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Although downstream efficiency still lags behind upstream, engineering-driven innovation is breaking through boundaries.
The biopharmaceutical market is challenged by growth, restructuring, and time-to-market pressures. For conÂtract development and manufacturing organizations (CDMOs), development timelines for standard vanilla antibodies have been cut in half over the past few years, says Uwe Gottschalk, chief scientific officer at Lonza. “That means going from DNA to clinical trials within one year,” he says, “and development times for all the individual steps entailed, from cell-line construction to a new manuÂfacturing process, have all been shrinking dramatically.”
For downstream bioprocessing, the greatest test has been addressing ongoing productivity gains upstream. “There is a gap, and it is widening, between what is coming out of the fermenters and the capabilities of downstream processing systems,” says Gottschalk. While upstream processing is volume-driven, unit operations downstream are mass driven, he says. One can make a kilogram of antibodies in a 1000-L bioreactor, or increase the output to 10 kilograms by increasing expression levels and overall yield, says Gottschalk. But to handle the larger amount downstream, at any capacity, will require chromatography columns that are 10 times larger, or that are run in 10 cycles, he says.
Market pressures have driven improved process technologies, which some observers refer to as “next-gen” bioprocessing, a catÂegory that includes process intensification and the connection of different unit operations, as well as continuous processing, both up and downstream. “These improvements allow manuÂfacturing to run more efficiently and produce higher yields in less time and space, reducing capital investment,” says Andrew Bulpin, head of process solutions at MilliporeSigma. “By 2020, we expect that approximately 20% of today’s molecular pipeline will be manufactured using elements of next-generation bioproÂcessing or continuous manufacturing,” he says.
Downstream, these improvements include combined unit operations, alternatives to chromatography, new process modelÂing techniques that allow for the visualization of “the golden batch,” and analytical approaches including greater use of process analytical technologies (PAT). While the gap with upstream may still be there, it is being addressed. This article highlights trends.
Behind many of the new advances is process intensification, which improves facility productivity by focusing on terms of kilograms of product manufactured per year, per square meter of facility footprint, says Peter Levison, executive director of business development at Pall Corp. “Chromatography is a potential bottleÂneck downstream because sorbents have a finite binding capacity, but process intenÂsification allows feed to be pre-concenÂtrated prior to adsorption,” he says. “In addition, one can move from batch to continuous chromatography to maximize the adsorptive capacity of the sorbent,” he says, noting the benefits of single-pass tangential flow filtration (TFF). As higher concentration formulations become the rule, filtration systems will have to handle concentrated and more viscous solutions without damaging product, he says, and virus and sterile filters are being developed to address these challenges.
Bulpin also notes the importance of process intensification “Our work with customers has shown that facility throughput can increase by up to 75%, simply by converting one- or two-unit operations,” he says.
Alex Chatel, product manager at Univercells, singles out TFF systems as a major process-intensification-based advancement. TFF enables a large increase in concentrations in a single pass, so that feed streams needn’t be recycled many times over through the membrane, he says. Bulpin recalls one case in which a manuÂfacturer installed a single-pass TFF device, which now provides inline feed stream concentration and/or dilution, elimiÂnating the need for holding tanks and intermediate process steps. “With slight modifications to a couple of unit operaÂtions, companies can incorporate a more continuous approach while reducing their manufacturing footprint and scale,” he says.
Using process intensification, unit operations are being integrated so that, for example, cell removal and early clarificaÂtion may be captured in one step, instead of the three or four that were previously required (i.e., typically centrifugation folÂlowed by depth filtration, sterile filtration and capturing column), says Gottschalk. As a result, some downstream processes have been reduced from 10 to three or four steps, and, in the future, he believes, two or three steps will be possible.
The move to continuous chromatogÂraphy (i.e., having multiple columns running side by side) has also been a sigÂnificant advance, says Chatel. Univercells, for example, is developing continuous-process-based platforms and is working on virus manufacturing platforms in projects sponsored by the Gates Foundation. “All are batch processes because they depend on binary loops and they need regeneration, but they are run so that when one is purifying product, the other column is being regenerated, resulting in a continuous stream of prodÂuct output. Advances in engineering and mathematics enabled this to happen, and have drastically reduced the amount of resin required,” he says.
The result of all these improvements has given manufacturers access to a “scaleÂable toolbox” that can enable batch, conÂtinuous, or hybrid process development and scale up using stainless steel or single-use systems, says Levison.
Examples he cites include, not only processes that enable continuous chroÂmatography, but depth filters designed to clarify higher cell density fed batch culÂture; continuous diafiltration and continÂuous concentration; and the introduction of large-scale prepacked chromatography columns and new single-use chromatogÂraphy skids to complement them. “With associated products such as sterile conÂnectors, biocontainers, and single-use filÂters, a closed, fully connected downstream processing train can now be assembled, allowing for rapid scale-up and process implementation,” says Levison.
There has been a shift to single use downstream, Gottschalk says. In some cases, single-use systems can run at close to 100% variable cost eliminatÂing the need to depreciate costs for the facility and stainless-steel equipment, he says. Gottschalk sees increased use of convective, instead of throughput-limited chromatography media, as a key trend. “Filters are developed with the ligands that would normally go on chromatography resins, and used in a membrane, monolith, or depth filter,” he explains.
Although some research groups and companies, most notably Merck & Co., have been exploring end-to-end continuÂous biomanufacturing, more manufacÂturers are using continuous processing strategically, both up and downstream. Univercells has developed a continuÂous production system in which cells are grown in batch mode but different process steps are run continuously. “We need to get away from the old approach, in which you send all the contaminants downstream,” says Chatel. As Gottschalk says, continuous processing was always used upstream for sensitive molecules that could not endure conditions in the fermenter (e.g., enzymes such as Factor A for hemophilia treatments). Perfusion fermentation was essential in order to keep the molecule intact, but the approach has since been used with other more stable molecules such as antibodies, and is now moving into the broad bioÂpharma world, he says.
