Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques

Figures & data

Table 1. Commonly used HPLC detectors (Locatelli et al., 2012; Knol et al., 2021).

Type	Features	Sensitivity
UV/VIS detector	Most common absorbance detector Easy to operate and provide good stability Wide range of wavelength selection, covering both UV and VIS ranges (typically 195–700 nm)	1 pg–1 ng
Photo diode array (PDA) detector	Detect an entire spectrum simultaneously and save time Convenient to determine the most suitable wavelength without repeating analyses Visualize the result in three dimensions (light intensity, wavelength, time)	1 pg–1 ng
Fluorescence detector	Detect fluorescent absorbance Possible to use fluorescence derivatives such as dansylchloride for some compounds having no fluorescence absorbance or low absorbance Provide high selectivity, sensitivity, and specificity Easy to operate and relatively stable	10 fg–10 pg
Mass spectroscopic detector	Technique that combines physical separation capabilities of HPLC with detection analyzer of mass spectroscopy. Provide high selectivity and sensitivity Fast detection without the need of retention time validation Useful for structure identification as well as quantitative analysis	100 ag–1 ng

Table 2. Selected examples of protein quantification using LC-MS/MS.

Drug	Biological matrix	Sample preparation	Column	Mass system	References
NVS001	Human serum	Immunoaffinity capture and protein digestion	C18 (2.7 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Fu et al., 2018)
Recombinant FGF21	Human serum	Immunoaffinity capture and protein digestion	C18 (2.6 µm) 50 × 2.1 mm	ESI-triple quadrupole	(Zhao et al., 2018b)
Nivolumab	Human serum	Immunoaffinity capture and protein digestion	C18 (1.6 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Millet et al., 2021)
Thyroglobulin	Chicken serum	Immunoaffinity capture and protein digestion	C18 (1.8 µm) 50 × 1.5 mm	ESI-triple quadrupole	(Shi et al., 2022)
Insulin like growth factor - I, II	Human plasma	Protein precipitation	C18 (1.6 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Pratt et al., 2021)
Denosumab	Human serum	Protein digestion	C18 (2.6 µm) 150 × 3 mm	ESI-triple quadrupole	(Shida et al. 2018)
Insulin like growth factor - I	Human serum	Solid phase extraction	C18 (1.6 µm) 50 × 2.1 mm	ESI-triple quadrupole	(Tanna et al. 2020)
PEG-Interferon-α-2b	Human serum	Protein precipitation and protein digestion	C18 (3 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Kusuma et al., 2021)
Tocilizumab	Human serum	Protein digestion and solid phase extraction	C18 (2.6 µm) 150 × 3 mm	ESI-triple quadrupole	(Mochizuki et al., 2022)
Parathyroid hormone-Fc	Human serum	Immunoaffinity capture and protein digestion	C18 (2 µm) 50 × 0.3 mm	ESI-triple quadrupole	(Wang et al., 2022)
Cardiac troponin I	Human plasma	Immunoaffinity capture and protein digestion	C18 (3.5 µm) 150 × 2.1 mm	ESI-triple quadrupole	(Schneck et al., 2018)
Apolipoproteins	Human serum	Protein digestion	C18 (2.7 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Toth et al., 2017)
8-iso-Prostaglandin F2 alpha	Human plasma	Liquid-liquid extraction	C18 (2.6 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Tomov et al., 2021)
Estrogens	Human saliva	Liquid-liquid extraction	C18 (2.5 µm) 100 × 2.1 mm	ESI-triple quadrupole	(Li et al., 2018)

Table 3. Advantages and disadvantages of ELISA and LC-MS/MS.

Method	Advantages	Disadvantages
	Ease-of-use Low equipment cost High throughput (microplate) High level of sensitivity	Cross-reactivity Relatively high sample volumes (100–200 µL) High cost-per-sample Low reproducibility Relatively long assay time
	High level of selectivity and sensitivity High throughput Low sample volumes (<5 µL) Relatively low cast-per-sample High reproducibility Multiplexing	High equipment cost Complexity in the optimization of assay conditions Potential matrix effect (lowering the assay sensitivity)

Figure 1. Sample preparation methods prior to the quantification of protein drugs in plasma using LC-MS/MS.

Figure 2. Workflow and critical factors in LC-MS/MS analysis of protein samples.

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