Search

Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.

Freeze-drying is the process of removal of water or organic solvent from a desired product by means of sublimation at a low temperature and low pressure. It is commonly employed for drying samples which are heat labile and require sensitive treatment, and is mainly used in the pharmaceutical and food industries. It is an expensive process, requiring vacuum, refrigeration and long cycle times, but does yield quality benefits due to the low temperatures involved and the porous nature of the product. Reducing drying times is important to manufacturers, and this depends on optimising rates of heat and mass transfer in the system without the sample losing its porous structure. However, freeze drying is difficult to study experimentally due to the low temperatures and pressures involved. The quality of the final product mainly depends on the sublimation rate and an optimum lyophilisation requires identification of the parameters which influence the process. The main aim of this study is to employ freeze drying microscopy (FDM) as a useful tool to identify these process parameters and help optimise primary drying phase of the freeze drying process for two systems: lactose (relevant to pharmaceuticals) and coffee (the most widely freeze-dried food product). This equipment allows the movement of sublimation fronts to be directly visualised in-situ under carefully controlled (and isothermal conditions), but has scarcely been used in the literature for this purpose. An image analysis method is developed to automatically track the movement of sublimation fronts, and the frontal data fitted to a simple mass transfer model employing surface and bulk resistances. Initial experiments with lactose solution show poor reproducibility in nucleation temperatures during the freezing step and thus primary drying rates. To improve reproducibility, a small amount of silver iodide (AgI) was added to samples which acts as a nucleating agent and increases the nucleation temperature. This addition of AgI also increases the mean ice crystal size in the samples and are easily visible under the freeze-drying microscope, and in many cases show a distinct orientation with respect to direction of sublimation front. Furthermore, the orientation greatly influences sublimation rates, being approximately factor of two faster when crystals are oriented in the direction of mass transfer. FDM experiments with coffee were less straightforward as nucleation temperatures could not be reliably controlled, even with AgI added. Nevertheless there was a clear decrease of bulk resistance with increasing nucleation temperature. An experimental programme was then undertaken to examine the impact of initial solid content, cooling rate, the addition of an annealing step, freeze drying temperature and aeration (for coffee samples). Frontal data were fitted to a simple mass transfer model comprising surface and bulk (per unit depth) resistances and good fits to data were obtained. FDM experiments with lactose and coffee clearly showed the presence of a surface resistance which could also be seen as a surface layer which was devoid of ice crystals (and hence not porous when sublimed). The edge resistance first increased and then decreased with solids content. The resistance per unit depth increased exponentially with solids content, so much so that there is an optimal solids content (around 10% solids) in relation of the rate of production of dried material. Cooling rates were mainly found to affect the surface resistance rather than bulk resistance and this may be due to different levels of surface drying when the samples are being cooled for different lengths of time. Annealing substantially changed the ice crystal sizes, and had a beneficial effect on freeze drying rates and had a similar effect to adding AgI. Freeze drying rates also increased with increasing temperature approximately in line with the saturated vapour pressure (SVP) of ice which is widely held to constitute the driving force for mass transfer. It was possible to make drying time calculations for conventional vial (lactose) and tray (coffee) drying using the frontal rate data obtained from FDM. For 10% lactose and 10% coffee (annealed) there was good agreement between the vial and tray data and predictions based on a microstructure oriented parallel to the direction of mass transfer. This was the only case where agreement was found, but also the only case where directionality was observed in FDM. The much faster drying times observed in the vial and tray experiments are thus attributed to directional solidification occurring in these systems, and this was borne out by SEM imaging. Aeration of the coffee samples was also found to substantially reduce drying times. The influence of microstructure on freeze drying rates is thus very clear.

Three-dimensional collagen/chitosan scaffolds fabricated by freeze-drying technique in 96-well polystyrene and PDMS plates were optimized during this study. Surface tension is, by and large, one of the most limiting factors in fabricating freeze-dried scaffolds in small format well plates. Traditionally, bowl-shaped top surfaces of collagen/chitosan scaffolds were common in polystyrene 96-well plate; whereas for PDMS 96-well plate, dome-shaped surfaces were formed. These surface tension phenomena are not desirable in cell studies especially during initial cell seeding. A combination of surface treatment and change of freeze-drying regime were developed to mitigate the surface tension problem in PS and PDMS 96-well plates respectively. Collagen/chitosan scaffolds of varying concentration and composition were experimented in both polystyrene and PDMS 96-well plates. Thin water film treatment with UV cross-linking was successfully used to eliminate meniscus in PS well plates; pre-cooling, on the other hand, was utilised to treat scaffold solutions in PDMS well plates. The resultant matrices all had flat top surfaces and average thickness of 1 mm. As expected, scaffolds with lower overall polymer concentration or, from a compositional perspective, scaffolds with high chitosan content generally had larger pores. Microscopic observation by multi-photon microscope was performed and chemical analyses were conducted to characterize the surface-treated scaffolds. In addition, scaffolds were tested in vitro using DLD-1 cells, hMSCs and fibroblasts for their biological performance. The purpose of this study was to address the problem of using small format culture wells for the fabrication of freeze-dried collagen-based scaffolds for studies of cell growth in 3D culture and in microfluidic perfusion bioreactors.

