Separation and characterization of glycoproteins and other biological materials: from reverse micelles to capillary electrophoresis

In this dissertation we review the use of liquid/liquid interfaces for the inhibition of biofilm formation, and explore their use for the decontamination of surfaces --Abstract, page iv

Effect of polymers, salts and their mixtures on the formation of dental plaque

Although some progress has been made towards the chemical removal of dental plaque, it is generally removed by mechanical means. The current research is aimed to further that progress. Our hypothesis is that it is possible to remove dental plaque by aqueous two-phase systems (ATPS’) (due to an equilibrium distribution of cells and/or proteins between an adsorbed phase and a liquid/liquid interface). This investigation details the preliminary work for using ATPS’ to remove dental plaque. The prevention or degree of reduction of Streptococcus sanguis (S. sanguis) biofilm formation on hydroxyapatite discs in the presence of ATPS’ was investigated. The idea is that removal of plaque would be likely only if a great reduction in bacterial attachment in the presence of these systems could be achieved. Dental plaque is a biofilm comprised of a diverse microbial community firmly attached to both root and enamel surfaces of teeth. Studies of in-vitro biofilms were performed using S. sanguis oral bacteria and hydroxyapatite discs (hydroxyapatite is a major mineral in human dental enamel so its use as the solid support is appropriate). Recognizing the limitations of in-vitro studies, the experimental conditions were chosen such that they mimic those encountered in the oral cavity. Seven day biofilms were grown in various concentrations of yeast tryptone (YT) media, YT media with polymer, YT media with salt, and YT media with aqueous two-phase system. The biofilms were then characterized by scanning electron microscopy (SEM) and scintillation counting. Two of the three ATPS’ investigated were found to reduce S. sanguis attachment to hydroxyapatite. The 10% YT media containing polyethylene glycol (PEG)/MgSO4 and all three concentrations of YT media containing PEG/Na2SO4 exhibited a reduction in bacterial attachment when compared to the systems containing the individual phase forming species and to the reference systems. These four systems can be further investigated for their effects on removal of young dental plaque --Abstract, page iii

Aggregation and Denaturation of Antibodies: A Capillary Electrophoresis, Dynamic Light Scattering, and Aqueous Two-Phase Partitioning Study

Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations

Decontamination of Surfaces by Lysozyme Encapsulated in Reverse Micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete

Distribution of Cells Between Solid/Liquid and Liquid/Liquid Interfaces

The use of aqueous two-phase systems (ATPSs) and each system\u27s individual phase-forming species to prevent Streptococcus sanguis attachment onto hydroxyapatite discs was explored. The strategy that we followed was to attach the cells to a solid surface in the presence of an additional interface. Conditions under which, simultaneously, the phase-forming species form two phases and the cells proliferate were identified. Growth curves were constructed in the presence of various polymers and salts commonly used to prepare ATPSs. Several aqueous two-phase systems were selected such that bacterial growth was comparable to that observed in pure medium. Cells were allowed to attach to hydroxyapatite discs for 7 days in the presence of varying concentrations of media, media with polymer, media with salt, and media with ATPS. Streptococcus sanguis attachment to the disks was evaluated by scanning electron microscopy. The addition of a PEG/Na2SO4 ATPS to high concentrations of yeast-tryptone (YT) media (\u3e65%) and of a PEG/MgSO4 ATPS to nutrient-limited media reduces surface coverage of S. sanguis to less than 10%. Comparison of the attachment levels for the systems containing PEG/Na2SO4 to media containing the individual phase-forming species and to the YT reference systems indicated that nutrient availability did not affect attachment

Poly (Ethylene) Glycol (PEG) Precipitation of Glycosylated and Non-Glycosylated Monoclonal Antibodies

The solubility of monoclonal antibodies (mAb) affects their production and their intravenous administration to patients. In this work, the solubility of a fully glycosylated and a non-glycosylated human mAb expressed in corn was studied by inducing their precipitation by poly(ethylene) glycol (PEG). The experiments were done using PEG 1450 and 8000 at concentrations ranging from 0% to 30% w/w, at different pHs and temperatures. Additional studies were performed in the presence of Griffonia (Bandeiraea) simplicifolia Lectin II, which binds to glycosylated proteins. These studies show that glycosylation increases the solubility of the antibody and that models based on excluded volume principles or on the statistical correlation of solubilities are unable to capture the effect of glycosylation on protein precipitation by PEG. PEG molecular weight controls the onset point of the precipitation curves but glycosylation is the main effect on PEG precipitation efficiency. A two-stages precipitation pattern was observed when a lectin and PEG were added to the glycosylated or non-glycosylated mAbs