LIGHT-ASSISTED DRYING (LAD) FOR THE STABILIZATION OF PROTEINS IN AMORPHOUS TREHALOSE
1 online resource (94 pages) : PDF
University of North Carolina at Charlotte
We have developed a new processing method, light-assisted drying (LAD), to create an amorphous trehalose matrix for the stabilization and storage of proteins. A challenge in the development of protein-based assays and therapeutics is preserving the structure of the protein during production and storage. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. LAD offers a relatively inexpensive method for drying samples. LAD immobilizes proteins in an anhydrous, amorphous solid that could potentially be stored at supra zero temperatures making this process attractive for use in low resource settings. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation accelerates drying and as water is removed the trehalose forms a protective matrix. Initially we investigated the effect of laser wavelength, processing power, and sample substrate to determine the optimal LAD parameters for fast processing times and low end moisture contents (EMC). We compared the effect of changing processing wavelength, power and resulting sample temperature, and substrate material on the EMC for two near-infrared (NIR) laser sources (1064 nm and 1850 nm). The 1850 nm laser resulted in the lowest EMC (0.03±0.01 gH2O/gDryWeight) after 20 minutes of processing on borosilicate glass microfiber paper. This suggests a storage temperature of 68.3°C. In the second study, LAD samples were optically characterized with polarized light imaging (PLI), scanning white light interferometry (SWLI), and Raman spectroscopy. PLI was used to look at crystallization kinetics of samples and determine optimal storage relative humidity (RH). PLI showed a 62.5% probability of minor crystallization during LAD processing and negligible crystallization during 14.3±0.5% RH storage. Scanning white light interferometry (SWLI) was used to measure sample thickness. SWLI measured an average maximum sample thickness of 90.81 ±6.53 µm. Raman spectroscopy was used to investigate trehalose distribution across LAD processed samples. No change in Raman spectra indicated that trehalose was present across samples in an amorphous form. The final study investigated the effect of varying protein concentration and protein size on EMC. Protein concentration and size (as defined by hydrodynamic radius) had no effect on the EMC reached by LAD for the same processing parameters. We also tested the functionality of a model protein, lysozyme after LAD processing with the 1064 nm laser at ~42°C compared to air drying, samples incubated at a temperature comparable to LAD, a control solution kept at 8°C and ambient temperature, and crystallized samples. Both the LAD – 1064 nm samples and various control and damaged samples did not experience any decrease in functionality. This implies that LAD has no unique negative effect on protein function. The LAD – 1850 nm study was performed above lysozymes melt temperature (~80°C). A sample was processed with LAD and also incubated in a water bath at a comparable temperature. The LAD processed sample showed negligible denaturation compared to the incubated sample which experienced partial denaturation.Overall, we found that LAD produced uniform samples with repeatable EMC that could enable storage of samples at supra zero temperatures without loss of protein functionality. LAD shows potential for use in applications that require the stabilization of proteins. These applications include protein based diagnostics such as micro arrays and rapid detection test strips. Proteins immobilized on these devices could be stored at ambient temperatures. LAD could also be used to stabilize protein therapeutics at ambient temperatures.
AMORPHOUS TREHALOSEBIOPRESERVATIONLIGHT-ASSISTED DRYINGNEAR INFRAREDPROTEIN STABILIZATION
Optical Science & Engineering
Trammell, SusanElliott, GloriaFried, NathanielBoreman, GlennLee, Charles
Thesis (Ph.D.)--University of North Carolina at Charlotte, 2018.
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). For additional information, see http://rightsstatements.org/page/InC/1.0/.
Copyright is held by the author unless otherwise indicated.