The Mobius CellReady family of products includes the bench-scale (3-L), small-scale (50-L) and pilot-scale (200-L) bioreactor systems that enable cell growth in volumes appropriate for early process development through clinical batch production. The small-scale Mobius CellReady 3-L bioreactor is a rigid, stirred-tank bioreactor (see 

) while the Mobius CellReady 50-L and 200-L bioreactor systems (see 

) are inflatable stirred-tank bioreactor process containers used in stainless-steel vessels. 

 outlines the features of all three. Both the Mobius CellReady 50-L and 200-L bioreactor process containers use the Mobius SensorReady technology for process monitoring and control. The Mobius SensorReady assembly is an external loop that is connected to the bioreactor process container that allows for a configurable number of probes and sensors to be used. 

The ability to scale up a biomanufacturing process is essential for process development and the production of biologics. Bioreactor process set points, acceptable ranges, and general operating parameters used at the large scale are commonly based on those developed at the benchtop or small scale where experimentation is more cost effective and efficient. Large-scale performance and production expectations are often established based on results obtained at smaller scales. It is, therefore, important that the process parameters developed at the small scale are readily transferrable to the larger scale.  

Figure 1: (A) 3-L Mobius CellReady bioreactor; (B) 50-L; and (C) 200-L Mobius CellReady bioreactor process containers. 

Challenges to bioreactor scale-up occur because even when two geometrically similar tanks are used, it is not possible to simultaneously maintain key bioreactor characteristics such as shear, mixing time, and oxygen mass transfer coefficient (k

a) identical in both the large and small tanks (1). Other variables, such as bubble size and distribution, nutrient regulation and delivery, and process control capabilities may also contribute to variable performance results across scales. Ultimately, successful scale-up is determined when comparable process performance endpoints such as cell growth, cell viability, protein production, and product quality are achieved. The probability of meeting these criteria can be increased when the bioreactor systems are well-characterized and the process design space is better understood. 

Table I: Comparison of the features of 3-L, 50-L, and 200-L CellReady bioreactors. 

In this study, several key engineering parameters k

a, power-per-unit volume, Reynolds number (Re), mixing time, and tip speed were characterized for the three different sized single-use bioreactor process containers. Chinese hamster ovary (CHO) cells were then cultured in each of these bioreactor systems based on maintaining equivalent power per unit volume as the primary scaling parameter. The results of this study define the characterization and process design space offered by the bioreactor systems and demonstrates the capability to achieve expected cell culture performance results across scales, thus demonstrating the scalability of the family of Mobius CellReady bioreactor systems. 

One of the most crucial scale-up parameters for bioreactors is mass transfer of gases. To provide an optimal environment for the cells, a bioreactor must be able to supply enough dissolved oxygen (O

) for efficient cellular metabolism as well as maintain an appropriate level of dissolved carbon dioxide (CO

/air delivery is required not only to support cell growth and protein production but also to prevent excessive CO

 accumulation in the media that can impact both of these critical performance endpoints (2). To assess the gas transfer efficiency of the Mobius CellReady bioreactor process containers, the volumetric mass-transfer coefficients (k

a) for oxygen were measured in each bioreactor process container using the static gassing out method. 

a values were determined by filling the bioreactor process containers to the maximum working volume with a mock media (1X phosphate buffered saline (PBS), 2 g/L Pluronic F-68, 50 ppm Anti-foam C), and setting the temperature to 37 °C. The dissolved oxygen was stripped from the bioreactors by supplying nitrogen gas via the microsparger. Once the dissolved oxygen (DO) concentration reached less than 2% air saturation, the nitrogen supply was turned off and air was sparged through the microsparger at flow rates ranging from 0.0025 vvm to 0.5 vvm at two different agitation rates. The DO concentration was recorded over time until the dissolved oxygen concentration in the media plateaued at the fully saturated value (100% air saturation). An air overlay gas was not used in these studies.  

a value for each trial was calculated from the linear portion of the DO vs. time graph. To avoid subjectivity in determining the linear portion, the uniform DO interval used for the calculation was between 10% and 90% air saturation. The k

a values shown represent the slope of the line created by plotting ln((C*–Ct

), where C* is fully saturated liquid, Ct

 is the percent air saturation at the initial time, Ct

 is the percent air saturation at time 2, and t

 are the initial time and time 2, respectively. 

Figure 2A: kLa Scalability of the 50-L and 200-L Mobius CellReady bioreactor process containers. Each bar represents the average kLa value of n=3 at each air-flow rate and impeller agitation rate tested. Error bars represent the standard deviation of triplicate determinations. 

, the impeller and sparger design and placement in the 50-L and 200-L bioreactor process containers are similar; however, the 3-L bioreactor is significantly different from the 50-L and 200-L bioreactor process containers. For example, in the 50-L and 200-L bioreactor process containers the membrane polyethylene microsparger is located directly beneath the impeller, while in the 3-L bioreactor a sintered polyethylene microsparger is located off to the side of the impeller. Also, the impeller blade shape differs between the bioreactors. A pitched blade impeller is found in the 50-L and 200-L bioreactor process containers while a marine impeller is found in the 3-L bioreactor. As shown in 

, the 50-L and 200-L bioreactor process containers, at the same power per unit volume, exhibit comparable k

a values. These two bioreactor process containers can achieve k

  at the lowest agitation and air flow rates tested to 60 hr

  at the highest agitation and air flow rates tested. Given the differences in the volume and design between the 3-L bioreactor and the 50-L and 200-L bioreactor process containers, it is not surprising that, at the same gas flow rates and power per unit volume, the k

a values are not immediately comparable. However as shown in 

, by adjusting the air flow rates, scalable k

a values between all three bioreactor systems at similar power per unit volumes can be achieved. 

Figure 2B: kLa Scalability of the family of Mobius CellReady bioreactor systems. Each bar represents the average kLa value of n=3 at each air-flow rate and impeller agitation rate tested. Error bars represent the standard deviation of triplicate determinations. 