Whey protein and sodium caseinate play a vital role in many food systems and exhibit multifunctional properties in food processing which enhance physical and nutritional values. The main functional properties, such as water holding capacity, oil holding capacity, foaming, and emulsification, are important charicteristics in food development and production. Among all of the commercially avaliable protein ingredients, both whey protein isolate and sodium caseinate, which are sourced from bovine milk, are widely used as protein ingredients for industial production, such as yogurt and baking products. These milk protein ingredients are nutritious, used for the building and repairing of tissues, fighting against infections, and providing energy for metabolism. Whey protein is obtained from cheese processing by-product-whey. Broadening whey protein’s application is beneficial for dairy industry sustainability and the valorisation of whey protein as a functional ingredient. The functional properties of these proteins are based on their biomolecular structures, which can be modified by several compositional and processing factors. Blackcurrant is abundant in health beneficial phytochemicals, such as vitamin C, polyphenols, and anthocyanins. Due to the intense sour taste, fresh blackcurrant fruits are always processed into juice concentrate for further food application. The bioactive compounds in blackcurrant juice concentrate are unstable in a free form, especially when they undergo further food processing conditions, and also in the human digestive systems. This can contribute to a low bioaccessibility and bioavailability for its health benefit components, especially anthocyanins. The development of further applications for blackcurrant juice concentrate in a food system is of vital importance to aid the sustainable development of the blackcurrant crop industry, and bringing the goodness of this fruit to a wider population. Combinations of common protein ingredients with fruits and vegetables concentrates or extracts are regarded as an effective ingredient to create novel protein products, to explore the application of concentrates or extracts from fruits and vegetables. Many studies have focused on the modification of protein structures, and the alteration of protein functionalities under controlled conditions. However, few studies have focused on the development of novel protein ingredients in a food-compatible manner, and their further incorporation in real food system. Whey protein isolate and sodium caseinate have been commonly used as delivery systems for functional components due to their wide availability, nutritional values, and protective effects on sensitive bioactive components. In this study, blackcurrant juice concentrate was encapsulated via spray-drying and freeze-drying strategies by utilising whey protein isolate or sodium caseinate as the wall material without applying any food additives, obtaining four different kinds of novel protein ingredients (SWB, FWB, SNaB, FNaB) with different physicochemical, functional, and nutritional characteristics. The nutritional, physicochemical and functional properties of these ingredients were determined and defined. The results indicated that these ingredients could be used as natural colourants, protein enhancers, antioxidants, replacement of carbohydrate. The novel functional protein powders were then further incorporated into real food matrix-cookie at various levels (0%, 5%, 10%, 15%) to develop functional snack (cookie). Dough and cookie physicochemical properties, and cookie nutritional qualities were determined. The protein ingredients gave cookie products with different colour and texture, increased antioxidant power, and most importantly generated hypoglyceamic effects. Collectively, these results illustrate that blackcurrant concentrate health benefits can be delivered to food systems via milk protein ingredients-based encapsulates

Abstract: Atmospheric spray freeze-drying (ASFD) is an alternative freeze-drying process to conventional vacuum freeze-drying. ASFD involves spray-freezing biologics of interest in a feedstock into a cold freezing chamber. Cold drying gas passes through the chamber at a high flow rate to sublime the water and ice in the powder with convective forces. ASFD is a seven-hour drying process compared to conventional freeze-drying which is in the order of days. Ertapenem, a regularly used antibiotic, and trehalose, a biologic stabilizer in freeze-drying, were freeze-dried at varying feedstock concentrations. Ertapenem was freeze-dried at 50, 100, 150, and 200 mg/mL; higher concentrations had pale yellow discoloration in both liquid and dried powder. Although ASFD ertapenem was porous compared to the lyophilized counterpart, higher feedstock concentrations yielded larger particles with lower porosity. Trehalose was freeze-dried at 100, 200, and 300 mg/mL. ASFD trehalose at lower concentration existed as a free-flowing powder whereas higher feedstock concentration yielded a thin brittle cake. Both trehalose and ertapenem powders at lower feedstock concentrations made powders with large pores and high void fraction, which have excellent wetting and aerodynamic properties. Bacteriophage D29 in solutions with varying concentration of mannitol and trehalose, were freeze-dried using ASFD. A solution of trehalose and mannitol at a mass ratio of 7:3 and a total feedstock concentration of 100 mg/mL had the lowest titer reduction of ~0.6 logs with a powder moisture content of 4.9±0.1 %. In comparison, pure trehalose and 1:1 ratio of trehalose and mannitol both had titer reductions larger than 1.5 logs. Raman spectroscopy of the powders showed that trehalose in the powder remained amorphous while mannitol completely crystallized, which are desirable for preserving phages in a dry solid form. The short drying process, improved wetting properties, and the ability to preserve bacteriophages makes ASFD an attractive alternative to conventional freeze-drying.

