Overview
In the pharmaceutical and biotechnology industry, single-use components originated in medical practice to reduce transfer of infectious material from one patient to another. More recently, single-use products have been developed and implemented by the industry for production of biopharmaceuticals. Disposable single-use filters, buffer bags, and final bulk product storage containers gained rapid market penetration and acceptance, while single-use bioreactors for cell culture, single pass TFF systems for harvest, and disposable options for downstream processing are only recently achieving widespread use. Obstacles have included regulatory concerns, technical challenges, and an established infrastructure of stainless steel bioreactors, glass column housing, and installed CIP/SIP systems that enable reliable re-use of these production assets. Despite these obstacles, the significant advantages of single-use technology in multi-product facilities such as contract manufacturing organizations and in enabling in-house production of early stage clinical material have enabled these technologies to gain a foothold in the bioproduction industry. In this article, the technology breakthroughs, regulatory concerns, and available single-use bioreactors and downstream processing equipment that has gained market acceptance for production of clinical and commercial biopharmaceuticals will be reviewed.
Single-Use Bioreactors
To date, single-use bioreactors have been developed for use in mammalian and insect cell culture by several vendors but no products are widely available to support bacterial production [1]. This is due to the high rate of heat generation by high-density bacterial production cultures and the high rate of oxygen consumption. Mammalian and insect cell culture are more amenable to the constraints of single-use technology since heat exchange and high oxygen flow is less critical to cell growth and productivity. Single-use bioreactors with increased oxygen supply options have also been developed to propagate yeast production strains [2]. Single-use bioreactors are available in volumes up to 2,000 L, and have been used in production at scales ranging from 20 L to 2,000 L.
To enable single-use bioreactor development, polymers and plastics suitable for cell culture had to be developed. These materials must be sterilizable by non-invasive methods such as gamma irradiation, support the pressure of the cell culture fluidic movement and agitation without tearing, enable gas exchange without compromising sterility, and be resistant to localized pH minimums or maximums. The original products that met these requirements were rectangular-shaped bags similar to blood collection bags or intravenous fluid bags used in medical practice. These reactors were self contained, and agitation/mixing was provided by an external platform rocker [3]. The platform rocker type production vessel contains ports for input of culture components such as the feed media for a fed-batch process, chemicals to control pH level, and antifoam. Sufficient aeration is generated by the rocking motion and mammalian cells can grow to densities up to 107 cells per mL. When they were first introduced in the mid-1990s these reactors were in the 20 L range and this small scale made them unsuitable for production. In recent years larger-scale platform bioreactors up to 500 L have been used as the production reactor for early stage production of clinical grade biopharmaceuticals and vaccines. In addition, these reactors are frequently used for culture of the seed train at increasing scales prior to inoculation of a large stainless steel production bioreactor [4].
One concern regarding use of platform rocker bioreactors is the non-comparability of the geometry, and therefore, a likely impact on product quality when production is switched to a more standard bioreactor configuration for larger-scale production. Any change in the culture conditions for a biopharmaceutical product can have an impact on glycosylation, aggregation, and proteolysis of the product as well as on host cell protein , DNA, and other process-related component levels in the harvest media. Many of these changes can be mitigated by changes in the downstream process, but it would be preferable to utilize scalable production geometry from the start of clinical production.
Several companies have developed single-use bioreactors that resemble the standard, cylindrical stirred-tank design that is prevalent in the installed base of stainless steel, and is the dominant geometry of large- scale bioreactors. For this type of single-use bioreactor, a fixed support containing the process control software is installed in the production facility, and the disposable bioreactor is provided as a bag that can be inserted into the fixed support structure [5]. Agitation is generally provided by an installed impeller that, like the bag, is single-use and that attaches to a motor in the support structure. Control of pH, feeding, and dissolved oxygen is through installed ports and probes. A recent study by Smelko et al. demonstrated that for high-intensity cell culture processes in CHO or NSO cells, the product from single-use bioreactors up to the 1,000 L scale in the standard configuration is comparable by all biochemical analyses to product from a 1,000 L stainless steel bioreactor [6]. In this study, the seed train, harvest, and all downstream processing steps were performed using identical equipment and methods so the only difference is the type of bioreactor.
