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Longitudinal monitoring of cell metabolism in biopharmaceutical production using label‐free fluorescence lifetime imaging microscopy
Author(s) -
Sternisha Shawn M.,
Mukherjee Prabuddha,
Alex Aneesh,
Chaney Eric J.,
Barkalifa Ronit,
Wan Boyong,
Lee Jang Hyuk,
RicoJimenez Jose,
Žurauskas Mantas,
Spillman Darold R.,
Sripada Sobhana A.,
Marjanovic Marina,
Arp Zane,
Galosy Sybille S.,
Bhanushali Dharmesh S.,
Hood Steve R.,
Bose Sayantan,
Boppart Stephen A.
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000629
Subject(s) - chinese hamster ovary cell , autofluorescence , biopharmaceutical , fluorescence microscope , confocal microscopy , fluorescence lifetime imaging microscopy , microscopy , cell culture , nicotinamide adenine dinucleotide , bioreactor , two photon excitation microscopy , plate reader , nad+ kinase , biochemistry , chemistry , fluorescence , biology , microbiology and biotechnology , enzyme , pathology , medicine , genetics , physics , organic chemistry , quantum mechanics
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.