Purpose: To characterize the phase behavior of mannitol and tertiary butyl alcohol (TBA) in TBA-mannitol-water ternary frozen solutions. Methods: Solutions of mannitol (2 and 5% w/w) in TBA-water systems of different compositions (5 to 30% w/w TBA) were prepared and cooled to -40°C. The frozen solutions were thawed to RT. The solutions were characterized, both during cooling and warming, by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), using both a laboratory source and synchrotron radiation (Argonne National Laboratory). Selected compositions were freeze-dried. Results: At and below the TBA-water eutectic composition (22.5% w/w TBA), mannitol crystallization was completely inhibited while TBA crystallized as a heptahydrate. When these frozen compositions were heated the thermal events observed were (i) two glass transitions, crystallization of mannitol and TBA heptahydrate, (ii) melting of binary eutectics of ice with each TBA dihydrate, TBA heptahydrate and mannitol along with (iii) ice melting. Irrespective of the initial TBA concentration, when freeze-dried with mannitol, the final lyophile consisted of δ-mannitol. Conclusions: While mannitol did not affect the phase behavior of TBA-water systems, TBA completely inhibited mannitol crystallization in the frozen state at compositions ≤ 22.5% w/w TBA. This provides an avenue for mannitol to serve as a cryoprotectants in frozen systems and as a bulking agent in lyophiles.

Clostridioides difficle infection (CDI) is the most common health-care associatedinfection in the United States. Annually, CDI infects nearly half a million patients, kills29,000 and costs the healthcare system an estimated US$6.3 billion. Although thispathogen can be treated with antibiotics, in 25% of cases, the bacterium does not respondto antibiotics and the patient develops a recurrent infection. Due to the hardy nature of thebacteria causing the recurrent CDI, additional, more costly treatment methods such asfecal microbiota transplantation must be employed. Alternatively, consumption of kefir, acommercially available fermented dairy beverage with live probiotic cultures, wasrecently found to be effective in curing recurrent CDI when consumed alongside theprescribed antibiotic treatment. Unfortunately, kefir has limited patient acceptance due toits strong acidic and fruity flavors resulting from the fermentation process. To combatthis, kefir may be further processed using freeze-drying or vacuum evaporation to removeundesirable volatile organic compounds (VOCs) that greatly contribute to flavor, but heatduring processing must be minimized to avoid reducing the population of beneficialmicroorganisms. In this study, effect of vacuum evaporation and freeze-drying on kefirVOC concentration, microbial viability and activity, and sensory quality was assessed.Commercially manufactured kefir was subjected to either a vacuum evaporation orfreeze-drying treatment, or was not further processed (control). Loss of volatilecompounds was monitored at the ppb level using selected-ion flow-tube mass spectrometry (SIFT-MS); kefir acceptability was evaluated by untrained panelists using a9-point hedonic scale, and differences among treatments was determined by comparingmean scores; microbial viability was assessed using selective media for enumeration ofLactobacillus spp. and Lactococcus spp.; and microbial activity was assessed bymeasuring fermentation rate of milk inoculated with treated and untreated (control) kefir.Vacuum evaporation and lyophilization treatments significantly diminished VOC contentin the kefir for 26 out of 27 compounds (P < 0.05). Cumulative VOC concentration forall 27 compounds was reduced by approximately 62% after both vacuum evaporation andfreeze-drying treatments. Despite the considerable difference in kefir VOC content,vacuum evaporation and lyophilization treatments did not significantly improve the likingof commercial kefir in sensory tests. The concentrations of Lactobacillus spp. andLactococcus spp. present in the kefir were significantly reduced by freeze-drying andvacuum evaporation treatments. Although significant, the population of lactic acidbacteria (LAB) in the kefir samples was still considered sufficient for probiotic products.Fermentation rates were slightly reduced after the experimental treatments, withlyophilization having a more significant effect.

