Abstract
Protein aggregation is a common biological phenomenon which is responsible for degenerative diseases and is problematic in the pharmaceutical industry. According to the rules provided by regulatory agencies, industry is supposed to assess the product quality regarding the presence of subvisible particles. Also, they should evaluate the technologies that are used to measure these particles. Therefore, US FDA and industry have been looking for methods capable of accurately characterizing the protein products. Four sizing techniques reviewed here are good candidates to be used for characterization of protein and their aggregates: dynamic light scattering, size-exclusion chromatography, electron microscopy and Taylor dispersion analysis. The first three are more established techniques while the last one is a more recent and growing technique.
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1 . An Introduction to Medicinal Chemistry. Oxford University Press, Oxford, UK (2013).
- 2 . Protein aggregation and its impact on product quality. Curr. Opin. Biotechnol. 30, 211–217 (2014).
- 3 . Role of protein aggregation. In: Neurodegenerative Diseases. Kishore U (Ed.). InTech Publishing, Croatia (2013).
- 4 . Toward understanding driving forces in membrane protein folding. Arch. Biochem. Biophys. 564, 297–313 (2014).
- 5 . Cellular strategies for controlling protein aggregation. Nat. Rev. Mol. Cell Biol. 11(11), 777–788 (2010).
- 6 . Protein aggregation and degradation mechanisms in neurodegenerative diseases. Am. J. Neurodegener. Dis. 2(1), 1–14 (2013).
- 7 . Introduction to current and future protein therapeutics: a protein engineering perspective. Exp. Cell Res. 317(9), 1261–1269 (2011).
- 8 Wang W, Roberts CJ (Eds). Aggregation of Therapeutic Proteins. John Wiley & Sons, Inc., Hoboken, NJ, USA (2010).
- 9 . Biopharmaceutical benchmarks 2014. Nat. Biotech. 32, 992–1000 (2014).
- 10 . Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Disc. 7(1), 21–39 (2008).
- 11 . Potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatography: Essential need to use orthogonal methods to assure the quality of therapeutic protein products. J. Pharm. Sci. 99(5), 2200–2208 (2010).
- 12 . Drugs: From Discovery to Approval. John Wiley & Sons, Inc., NJ, USA (2009).
- 13 . Simultaneous detection and analysis of protein aggregation and protein unfolding by size exclusion chromatography with post column addition of the fluorescent dye BisANS. J. Pharm. Sci. 101(2), 826–837 (2012).
- 14 . Aggregates in monoclonal antibody manufacturing processes. Biotechnol. Bioeng. 108(7), 1494–1508 (2011).
- 15 . Protein aggregation, particle formation, characterization & rheology. Curr. Opin. Colloid Interface Sci. 19(5), 438–449 (2014).
- 16 . Techniques for monitoring protein misfolding and aggregation in vitro and in living cells. Korean J. Chem. Eng. 29(6), 693–702 (2012).
- 17 . Classification of protein aggregates. J. Pharm. Sci. 101(2), 493–498 (2012).
- 18 . A critical review of methods for size characterization of non-particulate protein aggregates. Curr. Pharm. Biotechnol. 10(4), 359–372 (2009).
- 19 Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm. Res. 28(4), 920–933 (2011).
- 20 . Unraveling the early events of amyloid-β protein (Aβ) aggregation: Techniques for the determination of Aβ aggregate size. Int. J. Mol. Sci. 13(3), 3038–3072 (2012).
- 21 . Light scattering as spectroscopic tool for the study of disperse systems useful in pharmaceutical science. J. Pharm. Sci. 97(5), 1703–1730 (2008).
- 22 A colloidal silica reference material for nanoparticle sizing by means of dynamic light scattering and centrifugal liquid sedimentation. Part. Part. Syst. Charact. 27(3–4), 112–124 (2010).
- 23 . In vivo quantum-dot toxicity assessment. Small 6(1), 138–144 (2010).
- 24 . On the measurement of gold nanoparticle sizes by the dynamic light scattering Method. Colloid J. 73(1), 118–127 (2011).
- 25 . Hydrodynamic radius of polyethylene glycol in solution obtained by dynamic light scattering. Colloid J. 72(2), 279–281 (2010).
- 26 Lipid nanoparticle vectorization of indocyanine green improves fluorescence imaging for tumor diagnosis and lymph node resection. J. Biomed. Nanotechnol. 8(5), 730–741 (2012).
- 27 . Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics. Wiley-Interscience, New York, NY, USA (1976).
- 28 Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol. In Vitro 23(5), 927–934 (2009).
- 29 . Light Scattering, Size Exclusion Chromatography, and Asymmetric Flow Field Flow Fractionation: Powerful Tools for the Characterization of Polymers, Proteins, and Nanoparticles. John Wiley, Hoboken, NJ, USA (2011).
- 30 . Making sense of brownian motion: colloid characterization by dynamic light scattering. Langmuir 31(1), 3–12 (2015). •• Gives a comprehensive overview for dynamic light scattering (theory and application).
