Single- and limited-use technologies (also termed “disposables”) have had a transformative impact on the development and production of biopharmaceuticals. In the midst of this revolution is a good time to look back at where we have been and where we are now, in order to get some idea of where it is going, much like dead-reckoning navigation in sailing. As we see, one can expect continued penetration of the technology, especially in downstream processing, commercial applications, and early development activities. 

Brief history

Single use technology started with the use of membrane filters, which have been in use in biopharmaceutical processing since the earliest days of the industry and their use can be traced to the mid-1970s, when the United States Food and Drug Administration required non-fiber releasing filters to be used in the production of injectables. However, the innovation for other unit operations started with the development and introduction of single-use bags for the storage and transport of buffer and media by Hyclone (Logan, UT- now part of Fischer Scientific) in the 90’s. The popularity of single-use membrane filters and single-use bags set the stage for innovation for other unit operations, starting with the single-use bioreactor (SUB).

Upstream operations

Single-use bag technology enabled development and introduction of the Wave bioreactor system by Dr. Vijay Singh in mid-1999. Mass transfer is achieved by rocking the media in a partly filled plastic bag. The Wave system was quickly adopted into R&D and manufacturing in the first half of the last decade for culturing both suspension and attached cell lines. The Wave technology apparently has in mass transfer limitations since the largest working volume available is 500L. Most manufacturing applications used the technology as part of an inoculum seed train or for clinical manufacture. The technology, like most single-use technologies, offers the advantage of lower initial capital cost, fast turn-around, simpler operations, and reduced utility requirements, and it was these benefits, that led to its quick uptake. The success of this technology spurred innovation of other single-use bioreactor (S.U.B) designs, especially ones using more traditional impeller-style mixing. These SUBs are being used in some commercial applications.

The impeller based SUBS have increased in size up to 2000L, such as those offered by Xcellerex (Marlborough, MA). Technical difficulties in reaching a bioreactor of this size included solving heat transfer problems and developing mixing systems that are economical, yet robust enough to handle the mixing requirements. Some industry observers think 4000L or thereabouts is the upper limit of what would be technically, operationally, and economically viable. The need for single-use bioreactors of this size would most likely be driven by block-buster type antibody therapeutics.

The successful introduction of 2000L SUBs has implications for the commercial bioprocessing facility of the future. It is likely that SUBs will get increasing consideration for use in commercial applications as a result of having already been introduced into some commercial applications and increasing cell culture titers. This can be illustrated by the case of a state-of-art monoclonal antibody production process. A 2000L SUB producing 7 g/L of a monoclonal antibody is capable of producing significantly more than a 10,000L stainless bioreactor producing 1 g/L. This is remarkable since 1 g/L was a benchmark titer not that many years ago. Titers of 7 g/L or more in fed-batch culture are becoming increasing common today. A 2000L SUB producing 7 g/L of monoclonal antibody can produce 200 kg of purified drug substance a year. Some predict cell culture titers for antibodies will routinely reach titers of >10 g/L and possibly as high as 15-20 g/L. Thus, one or two 2000L SUBs would be capable of meeting the market demand for many monoclonal antibody products. Thus, only blockbuster antibodies products with production requirements greater than 1000 kg will drive the need for SUBs larger than 2000L. The use of SUBs commercially will initially find most acceptance for niche therapies (such as orphan drugs); monoclonal antibodies with relatively modest annual production requirements; and vaccines that need rapid deployment or have relatively small volume requirements that can readily be met by SUBs. Indeed, SUBs are currently being used in the commercial production of vaccines.

The 2000L SUB is also a size amenable to clinical production. Using the same size bioreactor for both clinical and commercial applications would have the advantage of avoiding the need to scale-up for validation and commercial production. Using a 2000L SUB as opposed to smaller bioreactor means fewer batches. This loss of process characterization data can be addressed with appropriate studies using qualified scale-down models and additional at-scale process engineering and characterization runs at scale. The latter would benefit from the improved economics of single-use technology. 

Today, most biological therapeutics are produced using fed-batch mode. Perfusion is an alternative culturing mode based on continuous medium feed and culture broth withdrawal. On paper, the productivity gains of perfusion operation over fed-batch can be very appealing and can be approximated by a factor of 10 over fed-batch productivity levels and, on the surface, make smaller single-use systems appear even more desirable. Perfusion processes require more development work that fed-batch to characterize and needs to include demonstration of product comparability at all phases (e.g., early versus late) of a single perfusion batch. Varying cell densities, environmental conditions, and cell stability factor can alter product quality. Furthermore, perfusion processes are more complex operationally since their high media requirements require exact and timely media make-up and they are more susceptible to operational upsets, including contamination from adventitious agents and plugging of cell retention devices. The decision to pursue perfusion based processing to the exclusion of fed-batch development needs to be carefully weighed.

 Downstream operations

Single-use technology has not had as large of an impact on downstream purification processing as it has for upstream processing. This can largely be attributed to the expensive cost of most resins used in purification chromatography. Nevertheless, in-roads are being made to garner the benefits of single-use and limited-use technology for downstream processing. GE Healthcare (Little Chalfont, UK) now offers its ReadytoProcess™ chromatography columns containing GE resins.  These columns act much like conventional chromatography columns and come prepacked, prequalified, and presanitized. While it is certainly possible to use this format in a single-use application, many will likely cycle the columns through more than one use as a limited-use application. Conventional chromatography technology has some inherent limitations which have not allowed them to readily adapt to increasing titers upstream. These limitations include loading capacity; physical limits to the size of the column and bed height; and batch processing which as low efficiency for resin usage. Advances are being made to address some of the limitations of conventional technology. This is especially important, since increasing cell culture titers have created bottle-necks in downstream processing for the industry.

