Advertisement

Applied Microbiology and Biotechnology

, Volume 104, Issue 3, pp 1097–1108 | Cite as

Zinc supplementation improves the harvest purity of β-glucuronidase from CHO cell culture by suppressing apoptosis

  • Ryan J. Graham
  • Stephanie Ketcham
  • Adil Mohammad
  • Bandaranayake M. B. Bandaranayake
  • Ty Cao
  • Bidesh Ghosh
  • James Weaver
  • Seongkyu Yoon
  • Patrick J. Faustino
  • Muhammad Ashraf
  • Celia N. Cruz
  • Chikkathur N. MadhavaraoEmail author
Biotechnological products and process engineering
  • 49 Downloads

Abstract

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.

Keywords

Trace metal variability CHO culture Zinc supplementation Apoptosis Lysosomal enzymes Specific activity 

Notes

Acknowledgments

The authors thank Dr. Kristina Howard for help with the flow cytometry measurements and Alan Carlin for critical reading of the manuscript and useful suggestions on presentation style.

Authors contributions

RG, SAK, AM, BMBB, JW, and CNM performed the experiments. BG and TC assisted with bioprocessing and purification. RG, CNM, SAK, AM, JW, and BMBB analyzed the data. RG, CNM, SAK, BMBB, SY, MA, CNC, JW, and PFJ wrote the manuscript. RG, AM, and CNM conceptualized the work.

Funding information

This study was intramurally funded by the Center for Drug Evaluation and Research, USFDA, for “Improved Understanding of Bioprocessing” and “Product Quality and Biopharmaceutics of Complex Dosage Forms”. RG, SAK, BG, BMBB, and TC received ORISE fellowships from CDER.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Disclaimer

This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.

Supplementary material

253_2019_10296_MOESM1_ESM.pdf (251 kb)
ESM 1 (PDF 250 kb)

