Recent advances in molecular biology have led to an explosive growth in the number of peptide and protein drugs derived from both recombinant technology and conventional peptide drug design. However, development of peptide and protein therapeutics has proven to be very challenging because of inadequate physical and chemical stability. In recent years, particle engineering processes have become promising approaches for enhancement of protein stability as well as provide options for more delivery routes. In this research program, spray freezing into liquid (SFL) process was developed and optimized in order to achieve broad platform and application in protein and peptide drug delivery systems. The overall goal of this research was to produce stabilized protein and peptide microparticles for various drug delivery systems by using SFL particle engineering technology. Firstly, the use of the SFL process to produce peptide microparticles was investigated. Insulin microparticles produced by the SFL process were highly porous, low tap density and narrow particle size distribution. The influence of the SFL process parameters and excipients on the physicochemical properties of peptide microparticles was determined and compared to the widely used particle formation technique--freeze-drying. The SFL process was further used to produce protein microparticles. In the study, bovine serum albumin (BSA), a medium sized protein, was used as a model drug. The influence of SFL process parameters and excipients on the stability of BSA was studied. Very low monomer loss of BSA was found in this study even though the specific surface area of the powder was very high. Results also demonstrated that the SFL process had minimal influence on protein structure. The SFL process was further investigated by comparing the SFL process to spray freeze drying process (SFD), which is a relatively new process to produce protein and peptide microparticles. The influence of atomization, freezing and drying on the stability of lysozyme was investigated for both the SFL and SFD process. This study tested the hypothesis that the SFL process is a better process than SFD process because of avoiding air-liquid interface and minimum interfacial surface absorption of protein in SFL process. The particle size of protein and peptide microparticles produced by SFL process was further reduced to nanoparticles by sonication or homogenization processes in organic solvent. In this study, the influence of process parameters on the particle size and enzyme activity of lysozyme was investigated. The results showed that sonication or homogenization did not influence the enzyme activity of lysozyme. Lastly, insulin and insulin/dextran microparticles produced by SFL the process was encapsulated into polymer microspheres for oral delivery. Complexation and polymer composition was studied in order to optimize release and stability of insulin. Insulin nanoparticles in microspheres minimized the release of insulin in acid with high drug loading compared to other studies. The stability of insulin was decreased by complexation to dextran sulfate. The results of this research demonstrated that the SFL process offers a highly effective approach to produce protein and peptide powders suitable for different drug delivery systems. The microparticles produced by the SFL process had desirable characteristics such as narrow particle size distribution and high porosity. The stability of protein and peptide was well maintained through the SFL process. Therefore, SFL process is an effective particle engineering process for protein and peptide pharmaceuticals. / text

In biosensors, printing involves the transfer of materials, proteins or cells to a substrate. It offers many capabilities thatcan be utilized in many applications, including rapid deposition and patterning of proteins or other biomolecules.However, issues such as stability when using biomaterials are very common. Using proteins, enzymes, as biomaterialink require immobilizations and modifications due to changing in the structural conformation of the enzymes, whichleads to changes in the properties of the enzyme such as enzymatic activity, during the printing procedures andrequirements such as solvent solutions. In this project, an innovative approach for the fabrication of proteinmicroparticles based on cross-linking interchange reaction is presented to increase the stability in different solvents.The idea is to decrease the contact area between the enzymes and the surrounding environment and also preventconformation changes by using protein microparticles as an immobilization technique for the enzymes. The theory isbased on using a cross-linking reagent trigging the formation of intermolecular bonds between adjacent proteinmolecules leading to assembly of protein molecules within a CaCO3 template into a microparticle structure. TheCaCO3 template is removed by changing the solution pH to 5.0, leaving behind pure highly homogenous proteinmicroparticles with a size of 2.4 ± 0.2 μm, according to SEM images, regardless of the incubation solvents. Theenzyme model used is Horse Radish Peroxidase (HRP) with Bovine Serum Albumin (BSA) and Glutaraldehyde (GL)as a cross-linking reagent. Furthermore, a comparison between the enzymatic activity of the free HRP and the BSAHRPprotein microparticles in buffer and different solvents are obtained using Michaelis-Menten Kinetics bymeasuring the absorption of the blue product produced by the enzyme-substrate interaction using a multichannelspectrophotometer with a wavelength of 355 nm. 3,3’,5,5’-tetramethylbenzidine (TMB) was used as substrate. As aresult, the free HRP show an enzymatic activity variation up to ± 50 % after the incubation in the different solventswhile the protein microparticles show much less variation which indicate a stability improvement. Moreover, printingthe microparticles require high microparticle concentration due to contact area decreasing. However, usingmicroparticles as a bioink material prevent leakage/diffusion problem that occurs when using free protein instead.