Quest for Fully Disposable Process Stream

Flexible Manufacturing that Easily Transitions between Products and Projects Is Coveted

The disposable bioprocessing market, which began with plastic bags designed for buffer preparation and quickly evolved to disposable bioreactor bags on rocking platforms, has blossomed into a range of products for cell culture and the processes upstream and downstream of the bioreactor itself.

In fact, there is a growing perception that a completely disposable bioprocess stream is feasible and even within reach. The biggest hurdles remaining relate to disposable technology development in the areas of biosensors and chromatographic purification. Progress is being made in these areas, though, and the buzz at recent scientific meetings has shifted from the challenges to an emphasis on the opportunities disposable products provide, especially in terms of ease of use, flexibility, maximizing infrastructure utilization, and, perhaps most notably, cost savings and environmental impact.

A key advantage of disposable components is the ability to build flexible manufacturing schemes that can readily be transitioned between products and projects. This is an especially important advantage for contract manufacturers (CMOs), which can maintain versatile production facilities without the need for extensive cleaning and cleaning validation protocols, and with greatly reduced risk of cross-contamination between product streams. Flexible manufacturing that enables rapid scale-up is particularly critical in areas such as vaccine production.

Disposables can greatly reduce the overall regulatory burden, the demand for energy and water to produce steam for steam-in-place operations, and the need for harsh cleaning chemicals and their removal during clean-in-place functions. These factors are not only economic drivers, they also represent sound environmental reasons for the use of disposables.

How close the bioprocessing industry is to a fully disposable process stream depends, in part, on scale. For small clinical batches, highly bioactive compounds, or personalized medicines, such as patient-specific vaccines, production can be done in disposable systems from start to finish. Large-scale manufacturing—for example, production of monoclonal antibodies in 10,000–20,000 liter stainless steel tanks—cannot be done economically with today’s disposable technology. For working volumes between these small-scale and large-scale applications, hybrid systems are pieced together, combining conventional and disposable elements.

Environmentally Positive

The first reaction is often that an increase in solid waste associated with a disposable flow path is antigreen; yet, notes Jerry Martin, svp, scientific affairs at Pall Life Sciences, the significant reductions in energy use, purified water use, and toxic cleaning chemicals realized with a switch to disposable process streams, results in “an environmental positive.”

Although the growing consensus is that disposables are ultimately both green and economical, there remains the issue of solid-waste production, as most of the single-use products employed in biomanufacturing are not recyclable given the technology available at present. Whether disposal in a landfill or incineration is the better option is open to debate, says Martin, who describes efforts under way to develop methods for burning plastics as a cogeneration source for energy production.

A substantial effort is being devoted to assessing the potential economic and environmental gains to be realized by implementing disposable technology. Nigel Darby, GM of biotechnologies at GE Healthcare, says that, although it is “too early to predict the outcomes of these studies, early data suggest that environmental impact will not be rate-limiting in the adoption of this technology and may even provide a net environmental benefit.” Furthermore, “disposables tend to decrease the amount of capital investment,” providing an economic incentive. Although a company will spend more on consumables with a disposable process stream, it appears that the overall result is a cost saving.

Regulatory Green Light

“FDA has looked at single-use manufacturing and does not see the need for any new regulations or guidance to cover it,” says Martin. In fact, disposables offer advantages from a regulatory perspective; they represent single-use, dedicated equipment, providing better containment, minimizing the risk of batch-to-batch contamination, and eliminating the need for cleaning and cleaning validation. In fact, an FDA guidance that favors single-use equipment for producing clinical batches “has been very enabling for CMOs,” Martin says.

Regulatory acceptance has been accompanied by heightened activity in testing to detect and measure potential extractables and leachables from the plastic materials used to make disposable products.

Pall’s Allegro™ line of disposable products for small- to large-scale processing includes Kleenpak™ Nova capsule filters. Single-use Kleenpak Connectors, each composed of a male and a female connector, are used to link two separate presterilized fluid pathways. The company’s Allegro 2-D biocontainers range in size from 50 mL to 50 L, while Allegro 3-D biocontainers and accompanying front-loading stainless steel totes are available in 100 L, 200 L, and 500 L sizes and contain a sampling port and two inlet top ports.

