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Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations

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Abstract

Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.

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References

  1. Volkin DB, Sanyal G, Burke CJ, Middaugh CR (2002) Preformulation studies as an essential guide to formulation development and manufacture of protein pharmaceuticals. Pharm Biotechnol 14:1–46

    Article  CAS  PubMed  Google Scholar 

  2. Deechongkit S, Aoki K, Park S, Kerwin B (2006) Biophysical comparability of the same protein from different manufacturers: a Case Study using Epoetin Alfa from Epogen1 and Eprex1. Int J Drug Dev Res 95:1931–1943. https://doi.org/10.1002/jps

    Article  CAS  Google Scholar 

  3. Murphy BD (2012) Equine chorionic gonadotrophin: an enigmatic but essential tool. Anim Reprod 9:223–230

    Google Scholar 

  4. Legardinier S, Cahoreau C, Klett D, Combarnous Y (2005) Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells. Reprod Nutr Dev 45:255–259. https://doi.org/10.1051/rnd:2005018

    Article  CAS  PubMed  Google Scholar 

  5. Legardinier S, Duonor-Cérutti M, Devauchelle G et al (2005) Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and mimic insect cell lines. J Mol Endocrinol 34:47–60. https://doi.org/10.1677/jme.1.01624

    Article  CAS  PubMed  Google Scholar 

  6. Mastrangeli R, Satwekar A, Cutillo F et al (2017) In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f). PLoS ONE 12:1–15. https://doi.org/10.1371/journal.pone.0184139

    Article  CAS  Google Scholar 

  7. Casarini L, Santi D, Brigante G, Simoni M (2018) Two hormones for one receptor: evolution, biochemistry, actions, and pathophysiology of LH and hCG. Endocr Rev 39:549–592

    Article  PubMed  Google Scholar 

  8. Gifre L, Arís A, Bach À, Garcia-Fruitós E (2017) Trends in recombinant protein use in animal production. Microb Cell Fact 16:40. https://doi.org/10.1186/s12934-017-0654-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Alvarez RH, Natal F, Almeida B et al (2017) Biological Activity of different batches of equine Chorionic Gonadotropin as determined by reversed-phase high-performance Liquid Chromatography and in vivo assay. J Adv Med Pharm Sci 12:1–9. https://doi.org/10.9734/JAMPS/2017/31956

    Article  Google Scholar 

  10. Villarraza CJ, Antuña S, Tardivo MB et al (2021) Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells. Theriogenology 172:8–19. https://doi.org/10.1016/j.theriogenology.2021.05.013

    Article  CAS  PubMed  Google Scholar 

  11. Harlow E, Lane D (1988) Antibodies a LABORATORY MANUAL. Cold Spring Harbor Laboratory Press, New York, USA, pp 53–137

    Google Scholar 

  12. Sievers F, Higgins DG (2018) Clustal Omega for making accurate alignments of many protein sequences. Protein Sci 27:135–145. https://doi.org/10.1002/pro.3290

    Article  CAS  PubMed  Google Scholar 

  13. Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898. https://doi.org/10.1093/oxfordjournals.jbchem.a133168

    Article  CAS  PubMed  Google Scholar 

  14. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132. https://doi.org/10.1016/0022-2836(82)90515-0

    Article  CAS  PubMed  Google Scholar 

  15. Alvarez RH, Natal FL, Nogueira C, Pinto Ribela MT, de Almeida BE, de Oliveira J, Ezequiel, Bartolini P (2016) Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin. 17:459–465. https://doi.org/10.4314/ajcem.v12i3

  16. Hermentin P, Witzel R, Kanzy EJ et al (1996) The hypothetical N-glycan charge: a number that characterizes protein glycosylation. Glycobiology 6:217–230. https://doi.org/10.1093/glycob/6.2.217

    Article  CAS  PubMed  Google Scholar 

  17. Ribela M, Gout P, de Oliveira J, Bartolini P (2006) HPLC Analysis of Human Pituitary Hormones for Pharmaceutical Applications. Curr Pharm Anal 2:103–126. https://doi.org/10.2174/157341206776819300

    Article  CAS  Google Scholar 

  18. Murphy BD, Martinuk SD (1991) Equine chorionic gonadotropin. Endocr Rev 12:27–44. https://doi.org/10.1210/edrv-12-1-27

    Article  CAS  PubMed  Google Scholar 

  19. Almeida BE, Oliveira JE, Damiani R et al (2011) A pilot study on potency determination of human follicle-stimulating hormone: a comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay. J Pharm Biomed Anal 54:681–686. https://doi.org/10.1016/j.jpba.2010.10.018

    Article  CAS  PubMed  Google Scholar 

  20. Alvarez RH, Natal FLN, Almeida BE et al (2016) Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin. Anim Reprod 13:750–755. https://doi.org/10.21451/1984-3143-AR781

    Article  Google Scholar 

  21. Callis PR (2014) Binding phenomena and fluorescence quenching. II: photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation. J Mol Struct 1077:22–29. https://doi.org/10.1016/j.molstruc.2014.04.051

    Article  CAS  Google Scholar 

  22. Garidel P, Hegyi M, Bassarab S, Weichel M (2008) A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy.1201–1211. https://doi.org/10.1002/biot.200800091

  23. Albrecht CJ, Lakowicz JR (2008) Principles of Fluorescence Spectroscopy Principles of Fluorescence Spectroscopy. 3rd edition. Anal Bioanal Chem 390 https://doi.org/10.1007/s00216-007-1822-x

  24. Gifre L, Arís A, Bach À, Garcia-Fruitós E (2017) Trends in recombinant protein use in animal production. Microb Cell Fact 16:1–17. https://doi.org/10.1186/s12934-017-0654-4

