Bioreactors are the most important tool in bio-materials manufacture. Here’s a guide to the latest developments in bioreactor design.
Bioreactors are vessels where microorganisms, stem cells, or specialised cells are cultivated into functional materials. The oldest and simplest examples are containers used for baking bread or fermenting wine. Over the last twenty years, however, they have become sophisticated instruments whose internal environments can be precisely manipulated to formulate advanced biomaterials. Different models are capable of incubating proteins for the vegan meat market, bio-based textiles, stem cells, biofuels, polymers, or bacterial solutions for wastewater treatment.
Inside bioreactors, cells are grown in a medium that supplies nutrients like phosphorus and nitrogen as well as gases like oxygen, nitrogen, and carbon dioxide. The temperature and Ph of this medium also affect cell growth. Adjusting these variables elicit particular enzyme reactions or chemical transformations.
Traditionally, bioreactor production has relied on a batch system. In this method, cells are removed from vessels after they have consumed all the nutrients in the medium. Normally, this takes 7 to 21 days. Recently, there has been a move toward perfusion bioreactors that automatically tops up media and removes any waste. This extends the running time of cell growth to weeks and months. Cellular Agriculture Ltd. is leading research into how these machines might be scaled for the cultured meat industry.
The costs of bioreactor operation
Scaling in the bio-materials industry depends heavily on minimising operation costs for bio-reactors. Estimating operation costs needs to account for all upstream processes: the number of starting cells needed to achieve a target volume, how many of these cells remain viable and grow, how much media is needed to feed them, and the scaffolding material. Downstream processes also need to be factored in. These are the financial and time costs of sterilisation and methods for purifying target materials from the solution once they have finished growing.
Another cost factor is finding readily available nutrients that will feed cell growth inside the bioreactor. One popular solution has been to use industrial wastewater to feed microalgae and other substances. This simultaneously produces clean water and repurposes any waste into inputs for high-value bio-products such as biogas.
The cost of the eventual bioproduct is heavily influenced by the final concentration of target material within the gas and nutrient solution, which becomes a waste byproduct. Highly diluted solutions are cost-inefficient since it ramps up the need for post-growth separation. Ideally, the desired materials should be present at very high concentrations in the solution to cut down on downstream processing.
Achieving highly concentrated mixtures poses its own technical problems. The usual method for separation has been depth filtration but filters for this may be too coarse when the target product is present at extremely high densities in the solution. Lately, more fine-tuned filters have been developed such as Pall’s Tax mAx clarification platform launched in 2019. As with many bioreactor innovations, this tech has been designed for the pharma industry for concentrated cell cultures of up to 2000 litres.
Controlling growth: digital systems
Identifying and sustaining the right levels of nutrients, gases, temperature, and Ph is central to industrial bioproduction. In their early days, fine-tuning these parameters rested on the experience of the human operator. Now, monitoring and adjustment are done through digital control systems. These range from basic software for small bench-top bioreactors and personal computers to more complex systems from suppliers like Biostat, BioFlo, and Biobundle. Eli Lilly and Company supplies the most advanced and dynamic digital bioreactor control systems designed to supervise the entire manufacturing process at large facilities.
All digitally controlled bioreactor networks rely on sensors and actuators that track and alter the bioreactor’s internal conditions. Sensors transform data points about biochemical and physical conditions into digital signals. These are fed into algorithms that then regulate growth conditions by controlling the motion of actuators. Nowadays, sensor innovation focuses on non-invasive tools, such as German company PreSens GmbH’s optical device for measuring pH and dissolved oxygen. Applikon Biotechnology has created gel-filled miniature pH sensors and oxygen centres that are only 6 mm in diameter. Emerson Rosemount has made an electrochemical sensor that is fully disposable and requires very little calibration and setup. Both of these are specifically designed for the miniaturised, disposable bioreactors that have become increasingly popular in pharmaceuticals.
Automating environmental control and adjustment is the holy grail of bioreactor design. Manufacturing such devices on a large scale would go a considerable way towards allowing cultured meat and stem cell cultivation to scale. To this end, ongoing research into bioreactor control systems has focused on developing smart actuators and smart sensors. Traditional actuators control bioreactor conditions through simple mechanical adjustments dictated by a higher computer. Smart actuators on the other hand each contain a microprocessor that allows it to self-diagnose and rectify issues with the bioproduction process. Similarly, smart sensors can process the signals that they are registering.