These disposable biocontainers are made of a polyethylene film that is both clear and low in extractables and leachables, according to Martin. The Allegro 2-D biocontainers feature rounded corners to minimize product loss. The company designed a tapered manifold to connect the plastic sheets to incoming and outgoing tubing to reduce the chance of leaking.

Sartorius Stedim Biotech recently added to its BioStat® CultiBag family of products, introducing three new products, including the CultiBag RM 600, a 600 L disposable bioreactor with a 300 L working volume. The CultiBag RM relies on the motion created by a rocking platform for mixing. It is designed for seed production or GMP manufacturing in batch or fed-batch mode. The 3-dimensional bag contains optical chemical sensors for pH and dissolved oxygen in the form of immobilized, disposable sensor patches. Also new from Sartorius is the CultiBag RM Twin-Rocker, which consists of two rocker units that can be controlled independently by a single BioStat RM Control Tower.

With the introduction of the CultiBag STR, Sartorius Stedim Biotech brings to the market a single-use stirred tank bioreactor. This new product line debuts with a disposable bioreactor chamber that has a maximum working volume of 200 L. The chamber sits inside a stainless steel support housing; the system includes a controller and disposable sensors.

Holger Bromm, director of marketing and product management for filtration and  fermentation technologies at Sartorius Stedim Biotech, identifies the major benefit of this reactor type. “It is as close as possible to a stainless steel reactor, with identical control, agitation, sparking, and design.” The company plans to introduce models with working volumes of 50 L, 500 L, and 1,000 L within the next 6–12 months.

The Hydrocyclone from Sartorius Stedim Biotech is a perfusion device for cell separation designed for use in combination with a large-scale disposable bioreactor being operated in continuous product collection mode. It functions as an inverted fixed-wall centrifuge without rotating parts.

Hydrodynamic energy-driven centrifugal forces move cells downward, while smaller particles, dead cells, and cell debris are driven toward an exit port at the top by a countercurrent inner secondary vortex. The Hydrocyclone has a maximum perfusion performance of up to 720 L/day; larger-scale perfusion can be achieved by operating multiple systems in parallel.

Other new disposable offerings from Sartorius Stedim Biotech include Sartoclear L-Drums for large-scale cell harvesting, which are an extension of the Sartoclear P product family. Composed of a double layer of cellulose-based depth filters, the Sartoclear drum can be used singly for cell clarification after centrifugation, or two drums can be used in series for cell harvesting, eliminating the need for centrifugation or spin filters, the company reports. The L-Drum will initially be available in four different multilayer combinations of filters, each with a surface area of 2 m2.

For single-use filtration applications, Sartorius Stedim Biotech completed its Sartopore®  2 polyethersulfone (PES) membrane filter product portfolio with the launch of the Sartopore 2 XLG MidiCaps and MaxiCaps disposable capsules for sterile filtration. The XLG and existing XLI versions of the MidiCaps and MaxiCaps exceed the total throughput performance of most commercially available PES filter membranes by 80–100% in application such as media filtration, sterile filtration after cell harvest, and filtration of downstream intermediates, according to Bromm.

Applikon’s AppliFlex single-use bioreactor is made of JMS Flex Film. The Appliflex product range has built-in fluorophor sensors for pH and dissolved oxygen; these parameters can also be controlled using classical pH, dissolved oxygen, and temperature sensors. The AppliFlex Rocker ensures reliable mixing.

Next to join the Appliflex product range later this year will be a new single-use bioreactor system being developed by Applikon in cooperation with Millipore.

SAFC offers a family of single-use products for cell culture and bioprocessing under the BIOEAZE™ trade name. The company’s ethyl vinyl acetate bags, polyethylene bags, sterile tank liners, EZ Top and asceptic connecting devices, and disposable tubing can be purchased separately or as an assembled, customized bioprocessing system.

Seeking Standardization

A lack of standardization around disposable ports and connectors is “a shared frustration” in the bioprocess industry, remarks Geoffrey Hodge, managing director of technology development at Xcellerex. CMOs and other producers can try to standardize their systems on a few select tubing sizes or rely on customized products, but it is more likely that they will have to work with a dozen or more manufacturers of disposable products to meet all of their needs.

