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BioPharm International, November 2010 Supplement (PDF)

In a multiproduct cGMP clinical manufacturing facility, flexibility and short processing times are important operating attributes. A critical aspect of a multi-product facility is the procedures used to minimize product cross-contamination. Single-use (SU) technology enables flexibility, short process times, and limited chances for cross-contamination. An SU tangential flow filtration (TFF) system was implemented in a cGMP clinical manufacturing facility. In this article, we evaluate the performance, control of operation, productivity, and overall cost savings of the system.

Improving the time it takes to bring new drugs to the market continues to be an important goal for pharmaceutical companies. There are several approaches that are taken with the overall goal of bringing more drugs into commercialization phase.

  Single-use (SU) technology is one of the strategies being adopted to reduce overall drug development time. SU technology brings significant advantages of reduced capital costs, faster construction and installation, reduced processing cycle times, and elimination of the need for post-use equipment cleaning and verification.

 Starting with the use of plasticware such as pipettes, petri dishes, and t-flasks, disposable components are being increasingly incorporated in the laboratory environment. With the development of SU bioreactors at the 2,000 L scale (Xcellerex XDR), chromatography systems (GE ReadytoProcess), and systems for micro- and ultrafiltration, disposable equipment continues to replace fixed stainless steel equipment in manufacturing plants. 

Demonstrating that process equipment is adequately cleaned is a critical aspect of any pharmaceutical plant operation. Following some type of post-use cleaning, the presence of residual protein and cleaning agent is typically assessed by subjecting equipment rinse solutions and swab samples to test methods including pH, conductivity, and fluorescamine. These procedures are time consuming and reduce equipment utilization. In a multiproduct facility, when equipment is shared between products with high and low potency, meeting the low acceptance criteria for residual protein can be very challenging. 

  To meet the demand of reducing production cycle time and improving productivity, a general downstream production process review was performed to identify the bottlenecks that affect the overall process efficiency. This process review identified that an existing stainless steel (SS) ultrafiltration–dialfiltration (UF–DF) system used for an intermediate UF–DF process in the facility was a bottleneck and presented an opportunity to evaluate an SU UF–DF system. The SS UF–DF system was designed for process development and scale-up rather than GMP production and had a limited retentate tank capacity (25 L). Additionaly, the clean-in-place (CIP) and post-CIP swabbing of the SS UF–DF system are very complex and time-consuming.  

Criteria for Evaluating SU UF–DF Systems 

Currently there are several SU UF/DF systems available. An evaluation was made to choose an appropriate system based on GMP production requirements, operational needs, budget, and timeline. These criteria included:  

 The system should have the capacity to handle 5 m

  membrane size, 10–50 L retentate tank working volume, >80 L/h/m

 (LHM) feeding flux, and differential pressure (ΔP) and transmembrane pressure (TMP) controls at 0–20 psi. The retentate tank should have a mixing device to avoid localized concentration during the operation.

Process monitoring, control, and data management

: The process should be able to be controlled by constant  TMP  and ΔP.  The pressures, flow rates, process phases, and other process parameters should be able to be monitored and recorded in real time. Data management should meet 21 

: There should be no or a limited number of bio-compatibility issues for any disposable parts that contact product and process buffers. The levels of leachables and extractables should be in the acceptable safety range for clinical drug substances. 

Equipment availability and vendor support

: The product should be readily available. An integrated and off-the-shelf system is preferred because it saves time on equipment validation and meets short project timelines. The manufacturer should have a good record for on-time equipment delivery and reliable technical support. The manufacturer should have the capability to consistently supply high quality accessories and consumable items. 

: The price of the equipment and disposable items should be reasonable to reduce the overall cost of production when implementing a new UF–DF system in a GMP production facility.

The Millipore SU Mobius FlexReady Solution for TFF model TF2 system was selected after comparing three different SU UF–DF systems that are currently on the market. An ultrasonic permeate flow meter and a retentate pressure control valve (PCV) were included to enhance process control. The length of the rods on the cassette holder was extended to increase the holder capacity from 2.5 to 5 m

Operational Performance of the Single-Use System

A typical UF–DF operation includes flow path and membrane installation, pre-use CIP, integrity test, UF, DF, product recovery, and post-use CIP. Figure 1 compares a SS UF–DF system and a single-use UF–DF system. The implementation of the Millipore SU Mobius FlexReady Solution for TFF offers some key advantages over the existing SS UF–DF system in terms of preparation, membrane integrity testing, UF–DF general operation, critical parameters (feed flow rate, efficiency of mixing in retentate tank), and product recovery. 

