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Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Ng, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 206-211). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vii / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter / Chapter 1. --- Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Historic discovery of growth hormone --- p.1 / Chapter 1.1.2 --- Structural and functional study of GH --- p.1 / Chapter 1.1.2.1 --- Molecular evolution of GH --- p.1 / Chapter 1.1.2.2 --- Two-dimensional and three dimensional structures --- p.5 / Chapter 1.1.2.3 --- Heterogeneity of GH --- p.8 / Chapter 1.1.2.4 --- Regulation and secretion pattern of GH --- p.9 / Chapter 1.1.2.5 --- Circulation of GH in blood --- p.11 / Chapter 1.1.2.6 --- Biological activity of GH in human --- p.12 / Chapter 1.2 --- GH receptor and signal transduction --- p.12 / Chapter 1.3 --- GH disorder --- p.15 / Chapter 1.4 --- Treatment for GH disorder --- p.16 / Chapter 1.5 --- GH assay --- p.17 / Chapter 1.6 --- Aims of study --- p.19 / Chapter 2. --- SUMO-hGH expression vector construction / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Fusion partner - SUMO --- p.23 / Chapter 2.3 --- Materials --- p.24 / Chapter 2.3.1 --- Reagents for bacterial culture --- p.24 / Chapter 2.3.2 --- Reagents for agarose gel electrophoresis --- p.26 / Chapter 2.3.3 --- 2'-deoxyribonucleoside 5'-triphosphate mix for polymerase chain reaction --- p.26 / Chapter 2.3.4 --- Sonication buffer --- p.26 / Chapter 2.3.5 --- Modified solubilization buffer --- p.27 / Chapter 2.3.6 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis --- p.27 / Chapter 2.4 --- Methods --- p.29 / Chapter 2.4.1 --- General techniques in molecular cloning of hGH gene --- p.29 / Chapter 2.4.2 --- Expression of SUMO-hGH fusion protein - small scale --- p.42 / Chapter 2.4.3 --- General protein analysis --- p.43 / Chapter 2.5 --- Results --- p.45 / Chapter 2.5.1 --- Molecular cloning of hGH gene into expression vector --- p.45 / Chapter 2.5.2 --- Expression of SUMO-hGH --- p.46 / Chapter 2.5.3 --- Modification of the expression conditions --- p.46 / Chapter 2.6 --- Discussion --- p.50 / Chapter 2.6.1 --- Expression vector --- p.53 / Chapter 2.6.2 --- Protein expression --- p.53 / Chapter 2.7 --- Conclusion --- p.54 / Chapter 3. --- SUMO-hGH purification and downstream processing / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Immobilized-metal affinity chromatography --- p.55 / Chapter 3.3 --- SUMO protease --- p.57 / Chapter 3.4 --- Materials --- p.59 / Chapter 3.4.1 --- Reagents for IMAC purification of SUMO-hGH fusion protein --- p.59 / Chapter 3.4.2 --- Reagents for IMAC purification of mature rhGH --- p.60 / Chapter 3.4.3 --- Reagents for Western blotting --- p.60 / Chapter 3.4.4 --- Gel filtration running buffer --- p.62 / Chapter 3.5 --- Methods --- p.62 / Chapter 3.5.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.62 / Chapter 3.5.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.63 / Chapter 3.5.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.64 / Chapter 3.5.4 --- Purification of rhGH by size exclusion chromatography - gel filtration chromatography --- p.64 / Chapter 3.5.5 --- General protein analysis --- p.65 / Chapter 3.6 --- Results --- p.67 / Chapter 3.6.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.67 / Chapter 3.6.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.69 / Chapter 3.6.3 --- Digestion efficiency of different constructs of SENP1C --- p.73 / Chapter 3.6.4 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.77 / Chapter 3.6.5 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.78 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.81 / Chapter 3.7.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.82 / Chapter 3.7.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.82 / Chapter 3.7.4 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.85 / Chapter 3.8 --- Conclusion --- p.85 / Chapter 4. --- Fermentation expression of SUMO-hGH and scale-up of downstream process / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Bioreactor system for E.coli host cultivation --- p.87 / Chapter 4.3 --- Mechanical cell disruption for cell --- p.88 / Chapter 4.4 --- rhGH binding assay --- p.88 / Chapter 4.5 --- Materials --- p.89 / Chapter 4.5.1 --- Reagents for bacterial culture by fermenter --- p.89 / Chapter 4.5.2 --- Reagents for HEK293 Hi cultivation --- p.91 / Chapter 4.5.3 --- Reagents for Dual-Luciferase® Reporter Assay System --- p.92 / Chapter 4.5.4 --- Reagents for silver stain of SDS-PAGE mini-gel --- p.93 / Chapter 4.6 --- Methods --- p.94 / Chapter 4.6.1 --- Bioreactor system and fixed volume