Live Interview from the 2008 BIO International Convention

 The adoption and acceptance of disposable biorocessing equipment today seems to be growing. What approach are most companies taking? Are they mostly using disposables in new processes or are they integrating them into existing processes?

 I would say it's really a combination of the two. Historically, with capital already invested into facilities, changing out 100% doesn't make sense. You can use those stainless-steel systems that are in place and integrate specific modules of disposable technology. Steps such as media and buffer addition are easy to implement fairly quickly. Seeding bioreactors are typically what we see integrated into an existing stainless-steel process. So it's really a combination of the two.  

Are the considerations very different depending on whether you are developing a new manufacturing process or integrating disposables into an existing process?

There are a couple of areas that have to be considered. One in particular is that if you have got an existing system in place that is constructed of stainless steel, most likely that will require steam sterilization, so some of your connector technologies to integrate will have to be different. It will have to be able to take that pressure, steam, and heat that it would be subjected to. So some of the connectology will have to be different as well as the design of construction in general. 

 What do you think is the biggest hurdle that most companies face when considering a transition to disposables?

 I think one of the most challenging points is just the approach to validation because it is different than stainless steel. For stainless steel you have got steam sterilization, which most companies are familiar with. And with single-use technologies, typically what you would see is gamma irradiation, something that suppliers are taking responsibility for. So the validation approach is different. Another topic would be extractables and leachables. That's more of a concern with the plastic technologies that are being used in single-use versus stainless-steel systems.  

 Is standardization an issue in terms of material for construction or things like the size compatibility of connectors?

Well, I would say that in terms of materials of construction, historically, if you look at filters, mostly the same base materials are present in a variety of different configurations. The films of bags tend to mostly be of a similar construction also. So there is some more standardization that I have seen over the years. Connectors are one that, again it depends on that duty cycle; if it is going to be steam sterilized, your construction materials will be different than if you are doing irradiation. 

How are biopharmaceutical manufacturers handling the disposal of disposables? Has anyone measured the environmental impact at all?

There have been some presentations that I have seen recently at the IBC conference a couple of weeks ago, where there was a presentation from, I believe it was Genentech, that spoke about this issue. So there are some companies that are evaluating it. There are also some guidance documents that the Bioprocess Systems Alliance (BPSA) has provided to give some general guidelines on how customers can consider disposing of those components. I think it's an issue that will come up more and more, but you have to also weigh that with the energy consumption for producing WFI and also the water usage. So really the balance is how does the disposal question fit into the benefits you can receive, the savings on energy consumption. 

 How are the US and European regulatory authorities responding to disposables?

Well, you tend to see more and more adoption of the technology, so they are becoming more familiar with it. The main concerns usually are the approach toward extractables and leachables. More recently, BPSA had a group meeting with the FDA to help the education from the suppliers' perspective on guidelines for extractables and leachables testing that suppliers typically do today. So I think it's a matter of time and exposure, and I have not heard of any issues where people are saying this is not a technology we should move forward with.  

Are there any best practices that companies can follow for transitioning to disposables?

Definitely there are more and more that we are seeing in the industry and BPSA is a group that does promote the adoption of the technology, and the supplier base has come together to put together guidance documents and you can see those on our web site. It is a great forum to help with that process. 

Do you think it is feasible to have a fully disposable manufacturing process, and if so, how soon do you think it will happen?

Well I think that really depends on the scale. Today, at a small scale you can have a fully disposable process. At the larger scale, at a 20,000-L bioreactor process, right now that's not really feasible. Over time, perhaps, we will get there but it's really going to be driven by the customer base and what their requirements are and if it is a value for them going forward. 

is the director of marketing at Charter Medical Ltd. 

Articles

Transitioning from stainless steel technology to disposable technology in bioprocessing.

Disposables for Bioprocessing: Making the Transition

The choice of single-use versus stainless-steel systems depends on a variety of process and other parameters such as bioreactor scale, product titer, and product changeover frequency. Computer-aided process design and simulation tools facilitate analysis and evaluation of process alternatives and assist scientists and engineers in their decision-making process. This article describes the steps required to build a comprehensive model in a batch process simulator for a process that uses single-use systems for buffer preparation and storage. The process is subsequently compared to a traditional method using stainless-steel tanks. The impact of single-use systems on production costs, demand for cleaning materials and consumables, and the cycle time of the process is thoroughly evaluated. 

As the number of biopharmaceutical molecules entering clinical trials is rising, there is an increased demand for technologies that can expedite the commercialization process. Disposables or single-use systems constitute such an enabling technology. They are commonly used for inoculum expansion using Wave rocking bioreactors that are available for working volumes of up to 500 L.

  More recently, stirred-tank disposable bioreactors have become available with working volumes of 1,000 L and 2,000 L, aimed at replacing small- to medium-scale stainless-steel bioreactors.

  Preparation and storage of cell-culture media and product purification buffers in disposable bags is another common application.

  The use of disposable bags greatly reduces the need for piping, clean-in-place (CIP) and steam-in-place (SIP) infrastructure, and the consumption of cleaning materials.

  This reduces the requirements for upfront capital investment and speeds up the commercialization process. These attributes of single-use systems make them particularly attractive to start-up companies that are short on capital and are under pressure to meet development milestones.  

Single-use systems, however, result in increased cost of consumables and their application ceases to be advantageous beyond a certain scale of production. Detecting the turning-point scale is a challenging task that depends on process and other parameters. Process simulation and other modeling tools can play an important role in this task by facilitating the analysis and evaluation of alternatives at various scales. The focus of this article is on the role of such tools in the evaluation of process alternatives and, in particular, the evaluation and comparison of single-use versus the traditional stainless-steel systems. 

The evaluation is done for a typical monoclonal antibody (MAb) facility at clinical manufacturing scale. Two process alternatives are evaluated in detail. In the first option, production buffers and media are prepared and stored in traditional stainless-steel tanks. In the second option, buffers and media are prepared and stored in single-use bags. 

Process simulation tools can assist in the evaluation of process alternatives in all the stages of process development and product commercialization by facilitating the following and other related tasks:

calculation of material and energy balances 

The cost analysis and resource tracking capabilities of such tools are predominantly used in this case study. Capital and operating costs are used to compare the two alternatives at various production scales. The impact of single-use systems on the demand for cleaning materials, CIP skids, labor, and utilities are also considered.  

Computer-aided process design and simulation tools have been used in the chemical and petrochemical industries since the early 1960s. Simulators for these industries have been designed to model continuous processes and their transient behavior for process-control purposes. Most biopharmaceutical products, however, are manufactured in batch and semi-continuous mode. Such processes are best modeled with batch-process simulators that account for time dependency and sequencing of events. In the mid 1990s, Aspen Technology (Cambridge, MA) introduced Batch Plus, a recipe-driven simulator that targeted batch pharmaceutical processes. Around the same time, Intelligen, Inc. (Scotch Plains, NJ) introduced SuperPro Designer. The initial focus of SuperPro Designer was on bioprocessing. Over the years, its scope has been extended to include modeling of small-molecule active pharmaceutical ingredient (API) and secondary pharmaceutical manufacturing processes. 