In response to demand for larger-volume processing, Millipore introduced the 1-inch Lynx® ST (Steam-To) connector, designed for the transfer of sterile fluids between steamable, hard-piped processing systems and sterilized disposable flow paths. Also new to the company’s Mobius®  family of single-use bioprocessing solutions is the MIX500 disposable mixing system, which contains a 500 L carrier, a single-use magnetically driven impeller inside a disposable Mobius process container, and an electronic drive unit. Mobius single-use mixing systems also come in 100 L and 200 L volumes.

ReadyMate™ genderless disposable aseptic connectors enable a safer, more economic, and simpler connection to assembly components in biopharmaceutical production, reports GE Healthcare.

GE Healthcare offers its ReadyMate™ line of genderless disposable connectors. By eliminating the need to stock both male and female connectors, inventories can be reduced by half, notes Darby. “Furthermore, the range of flow path sizes provides scientists with more flexibility in line tubing reductions or increases by using the same ingenuous aseptic connector,” he says.

In addition to the direct cost savings possible using disposable components, another way to look at their economic impact is how they affect a company’s productivity. With a quicker turnover rate from batch to batch and product to product, a company can move more products through a facility, advance more products into clinical trials, and shorten the time to market.

It is not unrealistic to realize a 20–30% increase in the number of projects a facility can manage with a switch to disposables, says Darby. “An extra year of a drug on the market has a considerable economic value,” he adds.

Scale Trends Downward

“An interesting trend is that improvements in expression technology are making it more feasible to work with the new disposable technology,” observes Darby.

Needs and capabilities are now converging. Even as the demand for larger-scale disposable options grows, there is an emerging recognition that as gene-expression levels continue to increase, large-scale bioreactors may not be necessary to produce the quantities of biomolecules needed for biotherapeutic and vaccine manufacturing.

An engineer performs mixing studies in the XDR-2000 disposable bioreactor in Xcellerex’ pilot plant.

In fact, Hodge predicts declining popularity of 10,000–20,000-liter bioreactors, with a shift in scale to 1,000, 2,000, and 5,000 L volumes. Xcellerex recently added a 2,000 L XDR™ bioreactor to its FlexFactory™ biomanufacturing platform. XDR bioreactors are also available in 200, 500, and 1,000 liter working volumes and smaller-volume units are in development. Xcellerex also offers XDM QUAD™ single-use mixing systems.

As cell culture systems are able to generate more product per batch, the requirements for the bioreactor technology become more stringent and include the need for a broader range of sensors and probes, online monitoring, and real-time feedback control capabilities—the types of process controls that are available on traditional stainless steel systems.

Thermo Fisher Scientific’s open-architecture design approach allows customers to select one of the company’s single-use bioreactors (S.U.B.) and couple it to their choice of control systems, probes, and fittings. “Customers seek this flexibility to incorporate new disposable technology such as sensors into their single-use bioreactor without restriction to the manufacturer of the technology,” says Brandon Pence, associate director of market management at Thermo. The HyClone S.U.B. system consists of a reusable stainless steel out support container and a single-use BioProcess Container and is available in   50, 100, 250, and 1,000 L maximum working volumes.

The HyClone Single-Use Bioreactor is available in 50-, 100-, 250-, and 1,000-liter maximum working volumes.

HyClone Single-Use Mixers (S.U.M.) incorporate the same stirred-tank design used in the S.U.B. They range in volume from 10 L to 1,000 L and are suitable for hydration of powdered media, preparation from liquid concentrates, and other liquid applications. The new S.U.M. docking station facilitates storage of materials after mixing while freeing the mixing station to process a new batch. The docking station detaches from the mixing vessel and BioProcess Container.

Challenges Ahead

Most of our customers have, until now, run the single-use processing equipment manually, says Mani Krishnan, director of process systems, downstream processing business unit of Millipore. “For product contact applications, however, there is an interest in making the process less operator intensive—automation and data acquisition are now being requested. This will drive single-use manufacturers to provide not only the disposable flow path but also the necessary hardware to operate the single-use flow path. These high-value applications require the single-use systems to be designed with sound engineering principles—merely connecting a bag to a piece of tubing and irradiating the assembly is not where the value is.”

