Protein particles and aggregates can form under various stresses, and it is always challengeable to detect and quantify particles and protein aggregates in protein formulations. Current technologies for measuring particle and protein aggregate levels are not satisfied with real-time measurement. In this work, we investigated mechanisms of particle and protein aggregate formation under different stresses using various assays we developed.
Extrinsic fluorescence dye such as 4,4’-Dianilino-1,1’-binaphthyl-5,5’-disulfonate (bis-ANS) was used to rapidly quantify the presence of protein particle and aggregate in protein formulation. Protein particles and aggregates were formed under mechanical and freeze-thaw cycle stresses. We obtained a linear correlation between fluorescence intensity and particle and insoluble protein aggregate levels, which could be used as a calibration curve to estimate unknown particle and protein aggregate concentration. Finally, the assay also had a reasonable capacity for quantifying protein particle concentration in high concentration protein formulation.
ThioflavinT (ThT) was used to detect the formation of insulin fibril formation following mechanical shock treatment. Various amounts of pre-formed insulin aggregate(pH=7.4) generated by probe sonicating were spiked into unstressed insulin solution to obtain a calibration curve to estimate unknown amounts of insulin aggregate. In addition, the mixtures were also incubated at 40 oC to accelerate the insulin fibril formation. We showed that the rate of fluorescence intensity increase over time was linearly correlated to the initial concentration of insulin aggregate concentration, and this correlation could also be used to predict unknown amounts of insulin aggregate in insulin solution. We dropped insulin solution (pH=7.4) by drop tester and cavitation was observed when vials were dropped on the hard surface. Transmission electron microscopy (TEM) images also showed the formation of fibril in solutions after dropping. Both dropped and unstressed insulin solution were spiked into unstressed (pH=3) and incubated at 40 oC to quantify the formation of insulin aggregate. Elevated fluorescence intensity was observed in samples with the presence of dropped insulin solution, and the rate of fluorescence intensity growth was used to estimate the concentration of insulin aggregate.
Cavitation bubbles were found in protein solutions when they were experienced mechanical shock by dropping. We observed that cavitation played an important role in sub-visible particle formation in protein solutions following the treatment of mechanical shock. We also found that air bubbles that were trapped over vial surface crevices were the primary cavitation sites which could nucleated cavitation bubbles during dropping. To investigate surface properties on cavitation incidence, we dropped different types of vials that had different roughnesses and we found smoother vial surfaces had lower cavitation incidence. Finally, we found that addition of PS20 in the protein solution could reduce the occurrence of cavitation and sub-visible particle formation when vials were dropping on the hard surface.
Finally, sub-visible particle and protein aggregate were generated during fill-finish operation. Through modifying the pump chamber surface charge, we investigated the effect of electrostatic interaction between pump chamber surface and protein formulation on particle and protein aggregate formation during the fill-finish process. We found that the positively charged surface could reduce the adsorption rate of protein on the surface due to the repulsion electrostatic interaction, which decreased the particle generation.
|Advisor:||Randolph, Theodore W|
|Commitee:||Medlin, Will, Kaar, Joel, Goodwin, Andrew, Carpenter, John|
|School:||University of Colorado at Boulder|
|Department:||Chemical and Biological Engineering|
|School Location:||United States -- Colorado|
|Source:||DAI 81/11(E), Dissertation Abstracts International|
|Keywords:||High-throughput protein aggregate assay, Protein aggregation, Protein formulation fill-finish process, Protein Formulation Stability|
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