Industry Experts Convene in New York to Discuss Latest Innovations: A BPI Special Report

As the biopharmaceutical industry continues to mature and grow, so too does the need to educate a broader audience of biopharmaceutical professionals interested in hearing, understanding, and applying the latest science and technology trends that support and in many cases are transforming today’s bioprocesses. To reach this extended and engaged audience, BioProcess International created the BPI Theater Series: a live, interactive program that provides bioprocessing content to traditional, noncore biopharmaceutical conference programs. It provides attendees with the opportunity to interact with leading biopharmaceutical industry experts as they present, discuss, and debate the impact of the latest scientific and technology trends related to the three pillars of biopharmaceutical development: upstream processing, downstream processing, and manufacturing.

BioProcess International originally partnered with BIO to launch the BPI Theater series in 2007. Attendance has grown every year, and today it has become the meeting place for bioprocessing professionals. In 2014, BioProcess International expanded the BPI Theater Series to include INTERPHEX 2014, which took place on 18–20 March at the Jacob Javits Center in New York City. The BPI Theater @ INTERPHEX was a tremendous success. Standing-room-only audiences experienced three interactive roundtable presentations addressing the harmonization of single-use systems, continuous bioprocessing, and new approaches to downstream processing. In addition, conference attendees enjoyed 12 individual presentations introducing key challenges and solutions that are changing approaches to today’s bioprocesses.



Requests for access to these presentations have been so overwhelming that we decided to publish abstracts of every one here and provide our 30,000+ readers with the opportunity to listen to the full presentation podcasts. This special report summarizes each presentation and roundtable (in chronological order) and provides you with dedicated links to each presentation podcast. Please make plans to visit the 2014 BPI Theater @ BIO, 2426 June in San Diego! See our ad on page 31.

Brian Caine, Publisher,

BioProcess International

Roundtable: Harmonization of Single-Use Systems — Coordination of SUS Standards and Best Practice Efforts


James Vogel,
founder and director, The BioProcess Institute


Jay Ankers,
director of technology, M&W Group (representing ASME-BPE)

Jerold Martin,
senior vice president, Global Scientific Affairs, Pall Corp (representing BPSA)

James Vogel introduced the session by commenting that single-use technologies are gaining in use. Their key benefit is that the processes are closed, nearly eliminating both microbial and cross-product contamination. The main concerns continue to be the presence of particulates, extractables and leachables, and the ability to control the supply chain.

As single-use technologies continue to be developed and adopted, regulators, professional societies, industry groups, and companies are developing standards for them. The goal is to provide safe equipment and processes leading to safe products. Many groups have proposed their own standards, but those are not yet harmonized with one another. To facilitate that harmonization, some groups started getting together last year at a town hall during the BioProcess International Conference. Representatives now participate in a monthly phone call and publish a newsletter.

Vogel suggested that the process of standardization resembles building a pyramid: trade and operators’ groups form the foundation by gathering best practices and consensus of their members. The next level consists of organizations that publish technical reports. From those reports, groups develop consensus standards that they agree to follow. Those standards are in turn recognized by governments and ultimately published in compendia that are referenced into law.

The panelists represented a number of the organizations involved, and each described the progress being made toward reaching consensus.

Jay Ankers represented ASME-BPE (American Society of Mechanical Engineers–BioProcess Equipment). This group publishes a book of standards every two years. In 2012, it was asked to make that book global in its extent, use terms that are understood globally, and be usable by regulatory agencies. Published standards often become law. ASME’s standards focus on hygienic processes and the design of systems that limit bioburden. ASME is working with BPSA and BPOG (see below) on extractables and leachables, particulates, change control, and process and product contact.

Jim Vogel spoke about ASTM International (American Society for Testing and Materials) and BPOG (Biophorum Operators Group). ASTM has 30,000 technical experts in 150 countries. This international standards organization develops and publishes technical standards through voluntary consensus on a wide range of materials, including those for medical applications. It tests methods, specifications, guides, and practices. Significant activities related to single-use include writings about supply-chain integrity; discussions under way on particulates, extractables and leachables; and change control. The organization is also working on dimensional standards for components, gamma radiation, and validation methods.

BPOG (Biophorum Operators Group) is a collaboration of end-user (only) biomanufacturing companies (23 members, 700 representatives). Its collective user view enhances these discussions. Ideas are forwarded to the other agencies and groups. BPOG currently has one team working on an extractables protocol; a second group is forming to look at supply chains.

