Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme β-glucuronidase produced from Chinese hamster ovary cells

  • Hamideh Parhiz
  • Stephanie A. Ketcham
  • Guozhang Zou
  • Bidesh Ghosh
  • Erica J. Fratz-Berilla
  • Muhammad Ashraf
  • Tongzhong Ju
  • Chikkathur N. MadhavaraoEmail author
Biotechnological products and process engineering


β-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.25 mM butyrate addition, a temperature shift (from 37 to 32 °C), and a pH shift (from 7.0 to 6.7) along with a control (pH 7, temperature 37 °C, and no additive) on the production and quality of human recombinant β-glucuronidase (GUS) by a Chinese hamster ovary (CHO) cell line. The study was performed as two independent runs (2 bioreactors per treatment per run; n ≤ 4). Although statistically not significant, protein production slightly increased with either 0.25 mM butyrate addition (13%) or pH shift (7%), whereas temperature shift decreased production (12%, not significant). Further characterization of the purified GUS samples showed that purification selectively enriched the mannose-6-phosphate (M6P)–containing GUS protein. Noticeably, a variation observed for the critical quality attribute (CQA) of the enzyme, namely M6P content, decreased after purification, across treatment replicates and, more so, across different treatments. The dimer content in the purified samples was comparable (~25%), and no significant discrepancy was observed in terms of GUS charge variants by capillary electrophoresis analysis. MALDI-TOF/TOF analysis of released N-glycans from GUS showed a minor variation in glycoforms among the treatment groups. Temperature shift resulted in a slightly increased sialylated glycan content (21.6%) when compared to control (15.5%). These results suggest that bioreactor processes have a differential effect, and better control is required for achieving improved production of GUS enzyme in CHO cells without affecting drastically its CQAs. However, the purification method allowed for enrichment of GUS with similar CQA profiles, regardless of the upstream treatments, indicating for the first time that the effect of slight alterations in upstream process parameters on the CQA profile can be offset with an effective and robust purification method downstream to maintain drug substance uniformity.


Enzyme replacement therapy Lysosomal storage disorders Parallel bioreactors Glycan content Mannose-6-phosphate β-Glucuronidase Chinese hamster ovary cells Critical quality attributes 



The authors thank Sarah Johnson and Rukman DeSilva of the Office of Biotechnology Products/CDER for the critical reading and comments on the manuscript. All authors acknowledge the critical reading, feedback, and support by Celia Cruz.

Author contributions

HP, SAK, BG, GZ, and EJFB performed the experiments and wrote the manuscript. MA and TJ wrote the manuscript. CNM conceptualized the work, performed the experiments, and wrote the manuscript.


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.” HP, SAK, BG, GZ, and EJFB were recipients of 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.


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

Supplementary material

253_2019_9889_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1162 kb)


