Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

Abstract

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…Despite the use of a lectin specific affinity chromatography we also identified a band at ~53 kDa as a host cell protein, most likely ribulose‐1,5‐bisphosphate carboxylase oxygenase large subunit by means of western blot (Figure S1b). A distinct band of ~26 kDa (Figure S1a) was not detected by a host cell protein‐specific polyclonal antibody cocktail (Arfi, Hellwig, Drossard, Fischer, & Buyel, ) or A‐chain‐specific antibody TA‐5. We concluded that this impurity was likely a viscumin degradation product.…”

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

“…We used pre-cast lithium dodecyl sulfate polyacrylamide gels (Thermo Fisher Scientific, Waltham, Massachusetts) and stained proteins in the gels with Coomassie Brilliant Blue G250 as previously described (Arfi et al, 2016).…”

Unexpected synergistic HIV neutralization by a triple microbicide produced in rice endosperm

“…Cereal plants such as rice expressing multiple HIV-neutralizing proteins in GRAS-status seeds offer an inexpensive platform for the production of microbicidal mixtures with the added benefit that dry seeds can be stored indefinitely under ambient conditions ( 48 , 49 ). Rice has been developed as a production platform for proteins that can be administered in minimally processed seed extracts, such as oral vaccines and topical prophylactics, without the need to remove toxic metabolites or endotoxins ( 33 , 34 , 49 – 53 ). Although there are many reports of plants expressing multiple recombinant enzymes for metabolic engineering ( 54 , 55 ), the only previous reports of multiple microbicidal components have involved fusion proteins such as b12/CV-N ( 45 ) and CV-N/12pi ( 56 ).…”