Process Analytical Approach towards Quality Controlled Process Automation for the Downstream of Protein Mixtures by Inline Concentration Measurements Based on Ultraviolet/Visible Light (UV/VIS) Spectral Analysis

摘要

Downstream of pharmaceutical proteins, such as monoclonal antibodies, is mainly done by chromatography, where concentration determination of coeluting components presents a major problem. Inline concentration measurements (ICM) by Ultraviolet/Visible light (UV/VIS)-spectral data analysis provide a label-free and noninvasive approach to significantly speed up the analysis and process time. Here, two different approaches are presented. For a test mixture of three proteins, a fast and easily calibrated method based on the non-negative least-squares algorithm is shown, which reduces the calibration effort compared to a partial least-squares approach. The accuracy of ICM for analytical separations of three proteins on an ion exchange column is over 99%, compared to less than 85% for classical peak area evaluation. The power of the partial least squares algorithm (PLS) is shown by measuring the concentrations of Immunoglobulin G (IgG) monomer and dimer under a worst-case scenario of completely overlapping peaks. Here, the faster SIMPLS algorithm is used in comparison to the nonlinear iterative partial least squares (NIPALS) algorithm. Both approaches provide concentrations as well as purities in real-time, enabling live-pooling decisions based on product quality. This is one important step towards advanced process automation of chromatographic processes. Analysis time is less than 100 ms and only one program is used for all the necessary communications and calculations.