- 31 . Protein analysis by dynamic light scattering: methods and techniques for students. Biochem. Mol. Biol. Edu. 40(6), 372–382 (2012).
- 32 . A modified Stokes–Einstein equation for Abeta aggregation. BMC Bioinform. 12, S13 (2011).
- 33 . Instant Notes in Physical Chemistry. Bios Scientific Publishers, Milton Park, UK (2000).
- 34 . Measuring the lateral size of liquid-exfoliated nanosheets with dynamic light scattering. Nanotechnology 24(26), 265703 (2013).
- 35 . Monitoring protein aggregation kinetics with simultaneous multiple sample light scattering. Anal. Biochem. 437(2), 185–197 (2013).
- 36 . The mysterious unfoldome: structureless, underappreciated, yet vital part of any given proteome. J. Biomed. Biotechnol. 2010, 568068 (2010).
- 37 . Instrumental Analysis Of Intrinsically Disordered Proteins: Assessing Structure And Conformation. John Wiley & Sons, Hoboken, NJ, USA (2010).
- 38 . Dynamic and static light scattering of intrinsically disordered proteins. Methods Mol. Biol. 896, 137–161 (2012).
- 39 . Introduction to protein crystallization. Acta Crystallogr. F. Struct. Biol. Commun. 70, 2–20 (2014).
- 40 . Application of dynamic light scattering in protein crystallization. Curr. Protoc. Protein Sci. 17(17), 10 (2010).
- 41 . High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Anal. Biochem. 399(1), 141–143 (2010).
- 42 . Measuring the hydrodynamic size of nanoparticles in aqueous media using batch-mode dynamic light scattering. Methods Mol. Biol. 697, 35–52 (2011).
- 43 . Accuracy of protein size estimates based on light scattering measurements. Open J. Biophys. 4, 83–91 (2014).
- 44 . Viscosity behavior of high-concentration protein mixtures. J. Pharm. Sci. 101(3), 1012–1020 (2012).
- 45 . Utilizing dynamic light scattering as a process analytical technology for protein folding and aggregation monitoring in vaccine manufacturing. J. Pharm. Sci. 102(12), 4284–4290 (2013). •• Is a good example of using dynamic light scattering for protein aggregation studies.
- 46 . Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study. Int. J. Biol. Macromol. 53, 133–137 (2013).
- 47 . Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations. Anal. Biochem. 472, 7–20 (2015).
- 48 . Combined dynamic light scattering and Raman spectroscopy approach for characterizing the aggregation of therapeutic proteins. Molecules 19(12), 20888–20905 (2014).
- 49 . Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics. PLoS ONE 8(1), e54541 (2013).
- 50 . Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm. Res. 27(5), 796–810 (2010).
- 51 . Modern Size-Exclusion Liquid Chromatography: Practice Of Gel Permeation And Gel Filtration Chromatography, (2nd Edition). John Wiley & Sons, Hoboken, NJ, USA (2009).
- 52 . Improving size-exclusion chromatography separation for glycogen. J. Chromatogr. A 1332, 21–29 (2014).
- 53 . Size-exclusion chromatography (SEC) of branched polymers and polysaccharides. Anal. Bioanal. Chem. 399(4), 1413–1423 (2011).
- 54 . Size separation and size determination of liposomes. J. Sep. Sci. 34(20), 2861–2865 (2011).
- 55 . Polydispersity characterization of lipid nanoparticles for siRNA delivery using multiple detection size-exclusion chromatography. Anal. Chem. 84(14), 6088–6096 (2012).
- 56 . Size-exclusion chromatography in biotech industry. J. Microbiol. Biotechnol. 3(4), 34545 (2014).
- 57 . Comparison of size-exclusion chromatography and flow field-flow fractionation for separation of whey proteins. Bull. Korean Chem. Soc. 32(4), 1315–1320 (2011).
- 58 . Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J. Pharm. Biomed. Anal. 101, 161–173 (2014).
- 59 . The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals. J. Pharm. Sci. 99(4), 1674–1692 (2010).
- 60 . Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J. Liq. Chromatogr. Relat. Technol. 35(20), 2923–2950 (2012). • Evaluates the use of size-exclusion chromatography in more detail for studying protein aggregation.
- 61 . Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway. J. Pharm. Sci. 100(2), 492–503 (2011).
- 62 . Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2 μm particles. J. Pharmaceut. Biomed. 78–79, 141–149 (2013).
- 63 . Characterization of small protein aggregates and oligomers using size exclusion chromatography with online detection by native electrospray ionization mass spectrometry. Anal. Chem. 86(21), 10692–10699 (2014).
- 64 . Therapeutic Proteins Methods and Protocols: Methods in Molecular Biology. Springer, Berlin, Germany (2012).
- 65 . A universal calibration for gel permeation chromatography. J. Polym. Sci. Part C Polymer Lett. 5(9), 753–759 (1967).
- 66 Transmission electron microscopy as an orthogonal method to characterize protein aggregates. J. Pharm. Sci. 104, 750–759 (2014).
- 67 . Correlative Light And Electron Microscopy. Academic Press, Elsevier, Amsterdam, The Netherlands (2014).
