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Fluid Suspensions & Emulsions

Summary:

Our primary interest is protein solution rheology and stability, involving bulk and interfacial properties and complemented by model solution measurements. By developing measurement and data analysis methods (for rheology of protein and model solutions), we are trying to help take colloidal rheology understanding beyond uniformly charged spheres to particles with more complex interactions.

Therapeutic protein solutions are highly concentrated during manufacturing and in finished dosage. At such concentrations, the fluid rheology is 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 method probes concentrated protein films adsorbed to interfaces where aggregation may take place, and 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 development of an antibody SRM.

Description:

Intended Impact

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.

Protein charge distribution 
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. 

Objectives

  • 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. 

Technical Approach

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.

Fluid Suspensions & Emulsions ProjectPlanb

Our approach in each of these areas is described below.  

Protein rheology
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.

Silica cubes with depletant
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.   

Major Accomplishments:

  • A versatile and accurate micro-viscometer has been developed at NIST and now duplicated at MedImmune. This instrument uses less than 10 uL of fluid, is accurate and precise to a few percent, can measure a two-decade span of shear rate in approx. 10 minutes, and is temperature controlled ((0 – 80) °C). Using this instrument, we demonstrate that various protein antibody solutions may exhibit Newtonian or non-Newtonian rheology, emphasizing the need to measure at a range of shear rate, temperature and other solution conditions.
  • Theoretical calculations of droplet dynamics determine properties from velocity measurements and thus lay a foundation for a new interfacial rheology method.

Previous Accomplishments

  • MEMS rheometer: Unique capability of direct microscopic sample visualization and multi-frequency rheometry of nL samples. G. F. Christopher et al., Lab Chip 10, 2749 (2010).
  • Droplet-based interfacial characterization:
    - By measuring dynamic interfacial tension, surfactant kinetic sorption coefficients have been determined. J. D. Martin et al., New Journal of Physics 11, 115005 (2009).

Start Date:

October 1, 2012

Lead Organizational Unit:

mml

Source of Extramural Funding:

 MedImmune, in kind support of postdoctoral fellow.

Customers/Contributors/Collaborators:

  •  MedImmune (J. Pathak and P. Sarangapani), Small volume viscometry and protein rheology.
  • Pfizer (S. Kar), Protein stability.
  • U. Delaware (J. B. Rovner, C. J. Roberts, and E. M. Furst), Protein aggregation and interfacial rheology.

Pharmaceutical companies are crucial contributors, with a deep knowledge and inventory of stable and unstable protein solutions. Rheology, scattering and particle analysis involves other collaborators at NIST.

Facilities/Tools Used:

  • The microviscometer that we have developed here is versatile and is being used to test the rheology of solutions for: protein therapeutics, organic photovoltaic manufacturing, tissue engineering scaffold materials, body armor, and measuring the effect of directional interactions between suspended particles. Please inquire to pursue an interest.
  • Our main tools are theory, optical microscopy, high-speed imaging, particle tracking and velocimetry, microfluidics, and neutron scattering. Desired future capabilities are better optics and higher-resolution tracking.

Staff:

Steven Hudson – Project Leader
George Burton
Frederick Phelan Jr.
Kathleen Weigandt
Debra Audus – NRC postdoc
John Royer – NRC postdoc
Paul Salipante – NRC postdoc
Gao Yuan – NIH-NRC postdoc
Joel Rovner – U. Delaware
Prasad Sarangapani – MedImmune

Staff from other projects
Jack Douglas
Ronald Jones

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