Facilitating Clinical Manufacturing of Recombinant Proteins with Large-Scale Single-Use Fermentation Technology

In conversation with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D., BIOVECTRA’s Scott Doncaster, Neil Morrison, and Cameron Graham discuss the company’s addition of single-use fermenter technology at its site in Windsor, Nova Scotia, Canada. The seamless integration of ABEC’s Custom Single Run (CSR) fermentation solutions with working volumes up to 1,000 liters will enhance BIOVECTRA’s high-growth biologic microbial fermentation processes and provide fast turnaround product-to-product and between batches, providing BIOVECTRA customers with greater flexibility, faster turnaround times, and higher capacity utilization.

David Alvaro (DA): Can we start with a primer on BIOVECTRA’s history with regard to fermentation?

Scott Doncaster (SD): BIOVECTRA entered the microbial space around 2005, starting with small-molecule microbial fermentation and purification. We rapidly doubled process titers and yields and created additional capacity. Building on these process science achievements, BIOVECTRA entered the clinical biologics fermentation space.

Producing both small and large molecules in the same space was quite challenging, however, due to containment needs for certain chemical compounds and the regulatory cleaning requirements for multi-product and multiple campaigns per year. The changeover between batches causes a significant loss of capacity due to the downtime during change-outs.

The decision was made to have two separate spaces, one for small molecule fermentation up to 15,000 liters alongside conventional organic synthesis capabilities, and a separate one for the production of microbial recombinant biologics via fermentation in two 17,000-L fermenters in Nova Scotia. This new project will address the gap in our capacity needs for the supply of clinical projects with 1000-L and smaller single-use (SU) fermenters. 

DA: What are the relative advantages of fermentation in stainless-steel (SS) versus SU systems?

SD: The key advantage with SS is the ability to achieve ideal fermentation conditions at a very large scale, even up to 100,000-L. Because SS units are permanent, however, they require the use of validated steam-in-place (SIP) / clean-in-place (CIP) processes to ensure that the sterile envelope, including the body of the fermenter, is maintained, which requires high upfront investments and ongoing costs in terms of time and labor.

In addition, SS systems carry significant operational burden, because there can be many manual operations, such as valve turns, that must be performed. New SS units are more automated, but there are still risks of failing the validated cleaning process of SIP/CIP if just a single solenoid valve fails. 

With SU technologies, there is no need for SIP/CIP because the systems come pre-sterilized and are only used once. This reduces the upfront and operational costs and setup/turnaround times. These systems also have a smaller footprint and tend to be more automated, with fewer manual interventions required — essentially the bag is changed and inlets and outlets reconnected — reducing risk of operator error, cross-contamination, and time on plant for cleaning.

Neil Morrison (NM): There has been a challenge with commercial-scale SU fermenters with regard to achieving the optimum process conditions. The key constraints have involved the cooling capacity and oxygen transfer rate (OTR) that can be achieved using SU systems. Escherichia coli, the most common microorganism used for fermentation, typically requires high OTRs that can only be reached with a suitable oxygen transfer coefficient or kLa. The kLa is a function of the power per volume and superficial gas velocity. Historically, in SU bags, it has been difficult to achieve the necessary mixing and power per volume to support high-growth microbial organisms such as E. coli. This is the limiting factor for the maximum scale of SU fermenters versus SU cell culture reactors.

DA: What specifically drove BIOVECTRA’s interest in implementing an SU fermenter for your clinical-scale operations?

NM: Looking at new technologies is part of BIOVECTRA’s culture. Our in-house engineering department is always analyzing innovative technology. We use science-based decisions to make our manufacturing operations as efficient as possible as we grow, and SU technologies have been one approach. For the new clinical biologics fermentation production line, we leveraged SU equipment for the rest of the process train and were looking for SU fermenters that could maximize the flexibility of disposable solutions for the clinical area.

If you can employ SU technologies across the entire process train, you basically eliminate the need for cleaning validation and significantly reduce the time for changeover between one product and the next. The more we convert, the faster we see the batch cycle time reduced, and the better production efficiencies we achieve.

Cameron Graham (CG): To identify possible SU fermenters, we expanded our own internal engineering resources to focus on the particulars of mixing and oxygen transfer rates in SU fermenters to ensure the selection of the vessel or the selection of the parameters that will give us the best chance of first-time success. We had established internal specifications but were not initially able to find any SU fermenters that quite matched them.

SD: We were challenged internally by the manufacturing, product transfer, and tech transfer groups to ensure that we could achieve the desired oxygen transfer rates and meet specific media feed requirements. We absolutely needed to satisfy those groups with real data to support the decision to implement an SU fermenter.