Discrete-event simulators have also found applications in the pharmaceutical industry, especially in the modeling of secondary pharmaceutical manufacturing processes. Established tools of this type include ProModel from ProModel Corporation (Orem, UT), Arena and Witness from Rockwell Automation, Inc. (Milwaukee, WI), and Extend from Imagine That, Inc. (San Jose, CA). The focus of models developed with such tools is usually on the minute-by-minute time-dependency of events and on animations of the process. Material balances, equipment sizing, and cost analysis tasks are usually out of the scope of such models. Some of these tools are customizable and third-party companies occasionally use them as platforms to create industry-specific modules. For instance, BioPharm Services, Ltd. (Bucks, UK), has created an Extend-based module with emphasis on biopharmaceutical processes.  

Microsoft Excel is another common platform for creating models for pharmaceutical processes that focus on material balances, equipment sizing, and cost analysis. Some companies have even developed Excel applications that capture the time-dependency of batch processes. This is typically done by writing extensive code (in the form of macros and subroutines) in visual basic for applications (VBA) that comes with Excel. The K-TOPS tool from Biokinetics, Inc. (Philadelphia, PA), belongs to this category. 

Building a model in a batch-process simulator

The article uses SuperPro Designer to illustrate the modeling and evaluation of MAb manufacturing process alternatives. The first step is to create a flow diagram of the overall process (Figure 1). The various equipment-shaped icons, called unit procedures, represent the processing steps required for making a batch of a certain product. The lines that connect the unit procedures represent material transfers. Batch process simulators usually come with a library of unit procedures. A unit procedure represents a set of activities or operations that are carried out in a piece of equipment during a processing step. A unit procedure may include any number of operations. The fermentation procedure (P-11) of Figure 1 includes the following operations: SIP-1, SET UP, TRANSFER-IN-1, TRANSFER-IN-2, FERMENT-1, TRANSFER-OUT-1, and CIP-1. The combination of unit procedures and operations enables the user to represent and model the various activities of batch-processing steps in detail.  

For every operation of a unit procedure, the simulator includes a mathematical model that performs material and energy balancing, and equipment-sizing calculations. If multiple operations within a unit procedure dictate different sizes for the equipment, the program reconciles the different demands and selects an equipment size that is appropriate for all operations. If the equipment size is specified by the user, the simulator checks to make sure that the vessel is not overfilled. In addition, the tool checks to ensure that the vessel contents will not fall below a user-specified minimum volume (e.g., a minimum stir volume) for applicable operations. 

In terms of cost analysis, simulation tools facilitate the process of estimating capital as well as operating costs. Some tools are equipped with built-in functions and databases for estimating equipment cost as a function of size, material of construction, operating pressure, and other parameters.

  The tools also may include databases for materials (pure components and mixtures), utilities, consumables, and other resources. The size and unit cost of single-use systems is stored in the consumables database. The user associates consumables with a processing step and the tool calculates the number of units and the cost.  

Because biopharmaceutical processes operate in batch mode, the simulator also must facilitate process scheduling and cycle-time analysis. The results of process scheduling are typically visualized with Gantt charts that display equipment occupancy as a function of time (Figure 2).  

Sensitivity and parametric analysis are other common benefits of using such tools. Input parameters can be varied manually or automatically and their impact can be evaluated quickly. If statistical data are available for certain input parameters, their impact on output (decision) variables can be evaluated using Monte Carlo simulation.

It is important to note, however, that the garbage-in, garbage-out (GIGO) principle applies to all computer models. If critical assumptions and input data are incorrect, the simulation outcome will also be incorrect. Consequently, a certain level of model validation is necessary. In its simplest form, a review of the results by an experienced engineer can play the role of validation. 

Figure 1 displays the flow diagram of the overall process (the way it is represented in SuperPro Designer). Please note that for simplicity, the buffer preparation and holding activities are omitted from Figure 1. Those activities, however, were taken into account in the detailed models that were built for evaluating the alternatives. The simulation steps are represented by codes that appear in parentheses (e.g., MP-103) throughout the article. 

The inoculum is initially prepared in 225- mL T-flasks. The material is first moved to roller bottles (2.2 L), then to 20 L, and subsequently to 100-L disposable-bag (Wave rocking) bioreactors. The broth is then moved to a stirred-tank seed bioreactor. The media solution for the seed bioreactor (165 L per batch) is prepared in a tank (MP-101) and then sterilized and fed to the reactor through a 0.2-μm dead-end filter (DE-101).  

Serum-free, low-protein media powder is dissolved in water for injection (WFI) in a stainless-steel tank (MP-103). A diluted medium (3%) of 1,628 L is prepared per batch. The solution is sterilized using a 0.2-μm dead-end polishing filter (DE-103). A stirred-tank bioreactor (PBR1) is used to grow the cells that produce the therapeutic MAb. The production bioreactor operates under a fed-batch mode. High media concentrations are inhibitory to the cells so half of the media is added at the start of the process and the rest is fed at a constant rate during fermentation. The concentration of media powder in the initial feed solution is 17 g/L whereas the concentration of the medium added during the fed-batch phase is 156 g/L. The fermentation time is 12 days. The volume of broth generated per bioreactor batch is approximately 2,000 L, containing roughly 4 kg of product. The product titer is approximately 2 g/L. 

The generated biomass and other suspended compounds are removed using a disc-stack centrifuge (DS-101). During this step, roughly 2% of the product is lost in the solid-waste stream. The bulk of the contaminant proteins are removed using a 62-L protein-A affinity chromatography column (C-101) that operates in four cycles. The product yield for this step is 90%. The protein solution is then concentrated 5x and diafiltered 2x (in P-21/DF-101). The total membrane filtration area for the diafilter is 2.6 m

. The product yield is 97%. The concentrated protein solution is then chemically treated for 1.5 h with polysorbate 80 to inactivate viruses (in P-22/V-111). An ion-exchange (IEX) chromatography step follows (P-24\ C-102) with a product yield of 90%. The IEX column has a volume of 28 L and the batch is processed in three cycles. Ammonium sulfate is then added to the IEX eluate (in P-25\V-109) to increase the ionic strength for the hydrophobic interaction chromatography (HIC) step (P-26\C-103) that follows. The recovery yield of the HIC step is 90%. The HIC column has a volume of 25 L and it processes a batch in three cycles. A viral exclusion step (DE-105) follows. This is a dead-end type of filter with a pore size of 0.02 μm. Finally, the HIC elution buffer is exchanged for the product bulk storage buffer (PBS) and concentrated 1.5-fold in DF-102. Approximately 100 L of the final protein solution is stored in a 200-L disposable storage bag (DCS-101). The amount of purified product produced per batch is 2.5 kg. The overall yield of the downstream operations is approximately 63%. 

Several buffers and media are required at different quantities. These have to be prepared and transferred to the use points in time for processing. Two alternatives for the buffer and media preparation activities are investigated.  

The first option uses single-use preparation and storage systems. Media are prepared in 200-L single-use bags and transported to the use point. The various buffers are prepared in 500-L and 1,000-L bags (using a skid) and then transferred through sterile filters into 200-L storage bags. The 200-L bags are subsequently moved to the point of use. Specifying single-use bags in SuperPro Designer is a simple process. The first step involves specifying the type of bag, capacity, purchase, and disposal costs and other properties in the consumables database. The bag can then be allocated to a unit procedure that represents buffer preparation or storage. The tool calculates the number of bags required per batch and campaign during simulation. Other consumables, such as chromatography resins and membrane cartridges, are specified and calculated in a similar manner. In this case study, the three types of bags used have working volumes of 200 L, 500 L, and 1,000 L and their assumed purchase costs are $300, $400, and $570 per item, respectively. 

