Publikationsserver der RWTH Aachen University
Expression and characterisation of biopharmaceuticals in heterologous expression systems
Abstract
dc:descriptionThe production of two complex human proteins, the therapeutically relevant anti-HIV full-size IgG1/kappa antibody, (BY-2)2F5, and a soluble form of the high-affinity receptor for human IgG, Fc-gamma-RI (CD64), were successfully established and optimised for heterologous expression in plants, bacteria and mammalian. The first aim of this thesis was the establishment of a protocol for the expression and purification of the anti-HIV antibody, 2F5, in plant suspension cultures (BY-2). Intact and pure antibody could be purified at levels of 1 mg/L per suspension culture out of 340 g BY-2 pellet. ELISA, EMSA and SPR proved comparable antigen binding capacity of the BY-22F5 antibody to the CHO derived control antibody. Neutralisation assays were performed in Vienna with this purification showing no negative influence of plant derived glycosylation. Initial fermentation experiments have shown that BY-2 cells can be grown without limitations on 100-L scale, but conditions for stable antibody production and downstream processing have to be further investigated. For GMP implementation and expression in large-scale reactors under defined conditions protocols and procedures must be established first. Based on the experience in the 100-L downstream processing, feasible equipment was suggested for a purification procedure of 100-L BY-2 culture in approximately two days in compliance with GMP regulations. The second aim of this thesis was to express Fc-gamma-RI as soluble recombinant protein in an appropriate expression system. Therefore, the extracellular domain of Fc-gamma-RI was generated and cloned into vectors for the expression in E.coli, mammalian and plant cells. All three systems were investigated for their ability to produce functional rsCD64. Highest expression levels were reached in the mammalian expression system yielding 1 mg/L. The expression and purification protocol was optimised and purity as well as integrity of rsCD64 was confirmed. Proof of functionality was confirmed by IgG binding activity in ELISA and SPR studies. Purified rsCD64 was functional as demonstrated by this IgG binding, and the binding characteristic was comparable to that of the original membrane bound receptor. Functional, soluble protein is one of the prerequisites for intensive characterisation of the Fc-gamma-RI. This characterisation, will lead to a better understanding of Fc-gamma-RI functionality and development of new specific Fc-gamma-RI targeted therapeutics.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2005
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Pätz, Antje
- Contributors dc:contributor
-
- Fischer, Rainer
Subjects
dc:subject × 15Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng