Innovation in biopharmaceutical manufacturing is accelerating due to a combination of forces. One of these forces corresponds to the development of biosimilars, which is growing in importance as patent exclusivity rights expire. Another force corresponds to therapeutic modalities beyond monoclonal antibodies.
To adapt to changing circumstances—product changes and capacity expansions—biopharma firms are exploring novel production modalities. As part of this effort, biopharma firms are considering modalities that incorporate single-use technologies. Single-use technologies can allow bioprocessing to become more continuous by facilitating subprocesses such as high-cell-density perfusion. With single-use technologies, bioprocessing can become more intense, as well as simpler and more streamlined.
Realizing such possibilities would soothe the most anxious biomanufacturer, but when new technoloiges are deployed, there’s always a fly in the ointment. In the case of single-use technology, an especially pesky problem is the release of degradants, side products, or residues into product streams. Before these impurities can be swatted away, they must be identified and quantified.
Revamping product lines and supply chains
“Single-use technology eliminates a lot of the work needed to switch from one product to another, to switch a facility to make larger scales or smaller scales,” says Gregory Frank, PhD, principal engineer at Amgen. Large stainless-steel vessels, for example, require significant infrastructure for clean-in-place (CIP) and sterilize-in-place (SIP) systems, which are obviated by single-use technology. Also, he notes, because you’re installing a single-use bag into a stainless-steel shell, you don’t risk product carryover. In addition, changeover from one process to the next can be executed in hours rather than days—increasing the utilization of the plant and reducing the need to build new plants.
It might take up to five years, and $450 million, to go through the design, planning, and building of a new facility using the older technology—often with no assurance of regulatory approval. A single-use technology facility, on the other hand, might cost 60–70% less than a stainless-steel facility, and it could be built in a couple of years, Frank says. The same considerations apply to retrofitting, with all its support utilities. “If it’s single use,” he notes, “you can virtually wheel equipment out and wheel new equipment in.”
Single-use technology can also improve supply chain management. To support this point, Frank describes a representative scenario: “With single-use technology, I just get in touch with the manufacturer of the single-use bioreactor and say, ‘I want to put some new ports in here.’ … You get them in a relatively short period of time, you complete a straightforward installation, and you’re good to go with your new perfusion process. … And you’ve also shortened any validation timelines and resources.” You also develop an ongoing collaboration with your continuous supplier, which can then help you manage your supply chain.
Looking into Russian doll bioreactors
One of the realities of bioprocessing is the seed chain, which takes a starter culture—perhaps a 1-mL preparation growing in a plate or a flask—and scales it up incrementally until it’s ready for the production bioreactor. Currently, single-use stirred-tank bioreactors allow for only a fivefold expansion (called a “turndown ratio”), necessitating multiple transfer steps before achieving production volume. This, in turn, requires intermediate equipment, along with its concomitant floor space and contamination risks.
Momen Amer, as part of his PhD dissertation in chemical engineering under the tutelage of Joshua D. Ramsey, Ph.D., at Oklahoma State University, created a proof-of-concept single-use two-chambered bioreactor boasting a 40-fold greater turndown ratio than existing commercial technologies. Amer likens the two-chambered bioreactor to a set of Russian dolls. Like the dolls, which are placed inside each other in order of decreasing size, the bioreactor chambers of different sizes may be nested.
Unlike a set of Russian dolls, which typically includes five or six dolls, the proof-of-concept nested bioreactor has just two chambers, or bags. A single shaft travels from the top of the smaller bag through the bottom and into the larger bag. “You need only one support structure for the bigger chamber size, and only one control unit,” Amer points out, “which adds to the benefits of saving footprint and equipment costs.”
The nested bioreactor’s performance was described by a paper that appeared last year in the Biochemical Engineering Journal. According to this paper, engineering characteristics (such as mixing time, power input per unit volume, and oxygen mass transfer coefficient) for the nested bioreactor were in good agreement with those for extant bioreactors.
A second-generation prototype has since been constructed using flexible plastic and FDA-approved materials. Instead of requiring a peristaltic pump to transfer the culture between the chambers, the new prototype relies on gravity to transfer the culture. The only action required is the opening of a small clamp.
