Bioreactor environment-sensitive sentinel genes as novel metrics for cell culture scale-down comparability

Authors

  • Bhargavi Kondragunta,

    1. Center for Advanced Sensor Technology and Dept. of Chemical, Biochemical and Environmental Engineering, University of Maryland, Baltimore, MD 21250
    2. Div. of Cellular and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892
    3. Div. of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20903
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  • Bharat H. Joshi,

    1. Div. of Cellular and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892
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  • Jing Han,

    1. Div. of Cellular and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892
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  • Kurt A. Brorson,

    1. Div. of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20903
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  • Raj K. Puri,

    1. Div. of Cellular and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892
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  • Antonio R. Moreira,

    1. Center for Advanced Sensor Technology and Dept. of Chemical, Biochemical and Environmental Engineering, University of Maryland, Baltimore, MD 21250
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  • Govind Rao

    Corresponding author
    1. Center for Advanced Sensor Technology and Dept. of Chemical, Biochemical and Environmental Engineering, University of Maryland, Baltimore, MD 21250
    • Center for Advanced Sensor Technology and Dept. of Chemical, Biochemical and Environmental Engineering, University of Maryland, Baltimore, MD 21250
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  • This article is dedicated to the memory of Professor James E. Bailey.

    The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the FDA. No endorsement of any vendor or technique is implied. G. Rao has an equity position in Fluorometrix.

Abstract

Scale-down of bioreactors is currently done based on matching one or more measurable parameters such as kLa and P/V, which could result in insufficient process comparability. Currently, there is a lack of genomic translational studies in cell culture scale-down, which could help delineate measurable cellular attributes for improved scale-down. In this study, we scaled-down from a typical bench-scale 5-L bioreactor to a novel high-throughput 35-mL minibioreactor based on matching oxygen transfer rate, which resulted in cell growth and product-related discrepancies using Sp2/0 cells. Performing DNA microarrays on time-course samples from both systems, we identified ∼200 differentially expressed transcripts, presumably because of bioreactor aeration and mixing differences with scale-down. Evaluating these transcripts for bioreactor-relevant cellular functions such as oxidative stress response and DNA damage response, we chose 18 sentinel genes based on their degree of difference and functionality, which we further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Tracking the differential expression of Sod1, Apex1, and Odc1 genes, we were able to correlate sparging-related damage and poor mixing, as possible causes for physiological changes such as prolonged culture in minibioreactors. Additionally, to verify our sentinel gene findings, we performed follow-up improved scale-down studies based on gene analysis and measured transcriptomic changes. As a result, qRT-PCR-based genomic profiles and cell growth profiles showed better convergence between the improved minibioreactor conditions and the model 5-L bioreactor. Our results broadly show that based on the knowledge from transcriptomic changes of sentinel gene profiles, it is possible to improve bioreactor scale-down for more comparable processes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

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