Tornado’s Raman analyzers can be used to resolve overlapping chromatographic elution profiles of individual proteins. This quantitative analysis was successfully applied to monitor the purification of BSA and Cytochrome C, and Beta lactoglobulin and Cytochrome C. The greatly increased spectral performance made possible with HTVS™ results in faster and more accurate measurement times for the quantification and characterization of proteins of interest in real time, providing the user with actionable information to facilitate improved process control and provide optimal purification efficiency. In this case study, learn how Tornado’s Raman Analyzer is a powerful tool for monitoring downstream protein purification process quality.
Downstream Protein Purification Optimization
Multivariate Calibration for the Analysis of Metabolites in Cell Culture Media
In this analysis, a design space was generated to produce a multivariate calibration set for a chemically defined growth medium which contained no correlations between variables. The samples were then synthesised in the laboratory and analysed using the HyperFlux™ PRO Plus 785 nm Raman Spectroscopy System.
Raman Spectroscopy has the ability to be used at a multicomponent sensor to monitor substrates and products of metabolism bioreactors. In this study, we used an experimental design that ensured orthogonality between variables. We believe we have demonstrated that with the HyperFlux™ PRO Plus Raman Spectroscopy System, it is possible to directly and reliably analyse many critical components down to at least 0.1 g/L (100 ppm) concentration in complex mixtures.
Tracking Polymorph and Crystal morphology
Crystallization is a commonly used process for solid-dose manufacturing. To improve the biological performance and economic benefits of a crystallization process, proper process control is necessary. However, crystallization can be difficult to control due to the complex relationship between thermodynamic and kinetic factors and processes such as nucleation, growth, and agglomeration. A thorough understanding of the process is required, which can be done through modeling, design of experiment stress testing and the use of process analytical technologies (PAT). Tornado’s Raman Spectroscopy has been shown to be a ubiquitous PAT technology ranging in application from the large molecule to the small molecule space. By coupling the HyperFlux™ Pro Plus Process Raman Spectrometer to a high-quality complementary tool such as the Crystalline® Parallel Crystallizer from Technobis Crystallization Systems, it is possible monitor your crystallization process and accelerate the development of drug products to market.
Residence time distribution (RTD) in the feed frame of a tablet press
Residence time distribution (RTD) has become an essential parameter to characterize pharmaceutical manufacturing equipment in continuous manufacturing. Utilizing Tornado’s HyperFlux™ PRO Plus with their High Throughput Virtual Slit (HTVS™) technology yielded low signal-to-noise(LoD) ratios and excellent performance predicting blend compositions as low as 1.5% w/w in the feed frame. These results indicate the robustness of the Tornado Raman system executing an extensive evaluation of a continuous manufacturing line sustaining the performance during ten days of data acquisition.
Analysis of Protein Glycosylation
Protein glycosylation is a fundamental process in nature that controls essential biological pathways, ranging from protein trafficking and cell adhesion to host–pathogen interactions. Tornado’s Raman spectroscopy has been demonstrated as a technique that can monitor protein aggregation, provide information on protein folding and monitor the glycosylation status of proteins.
The Tornado’s Raman Analyzers aid in interpretation of the molecular nature of particular post-translational modifications, which adds valuable process understanding in the development phase and also ultimately adds value in the process control phase.
Monitoring of Tangential-Flow Filtration (TFF) in Downstream Bioprocesses
Tangential-Flow Filtration (TFF) or ultrafiltration is a technique that is used in many different steps in downstream bioprocessing. TFF flow the feed stream parallel to the filter membrane. A pressure-driven cross-flow is applied and particulates which are small enough to pass through the membranes leave the feed stream, now called the retentate, and enter the cross-stream or permeate.
Tornado’s Raman analyzers with revolutionary spectrometer technology can be used as a process monitoring tool for TFF. Raman is considered to be one of the leading technologies in Process Analytical Technology in downstream processing. Raman spectroscopy is being used to monitor and intensify protein manufacturing including monoclonal antibody production. By using a single Raman probe, one can monitor protein concentration, concentrations of analytes in the feed stream, and any contaminants that are being removed. With another probe in the cross-stream or permeate, one can monitor the removal of undesired contaminants or desired biomolecules in real-time. In a buffer exchange process, one could monitor the incoming buffer, the outgoing buffer, and the protein concentration all with one measurement, eliminating the need for offline assays.
Monoclonal antibodies (mAbs) are biopharmaceutical products of high interest produced by culturing mammalian cell lines. In the last few decades, over 100 mAbs have been produced as marketed drug substances. They are therapeutically used in treating immunological disorders, alleviating drug effects, and fighting cancer to name a few of their uses. In-situ Raman spectroscopy is used extensively for monitoring the production and purification of mAbs. In upstream development, process Raman is used for monitoring key nutrients and metabolites, as well as the concentration of the mAb itself. Other key parameters such as cell density has also been tacked successfully by Raman. In downstream purification, process Raman is used to monitor the mAb throughout the chromatography steps while simultaneously looking for impurities. Other key uses of Raman for the capture chromatography process and other aspects of downstream processing have also been successful and proved highly advantageous to promote cost savings, yield optimization and process efficiency. Process Raman spectroscopy from Tornado Spectral Systems gives you real-time information in all stages of your mAb drug production process.
High-Throughput Raman Process Monitoring of Downstream Protein Purification
Raman spectroscopy is a proven tool for in-line process monitoring and control. It can be used to quantify concentrations of chemical compounds online and in real-time. High-performance Raman spectroscopy, made possible by Tornado Spectral Systems’ patented High-Throughput Virtual Slit (HTVS™), enhances Raman signal by increasing photon flux by 10x to 30x as compared to conventional spectrometers.
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– Sebastian, Analyticon Instruments GMBH