Jerold Martin spoke about BPSA (Bio-Process Systems Alliance), an industry-led corporate member trade association comprising 40 suppliers and six end users (including contractors). Its purpose is to encourage and accelerate adoption of single-use manufacturing technologies for production of biopharmaceuticals and vaccines. BPSA is the only organization focused exclusively on single-use manufacturing. Its significant activities include defining best practices while building consensus among users and suppliers. BPSA is developing a standard quality-agreement template to be used by end users and systems and component suppliers.

Other Groups to Watch

Jim Vogel described other groups working to define standards for single-use materials.

ELSIE (the Extractables and Leachables Safety Information Exchange) is a consortium of companies interested in consolidating leachable and extractable information for biopharmaceuticals and medical devices. Its reports and database of 340 compounds help companies conduct risk assessments.

PDA (Parenteral Drug Association), an organization involved with this industry since 1946, focuses on scientifically sound technical information and produces technical reports. It is currently working on a paper that captures current practices and recommendations for single-use.

PQRI (Product Quality Research Institute) is a nonprofit group that is working to coordinate efforts on drug product quality.

USP (US Pharmacopeial Convention) focuses primarily on products, but it provides good information on issues such as particulates. USP drug standards are enforceable by the FDA.

Take-Home Message

In the Q&A session, a majority of the audience agreed that the town halls are worthwhile. The panel encouraged everyone to work on a task force for best practices; it is one of the best things you can do to help the industry and your own career. You will gain greater knowledge and network with experts in the field. The moderator urged everyone to sign up for one task force within one of these organizations.

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Development of an Automated and Disposable Platform

Lexan Lhu,
applications engineer, Charter Medical

Charter Medical believes in the possibility of a completely automated, disposable perfusion system. To this end, Lhu described her company’s single-use bioreactor, perfusion device, and controller.

The CELL-tainer single-use bioreactor uses two-dimensional movement to create high mass-transfer rates and short mixing times. It is designed for mammalian and microbial fermentation. The rocker differs from a traditional rocker because the wave motion generated is not just vertical. Complex wave motion enables high mass-transfer rates and fast mixing times. This easy-to-use device features a sliding tray, system sensors, convection heating, direct cooling, and scalability up to 200 L.

Lhu shared data from Bioceros, comparing the CELL-tainer system with a stirred-tank vessel at 5 L. The former yielded a higher viability and titer of Chinese hamster ovary (CHO) cells. It can be inoculated with an amount as low as 160 mL. With no transfer steps, media can be added to the bag to scale up, enabling up to 20 L/bag. For E. coli fermentation, the CELL-tainer system is comparable to a stirred tank but produces the desired protein a bit earlier. Using E. coli, the small system produced 45 g/L in 35 hours, and the large system produced 45 g/L in 38 hours.

The CytoPerf disposable retention device for perfusion uses acoustic waves to separate cells from the supernatant. That reduces the potential for shear stress and creates no tangential forces. With no membranes in the fluid path, a vacuum draws material out of the bioreactor and into the acoustic cell. An acoustic field is applied to separate cells from supernatant, which is then poured off. Gas is used to backflush material into the bioreactor.

The CytoSys automated capacitance-based, software-driven compact controller offers five modes. It allows mammalian cells to be grown under perfusion conditions.

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Progress in Single-Use Systems: Microbial, Stem Cell, and Cell Culture BioProcess Scalability

Christopher McPhee,
bioprocess applications specialist, Eppendorf North America

McPhee highlighted two different systems of single-use vessels and their scalability. Reasons to have disposable bioreactor systems include preventing contamination and saving time and money. In this case, the savings in time and money involved one hour instead of 10 hours for set-up and tear-down time. Eppendorf’s two manufacturing centers are dedicated to two different product lines, one in Germany (DASGIP) and the other in Connecticut (NBS; New Brunswick Scientific is now part of Eppendorf).

DASGIP’s DASbox mini-bioreactor system is completely parallel and can be used for classic amino acid fermentation, E. coli work, and growing stem cells. It enables scale up from 5- to 50-L vessels. It comes sterilized and preconfigured for easy set-up, has angled impeller blades set to 45 degrees for low shear, and meets all USP standards for leaching. Controlled by enabling software, The system can perform four runs at one time, helping to speed time to market.

Experiments were conducted to test its ability to support microbial applications, scalability in microbial process development, and stem-cell expansion. In the first experiment, an E. coli strain was used to produce amino acids for comparing a benchtop 2-L system with a 250-mL DASbox system. For the next experiment, a DASbox system was tested for parallel development. The company evaluated four parallel bioreactors using a fed-batch process with E. coli k12 and then determined biomass. Finally, researchers grew stem cells from cord blood in a 0.3-L vessel.

