Downstream Process Control

Raman spectroscopy is emerging as a technique for real-time measurements for in-line downstream process control and monitoring. Raman can quantify multiple products and impurities simultaneously with measurement times taking seconds instead of minutes (or longer). 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. This significant advancement in technology enables faster measurements, lower limits of detection, and/or the use of intrinsically safe lower laser power, when needed.

Downstream processing refers to the recovery and the purification of biosynthetic pharmaceuticals from biological sources, such as a fermentation broth. Of particular interest is the production and purification of therapeutic monoclonal antibodies (mAb).

In October 2004, the US Food & Drug Administration (FDA) announced a Process Analytical Technology (PAT) approach for pharmaceutical manufacturers in their Guidance for Industry PAT — A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance. The framework highlighted a shift from traditional quality assurance methods and suggested a move to building in quality throughout the production/manufacturing process. Also included in the PAT framework were the principles of implementation of QbD (Quality by Design), which helps to promote quality assurance through the understanding and controlling of critical process parameters which affect a biological product’s critical quality attributes (CQAs). To ensure high-quality biological products, the understanding of this process must also be applied to downstream bioprocesses.

Rapid and non-invasive spectroscopic methods provide options for the monitoring and control of downstream bioprocess operations, including monitoring purification cycles in-situ, identifying buffers, identifying and quantifying protein products, quantifying impurities such as multimers, aggregates, and species with charge variance, and overall quality control.

Process Analytical Technology in Downstream Process Control

Monitoring the critical process parameters (CPP) with various sensors is the main application of PAT in downstream processes. Flow sensors/meters, pressure sensors, pH and conductivity probes, and spectroscopic sensors are some examples of such sensors. The measurement results derived from such sensors are analyzed by univariate analysis and operator knowledge. However, this is typically not enough to have an understanding concerning CQAs of the product. CQAs include product multimers and product charge variants, host-cell impurities such as host cell proteins, DNA and lipids, and resin leachables. These are typically monitored by at-line or online analyses.

Various HPLC techniques are used to monitor high and low molecular weight species content, charge and glycosylation variants, and aggregates; and they are typically employed as at-line analytical techniques in downstream processes. The typical monitoring technique for host-related impurities is immunological assays. All of these techniques require long analysis times and are still a bottleneck for real-time monitoring and real-time process control [1].

Spectroscopic techniques are great PAT solutions to accomplish these goals, and Raman is a powerful spectroscopic technique with great potential for future applications. Tornado’s HyperFlux™ PRO Plus is a powerful tool for monitoring downstream protein purification process quality. The HyperFlux™ PRO Plus equipped with HTVS™ technology produces highly detailed chemical information, and does so faster, with better signal-to-noise ratios and lower limits of detection compared to all other process Raman instruments on the market. The PRO Plus can be used to establish optimal purification end points, detect process excursions with root cause determinability, enhance process understanding and control of the manufacturing process, and increase yields and profitability.

Raman spectroscopy is already employed in downstream processing applications listed below:

Measurement of product concentration and product aggregation:

The product concentration estimation and monitoring of monoclonal antibodies (mAb) is critical in continuous perfusion processes. This part can be accomplished by UV spectroscopy without the need for advanced predictive mathematical modeling that Raman spectroscopy requires. However, Raman spectroscopy achieves one goal that UV spectroscopy cannot achieve. Raman spectroscopy with quantitative modeling can be used for the characterization and analysis of impurities, such as aggregates. Various concentrations of aggregate mAb types, for example, can be differentiated from each other due to having different Raman spectra.


Distinguishing levels of glycosylation in protein products is critical and is a subject of intense interest. Initial investigations of this type of measurement with Tornado instrumentation have proved very promising for achieving measurements of this type on-line.

Membrane fouling:

The fouling of membranes is a nearly-unavoidable situation when purifying proteins. Raman can be used to track protein concentration and even protein break-though during cross-flow filtration.


Tornado Raman offers reliable testing and methods of release for downstream processing. As a technique, Raman gives users a simple and accurate method of analysis for aqueous-based systems and offers chemical specificity for both excipients and drug products.  This, in turn, provides the efficacy of Raman technology to support a QbD manufacturing environment that requires real-time, in-situ monitoring of downstream bioprocesses.

Customers seeking to adopt Raman spectroscopy as a process analytic technology can request a copy of our whitepaper, High-Throughput Raman Process Monitoring of Downstream Protein Purification, or contact us at


  1. Goldrick, S.; Umprecht, A.; Tang, A.; Zakrzewski, R.; Cheeks, M.; Turner, R.; Charles, A.; Les, K.; Hulley, M.; Spencer, C.; Farid, S.S. High-Throughput Raman Spectroscopy Combined with Innovate Data Analysis Workflow to Enhance Biopharmaceutical Process Development. Processes 2020, 8, 1179. doi: 10.3390/pr8091179


Raman Spectroscopy in Bioprocessing: An Introduction to Spectroscopic Methods of Analysis & Applications in PAT PROAnalytics