August 1, 2010 (Vol. 30, No. 14)

Angelo DePalma Ph.D. Writer GEN

Distinct Advantages Result from Combining a Broad Array of Tools and Technologies

Scientists working in the bioprocess arena have a lot on their plates lately, including choosing between single-use and stainless steel equipment, integrating processes and instruments, and improving resource and facility utilization. Tool companies are scrambling to do their part to help customers by introducing enabling products that seek to ease some of the challenges and reduce many of the costs.

Andreas Muranyi, Ph.D., section manager of antibody processing at GE Healthcare, believes that GE’s Ready-to-Process™ (RTP) line of bioprocess equipment is a solution to several of the industry’s woes.

RTP encompasses the Wave Bioreactor™ and Cellbag disposable bioreactors; ReadyCircuit fluid-management bags, tubing, and connectors; aseptic connectors; filters; chromatography columns; and the ÄKTA ready chromatography system. Dr. Muranyi says that a fully disposable purification line utilizing his company’s RTP downstream components can provide greater than 80% yield while reducing aggregate levels by 90%.

GE’s approach leans heavily on PAT, QbD, and facility utilization, with an eye on time-to-market. “A large proportion of the expenses incurred during monoclonal antibody production are tied up in facility, floor space, installed equipment, and staff, which costs a lot of money whether you use them or not. The easiest way to reduce the cost per gram of active ingredients is to use the facility and related resources efficiently.”

RTP’s range reportedly extends over nearly every unit operation in a typical bioprocess, particularly downstream. During chromatography column changeover, the switch from conventional packed columns to prepacked ReadyToProcess columns saves about four hours (11 vs. 7), while use of the ÄKTA ready system reduces changeover time to just two hours, according to Dr. Muranyi.

“The change provides the equivalent of a full extra day for productive work.” Similarly, he says that changing from GE’s Kvick™ Lab filters to ReadyToProcess disposable hollow fiber membranes provides a time savings of about 30% (4.5 hours vs. 6.5 hours). Although the processing time is more than three times longer for RTP membranes, pre-use cleaning, membrane rinsing, and CIP/storage are eliminated for the disposable filters.

One question suppliers of single-use equipment enjoy answering more than any other is, “What is the sweet spot, in terms of scale or stage of development, for using disposables?” The answer, Dr. Muranyi wryly notes, is usually related to the sizes and scale of the equipment that a particular vendor sells. He then cites the futility in applying a uniform algorithm to all companies, processes, and molecules. Still, some generalities emerge.

GE Healthcare’s ReadyCircuit assemblies and ReadyKart mobile processing station are quickly configurable and assembled for sterile bioprocessing needs including crossflow and normal flow operations.

Stainless Steel Approach

Traditional stainless steel equipment is probably the best approach for large plants that produce a single, blockbuster monoclonal antibody product in ton quantities per year, with the cutoff for stainless vs. plastic somewhere in the 500–2,000 L working volume range.

Above 1,000 L, manufacturers must think seriously about factors other than simple scale, for example, legacy manufacturing systems, technology transfer from disposable to stainless, the likelihood that the molecule will be promoted, other pipeline drugs competing for manufacturing resources, and the related need for flexibility and agility. “There are lots of different scenarios and factors. You have to consider them all.”

GE has recently been pushing “smart” manufacturing approaches that combine stainless steel and plastic components and utilize those unit operations efficiently. Part of this effort has been “straight-through processing” enabled by QbD concepts and PAT technologies.

One straight-through strategy diverts column effluent directly from capture to ion-exchange columns, which eliminates the need for thousands of liters of buffer-storage capacity. Product from the ion-exchange column may then be filtered directly. Another approach involves mixing buffers directly, in-line, guided by precision dispensing pumps, sensors, and feedback control. A typical purification scheme taking three to four days can be reduced to 24 hours, Dr. Muranyi explains, and time savings are possible with all stainless, all disposable, or hybrid systems. 

“Engineers are becoming more broad-minded when it comes to stainless steel and plastic, mixing and matching them for their particular situations, and applying process understanding to create efficiencies.”

Lack of Integration

Detlev Szarafinski, global program manager at Sartorius Stedim Biotech, maintains a list of challenges driving adoption of single-use bioprocess equipment, including reductions in cost-of-goods and increased flexibility; specialized capacity needs across global markets, for example greenfield facilities in Asia; the after-effects of mergers and restructuring in Europe and North America; and adoption of technology platforms.

Despite its use in niche markets and at certain scales, disposable bioprocessing has achieved only a 13% penetration in biomanufacturing. Companies producing large-scale proteins (particularly monoclonal antibodies) generally shy away from single-use.

Even the 13% figure is open to interpretation, Szarafinski says, because the overwhelming majority are hybrid processes—particularly when upstream and downstream operations are viewed together. Most disposable manufacturing processes use both fixed-tank and plastic components. “The number of fully disposable facilities is really low at the moment, but I’m convinced that the number will increase.”

