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Protein Stability and Characterization

Abstract

Throughout the development process of protein therapeutics a broad array of analytical techniques is applied on a routine basis to measure various molecular attributes. These measurements are carried out in the context of the quality control system of the product (routine monitoring of the product quality e.g. regarding stability during long-term storage) or as various characterization studies aimed at broadening the knowledge of the molecule. All stages of product development rely on analytical support: cell line and purification development, formulation development, compatibility studies, comparability exercises and also assessment of stability during production, storage and shipping.

The successful design of product control and characterization strategies to support biotherapeutic development requires intimate knowledge of protein analytical chemistry: the potential modifications that may occur in the protein molecule and the analytical techniques capable of measuring these modifications.

Keywords

  • (Physico-chemical) protein analytical techniques
  • Protein degradation
  • Protein modifications
  • Protein chemistry
  • Bioassays

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Further Reading

  • Arbogast LW, Brinson RG, Marino JP (2015) Mapping monoclonal antibody structure by 2D 13C NMR at natural abundance. Anal Chem 87(7):3556–3561

    CAS  CrossRef  Google Scholar 

  • Berkowitz SA, Engen JR, Mazzeo JR, Jones GB (2012) Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov 11(7):527–540

    CAS  CrossRef  Google Scholar 

  • Butler JE (ed) (1991) Immunochemistry of solid-phase immunoassay. CRC Press, Boca Raton

    Google Scholar 

  • Cavanagh J, Fairbrother WJ, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice book, 2nd edn. Academic Press, San Diego

    Google Scholar 

  • Chirino AJ, Mire-Sluis A (2004) Characterizing biological products and assessing comparability following manufacturing changes. Nat Biotechnol 22(11):1383–1391

    CAS  CrossRef  Google Scholar 

  • Coligan J, Dunn B, Ploegh H, Speicher D, Wingfield P (eds) (1995) Current protocols in protein science. Wiley, New York

    Google Scholar 

  • Crabb JW (ed) (1995) Techniques in protein chemistry VI. Academic, San Diego

    Google Scholar 

  • Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312(5771):212–217

    CAS  CrossRef  Google Scholar 

  • Du Y, Walsh A, Ehrick R, Xu W, May K, Liu H (2012) Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs 4(5):578–585

    CrossRef  Google Scholar 

  • Dunbar BS (1994) Protein blotting: a practical approach. Oxford University Press, New York

    Google Scholar 

  • Fekete S, Guillarme D, Sandra P, Sandra K (2016) Chromatographic, electrophoretic, and mass spectrometric methods for the analytical characterization of protein biopharmaceuticals. Anal Chem 88(1):480–507

    CAS  CrossRef  Google Scholar 

  • Folzer E, Diepold C, Bomans K, Finkler C, Schmidt R, Bulau P, Huwyler J, Mahler H-C, Koulov AV (2015) Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molecule. J Pharm Sci 104:2824–2831

    CAS  CrossRef  Google Scholar 

  • Gahoual R, Beck A, Leize-Wagner E, François YN (2016) Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products. J Chromatogr B Analyt Technol Biomed Life Sci 1032:61–78

    CAS  CrossRef  Google Scholar 

  • Hames BD, Rickwood D (eds) (1990) Gel electrophoresis of proteins: a practical approach, 2nd edn. IRL Press, New York

    Google Scholar 

  • Henderson R (2018) From electron crystallography to single particle Cryo EM (Nobel lecture). Angew Chem Int Ed Engl 57(34):10804–10825

    CAS  CrossRef  Google Scholar 

  • Jiskoot W, Crommelin DJA (eds) (2005) Methods for structural analysis of protein pharmaceuticals. AAPS Press, Arlington

    Google Scholar 

  • Kahle J, Wätzig H (2018) Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis. Electrophoresis 39(20):2492–2511

    CAS  CrossRef  Google Scholar 

  • Kaltashov IA, Bobst CE, Abzalimov RR, Wang G, Baykal B, Wang S (2012) Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol Adv 30(1):210–222

    CAS  CrossRef  Google Scholar 

  • Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J (2008) Heterogeneity of monoclonal antibodies. J Pharm Sci 97(7):2426–2447

