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Biophysical Techniques for Protein Size Distribution Analysis

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Biophysics for Therapeutic Protein Development

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

Abstract

This chapter discusses the biophysical methods for size distribution analysis of biopharmaceutical protein products. Protein aggregates, conformational variants, reversible self-association, fragments, and glycosylation are common contributors to size heterogeneity in protein pharmaceuticals. Size distribution analysis is essential to product understanding and for ensuring product quality during development. The choice of specific techniques for determination of size heterogeneity may not be straightforward due to a broad range of possible sizes from protein fragments to visible particles. We discuss commonly used biophysical methods for the determination of protein size distribution including size-exclusion chromatography, field-flow fractionation, sedimentation, light scattering, microscopy, light obscuration, electrical zone sensing, and mass spectrometry. The advantages and limitations of each method are also presented. A combination of several methods is often necessary to obtain a comprehensive view of size heterogeneity.

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References

  • Attri AK, Minton AP (2005) Composition gradient static light scattering: a new technique for rapid detection and quantitative characterization of reversible macromolecular hetero-­associations in solution. Anal Biochem 346:132–138

    Article  PubMed  CAS  Google Scholar 

  • Bahr U, Rohling U, Lautz C, Strupat K, Schurenberg M, Hillenkamp F (1996) A charge detector for time-of-flight mass analysis of high mass ions produced by matrix-assisted laser desorption/ionization (MALDI). J Mass Spectrom Ion Process 153:9–21

    Article  CAS  Google Scholar 

  • Banyay M, Gilstring F, Hauzenberger E, Ofverstedt LG, Eriksson AB, Krupp JJ, Larsson O (2004) Three-dimensional imaging of in situ specimens with low-dose electron tomography to analyze protein conformation. Assay Drug Dev Technol 2:561–567

    PubMed  CAS  Google Scholar 

  • Barber TA, Lannis MD, Williams JG (1989) Method evaluation: automated microscopy as a compendial test for particulates in parenteral solutions. J Parenter Sci Technol 43:27–47

    PubMed  CAS  Google Scholar 

  • Bich C, Zenobi R (2009) Mass spectrometry of large complexes. Curr Opin Struct Biol 19:632–639

    Article  PubMed  CAS  Google Scholar 

  • Burg TP, Godin M, Knudsen SM, Shen W, Carlson G, Foster JS, Babcock K, Manalis SR (2007) Weighing of biomolecules, single cells and single nanoparticles in fluid. Nature 446:1066–1069

    Article  PubMed  CAS  Google Scholar 

  • Cao S, Pollastrini J, Jiang Y (2009a) Separation and characterization of protein aggregates and particles by field flow fractionation. Curr Pharm Biotechnol 10:382–390

    Article  PubMed  CAS  Google Scholar 

  • Cao S, Jiao N, Jiang Y, Mire-Sluis A, Narhi LO (2009b) Sub-visible particle quantitation in protein therapeutics. Pharmeur Bio Sci Notes 2009:73–79

    PubMed  CAS  Google Scholar 

  • Cromwell ME, Hilario E, Jacobson F (2006a) Protein aggregation and bioprocessing. AAPS J 8:E572–E579

    Article  PubMed  CAS  Google Scholar 

  • Cromwell MEM, Felten C, Flores H, Liu J, Shire SJ (2006b) Self-association of therapeutic proteins: implications for product development. In: Murphy RM, Tsai AM (eds) Misbehaving proteins: protein (Mis)folding, aggregation, and stability. Springer, New York

    Google Scholar 

  • Engel A, Muller DJ (2000) Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol 7:715–718

    Article  PubMed  CAS  Google Scholar 

  • Fechner P, Boudier T, Mangenot S, Jaroslawski S, Sturgis JN, Scheuring S (2009) Structural information, resolution, and noise in high-resolution atomic force microscopy topographs. Biophys J 96:3822–3831

    Article  PubMed  CAS  Google Scholar 

  • Filipe V, Hawe A, Jiskoot W (2010) Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res 27:796–810

    Article  PubMed  CAS  Google Scholar 

  • Follmer C, Pereira FV, Da Silveira NP, Carlini CR (2004) Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering. Biophys Chem 111:79–87

