Comprehensive, quantitative bioprocess productivity monitoring using fluorescence EEM spectroscopy and chemometrics.

This study demonstrates the application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative predictive analysis of recombinant glycoprotein production cultured in a Chinese hamster ovary (CHO) cell fed-batch process. The method relies on the fact that EEM spectra of complex solutions are very sensitive to compositional change. As the cultivation progressed, changes in the emission properties of various key fluorophores (e.g., tyrosine, tryptophan, and the glycoprotein product) showed significant differences, and this was used to follow culture progress via multiple curve resolution alternating least squares (MCR-ALS). MCR-ALS clearly showed the increase in the unique dityrosine emission from the product glycoprotein as the process progressed, thus provided a qualitative tool for process monitoring. For the quantitative predictive modelling of process performance, the EEM data was first subjected to variable selection and then using the most informative variables, partial least-squares (PLS) regression was implemented for glycoprotein yield prediction. Accurate predictions with relative errors of between 2.3 and 4.6% were obtained for samples extracted from the 100 to 5000 L scale bioreactors. This study shows that the combination of EEM spectroscopy and chemometric methods of evaluation provides a convenient method for monitoring at-line or off-line the productivity of industrial fed-batch mammalian cell culture processes from the small to large scale. This method has applicability to the advancement of process consistency, early problem detection, and quality-by-design (QbD) practices.

Performance monitoring of a mammalian cell based bioprocess using Raman spectroscopy.

Being able to predict the final product yield at all stages in long-running, industrial, mammalian cell culture processes is vital for both operational efficiency, process consistency, and the implementation of quality by design (QbD) practices. Here we used Raman spectroscopy to monitor (in terms of glycoprotein yield prediction) a fed-batch fermentation from start to finish. Raman data were collected from 12 different time points in a Chinese hamster ovary (CHO) based manufacturing process and across 37 separate production runs. The samples comprised of clarified bioprocess broths extracted from the CHO cell based process with varying amounts of fresh and spent cell culture media. Competitive adaptive reweighted sampling (CoAdReS) and ant colony optimization (ACO) variable selection methods were used to enhance the predictive ability of the chemometric models by removing unnecessary spectral information. Using CoAdReS accurate prediction models (relative error of predictions between 2.1% and 3.3%) were built for the final glycoprotein yield at every stage of the bioprocess from small scale up to the final 5000 L bioreactor. This result reinforces our previous studies which indicate that media quality is one of the most significant factors determining the efficiency of industrial CHO-cell processes. This Raman based approach could thus be used to manage production in terms of selecting which small scale batches are progressed to large-scale manufacture, thus improving process efficiency significantly.

Fluorescence excitation-emission matrix (EEM) spectroscopy for rapid identification and quality evaluation of cell culture media components.

The application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics.

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous-based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for "in-house" sample handling, tracking, and quality control.

Prediction of cell culture media performance using fluorescence spectroscopy.

Cell culture media used in industrial mammalian cell culture are complex aqueous solutions that are inherently difficult to analyze comprehensively. The analysis of media quality and variance is of utmost importance in efficient manufacturing. We are exploring the use of rapid "holistic" analytical methods that can be used for routine screening of cell culture media used in industrial biotechnology. The application of rapid fluorescence spectroscopic techniques to the routine analysis of cell culture media (Chinese hamster ovary cell-based manufacture) was investigated. We have developed robust methods which can be used to identify compositional changes and ultimately predict the efficacy of individual fed batch media in terms of downstream protein product yield with an accuracy of +/-0.13 g/L. This is achieved through the implementation of chemometric methods such as multiway robust principal component analysis (MROBPCA), and n-way partial least-squares-discriminant analysis and regression (NPLS-DA and NPLS). This ability to observe compositional changes and predict product yield before media use has enormous potential and should permit the effective elimination of one of the major process variables leading to more consistent product quality and improved yield. These robust and reliable methods have the potential to become an important part of upstream biopharmaceutical quality control and analysis.

Disaggregation of high-molecular weight species during downstream processing to recover functional monomer.

The use of chaotropic agents to recover functional monomeric material was investigated for the downstream purification of an Fc-fusion protein containing high levels of high-molecular weight (HMW) species. In batch studies, chaotropic agents irreversibly disaggregated a majority of the aggregated protein. An integrated processing mode, termed as on-column disaggregation, was developed in which the protein was captured on Protein A chromatography and then a chaotropic agent was used to simultaneously elute the bound protein and disaggregate the HMW species. On-column disaggregation process resulted in protein recoveries of >95% and aggregation reduction of approximately 50%. Analytical results are presented showing that the recovered monomeric material was comparable to the reference protein in biochemical, biophysical, and pharmacokinetic properties. The kinetic and molecular mechanisms governing protein aggregation and disaggregation will also be elucidated. For the Fc-fusion protein studied here, incorporation of the disaggregation strategy in both batch and on-column modes led to an increase of >10% in overall downstream yield.