These smart actuators and sensors are designed to work within a decentralised digital architecture, a burgeoning trend in bioreactor design. In the classical hierarchical control system, bioreactor sensor signals were bundled and fed into an industrial computer, which would then transmit data to a desktop interface. Researchers are now working on cutting out the intermediary industrial computer, instead wirelessly linking all individual sensors and actuators directly to a desktop or mobile interface. This is known as the ‘fieldbus control system’, or distributed control system. Its capacity for delivering rapid, high-precision adjustments makes automated bioreactor production a real possibility.
In the pharmaceutical industry, bioreactors are essential in supporting cutting-edge therapies. One class of valuable biological substances that bioreactors can manufacture in large quantities are plasmids, self-replicating DNA molecules found in bacteria, and some fungi. They are fundamental to genetic therapy, where genes in a patient’s cells are altered to treat disease. A small number of plasmids are inserted into microbial cells like E. coli or S cerevisae and placed into a specially calibrated bioreactor. Here, the microbial cells multiply along with the plasmids inside them. At the end, the plasmids are purified from their microbial hosts, ready to be injected into organs to replace defective proteins.
Traditionally, all bioreactors have been large and permanent stainless-steel installations. In recent years, the pharma industry has pivoted towards single-use bioreactors made up of small, sterilised bags. These nimble devices are increasingly being adopted by the biopharma industry to meet rapidly shifting market demand for different bio-compounds.
Evotec Biologics specialises in modular and disposable bio-manufacturing equipment. Many of the big bioreactor suppliers, such as Sartorius, now offer single-use culture vessels that fall in a range from 15 ml to 2000 litres.
Producing larger quantities of product using disposable bags means running many cultures at the same time. In bio-pharma, however, commercial success is not always synonymous with high volume production. More often, it is about achieving efficiency for a low-volume precision product with short market runs. Operating many small bioreactors in parallel makes it easier to test and observe conditions for optimal growth. Companies can quickly identify cultivation parameters that are worth scaling, reducing the time it takes to get new drugs to market. The adaptability of disposable bioreactors makes them an ideal solution for startups embarking on proofs of concept and early-stage process development.
At the other end of the spectrum is the cultured meat industry, bigger is most definitely better. This is a high-volume, low-value commodity. 1012-1013 cells are needed to generate ~10–100 kg of meat. Current estimates say that around 4000 factories containing 130 10, 000 litre stirred-tank bioreactors each would be needed to take a 10 percent share of the farmed meat market by 2030.
Production runs in the most established cultured meat companies are relatively small. The largest mammalian cell culture factory ever constructed was by Samsung Biologics, holding around 350, 000 litres of cell culture capacity. These mammoth capacities are the exception and more than 10, 000 litres of mammalian cell culture in the industry are rare.
Another problem in scaling the culture of food proteins is that growing specialised tissue from plant and animal cells is a more delicate process than cultivating bacteria and microorganisms. Cells are fragile and can break down unless the sterility, temperature, and nutrients inside the bioreactor are not perfectly controlled. Gases must be delivered to the cells in a way that doesn’t tear them, something that is a risk when bubbling methods are used.
One solution to deliver gases to cells is through silicone membrane gas exchanges. Gases permeate through the membranes without the disturbance involved in mechanical methods. PermSelect is one company that specialises in silicone membrane gas exchangers for bioreactors, although this technology is still more widely used in pharma than protein culturing.
Another consideration in meat culture is the bio-scaffold. In living bodies, cells grow embedded within a structural mass of proteins and polysaccharides. This is the extracellular matrix. Bioreactors must closely replicate this substance with 3-D substrates for the cells to grow along. These formations encourage cells to grow into long muscle-like strands, essential for recreating the authentic texture of farmed meat.
Research into bio-scaffolds is being buoyed by recent market interest in cell-based meat. In 2021, the German company Merck awarded funding to the Kaplan Lab at Tuft’s University to develop commercially scalable 3-D dibble bioscaffolds to support muscle and fat cells. The Kaplan Lab won out over 64 funding competitors from around the world. At the International Iberian Nanotechnology Laboratory in Portugal, Dr. Sara Oliveira is working on 3-D bioprinted bio-scaffolds.
Compared to pharmaceuticals, the cultured meat industry has been generally quite conservative when it comes to investing in novel bioreactor designs. So far, no meat-specific bioreactors have been commercialised. One way of scaling cultured meats may lie in bio-engineering mammalian cells so that they become as robust and prolific as microbial cells like yeast. This would enable higher volume cell cultures using bioreactors already on the market.
Future developments in bioreactor design for high-volume sectors like meat and polymers will likely borrow from pharma innovations. Innovations will tend to flow from established life science companies that have the expertise and the capital to invest in bioreactors that combine the precision of bio-pharma production with the high volume needs of food and industrial materials.