    CAS  CrossRef  Google Scholar 

  • McEwen CN, Larsen BS (eds) (1998) Mass spectrometry of biological materials, 2nd edn. Dekker, New York

    Google Scholar 

  • Moritz B, Schnaible V, Kiessig S, Heyne A, Wild M, Finkler C, Christians S, Mueller K, Zhang L, Furuya K, Hassel M, Hamm M, Rustandi R, He Y, Solano OS, Whitmore C, Park SA, Hansen D, Santos M, Lies M (2015) Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies. J Chromatogr B Analyt Technol Biomed Life Sci 983-984:101–110

    CAS  CrossRef  Google Scholar 

  • Müllertz A, Perrie Y, Rades T (eds) (2016) Analytical techniques in the pharmaceutical sciences. Springer, New York

    Google Scholar 

  • Pace CN, Grimsley GR, Scholtz JM, Shaw KL (2014) Protein stability. In: eLS. Wiley, Chichester

    Google Scholar 

  • Parr MK, Montacir O, Montacir H (2016) Physicochemical characterization of biopharmaceuticals. J Pharm Biomed Anal 130:366–389

    CAS  CrossRef  Google Scholar 

  • Ponniah G, Nowak C, Neill A, Liu H (2017) Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels. Anal Biochem 520:49–57

    CAS  CrossRef  Google Scholar 

  • Rathore D, Faustino A, Schiel J, Pang E, Boyne M, Rogstad S (2018) The role of mass spectrometry in the characterization of biologic protein products. Expert Rev Proteomics 15(5):431–449

    CAS  CrossRef  Google Scholar 

  • Ríos Quiroz A, Lamerz J, Da Cunha T, Boillon A, Adler M, Finkler C, Huwyler J, Schmidt R, Mahler H-M, Koulov AV (2016) Factors governing the precision of subvisible particle measurement methods—a case study with a low-concentration therapeutic protein product in a prefilled syringe. Pharm Res 33:450–461

    CrossRef  Google Scholar 

  • Salas-Solano O, Felten O (2008) Capillary electrophoresis methods for pharmaceutical analysis. Sep Sci Technol 9:401

    CAS  Google Scholar 

  • Shirley BA (ed) (1995) Protein stability and folding. Humana Press, Totowa

    Google Scholar 

  • Strege MA, Lagu AL (eds) (2004) Capillary electrophoresis of proteins and peptides. Humana Press, Totowa

    Google Scholar 

  • Thorpe R, Wadhwa M, Mire-Sluis A (1997) The use of bioassays for the characterisation and control of biological therapeutic products produced by biotechnology. Dev Biol Stand 91:79–88

    CAS  PubMed  Google Scholar 

  • Vlasak J, Ionescu R (2011) Fragmentation of monoclonal antibodies. MAbs 63:253–263

    CrossRef  Google Scholar 

  • Vlasak J, Ionescu R (2008) Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Curr Pharm Biotechnol 9:468–481

    CAS  CrossRef  Google Scholar 

  • Wei H, Mo J, Tao L, Russell RJ, Tymiak AA, Chen G, Iacob RE, Engen JR (2014) Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov Today 19(1):95–102

    CAS  CrossRef  Google Scholar 

  • Wild D (ed) (2013) The immunoassay handbook theory and applications of ligand binding, ELISA and related techniques, 4th edn. Elsevier, New York

    Google Scholar 

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Acknowledgements

This chapter is a re-work of part (including a number of figures) of the chapter “Biophysical and Biochemical Analysis of Recombinant Proteins”, by Tsutomu Arakawa and John Philo, which appeared in the previous editions of this book—cf. fourth edition, 2013. Despite the significant changes made to the original chapter, it still contains fragments of the excellent original text by Arakawa and Philo.

The author thanks Abbas Razvi and Marigone Lenjani for providing chromatograms and electropherograms shown in this chapter.

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Correspondence to Atanas Koulov .

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Koulov, A. (2019). Protein Stability and Characterization. In: Crommelin, D., Sindelar, R., Meibohm, B. (eds) Pharmaceutical Biotechnology. Springer, Cham. https://doi.org/10.1007/978-3-030-00710-2_3

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