Mixing is a crucial bioreactor performance characteristic because it is responsible for minimizing gradients and maintaining control within the cell culture environment. Good mixing in a bioreactor strives for sufficient fluid pumping and turnover throughout the system to effectively create a single homogeneous environment which can be accurately monitored and controlled. Good mixing should evenly distribute bioreactor contents, helping to minimize zones of uneven cell density, pH, temperature, dissolved gases, and nutrient or waste concentrations, while minimizing the shear stress imparted on the cells by the fluid dynamics or the mixing element itself. 

Table II: Tip speed, Reynolds number, and ungassed power-per-unit volume values for the 3-L Mobius CellReady bioreactor process container. 

Although no single parameter can guarantee comparable process performance between stirred tank bioreactor systems, choosing an agitation rate that matches energy dissipation or power per unit volume (W/m

 ) is a common first approach (3, 4). The impeller design, fluid density, and agitation rate are considered in the power per unit volume (P

 is ungassed power, V is liquid volume, Np is impeller power number, d is impeller diameter, n is impeller agitation rate and ρ is fluid density. Using this scaling method, agitation rate is varied to maintain similar ungassed power-per-unit volume across vessels. 

Table III: Tip speed, Reynolds number, and ungassed power-per-unit volume values for the 50-L Mobius CellReady bioreactor process container. 

The highest shear zones in a stirred tank bioreactor are often described as existing within the impeller zone. Because the outer edge of the impeller blades create shear as they rotate through the liquid, the impeller tip speed is often considered during bioreactor comparisons. The impeller tip speed calculation, π x d x n, where d is impeller diameter and n is agitation rate, however, does not take impeller design into consideration. Reynolds number (Re) can be considered when estimating fluid conditions at a given agitation rate by providing a ratio of the inertial forces to the viscous forces where Re = (ρ x n x d

 ) / µ. The fluid density is represented by ρ, n is agitation rate, d is impeller diameter, and µ is dynamic viscosity. The system is considered fully turbulent when Re > 10,000 (5, 6). The Reynolds number calculation is based on the assumption of a cylindrical tank with a centered rotating impeller and does not take impeller design into account. The Mobius CellReady 50-L and 200-L bioreactor process containers contain a bottom mounted, offset, pitched blade impeller and a baffle, which together increase the turbulence and improve overall mixing. 

Table IV: Tip speed, Reynolds number, and ungassed power-per-unit volume values for the 200-L Mobius CellReady bioreactor process container. 

 outline various tip speed, Reynolds number and ungassed power-per-unit volume values calculated for a range of applicable agitation rates for the 3-L, 50-L, and 200-L Mobius CellReady bioreactor process containers. 

Mixing was evaluated for the 50-L and 200-L Mobius CellReady bioreactor process containers by observing conductivity probe response curves measured at four locations (top, middle, bottom, and inserted in the probe port of the Mobius SensorReady loop) within the system. The average of these four probes is considered the system average. A salt solution was introduced at the liquid surface and the T

 mixing time was determined for each of the four probe locations as the time when conductivity profiles had reached 95% of its final value. Each trial was performed in triplicate and results are shown in 

. The Mobius CellReady 3-L bioreactor was evaluated in a similar manner, using just one conductivity probe inserted in the head plate probe port. 

Figure 3: System average mixing times of the family of Mobius CellReady bioreactor systems. Data points represent the system average mixing time of n=15 individual trials. Error bars represent 1 standard error for each data point. 

Results of these studies show that the Mobius CellReady 50-L and 200-L bioreactor process containers demonstrated similar system average mixing times at equivalent power-per-unit volume. Mixing times for the 3-L bioreactor are significantly shorter than mixing times observed in the larger bioreactor process containers for similar power per unit volume. This is to be expected as larger liquid volumes result in longer fluid paths. To achieve a well mixed condition in the same amount of time, the fluid velocity would have to increase for the condition in the larger volume tank. As a guide, fluid velocity is proportional to the square root of power per unit volume in turbulent conditions (i.e., when Re > 10,000) (5, 6). As a result, an increase in power-per-unit volume would be required to achieve similar mixing times at significantly larger scales. The system average mixing times for the Mobius CellReady bioreactor process containers, as determined by the multipleprobe experimental methods described above, do not dramatically increase with increasing power per unit volume across the agitation range tested. The 50-L and 200-L bioreactor process containers contain a single baffle, which coupled with the off-center, angled position of the bottom mounted impeller, provides good mixing dynamics even at lower power inputs. Empirical results show the mixing time to be 12–13 s from 2 to 15 W/m

  for the 3-L, 38–24 s from 5 to 30 W/m

  for the 50-L and 33–26 s from 1 to 25 W/m

  for the 200-L bioreactor process containers as displayed in 

Table V: Comparison of operating parameters for 3-L, 50-L, and 200-L CellReady bioreactors. 

Maintaining a homogenous environment within the bioreactor is the most crucial criterion for a successful cell culture process. While different agitation strategies may be used to scale up a biomanufacturing process, power-per-unit volume is the most often used scaling parameter (3, 4). The second most important criterion for a successful cell culture run is efficient delivery of oxygen to maintain cell growth and productivity. Using  these two criteria, a CHO cell culture batch process was performed in the 3-L, 50-L, and 200-L bioreactor systems using power per unit volume as the primary scaling parameter to demonstrate cell-culture scalability across the Mobius family of bioreactor systems. Further, gas flow rates were chosen to achieve similar k

a values for each vessel. The process parameters for each scale are outlined in 

Figure 4A: Viable cell density and viability vs. culture time for a batch culture process at the 2-L, 50-L, and 200-L working volumes. Data were obtained daily from the Vi-Cell XR (Beckman Coulter). 

To demonstrate scalable cell culture performance, parameters  including cell growth, viability and metabolism were compared For this study, samples were analyzed daily on a Vi-Cell XR (Beckman Coulter), a BIOPROFILE FLEX system (NOVA Biomedical) and a Blood Gas Analyzer (Siemens Rapidlab 248). As shown in 

, the viable cell densities and viabilities were comparable between all three scales. As shown in 

, the nutrient profiles and metabolic rates between all three scales were also comparable. The comparable cell growth, viabilities and metabolic profiles demonstrate that the cell culture performance in all three Mobius CellReady bioreactor systems is scalable. 

Figure 4B: Glucose and lactate Concentrations vs. culture time for a batch culture process at the 2-L, 50-L, and 200-L working volumes. Data were obtained daily from the BIOPROFILE FLEX system (NOVA Biomedical). 

