Therapeutic protein solutions are highly concentrated during manufacturing purification processes and in finished dosage. At such concentrations, the fluid rheology may be complex, creating the need for a convenient accurate method to measure viscosity. Here we develop rheology tools to help evaluate protein stability across a range of solution conditions and protein concentrations that are relevant to manufacturing and product formulation. One tool, is a capillary viscometer that requires only a few micro-liters of solution to probe the range of flow rates and temperatures of interest. Another tool to be developed will expand the dynamic range of rheo SANS at the NIST neutron user facility (NCNR), i.e. an instrument for simultaneous rheology and neutron scattering. A third method probes concentrated protein films adsorbed to interfaces where aggregation may take place, and where properties are suspected to correlate with solution stability. Teaming with collaborators, we test industrially relevant materials, evaluate structure and aggregation kinetics, and contribute to the characterization of an antibody standard reference material SRM.
Why does the health-care industry care about rheology and scattering of protein solutions?
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 quality 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.
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 or flow channel. 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 would be 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 (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 may 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. This result strongly motivates theoretical modeling and model experiment studies. Specifically, 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.
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.