DA: What is fundamentally unique about the ABEC fermenters that made the difference? 

CG: The new ABEC units alleviate the key concerns with SU fermenters around mixing and oxygen transfer. With ABEC’s latest Custom Single Run (CSR) design, the power per volume is comparable to what you’d expect to see in a SS vessel of that size. In addition, the aeration rates make it possible to reach a comparable superficial gas velocity. As a result, from a mathematical standpoint, the kLa is basically comparable across that board to an SS vessel. ABEC has also been able to solve the cooling issue, with their SU systems able to handle the heat generated during fermentation, at least up to the 1,000-L scale.

SD: An additional value-add for the ABEC SU system is the reduced installation complexity, which leads to less downtime in the existing facility before the units can be up and running. A traditional SS installation would require piping changes and welding and could add three months to the area downtime and overall schedule.   

DA: To what extent are other CDMOs leveraging SU fermenter technology at this scale?

NM: BIOVECTRA will be the first Canadian CDMO to offer single-use microbial fermentation at a 1,000-L scale.

SD: With the basic technologies from other equipment vendors still in beta development with potential end-users, I expect that, for the near term, deployment of larger SU fermenters will be quite niche. Eventually, they will become more common for applications where speed and proven production are needed, such as in clinical operations.

There are changes occurring in the industry that are impacting fermentation process conditions. Our clients, for instance, are developing more optimized processes with higher titers and higher cell densities (25–30 g/L cell mass vs. 15 g/L). The fermenters we offer must be able to accommodate the oxygen transfer and mixing rates in these very, very dense cell mixtures. Those improvements have challenged some of the available technology, such as centrifugation or microfiltration. 

Where high titers and high cell densities are needed at scales of 1000 L and above, SS will probably still be preferred until further advances in SU fermenter technology can be achieved. Until someone designs larger SUs bag that can be pressurized and appropriately cooled and provide the right mixing and oxygen transfer rates, there will be a role for SS. 

We are already seeing a switch to the use of SU technologies for large-scale mammalian biologics production in new facilities, with existing SS infrastructure still being used for established, high-volume products. It makes sense for mammalian cell culture, since they require low-shear mixing rather than turbulent mixing, and the power needs are much lower. In addition, mammalian cells manage the temperature more effectively. Even for these processes, we can expect a balance between SU and SS going forward, depending on the types and volumes involved.

DA: Can you tell me a little about the timeline for the clinical facility and the total scale of the new investment?

SD: We’re looking to have the units operational for Q3 of 2022, the timing of which is largely a function of the lead times on some of the components needed. Most of the facility work will be completed over the Christmas shutdown. The total investment is approximately U.S. $6.5 million, which includes an upgrade of the downstream processing area, with the addition of dedicated harvest, purification, and downstream processing equipment for individual products. 

DA: Are there any plans to go beyond this project and install additional SU fermenters in Nova Scotia or at other sites?

SD: The capacity of the new 100-L and 1,000-L fermentation suites is currently being sold. We have predicted approximately 25–26 runs per year — basically a two-week cycle on a campaign. If things go faster, then we can complete more batches, but that is our current planning model. If there is more demand for capacity, we will obviously look at additional installations. We can build on the experience of this project easily.

DA: How great a strain does the transition to SU technology place on process development or on customer acceptance of your ability to take their processes and advance them using SU fermenters?

NM: The impact that moving from SS to SU fermenters could have on tech transfer was definitely part of the discussion. We looked at all of the technical aspects in detail. We have tracked each of the operations and the overall oxygen transfer rates and found that the same growth profiles are maintained, leading to the same end results. Our studies demonstrated that a very similar tech transfer plan will be involved, whether linked to an SS or SU fermenter.

SD: I think that’s where ABEC plays a role. We can use their white papers and development work to show comparability between the output or performance in a 100-L SS versus a 100-L SU system. We will also be developing our own white papers to generically show the comparability of a 20-L tech-transfer batch to an SU batch at 100 or 1,000-L.

We recognize the importance of showing that the technology transfers from the benchtop — whether a glass, autoclavable benchtop fermenter or a 30-L stainless fermenter — into a higher-throughput SU system. We have a method in place to address that question. Finding a partner in ABEC that wants to solve the challenges faced by SU fermenters has been terrific.