    Article  CAS  Google Scholar 

  25. Akash MSH, Rehman K, Tariq M, Chen S (2015) Development of therapeutic proteins: advances and challenges. Turkish J Biology 39:343–358. https://doi.org/10.3906/biy-1411-8

    Article  CAS  Google Scholar 

  26. Bousfield GR, Liu W, Ward N (1987) Structural studies on equine glycoprotein hormones. J Biol 262:8610–8620

    CAS  Google Scholar 

  27. Hokke CH, Roosenboom MJH, Thomas-Oates JE et al (1994) Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin. Glycoconj J 11:35–41. https://doi.org/10.1007/BF00732430

    Article  CAS  PubMed  Google Scholar 

  28. DAMM JBL, HÅRD K, KAMERLING JP et al (1990) Structure determination of the major N- and O‐linked carbohydrate chains of the β subunit from equine chorionic gondotropin. Eur J Biochem 189:175–183. https://doi.org/10.1111/j.1432-1033.1990.tb15474.x

    Article  CAS  PubMed  Google Scholar 

  29. Min KS, Park JJ, Byambaragchaa M, Kang MH (2019) Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice. BMC Biotechnol 19:1–9. https://doi.org/10.1186/s12896-019-0550-6

    Article  CAS  Google Scholar 

  30. Legardinier S, Klett D, Poirier JC et al (2005) Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic™ insect cells. Glycobiology 15:776–790. https://doi.org/10.1093/glycob/cwi060

    Article  CAS  PubMed  Google Scholar 

  31. Lee S, Byambaragchaa M, Kim J et al (2017) Biochemical Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG), Using CHO Cells and PathHunter Parental Cells Expressing Equine Luteinizing Hormone /Chorionic Gonadotropin Receptors (eLH/CGR). 27:864–872

  32. Byambaragchaa M, Choi S-H, Joo H-E et al (2021) Specific Biological activity of equine Chorionic Gonadotropin (eCG) glycosylation Sites in cells expressing equine luteinizing Hormone/CG (eLH/CG) receptor. Dev Reprod 25:199–211. https://doi.org/10.12717/dr.2021.25.4.199

    Article  PubMed  PubMed Central  Google Scholar 

  33. Zhang L, Luo S, Zhang B (2016) Glycan analysis of therapeutic glycoproteins. MAbs 8:205–215. https://doi.org/10.1080/19420862.2015.1117719

    Article  CAS  PubMed  Google Scholar 

  34. Planinc A, Bones J, Dejaegher B et al (2016) Glycan characterization of biopharmaceuticals: updates and perspectives. Anal Chim Acta 921:13–27

    Article  CAS  PubMed  Google Scholar 

  35. Fares F, Azzam N (2019) Development of long-acting recombinant glycoprotein hormones by increasing the carbohydrate content. Drug Discov Today 24:1017–1022. https://doi.org/10.1016/j.drudis.2019.01.017

    Article  CAS  PubMed  Google Scholar 

  36. Ceaglio N, Gugliotta A, Tardivo MB et al (2016) Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG β-subunit. J Biotechnol 221:13–24. https://doi.org/10.1016/j.jbiotec.2016.01.018

    Article  CAS  PubMed  Google Scholar 

  37. Flintegaard T, Thygesen P, Rahbek-Nielsen H et al (2010) N-glycosylation increases the circulatory half-life of human growth hormone. Endocrinology 151:5326–5336. https://doi.org/10.1210/en.2010-0574

    Article  CAS  PubMed  Google Scholar 

  38. van Zuylen CWEM, de Beer T, Rademaker GJ et al (1995) Site-specific and complete enzymic deglycosylation of the native human chorionic gonadotropin α‐Subunit. Eur J Biochem 231:754–760. https://doi.org/10.1111/j.1432-1033.1995.0754d.x

    Article  PubMed  Google Scholar 

  39. Lapthorn AJ, Harris DC, Littlejohn A et al (1994) Crystal structure of human chorionic gonadotropin. Nature 369:455–461. https://doi.org/10.1038/369455a0

    Article  CAS  PubMed  Google Scholar 

  40. Crispo M, Meikle MN, Schlapp G, Menchaca A (2021) Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice. Theriogenology 164:31–35. https://doi.org/10.1016/j.theriogenology.2021.01.012

    Article  CAS  PubMed  Google Scholar 

  41. Thennati R, Singh SK, Nage N et al (2018) Analytical characterization of recombinant hCG and comparative studies with reference product. Biologics 12:23–35. https://doi.org/10.2147/BTT.S141203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina

    María Celeste Rodríguez, Pablo Esteban Mussio, Javier Villarraza, Diego Fontana & Natalia Ceaglio

  2. UNL, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Biotechnological Development Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina

    Claudio Prieto

  3. Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina

    María Belén Tardivo, Sebastián Antuña & Claudio Prieto

  4. Cellargen Biotech SRL, FBCB (School of Biochemistry and Biological Sciences), Biotechnological Development Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina

    Claudio Prieto

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  1. María Celeste Rodríguez
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Contributions

JV, DF and SA carried out the production of reCG in prefusion bioreactor; MBT carried out the first purification stage (the started material); JV performed the bioinformatic analysis; MCR and PEM designed and performed the experiments; MCR and NC wrote the manuscript. CP revised the manuscript. All the authors approved the manuscript.

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Correspondence to María Celeste Rodríguez.

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Rodríguez, M.C., Mussio, P.E., Villarraza, J. et al. Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations. Protein J 42, 24–36 (2023). https://doi.org/10.1007/s10930-023-10092-x

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