In all three cases, the DASbox system performed equivalently to a traditional system. The New Brunswick Scientific product line includes 5- and 50-L disposable vessels with built-in impellers. To show that the system can scale up from 5- to 50 L, the company experimented with growing different cell lines and measured the biomass generated. At high and low volumes, the growth rate was almost identical. The metabolics also behaved the same way in each system.

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Controlling Particulates Through the Supply Chain from the Perspective of a Plastics Manufacturer

Derek Masser
, director of sales, life sciences; and
Niraj Chandarana
, product development manager, life Sciences, both at Advanced Scientifics, Inc. (ASI)

Masser and Chandarana defined particulates, looked at the regulatory stance, and identified types of particulate sources from raw materials to fabrication and how to limit them.

Particulates are particles that can be visible or subvisible. Intrinsic particles are generated by a process itself; for example, while machining a block of plastic to form. Extrinsic particles originate externally, such as when pollen gets into a clean room.

Industry organizations have produced recommendations. End users should evaluate those and the standards that manufacturers are using to determine whether those will be acceptable to regulatory agencies. ASI bases its assessment of subvisible particulates for final product enclosures on USP standard <788>. The testing methodology is to flush a bag with purified water and then run a test to quantify the particle amount.

To reduce introduction of particulates from operators, those people need to understand gowning requirements. Human beings shed particles, especially while moving around. Limiting operators to one area can reduce particle shedding. To control/limit introduction of particulates in
cleanroom operations, a particle counter should be located next to the activity, not in a corner. Static electricity needs to be controlled by grounding equipment and dissipating the static (ionized air blowers dissipate charge).

To monitor material flow from raw materials to a finished product, use strong particulate controls as part of your audit schedule. Be aware that raw resin is heavy in particulates, so ensure that you have tight control over film manufacturing and processing — and take care in transferring products into your clean room. ASI uses shuttle systems to make a clean transfer without going through an airlock; final goods can leave a cleanroom the same way. Make sure you inspect 100% of the finished goods on a light table.

The audience asked which particulates that ASI trends, what its sampling plan is for its own products, and how to deal with extraction liquids that have soluble particles. ASI trends results and conducts testing according USP <788> and tests random products monthly. It recommends not using resins containing antistatic agents because those agents can be a major source of extractables. ASI uses electricity to control static instead.

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Single-Use Systems: Considerations That Extend Beyond the Bioreactor

Ann D’Ambruoso,
applications specialist, Applikon Biotechnology

As companies start adding single-use components and systems into their manufacturing processes, they should ask the right questions: Who is responsible for final delivery of the integrated product? How will it fit in with your current set-up? Who will provide service and follow-up for that installation? Are you going to reconfigure your hardware later, and do you have the flexibility to do so? Do you have all the components to your software that you need? How will it be validated once it is installed? Who provides the training on the system components?

Applikon is a privately owned company that has been in business since 1973. It started by manufacturing only bioreactors and controllers but has now expanded to supplying all bioprocessing equipment, including single-use components. Those elements include vessels with inputs and outputs, monitors, controller hardware and software, and IT infrastructure and field devices such as pumps, valves, motors, temperature controllers, thermocirculators, heaters, and sensors. Applikon’s probes and sensors use both traditional and next-generation sensor technologies. D’Ambruoso described the new sensors as reliable and noninvasive. They come presterilized and precalibrated; measure pH, dissolved oxygen and carbon dioxide; are disposable; and can be used in a bag or downstream. Controllers are available for a small, simple set-up or can be custom designed with requisite software to handle more complex systems.

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Identifying and Mitigating Risks Associated with Single-Use Tubing Sets for Product Sampling and Storage Applications

Tony Butler,
manager of single-use systems, New Age Industries/Advantapure

In his short presentation, Tony Butler emphasized the importance of your tubing and connections set-up. Manifolds comprise tubing, tees, zip ties, and other components. Although the manifold leakage rate is low (one in 1,000 to one in 10,000), it is a risk factor for process failure. He provided an example of a traditional, complex manifold with 37 parts. It had six vulnerable connections at which leakage could occur.

New Age Industries/Advantapure produces a molded manifold with only three connections, reducing the risk of failure. Reducing the number of connections will increase the success rate of your production.

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Process Requirements Met with Single-Use Pumps

Wallace Wittkoff

, director, global segment market, Quattroflow; and
Randy Cotter

, engineering manager, Cotter Brothers Corp.

The presenters advised looking first at pump performance curves to see what pump are designed to do — whether to keep constant flow, pulsate, or cause changes in pressure — and what the turndown is. Options include centrifugal, peristaltic, flow, and quaternary diaphragm pumps. The latter three work by positive displacement technology.

