High-throughput analysis of animal cell cultures using two-dimensional fluorometry.
We report a new method which combines fluorescence spectroscopy at microtiter plate scale with multivariate statistical analysis for rapid and high-throughput analysis of secreted recombinant protein and viable cell growth in animal cell cultures. The potential of the method is demonstrated by application to cultures of three Chinese Hamster Ovary (CHO) cell clones with distinct IgG(4) antibody yields. Supernatant samples collected throughout culture time were analysed by two-dimensional fluorometry; significant changes were observed in the regions of tryptophan, metabolic cofactors and vitamins. Partial least squares regression was then used to correlate the entire fluorescence map with measured concentrations of antibody and viable cells. For both target variables, a model was calibrated with representative data from the two less productive clones and validated with data from the best producer clone; this allowed viable cell density to be predicted for the validation clone with an average error of 10%; even better, the secreted antibody could be predicted with an average error of 7%, proving the predictive capacity of the model beyond the calibration region. All the main spectral regions were required to establish the best correlations for both targeted variables. In conclusion, this method effectively analyzes cellular productivity in 96-well plate format, shortening the time spent in early phases of bioprocess development.
Instituto de Tecnologia Química e Biológica - Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal. anat@itqb.unl.pt
Ana P
AP
Teixeira
Tiago M
TM
Duarte
Rui
R
Oliveira
Manuel J T
MJ
Carrondo
Paula M
PM
Alves
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002470:Cell Survival; D019295:Computational Biology; D006224:Cricetinae; D003412:Cricetulus; D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry; D016018:Least-Squares Analysis; D008954:Models, Biological; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D007074:Immunoglobulin G; D011994:Recombinant Proteins