The immune response of chickens immunized with β-N-acetylglucosaminidase was monitored in the egg yolks of the birds using an enzyme-linked immunosorbent assay. Significantly higher levels of specific antibodies were detected in the yolks of the birds immunized with the enzyme when compared with the yolks of a control bird collected over the same period significant differences also were found in the response within the immunized group of birds, indicating individual variability to the injections. Immunoglobulins were isolated from egg yolk after a preliminary purification using alginate to precipitate lipoproteins. A ten millilitre DEAE-Sephacel ion exchange chromatography column resulted in a final product containing 16 mg of IgG with a purity of 60% when 50 mL of an egg yolk supernatant was applied. Specific antibody activity toward the antigens β-lactoglobulin and E. coli lipopolysaccharide was higher in both cases in the isolated immunoglobulin fractions which contained lower purity (40%). Lower antibody activity was observed in the 60% purified fractions. Separation of specific antibodies from non-specific antibodies appeared to occur, possibly due to the given characteristics of the specific antibodies, or due to the differences exhibited by chicken IgG subpopulations. Using metal chelate interaction chromatography, a 10 mL copper-loaded column was able to yield 104 mg of IgG with a purity of 75% when 200 mL egg yolk supernatant was applied. Again, the heterogeneous nature of chicken IgG was illustrated. A comparison of the two chromatographic techniques indicated the advantages of metal chelate interaction chromatography over ion exchange chromatography under the conditions examined. Applications of the chicken IgG isolated by metal chelate interaction chromatography to an enzyme-linked immunosorbent assay for the detection of β-N-acetylglucosaminidase was attempted. Linear relationships were obtained when standard solutions of the enzyme were used in the assay. The results indicate that MCIC-isolated chicken immunoglobulins have excellent potential for use in analytical tests. / Land and Food Systems, Faculty of / Graduate

The objective of this investigation was to develop quantitative isocratic HPLC methods for the analysis of digoxin and digitoxin. An HPLC system that employs a reverse-phase column, UV detection at 220 nm and solvent systems consisting of various proportions of water, methanol, isopropanol and dichlormethane was developed for the separation of digoxin, digitoxin and their potential degradation products and metabolites. HPLC separations of the above compounds by isocratic, solvent switchover and gradient elution modes were carried out in chromatographic times of 27, 16 and 13 minutes, respectively. For purposes of monitoring the separation of dihydro metabolites of digoxin, a 100% fluid recovery system was developed for use in the HPLC analysis of digoxin and its metabolites after fluorogenic post-column derivatization using the air-segmentation process. As an evidence of selectivity, the isocratic HPLC systems were utilized for the separation of a mixture of ten closely related steroids and the isolation of digitoxin from Digitalis purpurea leaf. The isocratic HPLC systems were found to be applicable for the quantitative analysis of digoxin and digitoxin in their respective dosage forms. The HPLC assay of digoxin and digitoxin dosage forms was carried out in less than forty-five minutes. These methods were found to be precise, accurate, sensitive enough for single tablet assay, and capable of simultaneously monitoring the potential degradation products or metabolites of digoxin and digitoxin. A comparison of the assay of digoxin and digitoxin dosage forms by HPLC and USP methods indicated that: (a) the precision and accuracy of both methods were comparable and within acceptable limits; (2) analysis by HPLC can be completed in less than forty-five minutes whereas the USP methods require over four hours; and (3) the HPLC methods have the advantages of higher sensitivity, selectivity and simplicity over the USP methods. The HPLC methods were used for the stability study of digoxin and digitoxin in their respective dosage forms. Lanoxin and digitoxin tablets were found to be stable under all the conditions of storage used in this study. Natigoxin tablets, Lanoxin injection and elixir were found to be subject to varying degrees and patterns of degradation. On the basis of the stability results it was observed that the assortment of pathways that may be involved at different conditions and times of storage'would make it difficult to estimate digoxin shelf-life from data obtained by accelerated aging. From the results of this investigation, it was concluded that the isocratic HPLC methods were suitable for the assay of digoxin and digitoxin dosage forms as well as for purposes of stability testing and simultaneous monitoring of degradation products or metabolites. This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate

