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The industry revisits the pros and cons of microbial fermentation at scale for biotherapeutics.
Microbial fermentation has its place in the biomanufacturing industry, despite the prevalence of mammalian-based cell culture processes for the production of biologics. However, despite the lower costs and potential for substantial product yield with microbial fermentation, this process has challenges. This article discusses the advantages and challenges of microbial fermentation and looks ahead at what place fermentation may hold in the biomanufacturing industry moving forward.
Industry sources note that fermentation has played a role in manufacturing biologics for quite some time. “The first biopharmaceutical, insulin, was produced in the early days via microbial fermentation in Escherichia coli (E. coli), and is now also produced in yeast,” says Gregor Awang, PhD, director, Biologics Process Development, BioVectra, a contract development and manufacturing organization (CDMO).
In addition to insulin, many other biologic drug substances are now made via fermentation, including human growth hormone (HGH), enzymes, cytokines, monoclonal antibodies, antibody fragments, and bioconjugates, Awang enumerates. Furthermore, plasmid DNA, which is the intermediate needed to make messenger RNA (mRNA) for vaccines and therapeutics, is also produced in bacteria, and some bacteria have been modified as a product themselves to be used in nutritional supplements, Awang states.
“Fermentation is a key technology at the core of all biologics, whether it is antibodies, complex proteins, or genetic medicines,” says Jess Tytell, director of Technical Business Development, Ginkgo Bioworks, a US-based biotechnology company specializing in bioengineering microorganisms. Tytell explains that cell lines that have historically been employed for biologics production have been mammalian in origin, but with advances in cell engineering and development of new formats of biologics, microbial platforms will start to become more relevant.
In the meantime, more biosimilar products are expected to come onto the market in the next decade, so the question is whether fermentation will hold a place in the manufacture of these biosimilars. Tytell believes that fermentation will continue to remain a fundamental technology enabling the manufacturing of biosimilars as much as they have been for novel biologics. In the context of biosimilars, microbial platforms can enable the improvement of access to much-needed therapies because these platforms can help make the manufacturing of biologics much more cost effective, she notes.
Awang points out that, since most of the non-glycosylated biosimilars on the market—such as HGH and insulin—are made in either E. coli or yeast, fermentation will continue to be important in the manufacture of biosimilars. However, one limitation of fermentation for biosimilars is the proteins needing glycosylation, which adds polysaccharides to the protein to improve stability and potency. “Protein glycosylation is specific to mammalian cells, and microbes, as a rule, don’t add polysaccharides to proteins. If a product requires glycosylation, you would use mammalian cells. Specifically, you need to ensure human or human-like glycosylation to avoid an adverse immune response,” Awang explains.
Traditionally, biosimilars have been cultured in Chinese hamster ovary (CHO) cells, and, even when glycosylated, these protein products tend to be compatible with the human system—though there are human cell lines now available that provide fully human glycosylation.
“It is sometimes possible to make the protein with E. coli, then chemically add a carbohydrate moiety in vitro. This has the benefit of reducing costs of protein production as fermentation is less costly than mammalian cell culture. It depends on whether the protein is amenable to this approach, depending on the complexity of the required glycan. For example, some proteins can be PEGylated, which adds a relatively less complex carbohydrate,” Awang says.
Pichia pastoris (P. pastoris) is a yeast that combines high-yield production and is capable of some post-translational modifications, including glycosylation, Awang adds, noting that companies have engineered P. pastoris—and many other yeasts—to mimic the human glycosylation pathway.
As with most manufacturing processes, a particular process will have both its pros and cons when it comes to scale up, and microbial fermentation is no exception. When comparing fermentation to mammalian cell production, Cameron Graham, senior manufacturing and science technology (MST) process engineer, BioVectra says that scale up of a microbial process benefits from the fact that microbes are usually more resistant to shear than mammalian cells; however, microbial fermentation has the challenges of needing adequate oxygen transfer and mixing as well as the removal of excess metabolic heat.
“Typically, for microbial scale up, we’ll pursue a more robust scale up strategy in terms of the power per unit volume input and kLa (volumetric oxygen transfer coefficient), to maximize the efficiency of oxygen transfer into the fermentation broth. Particularly at larger scales (>1000 L) of high-growth cultures, the amount of heat can be more than what jacket cooling can handle since single-use bags don’t have the same conductivity as a stainless-steel surface. This can also be challenge in traditional stainless-steel vessels,” explains Graham.
Graham notes that there are options to lower the jacket coolant temperature, but the ratio of jacket heat transfer area to fermenter volume typically becomes more unfavorable as vessel volume increases. “In those cases, you have to explore internal surface area additions to the vessel itself. For example, we are exploring an upgrade to our 17,000-L stainless steel fermenter to include internal baffles that will allow us to achieve about 300 mmol/L/hour OTR [oxygen transer rate] at 30 °C broth temperature, and up to 450 mmol/L/hour OTR at 37 °C. Such an upgrade will allow us to maximize the microbial process while reducing the risk to product quality due to uncontrolled temperature rise,” Graham states.
