The University of Massachusetts Amherst
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PhD Defense, Whitney Blocher McTigue, “Encapsulation and Stabilization of Biomacromolecules”

Zoom link below to attend Whitney’s defense virtually https://umass-amherst.zoom.us/j/700447911

Date/Time: 

Wednesday, March 18, 2020 - 10:30am

Location: 

No longer LSL -- Zoom now

Details: 

Committee Chair:  Sarah Perry

Please use the Zoom link below to attend Whitney’s defense virtually next week!

https://umass-amherst.zoom.us/j/700447911 

ABSTRACT

Vaccines and other therapeutic cargoes are made, transported, and stored along a “cold chain,” a system designed to maintain the refrigeration of these fragile cargoes. However, if the vaccine or therapeutic falls outside this cold chain, the standard procedure is to throw it out, as it is challenging to check efficacy at point of administration. To combat this, methods for decreasing the reliance of these cargoes on the cold chain have garnered attention, with many efforts focusing on protein encapsulation strategies. Recent work in the area of protein encapsulation focused on purely aqueous techniques. Among them, complex coacervation has become a topic of discussion. Complex coacervation is a liquid-liquid phase separation phenomenon dominated by electrostatic interactions and entropy. The use of coacervates as protein encapsulants has earned consideration in the field, but there has been little headway in determining a set of design rules to accomplish this task. As not all proteins are strongly charged, we investigated the use of a two-polymer coacervate system for protein encapsulation. We explored the incorporation of three model proteins as a function of solution conditions, polymer properties, and the distribution of charges on the proteins. We determined that the net charge and the distribution of charges on both the protein and the polymers dominated protein incorporation. For example, the presence of a cluster of cationic residues on the surface of lysozyme resulted in several orders of magnitude higher protein incorporation than was observed for serum albumin and hemoglobin, which have a more isotropic distribution of charges. We confirmed this trend, comparing the encapsulation of two variants of caspase-6. The variant with a higher net charge yielded a higher encapsulation efficiency than the other.
In addition to facilitating aqueous encapsulation of proteins, we hypothesize that complex coacervation can help to enhance the thermal stability of protein cargo through a combination of physical crowding and “soft” chemical interactions that mimic the naturally crowded environment of the cytosol. We tested this hypothesis using two model viruses, porcine parvovirus (PPV), a non-enveloped virus, and bovine viral diarrhea virus (BVDV), an envelope-virus. Accelerated aging studies at 60°C over the course of seven days demonstrated that coacervate encapsulation allowed PPV to retain more than three log higher levels of activity as compared to free virus in solution. For BVDV we did not observe significant stabilization, although we posit that this may be due to the presence of the envelope, which might already provide such protection. Overall, these preliminary results, obtained without considering the chemistry of the polymers, indicate the potential for using complex coacervation to enhance the shelf life of vaccines and biologics. This work sets the stage for future efforts geared towards understanding the specific ways in which the coacervate environment can affect protein and/or virus activity, including the potential for solvent removal.

 
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