  1. Alley WR Jr, Madera M, Mechref Y, Novotny MV (2010) Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery. Anal Chem 82(12):5095–5106. Google Scholar
  2. Avello V, Tapia B, Vergara M, Acevedo C, Berrios J, Reyes JG, Altamirano C (2017) Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells. Electron J Biotechnol 27:55–62. Google Scholar
  3. Beckman-Coulter (2014) PA 800 Plus Pharmaceutical Analysis System. SDS-MW Analysis, Beckman, Coulter, Brea, CAGoogle Scholar
  4. Bones J, Mittermayr S, McLoughlin N, Hilliard M, Wynne K, Johnson GR, Grubb JH, Sly WS, Rudd PM (2011) Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment. Anal Chem 83(13):5344–5352. Google Scholar
  5. Bork K, Horstkorte R, Weidemann W (2009) Increasing the sialylation of therapeutic glycoproteins: the potential of the sialic acid biosynthetic pathway. J Pharm Sci 98(10):3499–3508. Google Scholar
  6. Buommino E, Pasquali D, Sinisi AA, Bellastella A, Morelli F, Metafora S (2000) Sodium butyrate/retinoic acid costimulation induces apoptosis-independent growth arrest and cell differentiation in normal and ras-transformed seminal vesicle epithelial cells unresponsive to retinoic acid. J Mol Endocrinol 24(1):83–94Google Scholar
  7. Chen F, Kou T, Fan L, Zhou Y, Ye Z, Zhao L, Tan W-S (2011) The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells. Biotechnol Bioprocess Eng 16(6):1157–1165. Google Scholar
  8. Cole KS, Steckbeck JD, Rowles JL, Desrosiers RC, Montelaro RC (2004) Removal of N-linked glycosylation sites in the V1 region of Simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3. J Virol 78(3):1525–1539. Google Scholar
  9. Cooper CA, Gasteiger E, Packer NH (2001) GlycoMod—a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 1(2):340–349.<340::aid-prot340>;2-b Google Scholar
  10. Cooper CA, Gasteiger E, Packer NH (2003) Predicting glycan composition from experimental mass using GlycoMod. Handbook of proteomic methods. Humana Press, Totowa, pp 225–231Google Scholar
  11. Ferrara C, Grau S, Jager C, Sondermann P, Brunker P, Waldhauer I, Hennig M, Ruf A, Rufer AC, Stihle M, Umana P, Benz J (2011) Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose. Proc Natl Acad Sci U S A 108(31):12669–12674. Google Scholar
  12. 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. Google Scholar
  13. Gehrmann ML, Douglas JT, Banyai L, Tordai H, Patthy L, Llinas M (2004) Modular autonomy, ligand specificity, and functional cooperativity of the three in-tandem fibronectin type II repeats from human matrix metalloproteinase 2. J Biol Chem 279(45):46921–46929. Google Scholar
  14. Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A (2010) Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 28:863. Google Scholar
  15. Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, Flynn GC (2011) High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 21(7):949–959. Google Scholar
  16. Grabowski GA, Hopkin RJ (2003) Enzyme therapy for lysosomal storage disease: principles, practice, and prospects. Annu Rev Genomics Hum Genet 4:403–436. Google Scholar
  17. 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. Google Scholar
  18. Gupta SK, Shukla P (2017a) Gene editing for cell engineering: trends and applications. Crit Rev Biotechnol 37(5):672–684. Google Scholar
  19. Gupta SK, Shukla P (2017b) Sophisticated cloning, fermentation, and purification technologies for an enhanced therapeutic protein production: a review. Front Pharmacol 8(419).
  20. Gupta SK, Srivastava SK, Sharma A, Nalage VHH, Salvi D, Kushwaha H, Chitnis NB, Shukla P (2017) Metabolic engineering of CHO cells for the development of a robust protein production platform. PLoS One 12(8):e0181455. Google Scholar
  21. Hawkins-Salsbury JA, Reddy AS, Sands MS (2011) Combination therapies for lysosomal storage disease: is the whole greater than the sum of its parts? Hum Mol Genet 20(R1):R54–R60. Google Scholar
  22. Hendrick V, Winnepenninckx P, Abdelkafi C, Vandeputte O, Cherlet M, Marique T, Renemann G, Loa A, Kretzmer G, Werenne J (2001) Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis. Cytotechnology 36(1-3):71–83. Google Scholar
