Innovative analytical solutions and quality by design approaches in preformulation studies of a recombinant protein as a biotherapeutic candidate

RODRÍGUEZ, M. C.; BÜRGI, M. M.; KRATJE, R. B.; OGGERO, M. R

Viña del Mar. Valparaíso

Simposio; X Simposio Latinoamericano de Tecnología de Cultivo de Células Animales (X SLATCC); 2024

Pontificia Universidad Católica de Valparaíso. Universidad de Chile. Universidad de Concepción.

IntroductionThe preformulation and formulation stages of biotherapeutic proteins are complex and time-intensive due to their intricate nature. Critical material attributes (CMAs) such as pH, buffering systems, and the selection of compatible excipients under stress conditions are commonly studied. High-throughput (HT) technologies and Quality by Design (QbD) approaches contribute significantly to the rational design of formulations, providing a clear path for drug product development. The aim of this work was to rapidly and effectively identify CMAs to ensure the quality of a recombinant molecule for the treatment of neurological disorders, combining HTP strategies such as differential scanning fluorimetry (DSF) and QbD approaches.MethodsFor proof of concept, two variants of the biotherapeutic candidate from alternative purification processes (n=6) were analysed using DSF. Among them, a promising variant was selected, and the impact of CMAs was studied under stress conditions like thermal stress. Additionally, fluorescence excitation-emission matrix (EEM) coupled with chemometric modelling (MCR-ALS) was used to monitor pH-induced changes in the protein's tertiary structure. Finally, a multivariate formulation procedure through design of experiment (DoE) was implemented to evaluate potential excipients and their interaction effects on protein stability. Independent assays (at least n=2) were performed to ensure the reproducibility and consistency of all the results.ResultsAt least 95%-pure variants from the alternative purification procedures were used for assessing. Tm values ranging from 48 to 55 °C were determined for the six batches evaluated by DSF analysis. Statistical significance between samples (p-value: 0.0092) was observed through a non-parametric test. The promising variant was then assayed for conformational stability under different pH conditions (3.0-9.0), performing DSF studies. At acidic pHs (3.0-4.0), melting curves indicated protein unfolding, with the highest Tm at pH 6.0 (47.5 ± 0.7 °C). This result was confirmed using EEM coupled with MCR-ALS modelling, providing spectral and abundance distribution profiles for monitoring pH-induced changes.Different excipients, either alone or in combination with buffer agents, were evaluated for their ability to stabilize the protein through DSF assays. The presence of NaCl and Pluronic® F-68 increased the Tm value, indicating higher thermal stability. A screening fractional factorial DoE was performed in accelerated stability studies to determine the effects of different excipients, identifying combinations that improve the molecule's stability.ConclusionsThis comprehensive and systematic approach represents a practical procedure that helps elucidating CMAs, and facilitates the development of the final dosage form of the biotherapeutic, for a better performance of its stability, efficacy, and safety.