Skip to main content
SpringerLink
Log in

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability.

Methods

The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram.

Results

The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA.

Conclusions

These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Abbreviations

ANS:

8-Anilino-1-Naphthalenesulfonic acid

BSA:

Bovine serum albumin

DLS:

Dynamic light scattering

EPD:

Empirical phase diagram

FACS:

Flow cytometry

FSC:

Forward scatter

NAPA:

N-acetyl-L-phenylalanine amide

NATA:

N-acetyl-L-tryptophanamide

NAYA:

N-acetyl-L-tyrosinamide

OD350nm :

Optical density at a wavelength of 350 nm

OVA:

Ovalbumin

SSC:

Side scatter

References

  1. Micklus A, Muntner S. Deal watch: Biopharma deal-making in 2015: changing the pharma landscape. Nat Rev Drug Discov. 2016;15(2):78–9.

    Article  CAS  PubMed  Google Scholar 

  2. Topp EM. Commentary: current perspectives on the aggregation of protein drugs. AAPS J. 2014;16(3):413–4.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Rawat A, Burgess DJ. Parenteral Delivery of Peptideds and proteins. In: Morishita M, Park K, editors. Biodrug Delivery systems: fundamentals, applications and clinical development. New York: Informa Healthcare; 2009. p. 50–68.

    Google Scholar 

  4. Ezan E. Pharmacokinetic studies of protein drugs: past, present and future. Adv Drug Deliv Rev. 2013;65(8):1065–72.

    Article  CAS  PubMed  Google Scholar 

  5. Joubert MK, Hokom M, Eakin C. Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses. J Biol Chem. 2012;287(30):25266–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chaudhuri R, Cheng Y, Middaugh CR, Volkin DB. High-throughput biophysical analysis of protein therapeutics to examine interrelationships between aggregate formation and conformational stability. AAPS J. 2014;16(1):48–64.

    Article  CAS  PubMed  Google Scholar 

  7. Telikepalli SN, Kumru OS, Kalonia C, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Structural characterization of IgG1 mAb aggregates and particles generated under various stress conditions. J Pharm Sci. 2014;103(3):796–809.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kiese S, Papppenberger A, Friess W, Mahler HC. Shaken, not stirred: mechanical stress testing of an IgG1 antibody. J Pharm Sci. 2008;97(10):4347–66.

    Article  CAS  PubMed  Google Scholar 

  9. Wang W. Advanced protein formulations. Protein Sci. 2015;24(7):1031–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hamrang Z, Rattray NJW, Pluen A. Proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation. Trends Biotechnol. 2013;31(8):448–56.

    Article  CAS  PubMed  Google Scholar 

  11. Mensink MA, Nethercott MJ, Hinrichs WLJ. Influence of miscibility of protein-sugar Lyophilizates on their storage stability. AAPS J. 2016;18(5):1225–32.

    Article  CAS  PubMed  Google Scholar 

  12. Platts L, Falconer RJ. Controlling protein stability: mechanisms revealed using formulations of arginine, glycine and guanidinium HCl with three globular proteins. Int J Pharm. 2015;486(1–2):131–5.

    Article  CAS  PubMed  Google Scholar 

  13. Lim JY, Kim NA, Lim DG, Eun CY, Choi D, Jeong SH. Biophysical stability of hyFc fusion protein with regards to buffers and various excipients. Int J Biol Macromol. 2016;86:622–9.

    Article  CAS  PubMed  Google Scholar 

  14. Thakkar SV, Joshi SB, Jones ME, Sathish HA, Bishop SM, Volkin DB, Middaugh CR. Excipients differentially influence the conformational stability and Pretransition dynamics of two IgG1 Monoclonal antibodies. J Pharm Sci. 2012;101(9):3062–77.

    Article  CAS  PubMed  Google Scholar 

  15. Abbas SA, Sharma VK, Patapoff TW, Kalonia DS. Characterization of antibody-polyol interactions by static light scattering: implications for physical stability of protein formulations. Int J Pharm. 2013;448(2):382–9.

    Article  CAS  PubMed  Google Scholar 

  16. Timasheff SN. Control of protein stability and reactions by weakly interacting Cosolvents: the simplicity of the complicated. Adv Protein Chem. 1998;51:355–428.

    Article  CAS  PubMed  Google Scholar 

  17. Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011;63(13):1118–59.

    Article  CAS  PubMed  Google Scholar 

  18. Arakawa T, Timasheff SN. Stabilization of protein structure by sugars. Biochemist. 1982;21(25):6536–44.

    Article  CAS  Google Scholar 

  19. Abbas SA, Sharma VK, Patapoff TW, Kalonia DS. Opposite effects of Polyols on antibody aggregation: thermal Versus mechanical stresses. Pharm Res. 2012;29(3):683–94.

    Article  CAS  PubMed  Google Scholar 

  20. Ohtake S, Kita Y, Arakawa T. Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev. 2011;63(13):1053–73.

    Article  CAS  PubMed  Google Scholar 

  21. Fukuda M, Kameoka D, Torizawa T, Saitoh S, Yasutake M, Imaeda Y, Koga A, Mizutani A. Thermodynamic and fluorescence analyses to determine mechanisms of IgG1 stabilization and destabilization by arginine. Pharm Res. 2014;31(4):992–1001.

    Article  CAS  PubMed  Google Scholar 

  22. Vagenende V, Han AX, Mueller M, Trout BL. Protein-associated Cation clusters in aqueous arginine solutions and their effects on protein stability and size. ACS Chem Biol. 2013;8(2):416–22.

    Article  CAS  PubMed  Google Scholar 

  23. Jing Z, Arya MB, James SN. A hydrophobic surface is essential to inhibit the aggregation of a tau-protein-derived Hexapeptide. J Am Chem Soc. 2013;135(18):6846–52.

