Bioprocesses are dynamic environments. As cell cultures grow and express the biological products required for medicines and vaccines, conditions within the bioreactor continually change. Understanding those changes in real time requires analytical technologies that can operate within the process itself.
In-line Process Analytical Technology (PAT) can provide manufacturers with real-time information about changing conditions within a bioprocess. Raman spectroscopy has considerable potential in this role because it can provide molecular information without removing samples for every measurement. However, moving Raman spectroscopy from the laboratory into a working bioreactor creates a different set of challenges. The probe must deliver useful spectral information from a complex biological sample. It must also fit within the physical constraints of existing systems. In deep-UV Raman, the optical design introduces further considerations. It must balance the benefits of fluorescence suppression and resonance enhancement with limited penetration depth and the potential for photodamage.
As part of the Innovate UK Sustainable Medicines programmes, Future Factories and InSPIREmed, Dr Yang Qian and Dr Will Brooks are addressing these challenges. They are developing a series of deep-UV Raman probes for in-line PAT applications.
For PAT, optimising spectral performance is only part of the challenge.
Bringing optical and mechanical design together
Yang leads the optical design, while Will develops the mechanical architecture needed to integrate the probes into existing bioreactor systems. The two areas are closely linked. Probe dimensions, access ports and positioning constrain the optical geometry. At the same time, optical system requirements influence the mechanical design.
Deep-UV Raman offers particular advantages for biological samples. Shifting the excitation into the deep-UV can significantly suppress fluorescence interference. Resonance enhancement can also increase the Raman response from particular functional groups. Together, these effects can yield cleaner spectra and improve sensitivity. This can be particularly valuable for samples that are difficult to characterise using conventional Raman excitation wavelengths.
Balancing spectral performance and photodamage
However, deep-UV excitation introduces its own design considerations. Biological material strongly absorbs UV radiation, which limits penetration depth. Excessive optical exposure can also cause photochemical changes in the sample. For an in-line PAT probe, this is critical. The measurement must provide useful analytical information without materially altering the cells, product or process under monitoring.
The risk of photodamage depends on both exposure time and the optical power density at the sample. Optical power and the dimensions of the laser excitation spot determine this power density. In turn, the probe’s mechanical geometry and physical constraints can influence the size of the excitation spot. The sample’s kinetic state may also matter. In a static solution, the same material can remain exposed to the excitation beam for longer. This could increase the likelihood of photodamage. In a continuously flowing system, material moves through the interaction region. This reduces the effective exposure individual cells or molecules experience. The team is currently investigating this as part of probe development and testing. The design must therefore consider several factors alongside the required spectral performance. These include optical power, excitation spot size, exposure time, probe geometry and sampling dynamics.
Developing in-line Process Analytical Technology for bioprocessing
In-line measurement does more than remove the need to take samples for *ex-situ* analysis. Conditions within a bioreactor change as cells grow, consume nutrients and produce target products. Measurements taken at intervals can provide important information. However, they capture only snapshots of a continuously evolving process. An in-line Raman probe can obtain spectral information directly from within that environment as the process develops. This could provide a more detailed understanding of what is happening inside the bioreactor. Ultimately, it could help manufacturers identify meaningful changes in the process as they occur.
Achieving this requires more than demonstrating that deep-UV Raman can generate useful spectra from a biological sample in the laboratory. A PAT instrument must also work within the physical and operational constraints of bioprocessing equipment.
Designing for existing bioreactor systems
Existing bioreactors already use probes and sensors to monitor parameters such as temperature, pH and dissolved oxygen. They also have established access points and mechanical interfaces. Designing Raman probes around these practical constraints provides a clearer route to integration. It allows manufacturers to introduce new analytical equipment into existing systems, rather than adapting those systems to the spectroscopic solution.
As part of the development process, Yang and Will have examined how existing probes integrate into industrial bioreactors. The mechanical constraints of these established systems now inform both the optical and mechanical design.
This also explains why no single probe configuration is likely to suit every application. Different bioprocesses present distinct analytical targets, sample environments and physical constraints. The optimum optical configuration depends on what needs to be measured. It also depends on the sample’s properties and the physical environment in which the probe must operate. Rather than developing a single universal configuration, the team is creating a series of probe designs tailored to specific measurement requirements. They treat each probe as an integrated optical and mechanical system. This approach allows them to balance competing requirements across different applications.
Engineering Raman for the real process environment
The work highlights an important aspect of developing new PAT instrumentation for bioprocessing. Optical performance cannot be considered in isolation from the environment in which the measurement takes place. For Yang and Will, the challenge is not merely to obtain good-quality Raman spectra. They must engineer a measurement system that delivers useful spectral information *in situ*. At the same time, the system must work within the practical constraints of a bioreactor or other downstream processing equipment.
By combining optical and mechanical design from the outset, the team is helping to move deep-UV Raman spectroscopy closer to practical PAT for real-world bioprocess monitoring.