Successful scale-up of a biomanufacturing process is dependent on several factors including gas mass transfer, mixing efficiency and shear effects. An in-depth understanding of the process design space of the three Mobius CellReady bioreactor systems has beed developed through a series of experiments aimed at characterizing several key engineering parameters of the bioreactors. The three bioreactor systems, despite design and volume differences, are capable of achieving equivalent k

a values by adjusting the air flow rates at equivalent power-per-unit volume set points. In addition, similar system average mixing times can be achieved for the 50-L and 200-L systems within their power per unit volume operating range. 

Figure 4C: Metabolic rates for culture days 1â5 for a batch culture process at the 2-L, 50-L, and 200-L working volumes. Data were obtained daily from the BIOPROFILE FLEX system (NOVA Biomedical). 

Based on an understanding of the design space of each of the bioreactor systems, process set points were chosen for CHO cell cultivation in each of these. Maintaining equivalent power per unit volume was chosen as the primary scaling parameter and gas flow rates were chosen based on achieving similar k

a values at each scale. During a 10-day batch culture, comparable cell growth, viability, and nutrient metabolism were achieved in each bioreactor system. 

The comparable cell culture performance results with all three bioreactor systems demonstrate the scalability of the family of Mobius CellReady bioreactor systems. The comprehensive characterization of several key engineering parameters resulted in a detailed understanding of the design space of each bioreactor. This understanding allows users to choose process set points that enable successful scale-up of biomanufacturing process across the Mobius CellReady family from the 3-L to 200-L scale. 

is global product manager for single-use bioreactors at EMD Millipore, Bedford, MA.  

 4.  S.D. Kaiser et al., CFD for Characterizing Standard and Single-use Stirred Cell Culture Bioreactors. In 

Computational Fluid Dynamics Technologies and Applications

 5.  Dalian University of Technology. Mixing, Chapter 6.  

http://ceb.dlut.edu.cn/uploads/soft/110415/7-110415154330.pdf

Articles

Features

Review the process-design space and scalability of a single-use, stirred-tank bioreactor.

Scalability of the Mobius CellReady Single-use Bioreactor Systems

Tubing is a crucial part of any single-use system and is required for secure fluid transfer. However, it also presents a significant contact area that can potentially contribute to adsorption, leachables, or particulates, so selection of appropriate tubing for each application is important. The best starting point is to consider the process fluid and conditions to which the fluid transfer tubing will be exposed. In this paper, I will also consider the potential impact on product, the types of tubing available to choose from, and other considerations in making a selection. 

Photo Credit: Michal Saganowski/Getty Images 

The recommendations below are based on my experience with single-use systems, along with suggestions by technical experts from three tubing suppliers: Steve Wilkowski of Dow Corning, John Stover of NewAge Industries, and Christopher Shields of Saint-Gobain.  

The first factor to consider when selecting tubing is the nature of the fluid you want to transfer. In single-use bioprocessing, fluids range from proteins or other biological solutions to strong pH adjusters and buffers. Biologicals may be sensitive to ultraviolet light or oxygen, and require opaque or low gas permeability tubing. Acids and bases must be used with chemically compatible tubing. In all cases, it is necessary to select tubing grades with low extractables to ensure minimum levels of leachables. 

Once biological stability and chemical compatibility requirements are defined, processing conditions should be considered, including process temperature, pressure, time, and sterilization conditions prior to use. Single-use bioprocesses are generally conducted at temperatures below 40 °C, so most tubing options are compatible. If tube welding and/or sealing is required on the system then meltable tubing will be needed, whereas the use of only quick-connects or sterile connectors eliminates this temperature requirement. Similarly, if the tubing is to be autoclaved, it should be resistant to at least 125 °C.  

The next process condition to consider is pressure. Although the lowpressure limits of single-use polymerfilm biocontainers generally limit pressure requirements for system tubing, tubing used upstream of sterilizing filters that are integrity tested prior to use is an exception. Depending on the grade of filter and integrity test method, test pressures can range from 40 psi (2760 mbar) to 70 psi (4825 mbar), or even higher with some mycoplasma or virus filters. To accommodate this, high durometer, braided or otherwise reinforced tubing must be incorporated upstream of the filters to ensure suitable pressure rating for integrity testing. Another exception is where peristaltic pumping is used to drive the process fluid. Tubing selection must consider the strength of the tubing to provide consistent flow and low generation of particulates. Tubing grades recommended for peristaltic pumping are typically qualified for much longer service than would be applied in most single-use applications, but failure to consider pumping suitability after sterilization can lead to significant problems.  

Sterilization conditions must be considered when selecting tubing. Most single-use applications are gamma irradiated for sterilization and consequently require gamma stable tubing (generally up to 50 kGy dosage), but it is not uncommon to apply autoclave sterilization for some tubing manifolds and filtration systems, especially during process development and early phase clinical batch manufacturing. The desired sterilization method must be considered in tubing selection. Information on gamma sterilization is provided in the BPSA Guide to Irradiation and Sterilization (1). 

The primary concern for the tubing's impact on process fluids are biocompatibility, leachables, adsorption–absorption, and permeability of gas and light (particularly ultraviolet light). Biocompatibility is typically assessed by applying the USP Biological Reactivity Tests to the tubing material after a "worst case" sterilization process, either 

 for Class VI (<88>, implantable) plastics (3). Other standard tests may include pyrogenicity. Recommended standard reference tests for tubing are described in the BPSA component quality test matrices guide (4). 

Potential leachables are initially assessed by considering extractables determined under exaggerated process conditions, such as higher temperatures, more aggressive solvents, and longer contact times. The maximum sterilization process conditions should also be included (typically >125 °C steam autoclave or 50 kGy gamma irradiation) because these factors can increase the level of process leachables from some tubing. Similarly, the impact of heat welding on leachables should also be included in extractables assessments. For more information on determination of tubing extractables data, see the 2008 and 2010 BPSA extractables guides (5, 6). 

