Entwicklung von Fermentationsprozessen zur Produktion rekombinanter Antikörperfragmente in Pichia pastoris und Nicotiana tabacum

Being partly funded by the EU-project "Processing technology for recovery of recombinant antibody produced in crop plants" (FAIR-PL-CT96-1039), the aim of this work was to provide approximately 500 mg of the recombinant single-chain antibody fragment 4813 to enable the development of a synthetic peptide based purification strategy for antibody fragments produced in recombinant plant systems. The fragment was to be produced either in a plant suspension culture or in a microbial host system. As a plant suspension culture the Nicotiana tabacum cv BY-2 cell was chosen. The methylotrophic yeast Pichia pastoris was chosen as a microbial host. Fermentation strategies were developed for both expression syststems. In the case of plant suspension culture, this included the analysis of fermentation conditions, growth kinetics and substrate uptake kinetics as well as experiments to stimulate expression in plant cells. For Pichia pastoris, the development of fermentation strategies focused on optimal feeding strategies of glycerol and methanol during the sequence of fed-batch growth and induction phases. Both expression systems were successfully scaled up to the 30-l stirred-tank reactor. 30-l fermentations for the production of scFv4813 in both systems (cell line R1-25, Pichia-clone N55) led to the decision to choose Pichia as the final production system, since scFv4813 was secreted into the medium at high levels. A purification strategy was developed (not part of this work) and ca. 500 mg of scFv4813 were purified from a single 30-l fermentation of Pichia pastoris using a 3-step purification scheme. The concentration and storage conditions of the product were investigated. The product purity was sufficient for subsequent crystallization studies.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2000

Author dc:creator

• Hellwig, Stephan

Contributors dc:contributor

• Kreuzaler, Fritz

• info:eu-repo/classification/ddc/660
• Technische Chemie
• Tabak
• Pichia pastoris
• Monoklonaler Antikörper
• Rekombinantes Protein
• Heterologe Genexpression
• Fermentation
• Reaktionstechnik

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121600%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:59855