Biomanufacturing might appear to be centered around the cell line, which does enjoy a certain pride of place, ensconced as it is in the bioreactor, where conditions are carefully monitored and exquisitely controlled. Yet the cell line exists to generate a product. So, one might argue that biomanufacturing is in fact centered around the product, whether it is an antibody, a recombinant protein, or a vaccine. Admittedly, the product suffers various indignities during downstream processing, such as being transferred from one unit operation to the next, enduring capture, intermediate purification, and polishing. But in its own way, the product is quite as pampered as the cell line. The product must not be unduly diminished in quantity—and certainly not in quality, not at all. The product’s integrity must be preserved.
To accommodate the product, biomanufacturers can take advantage of tools, such as chromatography columns and membrane filters, that can smooth the product’s downstream processing journey. Choosing the appropriate columns and filters can expedite purification, improve recovery, and increase productivity. Other tools include design of experiment (DOE) studies, which can improve the management of processing parameters, either through a traditional trial-and-error approach or through machine learning, which can predict how specific parameters may affect the entire process at different times under various conditions.
Finally, the product may follow the usual downstream itinerary, a series of synchronized steps, or it may have the luxury of a single, integrated, end-to-end process—or something in between. So, let the cell line have its bioreactor throne. The product may soon travel the royal road of purification on the way to fill and finish. To see how the product’s path is becoming smoother and more continuous, read about the advances discussed in this article, which conveys the insights of a retinue of downstream processing experts.
Fit for purpose
Large biotherapeutic molecules originate from a variety of sources—plasma, supernatant, or cell lysate, for example—with differing requirements for chromatographic separation. Size matters. But so do factors such as hydrodynamic radius, sensitivity to shear stress, propensity to aggregate or fragment, and the presence of closely related impurities, to name but a few. However, all of them, “suffer from slow diffusion and mass transfer kinetics and, as a result, low binding capacity,” Artur Stanczak, application specialist, process chromatography, Bio-Rad Laboratories, said in a recent webinar.
This need not always be the case. Dealing with large molecules requires modern resins that are fit for purpose. Stanczak discussed aspects of Bio-Rad’s Nuvia resin family members, such as a narrow distribution range, optimized ligand density, and rigid hydrophilic phase matrix. For example, he said that with Nuvia resins, even high flow rates “won’t impact DBC [dynamic binding capacity] dramatically because of the rigid polymer structure.” The idea is to reduce nonspecific binding and free up some binding sites. Some of the resins, such as Nuvia HP-Q, offer properties such optimized pore structure and surface extenders. These allow for high DBC at high flow rates, and thus an improved recovery of the target product and an increase in productivity.
DOE studies should be conducted to determine ideal conditions, including testing factors such as pH and conductivity.
A digital twin
Understanding critical process parameters is also important for optimizing the filtration process. For example, parameters such as concentration, crossflow, and transmembrane pressure (TMP) determine transmembrane flux, which directly impacts filter performance. Ordinarily, these parameters are assessed via TMP excursions, which involve measuring volume flow through a membrane at specific time points. “This approach, however, only provides snapshots; it’s not predictive,” explains Mark Duerkop, PhD, CEO of Novasign. “The current setup relies heavily on trial and error, lacking a straightforward, systematic method.”
Novasign’s intuitive software studio leverages machine learning to derive insights from an automated TMP excursion. From that training run, it automatically builds a model to simulate various scenarios in silico, guiding operators on optimizing their chosen parameters—whether the operators are interested in minimizing production time or managing diafiltration volume, pH, and excipient concentration. “Our digital twin tells you how to set up your process at laboratory scale and how to transition smoothly to pilot or manufacturing scale,” Duerkop asserts.
The smart machine learning tool removes the need for repeated validation runs for each parameter change by simulating different scenarios digitally. It assists in decisions about membrane selection and optimal operation, indicating the ideal switching points between ultrafiltration, diafiltration, and a second ultrafiltration stage. It also provides insights into performance within continuous single-pass tangential flow filtration. According to Duerkop, the studio delivers significant savings in time, labor, and product compared with traditional trial-and-error or DOE approaches.
Going (mock) viral
Chromatography and filtration steps in a downstream process are employed to separate the desired product from host cell proteins and other process-related impurities. During process development, each step is optimized for removal of impurities, typically by quantifying them via affordable and easy-to-use commercial kits. The end goal is the serial diminution of impurities through the purification process until they are undetectable, or at least within acceptable limits.