Adsorption, where target molecules bind to tubing contact surfaces, is a primary concern with protein molecules, but the relative smallsurface area to volume ratio typically limits protein concentration losses to only highly dilute solutions. Absorption, where target molecules migrate into the tubing solid phase itself, are a greater concern with small molecules such as preservatives, which are generally limited to final formulations, but should be assessed for tubing used at that stage of the process. Low permeability tubing (for gases and light) can also provide reduced absorption of small molecules. 

Although particles in tubing used in upstream processes (media, buffers and intermediates) upstream of filters have not been a major concern, applying single-use systems to aseptic vaccine manufacturing or in post-filtration formulation and filling of biopharmaceuticals has raised concerns about the potential impact of particles from tubing fill lines on final dosages. Extrusion of tubing is generally a low particle-generating process, but handling, cutting, joining, and environmental conditions for single-use system assembly can have an impact. Particle levels from tubing in systems used downstream of final filtration should be considered in critical applications. 

The most common types of tubing used in single-use applications are silicone and thermoplastic elastomers. Silicone tubing has a long history of safe use for a broad range of bioprocess fluids, and provides high temperature and gamma radiation stability. Other benefits include flexibility, translucency, biocompatibility, and smooth bore for low adsorption and particulates. Peroxide-cured grades have been successfully used in laboratory and clinical applications, but platinum-cured silicone is preferred for single-use systems because of lower extractables. Braided grades are available for high pressure applications. Silicone tubing cannot be thermo-welded and is limited to pre-assembled systems or connections using quick-connects or sterile connectors. 

For tube welding and sealing applications, thermoplastic elastomer (TPE) tubing is required. These are proprietary formulations, commonly referred to under trade names, and have a good record of safe use and are also preferred where reduced adsorption, absorption or gas permeability is desired, though they do not have the transparency of silicone tubing. Other specialty formulations of tubing are also available. 

Both silicone and thermoplastic elastomeric tubing are manufactured in numerous proprietary formulations, resulting in different extractables profiles and other properties. A case study was reported several years ago on the effect of tubing material on the bubble point values of downstream sterilizing-grade filters (7). Poly(dimethlysiloxane) silicone oil leached from some grades of tubing (both silicone and thermoplastic elastomer) and was adsorbed on downstream filter surfaces. This reduced the critical surface wetting energy of the filter membranes, thus impacting post-use bubblepoint values, causing false test failures. Tubing used upstream of sterilizing filters, and infinal filling, should be considered for low silicone oil, along with other leachables. 

It is common for new users of singleuse systems to consider choosing whatever grade of tubing they have experience with in their laboratory or clinicalbatch processes, but it is also important to consider the selection and sourcing of tubing by the single-use system integrator. It is also advantageous for integrators to limit their approved tubing sources, including inventory control, cost-efficiency, and generation of additional qualification studies (e.g., post irradiation extractables). Good integrators will also establish quality agreements for traceability and sourcing of raw materials (e.g., resins and elastomers), change notification protocols, and conduct supplier audits. Consequently, once your fluid and process requirements are defined, it can be beneficial to consider suitable grades among those already selected and qualified by the single-use system integrator.  

 is the senior vice-president of global scientific affairs at Pall Life Sciences, Port Washington, NY, US. 516.801.9086, 

 Jerold is also the chairman of the board and technology committee of the Bio-Process Systems Alliance. 

Guide to Irradiation and Sterilization of Single-use Systems

 24 General Chapter <87>, "Biological Reactivity Tests, 

 24 General Chapter <88>, "Biological Reactivity Tests, 

Guides to Extractables and Leachables from Single-use Systems

Jerold Martin considers the types of tubing available to the industry and how to make an informed selection.

Making the Best Choices in Single-Use Tubing

What started off more than a decade ago as a cutting-edge technology has now become mainstream; the arguments in favor of biopharmaceutical production using single-use equipment have largely been proven. There is an understanding that financial considerations are no longer a deterrent for adopting disposables, but more importantly, there is another realization concerning the use of single-use technologies. The industry has started to look beyond the immediate manufacturing implications and into how these technologies benefit end users (i.e., patients) through increased product visibility, better product quality, and patient safety. 

Patient safety is the most important topic of discussion in the biopharmaceutical industry today, and rightly so. Manufacturing companies are focused on finding ways to ensure that any accompanying risks are reduced to save patients from unnecessary risks to their health. Secondarily, manufacturers want to ensure that they are not losing batches because of contamination, which means lost money.  

But what does all of this really mean? Here we look into how single-use technology continues to offer new levels of performance to the industry and how it can benefit the patient.  

DISCOVERING AN UNEXPECTED  BENEFIT OF SINGLE-USE TECHNOLOGY

Although the economic model for using single-use technologies includes various benefits, the intangible benefit of increased patient safety has emerged and started a trend in the vast majority of multiproduct facilities. Many manufacturers are finding ways to implement single-use technologies into their process because patient safety, by way of avoiding cross-contamination, should trump cost savings.  

While the adoption of single-use technology has made great strides, it is often true that a single-use-only facility may not meet all the needs of drug manufacturers, nor is it always feasible. Mixed needs, therefore, have spurred a trend towards multi-use hybrid or flexible facilities, which combine the proven benefits of traditional stainless-steel and glass equipment with the promise of single-use equipment. The key is finding the right balance to fully maximize the potential of the facility by reducing validations, water use, and operator risk, and increasing productivity. The most attention should be paid to how to introduce these flexible facilities in a way that greatly reduces (with the ultimate goal of eliminating) cross-contamination potential between successive product batches.  

CROSS-CONTAMINATION CONCERNS INCREASE WITH POTENCY

The increasing potency of new medicines has further exacerbated the need to know what contamination risks are present. There are many new products that require only minuscule amounts of contaminants to cause a strong pharmacological reaction in patients. This potency is advantageous in dosing terms—maybe the dose will be one pill instead of three, or a biologic might need to be injected once a month rather than once a week. However, on the manufacturing side, it presents a new cause for concern. While a residual nanogram of aspirin may have no physiological effect, a microgram of a monoclonal antibody very well might have an effect that could be extremely harmful to the patient.  

For a manufacturer reliant on fixed stainless-steel vessels, there is always a risk that minute quantities of product will be left behind to contaminate the next batch, regardless of how carefully a vessel is cleaned. As drugs become more potent, the safe limits for residual product carryover become ever more challenging to achieve. Single-use equipment eliminates the potential for cross-contamination almost completely, because the process vessels are thrown away after use.  