The second option uses traditional stainless-steel tanks for buffer and media preparation and holding. Media are prepared and fed using a single tank (dedicated) per bioreactor. The buffer preparation area includes a number of preparation tanks connected to a group of holding tanks using a set of transfer panels. The holding tanks are connected with the main process using buffer delivery lines. The amount and number of buffers that need to be prepared determines the size and the number of the tanks. Table 1 displays the required tanks and their sizes. Three tanks are required for media preparation (MP-101, 102, and 103); five tanks are required for buffer preparation (PV-101, 102, 103, 104, and 105) and eight tanks are required for buffer holding (HV-101 to HV-108). Because the stainless-steel tanks are reused, CIP is essential after every buffer preparation batch.  

Table 1. Media and buffer prep and holding tanks for the stainless-steel option 

The fermentation time is 12 days; the facility is equipped with four production bioreactors resulting in a cycle time of 3.5 days. Eighty batches can be executed per year (20 per bioreactor). The product titer in the production bioreactors is 2 g/L. With a broth volume of approximately 2,000 L and a downstream yield of 62.5%, the amount of purified MAb produced per batch is 2.5 kg. 

Figure 2 displays the equipment occupancy chart for 13 consecutive batches of the 2,000-L stainless-steel process option. The activities (unit procedures) of each batch are displayed with a unique color. The various equipment groups are displayed on the chart. The occupancy of CIP skids is represented by the top six lines. The next two equipment groups correspond to the occupancy of the buffer preparation and holding tanks, respectively. Most of these tanks are reused within a batch. Reuse of a tank (e.g., HV-101) within a batch is represented by multiple rectangles of the same color (one rectangle for each unit procedure using that equipment). Reuse of tanks (for preparing and holding different buffers within a batch) reduces the number of vessels and consequently the capital investment for a new facility. However, it also increases their occupancy and cycle times making them scheduling bottlenecks more likely.  

The next equipment group corresponds to three seed (SBR1a, b, and c) and four production bioreactors (PBR1a, b, c, and d). Both the seed and production bioreactors operate in staggered mode (out of phase) to reduce the cycle time of the overall process to 3.5 days. A single downstream line (DSP group) handles all purification batches.  

The equipment occupancy chart enables users to visualize equipment utilization and readily identify equipment scheduling bottlenecks that determine the cycle time of the overall process. The production bioreactors (PBR1a, b, c, and d) have the longest cycle time and constitute the scheduling (or cycle time) bottlenecks for the base case. However, if the number of production bioreactors is increased, then the bottleneck will shift to the buffer preparation and holding tanks that have the next highest utilization. Scheduling bottlenecks linked to buffer preparation equipment is not a consideration for the disposables option unless the buffer preparation bag skids are reused.  

This article describes the steps required to build a comprehensive model in a batch process simulator for a process that uses single-use systems for buffer preparation and storage.

Systematic Evaluation of Single-Use Systems Using Process Simulation Tools

We have compared the environmental footprint of a traditional biopharmaceutical manufacturing facility using fixed-in-place stainless-steel equipment, and a facility implementing disposable technologies for cell culture, solution mixing and hold, product hold, and liquid transfer. We accounted for facility size, water consumption, energy use, and carbon emissions from all steps, including even steel manufacture, transporting plastics to and from the facility, plastic incineration, and employees driving to work. 

It is becoming increasingly common for individuals and organizations to calculate and publish their environmental footprints, both to understand the impact and to attempt to reduce it. The term 

 is now ubiquitous and colloquial. The less common 

 extends beyond carbon to include the usage of water and land associated with how individuals live or organizations operate. This paper will address such environmental footprints in the manufacture of standard monoclonal antibodies (MAbs) used as drug therapies.  

Traditional facilities with fixed-in-place stainless-steel fermenters, tanks, and associated piping and valves are still the prevalent manufacturing methodology to produce biological drug therapies. Many vendors, consultants, and drug companies are endeavoring to replace such traditional facilities with single-use systems to improve flexibility, cost, and also environmental footprint. It may seem paradoxical to claim that a single-use system can have a smaller environmental footprint than a traditional multiuse facility; however, the requirements for sanitization and cleanliness in biological drug manufacture place an extreme environmental burden on the multiuse traditional facility. Sanitization and cleaning is chemical, water, and energy intensive.  

Single-use systems do not require such intensive sanitization efforts; their environmental footprints are more a result of their plastic content. Plastics are essentially paraffin-like chemicals with repeating chains of CH

 molecules. Given this chemical nature, plastics are indeed fuels. The preferred disposal method for such fuels is incineration, with or without energy recapture.  

We have analyzed the cradle-to-grave carbon, water, and land footprints, calculated per single batch of a standard MAb, for a traditional fixed-in-place stainless-steel facility and a facility that relies on single-use equipment that is disposed of by simple incineration of all its plastic material. We have included the data from mining the iron ore through the diesel consumed in transporting plastic to the drug manufacturing facility as well as the transportation of waste plastic to the incineration facility, and the incineration of the plastic. 

We have seen a rapid uptake of disposable technologies in the biopharmaceutical industry. Much of this growth has been in the last five years, although disposables have been around much longer in hospital settings, where they are used extensively. This increasing interest in disposable technologies has naturally been followed by a growing concern about the solid plastic waste generated from their use. This has been an area of interest of the authors who, early in the development and application of disposables, have tried to assess the impact of disposables at the facility level.

The most common methods for disposing of single-use plastics are landfill and incineration, with incineration being more popular. In some cases, incineration can also result in significant energy savings through cogeneration techniques,

  in which a facility captures the energy generated from burning its waste and uses it to produce heat or electricity.  

The possibility of recycling has been assessed by some suppliers, but recycling opportunities are extremely limited, primarily because of the multilayer films of which disposable bag systems are made. Disposable bag films often combine polyethylene, polypropylene, ethylene vinyl acetate, and nylon. It would be more feasible to recycle the silicone tubing that is often attached to bag systems, but that would require segregating tubing and bag, the logistics of which are not simple. In addition, recycling may require pretreatment of biohazardous single-use materials, depending on the process step for which the disposable equipment was used. The combination of these factors makes it extremely difficult to develop an economically viable case for recycling disposable technologies in their current configuration. In a recent article, however, Millipore's Director of Sustainability David Newman suggested that homogenous or separable materials (in disposable filters, for example) could increase the feasibility of recycling.

  The environmental impact of disposables could also be reduced by packaging components in bulk, instead of individually, to decrease the amount of packaging.  

Taking into account the current state of disposable technology implementation and the disposal methods described above, we have compared the environmental impact of a commercial MAb process, working with either stainless steel or disposable technologies, looking specifically at the relative environmental footprints of both facilities. 

The environmental impact of the traditional stainless steel–equipped and disposables-based facilities has been evaluated using a model based on a commercial MAb process at 3 x 2,000 L scale.

  The main difference between the two manufacturing options is that one uses stainless-steel equipment and the other implemented disposable bags, tubing, and associated components for:  

The stainless-steel equipment requires cleaning after each use. The buffer vessels are cleaned using a simple rinse cycle. The cell culture bioreactors, product, and media vessels are cleaned using a more rigorous cleaning cycle, which includes a purified water (PW) rinse, caustic clean, acid clean, another PW rinse, and a final rinse with water for injection (WFI). Bioreactors and vessels that undergo a full cleaning cycle also require sanitization with steam.  

