moduli on the order of 10-3 Pa m. Shifting the creep compliance curves under different applied stresses forms master curves consistent with stress-time superposition of soft interfacial glasses. The interfacial rheology results are discussed in the context of the interface-mediated aggregation of AS-IgG1.

A Rapid, Small-Volume Approach to Evaluate Protein Aggregation at Air-Water Interfaces.

Non-native protein aggregation is a common concern for biopharmaceuticals. A given protein may aggregate through a variety of mechanisms that depend on solution and physico-chemical stress conditions. A thorough evaluation of aggregation behavior for a protein under all conditions of interest is necessary to ensure drug safety and efficacy. This work introduces a rapid, small-volume approach to evaluate protein aggregation propensity upon exposure to air-water interfaces (AWI). A microtensiometer apparatus is used to aerate a small volume of a protein solution with microbubbles for short periods of time (≤10 s). Sub-visible particles that form are captured and analyzed using backgrounded membrane imaging. This allows one to capture all particles in the solution while being sample sparing. The surface-mediated aggregation of two model monoclonal antibodies (MAbs) and a globular protein (aCgn) was tested as a function of pH and temperature. Temperature had a negligible effect under the rapid interface turnover time scales with this technique. Electrostatic protein-protein interactions, mediated by pH changes, were more influential for particle formation via AWI. Nonionic surfactants substantially reduced particle formation for all MAb solutions, but not aCgn. The results are contrasted with expectations when exposing samples to much larger air-water interfacial stress.

Kinetics and Competing Mechanisms of Antibody Aggregation via Bulk- and Surface-Mediated Pathways.

Non-native protein aggregation is a long-standing obstacle in the biopharmaceutical industry. Proteins can aggregate through different mechanisms, depending on the solution and stress conditions. Aggregation in bulk solution has been extensively studied in a mechanistic context and is known to be temperature dependent. Conversely, aggregation at interfaces has been commonly observed for liquid formulations but is less understood mechanistically. This work evaluates the combined effects of temperature and compression/dilation of air-water interfaces on aggregation rates and particle formation for anti-streptavidin immunoglobulin gamma-1. Aggregation rates are quantified via size-exclusion chromatography, dynamic light scattering, and microflow imaging as a function of temperature and extent of air-liquid interface compressions. Competition exists between bulk- and surface-mediated aggregation mechanisms. Each has a largely different temperature dependence that leads to a crossover between the dominant aggregation mechanisms as the sample temperature changes. Surface-mediated aggregation rates are pH dependent and correlate with electrostatic protein-protein interactions but do not mirror the pH dependence of bulk aggregation rates that instead follow trends for conformational stability. Mechanistic insights were informed by quiescent incubation of solutions before and after interface compressions. Detailed mechanistic conclusions require direct dynamic observation at the interface. Microbubble tensiometry is introduced as a promising tool for such measurements.