High-throughput analysis of animal cell cultures using two-dimensional fluorometry

Area of Science:

• Biotechnology
• Bioprocess Engineering
• Analytical Chemistry

Background:

• Monitoring cell growth and protein production is crucial for biopharmaceutical development.
• Current methods can be time-consuming and labor-intensive, especially in early-stage development.
• High-throughput screening is essential for optimizing cell culture conditions and clone selection.

Purpose of the Study:

• To develop and validate a novel, rapid, and high-throughput method for analyzing secreted recombinant protein and viable cell growth in animal cell cultures.
• To demonstrate the method's applicability using Chinese Hamster Ovary (CHO) cell clones producing IgG4 antibodies.
• To establish predictive models for cell density and antibody concentration using fluorescence data.

Main Methods:

• Combining microtiter plate-scale fluorescence spectroscopy with multivariate statistical analysis (Partial Least Squares regression).
• Analyzing supernatant samples using two-dimensional fluorometry to detect changes in tryptophan, metabolic cofactors, and vitamins.
• Correlating fluorescence spectral maps with measured antibody concentrations and viable cell densities.

Main Results:

• Significant spectral changes were observed in key biochemical regions during cell culture.
• A predictive model for viable cell density achieved an average error of 10% upon validation.
• A predictive model for secreted antibody concentration achieved an average error of 7% upon validation, demonstrating predictive capacity beyond calibration data.
• All major spectral regions were necessary for optimal correlation with target variables.

Conclusions:

• The developed fluorescence spectroscopy and multivariate analysis method enables effective analysis of cellular productivity in a 96-well plate format.
• This approach significantly reduces the time required for early-stage bioprocess development.
• The method offers a powerful tool for high-throughput screening and optimization in biopharmaceutical manufacturing.