    Article  PubMed  CAS  Google Scholar 

  • Fujishita O, Sendo T, Hisazumi A, Otsubo K, Aoyama T, Oishi R (1995) The evaluation of sizing accuracy of particle counters for parenteral drugs. PDA J Pharm Sci Technol 49:267–271

    PubMed  CAS  Google Scholar 

  • Gabrielson JP, Brader ML, Pekar AH, Mathis KB, Winter G, Carpenter JF, Randolph TW (2007a) Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J Pharm Sci 96:268–279

    Article  PubMed  CAS  Google Scholar 

  • Gabrielson JP, Randolph TW, Kendrick BS, Stoner MR (2007b) Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system. Anal Biochem 361:24–30

    Article  PubMed  CAS  Google Scholar 

  • Huang CT, Sharma D, Oma P, Krishnamurthy R (2009) Quantitation of protein particles in parenteral solutions using micro-flow imaging. J Pharm Sci 98:3058–3071

    Article  PubMed  CAS  Google Scholar 

  • Jossang T, Feder J, Rosenqvist E (1988) Photon correlation spectroscopy of human IgG. J Protein Chem 7:165–171

    Article  PubMed  CAS  Google Scholar 

  • Krutchinsky AN, Ayed A, Donald LJ, Ens W, Duckworth HW, Standing KG (2000) Studies of noncovalent complexes in an electrospray ionization/time-of-flight mass spectrometer. In: Chapman J (ed) Mass spectrometry of proteins and peptides. Humana Press, Totowa

    Google Scholar 

  • Kukrer B, Filipe V, Duijn EV, Kasper PT, Vreeken RJ, Heck AJR, Jiskoot W (2010) Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-­exclusion chromatography. Pharm Res 27:2197–2204

    Article  PubMed  Google Scholar 

  • Lee H, Kirchmeier M, Mach H (2011) Monoclonal antibody aggregation intermediates visualized by atomic force microscopy. J Pharm Sci 100:416–423

    Article  PubMed  CAS  Google Scholar 

  • Litzen A, Walter JK, Krischollek H, Wahlund KG (1993) Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography. Anal Biochem 212:469–480

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Andya JD, Shire SJ (2006) A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. AAPS J 8:E580–E589

    Article  PubMed  CAS  Google Scholar 

  • Liu H, Gaza-Bulseco G, Lundell E (2008) Assessment of antibody fragmentation by reversed-­phase liquid chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 876:13–23

    Article  PubMed  CAS  Google Scholar 

  • Mahler HC, Friess W, Grauschopf U, Kiese S (2009) Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98:2909–2934

    Article  PubMed  CAS  Google Scholar 

  • Manta B, Obal G, Ricciardi A, Pritsch O, Denicola A (2011) Tools to evaluate the conformation of protein products. Biotechnol J 6:731–741

    Article  PubMed  CAS  Google Scholar 

  • Moore JM, Patapoff TW, Cromwell ME (1999) Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor. Biochemistry 38:13960–13967

    Article  PubMed  CAS  Google Scholar 

  • Narhi LO, Jiang Y, Cao S, Benedek K, Shnek D (2009) A critical review of analytical methods for subvisible and visible particles. Curr Pharm Biotechnol 10:373–381

    Article  PubMed  CAS  Google Scholar 

  • North SJ, Hitchen PG, Haslam SM, Dell A (2009) Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr Opin Struct Biol 19:498–506

    Article  PubMed  CAS  Google Scholar 

  • Oliva A, Llabres M, Farina JB (2004) Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins. Curr Drug Discov Technol 1:229–242

    Article  PubMed  CAS  Google Scholar 

  • Pease LF 3rd, Elliott JT, Tsai DH, Zachariah MR, Tarlov MJ (2008) Determination of protein aggregation with differential mobility analysis: application to IgG antibody. Biotechnol Bioeng 101:1214–1222

    Article  PubMed  CAS  Google Scholar 

  • Philo JS (2006) Is any measurement method optimal for all aggregate sizes and types? AAPS J 8:E564–E571

    Article  PubMed  CAS  Google Scholar 

  • Philo JS (2009) A critical review of methods for size characterization of non-particulate protein aggregates. Curr Pharm Biotechnol 10:359–372

