IMPACT OF EXCIPIENTS ON MOBILITY AND STABILITY OF LYOPHILIZED BIOLOGICS FORMULATIONS
Creators: name: Tower, Cole
Publisher: name: Purdue University Graduate School
Year: 2024
Subjects: classificationCode: 321405
subject: Pharmaceutical sciences

description: Biologic drugs are a key defense against many health issues. In many cases, biologic drugs are not stable in the solution state and must be lyophilized. Lyophilization in the presence of excipients increases the stability of the drug by interactions with the excipients through hydrogen bonding, which will lower the local mobility of the drug. Key threats to stability include: inhomogeneity of the drug substance and excipients, high mobility, and crystallization. Solid-state nuclear magnetic resonance spectroscopy was used to identify crystallization, assess homogeneity, and measure the local mobility of lyophilized protein and mRNA/LNP systems. The impact of disaccharide type and concentration on protein stability was explored. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations, and solid-state nuclear magnetic resonance spectroscopy (ssNMR) <sup>1</sup>H T<sub>1</sub> and <sup>1</sup>H T<sub>1rho</sub> relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, and measure local mobility with <sup>1</sup>H T<sub>1</sub> relaxation times. HSA/sucrose systems had longer <sup>1</sup>H T<sub>1</sub> relaxation times and were slightly more stable than trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have <sup>1</sup>H T<sub>1</sub> relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared to binary systems. Phase separation was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions, a <sup>1</sup>H T<sub>1</sub> relaxation time below 1.5 s correlated with an unstable sample, regardless of disaccharide(s) used.The effect of mannitol on protein stability was studied. Human serum albumin was lyophilized in binary systems with mannitol, and in ternary systems with sucrose or trehalose and mannitol. The monomer content of the HSA was monitored over 36 weeks of storage at 50 C. The amount of mannitol in the system dictated the ability of mannitol to crystallize, and the polymorph that mannitol crystallized into. In HSA/mannitol systems, mannitol crystallization caused inhomogeneity of the matrix, determined by <sup>1</sup>H T<sub>1rho</sub> relaxation times. Adding a disaccharide to the matrix, however, increased the homogeneity of the matrix. Addition of mannitol to a HSA/disaccharide matrix resulted in less stability at similar HSA:disaccharide ratios.The impact of storage temperature on protein stability was investigated. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. <sup>1</sup>H T<sub>1</sub> relaxation times were measured by ssNMR and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.The effect of an RF-assisted lyophilization method on homogeneity, mobility, stability, and moisture content was explored. This method, utilizing 18 GHz microwave frequency to accelerate the lyophilization cycle, resulted in equivalent or better stability for attenuated live virus or protein formulations, respectively. ssNMR showed comparable amounts of homogeneity in the formulations, however mobility of the samples produced by RF-assisted lyophilization was slightly higher.A lyophilized mRNA/LNP formulation was prepared. Disaccharide type, disaccharide concentration, and freezing rate were found to alter critical quality attributes of the system. When mRNA/LNP formulations were stored at 4 C, solution formulations outperformed lyophilized formulations for at least 6 months. When mRNA/LNP formulations were stored at room temperature, solution formulations were superior for the first three months, however lyophilized formulations outperformed solution formulations after 6 months, with less growth in particle size and less loss of encapsulation efficiency. ssNMR was used to assess the interactions between the formulation components.