Recently, new single-use bioreactor systems have been developed that have new geometries and mechanisms of cell agitation, mixing, and control of nutrients and pH. Bioreactors with square or rectangular shapes are on the market, and have shown good ability to support mammalian cell growth and productivity [7]. In some cases, the impeller and motor is top-mounted or side-mounted to prevent formation of a low-mixing zone below the impeller [8]. An orbital shaker with production volumes up to 1,500 L was recently introduced, and when operated at suitable speeds this geometry provides adequate mixing and aeration without the use of any impeller [9]. All these reactor types consist of a fixed support shell with single-use, fully integrated bioreactor bags that fit into the support and that utilize the fixed controls. To our knowledge, these newer geometries are not widely used for production of biopharmaceuticals that are currently in the clinic but the industry is poised to evaluate the options and to select the one or more bioreactor types that provides the best ability to support mammalian and insect production cell culture.
Downstream Processing Applications for Single-Use Technology
The development of single-use downstream processing products capable of operating at bioproduction scales has lagged the development of disposable bioreactors, but significant efforts by suppliers to the bioprocess separations market is quickly reducing the gap. In contrast to 5-10 years ago, companies who wish to use disposable technologies in bioprocesses now have a wide range of scalable single-use purification products to choose from. As mentioned above, certain operations (normal-flow filtration and the use of buffer bags) have been supported with single-use products for many years; however, most purification operations have not.
Depth filters for clarification steps are now available from multiple vendors in multiple scales in a single-use format. As a result, bioprocess manufacturers now can choose whether to conduct clarification operations using multi-product stainless steel housings for conventional depth filters or single-use depth filter cassettes where all product-contact surfaces are disposable. Many commonly used depth filter media from several suppliers are now available in both conventional and single-use formats [10,11].
A similar situation exists for cross-flow filtration cassettes, which are frequently used in bioprocess applications for ultrafiltration and diafiltration operations. The first single-use UF/DF cassettes became available in the past decade. In addition to these devices, which operate with the same cross-flow processes as bioproduction manufacturers have used for decades, a new technology called “single pass ultrafiltration” is being developed, which allows for tangential flow concentration and diafiltration without recirculating the retentate solution. This approach has the potential to be converted to a disposable format in the future.
One of the most challenging unit operations to convert to single-use technologies has been chromatography, which is the heart of most biopharmaceutical purification processes. Several suppliers now offer pre-packed chromatography columns that are sold in plastic or low-cost glass housings and designed to be disposed after one or more cycles (because of the high cost of chromatography media, some manufacturers may choose to use these pre-packed columns for a campaign of several batches instead of disposing the column after each run). While this provides a workable solution up to a certain scale (approximately 20 L is a large pre-packed chromatography column), there is a practical limit to the achievable scale for a disposable chromatography column. To address this scale limitation, two technologies are being pursued more vigorously.
- Membrane Adsorbers. Membrane adsorbers are already widely used for “negative pass” chromatography where the product of interest flows through the column (i.e., the membrane adsorber), and impurities remain bound to the column. A common application is the use of anion exchange membrane adsorbers for virus, endotoxin, DNA, and host cell protein clearance in monoclonal antibody processes [12]. Unfortunately, current membrane adsorbers lack the capacity of chromatography media for chromatography steps where the product is bound to the media (i.e., so-called “capture” steps). As a result, new membrane adsorber technology that provides robust, scalable, high-capacity membrane adsorbers is required before these devices are likely to be used widely in commercial-scale bioproduction “capture” applications.
- Continuous Processing. Several firms are exploring the use of continuous chromatography, such as simulated moving bed (“SMB”) or related technologies to reduce the scale and capital cost of chromatography columns and systems. At least one firm is focused on establishing SMB using a fully disposable flowpath [13]. Since SMB, as with other continuous processes, is inherently more scale efficient, more material can be purified with this technology using current scale disposable chromatography columns than would be possible with a conventional batch operation.
Finally, technology is being developed for single-use operation of other downstream processing steps, including even centrifugation. Soon, we can anticipate the availability of many sound and feasible disposable-format purification process options for bioproduction applications.
Leachables and Extractables in Single-Use Systems
With the introduction of new polymers and chemicals into the process stream, companies and regulatory agencies must consider the impact of these chemicals on the safety of the final product that will be injected into patients. Extractables are defined as those chemicals that could potentially end up in the drug product, whereas leachables are those chemicals that actually do leach out from the equipment into the drug [14]. Vendors can provide data regarding extractables from their products based on analysis under various likely process conditions (temperature, agitation, various buffers, etc.), but leachables can only be confirmed by testing the drug product [15]. If leachables are present in the drug product, it is likely to be at trace levels compared to the biopharmaceutical product. The industry has created a trade organization, Bio-Process Systems Alliance (BPSA), to coordinate information generation and sharing and to develop a consensus on best practices for single-use system implementation, testing, and disposal [16]. While single-use technology implementation in a biopharmaceutical manufacturing process adds these additional requirements for risk assessment and final product testing, the benefits of single-use technology are substantial and have led to increasing use in upstream and certain downstream applications, and the market acceptance of these technologies is likely to continue to penetrate into larger-scale processes, more advanced clinical development and commmercial production, and additional downstream processing operations [17].
