Bioprocess development for therapeutical protein production

Download

index.pdf

Date

2008

Author

Çelik Akdur, Eda

Metadata

Show full item record

Item Usage Stats

267
views

174
downloads

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.

Subject Keywords

Chemical engineering.

URI

http://etd.lib.metu.edu.tr/upload/3/12610236/index.pdf
https://hdl.handle.net/11511/18171

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Chiral seperations by enzyme enhanced ultrafiltration : fractionation ofracemic benzoin Ölçeroğlu, Ayşe Hande; Yılmaz, Levent; Department of Chemical Engineering (2006) In this study, a methodology for separation of chiral molecules, by using enhanced ultrafiltration system was developed. Benzoin was the model chiral molecule studied. In the scope of developing this methodology, some parameters were investigated in the preliminary ultrafiltration experiments in order to set the operation conditions for enhanced ultrafiltration experiments. Due to the slight solubility of benzoin in pure water, 15% (v/v) Polyethylene glycol (PEG 400) and 30 % (v/v) Dimethyl sulfoxide (DMSO)...
Dynamic and steady-state analysis of oxidative dehydrogenation of ethane Karamullaoğlu, Gülsün; Doğu, Timur; Department of Chemical Engineering (2005) In this research, oxidative dehydrogenation of ethane to ethylene was studied over Cr-O and Cr-V-O mixed oxide catalysts through steady-state and dynamic experiments. The catalysts were prepared by the complexation method. By XRD, presence of Cr2O3 phase in Cr-O; and the small Cr2O3 and V2O4 phases of Cr-V-O were revealed. In H2-TPR, both catalysts showed reduction behaviour. From XPS the likely presence of Cr+6 on fresh Cr-O was found. On Cr-V-O, the possible reduction of V+5 and Cr+6 forms of the fresh sa...
Sythesis of zeolite beta for composite membranes Gür, Nadiye; Baç, Nurcan; Department of Chemical Engineering (2006) In this work, zeolite Beta was synthesized experimentally in order to be used as filler in fuel cell membranes in order to assess the proton conductivity of composite membranes. Effects of the Si/Al ratio, and synthesis time on yield, relative crystallinity, crystal size, and proton conductivity were investigated. Zeolite Beta with Si/Al ratio between 10 and 30 was synthesized with a batch formulation of 2.2Na2O:1Al2O3:ySiO2:4.6(TEA)2O:tH2O (where TEA≡ tetraethylammonium) at 150°C for 5-15 days of synthesis...
Bioreaction network flux analysis for industrial microorganisms: A review Çalık, Pınar (Walter de Gruyter GmbH, 2002-01-01) This review focuses on the analysis of the bioreaction networks of the microorgansims used in fermentation processes, by metabolic flux analysis that is the novel tool of biochemical reaction engineering, required for the quantitative analysis of cell metabolism which is under the control of the cell physiology. Metabolic flux analysis (MFA) is based on calculation of intracellular reaction network rates through various reaction pathways either theoretical or by using elaborate experimental data on uptake, ...
Purification and modification of bentonite and its use in polypropylene and linear low density polyethylene matrix nanocomposites Seyidoğlu, Tijen; Yılmazer, Ülkü; Department of Chemical Engineering (2010) The potential use of Reşadiye/Tokat bentonite as a reinforcement in polypropylene (PP) and linear low density polyethylene (LLDPE) polymer matrix nanocomposites filler was investigated. At first, organoclays (OC) were prepared by cation exchange reaction (CER) between the raw bentonite (RB) and three quaternary ammonium salts with long alkyl tails (QA): hexadecyl trimethyl ammonium bromide [HMA] [Br], tetrabutyl ammonium tetrafluoroborate [TBA] [BF4], tetrakisdecyl ammonium bromide [TKA] [Br] and one quater...

Citation Formats

E. Çelik Akdur, “Bioprocess development for therapeutical protein production,” Ph.D. - Doctoral Program, Middle East Technical University, 2008.