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In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.

No abstract

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Using a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements, we show that the analytical throughput for selective protein quantification can be increased significantly. An analytical assay based on this new methodology was shown to generate very precise results. Further, the assay was successfully applied as analytics for a resin screening performed in HTE mode. The increase in analytical throughput was obtained without decreasing the level of information when compared to analytical chromatography. This proves its potential as a valuable analytical tool in conjugation with high throughput process development (HTPD). Further, fast selective protein quantification can enhance process control in a commercial production environment and, hence, minimize the need for off-line release analysis.

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Recently, a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements was presented. Analytics based on the methodology was shown to increase analytical throughput for selective protein quantification significantly, however this was only demonstrated for one particular protein combination. In this work, the possibilities and limitations of the analytical method are investigated further. Principal component analysis (PCA) was performed on a broad range of absorption spectra to investigate their common characteristics and differences. The PCA was used both for cluster analysis and to define a measure for spectral similarity. For binary protein combinations, the calibration precision was shown to decrease exponentially with the defined spectral similarity factor. Knowledge of this correlation can be used to determine a priori whether a calibration will be successful or not. Calibration robustness was investigated by applying the analytics to liquid chromatography performed in HTE mode. Further it was shown, that a spectral difference of 0.6% was sufficient to sucessfully preform a spectral based calibration of two IgG1 monoclonals.