Characterization of monoclonal antibody fragments produced by plant cells

Abstract

Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120-and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.

Function and glycosylation of plant-derived antiviral monoclonal antibody

“…The expression level and appropriate posttranslational events (correct folding, glycosylation, and subcellular targeting) are important factors for the effectiveness of antibodies produced in plants (7,10,17,35). In our study, these factors were optimized by the combination of gene regulatory elements in a plant binary vector.…”

“…Although there are exceptions (14), in general, glycans attached to proteins containing a C-terminal KDEL sequence would be expected to be restricted mainly to the oligomannose type (13,15,16). ER retention of proteins in transgenic plants usually improves the production levels (9,17). However, because glycan processing can affect the stability of antibodies (18), it is unclear whether an mAb P with modified glycan structures would be active and able to confer effective systemic post-exposure prophylaxis.…”

Manufacturing antibodies in the plant cell

“…A detailed analysis of the antibody fragments produced by a secreted IgG in a root expression system showed that they were not caused by antibody degradation during protein extraction. Two smaller fragments of 50-and 80-kDa were identified as the products of protease activity in the apoplast, whereas two additional fragments of 120-and 135-kDa were most likely the products of proteolytic degradation along the secretory pathway outside the endoplasmic reticulum (ER); the carbohydrate residues of the 135-kDa antibody suggested that this fragment was formed between the ER and Golgi [61]. A deeper understanding of the plant protease activities responsible for antibody degradation is needed to minimize the detrimental effect of proteolysis in plant mAb production.…”

“…Deleted: [19,59,60] Deleted: of Deleted: [61] Deleted: MAb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 (Fig. 3).…”

Cosecretion of Protease Inhibitor Stabilizes Antibodies Produced by Plant Roots

“…Proteases released during plant tissue harvesting, extraction, and downstream protein purification often result in antibody degradation (Ma et al, 1994;Sharp and Doran, 2001). Using the nondestructive secretion process that provides high yields of recombinant proteins over the lifetime of a plant and facilitates downstream purification can circumvent this manufacturing challenge.…”