    Article  Google Scholar 

  24. Filipe V, Hawe A, Carpenter JF, Jiskoot W. Analytical approaches to assess the degradation of therapeutic proteins. Trends Anal Chem. 2013;49:118–25.

    Article  CAS  Google Scholar 

  25. Roberts D, Keeling R, Tracka M. Van der walle CF, Uddin S, Warwickert J, Curtis R. The role of electrostatics in protein-protein interactions of a Monoclonal antibody. Mol Pharm. 2014;11(7):2475–89.

    Article  CAS  PubMed  Google Scholar 

  26. Nishi H, Mathäs R, Fürst R, Winter G. Label-free flow Cytometry analysis of Subvisible aggregates in Liquid IgG1 antibody formulations. J Pharm Sci. 2014;103(1):90–9.

    Article  CAS  PubMed  Google Scholar 

  27. Alsenaidy MA, Jain NK, Kim JH, Middaugh CR, Volkin DB. Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles. Front Pharmacol. 2014;5:1–19.

    Article  CAS  Google Scholar 

  28. Joshi SB, Bhambhani A, Zeng YH, Middaugh CR. An empirical phase diagram-high throughput screening approach to the characterization and formulation of biopharmaceuticals. In: Jameel F, Hershenson S, editors. Formulation and process development strategies for manufacturing biopharmaceuticals. New Jersey: Wiley; 2010. p. 174–201.

    Google Scholar 

  29. Arakawa T, Ejima D, Tsumoto K. Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects. Biophys Chem. 2007;127(1–2):1–8.

    Article  CAS  PubMed  Google Scholar 

  30. Laurence JS, Middaugh CR. Fundamental structures and behaviors of proteins. In: Wang W, Roberts CJ, editors. Aggregation of therapeutic proteins. New Jersey: Wiley; 2010. p. 9–16.

    Google Scholar 

  31. Wen LL, Chen Y, Liao J, Zheng XX, Yin ZN. Preferential interactions between protein and arginine: effects of arginine on tertiary conformational and colloidal stability of protein solution. Int J Pharm. 2015;478(2):753–61.

    Article  CAS  PubMed  Google Scholar 

  32. Borzova VA, Markossian KA, Muranov KO, Polyansky NB, Kleymenov SY, Kurganov BI. Quantification of anti-aggregation activity of UV-irradiated-crystallin. Int J Biol Macromol. 2015;73:84–91.

    Article  CAS  PubMed  Google Scholar 

  33. Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res. 2007;25(7):1487–99.

    Article  Google Scholar 

  34. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98(9):2909–34.

    Article  CAS  PubMed  Google Scholar 

  35. Wang Y, Latypov RF, Lomakin A, Meyer JA, Kerwin BA, Vunnum S, Benedek GB. Quantitative evaluation of colloidal stability of antibody solutions using PEG-induced Liquid−Liquid phase separation. Mol Pharm. 2014;11(5):1391–402.

    Article  CAS  PubMed  Google Scholar 

  36. Saito S, Hasegawa J, Kobayashi N, Uchiyama S, Fukui K. Effects of ionic strength and sugars on the aggregation propensity of Monoclonal antibodies: influence of colloidal and conformational stabilities. Pharm Res. 2013;30(5):1263–80.

    Article  CAS  PubMed  Google Scholar 

  37. Lubich C, Malisauskas M, Prenninger T, Wurz T, Matthiessen P, Turecek PL, Scheiflinger F, Reipert BM. A flow-Cytometry-based approach to facilitate quantification, size estimation and characterization of sub-visible particles in protein solutions. Pharm Res. 2015;32(9):2863–76.

    Article  CAS  PubMed  Google Scholar 

  38. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003;20(9):1325–36.

    Article  CAS  PubMed  Google Scholar 

  39. Shukla D, Schneider CP, Trout BL. Molecular level insight into intra-solvent interaction effects on protein stability and aggregation. Adv Drug Deliv Rev. 2011;63(13):1074–85.

    Article  CAS  PubMed  Google Scholar 

  40. Charman SA, Mason KL, Charman WN. Techniques for assessing the effects of pharmaceutical excipients on the aggregation of porcine growth hormone. Pharm Res. 1993;10(7):954–62.

    Article  CAS  PubMed  Google Scholar 

  41. Serno T, Carprnter JF, Randolph TW, Winter G. Inhibition of agitation-induced aggregation of a IgG-antibody by Hydroxypropyl-β-Cyclodextrin. J Pharm Sci. 2010;99(3):1193–206.

    Article  CAS  PubMed  Google Scholar 

  42. Eronina TB, Chebotareva NA, Sluchanko NN, Mikhaylova VV, Makeeva VF, Roman SG, Kleymenov SY, Kurganov BI. Dual effect of arginine on aggregation of phosphorylase kinase. Int J Biol Macromol. 2014;68:225–32.

    Article  CAS  PubMed  Google Scholar 

  43. Burstein EA, Vedenkina NS, Ivkova MN. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973;18:263–79.

    Article  CAS  PubMed  Google Scholar 

  44. Haskard CA, Li-Chan ECY. Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes. J Agric Food Chem. 1998;46(7):2671–7.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3 Southern Renmin Road, Chengdu, Sichuan Province, 610041, People’s Republic of China

    Lili Wen, Xianxian Zheng, Xinyue Wang, Hairong Lan & Zongning Yin

Authors
  1. Lili Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xianxian Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xinyue Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hairong Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zongning Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zongning Yin.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wen, L., Zheng, X., Wang, X. et al. Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein. Pharm Res 34, 1378–1390 (2017). https://doi.org/10.1007/s11095-017-2152-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-017-2152-0

Key words

Search

Navigation