fed-batch fermentation --- p.94 / Chapter 4.6.2 --- Large scale mechanically disruption of cell membrane --- p.97 / Chapter 4.6.3 --- Downstream processing of SUMO-hGH --- p.97 / Chapter 4.6.4 --- Culture of HEK293 Hi cells --- p.97 / Chapter 4.6.5 --- Dual-Luciferase® Reporter Assay System --- p.98 / Chapter 4.6.6 --- Silver staining of SDS-PAGE mini-gels --- p.101 / Chapter 4.7 --- Results --- p.101 / Chapter 4.7.1 --- Fed-batch fermentation of E. coli BL21 --- p.101 / Chapter 4.7.2 --- Comparison on disruption methods and the purification of SUMO-hGH from cell lysate --- p.106 / Chapter 4.7.3 --- Optimization of His-MBP-SENP1C digestion condition --- p.108 / Chapter 4.7.4 --- Optimization of rhGH purification in 2nd round of IMAC --- p.110 / Chapter 4.7.5 --- Characterization of mature rhGH --- p.112 / Chapter 4.8 --- Discussion --- p.116 / Chapter 4.8.1 --- Fed-batch fermentation of E. coli BL21 --- p.118 / Chapter 4.8.2 --- Downstream processing of fermentation culture and characterization of rhGH --- p.120 / Chapter 4.8.3 --- M9 based defined medium fermentation study --- p.122 / Chapter 4.8.4 --- rhGH production yield estimation --- p.128 / Chapter 4.8.5 --- Comparison of our fermentation expression system to the published data --- p.130 / Chapter 4.9 --- Conclusion --- p.132 / Chapter 5. --- His-MBP-SENPIC expression and purification / Chapter 5.1 --- Introduction --- p.133 / Chapter 5.2 --- Materials --- p.134 / Chapter 5.2.1 --- Reagents for bacterial culture --- p.134 / Chapter 5.2.2 --- Reagents for immobilized metal affinity chromatography purification of His-MBP-SENP1C --- p.135 / Chapter 5.3 --- Methods --- p.136 / Chapter 5.3.1 --- Expression of His-MBP-SENP1C --- p.136 / Chapter 5.3.2 --- Semi-purification of His-MBP-SENP1C by Ni2+-NTA affinity chromatography --- p.138 / Chapter 5.4 --- Results --- p.139 / Chapter 5.4.1 --- Expression of His-MBP-SENP1C --- p.139 / Chapter 5.4.2 --- Digestion activity of His-MBP-SENP1C expressed --- p.139 / Chapter 5.5 --- Discussion --- p.141 / Chapter 5.5.1 --- Expression and purification of His-MBP-SENP1C --- p.141 / Chapter 5.5.2 --- His-MBP-SENP1C production yield estimation --- p.143 / Chapter 6. --- Production and characterization of monoclonal antibodies against rhGH / Chapter 6.1 --- Introduction --- p.145 / Chapter 6.2 --- Materials --- p.146 / Chapter 6.2.1 --- Reagents for Sp2/0-Ag14 cultivation --- p.146 / Chapter 6.2.2 --- Reagents for PEG fusion --- p.147 / Chapter 6.2.3 --- Reagents for enzyme linked immunosorbent assay --- p.149 / Chapter 6.2.4 --- Reagents for mAbs purification by HiTrap´ёØ Protein G HP Column --- p.150 / Chapter 6.3 --- Methods --- p.151 / Chapter 6.3.1 --- ELISA --- p.151 / Chapter 6.3.2 --- Immunization --- p.152 / Chapter 6.3.3 --- Culturing of myeloma fusion partner cells --- p.153 / Chapter 6.3.4 --- Isolation of splenocyte --- p.153 / Chapter 6.3.5 --- PEG fusion --- p.154 / Chapter 6.3.6 --- Limiting dilution --- p.155 / Chapter 6.3.7 --- Cryopreservation of hybridoma cell lines --- p.156 / Chapter 6.3.8 --- Mass production of monoclonal antibodies --- p.157 / Chapter 6.3.9 --- Purification of IgG mAbs from ascites --- p.157 / Chapter 6.3.10 --- MAbs isotyping --- p.159 / Chapter 6.3.11 --- Determination of kinetic parameters of mAbs --- p.159 / Chapter 6.4 --- Results --- p.162 / Chapter 6.4.1 --- Production of murine anti-rhGH monoclonal antibodies --- p.162 / Chapter 6.4.2 --- Characterization of anti-rhGH mAbs --- p.170 / Chapter 6.5 --- Discussion --- p.178 / Chapter 6.5.1 --- Mass production of mAbs --- p.179 / Chapter 6.5.2 --- Future works on mAbs --- p.179 / Chapter 6.6 --- Conclusion --- p.181 / Chapter 7. --- Development of sandwich ELISA for rhGH / Chapter 7.1 --- Introduction --- p.182 / Chapter 7.2 --- Materials --- p.184 / Chapter 7.2.1 --- Reagents for sandwich ELISA --- p.184 / Chapter 7.3 --- Methods --- p.184 / Chapter 7.3.1 --- Production of rabbit polyclonal antiserum against rhGH --- p.184 / Chapter 7.3.2 --- Sandwich ELISA --- p.185 / Chapter 7.4 --- Results --- p.186 / Chapter 7.4.1 --- Production of rabbit antiserum against rhGH --- p.186 / Chapter 7.4.2 --- Sandwich ELISA --- p.188 / Chapter 7.4.3 --- Optimization of sandwich ELISA --- p.190 / Chapter 7.4.4 --- Specificity of sandwich ELISA --- p.194 / Chapter 7.4.5 --- Cross reactivity of sandwich ELISA to E.coli cell lysate --- p.196 / Chapter 7.4.6 --- Measurement of SUMO-hGH with sandwich ELISA --- p.198 / Chapter 7.5 --- Discussion --- p.201 / Chapter 7.5.1 --- Application of sandwich ELISA --- p.203 / Chapter 7.5.2 --- Future works on sandwich ELISA --- p.205 / Chapter 7.6 --- Conclusion --- p.205 / References --- p.206 / Appendix - pJ2:G01458 nucleotide sequence --- p.213

