Antibody degradation in tobacco plants: a predominantly apoplastic process

Abstract

BackgroundInterest 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.ResultsIn 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.ConclusionsThe 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.

High-level transient expression of the N-terminal domain of IpaD from Shigella dysenteriae in four plant species transformed with different construct configurations

“…Notably, the lower levels of recombinant IpaD in the apoplastic space relative to ER-derived protein bodies in N. benthamiana and soybean may be due to the plenitude of proteases, as the same phenomenon has been also recorded by Benchabane et al (2008). Nevertheless, in both lettuce and tobacco (N. tabacum), the accumulations of ER-targeted and apoplast-targeted versions of recombinant IpaD were approximately equal in agroinfiltrated leaves, agreeing nearly with the observations of Hehle et al (2011). Furthermore, for different plant species, various levels of proteases are expected to be present in apoplastic spaces (Hehle et al 2011); in addition, some proteins fused with sorting signal peptides may correctly target in some tissues and/or falsely target in other organs of the same plant (Robinson et al 2005).…”

“…Nevertheless, in both lettuce and tobacco (N. tabacum), the accumulations of ER-targeted and apoplast-targeted versions of recombinant IpaD were approximately equal in agroinfiltrated leaves, agreeing nearly with the observations of Hehle et al (2011). Furthermore, for different plant species, various levels of proteases are expected to be present in apoplastic spaces (Hehle et al 2011); in addition, some proteins fused with sorting signal peptides may correctly target in some tissues and/or falsely target in other organs of the same plant (Robinson et al 2005). As a result, it is not unexpected that agroinfiltrated leaves of different plants would produce different levels of IpaD proteins.…”

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…When organised tissues such as hairy roots and shooty teratomas are used for protein production, secreted protein is retained to some extent in the apoplast and thus remains associated with the biomass, while a proportion may be released from the apoplast into the medium [46]. Although, in some studies, recombinant proteins targeted to the apoplast of whole plants have accumulated at relatively high levels [62,65,66], extracellular proteases have been shown to degrade antibodies in the apoplast of tobacco plants and hairy roots [67,68].…”

Antibody proteolysis: a common picture emerging from plants

“…3,4 In the case of antibodies, unintended proteolysis driven by this complex peptidase repertoire can affect the final yield of intact IgG, and may lead to almost complete degradation. 5 The presence of antibody fragments in plants has been ascribed to the following 3 phenomena: i) partial assembly intermediates; ii) extracellular peptidase activity after secretion 6 ; iii) activity of peptidases released during sample homogenization. 7 Additionally, fragments resulting from a first "opening cleavage" might be further processed into smaller fragments by plant proteases.…”