Employing Spectroscopic Tools in Downstream Process Control

Spectroscopic tools present an alternative method for reliable at-line process monitoring and control.
Mar 01, 2018
Volume 31, Issue 3, pg 24–26

Rabbitmildphoto/Shutterstock.comRecent advances in process analytical technology (PAT) for downstream process control include the development of spectroscopic tools, which have come into play as a reliable alternative or added process monitoring step that offers real-time process control (1). While early approaches to PAT in biological manufacturing were largely geared toward on-line analytical chromatography using high-performance liquid chromatography (HPLC), the growing use of continuous chromatography equipment in today’s biomanufacturing environment poses a challenge to capturing real-time deviations using at-line HPLC.

Recent advances

Among the most valuable steps to follow in downstream processes are chromatography, ultra-filtration, DNA fragmentation, and virus inactivation, according to Christophe Bonneville, CEO of RESOLUTION Spectra Systems, a supplier of optical analytical instrumentation. The main advances in PAT during those steps have consisted in equipment automatization and sensor improvements.

“For example, purification chromatography systems now include design of experiments (DoE) modules and a certain number of sensors providing valuable quantitative information on the different families of biological product (proteins, DNA, etc.),” Bonneville says.

Early approaches to PAT in downstream bioprocessing were largely geared toward on-line analytical chromatography, when batch processing was the predominant form of biological manufacturing. The move to a continuous manufacturing approach challenges on-line analytics.

“The main challenge is an overflow risk due to the huge number of off-line samples to control and thus a bottleneck at the analytical level. This is quite contradictory with the aim of continuous manufacturing implementation [which aims to be more precise and offers time-savings],” says Bonneville.

At-line HPLC has been implemented in the control of continuous chromatography equipment, and it provides high resolution of different product species. It requires, however, a complex setup, consisting of a device for sampling as well as the chromatograph itself (1). This poses an undesirable trait in the biomanufacturing environment where reliability might be an issue, according to Rüdt et al. in a 2016 study on advances in downstream bioprocessing (1).

“In addition, chromatography only allows to analyzing product families and not each product or molecule inside a family. For example, since both monoclonal antibodies (mAbs) and impurities contribute to absorption at 280 nm (A280), single wavelength measurements are not suitable as selective analytics,” notes Laure Pétillot, scientist at RESOLUTION Spectra Systems.

The role of spectroscopic tools

Other challenges to using at-line process controls include non-negligible time delays as a result of automated sampling and analytical separation. “Depending on the decision time of a unit operation, this may lead to late notice of process deviations or even completely prevent real-time monitoring,” Rüdt et al. said in their study.

“As far as we are aware of, not many solutions currently exist on the market to address these challenges, potentially also because some other challenges need to be addressed first,” Bonneville says.

In-line Raman analysis solutions, such as ProCellics, RESOLUTION Spectra Systems’ in-line, real-time bioprocess Raman analyzer launched in November 2017 (2), offers a real, selective analytical measure, such as enabling the distinction between individual sugars or proteins.

“Furthermore, continuous processes are specifically adapted to putting in place efficient multivariate calibration required to monitor in real time,” Bonneville adds. 

Spectroscopy is considered a practical alternative tool for process monitoring, and spectroscopic equipment carries similar investment costs ($20,000 to $200,000) as on-line HPLC (1). Spectroscopy offers fast measurement times, typically in the sub-second range up to a few minutes, which can often be readily performed in-line.

Having fast measurement times is an especially important factor in preparative chromatography, which is most commonly used in downstream processing. Because preparative chromatography processes are significantly non-linear and feature sharp concentration fronts, the critical quality attributes (CQAs) of the effluent, such as the mass fraction of impurities, tend to change quickly. This requires that the monitoring method used to detect the CQAs have short response times if such processes are to be reliably controlled (1).

Compared to at-line HPLC, spectroscopy has limited selectivity in providing signals for different components. Because of this limitation, information from spectroscopic measurements generally requires the use of a combination of multivariate measurements and mathematical tools for multivariate data analysis (MVDA) (1).

“With the Raman spectroscopy methods existing today, you can indeed obtain very reliable monitoring data as long as a very rigorous data process methodology implemented. Offering a true real-time solution requires a complex software solution,” says Bonneville.

“Raman analyzers enable the control of quality, purity, molecule conformation, and kinetics and can detect end-points. They also help solve the significant analytical control bottleneck problem previously mentioned,” Pétillot adds.

The company’s ProCellics Raman analyzer, for example, enables in-line and real-time analysis, and the software that goes with it was specifically developed to fit with bioprocess analytics, easing multivariate calibration. In addition, both the probe and the base unit can be sterilized, which allows them to comply with downstream environment requirements, according to Bonneville.

Generally, PAT for biologics production has been advancing toward real-time monitoring and controlling CQAs. To that end, spectroscopy-based PAT tools have been applied to a variety of applications with success. Spectroscopy-based tools offer fast measurement times, an important factor in real-time process monitoring and control, and present easy in-line implementation while maintaining comparable costs, according to Rüdt et al.

A major challenge for the future of PAT implementation will be how to have flexible instrumentation and different unit operations, especially with new formats of biologics emerging, such as antibody fragments, nanobodies, conjugated proteins and vaccines, and Fc-fusion proteins (1). 


1. M. Rüdt, T. Briskot, J. Hubbuch, “Advances in Downstream Processing of Biologics - Spectroscopy: An Emerging Process Analytical Technology,” Journal of Chromatography A, DOI: 10.1016/j.chroma.2016.11.010, Nov. 15, 2016.
2. RESOLUTION Spectra Systems, “ProCellics--First In-Line and Real-Time Bioprocess Raman Analyzer,” Press Release, Nov. 14, 2017.

Article Details

BioPharm International
Volume 31, Number 3
March 2018
Pages 24–26


When referring to this article, please cite as F. Mirasol, “Employing Spectroscopic Tools in Downstream Process Control,” BioPharm International 31 (3) 2018.


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