Abstract This study investigates the phase behavior of Poloxamer 188 (P188) in aqueous solutions under conditions typical of pharmaceutical lyophilization processes. We utilized heat flux sensors (HFSs) in a freeze-dryer and replicated lyophilization processes using a miniaturized setup in an XRPD climate chamber. HFS data from vials in the freeze-dryer indicated not only the primary crystallization of ice but also detected the secondary crystallization of the P188-water system. This was possible when primary ice nucleation was well-controlled and consistent across all vials, ensuring that primary crystallization signals didn’t overshadow those from secondary crystallization. Our findings reveal that P188 crystallization in aqueous systems is generally slow. Measurements in the XRPD climate chamber, involving a P188-based placebo solution, demonstrated that only a small portion of P188 crystallizes during the annealing step. The majority of P188 crystallizes during the primary drying step, concurrently with ice sublimation. Additional crystallization of P188 is observed even at the final secondary drying stage. These insights provide a valuable foundation for optimizing industrial lyophilization processes for Poloxamer-based lyophilizates. Graphical Abstract This study explores Poloxamer 188 crystallization during typical freeze-drying conditions for biologics. Heat flux sensors and an XRPD climate chamber reveal that P188 crystallizes quite slowly. Insights inform optimization of lyophilization processes of P188-containing formulations.

Abstract Background Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use. Methodology A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC). Results rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation. Conclusions Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins.

Abstract The mRNA lipid nanoparticle (mRNA-LNP) is a promising platform for vaccines and a variety of therapeutic areas, as demonstrated by the effective mRNA-LNP formulations in COVID-19 vaccines. While in-vitro transfection studies are crucial for optimizing mRNA-LNP formulations, few studies examine how cell line selection and reporter genes affect transfection efficiency. In our study, we investigated the in-vitro transfection efficiency of firefly luciferase mRNA-LNP on Jurkat cells, L-929 cells, and HEK 293 T cells. Jurkat cells, as a suspension cell line, displayed low transfection efficiency. The luciferase expression showed a non-linear relationship with mRNA dose, and cytotoxicity was observed with even low concentrations of mRNA. L-929 cells showed a linear relationship between bioluminescence and mRNA concentration, but only at low levels of mRNA, and their luciferase expression is limited. HEK 293 T cells are superior because of a strong linear dose–response and higher signal intensity. However, when using the luciferase-based assay for mRNA-LNP transfection, we observed high intra-group variations with signal fluctuated among technical replicates of the same formulation. In contrast, eGFP mRNA exhibited high reproducibility for the in-vitro transfection tests (coefficient of variation < 10%) and maintained strong linear correlation between mRNA concentration and eGFP expression (R2 > 0.95). This study highlights the importance of selecting a cell line model, a reporter gene, and analytical methods for developing a robust and reproducible in-vitro transfection assay for the development of mRNA-LNP formulation.

Abstract In vitro models used to investigate drug diffusion face certain limitations and challenges because they omit for mucus interactions that could influence diffusional transport. This study developed a simple mono-component mucin model using Mucin Type II from porcine stomach to predict the effects of the physicochemical properties of peptides on their diffusion through the intestinal mucus layer. The diffusion of octreotide and lanreotide through a mucin layer was compared with their respective Ala mutants replacing Lys (i.e., octreotide A5 and lanreotide A5). Ala mutants showed higher diffusion than their respective parent peptides, implicating that the charge interaction between positively charged, Lys-containing peptide and negative charge mucin override their hydrophobic interactions, thus hindering peptide diffusion. This finding was also supported by the faster diffusion of the negatively charged FITC-ADT10 compared to the positively charged FITC-HAV10 peptide. Thus, the interaction between the peptide’s positive and the negative charge of the glycans in mucin hinders peptide diffusion.. The neutral DTPPVK has the highest hydrophilicity and diffusion compared to negatively charged DTPPD, DTPPT, and ADTC5. Although DTPPD and DTPPT have about the same hydrophilicity, DTPPD has better diffusion than DTPPT because DTPPD with -2 charges has higher negative charge repulsion against mucin compared to that of DTPPT with -1 charge. Finally, ADTC5, with the lowest hydrophilicity, has the lowest diffusion through the mucin layer. This study found that the charge and hydrophilicity of peptides influence their diffusion across the mucin layer, and these studies correlate with the previous studies utilizing different in vitro models.

Abstract The paper investigates structure of freeze-dried formulations of a therapeutical monoclonal IgG1 with non-reducing disaccharides, sucrose and trehalose, as lyoprotectors. Formulations with variable sugar-to-protein ratios are manufactured using different freeze-drying protocols and post-drying temperature treatments. Small-angle X-ray scattering (SAXS) is applied to study packing arrangements of IgG1 molecules in the crowded solid-state environment. The retention of amorphous structure by the lyoprotectors is confirmed with the simultaneous wide-angle X-ray scattering (WAXS) tests. The new finding is the observation of two IgG1 SAXS peaks in the crowded environment, with position of one peak (peak A) changing with sugar-to-protein ratio, whereas the second peak (peak B) position is similar in all the formulations tested. The protein center of mass distance, which is calculated from the position of the peak A, increases with the increase in sugar content from 45 Å to 65 Å (sugar-to-protein mass ratio 0.79 to 7.95). Sugar molecules therefore serve as a physical barrier between IgG1 molecules. While the sugar-to-protein ratio significantly impacts protein separation, other factors, such as disaccharide type, presence of a surfactant, or drying process and post-drying thermal treatment, have minimal effect. The origin of the peak B has not been established yet, with three hypotheses considered. The results highlight applicability of SAXS for quantification of separation distances between protein molecules in freeze-dried formulations, thus improving the fundamental understanding of the stability of protein drugs. Graphical Abstract