Moisture sorption behavior, stability, and protein formulation characteristics of a trehalose choline chloride eutectic composition

1 online resource (50 pages) : PDF

2017

University of North Carolina at Charlotte

Protein based drugs and therapeutics are an important treatment for many life-threatening diseases but they can also be extremely expensive. The unique 3-dimensional structure of a protein is responsible for its function but also contributes to its sensitivity to environmental factors such as moisture, temperature, and pH. The challenges of manufacturing, storing, and delivering modern protein therapies require better solvents that increase manufacturability, extend shelf-life, and reduce cost. Recently, low melting point sugar-salt mixtures called Natural Deep Eutectics (NADES) have been explored as alternative solvents and stabilizers for proteins. These compositions are relatively easy and inexpensive to prepare and have the benefit of being in the liquid state at room temperature. The current study focuses on compositions based on trehalose, a disaccharide commonly used for stabilizing proteins, and choline chloride, a salt frequently used to formulate NADES. Solubility and thermal stability of the protein lysozyme were investigated as a function of water content for compositions with a trehalose:choline chloride molar ratio of 1 to 4. This ratio of components forms a clear liquid at room temperature with a very low moisture content and is the ratio used for similar sugar-salt eutectic compositions. The effect of varying the moisture content on the thermal stability of the model protein lysozyme was measured by differential scanning calorimetry. As the amount of water in the composition decreased, lysozyme showed an increase in thermal unfolding temperature, indicative of increased thermal stability. Experience has shown that changes in the water content can cause NADES compositions to become unstable and form undesirable crystals. Using saturated solutions with controlled humidity values, a sorption isotherm of the composition was constructed, and the compositions were monitored for evidence of crystallization at each tested relative humidity. The samples were observed for crystal formation over a period of approximately 3 weeks and weighed every few days to determine moisture uptake and to identify when samples achieved moisture equilibrium. The trehalose choline chloride composition remained stable and free of crystals when the moisture content was maintained between 0.1 and 0.2 grams of water per gram dry weight, corresponding to relative humidity values between 23% and 33%. Pure amorphous trehalose is characterized by a relative low level of absorption at low humidity values consistent with monolayer adsorption, with a transition to multilayer absorption at higher humidity values. The addition of choline chloride to the composition changed the isotherm to one that is characterized by a steady increase in moisture content over the entire humidity range without an observable monolayer sorption region. The shift in the sorption isotherm suggests that in the NADES composition the moisture is diffusing and distributing evenly throughout the bulk of the material. Lysozyme stored in this composition at controlled humidity levels of 23% and 33% for 5 weeks and diluted back into water, exhibited melting temperatures and enthalpies of unfolding consistent with freshly prepared protein solutions.

masters theses

Mechanical engineering
Materials science
Biochemistry

M.S.

Choline Chloride
Eutectic
Protein
Sorption
Stability
Trehalose

Mechanical Engineering

Elliottt, Gloria

Jacobs, Donald
El-Ghannam, Ahmed

Thesis (M.S.)--University of North Carolina at Charlotte, 2017.

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Burgin_uncc_0694N_11399

http://hdl.handle.net/20.500.13093/etd:243