(394e) 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.