  23. Hirano M, Totani K, Fukuda T, Gu J, Suzuki A (2017) N-Glycoform-dependent interactions of megalin with its ligands. Biochim Biophys Acta 1861(1 Pt A):3106–3118. Google Scholar
  24. Hunt L, Batard P, Jordan M, Wurm FM (2002) Fluorescent proteins in animal cells for process development: optimization of sodium butyrate treatment as an example. Biotechnol Bioeng 77(5):528–537. Google Scholar
  25. ICH-Q6B (1999) Specifications: Test procedures and acceptance criteria for biotechnological/biological products Q6B. p16.
  26. Jiang H, Lopez-Aguilar A, Meng L, Gao Z, Liu Y, Tian X, Yu G, Ovryn B, Moremen KW, Wu P (2018a) Modulating cell-surface receptor signaling and ion channel functions by in situ glycan editing. Angew Chem Int Ed Eng 57(4):967–971. Google Scholar
  27. Jiang R, Chen H, Xu S (2018b) pH excursions impact CHO cell culture performance and antibody N-linked glycosylation. Bioprocess Biosyst Eng 41(12):1731–1741. Google Scholar
  28. Kang P, Mechref Y, Klouckova I, Novotny MV (2005) Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun Mass Spectrom 19(23):3421–3428. Google Scholar
  29. Kang MJ, Yu H, Kim SK, Park SR, Yang I (2011) Quantification of trace-level DNA by real-time whole genome amplification. PLoS One 6(12):e28661. Google Scholar
  30. Kaplan A, Achord DT, Sly WS (1977) Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc Natl Acad Sci U S A 74(5):2026–2030Google Scholar
  31. Kaufmann H, Mazur X, Fussenegger M, Bailey JE (1999) Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol Bioeng 63(5):573–582.<573::Aid-bit7>3.0.Co;2-y Google Scholar
  32. Ketcham SA, Ashraf M, Madhavarao CN (2017) Direct quantification of protein glycan phosphorylation. Biotechniques 63(3):117–123. Google Scholar
  33. Kimchi-Sarfaty C, Schiller T, Hamasaki-Katagiri N, Khan MA, Yanover C, Sauna ZE (2013) Building better drugs: developing and regulating engineered therapeutic proteins. Trends Pharmacol Sci 34(10):534–548. Google Scholar
  34. Kornfeld S (1990) Lysosomal enzyme targeting. Biochem Soc Trans 18(3):367–374. Google Scholar
  35. 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. Google Scholar
  36. Kunkel JP, Jan DC, Butler M, Jamieson JC (2000) Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors. Biotechnol Prog 16(3):462–470. Google Scholar
  37. Lai T, Yang Y, Ng SK (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel, Switzerland) 6(5):579–603. Google Scholar
  38. 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. Google Scholar
  39. Meikle PJ, Hopwood JJ, Clague AE, Carey WF (1999) Prevalence of lysosomal storage disorders. JAMA 281:249–254Google Scholar
  40. Mihov D, Spiess M (2015) Glycosaminoglycans: sorting determinants in intracellular protein traffic. Int J Biochem Cell Biol 68:87–91. Google Scholar
  41. Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T (1997) Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues. Cytotechnology 23(1-3):47–54. Google Scholar
  42. Neufeld E (2006) Chapter 10: Enzyme replacement therapy—a brief history. Oxford PharmaGenesis, OxfordGoogle Scholar
  43. North SJ, Huang HH, Sundaram S, Jang-Lee J, Etienne AT, Trollope A, Chalabi S, Dell A, Stanley P, Haslam SM (2010) Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J Biol Chem 285(8):5759–5775. Google Scholar
  44. Oguchi S, Saito H, Tsukahara M, Tsumura H (2006) pH condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture. Cytotechnology 52(3):199–207. Google Scholar
  45. Parenti G, Andria G, Ballabio A (2015) Lysosomal storage diseases: from pathophysiology to therapy. Annu Rev Med 66:471–486. Google Scholar
  46. Parkinson-Lawrence EJ, Shandala T, Prodoehl M, Plew R, Borlace GN, Brooks DA (2010) Lysosomal storage disease: revealing lysosomal function and physiology. Physiology (Bethesda) 25(2):102–115. Google Scholar
  47. Peake RW, Bodamer OA (2017) Newborn screening for lysosomal storage disorders. J Pediatr Genet 6(1):51–60. Google Scholar
  48. Pereira CS, Ribeiro H, Macedo MF (2017) From lysosomal storage diseases to NKT cell activation and back. Int J Mol Sci 18(3):502–515. Google Scholar