In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&amp / #945 / A plasmid and the constructed system within the plasmid, pPICZ&amp / #945 / A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations. Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm / N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density.

The purpose of the study is to purify human growth hormone from the fermentation broth by affinity chromatography. For this purpose, human growth hormone specific oligonucleotide aptamers are selected among an aptamer library / selected oligonucleotides were synthesized and used as ligands. Effect of pH on ligand-human growth hormone complex formation was investigated and the highest complex formation was obtained at pH= 7.0. Human growth hormone is separated from the fermentation broth with 99.8% purity and 41% overall yield. The equilibrium data obtained was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are calculated as 0.338 mg hGH/&igrave / mol aptamer and 0.059 mg hGH/ml, respectively. Further, equilibriumdata obtained using aptamer affinity column was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are 0.027 mg hGH/&igrave / mol aptamer and 1.543 mg hGH/ml, respectively. It is possible that, selected aptamer can be used for purification of bulk amounts of recombinant human growth hormone by using aptamer affinity chromatography.

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations / expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.

In this study, the aim was to achieve high concentration of recombinant human growth hormone (rhGH) production by recombinant Pichia pastoris by investigating the effects of various operation parameters and to determine the suitable peptide ligand sequence that shows affinity and specificity to hGH. In this context, firstly the effect of temperature and Tween-20/80 addition on production and cell growth were investigated. While at T=30 and 32&deg / C, there was no difference, at 27 and 25&deg / C cell growth slowed down and production decreased significantly. The addition of Tween-20/80 in existence of co-substrate sorbitol did not affect the bioprocess while in absence of sorbitol Tween alone did not show the same positive effect on product formation and cell growth. Thereafter at T=30&deg / C, without addition of Tween, three sets of pilot scale bioreactor experiments were performed. In the first set, the effect of methanol feeding rate on bioprocess characteristics were investigated at the specific growth rates of &mu / =0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at &mu / =0.04 h-1, the highest rhGH concentration was achieved at &mu / =0.03 h-1. Secondly, conducting methanol feeding at &mu / =0.03 h-1, pH=5.5 experiment was conducted. The highest cell concentration, 45 g L-1 and maximum rhGH concentration 0.25 g L-1 were achieved at t=18 h of the process. Finally, the effect of batch sorbitol feeding on bioprocess was observed by the addition of 50 g L-1 sorbitol at t=0, 14 and 31 h of the production phase. It was shown that sorbitol addition to the medium increased process duration / hence cells enter stationary phase after a longer production phase. However, the protease concentration continued increasing with respect to time and at the end of the process reached twice the concentration it was obtained with single sorbitol addition case decreasing the rhGH concentration. In selection of the peptide sequence that shows affinity towards hGH, phage display method was conducted. Additionally the sequences from literature and computational design were used as alternatives. The interaction between these peptides and hGH was investigated by isothermal titration calorimetry and surface plasmon resonance.

