Structural Stability of Recombinant Human Growth Hormone (r-hGH) as a Function of Polymer Surface Properties

Research Paper
  • 87 Downloads

Abstract

Purpose

Despite the fact that r-hGH was first approved for use by FDA in 1995 and the conventional dosage form in the market has a limitation of daily subcutaneous injections, there remains a lack of sustained delivery system in the market. Nutropin depot, a long-acting dosage form of r-hGH was approved for marketing by FDA in 1999, however, it was discontinued in 2004. Since then, unabating efforts have been made to develop biodegradable polymer based formulations for r-hGH delivery. However, grey area is the comprehension of structural stability of r-hGH at an interface with the polymer and it is of utmost important to attain safe and efficacious sustained delivery system. The purpose of this study was to evaluate the changes in structure of r-hGH upon adsorption at biodegradable PLGA nanoparticles of different hydrophobicity as a function of pH.

Methods

DLS, fluorescence spectroscopy, and CD were collectively employed to evaluate structural changes in r-hGH.

Results

The studies revealed that r-hGH is most stable with low to high hydrophobicity PLGA grades under pH 7.2 followed by 5.3.

Conclusion

Overall, the nature and magnitude of structural changes observed has a strong dependence on the pH and differences and degree of hydrophobicity of PLGA.

KEY WORDS

circular dichroism spectroscopy (CD) dynamic light scattering (DLS) fluorescence spectroscopy nanoparticles poly (lactide-co-glycolide) (PLGA) recombinant human growth hormone (rhGH) secondary structure tertiary structure 