Figure 1. Operation comparison of a stainless steel ultrafiltrationâdiafiltration (SS UFâDF) system and a single-use (SU) system  

: The disposable flow path is gamma irradiated and ready for use when it arrives. With the SS UF–DF system, cleaning occurs in two steps: the UF–DF system and membrane. The gamma irradiated retentate bag and flow path have eliminated the system pre-use and post-use CIP, rinse samples and protein swabs, and storage steps, however cassette cleaning must still be performed. The disposable flow path is relatively easy to install, and typically can be done in 60 minutes or less. The flow path is discarded after each use, minimizing the potential for product cross-contamination.  

: The feed pump on the SS UF–DF system is a rotary lobe pump. An auxiliary peristaltic pump is required to perform membrane integrity testing for the SS UF/DF system. The SU UF–DF system eliminates the need for an auxiliary peristaltic pump for membrane integrity testing.   

: Four fully automated operations (initial fill, fed-batch, DF, and batch concentration) make the system user friendly. Process parameters are easy to input, and the resulting data are captured in our data acquisition system, eliminating the need to transcribe data into a paper-based GMP batch record. This facilitates trending or comparisons from batch to batch. Electronic data also simplify the technology transfer from pilot plant to commercial manufacturing.  

: One parameter with a significant impact on the UF–DF operation is the feed flow rate. To maximize the permeate flux, UF–DF operations typically require flow rates in the range of 240–360 LHM to maintain specific crossflow characteristics. With the SU Mobius FlexReady Solution, the peristaltic feed pump can achieve 20 L/min with 30 psi back pressure. The feed pump is either controlled by ΔP or fixed pump speed, which is correlated to the flow rate. The flow rate meets our current process feed flow rate specification, and the automated TMP control using the retentate PCV is relatively stable.  

: Mixing is critical during the diafiltration step to ensure a homogeneous solution for efficient buffer exchange. With the previous SS UF–DF system, retentate return flow distribution was the only method for mixing in the retentate tank. The SU Mobius TFF system design incorporates flow distribution by a retentate diverter plate and a magnetically coupled agitator for enhanced mixing in the retentate tank. Eliminating dead legs from the flow path is critical to achieving efficient buffer exchange. To this end, the design of the low dead-volume t-connectors for the pressure indicators in the disposable flow path assembly is important. In Figure 2, diafiltration from 1.1 M NaCl to 0.15 M NaCl was performed at a constant-volume diafiltration (50 L) and 9% agitation speed with a 5 m

  30 kD Millipore Biomax membrane. The maximum diafiltration volume with low agitation speed was considered the worst-case scenario. The experimental curve, is comparable to the theoretical curve indicating good mixing.  

Figure 2. Constant-volume diaï¬ltration in the Millipore single-use Mobius FlexReady solution for TFF. The solid line is experimental and the dotted line is theoretical: Retentate residue (%) = 100 * e (Râ1)*N, where R is the Donnan effect and N is diavolume.  

: To increase the percent recovery, a buffer flush step was performed to recover product retained in the system and membrane. With the SU Mobius FlexReady TFF system, buffer can be transferred to the retentate tank and weighed directly. This saves time and eliminates the step of weighing the flush buffer, which was required with the previous SS UF–DF system.      

To meet the requirement of process control, a retentate PCV was applied for constant TMP control. The retentate PCV position can be selected between 0 and 100% open for feed flow rate control. The 100% open position was chosen for PCV at the start of diafiltration. Table 1 summarizes the performance of constant TMP control at diafiltration phase for six GMP runs with a feed flow rate of 180 LHM. The diafiltration started with the feeding flow rate controlled by the pump speed. Once the feed flow rate was in the recommended operating range (ROP), constant TMP control was applied. The TMP setting was randomly selected in the TMP ROP range (<20 psi). It was found that TMP could be controlled at a narrow fluctuation range with a coefficient of variation (CV) less than 10% in these runs.  

The system uses an ultrasonic permeate flow meter to calculate cumulative diafiltration volume (DV), which is used to target the diafiltration process endpoint. During the factory acceptance testing, the accuracy of the permeate flow meter was tested using four solutions with different densities (2 M guanidine HCl, 2 M urea, 100 mM NaCl, and pure water). The results suggest that the solution densities have little impact on the flow meter reading (data not shown).  

