Discover Single-Use Fermenters for Biopharmaceutical Production

© Copyright BIOVECTRA 2022. All Rights Reserved. WHITE PAPER SINGLE-USE FERMENTERS FOR THE PRODUCTION OF BIOLOGICS Large-scale single-use fermentation technology using aerobic microbial expression systems offers an excellent complement to stainless steel bioreactors for clinical-to-commercial scale production of recombinant proteins. For decades, microbial fermentation has been used for the commercialization of biologics, with the efficient fermentation of such recombinant proteins as insulin in stainless steel (SS) bioreactors as large as 100,000 L. The biopharmaceutical industry is now at a pivotal stage due to the development and clinical success of a diverse range of new modalities— RNA therapeutics, plasmid DNA (pDNA) and mRNA vaccines, cell and gene therapy, and antibody fragments—that are being used for indications such as oncology, multiple sclerosis, and autoimmune disorders. While SS equipment continues to be vital for commercial manufacturing of many biologics, it comes with high upfront costs and the ongoing expense in time and labor required to operate it. This works well when a company is producing large amounts of drug substance but has limitations for the smaller volumes needed during development and clinical studies. The early stages of a drug clinical development program need processes that increase speed to market. What has been missing is a fast, cost-effective solution for fermentation studies that can be used during development and clinical-phase manufacturing. Recent innovations in single-use fermenters (SUFs) mean that startups and large biopharmaceutical companies alike can now produce biologics for tech-transfer scale up of recombinant proteins for phase 1 or early-phase 2 clinical trials, and even to treat orphan or rare indications. These closed, sterile SUFs—100 L and large-scale 1,000 L—are designed to provide mixing, oxygen transfer, and cooling capabilities comparable to SS bioreactors while being more agile and avoiding the high fixed costs of stainless steel. A valuable niche exists to manufacture personalized medicines, for which multiple discrete batches are required, and single use equipment would have a huge advantage over SS with respect to batch segregation and cross-contamination risks. Companies that want to start a clinical program and take it all the way to commercial launch are no longer faced with an either/or choice of single-use versus SS bioreactors. The combination of the two can be ideal for those engaged in clinical-to-commercial scale production. They would begin with SUFs for production of biologics needed for development, early-phase clinical trials, and orphan indications. Then, for large-scale production of successful drug products—mRNA vaccines or blockbuster biologics— manufacturers can transition to larger SS bioreactors (e.g., >1,000 L up to 17,000 L, or larger) to manufacture enough product for phase 3 clinical trials and commercialization. SUFs have overcome the challenges that existed in the early days, when single-use cell culture systems for microbial fermentation were simply repurposed from bioreactors designed for mammalian cell culture. Early versions demonstrated poor oxygen transfer, limited mixing, and insufficient temperature control. With engineering and design innovations, SUFs are now able to complement SS fermenters. The addition of SUFs improves process flexibility, leads to faster turnaround times, enhances quality, reduces regulatory burden, and lowers costs by enabling better use of facility capacity. © Copyright BIOVECTRA 2022. All 2 Rights Reserved. The addition of SUFs improves process flexibility, leads to faster turnaround times, enhances quality, reduces regulatory burden, and lowers costs by enabling better use of facility capacity. In this white paper we will discuss these benefits of SUFs and how they fit into a clinical-to-commercial scale program. Single-use fermentation technology is similar in many ways to conventional stainless-steel systems. The main difference is that SUFs use disposable sterile plastic bags that don’t require the traditional high-intensity preparation for cleaning, sterilization, and decontamination of SS bioreactors, which are expensive and time-consuming processes. SUFs come in various sizes, including 10–30 L process development fermenters, 100 L seed fermenters, and the industry-leading 1,000 L fermenter from ABEC. They can be used for all drug substances expressed in microbial cells, including pDNA, proteins, enzymes, Fabs, and bioconjugates. High-intensity mixing of microbial cultures, which optimizes oxygen transfer, is possible because of the generally low sensitivity of these cells to shearing. Mixing the cells within the SUF can be achieved by using a variety of different impeller styles and designs to ensure an optimal oxygen transfer rate (OTR). A complete biologics