Dean Stuart- Product Manager at Thermo Fisher Scientific
Raman spectroscopy is widely used in many applications – including biopharmaceutical manufacturing – that require analysis of the chemical composition of solid, liquid or gaseous materials. This non-destructive analytical technique can not only provide detailed information about the chemical structure, but also give insight into the phase and polymorphy, as well as crystallinity and molecular interactions. The drawback is that it often involves complex analytical workflows and requires the use of highly specialized equipment together with expert knowledge in Raman spectroscopy techniques. New technologies have been introduced to the market in recent years, making Raman technology more accessible to a wider public through analyzers with intuitive control and user-friendly interfaces. This article talks about the need for modern process analytical technologies that are accurate and reliable – while being more accessible to lab technicians – and how they can be used for biopharmaceutical process monitoring.
Biopharmaceutical manufacturing has revolutionized modern medicine through novel active ingredients that have enabled highly specific and effective treatments for a range of diseases, including cancer and autoimmune disorders. Biopharmaceuticals are created by host cells that are made to express the desired product, which is subsequently isolated, purified, and then formulated to ensure a consistently safe and efficacious medicine. When working with living organisms, the environment must be just right as even small variations in parameters such as pH, temperature, dissolved oxygen, feed composition and feed timing can affect the yield and quality. It is therefore crucial to understand every aspect of each step in the process to ensure optimal process control and avoid costly mistakes, including failed batches, inefficient use of resources and end products that do not pass quality control checks.
Careful Process Monitoring
Process production is a method used for bulk manufacturing of goods such as pharmaceuticals, foods and beverages, refined oil, gasoline, chemicals, and plastics. It makes use of a specific formula or recipe to create a product from a combination of ingredients or raw materials, involving numerous checks throughout the entire process, starting by establishing the quality of the raw materials and intermediate compounds, all the way through to testing the purity of the end product. The process analytical technologies (PATs) used for this purpose must be accurate and reliable, as well as adaptable, as they will be employed at different manufacturing stages. An example of a technology that is flexible enough to be used for process production is Raman spectroscopy.
Raman spectroscopy is a powerful analytical tool that provides rapid and precise analysis without being destructive to the sample. It has significant advantages over other spectroscopic methods, such as infrared (IR) and near-infrared (NIR) spectroscopy, as it offers specificity, compatibility with aqueous systems, and sampling flexibility, making it the method of choice for process monitoring.
Identifying Molecules
Raman spectroscopy helps extract information about the chemical structure, phase and polymorphy, crystallinity and molecular interactions by observing how laser light interacts with the matter. The laser beam is delivered to the sample using a fiber-optic cable with a probe at its end, and the incoming energy causes the molecules to vibrate and scatter the light, which is collected and interpreted by a detector. The scattering can be either elastic, with the energy of the molecule unchanged after interaction with the photon, or inelastic, where the molecule absorbs some of the energy and the scattered photon loses energy, resulting in a color change. The latter process is important as it provides valuable insight regarding the molecules present, generating a so-called Raman spectrum – a collection of peaks at certain photon frequencies that is unique to each molecule and can be used as a fingerprint to identify it. In this manner, it is not only possible to identify which molecules are present, but also in which amounts.
Small Footprint, Large Impact
Raman technology is non-destructive, which makes it ideal for continuous process monitoring with in-line or on-line analysis. It can be integrated directly into a production line and delivers results in a manner of seconds. The downside is that, up until now, this technique has demanded complex, bulky and expensive equipment, as well as a specialist technician to operate and maintain the instrument. These requirements compromised the reliability of this approach and increased the operational costs. The introduction of compact, easy-to-use, reliable and affordable systems changed the Raman technology landscape, making it accessible to a wider public. These new devices are not only smaller but also designed with less experienced operators in mind, offerring an intuitive user interface that allows non-experts to benefit from everything that Raman technology has to offer. Manufacturers can now easily integrate this technology into their production process, increasing efficiency and product quality.
