title: Process-relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics.
abstract: The biopharmaceutical industry has invested considerably in the implementation of single-use disposable bioreactors in place of or in addition to their stainless steel-counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene-film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035-0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label-free LC-MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547-1558, 2016.
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Kelly
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.
authors.forename: Shane
authors.initials: S
authors.lastname: McSweeney
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Orla
authors.initials: O
authors.lastname: Coleman
authors.affiliation: Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.|National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock Co, Dublin, Ireland.
authors.forename: Sara
authors.initials: S
authors.lastname: Carillo
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: Henry
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|School of Chemical Sciences, Dublin City University, Glasnevin, Dublin, Ireland.
authors.forename: Deepak
authors.initials: D
authors.lastname: Chandran
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.|School of Chemical Sciences, Dublin City University, Glasnevin, Dublin, Ireland.
authors.forename: Andrew
authors.initials: A
authors.lastname: Kellett
authors.affiliation: Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.|National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock Co, Dublin, Ireland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bones
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Clynes
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.
authors.forename: Paula
authors.initials: P
authors.lastname: Meleady
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.|Synthesis and Solid State Pharmaceutical Cluster, University of Limerick, Ireland.
authors.forename: Niall
authors.initials: N
authors.lastname: Barron
mesh_terms: D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D007074:Immunoglobulin G / Q000096:biosynthesis; D010755:Organophosphates / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013193:Stainless Steel / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D007074:Immunoglobulin G; D010755:Organophosphates; C000617831:bis(2,4-di-tert-butylphenyl)phosphate; D013193:Stainless Steel
keywords: CHO; bDtBPP; disposables; growth; leachables; proteomics