Centrifugal pumps often are used for cleaning in place (CIP). They allow you to close a valve without fear of rupturing the tubing or harming other parts. Peristaltic pumps are the workhorse of the industry and are used with bioreactors. They are a good choice when pulsation is not a factor and you don’t need linearity or turndown. Flow pumps are designed to have more linear flow; there is a direct relationship between the speed at which you run the pump and the flow rate you achieve. Flow pumps have been used with chromatography and filtration and have a turndown of 10 to 1. Quaternary diaphragm pumps can run from 1 L/hr to 150 L/hr using the same pump. They have very low slip even with low-viscosity solutions, get high turndown (80 to 1, sometimes 100 to 1), and offer low shear.

Not all pumps are yet made to work in single-use systems: No flow pumps and only one commercial centrifugal pump can do so. Both peristaltic pumps and quaternary diaphragm pumps can be used with single-use technology. The latter can work in either stainless steel or single-use systems and is convertible. Quattroflow pumps can be scaled from development to commercial processes.

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Roundtable: Continuous Bioprocessing — the Next Dominant Manufacturing Platform?


: John Bonham-Carter,
senior manager, bioprocess development, Refine Technologies


William G. Whitford,
senior manager, bioprocessing market, Thermo Fisher Scientific (now GE)

Dave Pollard,
head of process development, Merck

Eric Langer,
managing partner, BioPlan Associates

Paul Jorjorian,
senior scientific manager, purification development, Gallus BioPharmaceuticals

Peter Latham,
president, Latham BioPharm Group

Continuous processing is not a new concept, but within the biopharmaceutical industry interest is being revived by the creation of new markets, new types of drugs (notably biosimilars), new economic pressures, and new technical opportunities. Although fed-batch continues to dominate, the panelists examined how and to what extent that might be changing. The benefits of continuous processing are known to include reduced footprints, cost savings overall and/or for individual steps, the need for less material, higher yields, savings in the costs of labor and automation, lower capital costs, and the need for less downtime for changeovers. All those benefits are affected by scale and type of manufacturing (whether campaign, clinical manufacturing, R&D, and so on). Knowing where the benefits lie affects a company’s decision whether to explore continuous processing or not.

Carter said that companies have introduced continuous processes piecemeal within unit operations. But more people are now thinking about what an integrated continuous operation would look like and how that might affect designs of facilities and operational modes in the future.

One driving trend is the growing acceptance of single-use systems and modular designs; continuous processing becomes just one aspect of those. A drive toward smaller footprints is an ongoing opportunity, as is a drive toward end-to-end closed systems. Single-use technology allows connections in open air that prevent contaminations, reduce process interruptions, holding times, and risks overall. These are all main drivers toward a flexible manufacturing facility, and continuous processing is one aspect toward achieving that.

Langer pointed out, based on his annual surveys, that hesitancies about moving toward a continuous mode include confusion about which type of bioreactors to use — stainless steel or single-use, and whether in batch or perfusion modes. He said that the number of companies using perfusion bioreactors has jumped 20% since 2012: 15% up to 19.8% using stainless steel, and 32% to 36% using single-use. Those are not large increases, but they are acknowledged as a trend. He noted that earlier concerns about equipment led companies to decide that fed-batch processes were good enough. Companies may have considered using perfusion or expected it to play a bigger role in the future, but people in general thought it was too complex to incorporate. Those who have been using it, however, have solved the problems, so there is a “haves and have nots” level of information and experience. Although these are early days for continuous bioprocessing, some larger companies that have been working in perfusion (e.g., Bayer, Gallus, and Genzyme) consider such knowledge to be part of their everyday operations. That knowledge needs to be more widely distributed to counter the market perception that perfusion needs more work to succeed.

Contamination risk is an issue no matter what operating mode you choose, but does perfusion carry an inherently higher contamination risk? Although training issues and lack of experience might seem to indicate that the risk is higher, Jojorian told the audience that Gallus has run more than 160 60-day processes (more than 2,100 passages of material) since 2007 without a contamination event. So he believes that if you know what you are doing, there is no reason to think that perfusion inherently carries greater risk of contamination. Today’s increased understanding of perfusion processes and equipment should lessen concerns over contamination risks — but again, more training programs are needed.

The panel addressed the current state of the art in up- and downstream processing. They looked at how companies are integrating the two sets of operations and how to model that within a facility. Critical elements are the impact of such integration on economics, CoGs, and product quality. Also critical to consider are criteria posed by the regulatory agencies you are working under.

