Antibody degradation in tobacco plants: a predominantly apoplastic process.
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 IgG(1)κ 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.
Molecular Immunology Unit, Division of Clinical Sciences, St George's, University of London, UK.
Verena K
VK
Hehle
Matthew J
MJ
Paul
Pascal M
PM
Drake
Julian K C
JK
Ma
Craig J
CJ
van Dolleweerd
D019149:Bioreactors; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D030821:Plants, Genetically Modified; D059748:Proteolysis; D014026:Nicotiana / Q000737:chemistry / Q000378:metabolism*
D007074:Immunoglobulin G; D010936:Plant Extracts