To develop measurement and data analysis methods to help take colloidal rheology understanding beyond uniformly charged spheres. More complex particles may be anything from synthetic "patchy" particles to proteins.
An example from Ipsaro et al., Nature (2012) showing charge (red/blue) and shape anisotropy of a protein.
Proteins are of particular interest for their remarkable self-assembly and function.
Why does the health-care industry care about rheology and scattering of protein solutions?
1. Health & Safety. There is ongoing development of understanding among pharma and regulators of the relevance of protein aggregation. Aggregation of mAbs is a potential health risk, and viscosity and scattering measures are diagnostic of aggregation.
2. Operations. Viscosity is a critical and fundamental constitutive input for drug delivery, device design and manufacturing. For example, high viscosity leads to poor syringeability due to high injection force required.
This industry therefore needs a convenient method to measure viscosity that avoids potential problems with accuracy and excess sample volume, and which is conveniently part of their measurement system. It also needs techniques that can be used to predict protein solution stability, so as to inform molecular design and formulation development. This project is thus developing tools and analyses to measure properties of protein solutions and to identify measures that predict protein solution stability.
The self assembly of proteins is well known, motivating synthesis of patchy particles that can assemble likewise. Model solutions help simplify interpretation and help to identify the measures that are most effective in characterizing effects.
- Develop and demonstrate rheology methods and tests that are most predictive and indicative of protein solution instability.
- Certify viscosity of an antibody SRM (standard reference material) under development.
- Couple techniques together: rheology/fluorescence; rheology/particle characterization; rheology/neutron scattering.
In focusing on the rheology of protein solutions, we cover all bases: proteins of pharmaceutical interest and model solutions, and of both bulk and interfacial behavior.
Our approach in each of these areas is described below.
To measure the viscosity of very small quantities of protein solutions and pharmaceutics, we are developing a microcapillary viscometer. Capillary viscometry is a well-established and robust measurement technique. It basically involves measuring a driving pressure and a corresponding flow rate through a capillary. Here the challenge is to make testing very small volumes convenient. Specifically, to measure small flow rates ((0.1 to 100) nL/s), we developed a protocol to optimize the performance and statistics of a commercial microfluidic flow meter. And to quickly handle and load small volumes into the instrument, a micro-pipette simply dips into a standard sample vial. With minor modification, testing multi-well plate sample arrays is feasible. To measure a wide range of viscosity (from (0.5 to 2000) mPa s), we use different pressure sources capable of delivering from 3 Pa to 100,000 Pa, and we employ micro-pipettes of different size.
This method (when combined with scattering and other biophysical techniques) is now exploring correlations between viscosity, solution stability and cluster dynamics. Measuring the effects of pH, temperature, and salts is central to improving product yield during concentration of monoclonal antibodies (mAbs) in purification processes. Complex behavior is observed, and in some cases, flow can induce aggregation. The effects of pH and concentration are being measured over a much broader range than previously reported by others. The results demonstrate the failure of existing colloidal rheology models.
Model solution rheology
The failure of these models strongly motivates model solution rheology. Two approaches are in progress:
1.) model colloids that possess simple directional interactions are being tested to find out the properties that deviate from existing models and
2.) a new theoretical model for protein solutions is being developed.
Here cubic particles (1.66 um in size) attract weakly face-to-face through the addition of an invisible small soluble polymer.
Protein interfacial rheology
Air-water and oil-water interfaces are present throughout manufacturing and in final protein therapeutic products. The rheology of proteins at these interfaces is of special relevance to aggregation phenomena, because there proteins collect, may slightly denature and begin to aggregate. Interfaces therefore provide a natural model for instability. Unfortunately, interfacial rheology is not widely available, and data is thus sparse. To meet this need, a new method for dilatational interfacial rheology is being adapted for protein solutions, so that it will use smaller volumes and have wider dynamic range. These properties will be measured for a few proteins of interest under a wide range of solution conditions, and compared with bulk aggregation kinetics.
Model interfacial measurements
Just as colloids have thus far been remarkable models for phase transition behavior in 3D and for measuring statistics that are otherwise impossible, colloids will similarly be excellent models for sorption behavior.