From left: Dr Bi Xuezhi, Dr Tan Hwee Tong and Dr Sim Kae Hwan
Kae Hwan Sim 1, Lillian Chia-Yi Liu 1, Hwee Tong Tan 1, Kelly Tan 1, Daniel Ng 1, Wei Zhang 1, Yuansheng Yang 1, Stephen Tate 2 and Xuezhi Bi 1,3
1 Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
2 ScieX, Concord, Ontario, Canada
3 Duke-NUS Medical School, Singapore
Published in Scientific Data 2020 7(1): 263 (Online Version)
Chinese Hamster Ovary (CHO) cell lines are used for the production of almost 70% of all recombinant protein therapeutics, including monoclonal antibodies (mAbs). The production of mAbs is governed by regulatory authorities such as US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to ensure high product quality and safety. Mass spectrometry (MS)-based characterization of protein therapeutics and bioprocessing has become an essential analytical tool in biopharmaceutical industries. However, conventional data-dependent acquisition (DDA)-MS technique only selects limited precursor ions whose intensities exceed a predefined threshold for analysis. Thus, it favours the most abundant proteins and is limited by poor sensitivity, coverage and reproducibility for less abundant proteins.
Dr Bi Xuezhi and Dr Stephen Tate, scientists at A*STAR Bioprocessing Technology Institute (BTI) and SCIEX respectively, studied CHO cell lines and downstream processing of mAb samples using the state-of-the-art SWATH (sequential window acquisition of all theoretical fragment-ion spectra)-MS technique for high-throughput, robust and accurate protein quantification. SWATH-MS is a data-independent acquisition (DIA)-MS method which, in principle, creates a complete record of all precursor and fragment ions within any given sample, thus overcoming the inadequacies of DDA-MS. Analysis of SWATH-MS data, however, requires prior knowledge of the ions’ chromatographic and mass spectrometric behaviour coordinates in the form of a spectral library, and there is no publicly accessible CHO-specific spectral library with sufficiently in-depth coverage of proteome sequences for extensive protein identification and quantitation in a SWATH-MS workflow.
The team at A*STAR BTI addressed this by constructing a comprehensive CHO SWATH-MS spectral library with empirically curated SWATH-MS coordinates to enable reproducible and accurate quantitative measurements. The researchers generated the first publicly accessible CHO SWATH-MS spectral library that can measure more than 10,000 proteins. Robustness, reproducibility, and precision of the spectral library are also illustrated in the quantification of CHO-derived samples, including intracellular proteins, harvested cell culture fluid and downstream mAb process intermediates.
In addition, the CHO spectral library can be used in the identification and quantification of proteins across different CHO cell lines (CHO-K1, CHO-S and CHO-DG44), as well as monitoring removal of host cell protein contaminants during downstream processing. These findings further demonstrated applicability of SWATH-MS with the in-house CHO spectral library as a potential process analytical technology (PAT) for biopharmaceutical industries.
The A*STAR researchers contributing to this research are from the Bioprocessing Technology Institute (BTI).
Sim KH, Liu LC, Tan HT, Tan K, Ng D, Zhang W, Yang Y, Tate S, Bi X. A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins. Sci Data. 2020 Aug 11;7(1):263. https://doi.org/10.1038/s41597-020-00594-z