Genzyme provided some slides showing how a continuous process might work: That model is based on use of one platform, standardized equipment and training, flexibility in R&D, and lower labor and capital costs. The slides showed the simplicity of what that type of process might look like, including reduction of holding steps and a simplified flow of product. The conclusion is that you need to prove yourself to be in control of that product and process flow to the quality standard required by the FDA.

Quality: Discussion continued on trends and impacts from the quality side — and quality may be the true driver behind implementation. If you run a continuous process, your goal is real-time release, and that leads to how you intend to define a lot — whether by time, volume, or weight of products (among other criteria). The FDA doesn’t care how you do so but that you do it and keep quality consistent. Whether or not real-time release is a goal, if a batch is well defined, then you have greater flexibility to manufacture product for different needs (smaller numbers of lots for clinical stages, and so on).

The panel talked about regulatory pathways and how those may be influenced by current limitations in understanding of continuous processing. Upstream equipment these days may not be an impediment, but some equipment in downstream needs to be improved, especially for GMP compliance. Again, the question is what is actually a problem and what is simply unknown knowledge. How can existing state-of the-art knowledge be brought in from leading companies to define a standard process
design? How can this knowledge be passed along to universities and smaller biotech companies?

Other suggestions were for up- and downstream groups to design a process together, integrating teams so that they see how a change in one step creates changes or problems later on. Along with this discussion came the question about whether it is possible in a continuous mode to bring a slightly out-of-specification operation back “into spec” during the next unit operation. That hinges on clear understanding of critical quality attributes and what is desired for each unit operation. CQAs must be clearly defined so that a lot will be acceptable for release once it is moved back into spec.

At the end of the panel, the speakers summed up their viewpoints. The audience reflected a healthy level of skepticism. But all seemed to agree that continuous processing is called for by the demands of today’s industry. The key driver of the whole continuous bioprocessing area, said Bill Whitford at the close of the panel, is quality: the same quality everyday from a steady state upstream to the same quality and yield downstream; and being able to deliver increased product quality with decreased cost.

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Evaluations of Extractables and Leachables in the Manufacturing of Biopharmaceutical Products

Thomas C. Lehman

, manager, method development, Eurofins Lancaster Labs

Lehman explained how to design an extractables and leachables study. Such a study is needed to satisfy the Food and Drug Administration’s (FDA) regulations: “Drug product containers and closures shall not be reactive, additive or absorptive so as to alter the safety, identity, strength, quality or purity of the drug beyond the official established requirements”; and “Equipment shall be constructed so that surfaces that contact components and process materials or drug products shall not be reactive, additive or absorptive so as to alter the safety, identity, strength, quality or purity of the drug product.”

Extractables are chemical compounds that can be extracted from product-contact materials when exposed to solvents under exaggerated conditions — generally at time and temperature extremes. Leachables are more real-world compounds that can migrate into a product from a product-contact material under normal use or accelerated storage conditions.

Conducting Your Study: Lehman advises looking first at your entire process and identifying all product-contact materials. Then find out whether those materials have been characterized by their vendors and tested as appropriate for your proposed use. If they have not been, then you need to perform testing, develop a risk assessment for your process, identify the components that need to undergo an extractables study, design the study, develop analysis, and prepare your samples.

Staging the Study: A controlled extraction study should be followed by a simulation study that mimics the real use of material but uses the longest range of contact times. Choose analytical techniques such as gas chromatography that will yield the most information. And perform a risk assessment with a toxicologist. Based on such studies, you can then develop a safety threshold and determine whether any extractables or leachables found are a safety concern. Then develop a monitoring and testing program to be conducted during manufacturing or before releasing the product.

Lehman shared an example of a study showing all the above stages. When submitting new drug products to the FDA, expect to include your extractables study and toxicological risk assessment. One questioner wondered whether the product studied had excessive levels, and the answer was that levels weren’t excessive, but were high enough to concern a toxicologist. A second questioner asked whether a study would be relevant for suppositories, and the answer was yes because suppositories have direct contact with patients.

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Understanding Sources of Variation Allows Greater Control of Single-Use BioProcesses

Nick Hutchinson, market development manager, Parker domnick hunter

Hutchinson advised how to make a product more consistent by understanding its sources of variation. Such sources in a biomanufacturing process are biological variations (transcription and translation expression); raw materials and consumables; operational inputs including manufacturing methods; equipment; operator consistency when manufacturing occurs in multiple facilities; and the environment.

