Single-use fermenters (SUFs) have emerged as a promising technology in biopharmaceutical manufacturing, offering flexibility, cost-effectiveness, and reduced risk of cross-contamination when compared to traditional stainless-steel fermenters (SSF). Many biopharmaceutical companies are adopting SUFs, recognizing these cost reductions, faster setup and validation, and opportunities for automation.¹ Market forecasts suggest the value of single-use bioreactors is set to grow considerably from $4.5 billion in 2023 to $18.9 billion dollars by 2033.² However, concerns persist within the industry regarding their scalability and suitability for high-growth microbial applications. In this article we present practical insights and successful experiences gained over the past two years to meet the rigorous demands of high-growth microbial processes to optimize SUFs for biopharma manufacturing.

Our experience has demonstrated effective methods for scaling up from benchtop scale (10 L) to clinical scale (100 L), and further to commercial production scale (1,000 L) using SUFs.³ These scale-up strategies have proven to be feasible and effective, ensuring consistent performance and product quality. Engineering modifications and process adjustments to SUFs address the challenges associated with high-growth microbial organisms, particularly oxygen transfer rate (OTR) and cooling capacity.

SUFs offer an excellent complement to large, fixed equipment to optimize drug discovery, clinical production, and commercial manufacturing within one biopharmaceutical manufacturing facility.

Where do SUFs fit in biologics development and manufacturing?

More biotech startups, companies with early-phase drug products in clinical trials, and large pharmaceutical companies in commercial production are discovering the benefits of high-growth microbial fermentation to meet the demands of the burgeoning biologics market. Stainless-steel equipment has proven its value in large-scale commercial fermentations and harvesting of biologics. Yet, for smaller scale production of biologics (10–1,000 L), SUFs should be considered for their flexibility, cost-effectiveness, and speed. Benefits of SUFs include decreased utility costs through the elimination of cleaning and sterilization between batches, as well as the capacity to support the production of new modalities with quicker changeover between products. Moreover, high-growth expression systems in E. coli and Pichia pastoris enable the efficient production of a variety of bioproducts, including recombinant enzymes, proteins, peptides, antibody fragments, and nucleic acids such as pDNA and mRNA, which are crucial for vaccines and advanced therapeutics.

Microbes like E. coli can withstand much more vigorous agitation and have much faster growth rates than mammalian cells. BioVectra defines a high-growth organism as requiring an OTR of >300 mmol/L-h. Faster growth requires both adequate oxygen transfer and removal of excess metabolic heat. The current practical maximum SUF volume for high-growth microbial processes is 1,000 L due to cooling capacity constraints when limited to the heat transfer surface area provided solely by the vessel jacket. The cooling capacity can be increased by using low temperature glycol, yet larger fermenters (>1,000 L) may have insufficient jacket cooling to remove the heat generated by high-growth microbes and increases in internal surface area should be explored. Equipment suppliers have made modifications to larger SSFs (17,000 L), including internal cooling baffles to remove excess metabolic heat, that permit OTRs as high as 480 mmol/L/hour at 37°C.⁴

The information presented here shows that the SUFs used by BioVectra can support the necessary oxygen transfer and cooling capacity for high-growth organisms that require an OTR greater than 300 mmol/L-h.

Facility and process considerations when implementing SUFs

Increased agitation power to improve oxygen transfer and utility system upgrades to ensure cooling efficiency

High-performance fermenters are designed to ensure effective mixing, air flow, and oxygen transfer into the growth media by providing adequate agitation (i.e., power input per volume). To obtain sufficient mixing power and gas dispersion, SUFs should be equipped with at least one radial impeller. The impeller design is essential for achieving effective mass transfer when combined with a gas flow rate of one to two vessel volumes per minute (VVM), ensuring optimal conditions for high-growth fermentation processes. For example, the ABEC CSR 100 L and 1,000 L SUFs have a comparable agitation power per unit volume input to that of an equivalent sized SSF.

Attempts can be made to overcome insufficient mixing power by increasing the oxygen percentage in the sparging gas, but there’s a limit to how much oxygen you can effectively supply. At a certain point, excess oxygen will simply pass into the exhaust if there is insufficient dispersion of the sparged gas to the culture. For this reason, SUFs should also be capable of monitoring oxygen and carbon dioxide concentrations in the fermenter exhaust gas. This is not only a matter of process efficiency but also a significant safety concern, as high concentrations of these gases pose risks to workers and overall process safety. The measurements of oxygen and carbon dioxide in the exhaust gas are key inputs to the calculations for oxygen transfer/uptake rate and the carbon dioxide evolution rate. These measurements form the basis of the respiratory quotient calculation (RQ), which is indicative of the status of the culture. The OTR is a key indicator of scalability when BioVectra assesses processes and will drive decisions about vessel selection.

