In this interview, learn how Raman spectroscopy is used as a Process Analytical Technology (PAT) in bioreactor monitoring and control for cultivated meat production.
Why look at different ways to produce meat?
There are a number of reasons for revolutionizing the way that some meat is produced. Some meat is not entirely sustainable; for example, ongoing population growth means that trying to produce enough meat in the traditional manner can exacerbate already high land usage and water resource consumption.
Traditional meat has a negative impact on climate change, in large part due to the waste products produced. Intensive farming in particular can lead to the overuse of compounds such as antibiotics, and there are concerns around animal welfare. Another challenge with meat production is linked to the security of the food chain for humans and how easily this can be disrupted.
At Ivy Farm, we believe that cultivated meat is a good solution to these challenges. Cultivated meat involves growing in vitro cells with the aim of using these as part of the food chain. Growing cells in vitro to produce cultivated meat reduces land usage by growing cells in a bioreactor, while the controlled environment might help reduce emissions.
Similarly, the controlled nature of in vitro cultures will help avoid any negative issues surrounding the use of antibiotics. Cultured meat can also be produced continuously year-round.
Meat represents a large market with a lot of monetary value. A lot of people on the planet like to consume meat, and along with developing technology, we believe that successfully cultivating meat in vitro will gradually result in an increase in demand.
What is Ivy Farm doing to address the challenges of producing cultivated meat products?
One challenge in making cultured meat more accessible stems from the relative inexpensiveness of existing meat products. Even premium cuts and types of meat typically only cost around £30 per kilo, meaning we face a significant challenge in terms of reducing the cost of growing cells in bioreactors and developing processes to do this.
Ivy Farm is an R&D company at the moment. A lot of work goes into sourcing suitable animals in the first place, but the real R&D work starts with stages two and onwards where we look at how we can extract cells from tissue samples, isolate these, and then start to engineer, edit, and select the best lines for growing in in vitro culture before scaling up to large bioreactors.
We also perform our own media development in-house, ensuring we get the formulation right for these novel cell lines. We are working on process development and looking at scale up, evaluating stir tanks and bioreactors that are larger in volume than many standard cell culture processes.
We are also looking at developing food technology processes that allow us to turn cell suspensions into quality meat products. Some of our work is similar to existing processes that use cells and biotechnology, for example, the way that current protein alternatives are processed.
When developing these processes, we compare and contrast what happens between traditional meat and what we are proposing to do with cultivated meat.
For example, raising an animal takes several years, but we want to develop a more commercially viable process. We aim to revive cells from cryopreservation and start the production run within about three or four weeks. However, once we are working with terminal-scale reactors, we are looking at continuous bioprocessing to extend the manufacturing window. Post-reactor and post-processing would likely use a supply chain similar to the traditional meat supply chain.
In terms of our final product, growing cells in suspension and processing these can offer a good alternative to a traditional cut of meat. Many metrics that are measured in meat can be applied to a cell grown in suspension, and this has a very similar makeup when you consider the per-weight percent basis.
For example, its amino acids are very similar, cultured meat’s iron content is slightly higher, and omega-3 and omega-6 are improved in our cell suspensions. The ratios of saturated and unsaturated fats have also improved in our products.
We originally focused on pork cells, but Ivy Farm has moved toward working with bovine cells. The data sets for each type of animal cell were comparable, as were those for cells grown in suspension and cuts of meat currently available to the consumer.
In terms of creating an end product for the consumer, our product development team created a well-known product - Scotch eggs - by working closely with other food technology innovators. This product was widely reported and acted as a good proof of concept.
Much of our work uses standard bioprocessing techniques, and we are often able to increase the yield as we move from understanding the batch culture to employing feeds and continuous modes of operation, whether chemostat or perfusion.
Reducing immediate costs is a major enabling factor in developing a continuous culture. This will eventually allow us to create more up-to-date bovine lines to produce much more reliable amounts of cultivated meat products.
A lot of the bioprocessing we are currently doing is comparable to a cell therapy or biopharmaceutical production process. We look at the batch culture to understand what metabolites are being consumed, what complex proteins we might need to provide the cells to prompt them to divide, and how we can characterize the bioreactors themselves in terms of mass transfer kinetics.
Once we understand what our cells require to flourish, we can move towards continuous processes, allowing us to intensify the cells toward yields that will have a greater impact on producing total amounts of meat.
Image credit: tilialucida/shutterstock.com
How does process analytical technology help address challenges in bioprocesses like producing cultivated meat products?
Growing cells for cultivated meat is essentially a bioprocess (upstream cell culturing), but the principle applies to other bioprocesses as well, be it cell and gene therapy, nucleic acid manufacturing, or downstream processes. All bioprocesses have some critical process parameters (CPPs).