ABBREVIATIONS

% w/v

Percentage weight by volume

BSA

Bovine serum albumin

CD

Circular dichroism

Dh

Hydrodynamic diameter

DLS

Dynamic light scattering

g/cm3

Gram per cubic centimeter

kDa

Kilodalton

MES

2-(N-morpholine)-ethane sulphonic acid

mg

Milligram

min

Minute

ml

Milliliter

MW

Molecular weight

MWCO

Molecular weight cut off

nm

Nanometer

PDI

Polydispersity index

pI

Isoelectric point

PLGA 5050 1A

Poly (lactic-co-glycolic) acid uncapped polymer, ~ 10 kDa

PLGA 5050 5E

Poly (lactic-co-glycolic) acid ester endcapped polymer, ~ 50 kDa

PLGA 8515 3 CE

Poly (lactic-co-glycolic) acid ester endcapped polymer, ~30 kDa

PS

Polystyrene

r-hGH

Recombinant human growth hormone

SD

Standard Deviation

Trp

Tryptophan

Tyr

Tyrosine

UV

Ultraviolet

V

Volume

г

Surface Coverage

гPL

Plateau Surface Coverage

References

  1. 1.
    Cutfield WS, Derraik JGB, Gunn AJ, Reid K, Delany T, Robinson E, et al. Non-compliance with growth hormone treatment in children is common and impairs linear growth. PLoS One. 2011;6(1):e16223.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jahan ST, Haddadi A. Investigation and optimization of formulation parameters on preparation of targeted anti-CD205 tailored PLGA nanoparticles. Int J Nanomedicine. 2015;10(1):7371–84.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Castellanos IJ, Cruz G, Crespo R, Greibenow K. Encapsulation-induced aggregation and loss in activity of y-chymotrypsin and their prevention. J Control Release. 2002;81:307–19.CrossRefPubMedGoogle Scholar
  4. 4.
    Schellekens H. How to predict and prevent the immunogenicity of therapeutic proteins? Biotechnol Annu Rev. 2008;14:191–202.CrossRefPubMedGoogle Scholar
  5. 5.
    Buijs J, David WB, Hlady V. Human growth hormone adsorption kinetics and conformation on self-assembled monolayers. Langmuir. 1998;14(2):335–41.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Motheram R, Gupta PK. Behavior of Recombinant Human Growth Hormone at Solid/Liquid Interfaces. University of the Sciences in Philadelphia, ProQuest Dissertations Publishing. 2007;3264503. (These are internal data from the doctoral dissertation of a student in Usciences, not peer reviewed).Google Scholar
  7. 7.
    Kwak HH, Shim WS, Choi MK, Son MK, Kim YJ, Yang HC, et al. Development of a sustained-release recombinant human growth hormone formulation. J Control Release. 2009;137(2):160–5.CrossRefPubMedGoogle Scholar
  8. 8.
    Cai Y, Xu M, Yuan M, Liu Z, Yuan W. Developments in human growth hormone preparations: sustained-release, prolonged half-life, novel injection devices, and alternative delivery routes. Int J Nanomedicine. 2014;9(1):3527–38.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Herbert P, Murphy K, Johnson O, Dong N, Jaworowicz W, Tracy MA, et al. A large-scale process to produce microencapsulated proteins. Pharm Res. 1998;15(2):357–61.CrossRefPubMedGoogle Scholar
  10. 10.
    Lakowicz JR. Protein fluorescence. In: Lakowicz JR, editor. Principles of fluorescence spectroscopy. 2nd ed. New York: Kluwer Academic/Plenum; 1999. p. 445–86.CrossRefGoogle Scholar
  11. 11.
    Clark SR, Billsten P, Mandenius C-F, Elwing H. Fluorimetric investigation of recombinant human growth hormone adsorbed on silica nanoparticles. Anal Chim Acta. 1994;290:21–6.CrossRefGoogle Scholar
  12. 12.
    Bewley TA, Brovetto-Cruz J, Li CH. Human pituitary growth hormone. XXI. Physicochemical investigations of the native and reduced-alkylated protein. Biochemistry. 1969;8:4701–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Nutropin depot [Somatropin (rDNA origin) for injectable suspension], [package insert]. San Fransisco: Genentech, Inc. 2004. 21075_S008_annotated_OCT 2004.Google Scholar
  14. 14.
    Desai P, Gupta PK. Adsorption behavior of recombinant human growth hormone at moderately hydrophobic surfaces. University of the Sciences in Philadelphia, ProQuest Dissertations Publishing. 2010;3411393. (These are internal data from the doctoral dissertation of a student in Usciences, not peer reviewed).Google Scholar
  15. 15.
    Holzwarth G, Doty P. The ultraviolet circular dichroism of polypeptides. J Amer Chem Soc. 1965;87:218–28.CrossRefGoogle Scholar
  16. 16.
    Greenfield N, Fasman GD. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969;8:4108–16.CrossRefPubMedGoogle Scholar
  17. 17.
    Chapman D, Wallach DFH. In: Biological Membranes. Edited by Chapman D. London and N.Y: Academic Press. 1968. pp.125–202.Google Scholar
  18. 18.
    Bewley TA, Li CH. Human pituitary growth hormone: XXII. The reduction and reoxidation of the hormone. Arch Biochem Biophys. 1970;138:338–46.CrossRefPubMedGoogle Scholar
  19. 19.
    Horwitz J, Strickland EH, Billups CB. Analysis of the vibrational structure in the near-ultraviolet circular dichroism and absorption spectra of tyrosine derivatives and ribonuclease-a at 77 deg. K. J Amer Chem Soc. 1970;92:2119–29.CrossRefGoogle Scholar
  20. 20.
