Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Towards an improved prediction of concentrated antibody solution viscosity using the Huggins coefficient.

The viscosity of a monoclonal antibody solution must be monitored and controlled as it can adversely affect product processing, packaging and administration. Engineering low viscosity mAb formulations is challenging as prohibitive amounts of material are required for concentrated solution analysis, and it is difficult to predict viscosity from parameters obtained through low-volume, high-throughput measurements such as the interaction parameter, kD, and the second osmotic virial coefficient, B22. As a measure encompassing the effect of intermolecular interactions on dilute solution viscosity, the Huggins coefficient, kh, is a promising candidate as a parameter measureable at low concentrations, but indicative of concentrated solution viscosity. In this study, a differential viscometry technique is developed to measure the intrinsic viscosity, [η], and the Huggins coefficient, kh, of protein solutions. To understand the effect of colloidal protein-protein interactions on the viscosity of concentrated protein formulations, the viscometric parameters are compared to kD and B22 of two mAbs, tuning the contributions of repulsive and attractive forces to the net protein-protein interaction by adjusting solution pH and ionic strength. We find a strong correlation between the concentrated protein solution viscosity and the kh but this was not observed for the kD or the b22, which have been previously used as indicators of high concentration viscosity. Trends observed in [η] and kh values as a function of pH and ionic strength are rationalised in terms of protein-protein interactions.

Cluster Percolation Causes Shear Thinning Behavior in Concentrated Solutions of Monoclonal Antibodies.

High-concentration (>100 g/L) solutions of monoclonal antibodies (mAbs) are typically characterized by anomalously large solution viscosity and shear thinning behavior for strain rates ≥103 s-1. Here, the link between protein-protein interactions (PPIs) and the rheology of concentrated solutions of COE-03 and COE-19 mAbs is studied by means of static and dynamic light scattering and microfluidic rheometry. By comparing the experimental data with predictions based on the Baxter sticky hard-sphere model, we surprisingly find a connection between the observed shear thinning and the predicted percolation threshold. The longest shear relaxation time of mAbs was much larger than that of model sticky hard spheres within the same region of the phase diagram, which is attributed to the anisotropy of the mAb PPIs. Our results suggest that not only the strength but also the patchiness of short-range attractive PPIs should be explicitly accounted for by theoretical approaches aimed at predicting the shear rate-dependent viscosity of dense mAb solutions.

Determination of Protein-Protein Interactions at High Co-Solvent Concentrations Using Static and Dynamic Light Scattering.

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B22 from static light scattering (SLS) or the interaction parameter, kD from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B22 determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B22 measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B22 and kD is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B22 and kD measurements are used for screening excipient effects in liquid formulations.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Determination of Protein-Protein Interactions in a Mixture of Two Monoclonal Antibodies.

The coformulation of monoclonal antibody (mAb) mixtures provides an attractive route to achieving therapeutic efficacy where the targeting of multiple epitopes is necessary. Controlling and predicting the behavior of such mixtures requires elucidating the molecular basis for the self- and cross-protein-protein interactions and how they depend on solution variables. While self-interactions are now beginning to be well understood, systematic studies of cross-interactions between mAbs in solution do not exist. Here, we have used static light scattering to measure the set of self- and cross-osmotic second virial coefficients in a solution containing a mixture of two mAbs, mAbA and mAbB, as a function of ionic strength and pH. mAbB exhibits strong association at a low ionic strength, which is attributed to an electrostatic attraction that is enhanced by the presence of a strong short-ranged attraction of nonelectrostatic origin. Under all solution conditions, the measured cross-interactions are intermediate self-interactions and follow similar patterns of behavior. There is a strong electrostatic attraction at higher pH values, reflecting the behavior of mAbB. Protein-protein interactions become more attractive with an increasing pH due to reducing the overall protein net charges, an effect that is attenuated with an increasing ionic strength due to the screening of electrostatic interactions. Under moderate ionic strength conditions, the reduced cross-virial coefficient, which reflects only the energetic contribution to protein-protein interactions, is given by a geometric average of the corresponding self-coefficients. We show the relationship can be rationalized using a patchy sphere model, where the interaction energy between sites i and j is given by the arithmetic mean of the i-i and j-j interactions. The geometric mean does not necessarily apply to all mAb mixtures and is expected to break down at a lower ionic strength due to the nonadditivity of electrostatic interactions.

Models for Antibody Behavior in Hydrophobic Interaction Chromatography and in Self-Association.

Monoclonal antibodies (mAbs) form an increasingly important sector of the pharmaceutical market, and their behavior in production, processing, and formulation is a key factor in development. With data sets of solution properties for mAbs becoming available, and with amino acid sequences, and structures for many Fabs, it is timely to examine what features correlate with measured data. Here, previously published data for hydrophobic interaction chromatography and the formation of high molecular weight species are studied. Unsurprisingly, aromatic sidechain content of complementarity-determining regions (CDRs), underpins much of the variability in hydrophobic interaction chromatography data. However, this is not reflected in nonpolar solvent accessible surface enrichment at the antigen-combining site, consistent with a view in which hydrophobic interaction strength is dependent on curvature as well as on the extent of an interface. Sequence properties are also superior to surface-based structural properties in correlations with the high molecular weight species data. Combined length of CDRs is the most important factor, which could be an indication of flexibility that facilitates CDR-CDR interactions in mAb self-association. These observations couple to our understanding of protein physicochemical properties, laying the groundwork for improved developability models.

Focus group study of student physiotherapists' perceptions of reflection.

CONTEXT: The reflective practice module in the physiotherapy programme at the University of Limerick, Ireland represents the first incidence of the inclusion of such a module within physiotherapy curricula in Ireland. However, research examining the contribution of reflection as a means of learning is limited, particularly from the student perspective. OBJECTIVES: This study sought to explore students' perceptions of reflection and its potential contribution to their development before and after the module. METHODS: A qualitative research methodology using focus groups was employed to evaluate physiotherapy undergraduate students' perceptions of the module. Three focus groups were held in total. Two were held with Year 3 students, before and after their reflective practice module, respectively, to examine any changes in their perceptions of reflection. A third was held with Year 4 students to determine their perceptions after both the module and subsequent clinical placements. Sessions were audiotaped, transcribed and subjected to in-depth thematic analysis to resolve the significant themes that emerged from the data. RESULTS: Students reported a more advanced level of reflective ability post-module completion. They perceived personal and professional benefits to practising reflection and recognised these skills as strategies with which they could continue to facilitate their professional development. For students, time constraints in the clinical setting represented a barrier to reflection. CONCLUSIONS: Students support inclusion of the module in their training, acknowledging its role in improving their confidence and clinical reasoning, and facilitating continuing professional development. Further studies are required to generalise these findings to a wider population.