Jared Bee

I am investigating the interaction of proteins and surfaces relevant to biopharmaceutical protein production processes. There is some concern that nucleation and growth of aggregates are caused by small particulate impurities that can be introduced into the formulation.

Amber Clausi

Protein-based therapeutics often require a lyophilized formulation to ensure adequate long-term stability of the protein.  However, it has been reported that aluminum adjuvant salts (aluminum hydroxide and aluminum phosphate), which are an essential component of human vaccine formulations and aid in eliciting an immune response, aggregate upon freezing.  This aggregation usually results in the loss of potency of the vaccine, thus preventing lyophilization from being an adequate means of preservation of human vaccine therapeutics.  My research focuses on trying to understand the aggregation behavior seen in these particles as a function of the formulation parameters, most importantly buffer salts and excipient concentrations, and the processing conditions, specifically the rate at which the formulation is cooled to form a frozen solid.  We have observed that the aggregation can be minimized through faster cooling rates or by high concentrations of excipients, such as trehalose.  My research utilizes many particle characterization techniques, most importantly particle size analysis and zeta potential determination, as well as DSC and X-ray diffraction to characterize the frozen, dried, and reconstituted solids.   

Ryan Crisman

Protein aggregation is a major area of interest in the pharmaceutical industry because it is the main pathway leading to loss of valuable protein product.  High hydrostatic pressures (2000 bar) have been shown to be effective in reversing aggregation and reforming the native state.  To better understand the mechanisms involved in this process we are looking at the intermolecular interactions experienced by a protein during the pressure induced folding process.  We are currently using a novel technique, high pressure static light scattering, to study protein-protein interactions during this folding process.  It is our belief that this technique will provide a useful analytical tool to gain a fundamental understanding of high pressure effects on the protein aggregation process.

Amber Fradkin

The effect of soluble aggregates on immunogenicity

As therapeutic proteins become more prevalent there is a growing concern for evaluating the immunogenicty of these proteins before they are introduced to clinical trials.  When human proteins are introduced into the body, two recognized forms of an immune response may take place.  The first is that neutralizing antibodies bind to the protein thereby preventing its biological activity.  Neutralizing antibodies are the same antibodies responsible for neutralizing toxins that enter the body.  The second response is the formation of antibodies due to the breaking of the immune tolerance.  Both have potential to cause serious harm to human subjects in clinical trials. 

Sathish Hasige

To be used as therapeutics, an antibody needs to be delivered in very high concentrations. Unfortunately when you exceed the solubility limits proteins will aggregate. Preparation of very high concentration of these antibodies at a very low volume is critical. Without a proper formulation, even the best drug will be useless and sometime will be dangerous.  I am exploring the complementary application of different biophysical/biochemical approaches to address this problem of protein aggregation at high concentration dosage.  Data obtained will be used to correlate the effect of different formulation conditions and protein modifications on protein solubility and stability.

Brett Ludwig

The overall objective of my work is to gain a better fundamental understanding of protein aggregation in solution.  Our central hypothesis is that the kinetics and thermodynamics of protein aggregation are controlled by two main factors: conformational stability and colloidal stability.  Each of these factors can be manipulated by changing solution conditions (e.g., pH, ionic strength, presence of stabilizing or destabilizing excipients), protein structure, or both.  To test our hypothesis, we will manipulate protein conformational stability and protein-protein intermolecular interactions using recent advances in protein and colloid chemistry as well as well-established principles in solution thermodynamics, physical chemistry, and reaction kinetics.  As model proteins, we have chosen two proteins that have 98% sequence homology in order to minimize variability due to protein structural differences when comparing aggregation behavior.

Branden Salinas

Monoclonal antibodies (mAbs) are the focus of many investigational biopharmaceuticals due to their binding strengths and specificities.  Typically, antibodies have a high solubility and exhibit a resistance to aggregation which lends to the notion that they are stable molecules.  However, many antibody formulations are desired at relatively high protein concentrations for subcutaneous administration.  Traditional techniques for monitoring antibody unfolding or aggregation are not applicable at the desired dosage concentrations of >50 mg/mL.  Understanding non-idealities in antibody phase behavior; such as reversible associations, precipitation of native-like structures and non-native aggregation in solution, is at the core of this research.  Two investigational, therapeutic mAbs (~90% sequence homology) with drastically different phase behaviors are being compared to gain a better understanding of problems encountered during antibody formulation at high concentration.  Many of the phenomenological observations have been collected using Fourier Transform Infrared Spectroscopy (FTIR), Static Light Scattering (SLS) and free energy of unfolding measurements.  What is left to do is to determine the source of the discrepancies using a combination of structure modeling and high concentration phase behavior observations.  The structure modeling would allow for the development of charge maps as well as identifying regions that may be prone hydrophobic interactions.  High concentration techniques such as membrane osmometry in conjunction with FTIR can help discern the influences of charge-charge and other colloidal interactions between protein molecules as well as the effect of various solvent components on the colloidal and conformational stability of the mAbs.