Subcutaneous administration of monoclonal antibodies (mAbs) has gained importance in recent years due to the numerous advantages over intravenous administration, but it also has specific disadvantages. The limited volume of subcutaneous application requires the use of highly concentrated mAb solutions, which can lead to increased viscosity of formulation and reduced stability of these biological macromolecules.
The aim of this master’s thesis was to evaluate the effect of selected test excipients from four different classes, i.e., (i) amino acids, (ii) proline mimetics, (iii) di- and tripeptides containing proline and (iv) proline and nicotinic acid mimetics, on the viscosity of formulations containing a model IgG4 mAb and further their impact on the stability of the tested mAb. First, we optimized the basic formulation based on the calculation of the interaction diffusion coefficient (kD) and determined its optimal pH which was 5.5. Furthermore we demonstrated that the viscosity of formulations increases exponentially with increasing mAb concentration. In order to reduce viscosity, selected excipients were added at a concentration of 25 mM and NaCl in concentrations 50 and 150 mM to highly concentrated mAb formulations (210–310 mg/mL). Viscosity was measured using a chip-based viscometer, and it was found that all compounds (except S13, a dipeptide containing proline) reduced solution viscosity to some extent. The most effective compounds were those capable of forming multiple types of interactions with the mAb, such as hydrogen bonds, hydrophobic interactions, and π-π interactions. The appearance and growth of aggregates in formulations was also monitored using dynamic light scattering methods (DLS, MADLS) and size exclusion chromatography (SEC). Storage of samples at 4 °C did not result in a detectable increase in aggregates, regardless of the presence of test compounds. In contrast, three month storage at 40 °C revealed significant differences between samples with compounds S05 (a proline mimetic), S15 (a proline-containing dipeptide), and NaCl contributing to increased aggregation compared to the control, while others improved physical stability (S10, S11, both proline-containing dipeptide). Using the MADLS method, the presence of larger particles was detected in some formulations, which was later confirmed by SEC analysis. Statistical analysis showed a significant correlation between the two methods (r = –0.6796; p = 0.00378), indicating that MADLS is a suitable method for monitoring early signs of aggregation and thus a valuable tool in the development and optimization of mAb formulations.
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