Getting Real-Time Information from Biotechnological Processes

How Raman process monitoring is providing a spectra of benefits across the biopharmaceutical industry. Dean Stuart, Product Manager at Thermo Fisher Scientific

  • Traditionally, the main drawback preventing the more widespread use of Raman spectroscopy has been its reliance on expensive, complex and bulky equipment, requiring technical specialists to be on hand for operation and maintenance.
    Traditionally, the main drawback preventing the more widespread use of Raman spectroscopy has been its reliance on expensive, complex and bulky equipment, requiring technical specialists to be on hand for operation and maintenance.
  • Compact sized devices allow flexible real time monitoring of biopharmaceutical processes.
    Compact sized devices allow flexible real time monitoring of biopharmaceutical processes.

Biopharmaceutical processes involve the production of substances that are either manufactured in, extracted from, or semi-synthesized from biological sources. These methods are remarkably versatile, and capable of producing everything from vaccines to tissue and blood components. However, processes using live cultures can be much more challenging to control than those using chemical reactions alone, and even slight environmental perturbations can lead to wildly varying endproducts. The implementation of process analytical technologies (PATs) at multiple points in the production line can provide real-time information about the process, answering questions such as: Do the cells have the right amount of glucose? Are too many secondary metabolites building up? Are the cells beginning to produce the product of interest? How much product has been produced, and does it have the right characteristics?

RAMAN SPECTROSCOPY

Raman spectroscopy is one the most powerful and versatile PATs available today, offering rapid and accurate analysis whether a sample is in solid, liquid, gas, powder or slurry form. The technology relies on the Raman effect – the inelastic scattering of photons – to identify the individual constituents of a sample. It works by using a fiber-optic probe to direct a laser light at the sample, which energizes the covalent bonds of molecules, causing them to scatter the light. This can either be via an elastic process – with the energy of the molecule unchanged after interaction with the photon – or an inelastic one, where the molecule absorbs some of the energy and causes the scattered photon to change wavelength. Raman spectroscopy uses the inelastically scattered light – collected and interpreted by a detector – to generate a Raman spectrum. This ‘molecular fingerprint’ is unique to each molecule, and enables the qualitative identification of analytes present in the sample. In addition, because the relationship between spectral peak height and analyte concentration is linear, accurate predictions of constituent quantities can be made, even with small volume samples.

STATE-OF-THE-ART DEVICES TO SUIT MODERN PROCESSES

Raman spectroscopy measurements are nondestructive, rapid and – unlike many other forms of spectroscopy – suitable for the analysis of aqueous solutions, making them ideal for the continuous monitoring of biopharmaceutical processes. Since the composition of product streams in these types of manufacturing lines can vary from minute-to-minute, real-time data indicating the current state of the reaction can be invaluable. Traditionally, the main drawback preventing the more widespread use of Raman spectroscopy has been its reliance on expensive, complex and bulky equipment, requiring technical specialists to be on hand for operation and maintenance. However, recent technical developments have allowed for the introduction of more compact, affordable and user-friendly devices, such as the Thermo Scientific™ Ramina™ Process Analyzer. This new generation of instruments offers simplified user interfaces suitable for non-expert operators, and their compact size makes them viable for manufacturing lines and labs where space is at a premium.

ANALYSIS OPTIONS TO SUIT YOUR NEEDS

The exact process analytics requirements of each biomanufacturing workflow will differ considerably, depending on the input materials, microorganism, critical process parameters and target end-product. The flexibility of Raman spectroscopy as a PAT lends itself to use across various stages of the workflow, meaning that the installation and operation of Raman process monitoring equipment will depend on both the process and the bioreactor set-up. Regardless of the intended use, the implementation and analysis procedures will fall into one of four distinct categories: at-line, off-line, in-line and on-line. At-line and off-line measurements involve the removal of a sample to test outside of the product stream. While at-line analysis relies on nearby Raman spectroscopy stations for testing, off-line measurement involves the transfer of samples to a formal lab setting away from the production site. At-line or offline measurements are more than adequate for some processes, and compact analyzers with quantitative analysis capabilities – such as the handheld Thermo Scientific™ TruScan™ RM with TruTools – are ideal for these applications. However, a potential issue with these sampling methods is that, by the time the results are received, some processes may have become irreversibly out-of-tolerance, negating the possibility of any compensatory adjustments. This is especially true in the case of off-line measurements with a slow turnaround time, and can lead to failed batches that waste raw materials, time and money.

IN-PROCESS MEASUREMENT AND CONTROL

In-line measurements rely on the sampling device being inserted directly into the process flow, meaning that samples neither need to be removed from the bioreactor nor sent to a separate location for testing. Likewise, on-line measurements do not require sample removal, but divert a portion of the stream to an adjacent sampling loop in order to perform the Raman analysis. The sample can then either be returned to the process stream or discarded, depending on the application. These sampling and measurement methods allow for extremely rapid turnaround of results, making continuous monitoring of fast-evolving processes possible. When these analysis strategies are combined with control feedback loops, real-time automatic parameter adjustments can be made to keep the process within tolerance. Additionally, intelligent software solutions can use the extrapolation of data trends to predict the future state of the process, giving operators an added layer of confidence that the endproduct will in-spec.

USHERING IN A NEW ERA OF RAMAN MONITORING

Careful choice of PATs can help to ensure that raw materials, intermediate compounds and end-products are all of the required purity. Modern Raman spectroscopy systems are ideal for the monitoring of biopharma processes, providing non-destructive compositional measurements across a wide array of sample types, without requiring expert users for operation, maintenance or interpretation of results. Their compact size and relative affordability have also made them accessible to smaller and lower throughput facilities, for which traditional Raman solutions would not have been a viable option. In-line and on-line analysis approaches also enable the combination of real-time monitoring with automated process control, offering the potential to allow the manufacture of certain products which would not be commercially viable using at-line or off-line testing methods. Whether enabling the generation of new products, or simply optimizing the manufacture of existing ones, Raman analysis can provide biopharmaceutical manufacturers with the data required to make real-time, informed decisions to optimize their processes.