Skip to main content
SpringerLink
Log in

Direct Assessment of Oligomerization of Chemically Modified Peptides and Proteins in Formulations using DLS and DOSY-NMR

  • Original Research Article
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Protein higher order structure (HOS) including the oligomer distribution can be critical for efficacy, safety and stability of drug products (DP). Oligomerization is particularly relevant to chemically modified protein therapeutics that have an extended pharmacokinetics profile. Therefore, the direct assessment of protein oligomerization in drug formulation is desired for quality assurance and control.

Methods

Here, two non-invasive methods, dynamic light scattering (DLS) and diffusion ordered spectroscopy (DOSY) NMR, were applied to measure translational diffusion coefficients (Ddls and Dnmr) of proteins in formulated drug products. The hydrodynamic molecular weights (MWhd), similar to hydrodynamic size, of protein therapeutics were derived based on a log(Ddls) vs log(MWhd) correlation model established using protein standards.

Results

An exponent value of -0.40 ± 0.01 was established for DLS measured log(D) vs. log(MWhd) using protein standards and a theoretical exponent value of -0.6 was used for unstructured polyethylene glycol (PEG) chains. The analysis of DLS derived MWhd of the primary species showed the fatty acid linked glucagon-like peptide 1 (GLP-1) was in different oligomer states, but the fatty acid linked insulin and PEG linked proteins were in monomer states. Nevertheless, equilibrium and exchange between oligomers in formulations were universal and clearly evidenced from DOSY-NMR for all drugs except peginterferon alfa-2a.

Conclusion

The correlation models of log(D) vs. log(MWhd) could be a quick and efficient way to predict MWhd of protein, which directly informs on the state of protein folding and oligomerization in formulation.

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

Similar content being viewed by others

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon request.

References

  1. Rathore N, Rajan RS. Current Perspectives on Stability of Protein Drug Products during Formulation. Fill and Finish Operations Biotechnol Progr. 2008;24(3):504–14.

    Article  CAS  Google Scholar 

  2. Patro SY, Freund E, Chang BS. Protein formulation and fill-finish operations. Biotechnology Annu Rev. 2002;8:55–84.

    Article  CAS  Google Scholar 

  3. Edelstein S. Patterns in the quinary structures of proteins. Plasticity and inequivalence of individual molecules in helical arrays of sickle cell hemoglobin and tubulin. Biophysical Journal. 1980;32(1):347–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang D, Park J, Patil SM, Smith CJ, Leazer JL, Keire DA, et al. An NMR-Based Similarity Metric for Higher Order Structure Quality Assessment Among U.S. Marketed Insulin Therapeutics. J Pharm Sci. 2020;109(4):1519–28.

    Article  CAS  PubMed  Google Scholar 

  5. Lagassé HAD, Alexaki A, Simhadri VL, Katagiri NH, Jankowski W, Sauna ZE, et al. Recent advances in (therapeutic protein) drug development. F1000research. 2017;6:113.

  6. Knudsen LB, Lau J. The Discovery and Development of Liraglutide and Semaglutide. Front Endocrinol. 2019;10:155.

    Article  Google Scholar 

  7. Molineux G. Pegfilgrastim: using pegylation technology to improve neutropenia support in cancer patients. Anti-Cancer Drugs. 2003;14(4).

  8. Harris JM, Martin NE, Modi M. Pegylation. Clin Pharmacokinet. 2001;40(7):539–51.

    Article  CAS  PubMed  Google Scholar 

  9. Werle M, Bernkop-Schnürch A. Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids. 2006;30(4):351–67.

    Article  CAS  PubMed  Google Scholar 

  10. Maikawa CL, Smith AAA, Zou L, Meis CM, Mann JL, Webber MJ, et al. Stable Monomeric Insulin Formulations Enabled by Supramolecular PEGylation of Insulin Analogues. Adv Ther. 2020;3(1):1900094.

    Article  Google Scholar 

  11. Ugwu SO, Apte SP. The effect of buffers on protein conformational stability. Pharm Technol. 2004;28(3):86–109.

    CAS  Google Scholar 

  12. Zapadka KL, Becher FJ, Uddin S, Varley PG, Bishop S, Gomes dos Santos AL, et al. A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics. Journal of the American Chemical Society. 2016;138(50):16259–65.

  13. Patil SM, Keire DA, Chen K. Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products. Aaps J. 2017;19(6):1760–6.

    Article  CAS  PubMed  Google Scholar 

  14. Frederiksen Tine M, Sønderby P, Ryberg Line A, Harris P, Bukrinski Jens T, Scharff-Poulsen Anne M, et al. Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations. Biophys J. 2015;109(6):1202–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xie T, Fang H, Ouyang W, Angart P, Chiang MJ, Bhirde AA, et al. The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability. Sci Rep. 2020;10(1):2476.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Patil SM, Li V, Peng J, Kozak D, Xu J, Cai B, et al. A Simple and Noninvasive DOSY NMR Method for Droplet Size Measurement of Intact Oil-In-Water Emulsion Drug Products. J Pharm Sci. 2019;108(2):815–20.