When introducing single-use equipment, users—to their credit—are keen to monitor all manner of parameters they would not necessarily contemplate studying for a stainless-steel system. Particulates, extractables, and leachables all come under examination with single-use systems.  

The concern lies in making sure the product does not become adulterated during its manufacture. The vessels hosting production are made from plastic, so there is always the potential for extractables or leachables to find their way into the product from or through the film, or for gas permeation through the walls of the vessel. It's the same story in the final packaging because extractables, leachables, and gas permeation are all potential problems with plastic vials. And in general, particulates are a concern; they can either come from the equipment itself or from the atmosphere around the systems.  

In the past, a final product was inspected for particulate contamination—first by eye and later, as automated systems became available, by machine. Often, any particulates found in traditional systems were not given much attention; they were either not large enough for concern or there was not the right technology to isolate their source. Single-use products change this level of scrutiny because there is suddenly better product visibility. Suppliers are expected, and are now able, to tell manufacturers what particles might be present and where they potentially came from.   

This added material tracing and product-tracking capability brings an unexpected benefit of product quality and, thus, provides a patient safety benefit. Because the process is better studied, more traceable and more defined, the quality bar is automatically raised. The product-contact surfaces are better understood because they have been investigated in detail, and the validation process now generates more data than ever before.  

Biopharma producers rely on their suppliers much more than they have in the past. For a single-use-only facility, the suppliers are the new product, and often process, experts. Moreover, they essentially control the factory; if the bag and vessel suppliers ever cannot deliver, production could cease. All this reliance has created demand for greater support, supply-chain contingencies, and documentation than ever before. Companies that take advantage of single-use technologies are doing so because of the various benefits they offer. Undoubtedly, single-use technologies have allowed these companies to identify new ways to ensure quality of end product for patients. 

Single-use suppliers are working hard to characterize anything—particle or otherwise—that might be present, and identify if it originates from the bag, the tubing, or a fitting. The elimination of particulates has been a recent driver for the industry. Customers want to know whether the particles are native (i.e., originating from the single-use system) or foreign, such as from a piece of paper in the cleanroom. Ultimately, customers want suppliers to ensure that particles are eliminated. 

Being able to detect and quantify particles is the first step, and the next step in improving product quality is to eliminate them. Each step brings us closer to the vision of better protecting patients and ensuring the drug products they ingest have the intended effect, with no adverse risk factors from preventable causes.  

 is global product manager, Integrity single-use fluid technologies, at ATMI, 

Reduced carryover risk and close attention to particulate control result in greater patient safety through the use of single-use systems.

The Overlooked Benefits of Single-Use Technology: Improved Quality and Increased Patient Safety

Ten years ago, single-use systems were an emerging technology moving from common use as storage bags for cell culture media and serum, into broader applications that have developed into a major trend in clinical biopharmaceutical manufacturing.  Advances continue as more robust, better designed, and standardized technologies, with more inert materials, are developed for GMP applications. As single-use/disposable bioprocessing moves toward commercial-scale manufacturing applications, even more innovation is being demanded by the industry. 

In BioPlan's 10th annual report on the industry, four of the top 21 new product areas being sought by this industry were single-use related: disposable bags and connectors topped the list again this year, with 44% of the industry demanding better technology/products (compared with 40% last year), following were probes and sensors (39.6% this year, vs 34% in 2012) (1).  In comparison, stainless equipment once again hit the bottom of the list, with fewer than 5% indicating they would like new products developed in this area (see 

). The reason for this interest is partly because single-use devices allow lower overall facility costs, reduce contamination problems, provide faster changeovers, and reduce shutdown times. As such, they continue to grab the focus of the industry. 

Figure 1: Selected new technology and innovation areas demanded by the biopharmaceutical industry (a few of the 21 key areas indicated). 

In fact, a separate BioPlan study conducted in late 2012 of the 450 global subject matter experts and senior industry participants who make up BioPlan's Biotechnology Industry Council (BIC), asked participants to identify the key trends and factors they see in the industry. This year 22% of senior industry decision-makers pointed to single-use devices as a key trend, just behind downstream processing (24%) and analytical methods (24%) (2).  

While single-use devices remain a hot topic this year, much has changed from when they were first being evaluated by the industry. BioPlan's biomanufacturing industry study includes trends in the use of disposables and single-use systems. Data over a multi-year period offers insights into how the market for single-use equipment has evolved over time, and where it may be headed.  

BioPlan's 10th Annual Report indicates that adoption of single-use devices has continued to increase, and that some disposable devices are now almost universally used at one stage of bioproduction (R&D through commercial production) (1). When compared to adoption just seven years ago, the growth has been significant.  

Topping the list in market adoption this year are disposable filter cartridges, now used by 98% of respondents to the latest survey, up from a leading 93% last year, and 78% in 2006. The growth has been even more dramatic in other widespread devices:    

Tubing for disposable appli-cations (97% this year, up from 88% last year and 56% in 2006) 

Buffer containers (91% this year, up from 82% last year and 53% in 2006) 

Depth filters (90% this year, up from 84% last year and 57% in 2006). 

Even some of the lesser-adopted systems are showing strong growth. For example, this year, membrane adsorbers, the devices with the least amount of penetration, are still being used by 6 in 10 respondents (61%), up from 54% last year and just 13% in 2006. For many of the devices tracked, there was a big jump in adoption between 2006 and 2007, with growth rates slower since then; nevertheless, these trend figures show just how quickly the market for these devices has grown.  

It is also worth noting that quantities for these products were not indicated in the survey question, only incidence of usage at any stage. Thus, while market penetration and the number of purchasers for these products may be increasing substantially, these data do not provide direct information on the volume of sales increases. Separately, though, vendors have reported double-digit sales growth in materials and consumables for several years, most likely attributable to the increased adoption of single-use disposables and the introduction of new products in this sector of the market. 

While there have been dramatic changes in the adoption of disposable devices, some trends have remained remarkably steady over the years. BioPlan identified a series of more than 25 reasons for increasing use of disposables and asked respondents to indicate their single most crucial reason—fully 26.1% cited reduced capital investment in facility and equipment. This has been the top reason going back at least as far as 2009, increasing from 14.4% of respondents that year. Clearly, this has been one of the key benefits of single-use devices, and has become more important over time.  