Studies have been made of column efficiencies, extra-column effects, the liquid phase mass transfer term (C_l), and liquid phase distribution at ultra-low liquid loadings on glass bead gas-liquid chromatographic columns. The advantages of using lightly loaded columns are described in detail along with a critical evaluation of the limitations of this liquid phase reduction ad infinitum. Theories have been presented by Giddings^22,23,25,30,32 which predict the mass transfer term (C_l) as a function of two extreme liquid phase distributions. The first equation assumes 100% "capillary" liquid held at the glass bead contact points and represents a very inefficient column. This prediction is known to be valid for normal loaded columns in the 1.0 to 0.1 liquid percentage range^35,48. The second equation assumes a uniform liquid "adsorption" film around the glass bead which represents a very efficient condition. This equation has never been experimentally verified, but Hawkes^48 and Giddings^35 suggest that this condition is approached when the liquid phase is reduced and that a uniform film exists at loadings less than approximately 0.04%. The results of this research show that although a uniform film is approached with liquid load reduction, it is never reached, even at loadings as low as 0.004%. Data show that the capillary liquid assumption is valid for glass bead columns with liquid loadings greater than 0.05%, but fails to predict the proper efficiency term at the lower loadings. Two important transitions occur at about 0.03% liquid loading: (1) C_l ceases to decrease significantly with a liquid load reduction, and (2) C_l ceases to be the predominant (plate height controlling) efficiency parameter. Reductions below 0.03% do little to improve column efficiency and may cause adverse effects such as decreased resolution, increased adsorption, and a shorter column life. Data collected before and after some apparatus and procedure changes indicate the importance of minimizing extraneous peak broadening sources. It is suggested that a large amount of the literature is in error because of apathy in this area. The results suggest that a reduction in liquid load is an excellent method of increasing the column efficiency and reducing the analysis (sample retention) time.

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<p> Improved and efficient bioprocessing technology is a key requirement in the manufacture of biopharmaceuticals. The increasing need to reduce biopharmaceutical cost is driven by the business challenges such as the emerging biogenerics. The great advances in upstream technologies make bioseparation the major cost in bioprocessing. Developing efficient bioseparation technologies is therefore strongly desired. Membrane chromatography is a promising bioseparation technology which combines the advantages of membrane technology and chromatography, thus making high-throughput and high-resolution bioprocesses feasible. This thesis focuses on the novel applications and improvements of membrane chromatography: bioprocessing of transgenic tobacco derived monoclonal antibodies and PEGylated proteins, integrated bioprocessing for antibody fragmentation, study of antibody binding on membranes and the development of novel membranes.</p> <p> Membrane chromatography based bioprocesses were developed for purification of monoclonal antibody (mAb) from transgenic tobacco, primarily addressing the challenge of low mAb abundance in the feed material. PEGylated proteins were purified, demonstrating that high-throughput and high-resolution purification of low-binding-propensity proteins was feasible using membrane chromatography.</p> <p> Membrane chromatography based reactant adsorptive membrane bioreactor separator (RAMBS) systems were developed to integrate enzymatic fragmentation of human IgG with the purification of target fragment. This novel system facilitated the process intensification and led to higher IgG digestion than in liquid phase reaction.</p> <p> The mechanism of hydrophobic interaction based IgG binding on synthetic membranes was studied using the RAMBS system. The results showed that the binding took place primarily through a combination of the hinge and CH2 domain of Fc. This study provides a new approach for studying antibody interaction with membranes and surfaces and could help design membrane-based antibody purification, immunoassay and biomaterials.</p> <p> PEG grafted filter paper was developed as an inexpensive alternative to commercial synthetic membranes. These novel membranes possessed high permeability and low fouling tendency and demonstrated good selectivity and reusability in monoclonal antibody purification.</p> / Thesis / Doctor of Philosophy (PhD)

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