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Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials

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Abstract

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds—3,3′,5,5′-tetra-tert-butyl-2,2′-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)—were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that‚ even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated.

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References

  1. Eibl R, Eibl D. Disposable bioreactors. Heidelberg: Springer Heidelberg Dordrecht London New York; 2009.

  2. Zweifel H, Maier RD, Schiller M. Plastics additives handbook. 6th ed. Carl Hanser Verlag: München; 2010.

    Google Scholar 

  3. Crowson A. The effects of electron beam irradiation on additives present in food-contact polymers (Doctoral Thesis). Sheffield Hallam University (United Kingdom); 1991.

  4. El Makhzoumi Z. Effect of irradiation of polymeric packaging material on the formation of volatile compounds. In: Food packaging and preservation. Boston: Springer US; 1994. p. 88–99.

    Chapter  Google Scholar 

  5. Kawamura Y. Effects of gamma irradiation on polyethylene, polypropylene, and polystyrene. In: Irradiation of Food and Packaging. American Chemical Society; 2004. p. 262–76.

  6. Bourges F, Bureau G, Pascat B. Effects of electron beam irradiation on commercial polypropylene: kinetic study of antioxidant degradation. Packag Technol Sci. 2006;5(4):197–204.

    Article  Google Scholar 

  7. Peng J, Zhao Y, Hong Y, Burkhalter RS, Hogue CL, Tran E, et al. Chemical identity and mechanism of action and formation of a cell growth inhibitory compound from polycarbonate flasks. Anal Chem. 2018;90(7):4603–10.

    Article  CAS  Google Scholar 

  8. Hammond M, Marghitoiu L, Lee H, Perez L, Rogers G, Nashed-Samuel Y, et al. A cytotoxic leachable compound from single-use bioprocess equipment that causes poor cell growth performance. Biotechnol Prog. 2014;30(2):332–7.

    Article  CAS  Google Scholar 

  9. Hammond M, Nunn H, Rogers G, Lee H, Marghitoiu A-L, Perez L, et al. Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers. PDA J Pharm Sci Technol. 2013;67(2):123–34. Available from:. https://doi.org/10.5731/pdajpst.2013.00905.

    Article  CAS  PubMed  Google Scholar 

  10. Jurkiewicz E, Husemann U, Greller G, Barbaroux M, Fenge C. Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications. Biotechnol Prog. 2014;30(5):1171–6.

    Article  CAS  Google Scholar 

  11. Delaunay L, Jurkiewicz E, Greller G, Barbaroux M. How to design and qualify an improved film for storage and bioreactor bags. In: Eibl R, Eibl D, editors. Single-use technology in biopharmaceutical manufacture. 2nd ed. 2019. p. 229–34.

  12. Ding W, Madsen G, Mahajan E, O’Connor S, Wong K. Standardized extractables testing protocol for single-use systems in biomanufacturing. Pharm Eng. 2014;34(6):1–11.

    CAS  Google Scholar 

  13. Eibl R, Steiger N, Fritz C, Eisenkrätzer D, Bär J, Müller D, et al. Recommendation for leachables studies: standardized cell culture test for the early identification of critical films for CHO cell lines in chemically defined culture media. Frankfurt a.M: Dechema. 2014:1–22.

  14. Shah RR, Linville TW, Whynot AD, Brazel CS. Evaluating the toxicity of bDtBPP on CHO-K1 cells for testing of single-use bioprocessing systems considering media selection, cell culture volume, mixing, and exposure duration. Biotechnol Prog. 2016;32(5):1318–23 Available from: http://doi.wiley.com/10.1002/btpr.2322.

    Article  CAS  Google Scholar 

  15. Schwetlick K, Pionteck J, Winkler A, Hähner U, Kroschwitz H, Habicher WD. Organophosphorus antioxidants: part X—mechanism of antioxidant action of aryl phosphites and phosphonites at higher temperatures. Polym Degrad Stab. 1991;31(2):219–28.

    Article  CAS  Google Scholar 

  16. 21CFR177.2600 Code of Federal Regulations [Internet]. 2018 [cited 2019 Jun 28]. Available from: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=177.2600. Accessed 28 Jun 2019.

  17. Beißmann S, Grabmayer K, Wallner G, Nitsche D, Buchberger W. Analytical evaluation of the performance of stabilization systems for polyolefinic materials. Part II: interactions between hindered amine light stabilizers and thiosynergists. Polym Degrad Stab. 2014;110:509–17.

    Article  Google Scholar 

  18. Beißmann S, Reisinger M, Grabmayer K, Wallner G, Nitsche D, Buchberger W. Analytical evaluation of the performance of stabilization systems for polyolefinic materials. Part I: interactions between hindered amine light stabilizers and phenolic antioxidants. Polym Degrad Stab. 2014;110:498–508. Available from:. https://doi.org/10.1016/j.polymdegradstab.2014.09.020.