program, from development and clinical studies to commercialization, can include smaller process development bioreactors, SUFs for clinical studies drug production (in 100 L and 1,000 L fermenters), and SS for large- scale cGMP production of commercial drug products (in 100 L, 3,000 L, and up to 17,000 L fermenters). SUFs can grow with a clinical-tocommercialization program What is microbial fermentation? Microbial fermentation is the process of growing bacterial or yeast cells in liquid culture so the cells can express a drug substance, usually a recombinant protein such as insulin, human growth hormone, cytokines, monoclonal antibodies (mAbs), or antibody fragments (Fabs). When compared to mammalian cell culture, microbial fermentation has the benefits of less complex genetic engineering, simpler growth media, faster cell growth, and a much shorter production time. The main microbes used are the bacterium, E. coli, and the yeasts, S. cerevisiae and Pichia pastoris. © Copyright BIOVECTRA 2022. All 3 Rights Reserved. It takes one-third the time to qualify SUF equipment compared to SS systems. Typically, SUFs are provided with a base unit that contains the automation required toperform the process. Support systems can be easily linked to the base units. SS systems have more complex piping systems and utility connections, making the costof automation much higher, both for instruments and programming. The fermenter sterileenvelope is directly tied to the performance of the automated sequences, thereforeincreasing the risk of failure if there is any automation issue. Perhaps the main benefit of SUFs is the rapid changeovers that are possible withdisposable equipment. SS systems can be costly for a number of reasons—most notably,the costs of fixed equipment and labor—but the cleaning time between successivebatches may be the most significant cost differential when compared to SUFs.Changeovers of SS systems can take several weeks causing downtime that results insignificant loss of throughput. In contrast, switching to a new batch with SUFs can occurwithin one day. This means that a new production run can begin every five days, makingfacility utilization much higher. While the consumable costs of disposable fermenter bags, tubing, and filtration equipmentrecur for each SUF run, they are more than offset by higher throughput and capacity useper annum. Much of the lag between batches in SS fermenters occurs because the equipment mustundergo CIP and sterilize-in-place (SIP) before a new batch can be processed. This cantake as long as seven days to complete and requires water for injection (WFI) andspecialized cleaning chemicals, the supply of which is often constrained. Cleaning and sterilizing is unnecessary for SUF equipment as it is received as a sterileconsumable and is disposable. This reduces the need for CIP chemicals, WFI, and steamfor sterilization processing. SUFs are more automated Equipment qualification is faster Quicker changeovers between batches No need for cleaning and sterilization between batches © Copyright BIOVECTRA 2022. All Rights Reserved. 4 SUFs have lower utility requirements than SS bioreactors. For example, SUFs use one-tenth the water in the manufacturing process, including WFI and high-purity CIP solutions,significantly reducing daily operating expenses. Lower utilities costs It is possible to add more production SUFs per batch to increase or control output per cycle. Typically, SUF fermentation suites are designed to accommodate expansion of productivity. Compared to the much larger SS bioreactors used for cell culture, the maximum working volume of a SUF is currently capped at 1,000 L, due to conditions unique for microbial cell growth. Microbes like E. coli are capable of rapid, high-density cell growth, but this requires sufficient agitation and a larger head space in the bioreactor to supply the cells with the oxygen they need. In fermenters, this is measured as the oxygen transfer rate (OTR). In addition, many microbes, such as E. coli and P. pastoris, create excess metabolic heat that must be removed during fermentation. The ABEC CSR 1,000 L is designed to meet the cooling demands of high-growth microbial processes and allow an OTR that is similar to what is seen in SS systems of the same volume. However, at volumes greater than 1,000 L, SS bioreactors are still preferred due to the need to maintain aeration, OTR, and provide sufficient cooling at these higher volumes. Despite the many benefits of SUFs, the technology does come with some limitations worth considering. Limitations of SUFs Culture volume is capped Flexibility of production capacity Lower risk of cross-contamination The risk of contamination between batches of one product is significantly reduced, as is the risk when shifting from one product campaign to a different product campaign. This occurs because, not only are the fermenters single use, but their associated components