One exception has been the presence of viruses, which should not be detectable in the first place except as a contaminant. Instead, during scaled-down process validation, viruses are spiked in at each stage of the process and their removal tracked. This is typically carried out by a contract research organization using live virus under BSL2/3 conditions, which is expensive and can have long turnaround times. Regulatory agencies require data demonstrating the efficacy of the process to remove viruses before the product can be introduced into humans during clinical trials and commercial release.
There is an option to disrupt that paradigm, says David Cetlin, senior director, R&D, MockV products, Cygnus Technologies. “The MockV approach replaces live viruses with noninfectious particles that mimic the same properties of that original virus,” he says. “They’re physiochemically similar, meaning that analyses can be straightforward. For example, the data you generate from a MockV MVM [minute virus of mice] kit should align pretty well with the data that you would generate from a live MVM study.”
“Historically our customers were applying the kits predominantly for process development, resin screening, process optimization, and things like that,” Cetlin points out. But he adds that newly revised regulatory guidelines “enable customers to use our RVLP [retrovirus-like particle] kit product to actually validate their process and put that data into a regulatory submission.”
Synchrony
At most biomanufacturers, downstream processing is performed one step at a time, with deliberate transfers of the product from one intermediate stage, accomplishing tasks such as protein A affinity purification and viral inactivation. But at some biomanufacturers, downstream processing is semicontinuous or even continuous. “Typically, end users connect different chromatography and filtration systems to get all steps of the process running at the same time,” says Joanna Pezzini, CEO, PAK BioSolutions. “And to do that, you need to build automation to have the different steps talk to each other, to make sure that they’re running at the same rate.”
PAK BioSolutions offers a continuous end-to-end solution at three different scales from process development to commercial scale, allowing for purification of 2 to as many as 20,000 L per day. The company can connect multiple different steps of a biopharmaceutical purification process in a single system. “We can do diafiltration, chromatography, virus inactivation, inline concentration and filtration,” Pezzini remarks. “You can perform up to four unit operations simultaneously on a single system, and inline process analytical control technology assures that they run at the same rate.”
“If you can do four different purification steps at the same time, you can produce four times more product or run four times faster,” she adds. The other benefit she touts is resin savings: “We run a lot of chromatography cycles on very small columns instead of a few chromatography cycles on very big columns, so you’re purchasing smaller volumes of resin for products that only require a few batches each year.”
According to Pezzini, some companies that offer continuous manufacturing provide individual systems that perform one step in the process and leave it to the end user to build the automation that connects these different systems together. She says that unlike those companies, PAK BioSolutions is “creating a turnkey solution.”
Continuity for savings
Upstream and downstream processes can be connected to form an end-to-end continuous platform as well, helping to bring down manufacturing costs along the way. Such platforms have been constructed by Enzene Biosciences, a contract development and manufacturing organization focused on biologics production.
Enzene Biosciences has set out to reduce the cost of goods for innovative biologics manufacturing, aiming to reach $40 per gram by 2025. Until recently, the predominant technology for developing biologics has been—and largely remains—a fed-batch process. “With fed-batch, the only way to lower costs is by achieving a higher-titer clone or using an exceptionally large bioreactor,” explains Russell Miller, vice president of sales and marketing, Enzene Biosciences. “But even that may not be enough. We knew we needed to explore a different, more innovative technological basis.”
“The EnzeneX platform is a fully connected, continuous manufacturing platform from start to finish,” Miller asserts. Specifically, the “start” is a bioreactor step that incorporates perfusion technology, and the “finish” is the step just before nanofiltration.
The company’s website indicates that the Enzene platform “harnesses the power of intensified perfusion using alternate tangential flow and automated multicolumn chromatography to achieve continuous production of biologics.” Miller elaborates, “When you take the perfusion technology and you’re able to couple it with the elimination of hold tanks and run the process continuously [for 30 days], you enhance the opportunity to maximize productivity, which gives a commensurate reduction in cost of goods.”
The company has facilities in Pune, India, and in Hopewell, NJ, and it has started a phased rollout of its EnzeneX 2.0 technology. Among the innovations is an enhanced cell line capable of producing a titer of 8–10 g/L—double the previous cell line’s titer. According to Miller, the new technology will enable Enzene to scale up the bioreactor to 1,000 L. The next phase will bring online artificial intelligence–powered process analytical technology, with the final phase centered around optimization of culture media to further boost productivity and efficiency.