In GMP production, the accuracy of the flow meter was evaluated by comparing DV values using a flow meter versus those obtained form a weight scale. The comparison of results is summarized in Figure 3, where the dark columns represent DV results obtained from the weigh scale and the light columns represent DV values obtained from the permeate flow meter. The average difference between these two measurements is 3.3%. 

Figure 3. Comparison of measuring diaï¬ltration volume by permeate flow meter (light columns) versus by weigh scale (dark columns)  

System integration, product quality, and product safety impact factors (leachables, extractables, and biocompatibility) are major concerns. It is beneficial for a company to use a family of SU products from the same manufacturer to save the time and reduce the cost of safety evaluations and validation. 

Before introducing an SU UF–DF system to our facility, several other SU systems made by Millipore, including buffer and media mixing tanks and containers, were already evaluated and used in GMP production. The flowpath and associated retentate tank liner for the Millipore SU Mobius FlexReady Solution for TFF are made of the same film and material as those used for the associated containers for the Millipore mixing system, which are widely used in our facility. The risk of leachables and extractables affecting product quality and the compatibility of buffers and protein were previously assessed,

  saving both time and cost on those safety evaluations. Unlike SU UF–DF systems, the Mobius FlexReady Solution for TFF is a fully integrated system for operation, process monitoring and control, and data management. In the preliminary evaluation, two systems had the same assessment score based on operation specifications, process monitoring, and control. System integration and product quality and safety impact were the differentiating factors. Ultimately, the Millipore Mobius FlexReady Solution for TFF was chosen because of its better integrated system and the existence of previous safety evaluation assessment.

The operation of this single-use system is easier than our existing SS UF–DF systems and the performance is comparable. The disposable flow path pieces can be quickly installed, resulting in short equipment turnaround times. The retentate diverter plate and magnetically coupled mixing device helped avoid the concentration gradients in the retentate tank and improved the UF–DF performance. Product yield and purity of the intermediate UF/DF step using the  Mobius FlexReady Solution for TFF are comparable to the results from previous runs (data not shown). Also, the low system hold-up volume (0.6 L) and working volume (2.0 L) allowed for a wider operating range. The strong feed pump maintained a 20 L/m

  flow rate even at 30 psi back pressure. Because the whole flow path is disposable, the risk of cross-contamination during product changeover was minimized, and the time and effort for CIP were reduced in our multiproduct facility. 

The features of process monitoring, control, and data management enhance the process automation capability and reliability of data recording. The Allen Bradley Controllogix system and the associated human–machine interface (HMI) software were user-friendly. The PID-like display provided real-time information of recipe parameters and process data. The control system was interfaced with our production information management system for data collection and archiving. This feature enables the future implementation of electronic batch records in a GMP production environment. It also enables easy, batch-to-batch comparison for technology transfer.  

The PCV and permeate flow meter increased process monitoring and control capabilities. We observed fluctuations of TMP while using the PCV for constant TMP control. It was found that the fluctuations could be reduced to an acceptable level (Table 1) by initiating the operation with feed flow control by pump speed, then switching to constant TMP control by PCV once the feed flow rate is within the ROP range. During the diafiltration, total diafiltration volume target can be preset and monitored with the included permeate flow meter. Although there was a measurement difference of approximately 3% in the total diafiltration volume versus the weight scale measurement, the result is acceptable because the ROP for total diafiltration volume was quite wide and diafiltration completion was primarily based on the conductivity and pH of permeate filtrate.  

Table 1. Summary of constant transmembrane pressure control (TMP) control  

Implementing this single-use ultrafiltration-diafiltration system doubled the retentate capacity of the intermediate UF–DF system, and therefore shortened the unit process time and improved productivity. By eliminating the six pre- and post-use CIP steps, the usage of water for injection and caustic solutions for CIP also was reduced. Furthermore, the 16 corresponding rinse samples and swab sampling testing for CIP steps were eliminated. The overall cost saving will depend on actual utilization. We found the payback period to be 3.8 years when performing ten campaigns per year.  

