TY - JOUR
T1 - Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy
AU - Sternisha, Shawn M.
AU - Mukherjee, Prabuddha
AU - Alex, Aneesh
AU - Chaney, Eric J.
AU - Barkalifa, Ronit
AU - Wan, Boyong
AU - Lee, Jang Hyuk
AU - Rico-Jimenez, Jose
AU - Žurauskas, Mantas
AU - Spillman, Darold R.
AU - Sripada, Sobhana A.
AU - Marjanovic, Marina
AU - Arp, Zane
AU - Galosy, Sybille S.
AU - Bhanushali, Dharmesh S.
AU - Hood, Steve R.
AU - Bose, Sayantan
AU - Boppart, Stephen A.
N1 - This research was conducted in the Center for Optical Molecular Imaging at the Beckman Institute for Advanced Science and Technology on the campus of the University of Illinois at Urbana-Champaign and supported in part by funds from GlaxoSmithKline. We appreciate the supply of frozen cell vials, single-use bioreactors and control stations, analytical equipment, and consumables by the GlaxoSmithKline Biopharm Advanced Manufacturing Technology Group, Upper Merion, PA, USA, and their generous contributions of time and expertise to conduct these studies. We also acknowledge Pramthesh Patel, Diana Ritz, Ricardo Tecson, Supaporn Suwannakham, and Thomas Eyster for their guidance and support. The authors do not have any conflict of interest to declare.
This research was conducted in the Center for Optical Molecular Imaging at the Beckman Institute for Advanced Science and Technology on the campus of the University of Illinois at Urbana‐Champaign and supported in part by funds from GlaxoSmithKline. We appreciate the supply of frozen cell vials, single‐use bioreactors and control stations, analytical equipment, and consumables by the GlaxoSmithKline Biopharm Advanced Manufacturing Technology Group, Upper Merion, PA, USA, and their generous contributions of time and expertise to conduct these studies. We also acknowledge Pramthesh Patel, Diana Ritz, Ricardo Tecson, Supaporn Suwannakham, and Thomas Eyster for their guidance and support. The authors do not have any conflict of interest to declare.
PY - 2021/7
Y1 - 2021/7
N2 - 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.
AB - 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.
KW - bioreactor
KW - fluorescence lifetime imaging microscopy
KW - mAb production
KW - principal component analysis
KW - process analytical technology
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U2 - 10.1002/biot.202000629
DO - 10.1002/biot.202000629
M3 - Article
C2 - 33951311
AN - SCOPUS:85107120341
SN - 1860-6768
VL - 16
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 7
M1 - 2000629
ER -