In the disposables-based facility, single-use bag systems are used to prepare and store media, buffers, and products before further processing. The bag systems are provided presterilized, ready for process use, and are not used again; hence, no cleaning operations are required. 

In this study, we have focused mainly on the process and the key areas that are affected by choosing disposable equipment, such as facility size, utilities, consumables, and labor requirements. It was not our intention to evaluate materials that are common to both operations. In addition, we have not examined the heating and cooling loads associated with  heating, ventilation, and air conditioning (HVAC) systems, instead restricting our HVAC data to the loads needed to run fans. In this way, we eliminated regional factors resulting from climate differences, although this will be to the detriment of the disposables facility. The analysis has not taken into account general cleaning, clothing wash up activities, or the disposal of single-use garments. The single-use plant should have a smaller footprint in these areas because there are fewer employees. The study, therefore, only considers 

 in the use of consumables (e.g., disposable bags); we did not account for disposables that are assumed to be the same in both facilities. These include small-scale cell culture equipment (e.g., single-use flasks, filters, and pipette tips); filters (e.g., liquid sterile filters, depth filters, UF–DF cartridges); and other common disposable items (e.g., gloves, isopropyl alcohol wipes for cleaning, weigh boats).  

Table 1. Key parameters for comparing the environmental impact of disposables-based and stainless steelâbased facilities 

Table 1 provides a description of the key comparison parameters. The resulting data sets from the model are then used to evaluate the carbon footprint. The basis for the carbon footprint estimation is summarized in Table 2. The analysis will also take into account differences in carbon emissions that could result from different electricity sources, depending on whether the electricity was generated from: (1) firing coal, (2) firing natural gas in a combined cycle, or (3) the average mix of US sources (coal, gas, hydroelectric, nuclear, other).  

Table 2. Bases for estimating energy consumption and carbon emissions 

The analysis in this section identifies distinct differences (i.e., in utility requirements, materials, consumables, labor, space, electricity, steelwork, carbon) in the performance of the disposables-based facility when compared to the conventional stainless-steel plant. 

Figure 1 illustrates the water usage (PW and WFI) per batch for the two manufacturing options. Implementing disposable bag systems removed the use of stainless-steel bioreactors and product and solution vessels that require cleaning, thus reducing water usage. The model indicates that the disposables-based facility consumes 87% less water than the stainless steel–equipped facility.  

Given the reduced water requirements, smaller generators and storage vessels will be needed to operate the disposables-engineered facility. The WFI generator capacity required for the disposables-based facility is approximately 320 L/h, whereas the stainless steel–equipped facility requires a WFI capacity of 1,200 L/h to achieve the same production rate. Using a filling time of 10 h, storage vessels of 12,000 L and 4,000 L are needed in the stainless-steel facility and the disposables-based plant, respectively. 

Steam usage results mainly from autoclave activities and the sterilization of bioreactors and vessels. For the stainless steel–equipped facility, a substantial amount of steam (about 880 kg/batch) is needed to run sterilization operations. In the disposables option, the vessels are substituted by single-use bags, thus eliminating the need for clean steam.  

Table 3 summarizes the total amount of process materials and diluted cleaning solutions used per batch. As expected, the quantities used for process media and buffers are the same for both facilities. The single-use setup, however, has eliminated the need for chemicals (i.e., caustics and acids) associated with vessel cleaning operations because the bags are supplied presterilized and ready to use. The model estimates that the quantities of cleaning materials per batch are reduced by more than 95% in the disposables-based facility.  

Table 3. Comparison of material usage (L/batch) in stainless-steel and disposables-based plants 

The single-use nature of disposable technology has increased the volume of plastic waste. A total of about 880 kg of solid waste per batch is generated. The solid waste is normally incinerated, which may affect air quality, causing environmental concern, and may incur additional manufacturing cost.

  The exact cost will be linked to the disposal method used and whether pretreatment (decontamination autoclave, or kill with a dose of chlorine dioxide or other deactivator) is required before disposal. 

In the disposable case, because the demand to operate equipment cleaning and sterilization activities is significantly reduced, the labor required to support such tasks decreases. The disposables-engineered facility also may reduce labor requirements in other areas, such as quality. The set-up and turnaround time for disposable systems typically is shorter than for their stainless steel counterparts. All these factors result in a less labor-intensive facility. There is an overall 21% reduction in labor headcount in the disposables-based option compared to the traditional stainless steel–equipped facility. The bulk of the savings in labor headcount is derived from reduced clean-in-place (CIP) activities (Figure 2).  

The disposables-engineered facility simplifies hardware installation, design, and storage, leading to more efficient use of space and thus considerably reducing the square footage of the facility. Figure 3 illustrates the facility footprint in square meters for the various area classifications. Table 4 lists the amount of floor space needed in each area classification, based on the types of activities that take place in each. Because the two manufacturing options have the same inoculum preparation and downstream purification sequences, which take place in Class B and C areas, respectively, the floor spaces required for those two classes are the same in both instances. The savings in floor space are obtained from Classes D and U, where the cell culture, solution preparation operations, and utilities equipment are located, with the majority of the space savings coming from Class D. It can be seen that the facilities footprint of the disposables facility is reduced by 243.19 m

, or 38%, compared to the traditional facility.  

As a result of its smaller facility footprint, the disposables-based facility achieves process electrical savings of about 30%, indicating that such a plant is potentially more energy-efficient. The cubic feet per minute/kilowatt (cfm/kW) values for each class are listed in Table 4. Because the energy required to operate HVAC systems is directly proportional to the floor area, the electricity required to operate classes B and C is the same in the stainless-steel and disposables-based facilities. In the disposables-based facility, however, the total electricity consumed for HVAC operations is reduced by about 29%, which can be attributed to a smaller facility space required in classes D and U.  

Table 4.  HVAC energy consumption for each class of facility space. The disposables-based facility uses 38% less floor space in Class D and U areas, and as a result, the total energy consumption for HVAC is 29% lower in that setup. overall in the disposables facility. 

Can disposables reduce your facility's environmental footprint? We have compared the environmental footprint of a traditional biopharmaceutical manufacturing facility using fixed-in-place stainless steel equipment, and a facility implementing disposable technologies for cell culture, solution mixing and hold, product hold, and liquid transfer.

The Environmental Impact of Disposable Technologies

The decision to use single-use systems for manufacturing pharmaceuticals hinges on many factors, but the major driving force is a desire to save cost and time. Although there is no doubt that single-use systems reduce capital costs, this analysis shows that in many cases lifecycle costs increase. This article describes a methodology used during concept design to evaluate lifecycle costs of single-use systems. The methodology is implemented in a case study to determine the optimum mix of stainless-steel and single-use systems for a new biopharmaceutical manufacturing facility. Lifecycle costs, expressed as net present value (NPV), are evaluated for typical bioprocess applications including fermentation, recovery, purification, media and buffer prep, and buffer storage.

Single-use systems offer many advantages over conventional stainless-steel systems and have rightly gained wide acceptance in the biopharmaceutical industry. Advantages such as increases in batch success rate, eliminating potential cross contamination, more rapid changeover between campaigns, reductions in water and waste water requirements, and eliminating clean-in-place (CIP) and steam-in-place (SIP) validation have all been cited as reasons for using single-use systems. However, one of the primary drivers for implementing these systems continues to be the desire to reduce project cost and time.  