In Ivy Farm’s case, these are parameters such as pH, temperature, oxygen levels, nutrients, lactate, growth factor, and differentiation factor. These critical process parameters determine product quality and quantity, so it is important to maintain tight control of any process variation to ensure uniformity and quality of the final product.
The way a process is often controlled is highly dependent on laboratory characterization testing. This means that the sample is acquired at a specific time and taken to the lab for analytics to be performed, with final decisions made based on the result of laboratory testing. This type of offline analytics results in increased operational cost, time, and resources, however, and this type of sampling always carries with it a risk of contamination.
Bioprocesses are dynamic with wide-ranging variation, so the discrete sampling needed for laboratory characterization testing does not provide adequate information about process variations. This strategy is not reliable for process control of highly dynamic processes, potentially leading to the non-uniformity of products, quality, and quantity.
The ultimate objective of next-generation cultivated meat production is minimal reliance on characterization testing and a shift to real-time process monitoring and control. We can achieve these objectives by introducing advanced technologies, innovative processes, and digital transformation - three key features of process analytical technology (PAT).
Process analytical technology establishes a regulatory framework intended to facilitate the development and implementation of innovation in bioprocess development, manufacturing, and quality assurance. The key objective of PAT is to ensure that quality is built into the process by adopting quality-by-design principles.
Understanding a process and product knowledge are key principles, but the core of a PAT is quality by design. This means that quality control should be ingrained in a process rather than determined by testing a product after it is manufactured.
There are several key elements in process analytical technology. The first point is that this approach focuses on in-process monitoring. Bioprocesses are subject to a complex set of dynamics with wide variation, meaning that the only way to understand this process variation is by implementing a technology embedded in the process itself.
It is also essential to consider what tools can be used as a PAT, such as a spectrometer or a chromatogram. The term ‘analytical’ in PAT has a broad meaning, potentially including chemical, physical, microbial, mathematical, and risk analysis in an integrated manner. Overall, PAT is a concept designed to ensure uniformity in product quality and quantity.
A PAT instrument can be operated at-line, on-line or in-line. An at-line instrument means the sensor is not directly connected to a process. Instead, the sample has to be drawn from the process and introduced into the sensor. In on-line analysis, the sensor is connected to the process somehow, but the sensor is not in direct contact with the process. As its name suggests, in-line analysis means the sensor is integrated into the process.
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What are the benefits of using Raman PAT when working with complex bioprocesses like cultivated meat production?
Bioprocess parameters are complex and dynamic, and there is variation in the time domain, but Raman technology allows us to understand those processes. Using Raman sensors in-line generates continuous data that allows us to understand variations in our bioprocesses.
Thanks to its array of product features, the Thermo Fisher Scientific™ MarqMetrix™ All-In-One Process Raman Analyzer is ideally suited to the role of in-line process analysis.
The instrument’s probes are interchangeable, allowing us to measure various processes, whether samples are in dynamic flow or static, solid, liquid, or gaseous. The instrument is also factory-calibrated to simplify implementation without daily calibration.
The analyzer’s small footprint is important because manufacturing in bioprocessing applications typically occurs in a GMP environment where space becomes limited. A compact instrument helps to integrate process Raman analysis throughout the product development journey.
The MarqMetrix All-In-One process analyzer uses Raman spectroscopy, which works by scattering light off a molecule and analyzing it to identify the molecule's chemical structure. Biology is dominated by nonpolar covalent bonds, unsaturated bonds, aromatic bonds, and disulfides, all of which exhibit a strong Raman signature. Raman is a vital tool in the study of biosamples.
The intensity of Raman scattering depends on the intensity of the initial incident light used; the more energy we use, the stronger the spectrum. The intensity of the scattered light also depends on the number of molecules present and their concentration.
Raman spectroscopy has two key applications in the context of cultivated meat production: the identification of target molecules and the quantification of them. It is also important to note that Raman has a low water background signal, meaning that water in a sample has minimal impact on its analysis.
Every molecule has a unique fingerprint that gives it molecular specificity. The spectrum has few overtones and combination bands, making it easier to interpret. Raman also probes the molecule in its native state, providing useful information. It is an accurate, reproducible, rapid, and nondestructive method. Most importantly, a single scan allows us to monitor multiple critical quality attributes (CQAs)—a key consideration for in-line process monitoring.
All these features of Raman spectroscopy can be leveraged for identification and quantification as part of process monitoring and control.
What role do chemometric models play in analyzing cultivated meat products?
Chemometric models are extremely useful in Raman analysis because they use a sample's spectrum to predict some properties. However, generating training data to build a chemometric model to interpret a data set can take a long time.
In the case of Ivy Farm, we initially built the model using a different process and then transferred it to accommodate the organization’s cultivated meat production date. The two models used in these studies were the glucose and lactate core models.
