Hydroxylpropyl-ß-cyclodextrin as Potential Excipient to Prevent Stress-Induced Aggregation in Liquid Protein Formulations.

Due to the growing demand for patient-friendly subcutaneous dosage forms, the ability to increasing protein solubility and stability in formulations to deliver on the required high protein concentrations is crucial. A common approach to ensure protein solubility and stability in high concentration protein formulations is the addition of excipients such as sugars, amino acids, surfactants, approved by the Food and Drug Administration. In a best-case scenario, these excipients fulfil multiple demands simultaneously, such as increasing long-term stability of the formulation, reducing protein adsorption on surfaces/interfaces, and stabilizing the protein against thermal or mechanical stress. 2-Hydroxylpropyl-ß-cyclodextrin (derivative of ß-cyclodextrin) holds this potential, but has not yet been sufficiently investigated for use in protein formulations. Within this work, we have systematically investigated the relevant molecular interactions to identify the potential of Kleptose®HPB (2-hydroxylpropyl-ß-cyclodextrin from Roquette Freres, Lestrem, France) as "multirole" excipient within liquid protein formulations. Based on our results three factors determine the influence of Kleptose®HPB on protein formulation stability: (1) concentration of Kleptose®HPB, (2) protein type and protein concentration, and (3) quality of the protein formulation. Our results not only contribute to the understanding of the relevant interactions but also enable the target-oriented use of Kleptose®HPB within formulation design.

Simplified choice of suitable excipients within biologics formulation design using protein-protein interaction- and water activity-maps.

Still today, high-concentration protein formulations are often developed based on high-throughput experimental screening approaches. Although likely delivering working formulations, these approaches do not lead to a deep/mechanistic understanding of the protein phase behavior in solution. Within this work, we thus optimized and enhanced a recent approach for an initial low effort selection of potential excipients and excipient mixtures to be used in high-concentration protein formulations. This approach considers both: molecular interactions and thermodynamic determinants to access the phase behavior of the proteins in solution, as well as pharmaceutical engineering boundaries (such as osmotic pressure and osmolality) to deliver on optimal formulation conditions. Water activity coefficient γW-calculations (used to describe the protein environment in solution), unfolding temperature (conformational stability) and protein-protein interactions (colloidal stability) are used as determinants. Amino acids (20 proteinogenic amino acids), selected amino acid mixtures, as well as mixtures of amino acids and trehalose (l-arginine-trehalose; l-histidine-trehalose) are considered as model excipients. The approach is extends by studying the long-term stability of the predicted formulation conditions for a γ-globulin from human blood and denosumab. The results reveal, that by combining protein-specific experiments as well as model-based studies for the selection of excipient mixtures in high concentration protein formulations, the effort as well as the resource requirements can be reduced significantly.

Cosolvent and pressure effects on enzyme-catalysed hydrolysis reactions.

Thermodynamics and kinetics of biochemical reactions depend not only on temperature, but also on pressure and on the presence of cosolvents in the reaction medium. Understanding their effects on biochemical processes is a crucial step towards the design and optimization of industrially relevant enzymatic reactions. Such reactions typically do not take place in pure water. Cosolvents might be present as they are either required as stabilizer, as solubilizer, or in their function to overcome thermodynamic or kinetic limitations. Further, a vast number of enzymes has been found to be piezophilic or at least pressure-tolerant, meaning that nature has adapted them to high-pressure conditions. In this manuscript, we review existing data and we additionally present some new data on the combined cosolvent and pressure influence on the kinetics of biochemical reactions. In particular, we focus on cosolvent and pressure effects on Michaelis constants and catalytic constants of α-CT-catalysed peptide hydrolysis reactions. Two different substrates were considered in this work, N-succinyl-L-phenylalanine-p-nitroanilide and H-phenylalanine-p-nitroanilide. Urea, trimethyl-N-amine oxide, and dimethyl sulfoxide have been under investigation as these cosolvents are often applied in technical as well as in demonstrator systems. Pressure effects have been studied from ambient pressure up to 2 kbar. The existing literature data and the new data show that pressure and cosolvents must not be treated as independent effects. Non-additive interactions on a molecular level lead to a partially compensatory effect of cosolvents and pressure on the kinetic parameters of the hydrolysis reactions considered.