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The protein journal2023; 42(1); 24-36; doi: 10.1007/s10930-023-10092-x

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

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.
© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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Publication Date: 2023-01-18 PubMed ID: 36652139PubMed Central: 5336677DOI: 10.1007/s10930-023-10092-xGoogle Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study is about the physicochemical characterization of a recombinant equine chorionic gonadotropin (eCG), compared to commercial preparations of pregnant mare serum gonadotropin (PMSG). The research highlights the differences between the two, especially in terms of their glycosylation patterns, and emphasizes the importance of standardizing production processes.

Research Background

  • This research is centered around the hormone eCG, widely used in artificial ovulation and superovulation procedures to boost reproductive performance in livestock.
  • Until recently, the available eCG-based products for veterinary use had been partially purified versions of PMSG.
  • The study therefore aims to understand the structural and functional attributes of a bioactive recombinant eCG (reCG) that was produced in suspension CHO-K1 cells.

Methods Used

  • The recombinant eCG was purified using different chromatographic methods, including hydrophobic interaction chromatography and reverse-phase (RP)-HPLC, and then compared with a RP-HPLC-purified PMSG.
  • The researchers also executed a bioinformatics analysis to get a deeper understanding of reCG.
  • Several tests were carried out to determine the purity degree, aggregates, nicked forms, and higher order structures. Methods used included SDS-PAGE, RP-HPLC, and SEC-HPLC.
  • Glycosylation analysis was done through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC.

Research Findings

  • There were some differences found between the purified recombinant hormones. However, variations in the glycosylation pattern were observed between the recombinant molecules and PMSG.
  • Differences in sialic acid content were discovered between two commercial PMSG preparations, which could potentially lead to discrepancies in their biological potency.

Significance of the Study

  • The findings highlight the importance of standardizing production processes, especially in the bioprocess of recombinant proteins.
  • The results also underscore the significance of the glycan moieties on the structure of eCG and its biological activity, thereby preventing denaturing processes such as aggregation.

Cite This Article

APA
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C. (2023). Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations. Protein J, 42(1), 24-36. https://doi.org/10.1007/s10930-023-10092-x

Publication

The protein journal
ISSN: 1875-8355
NlmUniqueID: 101212092
Country: Netherlands
Language: English
Volume: 42
Issue: 1
Pages: 24-36

Researcher Affiliations

Rodríguez, María Celeste
  • 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. mcrodriguez@fbcb.unl.edu.ar.
Mussio, Pablo Esteban
  • 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.
Villarraza, Javier
  • 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.
Tardivo, María Belén
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
Antuña, Sebastián
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
Fontana, Diego
  • 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.
Ceaglio, Natalia
  • 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.
Prieto, Claudio
  • 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.
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
  • 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.

MeSH Terms

  • Pregnancy
  • Female
  • Animals
  • Horses
  • Gonadotropins, Equine
  • Chorionic Gonadotropin
  • Glycosylation
  • Recombinant Proteins / chemistry
  • Electrophoresis, Polyacrylamide Gel

