Advanced monitoring and control of pharmaceutical production processes with Pichia pastoris by using Raman spectroscopy and multivariate calibration methods

This contribution includes an investigation of the applicability of Raman spectroscopy as a PAT analyzer in cyclic production processes of a potential Malaria vaccine with Pichia pastoris. In a feasibility study, Partial Least Squares Regression (PLSR) models were created off-line for cell density and concentrations of glycerol, methanol, ammonia and total secreted protein. Relative cross validation errors RMSE_cvrel range from 2.87% (glycerol) to 11.0% (ammonia). In the following, on-line bioprocess monitoring was tested for cell density and glycerol concentration. By using the nonlinear Support Vector Regression (SVR) method instead of PLSR, the error RMSE_Prel for cell density was reduced from 5.01 to 2.94%. The high potential of Raman spectroscopy in combination with multivariate calibration methods was demonstrated by the implementation of a closed loop control for glycerol concentration using PLSR. The strong nonlinear behavior of exponentially increasing control disturbances was met with a feed-forward control and adaptive correction of control parameters. In general the control procedure works very well for low cell densities. Unfortunately, PLSR models for glycerol concentration are strongly influenced by a correlation with the cell density. This leads to a failure in substrate prediction, which in turn prevents substrate control at cell densities above 16 g/L.