Although there is no doubt that using single-use systems reduces capital costs, this article shows that in many applications, capital savings are offset by increased operating costs. Therefore, lifecycle costs for many single-use applications are higher than for conventional stainless-steel systems. Lifecycle costs vary depending on how single-use systems are implemented, and also with the location of the facility. Capital, labor, and utility costs vary greatly between the United States, Europe, and Asia. A single-use application that reduces labor hours by 20% may make economic sense in one location but not in another. Similarly, capital savings associated with single-use systems will depend on the geographic location of the facility, and whether the equipment is sourced locally or is imported.  

Generally, less complicated single-use systems such as simple storage bags for media and buffer have more favorable lifecycle economics than single-use technologies designed for more complex applications. This is because the low replacement cost of single-use components compare favorably with the cost to maintain and clean stainless-steel equipment. On the other hand, lifecycle economics for more complicated single-use applications such as bioreactors and fermenters is less clear because of the high cost of the single-use components. 

In this article, we describe a quick method for evaluating lifecycle costs for single-use systems compared with their more conventional stainless-steel counterparts. Because the decision to use single-use systems needs to be made at the beginning of any project, our evaluation is done at a level of detail suitable for concept design. The methodology is then implemented in a case study to determine the optimum mix of stainless-steel and single-use systems for a new biopharmaceutical manufacturing facility. Lifecycle costs are evaluated for typical bioprocess applications including fermentation, recovery, purification, media and buffer preparation, and buffer storage. 

Disposables Versus Stainless-Steel Systems

Decisions to use single-use systems should be made early in a project, typically during conceptual engineering. Single-use systems have a large impact on the layout of a facility and also may affect automation strategies, clean utility requirements, floor-to-floor heights, project timelines, procurement schedules, and even area classifications like heating, ventilation, and air-conditioning (HVAC) design.  Facilities that use only single-use processing sometimes realize substantial advantages over conventional designs, but tend to be limited in scale.

 Most biopharmaceutical facilities use a mixture of stainless-steel and single-use systems and for these facilities, an analysis of lifecycle economics can help determine the optimum mix.  

Capital costs for single-use systems are always lower than for conventional stainless-steel systems, so evaluating lifecycle economics comes down to comparing differences in operating cost versus the cost of capital. In conceptual design, capital costs are estimated from equipment cost using the Lange factor, which is an empirical multiplier that accounts for the cost of installing the equipment. Lange factors vary depending on the type of equipment and whether it arrives as a preassembled skid or as individual equipment items that are assembled in the field. The factor accounts for all direct costs associated with equipment installation including setting up the equipment, utility and process piping hookups, and automation.  

Table 1. Capital savings for single-use buffer bags 

Table 1 shows how the Lange factor is used to estimate capital-cost savings for a simple scenario involving replacing multiple stainless-steel buffer hold vessels with single-use bags. By doing so, not only are we able to delete the stainless-steel buffer hold vessels, but also we are able to delete two CIP skids and their corresponding infrastructure. The single-use case still has some capital cost (for bag holders) but its Lange factor is much lower than for stainless-steel vessels, reflecting the bags' simpler installation. The total direct capital cost savings for this alternative are just over eight million dollars.  

Table 2a. Estimated facility and HVAC costs (USD) 

Often, the use of single-use systems affects either the area required in a facility or the cleanliness classification of that area.  For the above alternative, using single-use bags eliminated 1,100 square feet of grade D space, which originally contained the stainless-steel buffer hold vessels but added back 1,075 square feet of controlled not classified space for storing single-use bags. These changes affect both capital and operating costs that are quickly estimated from rough order of magnitude benchmarks in Tables 2a and 2b. Experience suggests each added square foot of cleanroom space also contributes additional mechanical space, which is included in Table 2b. Capital and HVAC costs are calculated for a base location and discounted 25% to adjust for the local market. For this alternative, savings in facility costs are a relatively small ($58,000) with an annual savings in HVAC costs of $791.  

Table 2b. Difference in facility space costs as a result of implementation of single-use systems. Only ISO 9 (Grade D) and controlled not classified space are affected, along with space needed in mechanical areas as a result of decreased HVAC needs by reducing cleanroom space. 

Comparing operating costs for stainless-steel and single-use systems is more complex as can be seen in Tables 3a, 3b, and 3c. Operating costs are calculated for the first 10 years of plant operation beginning in 2010. Operating costs such as water, personnel, and utilities should be escalated at a percentage rate that reflects local market economic conditions.  

Table 3a. Rates used to estimate operating cost savings for single-use systems: Items for which costs are assumed to increase over time. 

Plant capacity (number of fermentation batches) increases over the 10-year period, which is typical for many facilities. Plant capacity change over time is an important part of operating cost evaluation because capacity directly affects the cost of the single-use systems. Many operating costs for conventional stainless-steel systems also increase with increasing plant capacity (water, CIP chemicals) but some, such as the cost of routine maintenance, commissioning, validation, HVAC, and capital, do not.  

Table 3b. Rates and quantities used to estimate operating cost savings for single-use systems: Items which are assumed to be constant or which only affect the first year. 

Moreover, the cost of WFI used in 2010 is five cents per liter. In much of the published literature, the cost of WFI is taken to be anywhere from 50 cents to one dollar per liter. Our experience is that these numbers overestimate the cost of WFI from a bulk generation and distribution system and unfairly slant many operating cost evaluations in favor of single-use systems. Five cents per liter is an all-in cost that includes the cost of capital, maintenance, validation, quality assurance, utilities, and waste-water discharge. It can be argued that the incremental cost of WFI is even lower because the facility is likely to have a bulk WFI generation and distribution system anyway to provide water for process as well as CIP of any remaining stainless-steel equipment. In this article, however, the price of WFI covers the total capital and operating costs of the bulk system.  

Table 3c. Estimated operating cost savings for single-use systems 

Significant savings are projected in commissioning and qualification for this alternative, which is typical for single-use applications. We apply this as a one time savings in 2010 only. Experience is that ongoing validation costs for single-use and stainless-steel systems are similar. 

The simple scenario described above, using single-use bags for buffer hold, realizes significant labor savings as a result of the way the single-use storage bags are used. For the stainless-steel case, each buffer was prepared for a single purification lot. In the single-use case, buffer is prepared in larger batches and aliquoted into single-use bags for multiple purification lots. This reduces the number of buffer preps required per week with corresponding savings in personnel, QA, CIP, and dispensing. We take credit for savings only when we have reduced operating hours sufficiently to eliminate a full time employee (FTE). The thinking is that saving only a few hundred hours doesn't reduce operating costs but merely lightens work schedules. 

As Table 3c shows, operating cost savings for this scenario vary by year with a maximum savings of $383,000 in 2010 and a loss of $57,000 in 2013. The high savings in 2010 reflects the one time credit for savings in commissioning and qualification. For this alternative, savings increase as plant capacity increases, indicating the replacement cost of the single-use systems is less than the CIP and labor costs of the stainless-steel systems. 

Table 4 indicates how capital and operating costs are combined to calculate a lifecycle cost expressed as net present value (NPV). The NPV reflects the sum of cash flows for each of the 10 years studied; each of the cash flows is discounted and expressed in today's dollars. The combined capital savings from Tables 1 and 2b show up as a negative capital outlay (capital savings) of $8,122,000. Operating costs and savings from Tables 2b and 3c are calculated for each of the 10 years studied. Capital cost is straight-line depreciated over 10 years. A corporate tax rate of 10% is used to adjust incremental profit or loss and a discount rate of 10% is used to account for the cost of money. These factors vary widely between companies and locations, especially tax rates which often reflect incentives from local governments. The calculated NPV for this alternative is an impressive savings of eight million dollars. This indicates the use of single-use bags to supply buffer is a clear winner over the stainless-steel buffer storage tanks.  

