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Chapter 6: High-Throughput Conformational and Colloidal Stability Screening of Biologic Molecules

  • Peter M. IhnatEmail author
  • Jun Zhang
  • Jianwen Xu
  • Kan Wu
  • Ralf Joe Carrillo
Chapter
  • 83 Downloads
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 35)

Abstract

A variety of biophysical techniques have been developed to characterize protein drug-like properties and assess aggregation propensity to support manufacturing unit operations and long-term storage.

Conformational and colloidal mechanisms comprise the two principal pathways involved in the aggregation of proteins in solution. Either of these two pathways may dominate depending on individual protein molecule and specific formulation and stress conditions. For a high-throughput drug candidate selection and developability screening platform, it is important to assess both conformational and colloidal stability. In this study, the relationship between indicators of thermal unfolding (Tonset-DSF) and oligomer formation (Tonset-SLS or Tagg) obtained from DSF/SLS analysis and aggregation behavior at a range of concentrations were investigated and compared with aggregation results from short-term accelerated storage. Also investigated was the comparison of results between two instruments. We evaluated a randomly selected group of monoclonal antibodies (mAb) and bispecific antibodies (DVD-Ig). Both Tonset-Agg-SLS and Tonset-DSF can be determined simultaneously by differential scanning fluorimetry (DSF) coupled with static light scattering (SLS). It was revealed that the mAbs in this study with a low aggregation propensity (Δmonomer loss ≤5% at 40 °C for 21 days) display a Tonset- SLS, agg larger than 60 °C and a Tonset-DSF larger than 55 °C. Similarly, both Tonset and Tagg for bispecific antibodies with lower aggregation propensities were approximately greater than 55 °C. We found no relationship between aggregation and thermal parameters for mAbs. However, an inverse relationship between aggregation and both Tagg and Ton was evident for the DVD-Igs. Both the Abs and DVD-Igs in this study comprised sample sizes that were likely too small to identify significant relationships between parameters if any did exist. We propose displaying the data on an aggregation risk assessment diagram divided into quadrants according to severity. The boundaries are defined by rationally defined temperatures and aggregation levels informed by experimentation, prior knowledge and experience. DSF/SLS is capable of determining conformational and colloidal stability indicators simultaneously using a small amount of protein sample (~ 0.1 mg). In the early discovery stage with the limited materials, these two parameters potentially can be considered as useful indicators for high-throughput drug candidate selection and developability screening.

Keywords

IgG1 monoclonal antibody Dual variable domain immunoglobulin Bispecific antibody Differential scanning fluorimetry Static light scattering Thermal analysis Aggregation Conformational stability Colloidal stability 

Bibliography

  1. 1.
    Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market. MAbs. 2015;7:9–14.CrossRefGoogle Scholar
  2. 2.
    Siedler M, Kumar V, Chari R, Saluja S, Fraunhofer W. Development of drug product formulations: molecular design and early candidates screening. In: Quality by design for biopharmaceutical drug product development: Springer; New York. 2015. p. 61–85.Google Scholar
  3. 3.
    Morar-Mitrica S, Adams ML, Crotts G, Wurth C, Ihnat PM, Tabish T, et al. An intercompany perspective on biopharmaceutical drug product robustness studies. J Pharm Sci. 2018;107:529–42.CrossRefGoogle Scholar
  4. 4.
    Rizzo JM, Shi S, Li Y, Semple A, Esposito JJ, Yu S, et al. Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity. J Pharm Sci. 2015;104:1632–40.CrossRefGoogle Scholar
  5. 5.
    Samra HS, He F. Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies. Mol Pharm. 2012;9:696–707.CrossRefGoogle Scholar
  6. 6.
    Laue T. Proximity energies: a framework for understanding concentrated solutions. J Mol Recognit. 2012;25:165–73.CrossRefGoogle Scholar
  7. 7.
    Laboratories U. Multiple simultaneous measurements provide improved characterization of protein stability. Technical Note.Google Scholar
  8. 8.
    Laboratories U. Versatility of the multi-parameter UNIT approach in characterizing a range of protein formulations. Application Note.Google Scholar
  9. 9.
    Sotltl F DJ, Garidel P, Blech M, Breitsprecher D. Analysis of formulation dependent colloidal and conformational stability of monoclonal antibodies. In: Technologies N, ed. Application Note, 2016.Google Scholar
  10. 10.
    Brader ML, Estey T, Bai S, Alston RW, Lucas KK, Lantz S, et al. Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies. Mol Pharm. 2015;12:1005–17.CrossRefGoogle Scholar
  11. 11.
    Goldberg DS, Lewus RA, Esfandiary R, Farkas DC, Mody N, Day KJ, et al. Utility of high throughput screening techniques to predict stability of monoclonal antibody formulations during early stage development. J Pharm Sci. 2017;106:1971–7.CrossRefGoogle Scholar
  12. 12.
    Temel DB, Landsman P, Brader ML. Orthogonal methods for characterizing the unfolding of therapeutic monoclonal antibodies: differential scanning calorimetry, isothermal chemical denaturation, and intrinsic fluorescence with concomitant static light scattering. Methods Enzymol. 2016;567:359–89.CrossRefGoogle Scholar
  13. 13.
    Jakob CG, Edalji R, Judge RA, DiGiammarino E, Li Y, Gu J, et al. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig) molecule. MAbs. 2013;5:358–63.CrossRefGoogle Scholar
  14. 14.
    Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol. 2007;25:1290–7.CrossRefGoogle Scholar
  15. 15.
    Hawe A, Filipe V, Jiskoot W. Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations. Pharm Res. 2010;27:314–26.CrossRefGoogle Scholar
  16. 16.
    Lang BE, Cole KD. Differential scanning calorimetry and fluorimetry measurements of monoclonal antibodies and reference proteins: effect of scanning rate and dye selection. Biotechnol Prog. 2017;33:677–86.CrossRefGoogle Scholar
  17. 17.
    Technologies N. What information do I get from the backreflection signal? Technical Note, 2017.Google Scholar
  18. 18.
    Chi EY, Krishnan S, Kendrick BS, Chang BS, Carpenter JF, Randolph TW. Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Sci. 2003;12:903–13.CrossRefGoogle Scholar
  19. 19.
    He F, Hogan S, Latypov RF, Narhi LO, Razinkov VI. High throughput thermostability screening of monoclonal antibody formulations. J Pharm Sci. 2010;99:1707–20.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2020

Authors and Affiliations

  • Peter M. Ihnat
    • 1
    Email author
  • Jun Zhang
    • 1
  • Jianwen Xu
    • 1
  • Kan Wu
    • 1
  • Ralf Joe Carrillo
    • 1
    • 2
  1. 1.Biologics Preformulation and Drug Development, Abbvie Bioresearch CenterWorcesterUSA
  2. 2.Sterile Product and Analytical Development, Merck & Co.KenilworthUSA

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