Disposable Biopharmaceutical Processes–Myth or Reality?

Biopharmaceutical processes typically require a significant investment in equipment—often a substantial obstacle for start-up companies. The risk of drug development failure is often high, further limiting access to the required capital. Flexibility and lower capital outlays are required not only by start-up companies, but also by research organizations with multiple product lines and by companies requiring quick capacity increases. Disposable technologies offer the highest potential for these companies to meet their business requirements. With lower capital requirements and increased flexibility, disposables are an important part of these companies' risk management strategy.

Development of single-use components is revolutionizing the approach to biopharmaceutical drug development and manufacturing. Disposables provide more solutions each year.

The Business Environment

Bioprocessing has historically been extremely capital intensive and fixed-cost driven, requiring dozens of separate unit operations, each typically involving many pieces of equipment. To minimize the risk of cross-contamination, equipment is often dedicated to a single drug product, even when the facility manufactures multiple products.

Capital investments exceeding $1 billion are not uncommon for cGMP biomanufacturing facilities.¹ Substantial capital requirements are a common barrier to entry for start-ups, smaller companies, private firms, and research organizations. Often, smaller companies that have discovered a promising drug must rely on other companies that have already invested in manufacturing plants, equipment, and competency. Arrangements include mergers and acquisitions, subcontracting arrangements to CROs or CMOs, and licensing arrangements. The number of companies that focus on drug discovery far exceeds the number of those capable of manufacturing commercial biologics, partly because of the enormous capital investment required for manufacture.

Biotechnology companies are also burdened with massive business risk, in part due to unknowns related to clinical results, cost-cutting trends in healthcare, and long lead times in marketing approval. To mitigate market risk, many companies strategically diversify their development pipeline, an approach that requires additional flexibility of equipment. Demand for the drug, and therefore capacity requirements, must also be scrutinized; the target population size may fluctuate and additional indications for the same drug may be approved later. Lastly, competitive forces are ever-increasing, including the threat of biogenerics, making time-to-market a critical success factor for most drugs.

In response to these trends and risks, biomanufacturers value solutions that reduce capital requirements, increase production flexibility, reduce risk, and increase speed to market.

Impact on Capital Investment

The value of disposable technologies continues to become more widely accepted, as they are integrated into increasingly more manufacturing processes. While disposables impact operating costs, for existing and new processes, this article focuses on the impact to new processes and the associated capital investment.

Stainless steel components for mixing, storage, and transportation require substantial space, whether they are in use or not, whereas single-use containers are a more efficient use of space during their useful lifetime. Inventory storage requirements are negligible, compared with stainless steel, requiring comparable space only during use. Whether full or empty, flexible containers are typically stacked with the aid of support structures (Figure 1), which themselves are collapsible when not is use. After use, single-use bags are discarded, not stored.

Figure 1. Support container for disposable storage bag (source: TC Tech)

As capital investments, the number of stainless steel vessels at a facility remains fixed. Bags are "just-in-time" consumables, existing only at the plant when needed. At any given time in a manufacturing facility, more steel vessels are typically sitting idle (or in some stage of cleaning) than are actually being used for processing.

Stainless steel components, particularly those in contact with the processing fluid, must be cleaned and sterilized before use. In addition to the 1.5 to 2.5 hours required to clean vessels from 100 L to 1,000 L,² massive volumes of WFI and steam are also required. These utilities are sometimes in high demand and can create an operational bottleneck. Vessel cleaning costs are in part a function of WFI cost per liter (varies by facility), vessel size, and flush volume required. Depending on these variables, WFI costs alone can be up to several thousand dollars per cleaning per vessel.

Since single-use components are typically presterilized by the supplier, utility systems and sterilizing equipment, such as autoclaves, can be reduced in number and size. This has a large impact on capital expenditures during new plant construction, not to mention commissioning and validation of the systems.