We maintain adequate jacket cooling for our 100 L ABEC CSR single-use fermenter using a chilled-water supply at 5°C, which supports top-of-the-range E. coli processes at this scale. As fermenter volume increases to 1,000 L, the ratio of available jacket surface area relative to the process heat load becomes less favorable and 5°C chilled water is no longer adequate to support a 300 mmol/L-h OTR, assuming a 30°C culture temperature. At the 1,000 L scale under these conditions, chilled glycol at -5°C is needed to achieve sufficient cooling.

Oxygen supplementation to maintain dissolved oxygen (DO)

The DO concentration is held relatively constant throughout most fermentations. For example, an SSF-based process may aim for 30% DO with a head pressure of 7.5 PSIG (0.5 barg) and a 30°C culture temperature. But the DO concentration set point needs to be adjusted when transitioning from a stainless-steel fermentation to a SUF—currently, single-use bags generally cannot be pressurized beyond approximately 0.5 PSI. Consequently, when transferring to a SUF, the DO set point should be adjusted to reflect this lack of head pressure and would increase to 45% DO, assuming the same head pressure/temperature conditions noted above. As SSFs can usually accommodate head pressures up to 14 PSIG (1 barg), the oxygen transfer capability of the system may be high enough to sidestep the need for oxygen supplementation. In contrast, when using an SUF for cultivating high-growth E. coli, the absence of head pressure often necessitates oxygen supplementation for high-growth microbial processes. The equation BioVectra used to calculate OTR is a function of the kLa (affected by agitation and gas flow) and the driving force (affected by pressure and oxygen content in the sparge gas).

A visual indication of similar kLa would be observed during the batch if the agitation profile reached its maximum at a similar point in the process when scaling up. In the case of similar head pressure across scales, it would be expected that the oxygen supplied relative to the air flow would be similar.

Scaling up with internal tech transfers

Given the number of process parameters that need to be considered when transitioning a process between stainless-steel and single-use fermenters, it can be helpful to have all options at hand. Having access to both technologies within a single facility offers manufacturers the flexibility to select the most suitable option for their specific production campaign. SUFs are particularly valuable for development phases and clinical scale manufacturing due to their efficiency and scalability.

Successful clinical trials can pave the way for scaling up to commercial production through an internal technology transfer. The data presented here demonstrates the feasibility of applying insights gained from scaling from a 10 L glass vessel to a 100 L SUF, and from a 200 L SSF to a 1,000 L SUF, highlighting the potential for seamless expansion in production capabilities.

Case study: Scaling from the lab (10 L) to the clinic (100 L)

BioVectra used the production of a high-growth E. coli process to demonstrate the scalability from a benchtop glass reactor to clinical production using an ABEC CSR 100 L SUF. Our tests looked at mixing, oxygen supplementation, and cooling capacity by measuring OTR, DO, temperature, pH, and agitation in rpm.

Results
We successfully scaled up the process, ensuring that the two key factors affecting the volumetric oxygen transfer rate (kLa)—gassed power per unit volume as a function of agitation, and superficial gas velocity as a function of total gas flow—remained consistent as the scale increased. The agitation rate reached its maximum level at a comparable time at both the 10 L and 100 L scales. Once maximum agitation rate was attained, oxygen supplementation was introduced, replacing air to maintain the total gas flow rate. At the 100 L scale, we used a higher proportion of oxygen relative to air, suggesting that the kLa at this scale was lower than that of the 10 L vessel, since the head pressure would have been similar to that of the 10 L glass vessel. However, it may be possible to reduce oxygen demand at the 100 L scale with increased agitation.

OTR of the SUF process was comparable to that of the 10 L process. Both broth temperature and pH were effectively controlled in the SUF at levels similar to that of the 10 L process. For this high-growth E. coli cell line, oxygen supplementation was required to maintain a constant DO of 35% ± 10% for the high-growth phase of the process. Scale-up from a 10 L glass reactor process led to comparable titers and processing time at the 100 L SUF scale.

