Page 18 - Shimadzu Journal vol.5 Issue1

Bioanalysis

Application of LC-MS/MS analysis for time-lapse
metabolomics in CHO cell culture

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Han-Hsiu Hsu , Tomohisa Hasunuma , Michihiro Araki , Takanobu Yoshida , Yoshimi Hori , Masahiro Murata , Akihiko Kondo * 2
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1ɹIGENOMIX JAPAN K.K., L Ningyocho 4F, 2-7-10 Nihonbashi Ningyocho, Chuo-ku, Tokyo, 103-0013, Japan
2ɹDepartment of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan
Abstract
Metabolic analysis of Chinese hamster ovary (CHO) cell culture plays an important role for the efficient production of biopharmaceutical industry.
Understanding CHO cellular metabolism including gene expression and coordination among various metabolic pathways and protein productions
is vital for the production of biopharmaceuticals. Recently, analytical methods for metabolic pathways such as glycolysis and TCA cycle have been
reported. This study further analyzed amino acids concerned with these pathways by LC-MS/MS, LCMS-8050 (SHIMADZU), for time-lapse
metabolic analysis of CHO-K1 cell batch culture. Temporal correlativity of each metabolite was calculated and visualize by pathway mapping
system, Pathpod, developed by Araki et al. Our results showed real-time changes of various metabolites during CHO culture, which may provide
extensively and speedy investigational analysis for genomic and metabolic modifications in biopharmaceutical or medical progresses.

1. Introduction of ice-cold methanol and 100 µl of chloroform, followed by vortex.
450 µl of cold methanol/3.8 mM tricine (9:1, v/v) mixture and 250 µl
Regulating the rate of protein production in Chinese hamster ovary of chloroform were added sequentially. Well mixed solution was
(CHO) cells is an important consideration in reducing the cost of centrifuged at 18,000 g for 20 min at 0°C, followed by transferring
biopharmaceutical industry. Methodologies such as microarrays and 300 µl of the methanol-aqueous layer into a new 1.5 ml tube. Sample
proteomics, which have been extensively reviewed (Griffin et al., extract was evaporated by drying by vacuum centrifugation for 8 h
2007), offer the prospect of examining the molecular phenotypes and diluted with 100 µl of 20% methanol-water solution (v/v). 3 µl of
underlying productivity in CHO and their application in bioprocess sample extract was injected into UHPLC-MS/MS system for analysis,
research. Moreover, liquid chromatography-mass spectrometry with conditions listed in Tables 1 and 2 (n×3). Whole acquisition
(LC-MS)-based intracellular metabolic analysis plays an important role method lasted for 35 minutes.
for CHO cell metabolic characterization for antibody production
(Chong et al., 2012) and host cell protein production (Yuk IH et al., Data processing and metabolic mapping
2015). Analytical methods for central metabolism pathways (CMPs) From the results of LC-MS/MS analysis, data of metabolites concerned
such as glycolysis and TCA cycle were reported (Judith Wahrheit et al., with amino acid metabolism were extracted and calculated as ratio
2014). In this study, we further analyzed intracellular amino acid and amounts to day 0, visualized by bar chart. Correlativity of each
metabolites concerned with CMPs by LCMS-8050 (SHIMADZU) in metabolite in earlier period (day 0 to 2, phase I) and later period (day
CHO-K1 cell batch culture. Time-lapse correlativity calculation was 3 to 5, phase II) was calculated by time-lapse Pearson's correlation
used to observe the changes of metabolite pool size from day 0 to 5. coefficient, and visualized by PathPod pathway mapping system
All data were visualized by Pathpod, a pathway mapping system (http://bp.scitec.kobe-u.ac.jp/pathpod/).
developed by Araki et al. We suggest that our result provide a useful
option for animal cell metabolic analysis.
3. Results
2. Experimental Cell proliferation
CHO-K1 cell proliferation was recorded by morphology and cell count
Cell culture and sampling (Fig. 1). Cells were seeded by 2×10 cells/flask and stand 24 h for
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CHO-K1 cells were cultivated in 75T Cell Culture Flask (BD Falcon) by adhesion (Fig. 1 A). 100% confluence was observed at day2 (Fig. 1 B).
Ham’s F-12 Nutrient Mixture medium (Gibco) with 10% fetal bovine Result of cell number showed that CHO cells proliferation increased at
serum (Biosera), 100U penicillin, and 100 µg/ml streptomycin day 0, 1, and 2, while decreased at day 3, 4, and 5 (Fig. 1 C).
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(penicillin-streptomycin mixture, Nacalai). 2×10 freeze stocked
(Cellbanker) cells were seeded for pre-culture, and medium was Changes of intracellular metabolites
changed at 24 h, followed by continuous cultivation at 37°C. Cell The relative amount of intracellular amino acid and metabolites
samples were collected after pre-culture (24 h), and each 24-hour concerned with CMPs was observed by time-lapse LC-MS/MS analysis
after medium change (day 1 to day 5). (Fig. 2). In this study, the results of following metabolites in Table 2
were extracted. Amino acids, including alanine, arginine, asparagine,
LC-MS/MS Analysis aspartic acid, citrulline, glycine, histamine, histidine, leucine, lysine,
Intracellular metabolites were analyzed by LCMS-8050 (SHIMADZU), phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
by reported methodology (William et al, 2012) with slight modifica- cysteine, and glutamic acid were observed and compared with
tions. Briefly, 2×10 CHO-K1 cells at day 0, 1, 2, 3, 4, and 5 of metabolites concerned with TCA cycle, glycolysis, amino acid
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cultivation were obtained and quenched with five volumes of ice-cold metabolism, and lactate metabolism such as adenylo-succinate,
150 mM NaCl, and centrifuged at 1,300 g for 3 min at -5°C. arginino-succinic acid, 3-phosphoglyceric acid, citric acid, fumaric acid,
Supernatant was discarded and the cell pellet was suspended in 200 µl lactic acid, malic acid, succinic acid, fructose 6-phosphate, glucose

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