title: 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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.|BSV Bioscience GmbH, Baesweiler, Germany.
authors.forename: Zulfaquar Ahmad
authors.initials: ZA
authors.lastname: Arfi
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hellwig
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Drossard
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. johannes.buyel@rwth-aachen.de.
authors.forename: Johannes Felix
authors.initials: JF
authors.lastname: Buyel
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D004731:Endotoxins; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000382:microbiology; D011112:Polymyxin B / Q000302:isolation & purification*; D011817:Rabbits; D012273:Ribulose-Bisphosphate Carboxylase / Q000172:deficiency*; D014026:Nicotiana / Q000235:genetics* / Q000276:immunology / Q000382:microbiology
chemical_list: D000911:Antibodies, Monoclonal; D004731:Endotoxins; D010936:Plant Extracts; D012273:Ribulose-Bisphosphate Carboxylase; D011112:Polymyxin B
keywords: Endotoxin removal; Molecular farming; Plant-derived biopharmaceutical proteins; polyclonal anti-host cell protein antibodies; quality control