• Application Note

Targeted Quantification of Cell Culture Media Components by LC-MS

Targeted Quantification of Cell Culture Media Components by LC-MS

  • WANG Xiaoxia
  • LIU YI
  • WANG Hui
  • JIA Zhengwei
  • Waters Corporation
Changing medium of a cell culture dish

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

An LC-MS/MS method library was developed for the quantitative analysis of components in cell culture medium and spent cell culture medium. This method, which covers a wide range of compounds including amino acids, vitamins, nucleoside pyrimidines and purines, nucleotides, organic acids, polyamines, and carbohydrates, enables the analysis of cell culture medium and the supernatant from the spent cell culture medium in multiple aspects. Meanwhile, its excellent reproducibility renders relative quantification of spent cell culture medium even when standards are not available.

Benefits

  • A fast and universal LC-MS/MS method for media component analysis of cell culture medium and spent cell culture samples.
  • Relative quantification of metabolites in spent cell culture medium can be achieved by this method when standards are not available.
  • No derivatization is required for sample preparation.

Introduction

Cell culture medium components and feeding strategy are important variables in upstream bioprocess development that must be optimized due to their direct impact on the quality and titer of recombinant therapeutic products. A comprehensive and quantitative understanding of cellular metabolic components could facilitate more robust bioprocess development that ensures productivity and quality. Thus, it is of great significance for the establishment of an efficient analytical method to monitor the changes of various components in cell culture medium and in spent cell culture medium during the cell culture process.

In order to quickly and comprehensively analyze the components of raw and spent cell culture medium, and intracellular substance, we developed a method library for the analysis of raw and spent cell culture medium, which comprises two LC methods and covers hundreds of compounds. Method one is mainly used for amino acids, vitamins, nucleoside pyrimidines and purines, organic acids, monophosphate nucleotides, polyamines, and carbohydrates, and method two is mainly used for nucleotides. Due to the diversity of these compounds and the large differences of their chemical properties, conventional assays are extremely complex requiring the use of different instruments and different sample preparation protocols, which makes it difficult to meet the throughput and standardization requirements of the biopharm industry. On the other hand, the ultra-high performance liquid chromatography-mass spectrometry system can provide a fast and highly specific method for the component analysis of cell culture medium, spent cell culture medium, and intracellular substance.

Experimental

EXPERIMENTAL PROCESS

EXPERIMENTAL PROCESS

Compound list

The compounds contained in the method library are listed in Table 1.

Compounds contained in the method library Table 1. Compounds contained in the method library.

Sample preparation protocol for cell culture medium: the culture medium sample was diluted by water (containing 0.1% acetic acid) before direct injection. The dilution factor was determined based on the concentration of the compound in the sample;

Sample preparation protocol for the supernatant of spent cell culture media: performing protein precipitation with 2 volumes of acetonitrile, centrifuge at 14000 rpm for 5 min, and take the supernatant and dilute 100 time with water (containing 0.1% acetic acid) before injection for analysis.

LC conditions

System:

ACQUITY UPLC I-Class

LC method one:

Column:

ACQUITY UPLC HSS T3 (2.1 x 150 mm, 1.8 μm)

Column temperature:

40 °C

Flow rate:

0.2 mL/min

Injection volume:

1 μL

Mobile phase A:

0.1% formic acid in water

Mobile phase B:

0.1% formic acid in acetonitrile

Gradient:

Gradient

LC method two:

Column:

ACQUITY UPLC BEH Amide (2.1 x 100 mm, 1.7 μm)

Column temperature:

25 °C

Flow rate:

0.3 mL/min

Injection volume:

1 μL

Mobile phase A:

ACN/H2O (v:v 95/5) with 10 mM Ammonium bicarbonate (pH=9)

Mobile phase B:

ACN/H2O (v:v 5/59) with 10 mM Ammonium bicarbonate (pH=9)

Gradient:

Gradient:

MS conditions

Mass spectrometer:

Xevo TQ-S micro

Acquisition mode:

ESI Positive/Negative switching

Capillary voltage:

2.5 kV

Ion source temperature:

150 °C

Nebulizer gas temperature:

500 °C

Nebulizer gas flow rate:

1000 L/h

Data processing software:

MassLynx v4.2 with TargetLynx

Results and Discussion

Figure 1 is the ion chromatograms of the compounds contained in the method library.

Chromatograms of the compounds contained in the method library Figure 1. Chromatograms of the compounds contained in the method library.

Chromatograms of two pairs of isomers contained in the analysis package, i.e., citric acid/isocitric acid and leucine/isoleucine, are shown in Figure 2.

Chromatograms of two pairs of isomers Figure 2. Chromatograms of two pairs of isomers contained in the analysis package, i.e., citric acid/isocitric acid and leucine/isoleucine.

Linear Result

Calibration curves were constructed for compounds that are commonly used in culture medium. The linear range, and reproducibility of retention time and peak area obtained from 6 consecutive injections are shown in Table 2 below.

Linearity and reproducibility results Table 2. Linearity and reproducibility results (to be continued on next page).

Reproduciblity of Sample Analysis

Relative quantification was carried out for the supernatant of the spent cell culture medium using the method library for 6 consecutive injections. Peak area RSDs obtained from the 6 consecutive injections were within 5% for all compounds, as shown in the figure below.

Relative quantification

Conclusion

  • An LC-MS/MS method library was developed for the quantitative analysis of components in cell culture medium and spent cell culture medium. This method, which covers a wide range of compounds including amino acids, vitamins, nucleoside pyrimidines and purines, nucleotides, organic acids, polyamines, and carbohydrates, enables the analysis of cell culture medium and the supernatant from the spent cell culture medium in multiple aspects. Meanwhile, its excellent reproducibility renders relative quantification of spent cell culture medium even when standards are not available.
  • The method library is imported by using Quanpedia database which contains information about compound retention time, MRM, mobile phase, elution gradient, and column. When performing method import, the LC gradient method, MS method, and quantitation method can be imported simultaneously in just three steps. The Quanpedia database is an open database that can be expanded at any time according to customer needs.

References

  1. Giuseppe Paglia1, James Langridge2, and Giuseppe Astarita3, Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC/QTof MS. 1Center for Systems Biology, University of Iceland, Iceland; 2–3Waters Corporation, Manchester, UK, and Milford, MA, USA.
  2. Sunil U. Bajada, Wenyun Lua, Elizabeth H. Kimballa, Jie Yuana, Celeste Petersonb, Joshua D. Rabinowitza, Separation and Quantitation of Water Soluble Cellular Metabolites by Hydrophilic Interaction Chromatography- Tandem Mass Spectrometry. aLewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton University, Princeton, NJ 08544, USA; bDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Received 13 January 2006; received in revised form 8 May 2006; accepted 10 May 2006; available online 6 June 2006.

720006759, October 2019

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