  49. Potelle S, Klein A, Foulquier F (2015) Golgi post-translational modifications and associated diseases. J Inherit Metab Dis 38(4):741–751. Google Scholar
  50. Rastall DP, Amalfitano A (2015) Recent advances in gene therapy for lysosomal storage disorders. Appl Clin Genet 8:157–169. Google Scholar
  51. Reuveny S, Velez D, Macmillan JD, Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods 86(1):53–59Google Scholar
  52. Rodriguez J, Spearman M, Huzel N, Butler M (2005) Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions. Biotechnol Prog 21(1):22–30. Google Scholar
  53. Sakurai T, Itoh K, Liu Y, Higashitsuji H, Sumitomo Y, Sakamaki K, Fujita J (2005) Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro. Exp Cell Res 309(2):264–272. Google Scholar
  54. Segatori L (2014) Impairment of homeostasis in lysosomal storage disorders. IUBMB Life 66(7):472–477. Google Scholar
  55. 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):249–257. Google Scholar
  56. Sola RJ, Griebenow K (2009) Effects of glycosylation on the stability of protein pharmaceuticals. J Pharm Sci 98(4):1223–1245. Google Scholar
  57. Sola RJ, Griebenow K (2010) Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 24(1):9–21. Google Scholar
  58. Staretz-Chacham O, Lang TC, LaMarca ME, Krasnewich D, Sidransky E (2009) Lysosomal storage disorders in the newborn. Pediatrics 123(4):1191–1207. Google Scholar
  59. Sung YH, Song YJ, Lim SW, Chung JY, Lee GM (2004) Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells. J Biotechnol 112(3):323–335. Google Scholar
  60. Sunley K, Butler M (2010) Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnol Adv 28(3):385–394. Google Scholar
  61. Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Muller D (2006) Process parameter shifting: part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng 94(6):1033–1044. Google Scholar
  62. USFDA (2014) Guidance for industry: immunogenicity assessment for therapeutic protein products. p 39.
  63. Vergara M, Becerra S, Berrios J, Osses N, Reyes J, Rodríguez-Moyá M, Gonzalez R, Altamirano C (2014) Differential effect of culture temperature and specific growth rate on CHO cell behavior in chemostat culture. PLoS One 9(4):e93865. Google Scholar
  64. Wang Y, Fang X, Cheng Y, Zhang X (2011) Manipulation of pH shift to enhance the growth and antibiotic activity of Xenorhabdus nematophila. J Biomed Biotechnol 2011:672369. Google Scholar
  65. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM (2003) Antibody neutralization and escape by HIV-1. Nature 422:307. Google Scholar
  66. WHO (2013) Guidelines on the quality, safety, and efficacy of biotherapeutic protein products prepared by recombinant DNA technology. Replacement of Annex 3 of World Health Organization Technical Report Series, No. 814.91
  67. Wilcox WR (2004) Lysosomal storage disorders: the need for better pediatric recognition and comprehensive care. J Pediatr 144(5 Suppl):3–14. Google Scholar
  68. Yoon SK, Kim SH, Lee GM (2003) Effect of low culture temperature on specific productivity and transcription level of anti-4-1BB antibody in recombinant Chinese hamster ovary cells. Biotechnol Prog 19(4):1383–1386.
  69. Zou G, Benktander JD, Gizaw ST, Gaunitz S, Novotny MV (2017) Comprehensive analytical approach toward glycomic characterization and profiling in urinary exosomes. Anal Chem 89(10):5364–5372. Google 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

  • Hamideh Parhiz
    • 1
  • Stephanie A. Ketcham
    • 1
  • Guozhang Zou
    • 2
  • Bidesh Ghosh
    • 1
  • Erica J. Fratz-Berilla
    • 2
  • Muhammad Ashraf
    • 1
  • Tongzhong Ju
    • 2
  • Chikkathur N. Madhavarao
    • 1
    • 3
    Email author
  1. 1.Office of Testing and Research, Center for Drug Evaluation and ResearchFDASilver SpringUSA
  2. 2.Office of Biotechnology Products, Center for Drug Evaluation and ResearchFDASilver SpringUSA
  3. 3.Division of Product Quality and ResearchOTR/OPQ/CDER/FDASilver SpringUSA

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