References

This article includes 41 references
  1. Volkin DB, Sanyal G, Burke CJ, Middaugh CR. Preformulation studies as an essential guide to formulation development and manufacture of protein pharmaceuticals.. Pharm Biotechnol 2002;14:1-46.
    doi: 10.1007/978-1-4615-0549-5_1pubmed: 12189723google scholar: lookup
  2. Deechongkit S, Aoki K, Park S, Kerwin B. 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.
    doi: 10.1002/jpsgoogle scholar: lookup
  3. Murphy BD. Equine chorionic gonadotrophin: an enigmatic but essential tool. Anim Reprod 9:223–230.
  4. Legardinier S, Cahoreau C, Klett D, Combarnous Y. Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells.. Reprod Nutr Dev 2005 May-Jun;45(3):255-9.
    doi: 10.1051/rnd:2005018pubmed: 15982452google scholar: lookup
  5. Legardinier S, Duonor-Cérutti M, Devauchelle G, Combarnous Y, Cahoreau C. Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines.. J Mol Endocrinol 2005 Feb;34(1):47-60.
    doi: 10.1677/jme.1.01624pubmed: 15691877google scholar: lookup
  6. Mastrangeli R, Satwekar A, Cutillo F. 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.
    doi: 10.1371/journal.pone.0184139google scholar: lookup
  7. Casarini L, Santi D, Brigante G, Simoni M. Two Hormones for One Receptor: Evolution, Biochemistry, Actions, and Pathophysiology of LH and hCG.. Endocr Rev 2018 Oct 1;39(5):549-592.
    doi: 10.1210/er.2018-00065pubmed: 29905829google scholar: lookup
  8. Gifre L, Arís A, Bach À, Garcia-Fruitós E. Trends in recombinant protein use in animal production.. Microb Cell Fact 2017 Mar 4;16(1):40.
    doi: 10.1186/s12934-017-0654-4pubmed: 28259156pmc: 5336677google scholar: lookup
  9. Alvarez RH, Natal F, Almeida B. 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.
    doi: 10.9734/JAMPS/2017/31956google scholar: lookup
  10. Villarraza CJ, Antuña S, Tardivo MB, Rodríguez MC, Mussio P, Cattaneo L, Fontana D, Díaz PU, Ortega HH, Tríbulo A, Macagno A, Bó GA, Ceaglio N, Prieto C. Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells.. Theriogenology 2021 Sep 15;172:8-19.
    doi: 10.1016/j.theriogenology.2021.05.013pubmed: 34082223google scholar: lookup
  11. Harlow E, Lane D. Antibodies a LABORATORY MANUAL. Cold Spring Harbor Laboratory Press, New York, USA, pp 53–137.
  12. Sievers F, Higgins DG. Clustal Omega for making accurate alignments of many protein sequences.. Protein Sci 2018 Jan;27(1):135-145.
    doi: 10.1002/pro.3290pubmed: 28884485google scholar: lookup
  13. Ikai A. Thermostability and aliphatic index of globular proteins.. J Biochem 1980 Dec;88(6):1895-8.
    doi: 10.1093/oxfordjournals.jbchem.a133168pubmed: 7462208google scholar: lookup
  14. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein.. J Mol Biol 1982 May 5;157(1):105-32.
    doi: 10.1016/0022-2836(82)90515-0pubmed: 7108955google scholar: lookup
  15. Alvarez RH, Natal FL, Nogueira C, Pinto Ribela MT, de Almeida BE, de Oliveira J, Ezequiel, Bartolini P. Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin. 17:459–465.
    doi: 10.4314/ajcem.v12i3google scholar: lookup
  16. Hermentin P, Witzel R, Kanzy EJ, Diderrich G, Hoffmann D, Metzner H, Vorlop J, Haupt H. The hypothetical N-glycan charge: a number that characterizes protein glycosylation.. Glycobiology 1996 Mar;6(2):217-30.
    doi: 10.1093/glycob/6.2.217pubmed: 8727793google scholar: lookup
  17. Ribela M, Gout P, de Oliveira J, Bartolini P. HPLC Analysis of Human Pituitary Hormones for Pharmaceutical Applications. Curr Pharm Anal 2:103–126.
    doi: 10.2174/157341206776819300google scholar: lookup
  18. Murphy BD, Martinuk SD. Equine chorionic gonadotropin.. Endocr Rev 1991 Feb;12(1):27-44.
    doi: 10.1210/edrv-12-1-27pubmed: 2026120google scholar: lookup
  19. Almeida BE, Oliveira JE, Damiani R, Dalmora SL, Bartolini P, Ribela MT. 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 2011 Mar 25;54(4):681-6.
    doi: 10.1016/j.jpba.2010.10.018pubmed: 21093191google scholar: lookup
  20. Alvarez RH, Natal FLN, Almeida BE. Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin. Anim Reprod 13:750–755.
    doi: 10.21451/1984-3143-AR781google scholar: lookup
  21. Callis PR. Binding phenomena and fluorescence quenching. II: photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation. J Mol Struct 1077:22–29.
    doi: 10.1016/j.molstruc.2014.04.051google scholar: lookup
  22. Garidel P, Hegyi M, Bassarab S, Weichel M. A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy. 1201–1211.
    doi: 10.1002/biot.200800091google scholar: lookup
  23. Albrecht CJ, Lakowicz JR. Principles of Fluorescence Spectroscopy Principles of Fluorescence Spectroscopy. Anal Bioanal Chem 390.
    doi: 10.1007/s00216-007-1822-xgoogle scholar: lookup
  24. Gifre L, Arís A, Bach À, Garcia-Fruitós E. Trends in recombinant protein use in animal production. Microb Cell Fact 16:1–17.
    doi: 10.1186/s12934-017-0654-4google scholar: lookup
  25. Akash MSH, Rehman K, Tariq M, Chen S. Development of therapeutic proteins: advances and challenges. Turkish J Biology 39:343–358.
    doi: 10.3906/biy-1411-8google scholar: lookup
  26. Bousfield GR, Liu W, Ward N. Structural studies on equine glycoprotein hormones. J Biol 262:8610–8620.
  27. Hokke CH, Roosenboom MJ, Thomas-Oates JE, Kamerling JP, Vliegenthart JF. Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin.. Glycoconj J 1994 Feb;11(1):35-41.
    doi: 10.1007/BF00732430pubmed: 8193552google scholar: lookup
  28. Damm JB, Hård K, Kamerling JP, van Dedem GW, Vliegenthart JF. Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin.. Eur J Biochem 1990 Apr 20;189(1):175-83.
    doi: 10.1111/j.1432-1033.1990.tb15474.xpubmed: 2331995google scholar: lookup
  29. Min KS, Park JJ, Byambaragchaa M, Kang MH. Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice. BMC Biotechnol 19:1–9.
    doi: 10.1186/s12896-019-0550-6google scholar: lookup
  30. Legardinier S, Klett D, Poirier JC, Combarnous Y, Cahoreau C. Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells.. Glycobiology 2005 Aug;15(8):776-90.
    doi: 10.1093/glycob/cwi060pubmed: 15814822google scholar: lookup
  31. Lee S, Byambaragchaa M, Kim J. 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 SH, Joo HE, Kim SG, Kim YJ, Park GE, Kang MH, Min KS. Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/CG) Receptor.. Dev Reprod 2021 Dec;25(4):199-211.
    doi: 10.12717/dr.2021.25.4.199pubmed: 35141446pmc: 8807129google scholar: lookup
  33. Zhang L, Luo S, Zhang B. Glycan analysis of therapeutic glycoproteins.. MAbs 2016;8(2):205-15.
    doi: 10.1080/19420862.2015.1117719pubmed: 26599345google scholar: lookup
  34. Planinc A, Bones J, Dejaegher B, Van Antwerpen P, Delporte C. Glycan characterization of biopharmaceuticals: Updates and perspectives.. Anal Chim Acta 2016 May 19;921:13-27.
    doi: 10.1016/j.aca.2016.03.049pubmed: 27126786google scholar: lookup
  35. Fares F, Azzam N. Development of long-acting recombinant glycoprotein hormones by increasing the carbohydrate content.. Drug Discov Today 2019 Apr;24(4):1017-1022.
    doi: 10.1016/j.drudis.2019.01.017pubmed: 30711574google scholar: lookup
  36. Ceaglio N, Gugliotta A, Tardivo MB, Cravero D, Etcheverrigaray M, Kratje R, Oggero M. Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG β-subunit.. J Biotechnol 2016 Mar 10;221:13-24.
    doi: 10.1016/j.jbiotec.2016.01.018pubmed: 26806490google scholar: lookup
  37. Flintegaard TV, Thygesen P, Rahbek-Nielsen H, Levery SB, Kristensen C, Clausen H, Bolt G. N-glycosylation increases the circulatory half-life of human growth hormone.. Endocrinology 2010 Nov;151(11):5326-36.
    doi: 10.1210/en.2010-0574pubmed: 20826563google scholar: lookup
  38. Van Zuylen CW, De Beer T, Rademaker GJ, Haverkamp J, Thomas-Oates JE, Hård K, Kamerling JP, Vliegenthart JF. Site-specific and complete enzymic deglycosylation of the native human chorionic gonadotropin alpha-subunit.. Eur J Biochem 1995 Aug 1;231(3):754-60.
    doi: 10.1111/j.1432-1033.1995.0754d.xpubmed: 7544284google scholar: lookup
  39. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW. Crystal structure of human chorionic gonadotropin.. Nature 1994 Jun 9;369(6480):455-61.
    doi: 10.1038/369455a0pubmed: 8202136google scholar: lookup
  40. Crispo M, Meikle MN, Schlapp G, Menchaca A. Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice.. Theriogenology 2021 Apr 1;164:31-35.
    doi: 10.1016/j.theriogenology.2021.01.012pubmed: 33529809google scholar: lookup
  41. Thennati R, Singh SK, Nage N, Patel Y, Bose SK, Burade V, Ranbhor RS. Analytical characterization of recombinant hCG and comparative studies with reference product.. Biologics 2018;12:23-35.
    doi: 10.2147/BTT.S141203pubmed: 29430170pmc: 5796461google scholar: lookup