are as well, greatly reducing maintenance-related batch contaminations. The risk of contamination between batches of one product is significantly reduced. © Copyright BIOVECTRA 2022. All Rights Reserved. SS fermenters are deliberately over-pressurized to increase the solubility of oxygen in theculture medium. In addition, P. pastoris may require methanol in its feed stream, adding thefurther complexity of using a flammable nutrient during fermentation. SS bioreactors havecontainment protection against overpressure events and fire, while SUFs do not. Microbial fermentation, usually in E. coli , is an efficient way to make large amounts ofplasmid DNA, even in a small facility. This DNA is used to transfect cells during theproduction of viral vectors (e.g., adenovirus, retroviruses, and all nine serotypes of adeno-associated virus (AAV)), for autonomous cell therapy, for in vitro transcription to makemRNA for vaccines, and is being developed for direct application in transfection vaccines. A 1 L fermenter makes enough plasmid for a 200 L cell culture transfection, sufficient fordevelopment purposes or for the production of a drug substance meant to treat ultra-raredisease indications. For commercial launch of biologics, such as an mRNA vaccine, theamount of plasmid needed requires scaling up to 1,000 L SUF. The ABEC CSR 1,000 Lfermenter can operate with working volumes of 200–1,000 L, making it flexible for differentbatch sizes. For viral vector manufacturing, operators run a separate batch for each of theplasmids used to transfect the mammalian cells—there can be multiple needed for eachtransfection. This means that, even for an indication like Crohn’s disease that afflicts millionsworldwide, it would only take as few as two batches per plasmid each year in a 1,000 L SUFto produce enough plasmid for the drug substance. Microbial fermentation at available SUF scales can be used to make small amounts ofrecombinant protein products in the one-kg range, such as enzyme reagents, highly potentprotoxins, or similar high-value, small-volume products. Current SUF scales are also ideally suited for rapid and flexible production for clinical trials,with the intention to switch to larger-volume SS fermenters later to produce the amountsrequired for commercial scale. This would require process modelling to validate that theprocess performs the same at both scales in the two fermenter types. The production of plasmid DNA and RNA The production of recombinant proteintherapeutics and reagents Lack of containment against high pressure and flammables © Copyright BIOVECTRA 2022. All Rights Reserved. 6 With faster changeovers, no need for validated cleaning, lower utilities costs, and reduced risk ofcross-contamination, single-use fermenters are a good fit for taking a promising DNA or high-potency recombinant protein candidate from the lab bench all the way to commercialization. For those looking to support their next biologics fermentation project, it is important to find a partnerthat offers the seamless integration of SUF with existing SS systems to expand the scale ofproduction from small-scale clinical amounts all the way to large-scale commercial production. Additionally, a CDMO partner with a range of microbial fermentation options, combined with processscience, solid engineering, cGMP manufacturing, analytical capabilities, and regulatory compliance,all under one company roof, ensures fast delivery, speed to market, and cost efficiency. Eliminating the need for chemicals and purified water used for CIP as well as the energy requiredfor the extensive SIP of SS vessels shrinks the environmental footprint of single-use fermentation.The microbial culture and other biological waste from the process is treated to kill any organisms,at which point it is no longer hazardous and is safe for discard. The single-use components canbe part of a recycling program for the secondary use of plastics once neutralized. Environmental sustainability What to look for in a CDMO for microbialfermentation BIOVECTRA has a track record of collaborating with our clients to solve the scale-up issues that come from difficult processes. This has resulted in robust, reproducible drug processes that have been filed and approved by the FDA and other regulatory agencies worldwide. WHITE PAPER To learn more, visit us online at: www.BIOVECTRA.com BIOVECTRA is a global biotech and pharmaceutical CDMO (contract development and manufacturing organization) that specializes in clinicalto- commercial scale production capabilities for: biologics, bioreagents, fermented small molecules, synthetic small molecules, and active pharmaceutical ingredients. Flexibility, creativity, process optimization and compliance are at the heart of our method. With over 550 employees and cGMP facilities in Prince Edward Island and Nova Scotia, Canada we assure our programs advance on time and with the highest quality outcomes. About US