    Article  CAS  PubMed  Google Scholar 

  17. D’Errico G, Ortona O, Capuano F, Vitagliano V. Diffusion coefficients for the binary system glycerol+ water at 25 C. A velocity correlation study. Journal of Chemical & Engineering Data. 2004;49(6):1665–70.

    Article  CAS  Google Scholar 

  18. Young C, and Bell. Estimation of Diffusion Coefficients of Proteins. Biotechnology and Bioengineering. 1980;1980; 22(5): 947–955.

  19. Dudás EF, Bodor A. Quantitative, Diffusion NMR Based Analytical Tool To Distinguish Folded, Disordered, and Denatured Biomolecules. Anal Chem. 2019;91(8):4929–33.

    Article  PubMed  Google Scholar 

  20. Wilke CR, Chang P. Correlation of diffusion coefficients in dilute solutions. AIChE J. 1955;1(2):264–70.

    Article  CAS  Google Scholar 

  21. Flory PJ, Volkenstein M. Statistical mechanics of chain molecules. Wiley Online Library; 1969.

  22. Tanford C. Protein Denaturation. In: Anfinsen CB, Anson ML, Edsall JT, Richards FM, editors. Advances in Protein Chemistry. 23: Academic Press; 1968. p. 121–282.

  23. Approved Labeling for Firazyr (NDA 022150). (https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/022150s000lbl.pdf):Accessed on 08.12.2022.

  24. Hinton DP, Johnson CS. Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions. J Phys Chem. 1993;97(35):9064–72.

    Article  CAS  Google Scholar 

  25. Approved Labeling for Saxenda (NDA 206321). https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/206321Orig1s000lbl.pdf:Accessed on 08.12.2022.

  26. Approved Labeling for Ozempic (NDA 209637). (https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/209637lbl.pdf):Accessed on 08.12.2022.

  27. Gallo M, Vanni D, Esposito S, Alaimo N, Orvieto F, Rulli F, et al. Oligomerization, albumin binding and catabolism of therapeutic peptides in the subcutaneous compartment: An investigation on lipidated GLP-1 analogs. J Pharm Biomed Anal. 2022;210: 114566.

    Article  CAS  PubMed  Google Scholar 

  28. Approved Labeling for Tresiba (BLA 203314). (https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/203314lbl.pdf):Accessed on 08.12.2022.

  29. Approved labeling for Pegasys (BLA 103964). https://www.accessdata.fda.gov/drugsatfda_docs/label/2002/pegihof101602LB.htm:Accessed on 08.12.2022.

  30. Approved Labeling for Neulasta (BLA 125031). (https://www.accessdata.fda.gov/drugsatfda_docs/label/2002/pegfamg013102LB.pdf):Accessed on 08.12.2022.

  31. Approved Labeling for Somavert (BLA 021106). (https://www.accessdata.fda.gov/drugsatfda_docs/label/2003/21106_somavert_lbl.pdf):Accessed on 08.12.2022.

  32. Dhalluin C, Ross A, Leuthold L-A, Foser S, Gsell B, Müller F, et al. Structural and Biophysical Characterization of the 40 kDa PEG−Interferon-α2a and Its Individual Positional Isomers. Bioconjug Chem. 2005;16(3):504–17.

    Article  CAS  PubMed  Google Scholar 

  33. Hilgenfeld R, Seipke G, Berchtold H, Owens DR. The evolution of insulin glargine and its continuing contribution to diabetes care. Drugs. 2014;74(8):911–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jacobsen LV, Flint A, Olsen AK, Ingwersen SH. Liraglutide in Type 2 Diabetes Mellitus: Clinical Pharmacokinetics and Pharmacodynamics. Clin Pharmacokinet. 2016;55(6):657–72.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This project was supported, in part, by an appointment (K.W.) to the Research Participation Program at the CDER administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy and the U.S. FDA. We thank David Keire for crtical reading of the manuscript.

Author information

Authors and Affiliations

  1. Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, 20993, USA

    Kai Wang & Kang Chen

Authors
  1. Kai Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kang Chen.

Ethics declarations

Disclaimer

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

Conflicts of Interests/Competing Interests

The authors declares no confict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 201 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, K., Chen, K. Direct Assessment of Oligomerization of Chemically Modified Peptides and Proteins in Formulations using DLS and DOSY-NMR. Pharm Res 40, 1329–1339 (2023). https://doi.org/10.1007/s11095-022-03468-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-022-03468-8

Keywords

Search

Navigation