This year, "eliminate cleaning requirements," cited by 13.6% of respondents, was the second-most crucial reason for increasing use of disposables, as it has also been since 2009. The percentage citing this factor has remained steady over the years. In a tie for third place this year, each at 9.1% of respondents, were "faster campaign turnaround time" and "flexibility of a 'modular' approach." The former retains its third position from 2010–2012, though has leveled off after rising growing steadily from 6.9% of respondents in 2009 to 12.4% last year. The latter appears to have grown more important, after ranging from 4.2-6.3% of respondents for the past four years.  

FACTORS RESTRICTING GREATER ADOPTION OF DISPOSABLES

While maturing, introduction of single-use, disposable systems is still relatively new, and as such, the number of factors decision-makers recognize as hurdles continues to evolve. While the market has burgeoned, there are some challenges that the industry still needs to overcome before the next stage of penetration can be reached. And in this area, we see some interesting trends.  

This year, when industry personnel were asked their single most important reason for not increasing use of disposables, of the 23 potential reasons identified, 1 in 5 respondents focused on one: leachables and extractables. Concern regarding leachables and extractables has grown steadily in the past few years, doubling from 10.4% of respondents in 2009.  

As recently as 2011, this was not the top concern hindering adoption. Instead, the most common reply from respondents was that they had already invested in equipment for their current system, hindering further use of disposables. Over the years, however, that reason has gradually become less of an issue, perhaps as current installed equipment has depreciated or worn out. There was a slight rebound in the percentage of respondents indicating this reason to be their primary concern this year (from 13.3% to 16.3%), but that this factor remains less of a worry today than leachables and extractables.  

Other trends seen include the declining proportion of respondents saying that the high cost of disposables (consumables) is their top hindrance—as can be expected with greater maturation of the market—and a general increase in the proportion who are concerned with not becoming vendor-dependent (single-source issues).  

One reason why the disposables market has been able to overcome the above obstacles is the continued pace of innovation in this area. Each year BioPlan asks the industry to identify the top five areas they want their suppliers to focus their development efforts on. And seemingly with every passing year, the industry responds that they want innovation in disposable products.  

As mentioned above, this year, a leading 43.8% of respondents indicated that disposable products, bags, and connectors are an area of interest, up from 40% last year and 36.5% the previous year. Interest in having suppliers improve their single-use devices is an indication of broader general adoption and a greater number of potential applications that are not being met with current technologies. Some of these concerns include the need for:  

Better materials with fewer particulates, and lower leachables and extractables

Stronger, more robust materials, tubing, films

Products able to withstand freeze/thaw cycles

Better probes and sensors to meet more varied applications

Suppliers generally have been answering the call: past surveys have indicated that disposable bioreactor bags/consumables, single-use bags/films, and disposable chromatography have consistently been among the top new technologies or product development areas suppliers have said their organizations are working on, and expending R&D resources to resolve.  

The industry sees great promise in the continued adoption of disposable devices. Entirely disposable upstream processes that can support large, commercial-scale production are becoming a reality, and now companies are focusing on cost-effective downstream single-use innovations. Today, even the older, more conventionally built facilities are looking to modify their infrastructure to support disposables for production. Over time, conventional processing equipment, in part or as integrated into overall process trains and modules, will be replaced by disposable designs or established as hybrid production modules.  

The future of innovation within disposables areas is likely to bring about an array of advances.  These will be driven by many important sub-trends that continue to shape this market. Industry experts from our BIC panel identified numerous such trends, including:   

Building quality into single-use operations to further reduce regulatory activities and oversight

Fixing disposable bioreactors that are creating inconsistent growth due to changes in resins, films, gamma irradiation, and cell-line specificity

The emergence of flexible and modular biomanufacturing facilities that reduce the need for expensive, stand-alone facilities for every drug product 

Single-use devices facilitating large-scale bioproduction in developing regions

Leachables and extractables standardization at clinical and commercial scale 

More new, scalable devices for downstream processing that get away from existing labor-intensive, costly technologies. 

Although, as yet, there are few, if any non-rigid single-use devices (e.g., bioreactor bag liners) used in GMP applications, this will likely change quickly as new products move through the development pipeline, into clinical-scale manufacturing, and on to regulatory approval for commercial GMP production. Leachables and extractables will remain a concern, but as data are developed and experience accumulates, the worries will diminish. The more vendors collaboratively work with customers, the greater the likelihood that disposable, single-use products will deliver sooner on their promise to change bioprocessing for the better.  

Further, as regulators gain familiarity with the safety profiles and materials used in these devices, necessary approvals for product manufacture will be facilitated. When this occurs, the market volume for single-use devices is likely to increase significantly. 

 is president and managing partner at BioPlan Associates, Inc., a biotechnology and life sciences marketing research and publishing firm in Rockville, MD, 

10th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production

BioPlan Associates' 2013 Biotechnology Industry Council Trends Analysis Study, 

BioPlan's Annual Report shows continued growth in the use of single-use technology.

Ten Years Later: Innovation Driving Single-Use Technology Advances

The biopharmaceutical industry has started to look more seriously, in the past five years, at the potential that single-use technology offers for commercial manufacturing of biologic drug substances. There are major advantages to be had by the introduction of these technologies, none more so that the reduction in turnaround time for the manufacture of biological drug substances, leading to reduction of product costs.  

Other tangible opportunities afforded by these technologies include reduced footprint of facility and energy reduction costs (i.e., reduction in sterilize-in-place [SIP] and clean-in-place [CIP] requirements and water usage). These opportunities have been well documented by various independent engineering and consultant firms. In addition, there are useful software models available to calculate what these opportunities provide from a cost perspective (1, 2).  

Despite the reduced operating costs, companies are still hesitant to adopt single-use technologies, in part because of concerns that have been raised over the control of the raw materials used in the production of single-use components and finished products as well as the cost of qualification activities. Implementing oversight on the manufacturing of single-use technologies through the use of quality management systems (QMS) on the supply chain can significantly reduce the overall risk to the product.   