References

  1. Assuncao Guimaraes C, Linden R (2004) Programmed cell deaths. Apoptosis and alternative death styles. Eur J Biochem 271(9):1638–1650.  https://doi.org/10.1111/j.1432-1033.2004.04084.x CrossRefPubMedGoogle Scholar
  2. Baek E, Noh SM, Lee GM (2017) Anti-apoptosis engineering for improved protein production from CHO cells. Methods Mol Biol 1603:71–85.  https://doi.org/10.1007/978-1-4939-6972-2_5 CrossRefPubMedGoogle Scholar
  3. Boustany RM (2013) Lysosomal storage diseases--the horizon expands. Nat Rev Neurol 9(10):583–598.  https://doi.org/10.1038/nrneurol.2013.163 CrossRefPubMedGoogle Scholar
  4. Clegg MS, Hanna LA, Niles BJ, Momma TY, Keen CL (2005) Zinc deficiency-induced cell death. IUBMB Life 57(10):661–669.  https://doi.org/10.1080/15216540500264554 CrossRefPubMedGoogle Scholar
  5. Eron SJ, MacPherson DJ, Dagbay KB, Hardy JA (2018) Multiple mechanisms of zinc-mediated inhibition for the apoptotic caspases-3, −6, −7, and −8. ACS Chem Biol 13(5):1279–1290.  https://doi.org/10.1021/acschembio.8b00064 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Fischer S, Handrick R, Otte K (2015) The art of CHO cell engineering: a comprehensive retrospect and future perspectives. Biotechnol Adv 33(8):1878–1896.  https://doi.org/10.1016/j.biotechadv.2015.10.015 CrossRefPubMedGoogle Scholar
  7. Franklin RB, Costello LC (2009) The important role of the apoptotic effects of zinc in the development of cancers. J Cell Biochem 106(5):750–757.  https://doi.org/10.1002/jcb.22049 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fratz-Berilla EJ, Ketcham SA, Parhiz H, Ashraf M, Madhavarao CN (2017) An improved purification method for the lysosomal storage disease protein beta-glucuronidase produced in CHO cells. Protein Expr Purif 140:28–35.  https://doi.org/10.1016/j.pep.2017.07.011 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gilbert A, Huang Y, Ryll T (2014) Identifying and eliminating cell culture process variability. Pharm Bioprocess 2(6):519–534.  https://doi.org/10.4155/pbp.14.35 CrossRefGoogle Scholar
  10. Graham RJ, Bhatia H, Yoon S (2019) Consequences of trace metal variability and supplementation on Chinese hamster ovary (CHO) cell culture performance: a review of key mechanisms and considerations. Biotechnol Bioeng.  https://doi.org/10.1002/bit.27140 CrossRefGoogle Scholar
  11. Grubb JH, Vogler C, Levy B, Galvin N, Tan Y, Sly WS (2008) Chemically modified beta-glucuronidase crosses blood-brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII. Proc Natl Acad Sci U S A 105(7):2616–2621.  https://doi.org/10.1073/pnas.0712147105 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Han S, Rhee WJ (2018) Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture. Biotechnol Bioeng 115(5):1331–1339.  https://doi.org/10.1002/bit.26549 CrossRefPubMedGoogle Scholar
  13. Huber KL, Hardy JA (2012) Mechanism of zinc-mediated inhibition of caspase-9. Protein Sci 21(7):1056–1065.  https://doi.org/10.1002/pro.2090 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kaschak T, Boyd D, Lu F, Derfus G, Kluck B, Nogal B, Emery C, Summers C, Zheng K, Bayer R, Amanullah A, Yan B (2011) Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media. MAbs 3(6):577–583.  https://doi.org/10.4161/mabs.3.6.17959 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ketcham SA, Ashraf M, Madhavarao CN (2017) Direct quantification of protein glycan phosphorylation. BioTechniques 63(3):117–123.  https://doi.org/10.2144/000114587 CrossRefPubMedGoogle Scholar
  16. Kim BG, Park HW (2016) High zinc ion supplementation of more than 30 μM can increase monoclonal antibody production in recombinant Chinese hamster ovary DG44 cell culture. Appl Microbiol Biotechnol 100(5):2163–2170.  https://doi.org/10.1007/s00253-015-7096-x CrossRefGoogle Scholar
  17. Kumar N, Gammell P, Clynes M (2007) Proliferation control strategies to improve productivity and survival during CHO based production culture : a summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines. Cytotechnology 53(1–3):33–46.  https://doi.org/10.1007/s10616-007-9047-6 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kumar S, Zhou S, Singh SK (2014) Metal ion leachates and the physico-chemical stability of biotherapeutic drug products. Curr Pharm Des 20(8):1173–1181CrossRefGoogle Scholar
  19. Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100(8):3451–3461.  https://doi.org/10.1007/s00253-016-7388-9 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Luo J, Vijayasankaran N, Autsen J, Santuray R, Hudson T, Amanullah A, Li F (2012) Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process. Biotechnol Bioeng 109(1):146–156.  https://doi.org/10.1002/bit.23291 CrossRefPubMedGoogle Scholar
  21. Madhavarao CN, Agarabi CD, Wong L, Muller-Loennies S, Braulke T, Khan M, Anderson H, Johnson GR (2014) Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors. Biotechnol Appl Biochem 61(2):184–192.  https://doi.org/10.1002/bab.1151 CrossRefPubMedGoogle Scholar
