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Structural and functional integrities of formulated proteins are key characteristics that provide a better understanding of influencing factors and their adjustment during formulation development. Here, the procedures commonly used for protein analysis were applied and optimized to obtain a higher degree of accuracy, reproducibility, and reliability for the analysis of lysozyme extracts from hot-melt extrudates (HME). The extrudates were prepared with polyethylene glycol 20 000. The test lysozyme HMEs were subjected to extraction procedures and analytical methods following the International Council of Harmonization guidelines for testing the active protein ingredient Q 1 A (R2) in its pure and formulated form. Therefore, reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, and fluorescence-based activity measurements were applied to study lysozyme stability and function after formulation. Long-term accelerated stability studies were performed for the pure and formulated protein. Our findings revealed a high degree of stability for lysozyme toward different temperatures and storage times, confirming that HME is a suitable formulation alternative that preserves lysozyme's properties and stability. The presented methods and workflow are recommended to be exploited for further protein drugs to assess usability and compatibility concerning different pharmaceutical applications.

Selection of an appropriate formulation to stabilize therapeutic proteins against aggregation is one of the most challenging tasks in early-stage drug product development. The amount of aggregates is more difficult to quantify in the case of peptides due to their small molecular size. Here, we investigated the suitability of diffusion self-interaction parameters (kD) and osmotic second virial coefficients (B22) for high-throughput (HT) screening of peptide formulations regarding their aggregation risk. These parameters were compared to the effect of thermal stress on colloidal stability. The formulation matrix comprised six buffering systems at two selected pH values, four tonicity agents, and a common preservative. The results revealed that electrostatic interactions are the main driver to control colloidal stability. Preferred formulations consisted of acetate and succinate buffer at pH 4.5 combined with glycerol or mannitol and optional m-cresol. kD proved to be a suitable surrogate for B22 as an indicator of high colloidal stability in the case of peptides as was previously described for globular proteins and antibodies. Formulation assessment solely based on kD obtained by HT methods offers important insights into the optimization of colloidal stability during the early development of peptide-based liquid formulations and can be performed with a limited amount of peptide (∼360 mg).

The influence of supersaturation and solubilization on oral absorption was assessed independently from the dissolution process for the non-formulated model drugs celecoxib and telmisartan. In vitro, physicochemical characterization and biphasic dissolution were used to characterize the supersaturation and solubilization effects of three water soluble polymers (copovidone, methylcellulose and Soluplus®) on the drugs. While celecoxib precipitated in a crystalline form resulting in pronounced stabilization of supersaturation, telmisartan precipitated as a highly energetic amorphous form and the potential of the polymers to enhance its solubility was subsequently, limited. In vivo, for the crystalline precipitating celecoxib, supersaturation and solubilization increased its oral bioavailability up to 10-fold. On the contrary, the amorphous precipitating telmisartan did not benefit from the limited stabilization in terms of oral exposure. Amongst all investigated in vitro tests the biphasic dissolution test was the most predictive in relation to supersaturation. However, for the potential micellar solubilization and the respective impact in the aqueous/organic interface, prediction accuracy of the biphasic dissolution test was limited in combination with Soluplus®. Despite the hetergeneous micellar distribution in vitro and permeation in vivo, the biphasic approach could clearly show the supersaturation potential on bioavailability (BA) for celecoxib on the one hand and the inferiority of supersaturation on BA for telmisartan.

High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (kD). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the kD analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of kD was dependent on the plate position of the sample replicates and kD value. Positive kD values (57.0 mL/g, coefficients of variation (CV) 8.9%) showed a lower variability compared to negative kD values (−14.8 mL/g, CV 13.4%). The variability of kD was not reduced using more data points (120 vs. 30). A kD analysis exclusively based on center wells showed a lower CV (&lt, 2%) compared to edge wells (5–12%) or a combination of edge and center wells (2–5%). We present plate designs for kD analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).