Biotechnology companies often build pilot plants in order to manufacture the volumes of drug product required for clinical phases. If the drug shows commercial promise and receives marketing approval from the regulatory agencies, increased demand will often require a commercial-scale manufacturing facility. Unlike stainless steel equipment which depreciates rapidly, disposable components are customizable: designed and made for a specific use, at a specific volume, at a specific time. Scale-up costs are incremental, not capital intensive. If the process is scaled either up or down, disposable components can be redesigned and supplied ready-to-use in a matter of weeks, not years. Furthermore, revalidation requirements are minimal so long as the process conditions, drug product or intermediate, and product contact surfaces remain unchanged.

Disposable solutions result in faster design, construction, and commissioning of facilities, with the operational benefits of "flexible manufacturing." As disposables continue to be used in more applications, they are evolving from isolated solutions into a holistic value proposition for small and large biotech companies alike.

The Promise of Complete Disposability

In light of the trends discussed above, equipment suppliers are allocating increasingly more development resources to disposable technologies. A widely discussed question lately has been, "Is a completely disposable bioprocessing facility possible and practical?" At the current moment, disposable (product-contact) solutions are available for all unit operations at smaller scales (<50 L harvest reactor volume). Even at mid- or pilot- scale production, most unit operations remain practical, using single-use technologies. As processes scale up to commercial manufacturing volumes, certain unit operations become practical only with reusable operations. However, there is a noticeable trend toward larger disposable equipment availability; once a technology gains acceptance at smaller scales, suppliers commonly focus on development for larger volumes.

In order to discuss disposable solutions to individual unit operations, a simplified, 500 liter monoclonal antibody (MAb) process is considered (Figure 2). The equipment specified in this model is for the purposes of illustrating disposable capabilities and potential. Specific equipment selection and sizing is specific to the drug manufacturer's product and process. Proper selection can only be based upon scientific trials, data collected, and optimization studies.

Figure 2. Simplified MAb process with disposable unit operations shown in blue; reusable equipment shown in red.

Upstream Processing

Disposable mixing systems (Figure 3) for media preparation are currently available using impeller, rocking motion, stir bar, and perforated septum platforms. In all instances, the drive units, controllers, and weighing platforms are reusable, while all product contact surfaces are disposable. Systems are installed providing repeatable mixing results for media preparation from 20–1,000 liter working volumes. Sterile bags are provided in customizable designs to fit the specific application, and are delivered presterilized by gamma irradiation and ready-to-use. Moreover, the mixing units can be re-used without lengthy downtime, often being the bottleneck in the upstream process. Cleaning and set-up times are reduced to minutes instead of hours. Such fast turnaround supports equipment and facility utilization.

Figure 3. Example of a disposable mixing system

From the mixing system, media are then sterile filtered into a presterilized filter and bag assembly. The filter capsule can be supplied connected to either a single bag, or if media are required for future use, to a manifold of bags. Either way, the assembly comes gamma-sterilized and ready-to-use. Disposable filter and bag assemblies (Figure 4) eliminate the need for stainless steel filter housings and storage vessels, which require capital outlays, storage, and maintenance. Furthermore, cleaning and sterilization of stainless steel components are eliminated. Filter and bag assemblies are typically available in standard configurations stocked by the supplier, but are also highly customizable, with a full line of bag chamber and manifold options, filter capsules, tubing, sampling ports, outlet fittings, clamps, connectors, and so on.

Figure 4. Filter and bag manifold assembly

After filtration, individual media bags are removed by thermally sealing the tubing and cutting from the manifold (Figure 5). Media bags are released based on the filter passing a post-use integrity test. Later, the media storage bags are sterile welded onto bioreactor bags and the media are pumped into the bioreactor bag. The blades used to weld the tubing sections are single-use, and discarded after the 60–90 second welding process.

Figure 5. Welder and sealer systems for tubing connection

Cell Culture

Disposable cell culture options at the laboratory scale have been in use for decades. From volumes of several mLs to several liters, roller bottles, spinner flasks, and hollow fiber membrane bioreactors have been used in small-scale MAb applications. These systems have typically lacked the sensors and controls to create an environment optimal for cell growth.

