Therapeutic proteins, if not formulated well, are susceptible to physical (aggregation) and chemical degradation from small perturbations in their structures impacting the drug potency and leading to immune response in vivo due to protein aggregate formation. Therefore, overcoming any stability issues and maintaining the stability of therapeutic proteins to be commercially viable has been one of the main challenges for formulation development. The most commonly used approach to enhance protein stability and stabilize the drug product is to add excipients to the protein solution, and the most common composition of a biopharmaceutical formulation consists of a buffer (e.g., histidine, phosphate), stabilizer (e.g., carbohydrates, sugars, polyols, amino acids), surfactant (e.g., PS20, PS80) and antioxidant (e.g., EDTA, DTPA). The stability of the proteins significantly varies with protein concentration, pH, buffer, buffer concentration, and the type of excipients. Excipient compatibility screening and physicochemical stress studies at accelerated conditions are conducted for finalizing the excipient selection and composition. Lyophilization (freeze drying) of the protein formulation in selected excipients is also done to further enhance protein stability, especially for formulations with significant stability challenges.
Despite carrying out the many screening studies needed to choose the right excipients for a given protein, the mechanisms by which the excipients provide stability to the protein are not fully understood. In other words, why are some excipients better at preserving protein stability than others? Understanding why some excipients are better stabilizers of proteins can help with developing robust biopharmaceutical formulations in an accelerated manner. Moreover, having an analytical tool to quantify and rank the factors leading to their stability helps to make the excipient selection process systematic and practical. In the literature there are very few studies that provide a mechanistic understanding of the stabilizing effect of excipients to maintain protein stability. Preferential exclusion mechanism by carbohydrates is one of the most prevalent mechanisms in the literature by which protein can be stabilized, adding beneficial effects on aggregation and conformational stability of the protein. No direct evidence for protein-excipient interactions was identified, whether the interaction being specific or not specific to the protein. Though, Kim et al using isothermal titration calorimetry (ITC) showed proteins with a high binding affinity to the carbohydrates, probably due to the hydrogen bond formation between the protein binding sites with the carbohydrate molecules. Souillac et al elicited by Fourier transform Infra-Red (FTIR) spectroscopic studies that in the presence of carbohydrate, the secondary structure was replenished by hydrogen bonds formed between the polar groups on the surface of the protein and carbohydrate moieties during lyophilization process. The aim of this work is to provide direct experimental evidence of binding affinity of excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method, and therefore ranking a series of excipients with respect to their dissociation constant (KD) and non-specific binding constants (Ns). Molecular dynamic and Monte Carlo simulations were conducted in parallel, to rank the excipient proximity to the proteins and thereby corroborating the STD NMR results and ranking. The study also provided insights into potential specific binding sites on proteins and the preferential conformation of the excipient to the protein, especially for sucrose and mannitol. Finally, the excipient ranking by NMR was correlated to mAb stability, by comparing both Tm, which gives the conformational stability of the protein, and B22, which gives the magnitude of the interaction between two protein molecules in solution, with the chosen excipients. This approach has aided and accelerated the excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conducting a conventional excipient screen-ing study which is time consuming.