Once better manufacturing controls have been introduced to minimize leachable and extractable impact on drug products, the qualification concerns may be mitigated by integrating risk-assessment principles into the qualification of single-use systems and components.  

While control and management of the supply chain could be quite unwieldy, risk assessment can be performed to limit the qualification activities needed for disposable components. This article suggests techniques for mitigating risk and securing the supply chain for single-use components used for biopharmaceutical manufacturing and using the risk assessment to reduce the cost of their associated qualification activities.  

Single-use technologies have been around for almost two decades but have become more prominent over the past few years in biopharmaceutical manufacturing facilities as business leaders see the advantages with speed to market and cost of goods for drug products. Single-use technologies in the past included single, discrete components such as filters that were used once and then discarded. The evolution from single components to more complex assemblies incorporating containers, filters, sensors, and other appurtenances provides advantages that were not readily available to the industry until recently. As these technologies evolve, more capabilities will become apparent and will lead to more streamlined manufacturing operations. To facilitate the increase in use of single-use technologies, a robust supply chain of single-use items is necessary to maintain production capabilities. 

The use of QMS is recommended in the ICH Q10 guideline 

 (3). A QMS paired with risk-assessment tools can be used to establish the best approach for the adoption of single-use technologies within a biopharmaceutical facility. The use of QMS tools ensures that single-use components and systems are manufactured to defined quality standards. The keys to the successful implementation of a secure supply chain for single-use disposable technologies may be accomplished by establishing contracts, qualification metrics, agreeable audit criteria, and robust supplier quality and change-control programs.   

A crucial component in securing the supply chain is to establish a contract between single-use component suppliers and the end users. The contract should be explicit, clearly identifying terms, costs, service level agreements, and quality metrics that the end user expects the supplier to meet. The contract, for example, should define metrics arrived at by both parties that may be used to audit the manufacturing and delivery of single-use components to the end user. 

With established metrics, both parties should address audit scope and frequencies that are mutually amenable. The measures or corrective actions that are acceptable to both parties should be anticipated for common failure modes to ensure that neither party overreacts and over commits to resolutions that will have a detrimental effect on the relationship. Consideration should also be given to define dispute arbitration and resolution pathways.   

Single-use component and system manufacturers as well as suppliers are also well served to implement risk-based quality control and QMS programs to facilitate internal and external audit support and responses. Manufacturers specifically can leverage risk assessment to identify key critical process parameters (CPP) for control of single-use systems (SUS). It is imperative to establish a good working relationship between the supply chain and the end users. In effect, the contract supplier becomes an extension of the company's business model. Success or failure is dependent on this relationship. 

 depicts a product build map for the single-use assembly that can be used as a guideline during the risk-assessment phase. This map provides a framework to assist in the risk analysis of the supplier's product. Each leg or arm provides checkpoints in the manufacture process to evaluate CPPs. By following a risk-assessment process, safeguards can be established and implemented at various steps to mitigate risk. Appropriate risk-mitigation tools can be used throughout the assessment activity, for example failure modes effects analysis (FMEA) spreadsheets or fault tree analysis (FTA) flowcharts (refer to ICH Q9 

 available tools) as a way of identifying crucial issues with the manufacturing process of a single-use product.  

Figure 1: Single-use systems, process build map.			

As with many components used in the manufacture of therapeutics, a lifecycle management approach may also be adopted for single-use systems. The lifecycle management approach delineated in the ICH Q10 guideline discusses the application of various quality tools to the development, technology transfer, manufacturing, and discontinuation phases of a products lifecycle. This process is illustrated graphically in 

Figure 2: Pharmaceutical lifecycle management.			

The lifecycle management process is an iterative process with programs designed to capture changes to the product through both management review of performance data and through the quality system in which product quality is maintained through a corrective and preventive action program (CAPA). Manufacturers and suppliers should take into account the establishment of testing parameters, product transfer techniques, production parameters, and controls for GMP of sub assemblies. QMS/CAPA control schemes should be in place to review and control process. Storage and shelf-life management systems should be set up. A discontinuation program and obsolescence programming (supplying until an effective date) should also be considered. When vetting suppliers and manufacturers, these points will aid in the selection of the right source for the procurement of such critical-to-quality components.  

It is imperative that the framework of manufacturing control parameters be established early in the contract development process. Sourcing should be done from approved suppliers. Quality change-management systems should be established with the supplier so that any changes arising from supplier process improvements are documented and approved early in the change process. Setting these controls in place during contract establishment will facilitate a smooth approach to manage improvements to the supply process. Mitigating risk is to be viewed as the primary driver.  

Once a supplier has been established as a qualified supplier, defined by the supply change-management process, routine scheduled inspections of the supplier and subsuppliers will be required. Performance of these inspections and review of control parameters that have been established in the master contracts are important to reduce likelihood of out-of-specification components being produced. 

Once the single-use supply chain has been established, it is crucial to assess what qualification activities are needed to implement a single-use system. While it is common knowledge that leachable-and-extractables testing is a necessary part of a single-use component program, it's important to realize that leachable-and-extractables testing can be costly to perform. However, testing may be reduced depending on the overall risk to the product for each single-use item.  According to Destry M. Sillivan of FDA, manufacturers should "submit sufficient information to provide evidence that the product contacting material does not introduce contaminants into the product so as to alter the safety, identity, strength, etc." (5). If there is little to no relevant risk associated with the material in question, "vendor data can be cross referenced and a detailed justification for the applicability of these data and a justification for no additional testing should be submitted" (5).  

When assessing what qualification activities are needed for single-use component implementation, it is important to identify the product contact components that may present a higher leachable and extractable risk. A risk assessment is a valuable tool to determine what the crucial single-use materials are in a process. Those that surpass the tolerable amount of risk for an organization may then be slated for acquiring additional vendor information and further in-house testing. Multiple factors should be considered as part of the risk assessment including the nature of final product, how the process stream contacts the single-use component, the single-use component material properties, and the attributes of the process solvents. Once appropriate risk factors have been identified, a risk assessment should then be used to determine the extent of the qualification required for each component. Tools for performing a risk assessment as well as the general risk-based philosophy are outlined in ICH, Q9 

It is important to recognize that a single-use item is often composed of many component parts that may or may not be made out of the same materials. Vendor documentation should be closely inspected to determine the material of construction of each component. Not all of these component parts may be manufactured at the vendor's facility. A primary reason for this is that many vendors outsource different components that comprise their single-use assemblies. Accordingly, subvendors must be contacted to obtain their validation packages for the individual parts to complete the risk assessment.  