Assessing Various Combinations of Stainless-Steel and Single-Use Equipment

For our case study, this methodology was used to evaluate lifecycle economics for other single-use alternatives as shown in Table 5. For each scenario, NPVs were calculated for three different capacity scenarios, including the base projection from Table 3c, a more conservative scenario involving fewer batches (downside), and a more optimistic scenario (upside).  

Table 5. Lifecycle cost analysis of single-use scenarios 

Scenario 1 examines replacing a seed train consisting of a 50-L and 250-L stainless-steel fermenter with a single 250-L single-use fermenter. In this scenario, the single-use fermenter would be inoculated initially at a 50-L volume and then topped off with additional media to produce 250-L of seed. It was reasoned that spreading the cost of the single-use fermenter bag over the two seed steps would produce more favorable economics than paying for both a 50-L and 250-L single-use fermenter bag. This illustrates an important point in evaluating single-use alternatives, which is that you don't always need to consider a like-for-like replacement. What makes sense in a stainless-steel world may not be the optimum design in a single-use world. Similarly, in the earlier buffer hold example, the favorable economics for single-use bags results mainly from a change in operating philosophy to produce buffer for multiple purification lots at the same time. This makes sense in a single-use world because it doesn't increase capital costs. In a stainless-steel world, costs increase with the size and number of storage tanks. 

As Table 5 indicates, Scenario 1 results in positive NPVs ranging from $320,000 at the lowest capacity to over $1.6 million for the upside scenario, suggesting this alternative be incorporated into our design. However, an examination of operating costs indicates the costs are higher for the single-use scenario for all 10 years. This suggests the savings derive from combining the two seed fermentations into a single fermenter, and not from using single-use systems. Based on this information we proposed Scenario 2, which combines the 50-L and 250-L seed fermenters into a single 250-L stainless-steel fermenter. This scenario produces lower less capital savings than scenario one but lifecycle savings are greater, making it the clear winner between the two scenarios.     

Scenario 3 examines replacing the stainless-steel production fermenter with a single-use fermenter. Lifecycle costs for this option are very poor and there is a large technical risk because single-use fermenters of the required size are not yet available on the market. The negative economics result from the high cost of the single-use fermenter bags, which are close to $10,000 each. 

Scenario 4 examines replacing three large stainless-steel vessels, one of them agitated, and depth filters in the recovery process with single-use systems. At this volume, the cost of single-use mixing bags is quite high, driving the economics to favor stainless-steel. In Scenario 8, we examine the same recovery process but replace just the hold vessels with single-use bags, keeping the stainless-steel agitated vessel. The economics for this option are favorable. 

Scenario 5 examines replacing fixed stainless-steel buffer and media-preparation tanks with single-use mixing systems. Here again, the optimum design for single-use systems is very different than for stainless steel. The stainless-steel design includes separate media and buffer preparation suites with separate gowning and material airlocks and separate air handlers. In the single-use design, we combine media and buffer preparation into a single suite because the risk of cross contamination is eliminated. We also reduce the number of preparation systems from seven to four, reflecting the higher productivity of the single-use mixing equipment. This resulted in significant savings in capital costs, operating personnel, and HVAC costs. However, these savings were dwarfed by the high cost of single-use mixing bags, resulting in negative NPVs for all capacity scenarios. 

Scenario 6 is the buffer hold case study described earlier, and Scenario 7 uses single-use bags to hold intermediate product after various purification unit operations. Both of these scenarios use relatively inexpensive single-use storage bags and result in positive lifecycle economics. 

Table 5 shows the importance of evaluating lifecycle costs in determining the optimum mix of stainless-steel and single-use systems. In our case study, if we look at capital savings only, we might decide to incorporate all of the single-use scenarios listed in Table 5. The result is a rough order of magnitude capital savings of $26 million.  However, the NPV for these scenarios is a loss of $7 to $13 million. A more optimum solution is to combine scenarios 2, 6, 7, and 8.  This results in a respectable capital savings of $18 million and positive lifecycle savings of $11 to just over $16 million. 

A final and very important financial consideration is the impact of single-use systems on project timeline. In many instances, the financial impact of getting to market faster dwarfs the lifecycle savings we have been discussing so far. The problem is that it is often very difficult to determine the impact of replacing a given piece of stainless-steel equipment with single-use systems on the overall project timeline. The reduced delivery time, installation, commissioning, and qualification of single-use systems shortens project timeline. However, the overall project timeline might still depend on long lead times for stainless-steel equipment for which there is no suitable single-use replacement. 

For projects where time-to-market is an important consideration, we recommend performing the analysis described in this article, then evaluating the alternatives with negative NPVs based on their potential to affect the overall project schedule. For example, alternative five shows a negative NPV for single-use media and buffer preparation. Typically, stainless-steel media and buffer preparation equipment is not on the critical project path and replacing it with single-use mixing bags is not likely to accelerate the overall project timeline. Therefore, alternative five might still be rejected. However, stainless-steel fermenters are often on the critical project path, so alternatives one and three might be reconsidered if speed to market were an important driver. 

Lifecycle costs are an important consideration in evaluating single-use systems against conventional stainless-steel designs. Generally, the less expensive single-use components provide more favorable economics. Simple storage bags for media, buffer, or intermediate product generally have positive economics when compared to stainless-steel vessels for the same purpose. The high replacement costs for more complex single-use systems, such as large mixing bags or bioreactors, tend to offset any savings that might be realized. However, the economic benefits of getting to market faster can dwarf lifecycle costs, and therefore, the impact of single-use systems on the project timeline needs to be considered where time to market is a significant driver.   

 is associate director of process engineering, Biotech Technology and Engineering (BTE), Wyeth Biotech, 978.247.4746, 

is a senior manager of technology at PharmaBio, Jacobs Engineering. 

1. Robinson J, Bader R. Economics of vaccine production using virus like particles in insect cell culture. Interphex. Philadelphia, PA; 2008 Mar 26–28.  

This article describes a quick method for evaluating lifecycle costs for single-use systems against their more conventional stainless-steel counterparts.

Lifecycle Cost Analysis for Single-Use Systems

The focus in single-use bioreactor development is effective oxygen transfer, and the cultivation of cell culture processes and microbial cultures. Cell densities and productivities similar to conventional stirred tanks can easily be achieved in single-use bioreactors with the introduction of real process control. The Biostat CultiBag RM can reach KLa values of 43.2 h-1 and 12.9 h-1, in 2-L and 20-L cultures, respectively, at small scale. This article will show the productivities of Chinese hamster ovary cells (CHO), E. coli, and C. diphtheriae in the Biostat CultiBag RM using disposable sensor technology.