    Article  CAS  Google Scholar 

  19. United State Pharmacopoeia <1663> Assessment of extractables associated with pharmaceutical packaging/delivery systems. 2018;41:7910–24.

  20. Jenke D, Ruberto M. Using the correlation between material composition and extractables and leachables to forecast extractables and/or leachables profiles part 2: polyethylene and bromobutyl rubber case studies. Pharm Outsourcing. 2014. https://www.pharmoutsourcing.com/Featured-Articles/153795-Using-the-Correlation-Between-Material-Composition-and-Extractables-and-Leachables-to-Forecast-Extractables-and-or-Leachables-Profiles-Part-1-Concepts-and-Plasticized-Poly-vinyl-chloride-Case-Study/. Accessed 30 May 2018.

  21. Bolland JL, Gee G. Kinetic studies in the chemistry of rubber and related materials. II. The kinetics of oxidation of unconjugated olefins. Trans Faraday Soc. 1946;42(0):236–43.

    Article  CAS  Google Scholar 

  22. Allen DW, Clench MR, Crowson A, Leathard DA. Characterisation of solvent-extractable transformation products of high molecular weight hindered phenols in polypropylene subjected to ionising radiation in air or to thermal ageing. Polym Degrad Stab. 1993;39(3):293–7.

    Article  CAS  Google Scholar 

  23. Scheirs J, Pospíšil J, O’Connor MJ, Bigger SW. Characterization of conversion products formed during degradation of processing antioxidants. In: Polymer Durability. American Chemical Society; 1996. p. 24–359. (Advances in Chemistry; vol. 249).

  24. Yadav LDS. Organic spectroscopy. Dordrecht: Springer Science+Business Media Dordrecht; 2005.

  25. International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use - Impurities in New Drug Substances Q3A(R2). 2006;

  26. Fujii I, Hirayama N. Chiral space formed by (+)-(1S)-1,1′-binaphthalene-2,2′-diyl phosphate: recognition of aliphatic L-α-amino acids. Helv Chim Acta. 2002;85(9):2946–60.

    Article  CAS  Google Scholar 

  27. Crous R, Datt M, Foster D, Bennie L, Steenkamp C, Huyser J, et al. Rhodium hydride formation in the presence of a bulky monophosphite ligand: a spectroscopic and solid-state investigation. Dalton Trans. 2005;8(6):1108–16.

    Article  Google Scholar 

  28. Rueping M, Sugiono E, Azap C, Theissmann T, Bolte M. Enantioselective Brønsted acid catalyzed transfer hydrogenation: organocatalytic reduction of imines. Org Lett. 2005;7(17):3781–3.

    Article  CAS  Google Scholar 

  29. Ormerod MG, Paul F, Cheetham M, Sun X-M. Discrimination of apoptotic thymocytes by forward light scatter. Cytometry. 1995 [cited 2019 Sep 11];21(3):300–4. Available from: https://doi.org/10.1002/cyto.990210311

  30. Swat W, Ignatowicz L, Kisielow P. Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry. J Immunol Methods. 1991;137(1):79–87.

    Article  CAS  Google Scholar 

  31. Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. J Immunol Methods. 2000;243(1–2):167–90.

    Article  CAS  Google Scholar 

  32. Kelly PS, McSweeney S, Coleman O, Carillo S, Henry M, Chandran D, et al. Process-relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics. Biotechnol Prog. 2016;32(6):1547–58.

    Article  CAS  Google Scholar 

  33. Pahl I, Dorey S, Uettwiller I, Hoffmann C, Priebe P, Menzel R, et al. Development of a standardized extractables approach for single-use components - general considerations and practical aspects. Bioprocess Int. 2018;16(10).

  34. ASTM E3231-19. Standard guideline for cell culture growth assessment of single-use material [Internet]. ASTM International: West Conshohocken; 2019. Available from: www.astm.org. Accessed 10 Nov 2019.

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Author notes

  1. Dana Budde and Tanja Verena Maier contributed equally to this work.

Authors and Affiliations

  1. Sartorius Stedim Biotech GmbH, August-Spindler-Str. 11, 37079, Goettingen, Germany

    Dana Budde, Tanja Verena Maier, Elke Jurkiewicz, Ina Pahl, Armin Hauk & Roberto Menzel

  2. Universität Bielefeld, Universitätsstraße 25, 33615, Bielefeld, Germany

    Dana Budde & Thomas Noll

  3. Technische Universität Ilmenau, Weimarer Straße 25, 98693, Ilmenau, Germany

    Eric Täuscher

  4. Jena University, Lessingstraße 8, 07743, Jena, Germany

    Helmar Görls

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  1. Dana Budde
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Budde, D., Maier, T.V., Jurkiewicz, E. et al. Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials. Anal Bioanal Chem 412, 4505–4518 (2020). https://doi.org/10.1007/s00216-020-02736-z

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  • DOI: https://doi.org/10.1007/s00216-020-02736-z

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