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Techniques for Higher-Order Structure Determination

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Biophysics for Therapeutic Protein Development

Part of the book series: Biophysics for the Life Sciences ((BIOPHYS,volume 4))

Abstract

Maintaining the higher-order structure (HOS) of the protein is critical for obtaining robust processes and developing stable formulations for therapeutic proteins. Loss of HOS potentially contributes to increased aggregation, enhanced immunogenicity, and loss of function. The physical characterization of proteins requires precise analytical techniques that interrogate the primary sequence and HOSs. Techniques must provide sufficient detail to better understand the structure and integrity of protein therapeutic and assess product performance. This chapter provides an overview of a number of biophysical techniques that are commonly used to monitor HOS during biotherapeutics development. Potential applications that are discussed include absorbance, fluorescence, Fourier transform infrared (FTIR), circular dichroism (CD), vibrational optical activity (VOA), and Raman spectroscopy. In addition, calorimetric techniques, light scattering, characterization of size and shape, oligomeric state, aggregation and self-association by analytical ultracentrifugation, and rheological characterization are discussed. Special emphasis is placed on techniques that are unique with respect to their ability to monitor different degradation pathways of therapeutic proteins. Further, the chapter highlights the need for employing multiple and often complementary biophysical techniques during development since each technique offers insight into different aspects of the protein HOS.

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Acknowledgements

The authors would like to thank Dr. Lee Olszewski, Dr. Doug Nesta, Dr. Aston Liu, and Dr. Joseph Rinella at GlaxoSmithKline Biopharm Development for supporting this publication. We thank Dr. Rina K. Dukor from BioTools Inc. and Prof. Laurence A. Nafie from Syracuse University for their insights and helpful discussion.

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Authors and Affiliations

  1. Biopharmaceuticals Development, GlaxoSmithKline, 709 Swedeland Road, UW2108, King of Prussia, PA, 19406, USA

    James Kranz & Wasfi Al-Azzam

  2. TechnoPharmaSphere LLC, 822 Geddy Lane, Downingtown, PA, 19335, USA

    Fatma AlAzzam

  3. Drug Product Science & Technology, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ, 08903, USA

    Atul Saluja

  4. Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320, USA

    Juraj Svitel

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  1. James Kranz
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  2. Fatma AlAzzam
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  3. Atul Saluja
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  4. Juraj Svitel
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  5. Wasfi Al-Azzam
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Correspondence to Wasfi Al-Azzam .

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  1. , Research & Development, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, 91320, California, USA

    Linda O. Narhi

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Kranz, J., AlAzzam, F., Saluja, A., Svitel, J., Al-Azzam, W. (2013). Techniques for Higher-Order Structure Determination. In: Narhi, L. (eds) Biophysics for Therapeutic Protein Development. Biophysics for the Life Sciences, vol 4. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-4316-2_3

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