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Characterization of DNA in cell culture supernatant by fluorescence-detection size-exclusion chromatography

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Abstract

A fluorescence-detection size-exclusion chromatography (FSEC) method was developed to characterize DNA in cell culture supernatant. Samples stained with Picogreen were fractionated by size-exclusion chromatography (SEC) and monitored simultaneously by UV absorbance and fluorescence. SEC provided a size-characterization capability absent from bulk fluorescent assays, and was also free from interference from other fluorescent and UV-absorbing small-molecule cell culture components. FSEC revealed that DNA in mammalian cell culture supernatant exists mostly in the form of nucleosomal arrays. FSEC combined with agarose electrophoresis revealed spontaneous degradation of DNA in mammalian cell culture supernatant over a 30 day period at 4 °C: from arrays containing up to ~40 nucleosomes, down to arrays containing three or fewer nucleosomes. It also detected nucleosomal DNA in wheat, soy, and yeast hydrolysates commonly used to enhance cell culture productivity.

Detection of host DNA in IgG-containing cell culture supernatant by fluorescence-detection size-exclusion chromatography

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Acknowledgments

The authors gratefully acknowledge Hui Theng Gan, Xinhui Wang, Chiew Ling Lim, Han Ping Loh, Jake Chng, and Lu Zheng for providing cell culture harvests and performing experiments and analyses. We equally acknowledge the generous support of Exploit Technologies Pte. Ltd., Validated Biosystems, and the Biomedical Research Council of the Agency for Science, Technology and Research, Singapore.

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Authors and Affiliations

  1. Bioprocessing Technology Institute, A*STAR, Downstream Processing Group, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore

    Lihan Tan, Veronica Yeo, Yuansheng Yang & Pete Gagnon

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  1. Lihan Tan
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  2. Veronica Yeo
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  4. Pete Gagnon
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Correspondence to Lihan Tan.

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Tan, L., Yeo, V., Yang, Y. et al. Characterization of DNA in cell culture supernatant by fluorescence-detection size-exclusion chromatography. Anal Bioanal Chem 407, 4173–4181 (2015). https://doi.org/10.1007/s00216-015-8639-9

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  • DOI: https://doi.org/10.1007/s00216-015-8639-9

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