Technology Redefines Continuous Processing Efficiency

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As perfusion-enabled bioreactors become better established, upstream biopharmaceutical manufacturing is shifting from fed-batch to continuous mode, harnessing improvements that have been made to alternating tangential flow (ATF) cell retention devices and tangential flow filtration (TFF). “The ratio between fed batch and perfusion is changing, and there is more interest in continuous due to the reduction in operating scale and the efficiency improvements that perfusion approaches bring,” says Christine Gebski, vice president and general manager of cell culture at Repligen Corp.  The company has been supporting perfusion cell culture approaches for nearly a decade with its XCell ATF and KrosFlo TFF cell-retention systems. 

The key benefit of operating upstream continuously in perfusion mode stems from the increase in productivity per liter of bioreactor volume that it allows, which can be 10 times that of a fed-batch bioreactor run, says Andrew Bulpin, head of process solutions at MilliporeSigma. “The cost for this additional productivity comes largely from the need for additional cell culture media. Most perfusion bioreactors are run at rates of one- to two-vessel volumes per day, which requires a significant increase in media production and downstream volume processing,” he says. However, Bulpin predicts that leveraging automation in the preparation of cell culture media and intensification of the downstream capture chromatography step will significantly debottleneck overall operations and lead to broader use of perfusion. 

Applications beyond traditional biopharm

Increased use of the technology is already being seen, not only for traditional monoclonal antibody and recombinant biopharmaceutical development but also for viral vectors and vaccines, says Gebski. “The technology has penetrated contract manufacturing and contract development and manufacturing organizations (CMOs and CDMOs) and Tier 1 to 3 producers and is finding less resistance as more people understand the process and manufacturing benefits,” she says. 

Continuous platforms are also being adapted for use in manufacturing innovative therapies by companies such as Biosana, an Australian company with cell therapies in Phase I clinical trials. In March 2019, The New Jersey Institute of Technology’s New Jersey Innovation Institute and Pall Corp. set up a venture that will establish two new centers for continuous process development of cell and gene therapies (1), augmenting a previous $1.9-million collaboration launched in November 2018, in which Pall, Cobra Biologics, and the Cell and Gene Therapy Catapult in the United Kingdom will develop new continuous cell and gene therapy manufacturing processes (2). 

Fundamental breakthroughs

A number of technology advances are driving the industry to improved continuous processing, including use of perfusion upstream.  One area of focus has been in optimizing approaches to allow for cell retention in single-use bioreactors, says Melisa Carpio, global technology consultant for cell culture technologies at Sartorius Stedim Biotech. 

Within the past year, she says, a number of enhancements have been made to existing single-use bioreactor systems to enable continuous bioprocessing. At the small scale, she says, both the ambr 15 and ambr 250 have additional options that can be added to existing systems to enable perfusion.  The ambr 15 cell culture system has two new tools that help perform perfusion mimic: a rapid vessel drain option and custom centrifuge adaptors that allow direct centrifugation of ambr 15 vessels.  The ambr 250 high-throughput system now also features a perfusion option that allows for true perfusion (both ATF and TFF) to be run at the 100 – 250 mL scale.  At larger scales, Carpio says, both the rocking motion (BIOSTAT RM) and stirred tank reactors (BIOSTAT STR) now incorporate features that permit perfusion process scale-up.  A new BIOSTAT RM bag with integrated perfusion membrane was launched at the 200-L scale (with 100-L of working volume) so that perfusion can be run in rocking motion bioreactors ranging from 1-L to 200-L.  The BIOSTAT STR now also features a bag configuration that can directly connected to cell retention devices such as Repligen’s ATF system, Carpio says.  

Atul Mohindra, R&D director for biomanufacturing at Lonza Pharma & Biotech notes that technology advances and greater process knowledge have been key breakthroughs driving use of perfusion. 

Improvements in disposable technology offerings, particularly in the cell-retention devices that are fundamental to perfusion have been instrumental, he noted. “Until now, this unit operation had not been robust, but the new generation of ATF and TFF devices are far more robust and perform better than previous technologies,” Mohindra says.

He also cites significant advances in sensors for at-line and in-line testing, which allow manufacturers to have better control over critical quality attributes (CQAs). At Lonza, Mohindra says, R&D has focused on a number of in line sensing technologies including the use of Raman spectroscopy to enable very precise measurement of viable cell concentration and individual metabolites such as glucose and lactate. 

Changes in cell line selection criteria allowing production of more stable clones better suited to perfusion processes also has advanced adoption, noting that Lonza is also looking to develop small-scale models which allow us to assess and select clones which are best suited for continuous processing.”  

In addition, Mohindra notes, better understanding of the interactions between cells, media components, and disposable technologies, permit the development of new engineering solutions that enable easier handling of media for perfusion processes.