Citations

This article has been cited 6 times.
  1. Byambaragchaa M, Park SH, Park MH, Kang MH, Min KS. Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells. Biomolecules 2025 Feb 14;15(2).
    doi: 10.3390/biom15020289pubmed: 40001592google scholar: lookup
  2. Lösle M, Lin CW, Beil-Wagner J, Aebi M, Buch T. Comparison of pregnant mare serum gonadotropin products with surprising differences in protein content. Sci Rep 2025 Feb 25;15(1):6824.
    doi: 10.1038/s41598-025-90833-3pubmed: 40000800google scholar: lookup
  3. Min KS, Park SH, Jeong HR, Park JH, Byambaragchaa M. Signal Transduction of Constitutive Activating and Inactivating Eel Lutropin/Choriogonadotropin Receptor (Eel LH/CGR) Mutants by Recombinant Equine Chorionic Gonadotropin (Rec-eCG). Dev Reprod 2024 Dec;28(4):141-151.
    doi: 10.12717/DR.2024.28.4.141pubmed: 39845510google scholar: lookup
  4. Amberger L, Wagner D, Höflinger S, Zwicker F, Matzek D, Popper B. Evaluation of Synthetic GnRH-Analog Peforelin with Regard to Oocyte Differentiation and Follicular Development in C57BL/6J Mice. Animals (Basel) 2024 Oct 4;14(19).
    doi: 10.3390/ani14192866pubmed: 39409815google scholar: lookup
  5. Byambaragchaa M, Park SH, Kim SG, Shin MG, Kim SK, Park MH, Kang MH, Min KS. Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells. Int J Mol Sci 2024 Jul 2;25(13).
    doi: 10.3390/ijms25137282pubmed: 39000389google scholar: lookup
  6. Byambaragchaa M, Park SH, Kim SG, Shin MG, Kim SK, Hur SP, Park MH, Kang MH, Min KS. Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells. Curr Issues Mol Biol 2024 Jun 15;46(6):6085-6099.
    doi: 10.3390/cimb46060363pubmed: 38921034google scholar: lookup
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