Impact of tubing material on stability and filling accuracy of biologic drug product.

This article is presenting completely new observations linked to Polysorbate 80 (PS80) oxidation in biologics drug product. Indeed, we observed that, in the drug product exposed to long contact time (∼ 1 h) in platinum-cured silicon tubing during the filling, the oxidation of PS80 is dramatically accelerated compared to short contact time. The phenomenon was observed in presence of iron traces (20 ppb), but not in absence of iron (< 2 ppb) or in presence of a chelator like EDTA. Electron Paramagnetic Resonance (EPR) measurements demonstrated the presence of radicals formed during the oxidation. It was deduced that platinum-cured silicon tubing is leaching some radical initiators, most probably peroxides decomposed by the iron. Alternative filling sets made of ThermoPlastic Elastomer (TPE) were investigated, both for the impact on PS80 stability and the filling performance using a peristaltic pump. The results showed that these filling sets were indeed not causing accelerated PS80 degradation but the process was not robust enough; these filling sets being too rigid for the constraints of the peristaltic pump rollers. These results show that there is no practical tubing alternative to platinum silicone cured tubing. To avoid the impact on PS80 oxidation the potential remediations presented in the article are to avoid any trace of iron or to add a chelating agent, or to discard the vials having experimented a filling stop (> 5 min).

Inhaled IgG1 antibodies: The buffering system is an important driver of stability during mesh-nebulization.

In the past decade, oral inhalation has been a thriving focus of research to administer antibody directly to the lungs as an aerosol, for local treatment of respiratory diseases. Formulation of inhaled antibodies is central for the stability of antibody, lung safety and to ensure inhaler performances. Surfactants have already been shown to prevent antibody degradation during aerosolization, but little is known about the impact of other components of liquid formulations on the structural stability of antibodies. Here, we report for the first time to the best of our knowledge, a significant effect of the buffering system on monoclonal antibodies stability, during mesh-nebulization. While the monoclonal antibody extensively aggregated in citrate buffer after nebulization and required high concentration of polysorbate 80 (PS80) to maintain protein integrity, acetate and histidine buffers resulted in a slight to moderate aggregation without PS80 and low concentration of PS80 was sufficient to stabilize antibody during mesh-nebulization.

Mixing of Monoclonal Antibody Formulated Drug Substance Solutions in Square Disposable Vessels.

Fill & finish manufacturing processes of biologics drug product involve multiple unit operations. In particular they often include a mixing step to reduce non-uniformities in fluids by eliminating gradients of concentration and pH may occur during freezing. This step should be conducted carefully to avoid any degradation of the protein under mechanical stress. This study was aimed at characterizing disposable vessels of square cross-section such as Levmixer® from Sartorius Stedim in terms of fluid dynamics and mixing in turbulent regime. The investigation included two tree large vessels (50, 200 & 650-l) and one 4-l vessel designed in house. For that purpose, the impact of stirrer speed, filling volume and duration of mixing on product quality attributes were studied, using a surrogate. Moreover, a scale-up rule, based on first principle, was established and allows prediction of the mixing time as a function of stirring speed and filling volume. A lab-scale test, using drug product, was performed at the same stress intensity but for a much longer duration than the commercial operation and did not reveal any trend to aggregation. Finally, based on the correlation, lab scale stress test and a single verification test at large scale, a design space within which the product can be processed without altering product quality was proposed.

Mechanistic understanding of metal-catalyzed oxidation of polysorbate 80 and monoclonal antibody in biotherapeutic formulations.

Surfactants are commonly used in biotherapeutic formulations to prevent the formation of aggregates and protect proteins from denaturation. Among them polysorbates are the most widely used. However, they are known to be prone to degradation, mainly via enzymatic hydrolysis and oxidation. In this study, the impact of different conditions and factors on the oxidation of polysorbate 80 (PS80) and of a monoclonal antibody (mAb) was evaluated. In particular, the role of different formulation components (e.g., mAb concentration, pH, buffer, surfactant grade, chelators) was investigated in the presence of iron as transition metal contaminant. The results of our studies demonstrated that PS80 oxidation was accelerated even in the presence of iron levels as low as 20 ppb. In addition, the results showed that the oxidation of a specific solvent-exposed mAb methionine increased with PS80 oxidation, in particular under accelerated stress conditions and that the oxidation phenomenon was hindered in absence of iron or after addition of EDTA. Our results showed that PS80 "all oleate" (PS80-AO) was more sensitive to oxidative degradation than PS80 "multi-compendial" (PS80-MC). Contrary to acetate and citrate buffers, the results showed that the kinetics of PS80 oxidation was pH-dependent in presence of histidine buffer. It was also demonstrated that, when increasing its concentration, the mAb exhibited a protective effect against metal catalyzed PS80 and methionine oxidation. Our systematic studies on the role of the formulation components and potential contaminants (i.e., iron) demonstrated the complexity of the oxidative mechanism and the importance of different competitive systems, including pro-oxidant factors (e.g., iron, pH, PS80 quality) and antioxidant factors (e.g., protein concentration, EDTA, citrate) that may occur in biologic formulations containing PS80.

Y-shaped mPEG-PLA cabazitaxel conjugates: well-controlled synthesis by organocatalytic approach and self-assembly into interface drug-loaded core-corona nanoparticles.

A well-defined poly(ethylene glycol) methyl ether-b-poly(lactic acid) copolymer (mPEG-PLA) featuring a new, Y-shaped, architecture with a hydroxyl functional group between the two blocks has been prepared and thoroughly characterized. The functional copolymer was then readily coupled to diglycolyl-cabazitaxel. The resulting copolymer conjugates assembled into stable and monodisperse nanoparticles (NPs) in aqueous suspension. The architecture of the copolymer conjugate is shown to impact the spatial distribution of the drug within the nanoparticles. With the Y-shaped architecture, cabazitaxel was found localized at the interface of the hydrophobic PLA core and the hydrophilic mPEG corona of the NPs, as substantiated by variable temperature NMR analysis of the nanoparticles in D2O. Preliminary in vitro release studies reveal dependence on the architecture of the copolymer conjugate. This new approach offers promising perspectives to finely tune the position of the active ingredient in polymeric nanoparticles.