In this study, regulatory gene effects on recombinant human growth hormone (rhGH) production by Bacillus subtilis were investigated. For this purpose, firstly Bacillus strains, which are deficient in abrB, aprE, degQ, degS, degU, scoC, sinI, sinR, and spo0A genes, were selected according to the regulatory gene network of aprE gene (serine alkaline protease gene of B. subtilis) since due to the degQ promoter and the pre-signal sequence of subC gene cloned in front of the hGH gene, hGH is produced by mimicking the serine alkaline protease synthesis. R-Bacillus strains were constructed by transformation of pMK4::pre(subC)::hGH plasmid to the selected strains. Thereafter, by the laboratory scale experiments, strains having the highest hGH production capacity were determined as scoC, aprE, sinR, and degU knockout strains. Using these strains, fermentation experiments were carried out in pilot-scale bioreactor in defined medium. Effect of pH control was also investigated and the highest cell and hGH concentration was obtained by scoC knockout strain in pH controlled operation as 1.62 kg m-3 and 126 g m-3, respectively. By this strain, the overall product and cell yield on total substrate were found as 16.12 g kg-1 and 0.15 g g-1, respectively. Furthermore, the highest total protease activity was attained by degU knockout strain as 65 U cm-3. On the other hand, maximum total organic acid secretion was determined as 1.31 kg m-3 in aprE knockout strain.

In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreactor operations were designed and per-formed. In general, the designed strategies are fundamentally based on simulta-neous feeding of the two substrates starting at t=0 h of the production phase, i.e., batch-wise 50 gL-1sorbitol feeding, together with fed-batch methanol feeding with a specific growth rate of &mu / 0=0.03 h-1 or &mu / 0=0.04 h-1, and fed-batch sorbitol feeding with a specific growth rate of &mu / 0=0.025h-1 which was calculated based on the specific consumption rate qS=0.152 g g-1h-1 of sorbitol. Consequently, sorbitol concentration was kept constant at 50 gL-1 within t=0-15h of the production phase / where, sorbitol feeding was terminated at t=15h. Amongst, in the first strategy (SSM1), methanol was fed to the system with the specific growth rate of &mu / 0=0.03 h-1, and the H+ concentration (pH) in the bioreactor was kept constant at pH=5.0. In the second strategy (SSM2), pH was kept constant at 5.5 until t=24h of the induction phase (production phase), thereafter, was reduced to pH= 5.0 / where methanol was fed to the bioreactor with the specific growth rate of &mu / 0=0.03 h-1. In the third strategy (SSM3), methanol was fed with the specific growth rate of &mu / 0=0.04 h-1, and the pH in the bioreactor was kept constant at pH 5.0. The highest rhGH production and cell concentration were achieved in the first strategy SSM1 as CrhGH=640 mg L-1 and CX=105.3 g L-1, and the overall cell and product yields on total substrate were calculated as YX/S =0.21 g g-1 and YCrhGH/S =1.83 mg g-1. In the second part of this study the two-substrates sorbitol and methanol were fed simultaneously in a solution compose of 1.37 mol sorbitol and 6.21 mol methanol in 13.88 mol water, which is named as SM. In this strategy (SM), the two-substrate solution was fed to the medium with the specific growth rate of &mu / 0=0.03 h-1 on sorbitol until t=30h / thereafter, only methanol was fed to the bio-reactor with the specific growth rate of &mu / 0=0.03 h-1. The highest cell and rhGH concentrations obtained in SM were, respectively, Cx=104.7 g L-1 and CrhGH=124 mg L-1 / and the overall cell and product yields on the total substrate were calcu-lated as YX/S=0.21 g g-1 and YCrhGH/S=0.39 mg g-1. Although the highest cell con-centration obtained at SM is close to that of the SSM1, the rhGH concentration obtained at SM is 5.2-fold lower than that of the strategy SSM1.

Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.