To address biological variations, consider that a monoclonal antibody is about 1,000 times larger than a molecule of aspirin, with a complex 3D structure. That structure determines how a MAb will work inside the body. Sometimes during transcription and translation by mammalian cells, a mistake in expression occurs, and the final form reveals modifications. For example, a company developing a vaccine for malaria found that under normal conditions, seven variants were expressed, but not all of them were therapeutic. To solve that problem, the company used genetic modification to overexpress a chaperone protein and was able to go from seven to two variants — resulting in a better quality of protein coming out of the bioreactor.

To prevent variations caused by raw materials and consumables, develop a procedure for rejecting or accepting raw materials, audit raw material suppliers, audit manufacturers of products used (such as single-use suppliers), and have a suitable change-control process for materials.

Use of process analytical technology (PAT) can curb variations stemming from operational inputs, including
manufacturing methods. The FDA defines PAT as “a system for designing, analyzing, and controlling manufacturing by making timely measurements of critical quality and performance attributes of raw and processed materials and process.” Hutchinson shared two examples of the positive effects that using PAT can have.

Another experiment compared manual with automatic feeding strategies of medium to cell cultures in the bioreactors. Automation can reduce some variability in a process by reducing the chance for human errors.

The final cause of variation is the environment, and its major cause is a contamination event. A closed system, as with single-use technologies, reduces such risk.

In summary, Hutchinson defined the four sources of variation and offered ways to control them, including process development tools (genetic engineering) and manufacturing strategies such as increased use of automation.

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Single-Use Solutions to Meet High Efficiencies in Different Cell Culture Applications

Hélène Pora

, vice president, single-use systems, Pall Life Sciences

Pora discussed ways to combine single-use technologies with automation to improve efficiencies. She began by commenting that single-use technology has gained wide acceptance at the clinical scale, where its use is growing at a steady rate. However, in commercial manufacturing, only ~15% of the market uses disposables. As the technology continues to develop, people are starting to consider single-use for other commercial applications.

One concern has been a lack of reproducible results, often due to operator variability. A solution is to combine the benefits of single-use with those of a highly automated stainless-steel process to create an automated single-use process. Such a system would enable excellent process control, reproducibility, and improved yields.

Pora’s examples focused on bioreactors and media preparation. Pall Life Sciences has both a rocking platform and a stirred-tank bioreactor. Rocking platforms traditionally rocked only up and down, but Pall has produced one that can move in three dimensions — a more complex motion. The company found that using three-dimensional rockers and bags increased cell productivity by 30%.

The stirred-tank bioreactor can be used for fed-batch processes for vaccines, monoclonal antibodies, and cell therapies. The mixing time is short: The mixing mechanism is a bottom-driven impeller with adjustable speeds, even at slow rotations, so as to reduce shear and cell damage. In a scale-up process from 10 L to a commercial scale (200 L) using Chinese hamster ovary cells, the cell viabilities nearly matched one another.

Pall Life Sciences offers an automated system that uses a single platform for multiple operations. From the process controller, you can control pH, temperature, pressure, conductivity, flow, and other variables of interest. It can be used upstream as well as downstream for sterile filtration, harvesting, virus filtration, virus activation, and so on.

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Single-Use Perfusion for Continuous Biomanufacturing

William Whitford,
senior market manager, cell culture, Thermo Fisher Scientific (now senior technical market manager, GE Healthcare Life Sciences)

One hundred years ago Henry Ford proposed making cars using continuous processing. The concept is now well-established for all kinds of manufacturing, from cars to chemicals to pharmaceuticals. In the pharmaceutical industry, continuous processing is supported by many ongoing elements: regulators, process analytical technologies (PAT), and quality by design (QbD) friendly. It heightens consistency of process parameters by providing a steady state, increases process efficiency (supports integrative processing with fewer steps, fewer holds, more efficient movements), and reduces operator intervention and product loss. Smaller footprints amendable to closed processing increase overall facility capacity, allow expansion of production without facility expansion, and increase profitability.

Continuous processing contributes to a sustainable “green initiative.” Single-use perfusion is thought to be more difficult than other methods, but once such a platform is established, process development is faster. It uses the same equipment as that used in manufacturing, which reduces technology transfer. Other benefits are that the cells are not spinning as in a batch process and aren’t exposed to as many enzymes. Single-use perfusion operates at a higher molecular efficiency and with lower reaction volumes.

Examples of single-use continuous bioreactors include the WAVE bioreactor by GE Healthcare and hollow-fiber perfusion bioreactors (made by a number of companies). New developments complement perfusion and continuous biomanufacturing, such as specific Chinese hamster ovary (CHO) cell lines that work well in perfusion bioreactors. You can also manufacture vaccines and produce stem cells.

So continuous processing supplies many features and benefits to biomanufacturing. Single-use systems complement the benefits, and companies are accomplishing more and more with it.

