Table 4.
Analytical techniques for physicochemical characterization of protein
| Methods | Principles | Instability | Applications | Proteins & peptides | Reference |
|---|---|---|---|---|---|
| DLS | Hydrodynamic size, Polydispersity | Physical | Aggregation (1 nm–10 µm) | IgG, Etanercept | Sarciaux et al. (1999), Ahrer et al. (2006), Hawe et al. (2009), Menzen et al. (2014), Emami et al. (2018a, b), Kim et al. (2014), Kim et al. (2016a, b) |
| NTA | Concentration, Arithmetic size | Physical | Aggregation (30–1000 nm) | IgG, Lysozyme | Nuchuchua et al. (2014) |
| SLS (Light Obscuration) | Average Mw | Physical | Aggregation (1–600 µm) | IgG | Hawe et al. (2009), Emami et al. (2018a, b) |
| Turbidity | Optical density (λ ˃340 nm), Aggregation index | Physical | Aggregation | IgG, Lysozyme | Sarciaux et al. (1999), Ahrer et al. (2004), Jovanovic et al. (2008a, b), Hawe et al. (2009), Menzen et al. (2014), Jovanovic et al. (2006), Jovanovic et al. (2008a, b) |
| SEC | Hydrodynamic size, Mw | Chemical, Physical | Fragmentation, (Disulfide) oxidation, Aggregation, dimer (1–100 nm) | IgG, Lysozyme, Etanercept | Sarciaux et al. (1999), Ahrer et al. (2004), Schüle et al. (2007), Sahin et al. (2010), Garidel et al. (2015), Emami et al. (2018a, b), Nuchuchua et al. (2014), Kim et al. (2014), Kim et al. (2016a, b) |
| IEX | Charge | Chemical | Deamidation ((1–100 nm) | IgG | Wang et al. (2007) |
| RP-HPLC | Hydrophobicity | Chemical | Fragmentation, Deamidation, (Disulfide) Oxidation (1–100 nm) | Growth hormone | Kim et al. (2014), Kim et al. (2016a, b) |
| Analytical Ultracentrifuge | Mw | Physical | Aggregation | – | Jiskoot et al. (2005) |
| Electrophoresis, Native-PAGE, SDS-PAGE, CE-SDS, MCGE | Mw, Charge | Chemical, Physical | (Disulfide) oxidation, Fragmentation aggregation, Dimer, Isomerization | IgG, Phosphopeptide, Lysozyme, Anthrax vaccine, Influenza vaccine, Adalimumab | Alexander et al. (1995), Hawe et al. (2009), Awotwe-Otoo et al. (2015), Wang et al. (2015), Emami et al. (2018a, b), Ollikainen et al. (2016), Wang et al. (2007), Jovanovic et al. (2006), Wang et al. (2012), Maa et al. (2004), Emami et al. (2019) |
| LC–MS | Mass | Chemical | Deamidation | β-Lactoglobulins | Monaci et al. (2007) |
| Mass spectrometry, MALDI-TOF, ESI-TOF | Mass/Charge | Chemical, Physical | Fragmentation, Low Mw aggregation | IgG, Adalimumab | Alexander et al. (1995), Sandra et al. (2014), Zhang et al. (2014), Emami et al. (2019) |
DLS dynamic light scattering, SLS static light scattering, NTA nanoparticle tracking analysis, IEX ion exchange chromatography, SEC size exclusion chromatography, RP-HPLC reverse phase- high performance liquid chromatography, Native-PAGE native polyacrylamide gel electrophoresis, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis, CE-SDS capillary electrophoresis sodium dodecyl sulfate, MCGE microchip capillary gel electrophoresis, Mw molecular weigth, LC–MS liquid chromatography-mass spectrometry, MALDI-TOF matrix-assisted laser desorption/ionization-time of flight, ESI-TOF time-of-flight mass analyzer with an electrospray ionization