Objective: To optimize an LC-Q TOF MS method for the determination of target proteins expressed in recombinant cells from a cell bank. Methods: The recombinant Chinese hamster ovary (CHO) cell line expressing antibodies was selected. The supernatant of recombinant CHO cell suspensions was retained after 12 500 r · min-1centrifuging for 10 min and treated with 50 mmol · L-1 NH4HCO3 solution. Chymotrypsin was added for enzymatic digestion, and the comparative analyses were carried out using two LC-Q TOF MS with Advance Bio Peptide (100 mm×2.1 mm, 2.7 μm) column, the Sciex TRIPLE TOF 5600+ and the Agilent Q TOF G6545, respectively. To establish the database, the amino acid sequences of the characteristic peptides of the protein heavy and light chains were entered into SCIEX BioPharmaView Version 3.0 and Agilent MassHunter BioConfirm 12.0 software, respectively. The peptides detected in the samples were searched to identify the target peptides. Results: The samples were analysed by a Sciex TRIPLE TOF 5600+ instrument to obtain 8 characteristic peptides with 100% amino acid sequence coverage and the samples were analysed by an Agilent Q TOF G6545 instrument to obtain 12 characteristic peptides with 100% amino acid sequence coverage. Conclusion: The method established in this study is simple to operate, has high coverage and good stability, can be used to identify the expression of target proteins in recombinant CHO cell lines. It also provides a reference for the expression identification of target proteins in similar cell lines.
KANG Zhao-fei
,
WANG Ke-xin
,
HAN Chun-le
,
PANG Lin
,
RAO Chun-ming
. Study of liquid chromatography-mass spectrometry identification of target protein expression in recombinant CHO cell lines[J]. Chinese Journal of Pharmaceutical Analysis, 2025
, 45(1)
: 99
-103
.
DOI: 10.16155/j.0254-1793.2024-0285
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