Consider, for example, a single-use bag system; there are the bag films, the connectors, the tubing, the capsule filters, and sampling connections as shown in 

As noted above, each of these items may be made out of different materials that all need to be considered for potential risk to product. 

Figure 3: Single-use storage bag example risk assessment.			

This example of a risk assessment performed on a storage bag illustrates how different components of a single-use bag can have different risk scores. The storage bag itself is rated at a higher risk relative to the rest of the component parts because it has a larger surface-area-to-volume ratio and the film is in contact with the storage material for an extended period of time. Where this bag is used in the production process will determine the overall outcome of the risk assessment. If the storage bag was used in upstream process the risk is low; however, if the storage bag was used in late stages of the process it becomes a much higher risk and would require leachable testing. 

What the drug product is intended to treat, the intended route of administration, and where the single-use item is used within the process stream are the leading risk factors. The longer the product is in contact with the single-use item, the greater the risk that leachables will be transferred into the process stream. When considering this risk factor, it is important to recognize that the tubing on a component part may only see the process stream for a fraction of the time that a bag may see. This and the varying materials of construction are the reasons that component parts of each assembly should be considered separately. The proximity of the single-use item to final fill will increase the risk because there is less opportunity for the leachable (or particulate) to be mechanically or chemically removed via filtration or chromatography.  The volume of product stream when compared with the surface area of the single-use item is also an important risk factor because the lower the volume is, the more potentially leaching surface area that it may contact. Consider the product contact area of a ultrafiltration/diafiltration (UF/DF) filter; the exceptionally high surface area as well as its proximity to the end of the process makes it a much higher risk to the product than an upstream storage bag.  

The composition of the process stream will also contribute to the overall risk of leachables and extractables occurring. Because plastics are not typically compatible with all chemicals, the process stream extraction risks can be higher depending on the solvent and chemical concentration of specific components.  First and foremost, the chemical compatibility of each of the single-use components must be understood to ensure that the process stream does not contain chemicals that will adversely affect the disposable item. Additionally, as the pH moves away from neutral to either alkaline or acidic, the risk increases for leachables to occur. For example, a 1N NaOH solution will pose higher risk than one of 0.1N.  

In addition to concentration and material incompatibility, the temperature of the process stream can affect the risk of leachables and extractables being transferred into the product. Typically, the extractables qualification tests include elevated temperatures because, as temperature increases, the extraction risk increases as well. Conversely, lower process temperatures provide the least risk of this occurring. Additionally, the single-use item may be exposed to heat during the serilization process. If the item is heat-sterilized, there may be a greater risk of transferring extractables to the process stream.  

How the drug product will be used will also play into the overall risk of leachables and extractables. Although typically how the drug product is used cannot be adjusted to reduce the risk, the qualification activities may be increased to ensure that the single-use item is safe to use for the process. The risk assessment should consider what the final dosage will be and how often the patient will receive the drug. The more of the drug product the patient receives, the greater the exposure to potential leachables and extractables; therefore, the risk is greater. Route of administration needs to be considered. If the product is an injectable then the risk is much higher than if the product is a topical agent. 

What is already known about the single-use item should be used in the risk assessment. Vendor-supplied data should be analyzed for the material safety risks. Material safety risks serve to document what is currently known about the materials of construction. Again, the risk assessment needs to ensure that all component parts on the single-use item are accounted for within the vendor qualification package. Toxicity and/or biocompatibility (USP class VI classified) testing should have already been performed and documented. All components should be Animal Derived Component Free (ADCF) or EMEA 410/01 compliant. A great advantage to the risk assessment for implementing single-use items is that vendors will have their own extractable studies that can be referenced. A toxicologist should review the extracted substances to determine if they are acceptable. The conditions under which the extractable tests were performed should be observed; the manufacturing process might not get anywhere near these conditions but keep in mind that these tests are performed at worst-case conditions to see what can be extracted. Another thing to look for in the vendor qualification package is to see if the plastics are certified under 

) 21. 177. Although this certification is for food contact surfaces, it can reduce the overall risk because the plastics were derived from a more controlled process. A summary of key risk factors is illustrated in 

Table I: Key risk factors for single-use components.			

Qualification activities maybe reduced depending on risk to the process/product. Each qualification activity should be looked at individually with respect to how the single-use item will be used in the process and how high it scored on the risk assessment.  Not every single-use item will need to undergo leachable testing if the risk score is low enough, which saves time and capital for the single-use item implementation. Keep in mind that if one component on the single-use assembly scored higher than the other component parts, it may be possible to have the suggested qualification activities performed on just that part and not the entirety of the assembly. 

Single-use technologies will continue to be adopted over the next decade within the biopharmaceutical industry in an ever-expanding capacity (4). Establishing controls and processes that follow an ICH Q10 risk-based approach is one suggested methodology to help establish best practices and controls for introduction and security of these single-use technologies.  

With the movement of the industry towards single-use components, many companies have started to address the impact of single-use equipment on product quality. The use of risk analysis is a powerful tool for implementing a single-use program.  

 is Sr. Director of Technology, all at Hyde Engineering + Consulting, Inc, Boulder, CO. 

 1. BioSolve Software Modeling, BioPharm Services, Chesham, Buckingham, HP5 1DG, UK, 

 2. Intelligen Software Services, Intelligen, Scotch Plains, NJ 07076 USA, 

 4. Single Use Bioreactors for Pharma; World Market 2012-2022, Vision Gain 

The authors suggest techniques for mitigating risk and securing the supply chain for single-use components used in biopharmaceutical manufacturing.

BioPharm International-04-02-2013

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The Overlooked Benefits of Single-Use Technology: Improved Quality and Increased Patient Safety

Ten Years Later: Innovation Driving Single-Use Technology Advances

Securing and Qualifying Single-Use Technologies

When the Process Becomes the Product: Single-Use Technology and the Next Biomanufacturing Paradigm