This approach of transferring existing models allowed Ivy Farm to save time and costs because there was no need to collect training data on their specific processes. We used different runs to build the chemometric training models, including different cell types, media, skills, modes of bioreactor operation, and processes.
A PLS algorithm was used to build the models, with five latent variables selected based on a press plot and a VIP score plot. The VIP score plot showed us which variables were important in building this model.
Although cultivated meat process data was not used to train these models, the models were transferable to these processes. All the models were developed using training data from a CHO cell line, which is generally used to manufacture monoclonal antibodies. In this study, because the models were built on a specific molecular signature of glucose and lactate, they were transferable directly from one process to another.
In addition to ensuring the transferability of our model across processes, we were also interested in testing whether this model could be scaled up. We built a model using a 5-liter small-scale bioreactor and then tested this in a 500-liter bioreactor using the glucose and lactate models.
We found that our Raman models were scalable from a small volume to a larger volume, which is highly beneficial considering a product's lifecycle, which starts from bench skills before moving to manufacturing in large volumes. We also tested this model using different cell lines in different processes, confirming that it is transferable across different cell lines and media.
How can Raman spectroscopy be used in maintaining tight process control?
The strength of Raman spectroscopy lies in monitoring, and its capacity for in-line measurement enables tight process control. To demonstrate this, we used a glucose model to predict the glucose concentration and hold this as a desired set of values.
We tested these capabilities by starting a bioreactor run at six grams per liter and setting a glucose control point at four grams per liter. Initially, the glucose was consumed, and once the glucose was below three grams per liter, we triggered the logic process to hold at four grams per liter for the entire run.
The Raman instrument was integrated with a bioreactor in this scenario, and core models were loaded into the Thermo Scientific™ Lykos™ PAT software. The predicted values from Raman were communicated to Thermo Scientific™ TruBio™ Bioprocess Automation and Control Software using the OPC UA platform, which controlled the pump. This feedback control maintained the glucose at four grams per liter throughout the bioreactor runs.
Process Raman analysis is fast, reliable, and data-rich, making it suitable for monitoring, controlling, and automating bioprocesses occurring in the aqueous phase.
Accurate and reliable monitoring and control of critical process parameters during cultivated meat production can improve the quality and quantity of products.
We have seen that accurate, reliable, transferable glucose and lactate core models facilitate quick adoption of technology, allowing a user to leverage the unparalleled benefits of real-time measurement of process Raman analysis as a PAT solution.
Monitoring several parameters in a single scan also allows automated multimodal feedback control for human-free operation, providing tighter process control and product quality uniformity.
About the interviewees:
Nimesh Khadka is a Senior Product Applications Specialist at Thermo Fisher Scientific, specializing in analytical biochemistry, spectroscopy, and chemometrics. Driven by a passion for innovation, he is currently championing Raman spectroscopy as process analytical technology (PAT) for developing protein and nucleic acid therapeutics.
Nimesh completed his Ph.D at Utah State University with focus in enzymology, spectroscopy, and alternative bioenergy. His postdoctoral training was conducted at Case Western Reserve University, where he dedicated his research to the study of structural biology in biomolecules associated with eye pathologies. This extensive background has provided Nimesh with a deep understanding of the intricacies of biomolecular structures and their applications in various fields. As an active member of the American Chemical Society, Nimesh remains at the forefront of emerging technologies and is committed to driving innovation to meet customer’s need.
About Thermo Fisher Scientific – Portable and Handheld Raman Spectroscopy
Thermo Fisher Scientific offers innovative solutions that help our customers solve complex analytical challenges, accelerate life sciences manufacturing, deliver medicines to market, and increase laboratory productivity. Our Thermo Scientific portable and handheld process Raman analyzers enable accurate, real-time results for process monitoring.
Our Thermo Scientific™ MarqMetrix™ All-In-One Process Raman Analyzer is an all-in-one system purpose-built for rapid deployment, ease of use, and scalability in markets where time-to-results is critical. The Ramina Process Analyzer is designed for:
- Analysis without sample preparation, delivering Raman spectral results in real-time
- Easy setup and deployment by non–Raman spectroscopists
- Non-destructive workflows to protect precious samples
- Non-invasive handling to minimize contamination of samples
- Small footprint for convenient deployment
- Factory calibration for hardware stability and portability
Our Thermo Scientific TruScan G3 Handheld Raman Analyzer includes state-of-the-art optics paired with a patented multivariate residual analysis that offers an effective chemometric solution for material identification, with two spectral pre-processing options. The analyzer’s non-destructive point-and-shoot sampling principle facilitates rapid verification of a broad range of chemical compounds, including cellulose-based products.
The Thermo Scientific TruScan G3 Handheld Raman Analyzer takes pharmaceutical manufacturing QA/QC to the next level with:
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