    Article  PubMed  CAS  Google Scholar 

  • Rambaldi DC, Reschiglian P, Zattoni A (2011) Flow field-flow fractionation: recent trends in protein analysis. Anal Bioanal Chem 399:1439–1447

    Article  PubMed  CAS  Google Scholar 

  • Rivas G, Stafford W, Minton AP (1999) Characterization of heterologous protein-protein interactions using analytical ultracentrifugation. Methods 19:194–212

    Article  PubMed  CAS  Google Scholar 

  • Roux KH (1999) Immunoglobulin structure and function as revealed by electron microscopy. Int Arch Allergy Immunol 120:85–99

    Article  PubMed  CAS  Google Scholar 

  • Sandin S, Öfverstedt L-G, Wikström A-C, Wrange Ö, Skoglund U (2004) Structure and flexibility of individual immunoglobulin G molecules in solution. Structure 12:409–415

    Article  PubMed  CAS  Google Scholar 

  • Sharma DK, King D, Moore PD, Oma P, Thomas D (2007) Flow microscopy for particulate analysis in parenteral and pharmaceutical fluids. Eur J Parenter Pharm Sci 12:97–101

    Google Scholar 

  • Tatford OC, Gomme PT, Bertolini J (2004) Analytical techniques for the evaluation of liquid protein therapeutics. Biotechnol Appl Biochem 40:67–81

    Article  PubMed  CAS  Google Scholar 

  • Thomson NH (2005) Imaging the substructure of antibodies with tapping-mode AFM in air: the importance of a water layer on mica. J Microsc 217:193–199

    Article  PubMed  CAS  Google Scholar 

  • Vendruscolo M (2007) Determination of conformationally heterogeneous states of proteins. Curr Opin Struct Biol 17:15–20

    Article  PubMed  CAS  Google Scholar 

  • Wang YJ, Shahrokh Z, Vemuri S, Eberlein G, Beylin I, Busch M (1996) Characterization, stability, and formulations of basic fibroblast growth factor. Pharm Biotechnol 9:141–180

    Google Scholar 

  • Wang G, Johnson AJ, Kaltashov IA (2011) Evaluation of electrospray ionization mass spectrometry as a tool for characterization of small soluble protein aggregates. Anal Chem 84:1718–1724

    Article  Google Scholar 

  • Washabaugh MW, Collins KD (1986) The systematic characterization by aqueous column chromatography of solutes which affect protein stability. J Biol Chem 261:12477–12485

    PubMed  CAS  Google Scholar 

  • Wen J, Arakawa T, Philo JS (1996) Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem 240:155–166

    Article  PubMed  CAS  Google Scholar 

  • Wenzel RJ, Matter U, Schutheis L, Zenobi R (2005) Analysis of megadalton ions using cryodetection MALDI time-of-flight mass spectrometry. Anal Chem 77:4329–4337

    Article  PubMed  CAS  Google Scholar 

  • Wu G, Hummel D, Herman A (1997) Analysis of the solution behavior of protein pharmaceuticals by laser light scattering photometry. ACS Symp Ser 675:168–185

    Article  CAS  Google Scholar 

  • Wuchner K, Buchler J, Spycher R, Dalmonte P, Volkin DB (2010) Development of a microflow digital imaging assay to characterize protein particulates during storage of a high concentration IgG1 monoclonal antibody formulation. J Pharm Sci 99:3343–3361

    Article  PubMed  CAS  Google Scholar 

  • Yohannes G, Jussila M, Hartonen K, Riekkola ML (2011) Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles. J Chromatogr A 1218:4104–4116

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Analytical Development, Novavax, Rockville, MD, 20850, USA

    Ziping Wei

  2. Biopharmaceutical Development, Medimmune, 1 Medimmune Way, Gaithersburg, MD, 20878, USA

    Alla Polozova

Authors
  1. Ziping Wei
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  2. Alla Polozova
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Correspondence to Alla Polozova .

Editor information

Editors and Affiliations

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

    Linda O. Narhi

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Wei, Z., Polozova, A. (2013). Biophysical Techniques for Protein Size Distribution Analysis. 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_4

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