Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Shawn M. Sternisha, Prabuddha Mukherjee, Aneesh Alex, Eric J. Chaney, Ronit Barkalifa, Boyong Wan, Jang Hyuk Lee, Jose Rico-Jimenez, Mantas Žurauskas, Darold R. Spillman, Sobhana A. Sripada, Marina Marjanovic, Zane Arp, Sybille S. Galosy, Dharmesh S. Bhanushali, Steve R. Hood, Sayantan Bose, Stephen A. Boppart

Research output: Contribution to journalArticlepeer-review


Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

Original languageEnglish (US)
Article number2000629
JournalBiotechnology Journal
Issue number7
StatePublished - Jul 2021


  • bioreactor
  • fluorescence lifetime imaging microscopy
  • mAb production
  • principal component analysis
  • process analytical technology

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine


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