Abstract
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.
Highlights
Inline concentration measurements (ICM) of individual components in a mixture are critical for almost every unit operation in the field of chemical and biotechnological processes
The second example in this paper shows the application of the SIMPLS-algorithm for inline and real-time monomer and dimer concentration measurements of monoclonal antibody Immunoglobulin G (IgG)
The inline concentration measurements are based on Ultraviolet/Visible light (UV/VIS) spectra measured with a diode array detector (Smartline DAD 2600 from Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany) with 256 diodes and a wavelength range of 190 to 510 nm
Summary
Inline concentration measurements (ICM) of individual components in a mixture are critical for almost every unit operation in the field of chemical and biotechnological processes. Most processes are designed to match specific concentration and purity criteria, the actual values still have to be monitored inline or offline [1] This becomes even more important for batch operations, like chromatography, where concentration and purity vary over time. With prior knowledge of the extinction coefficients of each component ελ,i , this set of linear equations can be solved for the concentrations ci This can be done with several mathematical methods like the least-squares or non-negative least-squares (NNLS) [25,26] algorithm. The first example in this paper shows the application of the NNLS-algorithm by measuring the concentrations of three proteins from an analytical ion exchange chromatography Another approach for UV/VIS-diode array detector (DAD) based concentration measurements was introduced by Brestrich et al [18,27,28]. This work is rather a major milestone to achieving a fully automated and self-optimizing system for the prototype, enabling life pooling, purity-based column switching and advanced quality control
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