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Open Access Research article

Antibody degradation in tobacco plants: a predominantly apoplastic process

Verena K Hehle, Matthew J Paul, Pascal M Drake, Julian KC Ma and Craig J van Dolleweerd*

Author Affiliations

Molecular Immunology Unit, Division of Clinical Sciences, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK

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BMC Biotechnology 2011, 11:128  doi:10.1186/1472-6750-11-128

Published: 30 December 2011

Abstract

Background

Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem.

Results

In this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space.

Conclusions

The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.