Continuous processing is already a reality for single-use systems, Levinson says. “We can currently clarify cell culÂture media continuously using depth filÂtration; purify product using continuous chromatography; carry out continuous virus inactivation; and then complete the ultrafiltration and diafiltration stages using continuous single-pass filtration technologies,” he says. Trends for the future relate more to the modularity of the continuous downÂstream platform, with connectivity between each unit operation and inteÂgrated PAT and automation and control platforms, Levison says. For a truly conÂtinuous upstream process, a perfusion bioreactor could be used upstream, but it would require continuous cell culture fluid harvesting. Current cell-retention technologies based on hollow fiber filtraÂtion are prone to fouling, he says, so the composition of the harvest is variable and changes over time. One alternative could be to use acoustic wave separaÂtion (AWS), and platforms using AWS should be commercially available in the near future, he says.
The biggest change in bioprocess autoÂmation has been the introduction of the model-driven approach, says Bulpin, who explains that this approach allows users to define process, interactions, and contexts in form of parameterized data. This helps to reduce complexity, increase flexibility and reusability, and enable dynamic re-configuration of systems and processes. Model-driven design can lead to increased quality by increasing visÂibility, providing higher abstraction layers with less custom code, and empowerÂing the domain experts by focusing on the process rather than underlying techÂnology, Bulpin says. In the long term, he expects it will lower the sensitivity to change and help to bridge the gap between business and technical domains.
At the same time, analytical methÂods are improving, and use of PAT is increasing. Chatel has seen advances in sensors for pH, dissolved oxygen, and traditional measurements, that have allowed companies to adopt monitorÂing and control practices for bioreactors and other steps such as chromatography. “Future sensors will be able to detect metabolic concentrations in cell cultures (e.g., measure titers at line and provide feedback control over feeding strategies) to reduce burden of contaminants and ultimately lead to more lean processes and reduce processing time,” he predicts.
Noting the increased use of Raman spectroscopy-based methods, Gottschalk says, “We can now monitor the most relevant critical parameters online or at-line so that, after we have manufacÂtured the batch, we know whether it is acceptable and can be approved or not. In biopharma, we have one or two critiÂcal assays that need to be run at the end of production, such as the adventitious virus test, which cannot currently be brought online, but most other analytiÂcal tests can be run online,” he says.
Bupin sees artificial intelligence (AI), primarily machine learning, as drivÂing future improvements in PAT. “This approach should significantly improve quality and process robustness; combined with near-real-time data acquisition, it will enable closed loop for the process execution,” he says, improving commuÂnications and interaction between enterÂprise systems and manufacturing field I/O (inputs/outputs), including sensors, actuators, analyzers and drives.
As more bioprocessing steps are automated, the approach is also proving important for sampling, says Gottschalk. Lonza is working on data mining and new approaches using machine learning and other artificial intelligence concepts. “The goal is to use advanced concepts and multivariate data analysis to ensure that we run only the ‘golden batches,’” he says.
Over the next few years, Levison expects new inline or at-line tools to become available to monitor both critiÂcal quality attributes and critical process parameters. “Whether this will involve new sensing and detection technologies or multi-attribute measurements (MAM) based on existing technologies, or indeed a combination of both approaches, remains to be determined. What is clear is that as we move towards advanced anaÂlytics and PAT, more data will be generÂated more frequently so the demands on the data storage, management and handling will increase and this all needs to interact with the process control sysÂtem. AI and the Internet of Things will play a big part in these advances, which all fall under the umbrella of Industry 4.0,” he says.
One of the benefits of Industry 4.0 would be ability to use simulation (e.g., in the digital twin approach). “Provided that the digital twin can be shown to be a true twin of the real process, then its use in method development and optimization may become a reality (e.g., for determining process limits withÂout carrying out a detailed design of experiment). If the digital twin could predict batch-to-batch variability and process robustness then perhaps this could reduce time to market of new medicines directly impacting on patient health,” says Levison.
One practical area where downstream efficiencies are being improved is in buffer and waste management. “Single-use technologies offer benefits because they reduce cleaning and cleaning validation requirements,” says Levison. “With the introduction of single-use mixers and single-use biocontainers have come opportunities for storage of buffer concentrates with in-line dilution and/or conditioning to generate the buffer on demand at the point of use,” he says. Fluid waste can be collected in biocontainers which can then be aseptiÂcally disconnected and disposed of, he adds. However, as facilities become smaller, buffers continue to pose comÂplex storage and logistical challenges and can become a bottleneck for single use production, Levison says. One soluÂtion to increase facility buffer capacity is through the use of concentrates or stock solutions, both enabled by single-use mixers and single-use biocontainers. Managing buffers this way and delaying the dilution of these concentrated soluÂtions up to the point of use, allows sinÂgle-use facilities to increase their buffer capacity and increase their productivity.
In the area of buffer management and maintenance, MilliporeSigma recently launched the BioContinuum buffer delivery platform, Bulpin says, which uses buffer concentrates and in-line dilution to deliver buffer directly into the system. The company expects the platform to permit an 18% reducÂtion in cleanroom area requirements in a 2000-L bioreactor facility; reduction in buffer costs by up to 16%; and more than 50% reduction in labor and capital costs for a facility that uses five 2000-L bioreactors, he says.
Vol. 32, No. 7
When referring to this article, please cite it as A. Shanley, “Catching Up Downstream," BioPharm International 32 (7) 2019, pp 12-16.