Influence of Arginine Salts on the Thermal Stability and Aggregation Kinetics of Monoclonal Antibody: Dominant Role of Anions.

Thermal stability of the CH2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of arginine salts with acetate, glutamate (Glu-), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, Tm, an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the CH2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography. The effect of Arg+ on the thermal stability and aggregation kinetics of the antibody is shown to be strongly anion-dependent: both ArgAceate and ArgGlu improve the stability, while both Arg2SO4 and ArgCl decrease it. Furthermore, the addition of ArgCl and Arg2SO4 accelerates the aggregation kinetic, but to a lesser extent than the respective Na+ salt, suggesting that Arg+ binds to the antibody more strongly than Na+. However, the binding of Arg+ did not lead to more destabilization of the CH2 domain by the Arg+ salts at low concentrations, comparing to the respective Na+ salt. This finding indicates that Arg+ prefers the protein surface, rather than the exposed backbone upon unfolding. Furthermore, the change in the ranking for affecting the thermal stability and aggregation kinetics as the salt concentration increases implies the presence of other multiple mechanisms, e.g., cluster formation through the homoion pairing between Arg+ molecules and their preferential exclusion from the protein surface, and heteroion pairing between Arg+ and SO42-.

Characterization of the initial level and migration of silicone oil lubricant in empty prefilled syringes for biologics using infrared spectroscopy.

Glass prefillable syringes are lubricated with silicone oil to ensure functionality and a consistent injection for the end user. If excessive silicone is applied, droplets could potentially result in aggregation of sensitive biopharmaceuticals or clouding of the solution. Therefore, monitoring and optimization of the applied silicone layer is critical for prefilled syringe development. The hydrophobic properties of silicone oil, the potential for assay interference, and the very small quantities applied to prefilled syringes present a challenge for the development of a suitable assay. In this work we present a rapid and simple Fourier transform infrared (FTIR) spectroscopy method for quantitation of total silicone levels applied to prefilled syringes. Level-dependent silicone oil migration occurred over time for empty prefilled syringes stored tip-up. However, migration from all prefilled syringes with between 0.25 and 0.8 mg of initial silicone oil resulted in a stable limiting minimum level of between 0.15 and 0.26 mg of silicone in the syringe reached after 1 to 4 years of empty tip-up storage. The results of the FTIR assay correlated well with non-destructive reflectometry characterization of the syringes. This assay can provide valuable data for selection of a robust initial silicone oil target and quality control of prefilled syringes intended for biopharmaceuticals. LAY ABSTRACT: Glass prefillable syringes are lubricated with silicone oil to ensure functionality and a consistent injection for the end user. If excessive silicone is applied, droplets could potentially result in aggregation of sensitive biopharmaceuticals or clouding of the solution. Therefore, monitoring and optimization of the applied silicone layer is critical for prefilled syringe development. The hydrophobic properties of silicone oil, the potential for assay interference, and the very small quantities applied to prefilled syringes present a challenge for the development of a suitable assay. In this work we present a rapid and simple Fourier transform infrared (FTIR) spectroscopy method for quantitation of total silicone levels applied to prefilled syringes. Level-dependent silicone oil migration occurred over time for empty prefilled syringes stored tip-up. However, migration from all prefilled syringes with between 0.25 and 0.8 mg of initial silicone oil resulted in a stable limiting minimum level of between 0.15 and 0.26 mg of silicone in the syringe reached after 1 to 4 years of empty tip-up storage. The results of the FTIR assay correlated well with non-destructive reflectometry characterization of the syringes. This assay can provide valuable data for selection of a robust initial silicone oil target and quality control of prefilled syringes intended for biopharmaceuticals.