First sighted in the 1970s, turn-key, single-use bioreactors using the rocking motion platform (Figure 6) have been gaining popularity in recent years for medium-scale cell culture, at working volumes from 50 mL to 500 L. In the coming years, it is expected that these low shear systems will become available at larger volumes. Additionally, significant development continues on other platforms, including impeller-based disposable bioreactors.

Figure 6. Rocking motion bioreactor (Source: Wave AG)

With the exception of the controller and drive platform (not in contact with product), all components of these rocking motion bioreactors are disposable, including the bag chamber, vent filter(s), pH probes, dissolved oxygen probes, tubing, fittings, etc. (Figure 7). The culture bags are available in standard configurations, or customized to meet the exact needs of the application.

Figure 7. Single-use bioreactor chamber (Source: Wave AG)

Downstream Processing

While downstream purification processes vary from process to process, most of the unit operations typically found are available in disposable formats, including cell removal and harvest clarification, buffer preparation, pH titration and low pH hold viral inactivation, S- and Q-chromatography steps, viral filtration, ultrafiltration–diafiltration, and final formulation. Each of these operations will be discussed briefly.

Cellulosic depth filters for cell harvest and clarification are available in completely disposable capsules, eliminating the need for housing cleaning and validation.³ They can be simply and directly connected to the downstream processing line or to disposable bags, thereby minimizing product contact by the operator, reducing contamination risk and operator safety concerns. Depth filter capsules are available from small- to large-scale, in a range of pore sizes up to 20 mm. Cell-free and clarified harvest is typically then pumped through a sterilizing grade filter capsule to the subsequent integral unit operation or storage bag.

Centrifugal separation also allows for disposable product contact surfaces, but has direct scalability problems and is best suited for volumes smaller than those seen at production scale.

Figure 8. Load capacity 10 kg/L (source: BioPharm Services, London)

Figure 9. Load capacity 2 kg/L (source: BioPharm Services, London)

Robust chromatographic separation using membrane adsorption is proving to be an economical alternative to traditional resin and column-based operations.⁴ Microporous membrane with pore sizes of >3 mm drastically suppresses diffusion-related mass transfer effects and enables purification of large biomolecules (capturing), or the adsorptive removal of contaminants such as HCP, DNA, endotoxin and viruses in flow-through mode (polishing).⁵ Membrane-based chromatography allows for far accelerated flow rates and very low nonspecific product binding. In a typical polishing application, a 1.5 L disposable membrane chromatography capsule can remove the contaminants downstream of a 10,000 L bioreactor, replacing a resin column that is >100 times larger.⁶ While reusable resin columns require a complex periphery, including a packing station and a chromatography skid, this is not the case for disposable membrane cartridges, the impact of which can be substantiated with process cost models. Comparisons of a polishing step in a flow-through operation with anion exchange chromatography are shown in Figures 8 and 9. Unit operation costs of a reusable resin-based chromatography (100 cycles) were compared to disposable membrane chromatography. The maximum antibody load was limited by the contaminant levels in the feed. Figure 8 shows the typical scenario of polishing after cation exchange intermediate purification, where the load capacity was 10 kg/L. Figure 9 shows polishing after initial capture with Protein A, with a load capacity of 2 kg/L. (One liter of membrane corresponds to 3.6 m².)

In addition to cost advantages, however, there are many additional benefits, including significantly shorter cycle times and superfluous carryover studies in process validation, making adsorptive virus clearance much more straightforward.

A broad range of functional chemistries, including various ion exchange and affinity ligands, is currently available in disposable membrane formats with typical dynamic binding capacities of 30 g/L for proteins and up to 10 g/L for DNA. Protein A chromatography, while possible in membrane format, remains economically practical at production scales using traditional resin-based columns until small Protein A mimetic ligands allow for disposable alternatives.

The trend towards disposable chromatography has only just begun; it will lead to a complete replacement of reusable systems in polishing, with membrane capsules as part of the final filtration train.⁶ New formats of membrane chromatography include disposable direct capture devices allowing for processing of unclarified fermenter offload.