Krishnan reports an increasing number of requests from customers for single-use processing systems for product contact applications such as clarification, protein concentration/diafiltration, and final fill/finish. These are “primarily for phase I/II type molecules where the volumes are relatively small or at CMOs where the need for flexibility is the highest. We are also seeing request for medium-to-large scale processing hybrid equipment,” a single-use flow path, and stainless steel contact surfaces.

One of the last hurdles to realizing a totally disposable bioprocess stream is the chromatographic purification step. Large-scale manufacturing yields high-volume fluid streams, which necessitate huge columns and large amounts of specialty resins that are simply too expensive to throw away after a single use.

Pros and Cons

At CHI’s “Peptalk” conference, roundtable panelists including Leigh Pierce, president of PacificGMP, Scott Fulton, cofounder and scientific advisor of Tarpon Biosystems, and Geoffrey Hodge, of Xcellerex, discussed “The Pros & Cons of Disposables.”

PacificGMP has utilized a 100% disposable process stream since its inception four years ago. “We have no steel tanks, no skids,” says Pierce, but “we still need disposable columns and resins.” The increased flexibility that disposable components provide allows the company to “get up and running quickly. Our queues are shorter; our costs are less,” Pierce adds. “We don’t have a water purification system on site; our needs are reduced as we only use WFI for processes, not cleaning.”

PacificGMP is experimenting with continuous feed/perfusion processes to extract the maximum amount of product from a single-use bioreactor bag. The cells are recirculated in a hollow-fiber filtration system. With this continuous-feed approach the company has been able to process 15,000 liters of culture fluid in three runs using a 500 L disposable bag. “I think scale will cease to be an issue with the exception of the downstream component,” says Pierce.

There is a great deal of interest in the disposable chromatography technology being developed by Tarpon Biosystems, according to Fulton. BioSMB™ is a modified form of simulated moving bed (SMB) technology, which is in use in other industries and is now being developed for biopharmaceutical purification applications.

One of the issues in trying to adapt conventional SMB systems for use in an asceptic flow path is the number of valves these systems typically contain, explains Fulton.  A typical purification system might contain 8 to 12 columns, with each column having 8 to 9 valves. With the development of a disposable plastic valve block, SMB becomes an economically viable option for disposable chromatographic separation technology.

“With SMB you can maximize use of the resin,” says Fulton. The combination of a substantially larger loading capacity and more rapid cycling allows for purification of large volumes of process fluids in one or two batches, thereby reducing resin and buffer needs by 40–60%.

“SMB makes disposable chromatography feasible,” Fulton states. With cell-culture expression levels increasing to routine yields of 5 g/L and higher, the production bottleneck has shifted downstream to the purification step. “The advantages of SMB grow dramatically as expression levels go up,” says Fulton. Whereas an increase of 1 g/L to 10 g/L would necessitate a ten-fold increase in capacity for traditional chromatographic methods, with SMB, “you can just cycle it faster,” and only a small increase in scale is needed. Tarpon has produced a lab-scale prototype of the BioSMB that can handle the output of a 500–1,000 L bioreactor, he adds.

Limitations still exist in disposable sensor technology for online monitoring. Some sensors such as pressure gauges and dissolved oxygen proves are available, and pH sensors are coming along, according to Pierce, but other detectors such as UV flow cells are still far too costly for one-time use. SciLog recently introduced a family of disposable in-line sensors for monitoring conductivity (SciCon), pressure (SciPres), and temperature (SciTemp). These precalibrated sensors come in five different sizes, ranging from Luer, 3/8″ barb, 1/2″ barb,  3/4″ TC to 1.0″ TC.

According to Karl G. Schick, Ph.D., vp of R&D, these sensors accommodate the industry’s need for system scalability.

As part of a program funded by the Defense Threat Reduction Agency to develop technologies and strategies to accelerate biomanufacturing, Xcellerex has investigated an SMB process for purification and reports a reduction in buffer consumption by half or more, contributing to significant cost reductions. The goal of this program is to identify and optimize the technology needed to facilitate a rapid response to a biothreat, enabling quick production of millions of vaccine doses starting from a DNA sequence.

Xcellerex is currently in Phase II of the program, which involves developing a rapid response capability and then demonstrating the manufacture of a subunit vaccine or monoclonal antibody in less than 12 weeks in a live-fire scenario. The company’s FlexFactory™ single-use bioprocessing platform is scalable to a 2,000 L working volume.