Benefits and obstacles

Apart from reducing bioreactor size-and with it, both capital expenditures and footprint- continuous perfusion systems boost flexibility, Mohindra says, by allowing companies to manufacture diverse product types, a crucial ability for any CDMO. “Traditionally, perfusion processes have been used for labile products but there are also advantages for non-labile proteins. Customers come to us increasingly with more complex formats and perfusion can increase the robustness of manufacturing bispecific antibodies and other new molecular formats. Better control of CQAs means that we can achieve a more stable process and better regulate the final product,” he adds. As a result, duration of the process can be modulated to increase or decrease output in line with changes in demand, (e.g., if more is needed for clinical trials or if regulators grant expedited review of a drug candidate). 

However, Mohindra notes that perfusion doesn’t yet have the track record of fed-batch manufacturing. “Even certain terminology in regulatory and quality submissions is still geared towards fed-batch processes and needs to be adapted to include continuous processes,” he says. However, he notes that FDA has clarified that lot sizes may be defined based on either mass or time, and have stated that continuous manufacturing is fully consistent with the pharmaceutical quality-by-design concept.

Another challenge Mohindra notes is that continuous processes blur the lines between the skillsets required for upstream and downstream processing, which have traditionally been handled by separate groups. This means reskilling some teams and changing operational setups. At this point, however, only a few companies are trying to integrate continuous processing both upstream and downstream. 

“A lot of new technology has been brought to the market recently, so there is a much larger toolbox available to developers and manufacturers, and it’s not all about going straight to the fully continuous route,” says Peter Levison, executive director of business development at Pall. “Companies are considering a hybrid approach, in which they look at individual unit operations and replace the most troublesome ones, rather than going straight from batch to fully continuous,” he says. Generally, he notes, there is growing interest in perfusion and a lot of development work going on, but it will likely take years before it moves fully into large-scale upstream processes.

CDMOs invest in continuous, and N-1 seed train platforms

Nevertheless, continuous processing is moving into larger-scale projects and new therapeutic areas and CDMOs including Lonza,  MilliporeSigma, Fujifilm Diosynth Biotech, and Samsung Biologics have embraced perfusion-based continuous processing. In June 2019, Fujifilm Diosynth Biotech invested $10 million in a continuous processing facility in the UK, which is expected to come onstream in the autumn of 2019; the facility will use continuous upstream processing for pre-cGMP process development (3). 

Whether in stainless-steel or single-use environments, continuous systems are being scaled up and out, and integrated with automation and process control and improved media, to boost cell density and product yields. Seed train, or N-1 approaches, in which cell densities are first built up in one or more smaller bioreactors with ATF or TFF, and then moved to a full-scale bioreactor, have become more important as a way to speed processing.

In August 2019, Samsung Biologics scaled up the world’s largest ATF-and-perfusion installation to date, using an N-1 approach, at its facility in Songdo, South Korea (4). The company claims that the new installation, which uses a 15,000-L stainless steel bioreactor and Repligen ATF technology, will reduce production time by 30% and allow for a 10-fold improvement in cell culture while retaining viabilities of over 98% at the seed stage. Samsung Biologics has been able to maintain a perfusion rate of up to three-vessel volumes per day by using three 11-m2 X-Cell ATF-10 systems, developed by Repligen. The perfusion process was automated by integrating the ATF system with bioreactor control, the company reported, and validation and other tests were completed within six months.

For die-hard fed-batch users, Repligen introduced a High Productivity Harvest clarification application in 2019, Gebski says, which allows users to run their process in fed-batch mode, then utilize XCell™ ATF for a short duration harvest during which cell viability is maintained and cell density increases. The application allows the output of a fed-batch reactor to be doubled or the duration of the run 

Lonza has also realized benefits with upstream continuous processing. “We have been working on process intensification at the inoculum stage, which should allow us to significantly reduce the number of cell-expansion steps and improve the overall space and time yield in our facilities,” Mohindra says. “Simplifying the N-1 stage means that we can inoculate the production bioreactor at a higher cell density. Consequently, we can cut down the time required in the production bioreactor by about 50%, which brings significant time savings to our customers,” he adds. 

Another benefit he says has been improved ability to control CQAs, as a result of work that is underway at Lonza’s R&D labs in Bend, OR, Mohindra says. “We see an increasing number of new molecular formats in clinical programs-for example, bispecifics-and we are looking to re-tune the process to improve the consistency and quality of products. This also means linking new in-line spectroscopic technology (e.g., Raman sensors) with data handling and analytics.”

All of the company’s recent facility expansions, including that of its Ibex Solutions facility in Switzerland and its midscale facility in Portsmouth, NH have been designed to accommodate N-1 perfusion processes, Mohindra says.