The beauty of E. coli is that fermentation processes has been proven and can be run in SU bags that are permeable to air and that are rocked or shaken for 48 hours. Such a process won’t produce the protein of interest at a commercially acceptable titer as would be achieved in a highly stirred, highly oxygenated system. So, for us, the question with clinical products isn’t about pass or fail; it’s about getting a certain amount of that product manufactured as quickly as possible, and SU systems offer tremendous advantages in this scenario.

DA: Can you tell me a little more about the different contributions that ABEC is making beyond the physical hardware?

NM: We have seen ABEC breaking the boundary of the SU fermenter, applying some really interesting design techniques to reach new areas. And we see them expanding beyond just fermentation. For instance, they have large mixing bags and SU mixers, as well as new SU tangential-flow filtration skids, which is something we have seen constrained in the past.

SD: I think that, through our shared communication back and forth, we will continuously improve this system; not just the bag, but the process analytical technology and the integration into the control system. They are open to our input and that back-and-forth exchange rather than simply selling a product and moving on to find the next customer.

CG: During the research and exploratory process, ABEC was very open as far as our challenging the capabilities of the system. We went into it with a healthy bit of skepticism that it could meet our demands. They were very open with sharing the information and justification to basically align with what we were seeing from our own internal calculations to ensure that we were making the best decision as far as technology and supply of consumables goes.

DA: What impacts do you think this SU fermentation technology will have, both for BIOVECTRA as an organization and to individual customers?

SD: At a high level, ABEC’s technology enables simpler technology transfer and thus helps facilitate getting products to the client within the required timeline. Our customers have clinical trials lined up, and those dates are hard, set, and fast. SS fermenters are highly complex, with lots of interconnecting systems, so validated methods can be difficult. If we have one failure or issue or we have a problem with a cleaning method validation, that can be a huge issue. Eliminating the need for cleaning validation with the SU fermenters will avoid those risks and clearly benefit both BIOVECTRA and our clients. Most importantly, this expedited timeline has the potential to make life-altering impacts to patients.  

Additionally, we are treating this new suite as a separate unit operation from our commercial production area. There will be dedicated people and staff working on clinical projects that are significantly accelerated compared with processes involving commercial products with multiple campaigns per year. The know-how and efficiencies will be further added benefits for our customers.

DA: Where does the new SU clinical fermentation suite fit into BIOVECTRA’s overall plan and what comes next?

SD: Overall, our goal is to meet market and client needs. We have somewhat of a niche market in the products we produce: E. coli-based microbially fermented chemical APIs and recombinant proteins. We don’t use 100,000-L or even 50,000-L fermenters like some CDMOs. At the same time, however, our commercial scale isn’t just a thousand liters over and over again. This new installation fills that gap for tech transfer scale-up to feed more clinical programs for recombinant proteins. For successful projects, we can then offer commercial capacity at the same location. Furthermore, as demand grows, BIOVECTRA is committed to adding more clinical and commercial capacity.

DA: To close the interview, I want to use the widest lens. What do you see as the next big thing in pharma?

SD: Due to the COVID-19 pandemic, we have seen the general acceptance of and ease of regulatory filing for nucleic acid medicines in response to infectious disease. They are very elegant compared with traditional approaches that start with the use of a microorganism like E. coli containing plasmid DNA to produce a protein that may or may not be folded in a manner that leads to bioactivity, which must then be purified and formulated. With nucleic acid medicines, the body creates the protein, eliminating a lot of those steps and risks.

The technology has been proven, and it should be applicable for many existing biologics, including vaccines — stripping it back down to conversion of DNA into a protein within the body. Now it needs to be more widely accepted. Of course, there are limitations, but the key right now is acceptance.

Nucleic acid medicines are of particular interest to BIOVECTRA because plasmids are produced via fermentation.

In particular, it is important to note that large quantities of plasmids can be produced in a very small facility. You can take the plasmid DNA and amplify it. With mRNA, for instance, the amplification of a drug from a single piece of plasmid is huge. Only a small starting mass is needed to produce a much larger mass with the addition of more raw materials.

BIOVECTRA has projects underway to advance our vaccine and plasmid technologies. Our fermentation capability is well established. The challenge is to scale up downstream processing. Our goal is to develop an elegant solution to this problem. A lot of it at the end of the day is classical biochemistry, which is where we have an advantage, because we have 50 years of synthetic chemistry, continuous processing, and other technologies under our belt that we can apply to advancing plasmid manufacturing.

Scott Doncaster

Scott Doncaster earned a B.Sc. in biochemistry from Mount Allison University and completed a Master’s program in biochemistry at Queens University. Scott joined BIOVECTRA in 1995 where he served a series of roles before being appointed Vice President, Manufacturing Technologies & Engineering in 2014.