Disposable mixing systems, described previously, are ideally suited for buffer mixing, while filter capsule/storage bag manifolds systems are ideal disposable solutions for column wash, equilibration, sanitization, elution, strip, and storage.

Integrated disposable mixing, filter, and bag assemblies are used for pH titration and low-pH hold viral inactivation. Column eluate is titrated down to a low-pH by adding acid through a second filter to the eluate bag. The solution is then transferred via peristaltic pump to a second bag for low pH hold for viral inactivation. After the hold, a buffer is added to raise the pH.

Disposable virus filtration has been developed for the removal of relevant, as well as adventitious viruses. Virus filters are available in a variety of capsule sizes, so batch sizes of 1,000 L or more are possible. In order to protect the viral filter, a 0.1 mm viral prefilter is commonly used. The entire fluid path is disposable, including the viral prefilter, viral filter, collection bag, and all tubing. Single-use concepts in cross-flow applications have recently become very popular in vaccine manufacturing and are currently being evaluated in monoclonal antibody production also.

Virus filtrate is concentrated and diafiltered to exchange the existing buffer for the bulk formulation buffer which is prepared in a disposable mixing system. The purified bulk drug is collected in a disposable bag. Crossflow systems with entirely disposable fluid paths are standard or customizable depending on batch size, concentration factor, and required processing time. Crossflow cassettes have molecular weight cutoff ratings from 2 kD to 100 kD (Figure 11).

Figure 11. Scalable crossflow cassettes

The purified bulk bag is then welded onto the final formulation mixing bag. Sterile liquid excipient is added by welding the excipient bag to the formulation bag. Powder excipients may be added via a powder addition port in an aseptic hood.

Final product is then transferred to the filling isolator through a transfer port that uses a disposable presterilized connection device (Figure 12).

Figure 12. Transfer port with disposable connection device

Single-use final filling operations remain an area for future development. Currently, filling operations with disposable product-contact surfaces exist only at smaller scales. With the trend toward larger-scale solutions, this practice will likely change in the years to come.

Disposable connections and other accessories are critical in order to integrate components and realize the flexibility benefits. A myriad of aseptic and non-sterile connections are commonly available. Quick disconnect, luers, sanitary fittings, injection ports, clamps, and T- and Y-connectors, to name a few, are all available as disposables, typically integrated onto tubing sets and bag assemblies. Connections allow for plastic–plastic and stainless–plastic connection. Additionally, thermal welders and sealers allow for aseptic connection of tubing of the same diameter and material.

Validation Requirements

While disposable components eliminate the need for cleaning and sterilization validation, increased product contact with polymer surfaces raises questions about chemical compatibility, leachables, extractables, and nonspecific adsorption. In all cases, testing for these occurrences should be performed under actual process conditions, using actual product or a model solvent. Validation requirements do not vary from already existing needs, except that the scale of the testing, especially in regard to leachables and extractables, will be larger. Commonly, suppliers of disposable technologies are requested to, and able to, support such validation requirements.

Conclusion

Disposable solutions continue to develop in response to the biopharmaceutical industry's maturing needs. The disposable bioprocess is well within reach, having tremendous implications on capital requirements and facility design. The trend toward additional development in disposable solutions is clear; suppliers continue to strive to fill what few gaps still exist, at increasingly larger scales. As development continues, in-depth cost modeling analysis associated with manufacturing biological drugs will also evolve. This is necessary to evaluate the economic justification, and supports the decision to either invest in capital equipment or source disposable technologies. Completely disposable bioprocesses are very much a reality. Indeed, "early adopter" biopharmaceutical drug producers are already designing facilities around this concept.

Keith DiBlasi is the manager of technical services, Sartorius North America, Inc., Edgewood, NY. Maik W. Jornitz is the group vice president of bioprocess biosystems. Uwe Gottschalk, PhD, is the group vice president of purification technologies, Sartorius AG, Goettingen, Germany. Paul M. Priebe is head of product management process, Edgewood, NY.

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