One reason, Szarafinski explains, is the current lack of component integration within unit operations. “We talk about single-use components such as filters and bags, but these represent process steps. Our approach is to combine those steps into a process sequence or unit operation.” This general approach, he says, applies to single-use, hybrid, and reusable formats.

Sartorius Stedim Biotech’s FlexAct platform consists of custom-configured single-use products encompassing entire unit operations. FlexAct is based on a configurable, central operating module and specific components, for example, the currently available FlexAct BP for buffer preparation. Over the next year the company plans to introduce modules for media preparation, cell harvest, crossflow ultrafiltration/diafiltration, virus removal, virus inactivation, virus adsorption, polishing, formulation/filling, and formulation/ transfer.

Szarafinski raises important issues that are coming to the fore as industry adopts more single-use components and systems:

  • Platform technologies and pre-assembled, presterilized systems may help mitigate regulatory and validation issues. Systems that are not pre-assembled are not necessarily ready to use despite the fact that their components may have been individually validated.
  • Monitoring and control issues include the need to incorporate PAT into totally single-use and hybrid manufacturing systems.
  • Integration of upstream and downstream single-use technology is lagging behind adoption at either end of the processing train. In addition, the emergence of hybrid (steel/plastic) systems underscores the need for aseptic connectors and tubing. Upstream-downstream integration is complicated by rising titers, particularly for antibody manufacturing.
  • The supply chain can be problematic since single-use components are consumables and users must secure their supply throughout a campaign.
  • Sustainability is more complicated than people realize. Although numerous analyses have demonstrated the green aspects of single-use process equipment, users must still formulate recycling and waste disposal strategies.
  • Finally, cost savings have similarly been illustrated at certain scales and development stages, but users still must demonstrate process design and cost optimization for their specific process.

Platform technologies are of great interest to Dethardt Müller, Ph.D., group leader for technology development at Rentschler Biotechnologie. Dr. Müller says that modular combinations of “high-performance platform technologies enable robust, generic bioprocesses.” He is also interested in such operations as clone selection and expression through purification. According to Dr. Müller, the goals of such efforts are streamlining and reducing costs without adversely affecting yield or quality.

Resource and Facility Utilization

Integration and utilization of manufacturing resources are an ongoing concern. “Antibody processing is maturing and changing,” explains Neil Soice, Ph.D., principal research scientist at Millipore. The company is seeking to help bioprocessors in their struggle with upstream-downstream misfit.

Most bioprocess plants operating today were designed years ago to accommodate protein titers that were much lower than today’s. This has introduced limitations and constraints with respect to buffer and holding tanks, which, according to Dr. Soice, have become bottlenecks.

“The larger manufacturers have invested heavily in their facilities, and are trying to leverage that investment by getting as many drugs as possible to the clinic,” he says.

Millipore has introduced several purification products over the last three years that serve this end, specifically chromatography resins with high intrinsic capacity and flow rates. These include the ProSep® Ultra Plus capture resin, which provides 50 g/L of binding capacity for mAbs, the high-capacity ProRes™-S cation exchange media, and an anion-exchange membrane adsorber for polishing.

Millipore has found that by optimizing each step, it’s possible to elute directly from the capture column to the cation-exchange column without intermediate hold. “And if you’re smart about the cation-exchange step, you can elute directly onto the membrane adsorber.” Dr. Soice stresses that while this purification scheme works with both single-use and stainless steel equipment, it is not 100% ironed out for every product, particularly at large scale.

Will connected processing save time in a real-world, large-scale process? It certainly does at lab scale, but at manufacturing scale the hold tanks serve a purpose other than storing product between steps. Tanks enable process engineers to design the beginnings and ends of unit operations around the flow of human resources—to hold product between work shifts.

“Connected processing would force you to optimize processes completely differently,” Dr. Soice says. “It would force bioprocessors to break the existing paradigm, and not let things sit around because they’re waiting for the next shift to come through.”

ChromaSorb membrane adsorber provides robust impurity removal over a wide conductivity range, according to Millipore. This single-use device reportedly reduces validation requirements, capital equipment expenditures, and time and labor when compared to traditional column-based anion-exchange resins.

Sidebar: Applications for Ceramic Hydroxyapatite

At BIT’s “International Congress of Antibodies” held earlier this year in Beijing, Larry Cummings, consulting scientist for Bio-Rad Laboratories, discussed the use of CHT™ ceramic hydroxyapatite media for antibody purification. According to Cummings, the value of CHT in the downstream processing of therapeutic antibodies is derived from its ability to remove antibody aggregates, protein A, endotoxin, host-cell proteins, nucleic acids, and viruses from the target protein in a single step.

“The utility of this matrix derives from its mixed-mode binding, which combines both metal chelation and classical cation exchange,” explained Cummings, who added that Bio-Rad recently introduced two methods to further enhance the utility of CHT by extending column life through mitigation of pH drops that are often observed with salt elution on cation exchangers.

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