  22. Marreiro DD, Cruz KJ, Morais JB, Beserra JB, Severo JS, de Oliveira AR (2017) Zinc and oxidative stress: current mechanisms. Antioxidants (Basel) 6(2).  https://doi.org/10.3390/antiox6020024 CrossRefGoogle Scholar
  23. Mohammad MK, Zhou Z, Cave M, Barve A, McClain CJ (2012) Zinc and liver disease. Nutr Clin Pract 27(1):8–20.  https://doi.org/10.1177/0884533611433534 CrossRefPubMedGoogle Scholar
  24. Mohammad A, Agarabi C, Rogstad S, DiCioccio E, Brorson K, Ashraf M, Faustino PJ, Madhavarao CN (2019) An ICP-MS platform for metal content assessment of cell culture media and evaluation of spikes in metal concentration on the quality of an IgG3:kappa monoclonal antibody during production. J Pharm Biomed Anal 162:91–100.  https://doi.org/10.1016/j.jpba.2018.09.008 CrossRefPubMedGoogle Scholar
  25. Morrow T, Felcone LH (2004) Defining the difference: what makes biologics unique. Biotechnol Healthc 1(4):24–29PubMedPubMedCentralGoogle Scholar
  26. Parenti G, Andria G, Ballabio A (2015) Lysosomal storage diseases: from pathophysiology to therapy. Annu Rev Med 66:471–486.  https://doi.org/10.1146/annurev-med-122313-085916 CrossRefPubMedGoogle Scholar
  27. Parhiz H, Ketcham SA, Zou G, Ghosh B, Fratz-Berilla EJ, Ashraf M, Ju T, Madhavarao CN (2019) Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme β-glucuronidase produced from Chinese hamster ovary cells. Appl Microbiol Biotechnol 103(15):6081–6095.  https://doi.org/10.1007/s00253-019-09889-7 CrossRefPubMedGoogle Scholar
  28. Prabhu A, Gadre R, Gadgil M (2018) Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells. Appl Microbiol Biotechnol 102(14):5989–5999.  https://doi.org/10.1007/s00253-018-9064-8 CrossRefPubMedGoogle Scholar
  29. Qian Y, Khattak SF, Xing Z, He A, Kayne PS, Qian NX, Pan SH, Li ZJ (2011) Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells. Biotechnol Prog 27(4):1190–1194.  https://doi.org/10.1002/btpr.630 CrossRefPubMedGoogle Scholar
  30. Rabbani P, Prasad AS (1978) Plasma ammonia and liver ornithine transcarbamoylase activity in zinc-deficient rats. Am J Phys 235(2):E203–E206.  https://doi.org/10.1152/ajpendo.1978.235.2.E203 CrossRefGoogle Scholar
  31. Riggio O, Merli M, Capocaccia L, Caschera M, Zullo A, Pinto G, Gaudio E, Franchitto A, Spagnoli R, D'Aquilino E, Seri S, Moretti R, Cantafora A (1992) Zinc supplementation reduces blood ammonia and increases liver ornithine transcarbamylase activity in experimental cirrhosis. Hepatology 16(3):785–789CrossRefGoogle Scholar
  32. Samie MA, Xu H (2014) Lysosomal exocytosis and lipid storage disorders. J Lipid Res 55(6):995–1009.  https://doi.org/10.1194/jlr.R046896 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schneider M, Marison IW, von Stockar U (1996) The importance of ammonia in mammalian cell culture. J Biotechnol 46(3):161–185CrossRefGoogle Scholar
  34. Sha S, Agarabi C, Brorson K, Lee DY, Yoon S (2016) N-glycosylation design and control of therapeutic monoclonal antibodies. Trends Biotechnol 34(10):835–846.  https://doi.org/10.1016/j.tibtech.2016.02.013 CrossRefPubMedGoogle Scholar
  35. Sly WS, Quinton BA, McAlister WH, Rimoin DL (1973) Beta glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J Pediatr 82(2):249–257CrossRefGoogle Scholar
  36. Williamson J, Miller J, McLaughlin J, Combs R, Chu C (2018) Scale-dependent manganese leaching from stainless steel impacts terminal galactosylation in monoclonal antibodies. Biotechnol Prog 34(5):1290–1297.  https://doi.org/10.1002/btpr.2662 CrossRefPubMedGoogle Scholar
  37. Yuk IH, Russell S, Tang Y, Hsu WT, Mauger JB, Aulakh RP, Luo J, Gawlitzek M, Joly JC (2015) Effects of copper on CHO cells: cellular requirements and product quality considerations. Biotechnol Prog 31(1):226–238.  https://doi.org/10.1002/btpr.2004 CrossRefPubMedGoogle Scholar
  38. Zamorano F, Wouwer AV, Bastin G (2010) A detailed metabolic flux analysis of an underdetermined network of CHO cells. J Biotechnol 150(4):497–508.  https://doi.org/10.1016/j.jbiotec.2010.09.944 CrossRefPubMedGoogle Scholar
  39. Zhou S, Schoneich C, Singh SK (2011) Biologics formulation factors affecting metal leachables from stainless steel. AAPS PharmSciTech 12(1):411–421.  https://doi.org/10.1208/s12249-011-9592-3 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  • Ryan J. Graham
    • 1
    • 2
  • Stephanie Ketcham
    • 1
  • Adil Mohammad
    • 1
  • Bandaranayake M. B. Bandaranayake
    • 1
  • Ty Cao
    • 1
  • Bidesh Ghosh
    • 1
  • James Weaver
    • 3
  • Seongkyu Yoon
    • 2
  • Patrick J. Faustino
    • 1
  • Muhammad Ashraf
    • 1
  • Celia N. Cruz
    • 1
  • Chikkathur N. Madhavarao
    • 1
    Email author
  1. 1.Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and ResearchFood and Drug AdministrationSilver SpringUSA
  2. 2.Department of Chemical EngineeringUniversity of Massachusetts LowellLowellUSA
  3. 3.Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Science, Center for Drug Evaluation and ResearchFood and Drug AdministrationSilver SpringUSA

Personalised recommendations