Investigation of the Impact of Saccharides on the Relative Activity of Trypsin and Catalase after Droplet and Spray Drying.

While using saccharides as stabilizers for therapeutic protein drying is common, the mechanisms underlying the stabilization during drying remain largely unexplored. Herein, we investigated the effect of different saccharides, trehalose dihydrate (TD), dextran (DEX), and hydroxypropyl ß-cyclodextrins (low substitution-HP and high substitution-HPB), on the relative activities of the enzymes trypsin and catalase during miniaturized drying (MD) or spray drying (SD). For trypsin, the presence of saccharides, especially HP, was beneficial, as it significantly improved the enzyme activity following MD. The HPB preserved trypsin's activity during MD and SD. Adding saccharides during MD did not show a notable improvement in catalase activities. Increasing TD was beneficial during the SD of catalase, as indicated by significantly increased activity. Molecular docking and molecular dynamics simulations oftrypsin with HP or HPB revealed the influence of their substitution on the binding affinity for the enzyme. A higher affinity of HP to bind trypsin and itself was observed during simulations. Experimentally, activity reduction was mainly observed during MD, attributable to the higher droplet temperature during MD than during SD. The activities from the experiments and aggregation propensity from molecular modeling helped elucidate the impact of the size of protein and saccharides on preserving the activity during drying.

Impact of Different Saccharides on the In-Process Stability of a Protein Drug During Evaporative Drying: From Sessile Droplet Drying to Lab-Scale Spray Drying.

OBJECTIVES: Solid biopharmaceutical products can circumvent lower temperature storage and transport and increase remote access with lower carbon emissions and energy consumption. Saccharides are known stabilizers in a solid protein produced via lyophilization and spray drying (SD). Thus, it is essential to understand the interactions between saccharides and proteins and the stabilization mechanism. METHODS: A miniaturized single droplet drying (MD) method was developed to understand how different saccharides stabilize proteins during drying. We applied our MD to different aqueous saccharide-protein systems and transferred our findings to SD. RESULTS: The poly- and oligosaccharides tend to destabilize the protein during drying. The oligosaccharide, Hydroxypropyl ß-cyclodextrin (HPßCD) shows high aggregation at a high saccharide-to-protein molar ratio (S/P ratio) during MD, and the finding is supported by nanoDSF results. The polysaccharide, Dextran (DEX) leads to larger particles, whereas HPBCD leads to smaller particles. Furthermore, DEX is not able to stabilize the protein at higher S/P ratios either. In contrast, the disaccharide Trehalose Dihydrate (TD) does not increase or induce protein aggregation during the drying of the formulation. It can preserve the protein's secondary structure during drying, already at low concentrations. CONCLUSION: During the drying of S/P formulations containing the saccharides TD and DEX, the MD approach could anticipate the in-process (in) stability of protein X at laboratory-scale SD. In contrast, for the systems with HPßCD, the results obtained by SD were contradictory to MD. This underlines that depending on the drying operation, careful consideration needs to be applied to the selection of saccharides and their ratios.

Determination of nucleating agents in plastic materials by GC/MS after microwave-assisted extraction with in situ microwave-assisted derivatization.

This work demonstrates the analysis of sorbitol-based nucleating and clarifying agents (NCAs) used as additives for polyolefin-based materials by gas chromatography/mass spectrometry (GC/MS), employing a highly reliable and efficient sample preparation methodology applying microwave irradiation. A derivatization by silylation of the analytes was done to improve the GC-suitability of the analytes. After successful optimization of the conditions for a microwave-assisted derivatization (MAD), the microwave-assisted extraction (MAE) of the analytes from polymer samples was investigated. Tetrahydrofuran (THF) turned out to be the best extraction solvent, as poorly soluble analytes show the highest solubility in this solvent and THF supports the silylation after extraction. This two-step approach and subsequent chromatographic determination resulted in reproducibilities from approximately 2% up to 6% and recoveries from 95.8% up to 104.2% in real samples. In order to reduce the number of sample preparation steps a one-step approach was investigated and optimized, in which MAE and MAD were carried out simultaneously. The developed procedure resulted in remarkably better repeatabilities ranging from 0.05% up to 4% and reproducibilities of up to 10%. The recoveries matched those obtained with the two-step process. Linearities could be achieved in both approaches with R(2) better than 0.99 for all selected analytes over two orders of magnitude. All data indicate the suitability of both presented methods for the reliable determination of sorbitol-based NCAs in polyolefin materials.