Early disposable bioreactors designs did not have good control capability, and growth was generally equivalent to shake flasks. Simple cultivation of cells and product for small-scale operations were possible, but a reusable stirred-tank system was required for real process optimization studies, where data could be logged and process parameters could be controlled and modelled. This was mainly because of the lack of good pH and dissolved oxygen (DO) control or at least a level of control similar to traditional stirred systems. Another limitation of rocking disposable bioreactors have been the limited aeration and agitation rates that could be achieved in systems that mix the culture by rocking back and forth and provide gassing by surface aeration alone. Such systems were mainly suited to cell cultures that exhibit low biomass concentrations and oxygen uptake rates. Also, mammalian cells are more fragile, which prevents the use of vigorous aeration and agitation strategies, making a rocking platform an ideal cultivation vessel. However, many modern cell culture processes require more strenuous aeration and agitation, and cells are becoming more robust. Therefore, the ability to have a good gas mixing strategy with feedback control is important. Microbial processes have a high demand for oxygen and are processed to high biomass concentrations. Most disposable systems cannot offer high processing rates. However, disposable rocking devices can be just as effective at the seed stage when good gas mixing and controls are available.  

Recently, disposable stirred-tank bioreactor designs have been introduced that mimic traditional stirred-tank bioreactors (STR) and therefore gain more market acceptance. Most of these designs use reusable sensor technology with standard feedback control loops. The insertion of reusable sensors into a disposable system involves time-consuming tasks such as cleaning, sterilizing, and calibrating the sensors before aseptic insertion into the bioreactor. Disposable sensors are relatively new to the market and the market acceptance of such systems is slow because it requires detailed evaluation and validation. The operation of disposable sensors is different because no cleaning, sterilizing, or calibration is required at start-up. The sensors consist of membrane patches with an immobilized fluorescent dye that is able to detect the respective analyte (H+ or O

). The sensor patches are already part of the bag assembly. They come gamma irradiated and there is no break in the bag seal. An optical fiber transmits light of a specific wavelength to the sensor and returns the luminescence response from the sensor back to the measuring amplifier. This fiber optic works through the bag film, thereby maintaining sterility at all times. Standard feedback control loops are possible and recalibration functions to correct drift over time. 

The use of superior gas mixing systems with disposable sensors, and tighter process control, can be used to cultivate both modern cell culture and seed stage microbial cells successfully. Process monitoring and control of cell cultivation processes are required for culture reproducibility, modeling scale-up parameters, increasing achievable cell densities, extending batch age, and increasing productivity and yield. This article shows that using disposable sensor technology, comparable cell densities, viabilities, and titers were achieved in several cell lines, and in some cases, were even higher than in stirred tanks.  

The KLa-values for the CultiBag RM were determined by the gassing-out method for typical rocking speeds, angles, and gas flow rates. Ambient air and pure oxygen were used as process gasses.  

 BL21 (DE3), streaked out on LB agar plates, was used to inoculate two Sartorius-Stedim Biotech CultiFlask 50 disposable bioreactors (Sartorius-Stedim Biotech DF-050MB-SSH-4), each filled with 20 mL LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCL).  The seed culture was grown overnight at 37 °C and 150 rpm in an incubator (Incubator Sartorius Certomat). The BIOSTAT CultiBag RM 20 OPTICAL, part number DH-020LORM-1 using Cultibag RM 20L optical, part number DBO020L  was filled with 1 L of LB medium, preheated to 37 °C, and inoculated with the preculture to reach an optical density (OD600) of 0.15. Cultivation was started with the following process parameters: temperature 37 °C, rocking speed 42 rpm, rocking angle 10° and airflow 0.5 lpm. Ambient air was used for oxygen supply. Growth was monitored by measuring the optical density in regular intervals. For comparing the growth characteristics, a baffled Erlenmeyer flask filled with 200 mL sterile LB medium was inoculated with 

BL21 (DE3) to a final OD600 of 0.15 and incubated at 37 °C and 150 rpm in an incubator.  

Cultivation of Corynebacterium diphtheriae. 

The Biostat CultiBag RM 20 (Sartorius-Stedim Biotech DH-020-L-0-RM-1) was used for a feasibility study to replace a stainless-steel reactor for seed stage fermentation in vaccine production. The CultiBag RM 20-L (Sartorius-Stedim Biotech DBO020L) was filled with 10 L of CY-media (casamino/yeast) and inoculated with approximately 130 mL of a 

 culture grown in an aspirator bottle to an OD590 of 8.66 resulting in an optical density (OD590) of 0.123 at the start of fermentation. Cultivation was started using following process parameters: temperature 32 °C, rocking speed 12 rpm, rocking angle 5.9° airflow 0.3 lpm. During the course of the cultivation, the rocking rate was raised to 42 rpm, the angle to 10°, and the airflow to 0.55 lpm to enable high mass transfer. Ambient air was used for oxygen supply. The optical density was measured in regular intervals throughout the process. For comparison, a stainless-steel reactor filled with 20 L of medium was inoculated with approximately 270 mL of a 

 culture grown in an aspirator bottle to an OD590 of 8.66 resulting in an optical density (OD590) of 0.179 at the start of fermentation. The fermentation initial process parameters were: back pressure 5.0 psig, airflow 4.2 SLPM, and agitation 70 rpm to control the DO to ≥25%. During the fermentation, the cascade control increased the agitation to 250 rpm to maintain DO. 

A commercial CHO cell line cultivated in a serum-free fed-batch stirred-tank culture and compared with CultiBag RM using disposable sensors for process control, referred to as the X cell line for the purposes of this paper. Temperature, pH, and dissolved oxygen were controlled at constant, product-specific set points throughout the production cycle. The details of these cultivation parameters are not given because of confidentiality. 

A commercial process of a CHO cell line, referred to as the A cell for the purpose of this paper. Details of the cultivation parameters are not given because of confidentiality. The CultiBag was compared to four 2-L stirred-tank bioreactors and Wave Cellbags on Wave rocking machines. Seed cultivation was performed in shake flasks and Wave Cellbags. The inoculum conditions and feed-control strategy in the CultiBag and the Wave bioreactors was similar to the conventional stainless-steel tank bioreactors. The operational parameters of the CultiBag were similar to those for the Wave Bioreactor. The pH and DO set points for the CultiBag were similar to those for the stirred-tank bioreactors. For the stirred-tank bioreactors, pH was controlled by automatic addition of 1 M NaOH and sparging CO

 on demand. DO was controlled by sparging pure O

 on demand. For the CultiBag bioreactor, pH was controlled by continuous automatic surface aeration of CO

 automatic addition of 1 M NaOH. DO was controlled by continuous automatic surface aeration with the four-stage cascade control system. Cell culture performance was analyzed using CEDEX Cell Counter (Innovatis AG) for cell density and viability; YSI analyzer 2700 Select (YSI, Inc.) for cell culture metabolites: glucose/lactate and L-glutamine/L-glutamate; NOVA BioProfile 100 Analyzer (Nova Biomedical) for ammonium level; ABL-5 radiometer (Radiometer Medical) for pH/pO

; and RP HPLC for productivity (titer).  

Two different CHO sub clones, CHO-S and CHO-K1, were compared for their growth characteristics and metabolic profiles. The cells were cultured in repeated batch mode in the reusable bioreactor (Sartorius-Stedim Biotech Biostat B-DCU 2; 10-L UniVessel with pitched three-blade impellers and ring sparger) as well as in the single-use CultiBag RM 20-L. The CHO-S cultivations were run in a head-to-head comparison, while for the comparison of CHO-K1, two similar bioreactor runs were assessed. The CHO-S seed culture was grown in a stirred-tank bioreactor and 2,000 mL of the seed were used to inoculate the Biostat B-DCU reusable stirred tank bioreactor as well as the Biostat CultiBag RM. The final volume inside both bioreactors was 10 L of Power-CHO 2 (0.1% Pluronic, 6 mM L-glutamine, Lonza) media. The initial cell density was ~1 x 10

/mL. The process parameters used are listed in Table 1.  