Awang observes that media costs are lower for microbial fermentation, making scale up less expensive than with mammalian cultures, which can have up to 20 different ingredients—a few of which are extremely expensive and sensitive to certain conditions.
“Yields tend to be much better with mammalian cell cultures (up to 10 g/L), than for fermentation (1–3 g/L). However, fermentation process development is faster and less technically challenging since there are a lot of tried-and-true methods for optimizing the process in E. coli,” Awang says.
Awang continues that the key for both microbial fermentation and mammalian cell culture is getting the correct type of DNA inserted into the cells to ensure a high titer and yield of product. For example, transferring DNA into E. coli and selecting a suitable clone with the intact DNA is much simpler than transfecting mammalian cells. In the latter case, it is necessary to screen thousands of mammalian cell clones to get one that has the desirable combination of traits, depending on where in the cell genome the inserted DNA (transgene) has located, and how many copies. “In the past decade, DNA editing using CRISPR [clustered regularly interspaced short palindromic repeats] and other techniques has made inserting DNA precisely into stable ‘hot spots’ in mammalian cells much more precise, resulting in higher stability and product yield,” says Awang.
Fermentation, however, has shorter processing times. According to Awang, a good microbial process can take as little as one to three days, as opposed to the weeks necessary for mammalian cell culture. “This faster process has the added benefit of reducing the risks associated with contamination. Not only is the risk lower with fermentations, but if you do have a contaminated fermentation, the loss of worker hours is a lot less. A contamination two weeks into a mammalian culture can be a massive financial loss,” Awang emphasizes.
Tytell concurs that microbial fermentation advantages include significantly lower costs for microbial hosts, less expensive media, and fewer issues with contamination. The latter can mean less downtime and higher productivity. In addition, optimizing microbial hosts is faster, leading to a shorter turnaround time to develop the final production strain, which can decrease overall production timelines, she adds. In comparison, mammalian hosts require more screening (e.g., for viral infections).
The disadvantages of microbial fermentation that Tytell sees include the fact that post-translational modifications are different, and this difference is relevant for more complex biologics. In addition, there are fewer approved biologics from non-Chinese hamster ovary hosts, so there may be higher regulatory hurdles for newer biologics produced via microbial fermentation; however, these hurdles are expected to go away quickly after the first few approvals, says Tytell.
With a rich pipeline of emerging therapy drug candidates, what place will fermentation-based processes hold in the biomanufacturing industry as these more highly complex biomolecules move towards commercialization?
“Fermentation has a place in a range of therapeutics in development. For example, plasmid DNA produced in E. coli can be used as both a therapeutic or an intermediate to make mRNA vaccines, gene therapies, and for gene editing,” says Awang.
Conjugated protein therapeutics, in which a protein and another chemical entity, such as polyethylene glycol, are linked to improve the function of a biologic, is another area in which microbial fermentation has application, says Awang. “Bioconjugation had died out around the turn of the century but is coming back with a bang. Products in development include PEGylated hormones, such as GM-CSF [granulocyte-macrophage colony-stimulating factor], which can be made in bacteria or yeast, and is then PEGylated to improve its stability. Or mAb protein fragments made in E.coli, which can be conjugated to therapeutic payloads to direct the payload to specific disease cells,” Awang explains.
Tytell says that many current biologics do not require post translational modifications. For those biologics, and because of cost of goods and development timelines, microbial fermentation is poised to become a workhorse (e.g., nanobodies, aglycosylated antibodies, other protein based biologics, etc.). As microbial hosts continue to develop, they will become more valuable for complex biologics, she anticipates.
“As microbial engineering continues to develop and microbial strains can be produced that can mimic key mammalian features, such as glycosylation, these fermentation-based processes will become extremely valuable in more complex biologics. The faster turnaround to testing and higher titers will be especially valuable for the discovery and validation phases, which become more critical as molecules get more complex,” Tytell states.
Meanwhile, another current popular trend in biomanufacturing, single-use technologies, may have a place in fermentation processes, but stainless-steel systems will likely remain the core setup for microbial fermentation. Single-use microbial technologies can be conducive to fermentation-based biomanufacturing if certain criteria are met, Jeremy Kerrick, head of Process Development and Engineering at Ginkgo Bioworks emphasizes.
For instance, scale matters. If the output of a process is a commodity, then scaling up is often the approach that drives down cost of manufacturing and the final product. This typically means that the best option is stainless-steel, since there are currently no single-use fermentation options beyond 50 L, and these are only just now showing up on the market, says Kerrick. Thus, scaling up into a single use microbial fermentation-based platform to drive down cost is not currently possible.