Amer and Ramsy see the potential to go to three or even four nested chambers, with the largest chamber either feeding the production bioreactor, or itself being the production bioreactor. “Once you make the initial transfer, everything is contained within one system,” notes Ramsey. “But we haven’t tried this yet.”
Settling for clarification
Recent advances in single-use technology are also relevant to downstream processing. One such advance is the disposable inclined settler. The inclined settler was introduced to modern bioprocessing nearly three decades ago by Dhinakar S. Kompala, PhD, then at the University of Colorado, Boulder. It is being used in large-scale stainless-steel incarnations by the likes of Bayer and Roche to harvest supernatant while allowing viable cells to settle and recycle back to their perfusion bioreactors.
Kompala’s company, Sudhin Biopharma, has developed a compact version of the stainless-steel inclined settler. It has 6–10 times more settling area over the same footprint. According to Kompala, the compact stainless-steel inclined settler can clarify small cultures containing Pichia pastoris cells or CHO cells.
The company has also created the BioSettler, a single-use plastic version of its stainless-steel compact settler. Of course, there are other single-use perfusion devices, such as membranes, but they can clog. Also, membranes begin to retain proteins after two weeks or so, Kompala says. Sudhin’s devices, in contrast, are designed to achieve high densities and high productivities over months. “There is no barrier, no membrane in our settler,” Kompala asserts. “It doesn’t ever get clogged.”
The BioSettler has now been adapted to clarify cell culture broth from a fed-batch bioreactor to recover over 90% of secreted product. The BioSettler can reduce the turbidity by over 80%, replace a stainless-steel centrifuge, and reduce the area of depth filtration membrane prior to downstream purification processes. Other potential applications of the BioSettler include gentle separation of stem cells from organoids and microcarrier beads. The BioSettler has thus far been demonstrated at small scale. “We’re looking for people who would be interested in collaborating with us to demonstrate it at higher and higher scales,” Kompala remarks.
The company has also done some preliminary experiments using the single-use settler as a “column-free, nonchromatographic method” to capture monoclonal antibodies on Protein A beads and separate them from cells and debris at the end of a fed-batch run. “It is really easy for us to eliminate at least two different unit operations,” Kompala declares. “The results are really amazing.”
Finding extraneous matter and material differences
The rise of single-use technologies in biomanufacturing comes with downsides as well. The polymeric compounds that constitute most single-use devices are a potential source of extractables and leachables, which would not be generated by the stainless-steel devices that single-use devices replace. “If you can’t accurately identify and quantify extractables and leachables,” cautions Mark Jordi, PhD, president, Jordi Labs, “you’ve created an unsafe situation.”
General guidance, from industry working groups, the FDA, and others, suggests that mass spectrometry should be used for discovery of extractables and leachables, yet this guidance is short on specifics as to the exact methodologies that should be applied. This guidance is also unclear on other points, such as the thresholds for identification and the appropriate surrogate standards for quantification.
Jordi and his colleagues created a model system consisting of a single-use bioprocess bag, tubing, and a disk filter. Using this model system, the investigators performed a series of extractions on the system as well as on each individual component. Then the investigators analyzed the extracts stepwise using a variety of screening and mass spectrometry techniques, followed by computational analyses and database searches, to detect extractables exceeding the analytical evaluation threshold (AET)—the point above which a chemist should pay attention to the leached compound.
“I showed that depending on which standard I used, I would get different numbers of AET-level compounds,” Jordi recalls. Numbers of relevant compounds would change by orders of magnitude.
Everyone uses surrogates—especially when they are trying to identify unknowns such as degradants, side products, or residues. But surrogates may do a poor job of representing the materials in single-use devices. For example, surrogates may display very different ionization efficiencies or matrix effects.
Jordi believes that extractable and leachable analysis is important for the safety of single-use technology, but to get good testing, you need good science done by knowledgeable chemists. “This is not cookie-cutter testing,” advises Jordi. “Do not act like this is routine.”
