Quality prediction of cell substrate using gene expression profiling.
Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.
Han
Jing
J
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29B, Room 2NN20, 29 Lincoln Drive, Bethesda, MD 20892, USA.
Farnsworth
Richard L
RL
Tiwari
Jawahar L
JL
Tian
Jie
J
Lee
Hin
H
Ikonomi
Pranvera
P
Byrnes
Andrew P
AP
Goodman
Jesse L
JL
Puri
Raj K
RK
D000256:Adenoviridae / Q000235:genetics; D015415:Biomarkers; D018929:Cell Culture Techniques; D002460:Cell Line; D016000:Cluster Analysis; D015536:Down-Regulation; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D015999:Multivariate Analysis; D020411:Oligonucleotide Array Sequence Analysis; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D015854:Up-Regulation
D015415:Biomarkers