Table 1.  Process parameters used to compare B-DCU STR and CultiBag RM20 

After 72 h of cultivation, the culture was divided 1:5 in the same cultivation vessel, i.e., a repeated batch process was carried out. Eight L of media containing the cell suspension were harvested and replaced with 8 L of fresh media. Samples were taken in regular intervals and the viable cell number was determined. Lactate and glucose levels were measured using the Glucose/Lactate Analyzer YSI 7100. The cultivation of CHO-K1 was carried out in a similar manner. The only difference was that the B-DCU STR and the CultiBag RM were inoculated from individual seed cultures to an initial cell density of 5 x 10

/mL in the CultiBag, respectively. The temperature in the CultiBag was set to 37 °C.  

The kLa-values for the Biostat CultiBag RM at full-rocking speed, angle, and gas flow using air were 22.0 h-1 for the 2-L system and 6.0 h-1 for the 20-L system. Using pure oxygen as process gas, the KLa-values increased to 43.2 h-1 and 12.9 h-1, respectively.  

 was assessed (Figure 1). The optical densities of the cultures were measured and the DO was monitored in the CultiBag RM using the disposable optical DO probes. During the course of the cultivation, the DO dropped to around 25% because of the limited oxygen transfer by headspace aeration. However, the culture reached an OD590 of five in the CultiBag RM and 7.3 in the stainless-steel fermenter after an 8-h cultivation period, indicating that the BIOSTAT CultiBag RM is well suited for cultivation of 

A commercial CHO cell line was evaluated using standard rocking and cultivation parameters. The cell growth shown in Figure 2 was slightly lower than the STR but the viability was higher at the end (Figure 3). Figure 4 shows that the overall production was greater than stirred-tank bioreactors.  

To demonstrate the reliability of disposable sensor technology, the real time data plot is shown in Figure 5. The pH control is shown to be within +/–0.1 pH units and equivalent to traditional sensors.  

Overall cell culture performance of the CultiBag bioreactor was comparable to the STR (Table 2). The final RP HPLC titer value and the average specific productivity of CultiBag were higher than that of the STR. Temperature control was comparable to a conventional bioreactor (within +/- 0.5 °C). Cell culture performance of the CultiBag bioreactor was comparable to the STR's. Some early stage  proportional-integral-derivative controller (PID) loops were tuned to achieve better control of pH and DO. The average specific productivity of CultiBag was higher than that of the STR's. Without pH and DO control, the Wave bioreactor demonstrated different cell culture performance compared to the CultiBag bioreactor in terms of higher final cumulative viable cell count (CVC)  value, lower RP HPLC titer, and lower average specific productivity.  

Table 2. Cell culture performance of the CultiBag bioreactor compared to the STR 

The growth characteristics of the CHO-S and CHO-K1 were also similar, with both cell lines reaching comparable levels in both types of bioreactor (Figure 6). The CHO-S showed better growth, reaching higher cell densities then the CHO-K1, probably reflecting the better adaptation to suspension culture.  

Figure 7 shows the glucose and lactate profile of the CHO-S cultivation. In this instance, the glucose consumption of the cells grown in the B-DCU STR was higher. Consequently, the lactate build up was also higher. This observation is congruent with the slightly higher viable cell density reached in the B-DCU.  

The Biostat CultiBag RM is shown to reach KLa values of 43.2 h-1, and 12.9 h-1, respectively, at small scale for 2-L and 20-L systems. The reactor easily provides oxygen transfer rates sufficient for medium cell densities in seed stage microbial fermentation. 

, still the preferred microbial host for recombinant protein production, was shown to be successfully cultivated to medium cell densities. In addition, growth of the 

 reached an OD590 of 4.95 in the CultiBag RM and 7.26 in the stainless-steel fermenter after an 8 h cultivation period, indicating that it was successfully cultured in preparation of a seed inoculum despite a drop in dissolved oxygen concentration. A second trial confirmed these results. Despite the high demand for aeration and agitation in microbial cultures, we have demonstrated that the Biostat CultiBag RM is suitable for microbial seed stage cultivation. The superior gas mixing capability optimizes headspace aeration and creates a suitable vessel for microbial cultivation. 

When comparing a rocking bioreactor with and without process control as demonstrated by the CHO-A process, a different cell culture performance is demonstrated. The rocking device with control was the CultiBag system with feedback control loops for pH and DO to disposable sensors, and the rocking device without any pH and DO control was the Wave bioreactor. Both systems exhibit similar rocking motion mixing. The Wave bioreactor exhibits a rocking motion, which starts and stops, forcing the culture to create turbulent waves for mixing. The Wave bioreactor was gassed with a constant flow rate of air and CO

, without any feedback control mechanisms. A constant flow of CO

 can cause pH fluctuations to occur as the uncontrolled CO

 dissolves into the media and converts to carbonic acid. The CultiBag offers a gentler and continuous rocking motion with an inbuilt feedback control mechanism, adding CO

 on demand and only when the culture needs it. The constant flow of air at the liquid surface prevents CO

 accumulation and pH fluctuation. Both systems had similar rocking and temperature control. The Wave bioreactor was shown to have a higher final CVC value, a lower RP HPLC titer, and a lower average specific productivity, thereby indicating that process control offers many advantages for cell cultivation. 

When comparing the CultiBag side-by-side with a conventional stirred-tank bioreactor system in the CHO-A process, the overall cell culture performance of the CultiBag bioreactor was comparable to the STR. In this case, both systems had good control systems in place, but the STR used reusable sensors and the CultiBag used disposable sensors. This demonstrates the comparability between conventional and disposable sensor technology in a live process. Additionally there was no oxygen limitation in the rocking bioreactor compared with the stirred tank. In fact, the final RP HPLC titer value and the average specific productivity of CultiBag were higher than that of the STR.  

This is further demonstrated by the CHO-S and CHO-K1 process, which also shows comparable growth and productivity to the STR. Additionally, real time data plots show the online process control of the disposable sensors in the CHO-X evaluation, demonstrating that the process control of disposable sensors are similar to reusable sensors. We can therefore conclude that, not only do disposable sensors provide adequate control of processes, but that these rocking bioreactors can exhibit cell growth productivities similar to a traditional STR and, in some cases, even higher. With the added benefit of disposable systems in terms of cleaning, sterilizing, reduced cross contamination, and flexibility, it is obvious to see why the industry is focused on disposable systems. 

, is a product manager, disposable bioreactors, at Sartorius Stedim North America, Edgewood, NY, 631.254.4249, 

 is a senior technology scientist at Wyeth Vaccines, Sanford, NC. 

, is a senior scientist at Schering-Plough, Summit, NJ. 

 is manager of recombinant technology at CSL Behring, Preclinical R&D, Marburg, Germany. 

, is a scientist of R & D pharma process and 

, is an application specialist of fermentation technology, both at Sartorius Stedim Biotech GmbH, Goettingen, Germany. 

Productivities similar to those achieved with stirred tanks can be achieved with disposable bioreactors.

BioPharm International-11-02-2008

Disposables for Bioprocessing: Making the Transition

Systematic Evaluation of Single-Use Systems Using Process Simulation Tools

The Environmental Impact of Disposable Technologies

Lifecycle Cost Analysis for Single-Use Systems

Disposable Bioreactors for Cells and Microbes