References
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- http://pbsbiotech.com/products-technology/
- Stettler M. et al. Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol Prog. 2007 Nov-Dec;23(6):1340-6.
- Lutz H. et al. Considerations for scaling up depth filtration of harvested cell culture fluid. BioPharm Intl. 2009; 22(3):58-66.
- Muhl M. and Sievers D. Cell harvesting of biotechnological processes by depth filtration: Effects of system design and operating mode on process scalability. BioProcess Intl. 2011; Industry Yearbook: 86-88.
- Lajmi AR, Nochumson S, Berges A. Impact of antibody aggregation on a flow-through anion exchange membrane process.Biotechnol Prog. 2010 Nov-Dec;26(6):1654-61.
- Bisschops M. et al. Single-use, continuous, countercurrent, multi-column chromatography. BioProcess Intl. 2009; 7(S1): 18-23 .
- Extractables and Leachables Subcommittee of the Bio-Process Systems Alliance. Recommendations for extractables and leachables testing Part 1: Introduction, regulatory issues, and risk assessment. BioProcess Intl. 2007; 5(11): 36-44.
- Extractables and Leachables Subcommittee of the Bio-Process Systems Alliance. Recommendations for extractables and leachables testing Part 2: Executing a program. BioProcess Intl. 2008; 6(1): 44-52.
- http://www.bpsalliance.org/
- Ott, K.D. “Are single-use technologies changing the game?” BioProcess Intl. 2011; 9(S2):48-51.
Author Biographies
Susan Dana Jones is a seasoned biotechnology entrepreneur with experience in biopharmaceutical discovery, product development, and strategic planning. She co-founded two successful biotechnology companies and has managed multiple discovery and product development programs. Dr. Jones is a subject matter expert in cell line development and characterization and is a leader in applying the principles of Quality-by-Design (QbD) to early stage biopharmaceutical development programs. She currently advises clients on the advancement of product candidates from research through development. Dr. Jones also assists clients in the application of GMP regulations and preparation of regulatory submissions as well as performing technical due diligence in support of investors or mergers and acquisitions. Dr. Jones is a frequently invited spealer at industry conferences and symposia and also serves as a guest lecturer at Northeastern University. She is a member of the Board of Directors of Gene Solutions, LLC, the Symphogen A/S Scientific Advisory Board, the Editorial Advisory Board of BioProcess International. Prior to joining BioProcess Technology Consultants, Dr. Jones was Senior Vice President of Corporate Development at Serenex, Inc, where she worked with other senior managers to complete a $15M Series B financing. Prior to Serenex, she was Vice President of Product Development at Waratah Pharmaceuticals and previously held development and management positions of increasing responsibility at Peptimed Inc., Virus Research Institute (now Celldex Immunotherapeutics), IntraImmune Therapies Inc., and Dyax Corp. Dr. Jones received her Bachelor’s degree from Harvard University, holds a Ph.D. in Genetics from the University of California, San Francisco, and performed post-doctoral research at the Dana-Farber Cancer Institute of Harvard Medical School.
Tom Ransohoff has over 20 years of experience in the biopharmaceutical industry. Mr. Ransohoff’s areas of expertise include development and scale-up of biopharmaceutical processes; separations and purification technologies; cGMP manufacturing; and management of technology-based start-up ventures. While part of BPTC, Mr. Ransohoff co-founded Tarpon Biosystems, Inc and BioFlash Partners LLC, where he was President and CEO prior to successful sale of this business in 2010. Before joining BioProcess Technology Consultants in 2002, Mr. Ransohoff was Vice President, Operations at TranXenoGen, Inc., responsible for purification process development and operations. Prior to that he was Vice President, Bioseparations at Dyax Corp, where he was instrumental in managing a business unit to develop novel affinity separations products using phage display technology. At Repligen, Mr. Ransohoff was Senior Director, Manufacturing, responsible for cGMP pilot plant operations producing biopharmaceuticals for clinical trial use and critical reagents such as Protein A for commercial sale. He is a member of the Editorial Advisory Board of BioPharm Magazine and has served on a number of scientific and professional advisory boards. Mr. Ransohoff has a Bachelor’s degree from MIT and a Master’s degree from the University of California, Berkeley, both in Chemical Engineering.
This article was printed in the May/June 2011 issue of American Pharmaceutical Review - Volume 14, Issue 4. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit www.americanpharmaceuticalreview.com and subscribe for free.