Conclusion
This experiment successfully demonstrated the ability of a 100 L SUF to provide the conditions to promote rapid growth of organisms that would traditionally need stainless-steel fermentation, including temperature control and a sufficiently high OTR (>300 mmol/L/hour). The kLa data collected as part of BioVectra’s scale-up strategy provides insight into the effectiveness of the scale up and allows informed decisions to be made for possible improvements for future batches.

Case study: Scaling from a 200 L SSF to a 1,000 L SUF

We used production of a high-growth E. coli to demonstrate a scale-up strategy from a 200 L SSF to a 1,000 L ABEC CSR SUF. Data collected included OTR, DO, temperature, pH, and agitation in rpm. The same scale-up strategy that was used previously to maintain a target kLa as a function of gassed power per volume input and superficial gas velocity was used for this 200 L SSF to 1,000 L SUF application. The agitation, air flow, and oxygen flow profiles were compared between the scales as shown in Figures 1 and 2.



The agitation profile was similar between scales, which indicates that the kLa was maintained consistently for scale up as shown in Figure 3. The 200 L SSF process operated with 7.5 PSIG (0.5 barg) of head pressure whereas the 1,000 L SUF operated with 0.5 PSIG (0.03 barg) head pressure. Therefore, it was expected that an increased ratio of oxygen relative to air would be required at the 1,000 L scale to make up for the reduction in driving force in lieu of higher head pressure.



Results
Measurements of OTR, media temperature, pH, titer, and processing time of drug substance were comparable between the two processing scales. The DO profile was similar to the DO maintained slightly higher in the 1,000 L SUF to account for the lack of head pressure. The final titer of drug substance was higher for the 1,000 L SUF process.

Conclusion
These results show scale-up from a 200 L SSF to a 1,000 L SUF is possible while maintaining similar levels for all process parameters. The 1,000 L SUF can handle the necessary OTR (>300 mmol/L/hour) and cooling needs of the most demanding E. coli processes.

Advancing SUFs for Future Biopharma

Historically, when compared to SSFs, SUFs have lacked comparable agitation power, airflow rates, and cooling capacity needed for microbial fermentation. The two case studies presented here show that innovations with current SUFs on the market permit the necessary mixing, oxygen transfer, and cooling capabilities to support high-growth microbial cultures and replace stainless-steel vessels of a similar size. While the two experiments shared here demonstrate how SUFs can be effectively scaled up from benchtop research to clinical application, and further from clinical stages to early commercial production, it also shows the type of comparability tests routinely undertaken, and parameters measured to scale an existing process. Such tests provide a manufacturer with the range of each parameter needed to produce the drug substance with appropriate titer and quality.

There is a place for these two technologies to live side by side in the same facility, providing the flexibility for a single manufacturer to progress from R&D to clinical production, and then onto large-scale commercial production of a biologic drug substance. Combining SUFs and SSFs means that scale-up and technology transfer can occur within one organization, simplifying the process and reducing opportunities for problems to arise. Having options to use SUFs at the 100–1,000 L scale is a boon for biopharma manufacturers wanting to take advantage of high-growth microbial fermentation for the production of biologics.

References
1. S. Ghosh. Single-Use Bioreactors Gain Popularity Worldwide. Bioprocess Online. 29 September 2023. https://www.bioprocessonline.com/doc/single-use-bioreactors-gain-popularity-worldwide-0001
2. Future Market Insights. Single-Use Bioreactors Market Outlook (2023 to 2033). August 2023. https://www.futuremarketinsights.com/reports/single-use-bioreactors-market
3. Biovectra. Single-Use Fermenters for the Production of Biologics. 2023. https://go.biovectra.com/WP-Single-Use-Fermenters-For-The-Production-of-Biologics
4. C. Graham, G. Awang. High-Growth Microbial Fermentation for the Manufacture of Biologics. PharmTech. 21 February 2024. https://www.pharmtech.com/view/high-growth-microbial-fermentation-for-the-manufacture-of-biologics

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Cameron Graham, P.Eng., manager, manufacturing science and technology, BioVectra, offers nearly a decade’s worth of experience in chemical engineering, with the last eight years concentrated specifically on the bio-pharmaceutical industry. In his current role at BioVectra, Cameron leads technology transfer and facility fit for the Nucleic Acids business unit, from lab to commercial scale.

Neil Morrison, director, biologics, manufacturing science and technology, BioVectra, has more than 10 years of experience in the pharmaceutical and biotech industry, with a background in process engineering. In Neil’s current role, he is responsible for tech transfer, process scale-up, equipment design, and commercial process design for biological processes.