Quantifying Protein Viscosity As a Method for Determining Biopharmaceutical Degradation

Quantifying Protein Viscosity as a Method for Determining Biopharmaceutical Degradation

Katherine N. Clayton¹, Dong Hoon Lee¹, Steven T. Wereley¹, Tamara L. Kinzer-Ursem²

¹School of Mechanical Engineering, Purdue University

²Weldon School of Biomedical Engineering, Purdue University

Patients requiring protein-based biopharmaceutical prescriptions run the risk of inactivation of their biologic due to heating or short-shelf life. However, the maintenance of protein folding and activity levels of these biopharmaceuticals is essential for the safety and efficacy of patient treatment. Developing quick and efficient methods for determining protein degradation could be of use in current laboratory work flows. Here, we present Particle Scattering Diffusometry (PSD), a viscosity measurement technique that uses low sample volumes (< 4mL) that can be integrated into micro- or nanofluidic systems. Based on the fundamental principles of diffusion, particles (~200 nm) undergoing Brownian motion are imaged under fluorescence microscopy. The diffusion coefficient of the particles is calculated by correlating successive particle images (at time Dt) to one another (cross-correlation, s_c) and the particle image with itself (autocorrelation, s_a) at a magnification (M):

The viscosity of the solution is then calculated from the Stokes-Einstein equation.

In this work we use PSD to measure amounts of protein degradation of a common biologic used in diabetes treatment as a function of protein viscosity. We address insulin stability by comparing degraded and intact insulin samples between concentrations of 1 to 10 mg/ml. Particles introduced to the solution are imaged with PSD.  Solution viscosity is calculated from the measured diffusion coefficient of the particles. We find that the viscosity of denatured insulin exponentially increases with concentration, whereas intact insulin maintains a similar viscosity across a range of concentrations. Additionally, we combined denatured and intact insulin to quantify changes in the viscosity when a certain fraction of the insulin is denatured. Not unexpectedly, we find that there is a decrease in the viscosity of insulin with increasing concentrations of intact insulin. Importantly, we are able to detect small changes in solution viscosity when low amounts of denatured insulin is present within the samples. By achieving viscosity measurements of denatured insulin presence in sample, PSD can be used to test for biopharmaceutical sample stability for quality control in field testing.

In summary, we have established a rapid (~8 seconds) and sensitive technology for detecting biomarkers in very low sample volumes (microliters) using instrumentation and tools that are common to most laboratory settings (fluorescent microscope, CCD camera, and computer). Based on the success of using PSD for biopharmaceutical viscosity analysis, we expect to translate these techniques for use in a wide range of protein-based biopharmaceuticals. In this presentation we will to discuss the development of the PSD technique, our current results and challenges associated with PSD measurements, as well as future work to integrate this method into basic laboratory settings.