Table 7.

The list of analytical techniques to characterize medicinal proteins and peptides [39,253,264,265].

Technique		Output	Destructive	QC Method	General Comments
Conformational assessments	DSC	Thermal parameter (Tm, ΔG unfolding)	Yes	No	The best option for checking temperature-dependent parameters; not high throughput.
	CD	Secondary and tertiary statures	No	No	Sensitive to the polarity of the solution.
	Fluorescence spectroscopy	Tertiary statures/ general view on the protein structure	Yes	No	Sensitive to small structural changes of proteins and peptides; the need for relatively small amounts of material; relatively high speed; takes a general view of structural changes and cannot go into details.
	DSF	Tm, aggregation onset	Yes	No	During the thermal unfolding of a protein, a dye (commonly Sypro Orange) is added to the sample, which, by connecting to the unfolded parts, leads to an increase in its fluorescence emission.
	UV	Tertiary structure	No	No	Makes a general view of structural changes.
	Raman	Secondary structure/ chemical characterization	Yes	No	It has a high overlap with FTIR, but on the contrary, a wide range of solvents can be used in Raman analysis to examine samples.
	Infrared	Secondary structure/chemical integrity	Yes	No	By examining the amine-I and II regions of proteins and their deconvolution, it is possible to reach the percentage of the secondary structure in the protein (in solution and solid states) either in the form of fold and/or aggregation/fibrils. ATR facility requires very small amounts of substance without the need to prepare KBR tablets.
Oligomerization studies	AUC	Molecular weight/shape	No	Yes	Determining the size of particle aggregation.
	DLS	Hydrodynamic size		No	High range of particle detection (between 1nm to 5 µM), reliable within a certain range of polydispersity
	FAPS	Particles/Serum interactions		No	Extracting fluorescence data from the aggregated samples. One of its limitations is the use of dyes to identify aggregates, which may affect the protein structure.
	AF4	Hydrodynamic size	Yes	Yes	AF4 technique separates particles based on their diffusion coefficients.
	NTA	Hydrodynamic size	No		It can measure the size of particles, imaging and quantifying them.
	RMM	Concentration/size/mass	Yes	No	In a microfluidic way, it can calculate particle size.
	SEC	Hydrodynamic size	Yes	Yes	Determine the molecular weight, aggregation rate, and interactions between proteins.
	SDS-PAGE (all types)	Molecular weight/ interactions between proteins	Yes	Yes	Covalent interactions between proteins and also protein digests; in reducing and non-reducing types, it can observe disulfide bonds.
	Optical microscopy	Size/morphology	No	No	Detecting large particles (larger than 1 µm).
	Native MS	Fragments/aggregates	Yes	No	With principles similar to MS, it investigates non-covalent interactions and post-translational changes in proteins.
	Light obscuration	Concentration/size	No	Yes	This technique, which is also known as Single Particle Optical Sensing (SPOS), is not very sensitive to small sizes (detecting sizes more than 1 µm).
	Fluorescence microscopy	Particles/amorphous and morphous aggregates	Yes	No	A fluorophore molecule is needed that can provide the output signal. Some fluorophore dyes change fluorescence intensity by interacting with proteins and being buried in their structure, which can be a pattern of protein folding and even aggregation.
	Flow imaging	Concentration/size/morphology	No	Yes	The basis is similar to optical microscopy, except that it can provide data qualitatively.
	CE-SDS	Molecular weight	Yes	Yes	Advantages such as quicker analysis, the facility for quantification, full automation, the need for low sample, and better resolution
	Electrical zone sensing	Concentration/size	Yes	No	By applying electric force and migration of the two-pole magnifier, it can achieve the size of the particles.
	Turbidity	Optical density > 360 nm	No	Yes	It is a low-cost, rough method that can be used to detect large particles. Its high speed and simplicity are its positive points.
Chemical changes	RP-HPLC	Hydrophobicity	Yes	Yes	Sensitive to slight changes in surface hydrophobicity of proteins, requiring small amounts of samples.
	cIEF	Charge	Yes	Yes	Similar to IEF, it can separate proteins based on their PI. Compared to its traditional sample, i.e., IEF, it requires much less material, and due to its capillary nature, a higher voltage can be applied to the sample, which leads to a reduction in the test time. This test is successful in the case of samples higher than 150,000 Daltons that dissolve well in aqueous solutions.
	IEX chromatography	Charge	Yes	Yes	Separating proteins by considering their charge. High speed and acceptable accuracy.
	MS	By the difference in weight molecular changes	Yes	No	Differentiation of diverse components with considering mass-to-charge ratio (m/z).
	LC-MS	By the difference in weight molecular changes	Yes	No	In cases where a protein complex is present, initial separation by HPLC enables MS to obtain more details of the sample by removing noise.
	Zeta potential	Charge	No	Yes	Measuring the particle charges; the types of solvents and even the percentage of ions in water strongly affect the data.

Abbreviations: can: acetonitrile, AUC: analytical ultracentrifugation, DSC: differential calorimetry, Tm: melting temperature, CD: circular dichroism, DSF: differential scanning fluorimetry, UV: ultraviolet, DLS: dynamic light scattering, FAPS: fluorescence-activated particle sorter, AF4: asymmetrical flow-field flow fractionation, NTA: nanoparticle tracking analysis, RMM: resonant mass measurement, SEC: size-exclusion chromatography, SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MS: mass spectrometry, CE-SDS: capillary electrophoresis sodium dodecyl sulfate, RP-HPLC: reversed-phase high-performance liquid chromatography, cIEF: capillary isoelectric focusing, IEX: ion exchange chromatography, and LC: liquid chromatography.