Process control and automation

While efforts continue to improve data access and analytics, vendors are fine-tuning process-control systems and integrating them with ATF and TFF technologies, and perfusion systems. “A data-rich, continuous operation like perfusion cell culture is ripe for control optimization through process and data analytics, and several layers of integration offer value,” says Bulpin. “First, the use of real-time sensors (e.g., capacitance, Raman, and NIR types) provide a much better picture of the health of the cell, the concentration of nutrients and byproducts, and product and cell titers. Integrating the bioreactor and cell-retention system control strategy will allow us to use this data to maintain high productivity processes within a tight band of control,” he says, noting the potential for machine learning algorithms to be applied to optimize production and even utilize predictive control. 

Models from a number of other industries offer biopharma developers a glimpse of what is possible, says Lisa Graham, vice president of analytics engineering with Seeq, which offers a browser-based application. Connecting with data historians, the app makes data available for users to perform near real-time data analytics and monitoring. In biologics, she says, there is a great need for real-time data and feedback when shifting from batch to perfusion mode, in order to maintain a steady-state process, she says. 

A number of projects are also tackling better process control and automation. Sartorius Stedim and Repligen, for example, established a collaboration in 2018 to codevelop and integrated perfusion bioreactor. Zeroing in on single-use perfusion bioreactors in the 50­–2000-L range and using next-generation ATF technology, the goal is to integrate XCell ATF hardware and control logic directly into the bioreactor. The goal, says Carpio, is to integrate the bioreactor and cell-retention controller into a single interface that would allow users to control cell growth and perfusion rates in continuous and intensified bioprocessing. 

At Sartorius Stedim, work is ongoing to enhance continuous processing capabilities via integrated analytics and advanced recipe control, says Carpio. In one recent example, the company’s Corporate Research team automated inoculation from an n-1 intensified culture in the BIOSTAT RM to the production BIOSTAT STR. Using an integrated biocapacitance reading (via Sartorius’ BIOPAT Viamass), the team programmed the controller to transfer culture from the seed to the production reactor automatically, once the Viamass reading reached a specified threshold. Data showed that the growth and productivity of the automatically inoculated bioreactor was comparable to that of a bioreactor inoculated using the traditional manual inoculation method.  “Applying this approach to the manufacturing process will allow for shorter timelines as well as reduced error due to the automation,” Carpio says.

Mohindra sees advances in process control and data analytics as one of the most exciting drivers of continuous upstream bioprocessing. Driving these advances, he says, is the need for improvement in speed, cost, and quality. “Where we used to be able to measure basic parameters such as dissolved oxygen or temperature, we can now measure individual amino acids in line. The goal is to be able to measure conditions directly where the magic happens in the cell and this is really challenging the industry,” he says. 

He expects the industry to be able to test by exception, to reduce the need for lab testing significantly. “Here, we’re lifting some of the tools and data-handling experience from small-molecule manufacturing, where real-time release (RTR) is happening. Even though I don’t think we’ll realistically get to RTR in mammalian cell-based biopharmaceutical manufacturing, there is the potential to improve timelines significantly,” he says. Ultimately, he expects to be using machine learning and predictive analytics, along with strong data sets based on past projects, to ensure process improvement.

The building blocks needed for end-to-end continuous operation are already there, says Levison, but process control and analytical systems are lagging behind. “There is a need for unit operations to be controlled in a way that they talk to each other, and facilitate feedback control so that, for example, if a single component needs changing this can be ascertained online,” he says. New analytical methods are available that will require the ability to store and analyze vast amounts of data, and more automation and control systems will be needed to do this. However, he notes, great strides have been made over the past 18 months, and he expects to see more in the near future.

Better media and media handling

Technology providers are also responding to the need for better buffer and media handling systems for continuous perfusion processes. This year, MilliporeSigma introduced the BioContinuum buffer delivery platform, which includes a configurable offering of buffer concentrates, a precision dilution system, and single-use fluid management assemblies, says Bulpin. Its aim, he says, is to reduce the floorspace, labor, and process time required for process buffer preparation. The company also introduced EX-CELL Advanced HD perfusion media, specifically formulated for perfusion bioreactor processing, to improve results typically seen with recovered fed-batch media. 

References

1. Pall, “NJII and Pall Corporation Form Agreement to Advance Cell and Gene Therapy Manufacturing,” Press Release, March 19, 2019.
2. Cobra Biologics, “Cobra Biologics, Pall Corporation and the Cell and Gene Therapy Catapult Win £1.5M Innovate UK Grant to Investigate Continuous Manufacturing for Gene Therapies,” Press Release, Nov. 26, 2018.
3. Fujifilm, “FUJIFILM Corporation Announces the Introduction of a Novel, Fully Integrated Continuous Production System for the Manufacture of Biopharmaceuticals,” Press Release, June 5, 2019.
4. Samsung Biologics, “Samsung BioLogics Implements Large Scale N-1 Perfusion for Commercial Application” Press Release, Aug. 12, 2019.

Article Details

BioPharm International
Vol. 32, No. 10
October 2019
Pages: 12–16

When referring to this article, please cite it as A. Shanley, "Technology Redefines Continuous Processing Efficiency," BioPharm International 32(10) 2019.