The authors would like to thank the Millipore team for technical and logistic support for introducing this SU UF–DF to our facility. The authors also would like to acknowledge the Centocor pilot plant purification team for their support in implementing the SU UF–DF system for GMP production. 

 is the global head of supply execution, all in pharmaceutical development and manufacturing sciences at Johnson & Johnson, Inc, Spring House, PA, 215.628.5953, 

1. Paul MS, Mytelka SD, Dunwiddie TC, Persinger CC, Bernard H, Stacy MR, et al. How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nature Rev Drug Discov. 2010;9:203–14. 

2. Charles I, Lee J, Dasarathy Y. Single-use technologies—a contract biomanufacturer's perspective. BioPharm Int. Suppl. Guide to Disposables. 2007 Nov; 31–6. 

3. Millipore, Inc. PureFlex: Extractables, bioreactivity safety evaluation approach. Technical brief.  

4. Millipore, Inc. Extractables bioreactivity safety evaluation of PureFlex film for Centocor. 2007.  

5. Maigetter RZ, et al. Single-use (SU) systems. Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology. 2010: 1–39. 

Peer-Reviewed Research

A case study evaluates the performance, control of operations, productivity, and cost savings of a single-use system.

Implementing Single-Use Technology in Tangential Flow Filtration Systems in Clinical Manufacturing

Tangential flow filtration (TFF) is a common processing step in concentration and diafiltration (buffer exchange) operations in the downstream processing of biopharmaceutical products. Using a presanitized, disposable TFF membrane makes it possible to reduce the number of process steps and thus reduce labor by 50% or more and reduce buffer and water usage by 75% or more. In addition to the cost savings realized from the reduced labor and buffer usage, single-use TFF can increase productivity, by >45% in many cases. This article outlines an economic model for comparing the costs of reusable and single-use TFF in biopharmaceutical applications.

Over the last decade, the bioprocessing industry has recognized that single-use products can provide significant savings in time, labor, and capital. As a scalable and flexible technology, single-use systems have increased production capacity, eliminated clean-in-place (CIP) steps, cleaning validation, steam-in-place sterilization, and reduced the use of caustic chemicals and water for injection. In addition, single-use products can reduce the risk of cross contamination between batches or campaigns. These benefits apply to single-use tangential flow ultrafiltration as well. 

Tangential flow filtration (TFF) is a common processing step in concentration and diafiltration (buffer exchange) operations in the downstream processing of biopharmaceutical products. As shown in Figure 1, typically there are several ultrafiltration TFF steps in downstream processing, such as following the affinity chromatography, anion or cation exchange chromatography steps.  

Figure 1. In a typical downstream process, there are several ultrafiltration tangential flow filtration (TFF) steps following the affinity chromatography, anion or cation exchange chromatography operations  

 When TFF systems are operated as clean-and-reuse systems, they typically involve 10 major process steps (setup, CIP, flush, normalized water permeability [NWP], equilibration, processing, CIP, flush, NWP, and storage). Using presanitized, single-use TFF membranes, however, reduces the number of process steps from 10 to four (set up, equilibrate, process, CIP). 

As a result, single-use TFF can reduce labor and processing time by 50% or more. In addition, by eliminating many flush and CIP steps, single-use TFF can reduce water, CIP solution, and buffer consumption by 75% or more. By developing an economic model, companies can identify the costs savings associated with these reductions in labor, buffer, and water. This article will outline such an economic model for comparing the costs of reusable and single-use TFF in biopharmaceutical applications. 

A typical TFF process contains basic operations that include pre-use, process, and post-use activities. Figures 2a and 2b show the percentage of time required to perform each step. Typically, in a process based on reusable TFF, only 50% of the total process time is devoted to actual processing of the product. The remaining 50% is spent in preparing and cleaning the TFF system. In contrast, in a single-use TFF system, 80% of the total process time is devoted to processing the product, thus increasing process efficiency. 

Figures 2a & 2b. The percentage of time required to perform each step of a reusable tangential flow filtration (TFF) process. Typically, only 50% of the total process time is devoted to actual processing of the product. The remaining 50% is spent in preparing and cleaning the TFF system. In contrast, in a single-use TFF system, 80% of the total process time is devoted to processing the product, thus increasing process efficiency.  

 Two key factors that affect the economics of single-use TFF are membrane area and the number of annual process cycles. Figure 3 shows the relationship between the number of annual process cycles and the economic benefit (percent savings) associated with single-use TFF at several different process scales. As seen in the figure, single-use TFF technology is most beneficial at smaller scales, i.e., <5 m

. Larger-scale processes can benefit, however, when the number of annual process cycles is less than 20. 