Questions from the audience were about new products in development and the ability of the described set-up to produce and maintain stem cells. Whitford’s company does produce single-use bioreactors and other products that can be converted to work in a continuous processing set-up. Production of cell-based therapeutics is being explored, and there appear to be unique advantages to be gained from using these new technologies.

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Integrated Single-Use Purification Technologies Enabling Continuous MAb Processing

Michael Phillips,
director upstream and downstream technology, EMD Millipore

EMD Millipore has designed integrated, single-use purification technologies to enable continuous monoclonal antibody (MAb) processing. The company’s goal was to develop a faster, cheaper, more flexible process that minimized risk, met purification targets, was safe for personnel and the product (closed system), and minimized financial risk. For its program, the company used protein A chromatography (other techniques didn’t work as well or were more expensive).

The purification process has two steps comprising four technologies: a new activated carbon-based flow-through system combined with anion-exchange flow-through, which can work back-to-back; and a cation-exchange flow-through system designed to remove aggregates, host-cell proteins, and other impurities, followed by virus filtration (both use the same solution conditions).

Because the company wanted to use single-use technologies, the clarification step was a continuous precipitation technology followed by a depth filter designed specifically for that. Results from the activated carbon-based technology showed that it successfully removed small proteins, antibiotics, antifoam agents, pluronics, and some antibody fragments. The cation exchange was designed so that the higher-weight aggregates would bind more strongly, with elective binding of aggregates. It showed a high removal of aggregates, host-cell proteins, and protein A.

The company experimented using 15 different MAbs, with excellent results. After all components were tested, they were combined in a continuous process including in-line pH adjustment. EMB Millipore tested three MAbs with a cumulative yield around 90%. Host-cell proteins were not detected, and aggregates met the target of 1%.

Cost modeling was performed using 3-kg and 15-kg scales broken down into labor, consumables, materials, and capital. Going from a traditional process to an all-flow-through one at 3 kg saved about $12.8/gram or roughly a 21% decrease; and 15 kg saved $8/gram, with a 30% decrease in cost. Most of the savings were realized by using single-use.

So EMD Millipore has an integrated flow-through package with two new technologies that work synergistically to meet purification targets. Moving to a flow-through mode improves economics, minimizes solution conditions, requires less process development, reduces the use of buffers, and creates a smaller footprint. During questions Phillips further clarified the monomer and aggregate separation, explained why using protein A was the best choice, and confirmed that the company experimented with and produced the resin used in the anion exchange.

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Large-Scale Continuous Processing in the Age of Single-Use

Mikal Sherman,
application specialist, fermentation technologies, Sartorius-Stedim North America

Companies achieved success in the past using stainless steel; its attributes include good sensor technology, robustness, and reliability. Single-use systems are starting to gain those attributes but haven’t yet matured into becoming full replacements for steel. Challenges include the ability to use sensors, compatibility with existing equipment, aggressive processes (higher agitation rates), and scalability. However, recognizing the many advantages of single-use technologies, Sartorius has invested heavily in and has made strides forward in single-use systems. It has developed a single-use system that can interface with existing equipment, is compatible with existing controllers, and uses the same sensor technology as used for stainless steel.

Sartorius was the first company to bring a true stirred-tank reactor (STR) into the market. The company is introducing new bags to the market and has shown that cell cultures can be successfully grown within them. Special care goes into the packaging for the single-use bags to ensure that they are undamaged after shipping and ready to use.

Finally, one example of the company’s PAT technology is near-infrared batch fingerprinting, along with computers that conduct multivariate analysis to determine that runs are staying within parameters. Continuous processes have been used for traditional processing. Sartorius had success at the production scale using a continuous process (limited to a concentrated fed batch process). It was able to do this using a 2,000-L bioreactor with an alternating tangential flow (ATF) perfusion device.

Sartorius has worked to develop a 2,000-L bioreactor that can perform as much like stainless steel as possible. One challenge was to get enough oxygen into solution. The STR system can reduce total gas flow by nearly half. By doing this, the company has eliminated the over-pressure issue with single-use bioreactors that can lead to failure.

Sartorius’ single-use bioreactor is ready to accept difficult and aggressive processes and will perform them successfully. Scale-up models range from 2- to 2,000 L. The company is bringing forward new products to the market that are reliable and excel at cell growth and lot consistency.

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Roundtable: New Approaches to Downstream Processing — Thinking Outside the Column


Keith Wells,
senior consultant, BioProcess Technology Consultants


Jim Stout,
vice president, process sciences, Natrix Separations

Paul Jojorian,
senior scientific manager, purification development, Gallus Biopharmaceuticals

Mark Brower,
senior research chemical engineer, Merck & Co.

