Development of an immunoassay to detect tobacco host cell proteins during biopharmaceutical development
Year: 2015
Type: dissertation
Abstract: Recombinant proteins have been produced in plants for decades but regulatory approval (marketing authorization) for a recombinant biopharmaceutical protein produced in a plant-based expression system was only achieved in May 2012. This has opened the door for the production of biopharmaceutical proteins in plants, but the development of a comprehensive, standardized set of analytical methods is still underway. The quantitative analysis of HCPs in drug substances and drug products is a key regulatory requirement for all biopharmaceuticals because this ensures HCPs are reduced to acceptable levels in the final product (typically 10-100 ppm), and qualitative and quantitative immunoassays are required to achieve such sensitivity. The aim of the work described in this thesis was to develop a generic immunoassay for the quantification of tobacco-specific HCPs as a counterpart for the commercially available assays for more-established expression systems such as CHO cells and E. coli. The generation of polyclonal antisera against the majority of tobacco HCPs is prevented by the presence of RuBisCO. The first part of this thesis describes a rapid and inexpensive method to achieve the depletion of RuBisCO from tobacco extracts using PEG 4000. The other major challenge was to produce the endotoxin-free antigens before immunization. The low endotoxin load was easily removed from wild-type tobacco plants (N. tabacum), which are used for stable expression, using Polymyxin B agarose. However, the extracts of agroinfiltrated tobacco plants (N. bethamiana), which are used for transient expression, contain much higher endotoxin levels and a two-step process was required, involving a FiberFlo EN filter to remove most endotoxins followed by Polymyxin B agarose or EndoTrap blue columns to remove residual amounts. In the second part of the thesis, unpurified and purified antibodies were tested against their corresponding antigens by 1-D and 2-D western blot and ELISA. Western blots of N. tabacum and N. benthamiana HCPs confirmed wide coverage by the anti-HCP antibodies, although antibodies generated against RuBisCO-depleted extracts and non-depleted extracts each detected unique spots. In the third part of the thesis, an ELISA was set up for N. tabacum and N. benthamiana HCPs allowing qualitative as well as quantitative detection. Finally, this immunoassay setup was applied in the downstream processing of a plant-derived biopharmaceutical to confirm the log reduction in HCP levels achieved during different process steps. The anti-HCP antibodies detected HCPs which had bound and eluted during protein A affinity chromatography, confirming that the assay was suitable for deployment during process development.
Source: RWTH Publications (RWTH Aachen)
Language: English
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2780195043