(389h) Selective Protein Quantification on Continuous Chromatography Equipment with Limited Absorbance Sensing: A PLS and Statistical Wavelength Selection Solution

Monoclonal antibodies are a class of protein that provide targeted and effective treatments for a wide range of diseases. Continuous chromatography is emerging as a vital component of the monoclonal antibody manufacturing process. The long-term operation of continuous chromatography processes relies on real-time estimation of protein concentrations, like that of monoclonal antibodies or host cell protein impurities.

Recently, partial least squares models fit with spectroscopic UV-Vis absorbance data have demonstrated the ability to selectively quantify proteins. However, the practical adoption of such models has been hindered as standard chromatography equipment is only capable of measuring UV-Vis absorbance at a few user-defined wavelengths, while existing demonstrations rely on complete spectral data. To address this limitation, we propose a method for selecting wavelengths that can selectively quantify proteins using continuous chromatography equipment.

First, we create a set of independent protein mixtures and then we utilize a plate reader to measure their absorbance spectra. The spectral data is refined by removing wavelengths that do not follow the Bouguer-Beer-Lambert law. We compare the wavelength selection orders determined by sequential forward search, variable importance to projection scores, and the least absolute shrinkage and selection operator. Finally, the selected wavelengths selected are tried on the Sartorius BioSMB PD continuous chromatography system. We show the selected wavelengths can selectively quantify two proteins with as few as two wavelengths, and the prediction performance is superior to that produced by a set of base-case wavelengths of 230, 260 and 280 nm. The method is shown with different protein concentration ranges and different path lengths.