Tracy Thompson,
founder and CEO, PolyBatics

Robert Gronke,
senior principal scientist, Biogen Idec

The downstream process is becoming a bottleneck in the entire process of producing biopharmaceuticals. Increased productivity upstream puts pressure on downstream productivity and throughput. Cell culture processes are driven by volume, so every increase in titer leads to an increase in productivity. If you double the titer, then you double productivity without having to change equipment. That is not the case in purification processes, which are mass driven. Right now the downstream process relies heavily on chromatography. That technology has been very effective over the years for meeting product-quality requirements but is increasingly becoming one of the bottlenecks. It will require larger columns and alternative strategies to keep up with the upstream productivity. Industry trends are moving toward higher- yielding processes, biosimilars, new markets, price pressures, new types of products, and the advent of single-use and its potential and lower costs.

Jim Stout (Natrix): Some of Natrix’s solutions for improving downstream performance are continuous processing, multistage filtration technology, phase-separation and precipitation modalities for reducing impurities, simulated moving bed, expanded bed, and concurrent chromatography (uses less resin and has more columns for efficiency). The company’s high-density (HD) membranes are well suited for all those challenges. They provide high capacity, fast kinetics, and high resolution, with reproducible results cycle after cycle. The HD membrane has a flexible, reinforced fiber mesh filled with functional porous hydrogel, which provides binding groups and functional pore structure. The NatriFlo HD-Q product for polishing will soon be joined by the HD-C weak cation-exchange membrane for capture and polish.

Paul Jojorian (Gallus Biopharmaceuticals) spoke about his company’s fully disposable downstream process. Gallus has used disposable downstream technologies for 12 years, running multiple commercial processes. Multiple benefits for a CMO using disposable technologies include lessening the risk of cross contamination between customers’ products. A fully disposable process includes clarification by depth filtration (with disposable prepacked columns), a number of different membrane absorbers, a disposable chromatography system, and disposable viral filtration.

Mark Brower (Merck & Co) spoke about continuous chromatography downstream. Columns can be used differently to prevent the bottleneck. Traditional titer and processing takes ~60 hours for purification; Merck hopes to condense that to 1624 hours. With continuous chromatography, you can break down a one-batch purification column into several columns running in parallel, with different steps running in each column. The fully disposable system includes single-use sensors and pump heads and has proved to be reliable. It offers complete antibody capture, high aggregate and HCP clearance. And it operates at high flow rates while achieving desired purity.

Tracy Thompson (PolyBatics) spoke about the use of biopolyester beads. They are grown inside bacteria, and the enzyme that forms those beads remains covalently attached. The company can produce a single bead with specific proteins on its surface and engineer such particles to the desired functionality. Materials can be produced quickly and cheaply, producing a custom affinity chromatography medium in 50 days with high binding capacity and required specificity. The beads are tiny and compressible, so if used in a packed column, they can be encapsulated in an inert matrix or be combined with silica to form a rigid silica structure with the particles. Polybatics’ goal is to make a rapidly customizable, disposable medium that can be used in production of new drugs coming to the market.

Robert Gronke (Biogen Idec) detailed polyethylene glycol precipitation. The company aimed to speed up downstream processing by replacing the low-capacity protein A step but still achieving comparable quality, purity, and yield. The continuous precipitation platform works well with monoclonals, but not with FC fusion proteins or smaller molecules. The process scales to 1,000 L, and the company expects to easily reach 2,000-L.

Questions Posed By the Moderator

What are the biggest barriers to any of these technologies?
The biggest barriers are the risk of doing something new and having facilities available for trying new things.

Do you have an internal cost target with a relative yield (dollars/gram)? A different way to look at it is per liter of bioreactor fluid because that is the element that drives the cost. Also, flexibility might be more important than cost when you can be more efficient (faster) in getting new molecules into the clinic for testing.

What is the cost/benefit analysis for using all of these downstream disposables? The cost to pack a column including testing is not cheap ($12,000 to $20,000 per column, and it can take a week to qualify a column), so buying a disposable column can cost less. For disposables, the start-up cost is lower, but the operational cost is higher (according to one study). You have to assess all the variables for your own situation.

With the new technologies coming along, what do you see as the role of protein A in the future? Will there be other choices that are faster and as good? Protein A makes life easy in purification. If we could design ways to use protein A more efficiently, people won’t be as interested in new technologies.

What do you see as the biggest opportunity in the downstream market? Opportunities exist in emerging markets such as India and China, increased use of single-use technologies, and right-sized processing at different scales for any type of protein.

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