Biodegradable poly(D,L-lactide-co-glycolide) PLGA nanoparticles (NPs) have been extensively investigated for drug delivery. Protein conjugated NPs provide the advantage of active targeting. To overcome the instability associated with storage of aqueous dispersions, NPs are usually lyophilized. However, the freezing and freeze-drying stress can lead to nanoparticle aggregation and protein denaturation. As such, sucrose is widely used as a cryoprotectant and minimal ‘cryoprotectant to particle ratio’ is required. In this study, we used trypsin as a model protein to conjugate on the PEGlyated NPs. The effects of the freezing and freeze-drying process on trypsin conjugated NPs was studied by comparing the size of the NPs, the morphology of the NPs under fluorescent images and the trypsin activity after of the fresh made, freeze-thawed and lyophilized NPs. A minimum sucrose to NPs ratio was founded to provide the complete protection of the trypsin conjugated NPs.

Porous materials have attracted massive scientific and technological interest because of their extremely high surface-to-volume ratio, molecular tunability in construction, and surface-based applications. Through my PhD work, porous materials were engineered to meet the design in selective binding, self-healing, and energy damping. For example, crystalline MOFs with pore size spanning from a few angstroms to a couple of nanometers were chemically engineered to show 120 times more efficiency in binding of large molecules. In addition, we found building blocks released from those crystals can be further patched back through a healing process at ambient and low temperatures down to -56 ºC. When building blocks are replaced with graphenes, ultra-flyweight aerogels with pore size larger than 100 nm were made to delay shock waves. More stable rigid porous metal with larger pores (~um) was also fabricated, and its performance and survivability are under investigation. Aside from experimental studies, we also successfully applied numerical simulations to study the mutual interaction between the nonplanar liquid-solid interface and colloidal particles during the freezing of the colloidal suspensions. Colloidal particles can be either rejected or engulfed by the evolving interface depending on the freezing speed and strength of interface-particle interaction. Our interactive simulation was achieved by programming both simulation module and visualization module on high performance GPU devices. Advisor: Li Tan

Inferring paleoclimate from ancient fluvial strata can be challenging,

Freeze-drying (lyophilization) is a drying process which is used to remove water from heat sensitive products, usually for the purpose of preservation. By removing water, the product becomes more stable at room temperature. This is a common process in the pharmaceutical industry because freeze-drying offers the advantage of drying at low temperatures and producing very low residual moisture contents. Often the materials dried in this manner are heat sensitive and require the highest possible quality. However, freeze-drying is a very slow process, often requiring 24 to 48 hours. During the process, vacuum pumps and refrigeration systems run continuously, making freeze-drying a very expensive process. The goal of this project was to show that volumetric heating can be used in pharmaceutical freeze-drying and that this mode of heating offers some advantages. There were two approaches taken to the work, one experimental and one analytical. The experimental approach was broken into two phases, one focused on comparing microwave and conventional freeze-drying and the other focused on demonstrating the advantages of volumetric heating. In the analytical approach, a mathematical model was used to confirm the trends observed in phase II of the experimental work. Experiments were conducted in a conventional laboratory freeze-dryer and the drying rate results were compared to the results obtained with an experimental microwave freeze-drying apparatus. Experiments were also conducted with the vaccine strain A. pleuropneumoniae. A viability study was conducted, comparing the viability loss caused by each process. The viability study showed a slightly higher viability loss for the microwave process. A comparison of drying curves showed that the microwave process resulted in a slight improvement in primary drying time: 2.5 hours for the microwave process compared to 3 hours for the conventional process. There was a significant difference in overall drying times: 4 hours for the microwave process compared to 11 hours for the conventional process. This result was caused by a lower residual moisture content at the start of secondary drying and a higher secondary drying temperature for the microwave process. Experiments were also conducted to show that using lower chamber pressure results in higher drying rates. This is not the case in a conventional freeze-dryer since heating is dependent on the chamber pressure in the low pressure environment of freeze-drying. Thus, an advantage of volumetric heating was demonstrated. The results show that a modest increase in pressure, from 0.05 to 0.3 Torr, caused a one third reduction in primary drying time. The mathematical model developed in the analytical work relied on the D'Arcy equation to describe the flow of vapor in the porous dried layer. The results of the model confirm trends seen in the measured temperature and weight profiles. Analyzing the effect of varying the chamber pressures shows that lowering the pressure in the range of 1 to 0.01 Torr results in a significant increase in drying rate giving as much as a two thirds reduction in drying time for the case studied. A model incorporating mass transport equations derived from the dusty gas model was also presented. This model offers the benefit of a more accurate prediction of mass transport through the porous dried layer. NOTE: (09/2008) An updated copy of this ETD was added after there were patron reports of problems with the file.