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.

Binding of excipients is a poor predictor for aggregation kinetics of biopharmaceutical proteins.

One of the major challenges in formulation development of biopharmaceuticals is improving long-term storage stability, which is often achieved by addition of excipients to the final formulation. Finding the optimal excipient for a given protein is usually done using a trial-and-error approach, due to the lack of general understanding of how excipients work for a particular protein. Previously, preferential interactions (binding or exclusion) of excipients with proteins were postulated as a mechanism explaining diversity in the stabilisation effects. Weak preferential binding is however difficult to quantify experimentally, and the question remains whether the formulation process should seek excipients which preferentially bind with proteins, or not. Here, we apply solution NMR spectroscopy to comprehensively evaluate protein-excipient interactions between therapeutically relevant proteins and commonly used excipients. Additionally, we evaluate the effect of excipients on thermal and colloidal protein stability, on aggregation kinetics and protein storage stability at elevated temperatures. We show that there is a weak negative correlation between the strength of protein-excipient interactions and effect on enhancing protein thermal stability. We found that the overall protein-excipient binding per se can be a poor criterion for choosing excipients enhancing formulation stability. Experiments on a diverse set of excipients and test proteins reveal that while excipients affect all of the different aspects of protein stability, the effects are very much protein specific, and care must be taken to avoid apparent generalisations if a smaller dataset is being used.

Evaluation of a Biologic Formulation Using Customized Design of Experiment and Novel Multidimensional Robustness Diagrams.

Formulation development includes selection of appropriate excipients to stabilize the active pharmaceutical ingredient throughout its recommended shelf life, against potential excursions in its life cycle and sometimes to aid in the delivery of therapeutics into the patient. Identity and quantity of every ingredient in a therapeutic formulation are critical to achieve their intended purpose. Deviations from a target composition can result in manufacturing, safety, and efficacy challenges. It is mandatory to establish robustness of a formulation for the expected changes in its composition arising from the qualified "process variability" of the impacting process steps during manufacture. The approach for carrying out a robustness study evolved through improved understanding of a therapeutic stability and exploration of new tools, including the quality by design elements strongly recommended by regulatory agencies. An approach is presented here to study formulation robustness in multidimensional space using a customized experimental design and novel multidimensional diagrams, which present a unique way of identifying robustness limits. The concept is universally applicable to any multivariate analysis and such diagrams would be useful to comprehend the outcome on all variables at a glance. Interpretation of these diagrams is discussed, some of which are applicable in general to any statistical design of experiment.

Identification of Protein-Excipient Interaction Hotspots Using Computational Approaches.

Protein formulation development relies on the selection of excipients that inhibit protein-protein interactions preventing aggregation. Empirical strategies involve screening many excipient and buffer combinations using force degradation studies. Such methods do not readily provide information on intermolecular interactions responsible for the protective effects of excipients. This study describes a molecular docking approach to screen and rank interactions allowing for the identification of protein-excipient hotspots to aid in the selection of excipients to be experimentally screened. Previously published work with Drosophila Su(dx) was used to develop and validate the computational methodology, which was then used to determine the formulation hotspots for Fab A33. Commonly used excipients were examined and compared to the regions in Fab A33 prone to protein-protein interactions that could lead to aggregation. This approach could provide information on a molecular level about the protective interactions of excipients in protein formulations to aid the more rational development of future formulations.