The availability of single-use platforms is also a factor. High-value mammalian-based therapeutics such as CGTs rely heavily on single-use reactors. Yet, these single-use reactors typically scale to up to 2000 L. “While they may incrementally increase in size in the future and perhaps marginally drive down the cost of manufacturing, it seems unlikely that they can get much larger than what is on the market now. It seems logical that once single-use technology reaches this scale for microbial fermentation, it will also be limited, and then the economics of scaling will again steer one toward stainless steel,” says Kerrick.
In terms of titer, novel approaches to increasing the expression of the modality of interest could swing the pendulum back to smaller-sized fermenters and, thus, to single-use technology. According to Kerrick, superior producer cell lines, induction or transfection advances, and genetically modified organisms are all means being explored across industries to increase titers. He explains that, if a process currently provides 2 g/L of product and is produced in a 20,000-L reactor, which suddenly sees an increase of 10 fold titer, then 1000-L or 2000-L single-use fermenters are a viable option. Further, single-use technology has some advantages over stainless-steel at this scale, which could make single-use the superior choice.
Meanwhile, development at the bench and pilot scale needs to be taken into consideration. Until recently, mammalian and microbial development laboratories were dominated by glass and stainless-steel reactors, Kerrick points out. With the introduction of high throughput bioreactor systems many mammalian development teams have made the move from stainless-steel to single-use technologies.
“Breaking down a dirty reactor, autoclaving, reassembly, cleaning in place (CIP), and steaming in place (SIP) are required [with stainless-steel bioreactors], which are time consuming and lead to downtime. Systems are often contaminated because a single step in the cleaning process was not done perfectly, leading to more downtime. Single use systems are typically provided in gamma-irradiated bags and arrive sterile. Single use systems greatly reduce the need for laborious cleaning and downtime. As more and larger single use microbial fermentation technologies are introduced onto the market, more microbial teams will follow the trend of their mammalian counterparts,” Kerrick states.
Meanwhile, Awang notes that single-use fermenters have many benefits, including the rapid changeovers between batches (which can occur within one day) as well as the previously mentioned decreased risk of contamination and the elimination of costly and time-consuming cleaning and sterilization between batches that is essential with stainless-steel equipment. “Single-use processes can also be large enough for a client that wants to do some fast clinical runs, since it will be much less expensive, and there are efficiencies with single-use up to 2000 L that aren’t available with stainless-steel systems,” Awang says. One caveat being, however, that for biologics produced in the range of tons per year, stainless steel remains the preferred option.
Graham points out that biopharmaceuticals made by single-use fermentation units require purpose-built fermenters with a bottom radial impeller for adequate mixing/power per unit volume input. These fermenters must also be capable of one to two vessel volumes per minute at commercial scale. “It is also advised that single-use fermenters have oxygen supplementation available for increased driving force in lieu of the lack of head pressure capability for single-use fermenters to optimize microbial growth and, ultimately, yield of drug substance,” he states.
Choosing the best equipment will depend on scale and the type of product being manufactured, Awang continues. “A good example is the autologous therapeutics used in personalized medicine. These require small-scale runs of 50 L or less with batch segregation. For these, single-use fermentation is great because one vessel is dedicated for each patient. There is no need to clean it, leading to faster turnarounds. This increases speed, reduces costs, and, most importantly, prevents cross-contamination,” empasizes Awang.
In addressing the current viability of microbial fermentation for the anticipated influx of new emerging therapies, Graham points to work BioVectra has been doing. The company has been testing a new 100-L single-use fermenter to verify their ability to deliver suitable power per volume and mixing. “In our experience, we can increase oxygen supplementation, but, without sufficient mixing to properly disperse the oxygen and achieve the desired transfer rate, oxygen supplementation can have limitations. Recently, we were excited to test a 400 mmol/L/hour process and completed the run while maintaining fermentation broth temperature within the expected specification. This is an important consideration for our clients in terms of the feasibility of single-use fermenters for use in high growth microbial processes,” Graham explains.
A final selling point for using single-use systems is that they also tend to have lower utility needs, and single-use has faster equipment qualification than stainless-steel bioreactors, Graham concludes.
While fermentation has roots in ancient history, it has a bright future in the world of emerging therapeutics. Depending on the product being considered, the scale required and the need to speed a process to market, fermentation-based manufacturing—when reconfigured for nuanced requirements—holds a valuable and flexible position within the manufacturers toolbox.
Vol. 35, No. 10
When referring to this article, please cite it as F. Mirasol, “Weighing the Benefits of Fermentation for New Biotherapies,” BioPharm International 35 (10) 18–22 (2022).