Microrheology for protein therapeutics development
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Monoclonal antibody (mAb) protein therapeutics are a rapidly growing class of pharmaceuticals. High protein concentrations are required for long-term storage and subcutaneous administration, but these concentrations often result in significantly higher solution viscosities. Viscosity of mAb solutions is used as a development-risk screening factor during early stage development. However, proteins are scarce at this stage of development, and a formulation screen must be conducted quickly to “lock” a suitable formulation that mitigates potential viscosity issues for further clinical development. Multiple particle tracking (MPT) microrheology addresses these needs for rheological measurements of protein solutions with microliter sample volumes and rapid acquisition. In this work, the accuracy and precision of MPT methods are improved via new theoretical approaches, and MPT microrheology is implemented in a device for high-throughput characterization of mAb solution viscosities for early stage pharmaceutical development. ☐ In passive microrheology viscosity is typically determined by the logarithmic intercept of the mean-squared displacement (MSD). This approach biases the largest lag times τ, which are also those with the poorest statistics. A new method is de- scribed that identifies an optimal lag time τ from an MPT experiment to calculate the viscosity using the Van Hove self-correlation. Particle displacement follows a Gaussian distribution, and deviations from Gaussian behavior, quantified using the excess kurtosis α2, indicate which lag times are unsuitable for the viscosity calculation through the use of a test statistic Zα2 . This method ensures that sources of error in the MPT experiment are minimized, generating the most accurate and precise measurements. Particle tracking uncertainty, or static error, has typically been measured with immobilized particles in a gel, but the imaging conditions of the gel are not identical to those in the sample of interest. To estimate the static error ε under sample conditions, a new in situ method was developed. This method is also derived using the excess kurtosis α2, and it enables measurement of the static error using the same data collected during the MPT experiment. The correction of the mean-squared displacement by the in situ method is more reliable than the gel estimation, which overcorrects for the static error. With the true MSD now accessible at short lag times, experimental artifacts can now be distinguished from phenomena in solution, even in highly viscous (> 10,000 cP) solutions. ☐ These new methods are used to characterize viscosity profiles for therapeutic protein solutions with a high-throughput microfluidic device with multiple channels for 2 μL samples on a single microscope slide. Mounted on a temperature control stage, this device executes 72 temperature-concentration dependent viscosity measurements in less than 6 hours. An Arrhenius temperature dependence has been observed for the viscosities of two humanized immunoglobulin antibodies, mAb1 and mAb2. Remarkably, the two mAbs have different concentration dependence; even though they share 98% identical sequences, mAb1 has as much as ten times the viscosity of mAb2 at 90 mg/mL at 0.9°C, underscoring the importance of such screening in early stage development. ☐ Particle tracking microrheology also enables the first study and comparison of the viscosity profiles of bispecific mAbs (BsAb-A/B and BsAb-B/C) and their monospecific counterparts (mAb-A, mAb-B, and mAb-C). The viscosity of mAb-C is higher than that of mAb-A, and the Arrhenius mixing rule accurately predicted the mixture of (mAb-A + mAb-C) and the solution of BsAb-A/C. While mAb-A and mAb-B have similar viscosity profiles, their mixture and BsAb-A/B have significantly higher viscosity. Microstructure and protein-protein interactions are examined using light scattering and size exclusion chromatography, and possible mechanisms for the increase in viscosity are discussed. ☐ In summary, new algorithms have been developed to increase accuracy and precision of multiple particle tracking microrheology, and a new device has been developed for characterizing protein formulations for early stage pharmaceutical development. As part of this thesis work, multiple particle tracking microrheology has been implemented in an industrial laboratory to support early stage pharmaceutical development. With a sample size of 2 μL and with an acquisition time on the order of seconds, accurate and rapid characterization of a library of protein formulations can now be done at a fraction of the time and cost of bulk rheology.
Description
Keywords
mAb, Microfluidics, Microrheology, Monoclonal antibody, Protein therapeutics