Using Rayleigh-Mie scattered light and polarised multidimensional fluorescence emission in combination for protein quantification in a model clarified bioreactor harvest

Background
Accurate monitoring and control of bioprocesses require the measurement of critical parameters, such as product and interfering protein concentrations, particularly in the bioreactor harvest prior to during downstream purification. One promising approach is to combine polarised multidimensional fluorescence measurements of intrinsic emission with light scattering measurements. This offers a non-invasive and sensitive method for protein quantification that can be implemented using either polarised Excitation Emission Matrix (pEEM) or Total Synchronous Fluorescence (pTSFS) measurement modes which can deal with matrix interferences from the complex cell culture media which is also fluorescent.
Results
Polarised Synchronous Light Scattering (pSyLS), pTSFS, and pEEM measurements on a sample set of 45 unique samples with varying protein (Immunoglobulin G and Bovine Serum Albumin) and media (yeastolate) concentrations (the model for a clarified, multi-protein bioreactor harvest) were made. Spectral data were evaluated using Principal Component Analysis (PCA) and Partial Least Squares (PLS) regression used for quantifying IgG and total protein content. Best results were achieved with parallel polarised (||) measurements for both total protein content with prediction errors of 11 %, 14 %, and 14 % using TSFS||, EEM||, and SyLS|| measurements respectively, and IgG content, with prediction errors of 10 % for both TSFS|| and EEM||. Quantification was best when the Rayleigh-Mie scattered light signal was included. The primary limitations were a combination of low sample numbers (n = 45) along with a relatively low target analyte range (max 1.8 gL–1), with reference method accuracy being a secondary consideration.
Significance & novelty
Demonstrated for the first time that pSyLS can be used for protein quantification in a complex clarified bioreactor harvest model. This is also the first comparison between polarised TSFS, SyLS, and EEM measurements for estimating total protein and IgG content in a complex interfering matrix. For pEEM measurements the significant result is that for samples with a constrained particle size distribution, including Rayleigh-Mie scatter signal in the model enables better protein quantification. This shows the strong potential of pSyLS, pTSFS, and pEEM measurements for real time bioprocess monitoring of protein content.