    Gomez-Orellana I, Variano B, Miura-Fraboni J, Milstein S, Paton DR. Thermodynamic characterization of an intermediate state of human growth hormone. Protein Sci. 1998;1:1352–8.CrossRefGoogle Scholar
  21. 21.
    Sirsi SR, Schray RC, Wheatley MA, Lutz GJ. Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained influence of polymer behaviour in organic solution on the production of polylactide nanoparticles by nanoprecipitation. Release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides. J Nanobiotechnol. 2009;7:1.CrossRefGoogle Scholar
  22. 22.
    Aloj S, Edelhoch H. The molecular properties of human growth hormone. J Biol Chem. 1972;247(4):1146–52.PubMedGoogle Scholar
  23. 23.
    Bremer MGEG, Duval J, Norde W, Lyklema J. Electrostatic interactions between immunoglobin (IgG) molecules and a charged sorbent. Colloids Surf A Physicochem Eng Asp. 2004;250:29–42.CrossRefGoogle Scholar
  24. 24.
    Demanèche S, Chapel JP, Monrozier LJ, Quiquampoix H. Dissimilar pH-dependent adsorption features of bovine serum albumin and alpha-chymotrypsin on mica probed by AFM. Colloids Surf B. 2009;70:226–31.CrossRefGoogle Scholar
  25. 25.
    Höök F, Rodahl M, Kasemo B, Brzezinski P. Proc. Structural changes in hemoglobin during adsorption to solid surfaces: effects of pH, ionic strength, and ligand binding. Natl Acad Sci USA. 1998;95:12271–6.CrossRefGoogle Scholar
  26. 26.
    Rabe M, Verdes D, Seeger S. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interf Sci. 2011 Feb 17;162(1–2):87–106.CrossRefGoogle Scholar
  27. 27.
    Pace CN, Shirley BA, McNutt M, Gajiwala K. Forces contributing to the conformational stability of proteins. FASEB J. 1996;10(1):75–83. -11CrossRefPubMedGoogle Scholar
  28. 28.
    Chantalat L, Jones ND, Korber F, Navaza J, Pavlosky AG. The crystal structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept Lett. 1995;2:333.Google Scholar
  29. 29.
    Eftink MR. Fluorescence techniques for studying protein structure. Methods Biochem Anal. 1990;35:117–29.Google Scholar
  30. 30.
    Steiner R, Kirby E. The interaction of the ground and excited states of indole derivatives with electron scavengers. J Phys Chem. 1969;73:4130–5.CrossRefPubMedGoogle Scholar
  31. 31.
    Chen Y, Liu B, Yu HT, Barkley M. The peptide bond quenches indole fluorescence. J Am Chem Soc. 1996;118:9271–8.  https://doi.org/10.1021/ja961307u. [Cross Ref]
  32. 32.
    Adams P, Chen Y, Ma K, Zagorski M, Sönnichsen F, McLaughlin M, et al. Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides. J Am Chem Soc. 2002;124:9278–86.  https://doi.org/10.1021/ja0167710.CrossRefPubMedGoogle Scholar
  33. 33.
    Ghisaidoobe ABT, Chung SJ. Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Förster resonance energy transfer techniques. Schneckenburger H, ed. Int J Mol Sci. 2014;15(12):22518–38.  https://doi.org/10.3390/ijms151222518.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Chen Y, Barkley MD. Toward understanding tryptophan fluorescence in proteins. Biochemistry. 1998;Google Scholar
  35. 35.
    Vander Donckt E. Fluorescence solvent shifts and singlet excited state pk's. of indole derivatives. Bull Soc Chim Belg. 1969;78:69–75.CrossRefGoogle Scholar
  36. 36.
    Yu H-T, Colucci WJ, McLaughlin ML, Barkley MD. Fluorescence quenching in indoles by excited-state proton transfer. J Am Chem Soc. 1992;114:8449–54.CrossRefGoogle Scholar
  37. 37.
    Chen Y, Liu B, Barkley MD. Trifluoroethanol quenches indole fluorescence by excited-state proton transfer. J Am Chem Soc. 1995;117:5608–9.CrossRefGoogle Scholar
  38. 38.
    Saito I, Sugiyama H, Yamamoto A, Muramatsu S, Matsuura T. Photochemical hydrogen-deuterium exchange reaction of tryptophan. The role in nonradioative decay of singlet tryptophan. J Am Chem Soc. 1984;106:4286–7.CrossRefGoogle Scholar
  39. 39.
    Shizuka H, Serizawa M, Kobayashi H, Kameta K, Sugiyama H, Matsuura T, et al. Excited-state behavior of tryptamine and related indoles. Remarkably efficient intramolecular proton-induced quenching. J Am Chem Soc. 1988;110:1726–32.CrossRefGoogle Scholar
  40. 40.
    Buijs J, Hlady VV. Adsorption kinetics, conformation, and mobility of the growth hormone and lysozyme on solid surfaces, studied with TIRF. J Colloid Interface Sci. 1997;190:171–81.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Vlasova IM, Zhuravleva VV, Saletsky AM. Denaturation of bovine serum albumin initiated by sodium dodecyl sulfate as monitored via the intrinsic fluorescence of the protein. Russ J Phys Chem B. 2014;8:385–90.  https://doi.org/10.1134/S1990793114030154.CrossRefGoogle Scholar
  42. 42.
    Guanghui Ma, Zhi-Guo Su, Wang L, Yang T. Microspheres and microcapsules in biotechnology: design, preparation and application. Drug Loading methods. Boca Raton: CRC Press; 2013. pp. 271. International standard book number -13: 978-981-4364-62-1(ebook-PDF).Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.University of the Sciences in PhiladelphiaPhiladelphiaUSA

Personalised recommendations