Detailed Process Information
Raman spectroscopy can handle samples in different forms, including solid, liquid, gas, powder, aqueous solutions or slurry, and each peak in the spectrum provides detailed information about the substances that are present. This flexibility allows testing at various points in the production process to monitor the analyte of interest, which is especially beneficial when dealing with a bioreactor that often contains many types of molecules. Quantifying the amount of a certain substance from a Raman spectrum is straightforward as there is a linear relationship between the intensity of a peak and the concentration of the corresponding molecule. It is therefore easy to build quantitative models that accurately predict the concentration even when dealing with a relatively small sample set. These useful features open up a range of possibilities and can be used throughout the entire biopharma manufacturing process to verify the integrity of raw materials, monitor bioreactor processes in real-time and evaluate the end product. Raman spectroscopy can answer questions such as ‘Are the cells supplied with the right amount of glucose?’, ‘Are too many secondary metabolites building up?’, ‘Have the cells begun to produce the desired product?’ and ‘How much product has been produced, and does it have the right characteristics?’. As all the answers are provided in real time, it is possible to make continuous adjustments to optimize the processes.
Monitoring Across the Entire Production Chain
Process analysis involves several types of measurements that can be divided into four primary classes, defined by their location and whether the sample needs to be removed from the production line for testing:1
In-line measurement
During in-line measurements, a probe or sampling interface is placed either inside or in line with the process or product flow. This usually means inserting a probe directly into a flow system or bioreactor, to continuously monitor the product. Using Raman spectroscopy at this stage allows evaluations at several different locations in parallel to determine product consistency throughout the process. This is possible because neither probe nor sample needs to be removed during the measurement process.
On-line measurement
On-line measurement is similar to in-line monitoring, but with some differences; although the sample can still be measured without being removed, a part of the product is redirected for analysis. This means that measurements are performed on just a portion of the product, and the diverted sample can be re-introduced to the process stream or diverted to waste, depending on the application.
At-line and off -line measurement
Contrary to in-line and on-line measurements that do not require the sample to be removed for analysis, at-line and off-line measurements involve checks away from the production line. In case of at-line measurements, the tests are run in close proximity to the production facility, while for off-line measurements the sample is transported to a remote laboratory. Compact Raman analyzers – for example miniaturized handheld Raman analyzers with quantitative analysis capabilities – are ideal for effective measurements in either of these environments.
Optimizing Glucose Levels
Raman spectroscopy has proven to be useful for many different applications in biopharmaceutical production, and one example is modeling of the glucose content of a bioprocess. Glucose is required for cell reproduction and is subsequently added through a feeding cycle to keep a constant rate of cell production. Keeping glucose at the right level is of utmost importance for most processes as it is directly connected to the yield, and modeling its content can therefore help gain precise control of the production rate.
Modeling requires data which, in this example, has been collected from a bioprocess performed at varying global locations,2 using the same instrument set-up at each site (see Figure 1). The stability and accuracy of the analyzer ensured consistency of results between the different locations. Additionally, fundamental preprocessing methods were used to target and amplify the relevant signals within the Raman data. This means that, although the individual data sets were small, the gathered information could be combined, resulting in a precise predictive ‘global’ glucose model that could be used at any location using that specific set-up.
This powerful tool can off er real-time tracking of the glucose concentration throughout the bioprocess by analyzing the output. Application of this model to a fourth site is illustrated in Figure 2, showing how the glucose level steadily declines over time until a specified minimum value is reached, rising again after the addition of glucose. Using Raman spectroscopy this way ensured optimal process control.
Summary
Raman spectroscopy has proven to be useful in various applications in both research and manufacturing, including process control for biopharmaceuticals, where it offers enhanced, non-destructive compositional measurements for a variety of sample types. This technology has several advantages over other spectroscopic methods, such as infrared and near-infrared spectroscopy, providing specificity, compatibility with aqueous solutions, and sampling flexibility. In addition, modern analyzers based on this technology are easier to use compared to old models that required expert knowledge to operate. These new systems are also smaller, which makes them perfect for real-time monitoring of bioprocesses both in-line and on-line, as well as for at-line and off -line measurements, opening up the technology to more applications and facilities. When combined with data science tools, Raman analysis can help manufacturers gain precise control over their processes by monitoring relevant parameters such as glucose, lactate, glutamine and glutamate, amino acids, pH, cell viability, and cell volume. Ultimately, this will enable higher yield and quality of the end product, while minimizing waste and reducing costs.
References
- Application note. In-line, on-line, at-line, or off -line: how solid-state Raman analyzers can be adapted for any point of need. WP-RAMINALINE-0322 v01. Thermo Fisher Scientific.
- Application note. Using process Raman and data science to gain actionable information for glucose monitoring. AN-RAMINA-0522 v01. Thermo Fisher Scientific.
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