Near-infrared spectroscopy based monitoring of all 20 amino acids in mammalian cell culture broth.

ABSTRACT

The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0-24 mM), as well as concentrations of glucose (0-6.7 mg mL-1), lactate (0-2.7 mg mL-1), and trastuzumab (0-2.5 mg mL-1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000-11,000 cm-1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94-0.99) and prediction (Rp2 = 0.83-0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89-0.99, RMSE = 0.04-1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.