One of these technologies is simulated moving bed chromatography (SMB), invented by the petrochemical industry about a half century ago, is an established, proven technology that is already routinely used for chemical purification in other industries, such as food, petrochemicals, and traditional pharmaceuticals. It is being developed for use as a single- or limited-use technology for biologics processing and can be used with a variety of resins in a disposable-format chromatography cartridges for multi-column applications. The technology has several key advantages over conventional batch chromatography processing, which, taken together, can significantly improve the economics of purification: decreased buffer/solvent use; reduced separation media usage per batch; and more continuous purification processing. These advantages can increase plant through-put; reduce capital requirements; and lower direct labor and material costs. The technology can readily accommodate increasing titers by simply adding more sanitary disposable valve cassettes and cartridges to the same system hardware.

Another single- or limited use purification technology that has been introduced is membrane ion exchange chromatography, such as the Mustang® Q from Pall Corporation (Port Washington, NY); it is offered in both strong anion or cation exchange forms. While this technology can find application at moat any stage of purification, it has found most application as a polishing step. Membrane columns offer high dynamic binding capacities, high flow with low pressure drops, and reduced buffer usage, but have suffered from low binding capacities. They have been applied to the purification of DNA, IgGs, plasmids, viruses, and other high molecular weight products. Process robustness often requires a reduction of endotoxin and DNA to low levels in the final, polishing purification step, a task usually well suited for membrane chromatography.

Since their introduction in the early days of the industry, membrane filters have evolved to include viral nano-filtration filters, which are one of the most important components for viral safety in biologics processing. Nano-filtration filters are especially important in providing higher safety margins for small, non-enveloped viruses, which are among the most resistant viruses to chemical inactivation.

Other single-use downstream unit operations include tangential flow filtration (TFF) and depth filtration units that are capable of covering development through commercial manufacturing needs.

 Drug product operations

Single-use technology and concepts are also changing fill manufacturing operations. Solvay Pharmaceuticals was able to design a fill operation that required no class-A laminar flow, through the use of single-use technologies (disposable bags, transfer sets, connectors, and rapid-transfer ports) that are delivered preassembled and gamma sterilized, ready for aseptic fluid transfer.[1] Aseptic Technologies (Gembloux, Belgium) offers a closed vial product, Crystal®, for the aseptic filling of injectable drugs. The vials are ready-to-fill and require no pretreatment or preparation of the vial components in advance of the fill. A non-coring needle is used for filling and the septum is sealed with a laser immediately afterwards. The technology can be applied also to lyophilized and potent or bio-hazardous materials. Care must be exercised in the use of such of single use technologies in fill operations in order to insure no undesirable surface or extractable interactions occur with the drug product. This can be accomplished by designing product specific studies and performing appropriate risk analyses. This is especially important for fill operations because of the direct linkage to patient safety.

 Process development

Single-use technology is also penetrating early development activities as well, particularly for upstream processes. Such innovation promises to speed development efforts and reduce labor requirements in conducting experiments. Innovation here fits quite nicely with the Quality by Design (QbD) precepts and related initiatives by regulatory authorities. Such upstream technologies can lead to better clone selection and speed process characterization. Some industry participants have generated data indicating that better clone selection is made if final clone selection considers data from actual bioreactors (or culturing systems that closely replicate them) operated in the same mode (e.g., fed-batch) as the ultimate manufacturing process. Obviously, experimental methods that allow a more rapid exploration of process parameters would increase the odds of selecting the best clone. Such speed also can aid process characterisation and development by allowing a more comprehensive and rapid exploration of the process design space with fewer resources. A couple of recent technological innovations highlight this trend. The Advanced Microscale BioReactor (ambr™) from The Automation Partnership (Hertfordshire, UK). The ambr™ is a mini-scale bioreactor technology platform that mimics the physical characteristics of traditional, much larger bioreactors at a mini-scale (10-15ml) using disposable reactor cartridges. A related product concept, but based on a much different design, is the SimCell™ technology (Seahorse Bioscience, North Billerica, MA). This technology uses microplate sized disposable plastic cards containing six 700uL cell culture chambers that mimic the environment in a bench-scale bioreactor. Data has been developed that demonstrate that mini-bioreactors can replicate bench-top bioreactors, including glycosylation patterns in response to environmental conditions. 

The Mobius(R) CellReady 3L Bioreactor is a single use, stirred tank bioreactor designed for the cultivation of mammalian cell lines in suspension. It was developed in partnership with Applikon Biotechnology and designed to meet or exceed the performance requirements of standard Applikon 3L glass bioreactors. It is ideally suited for process development applications. The CellReady bioreactor ensures maximum operational flexibility, with pre-fitted weldable tubing and vent filter, two sparging options, and compatibility with most standard bioreactor controller configurations. CellReady Bioreactors are offered exclusively by Millipore while Integrated Systems (including ezControl and CellReady) may purchased through both Applikon and Millipore.

 Business model impact

Single-use technology is changing business models and the approach to development and manufacturing at some companies. The technology has enabled some smaller biotechs to keep more control over their clinical manufacturing by keeping it in-house. The facility and infrastructure needed to support a single-use based manufacturing process is considerably less than that needed for an all stainless facility. Some CMO’s service offering is largely based on single-use technologies, which gives them the advantage of lower capital requirements and flexibility, especially quicker batch change-overs between runs and products. High plant utilization and plant through-put are especially key drivers for their business models.

Concluding thought

The transformative effects on bioprocessing from single- and limited- use technologies is far from over. Going forward, the most transformative effects will be felt in downstream processing, commercial applications, and development activities. The drivers for their continued success is speed and flexibility, and reduced capital costs.

[1] J-E Zandbergen and M. Monge. Disposable Technologies for Aseptic Filling- A Case Study. BioProcess International- Supplement. June, 2006:48-51.