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