- 68 . Analytical Electron Microscopy For Materials Science. Springer, Berlin, Germany, 152 (2002).
- 69 . Physical Principles of Electron Microscopy: An Introduction to TEM SEM and AEM. Springer, Berlin, Germany, 211 (2008).
- 70 . Transmission electron microscopy characterization of fluorescently labelled amyloid β 1–40 and α-synuclein aggregates. BMC Biotechnol. 11, 125 (2011).
- 71 . Radiation damage in the high resolution electron microscopy of biological materials: a review. J. Microsc. 113(2), 113–129 (1978).
- 72 Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy. J. Lipid Res. 52(1), 175–184 (2011).
- 73 . Viral Nanotechnology. CRC Press, Boca Raton, FL, USA (2016).
- 74 . Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy. Proc. Natl Acad. Sci USA 111(32), 11709–11714 (2014).
- 75 . Cryo electron microscopy structures of Hsp100 proteins: crowbars in or out? Biochem. Cell Biol. 88(1), 89–96 (2010).
- 76 Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics. Nature 497, 643–646 (2013).
- 77 . Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems. Int. J. Pharm. 417(1–2), 120–137 (2011).
- 78 Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes. Int. J. Nanomedicine 9, 1367–1377 (2014).
- 79 . Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat. Protoc. 7(9), 1716–1727 (2012).
- 80 . On the dispersion of a solute in a fluid flowing through a tube. Proc. R. Soc. A 235, 67–77 (1956).
- 81 . Dispersion of soluble matter in solvent flowing slowly through a tube. Proc. R. Soc. A 219, 186–203 (1953).
- 82 . Polydispersity analysis of Taylor dispersion data: the cumulant method. Anal. Chem. 86(13), 6471–6478 (2014).
- 83 . A Taylor dispersion analysis method for the sizing of therapeutic proteins and their aggregates using nanolitre sample quantities. Int. J. Pharm. 416(1), 394–397 (2011).
- 84 . Physicochemical characterization of a PEGylated liposomal drug formulation using capillary electrophoresis. Electrophoresis 32, 738–748 (2011).
- 85 . Determination of individual diffusion coefficients in evolving binary mixtures by taylor dispersion analysis: application to the monitoring of polymer reaction. Anal. Chem. 82(5), 1793–1802 (2010). •• Covers Taylor dispersion data (TDA) theory and its application for mixtures. Very good paper for learning how to use TDA technique.
- 86 . A nanolitre method to determine the hydrodynamic radius of proteins and small molecules by Taylor dispersion analysis. Int. J. Pharm. 411(1–2), 64–68 (2011).
- 87 . Characterization of asphaltenes by nonaqueous capillary electrophoresis. Energy Fuels 25(1), 208–214 (2011).
- 88 . Taylor dispersion analysis in coiled capillaries at high flow rates. Anal. Chem. 85(8), 4051–4056 (2013).
- 89 . Size and charge characterization of polymeric drug delivery systems by Taylor dispersion analysis and capillary electrophoresis. Anal. Bioanal. Chem. 405(16), 5369–5379 (2013). • Gives example of diverse application of TDA.
- 90 . Taylor dispersion analysis of mixtures. Anal. Chem. 79, 9066–9073 (2007).
- 91 . Determination of dendrigraft poly-L-lysine diffusion coefficinets by taylor dispersion analysis. Biomacromolecules 8(10). 3235–3243 (2007).
- 92 . Size-based characterization of nanoparticle mixtures by the inline coupling of capillary electrophoresis to Taylor dispersion analysis. J. Chromatogr. A 1426, 220–225 (2015).
- 93 . Measurement of drug diffusivities in pharmaceutical solvents using Taylor dispersion analysis. J. Pharm. Biomed. 61, 176–183 (2012).
- 94 . Evaluating the inter and intra batch variability of protein aggregation behaviour using Taylor dispersion analysis and dynamic light scattering. Int. J. Pharm. 453(2), 351–357 (2013).
- 95 . Insulin diffusion and self-association characterized by real-time UV imaging and Taylor dispersion analysis. J. Pharm. Biomed. 92, 203–210 (2014).
- 96 . Polymers for drug delivery systems. Annu. Rev. Chem. Biom. Eng. 1, 149–173 (2010).
- 97 . Physico-chemical characterization of polymeric micelles loaded with platinum derivatives by capillary electrophoresis and related methods. J. Control. Release 196, 139–145 (2014).
- 98 . Size characterization of commercial micelles and microemulsions by Taylor dispersion analysis. Int. J. Pharm. 492, 46–54 (2015).
- 99 . Hydrodynamic size characterization of a self-emulsifying lipid pharmaceutical excipient by Taylor dispersion analysis with fluorescent detection. Int. J. Pharm. 513, 262–269 (2016).
- 100 . Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection. MAbs 7(1), 77–83 (2015).
- 101 . Measuring arbitrary diffusion coefficient distributions of nano-objects by Taylor dispersion analysis. Anal. Chem. 87(16), 8489–8496 (2015).