Figure 3. The relationship between the number of annual process cycles and the economic benefit (percent savings) associated with single-use tangential flow filtration (TFF) at several different process scales. Single-use TFF technology is most beneficial at smaller scales, i.e., 

Single-use TFF offers the greatest savings in clinical and contract manufacturing, where the scale is low and changeovers are frequent.

An Economic Analysis of Single-Use Tangential Flow Filtration for Biopharmaceutical Applications

Disposables historically have been used in biotechnology processes for the past three decades, initiated with the use of single-use plastic support for cell culture (e.g., vials, shakers, T-flasks, and roller bottles). Another step was made more recently by the implementation of plastic bags into these processes, either used in the process itself or in supportive steps, such as media and buffer preparation and storage.

One of the key trends for biotech manufacturing is the development of disposable bioreactors. This trend was initiated by the introduction of the Wave system. The implementation of this technology was in line with the use of disposable plastic bags. The Wave bag technology quickly captured the interest of the biotech community. These systems mainly are used for cell expansion to replace shake flasks or intermediate small- and pilot-scale bioreactors, simplifying the process. Despite many advantages, this technology presents some limitations for its use as a final production-scale bioreactor, such as scalability, specificity of the agitation, and the bioreactor geometry. 

Because of these limitations, an industrial need drove the development of more conventional and scalable disposable bioreactors. One of the first systems introduced on the market was the Single-Use Bioreactor (SUB) from Hyclone. The SUB opened a new range of applications because of its potential use either as seed vessels or as production vessels for cell-based processes. 

Today, single-use bioreactors are used extensively for the production of monoclonal antibodies (MAbs) and recombinant proteins. Based on this market trend, vaccine manufacturers such as GSK Biologicals decided to investigate the potential use of this emerging technology for vaccine manufacturing. The focus of this article will be on the use of disposable bioreactors in the context of viral vaccine production. 

Viral vaccine manufacturing processes present some specific constraints as compared to other biotech products linked to the cell substrate used and to the viral production. These specificities are:  

Multiple cell lines are used for these productions such as VERO, MDCK, MRC5, BHK, and CHO cells, making it more challenging to develop a platform process.

Cell substrates for viral production often are cell-anchored cell lines, such as VERO cells, requiring the use of micro-carriers for bioreactor process steps.

Viral production must be handled in the right biosafety containment, i.e., biosafety level 2 or 3 environments.

Production scales generally are smaller compared to MAb processes (ranging from 500 to 2,000 L).

Main Drivers for Implementing Disposable Bioreactors in Vaccine Production

There are many benefits of using disposable systems in biotech processes. Two of these benefits justify the evaluation of disposable bioreactors for viral production processes.  

Maximizing facility output because of the fast turnover of disposable systems (no clean-in-place and steam-in-place operations in production vessels).

The reduction of capital investments linked to the reduction and simplification of the facility design and to the reduction of equipment investment.

In addition to these points, other drivers specific to vaccine manufacturing were considered.  

Simplified biosafety level 2 and 3 production areas (because of the smaller footprint, lower ceiling height, and removal of water-for-injection and steam utilities).

Minimizing the harvest size to reduce the size of purification equipment and suites.

As previously mentioned, generic processes are difficult to define in the context of viral vaccine production. Therefore, a worst-case process that would cover all other company viral vaccine processes was defined. The disposable bioreactor technology selection based on this process will then be recommended as a standard single-use bioreactor platform for viral vaccine applications. 

The following set of parameters was used to define the worst-case process:   

animal-free media to decrease shear protection and nutritive support from serum containing formulations

cell cultures using micro-carriers (at a high concentration) because cells grown in this condition are more sensitive to shear stress compared to suspension cultures

The combination of these process criteria made this process challenging enough to cover a large range of current and future in-house processes. 

Figure 1. Disposable bioreactors from four companies were evaluated  

Based on market availability, system maturity, available scale, and mixing systems, four disposable bioreactors were selected (Figure 1).  

Cultibag STR from Sartorius Stedim Biotech

To evaluate these four disposable bioreactor technologies, several criteria were selected. These criteria can be divided in two sections:  

 such as cell culture and viral production performances, mixing and aeration characteristics, and scale-up predictability.

 such as film type, biosafety, procurement, assurance of supply, and price.

Mixing and aeration performance evaluation remains mandatory to ensure robust scale-up of cell culture processes, especially for adherent cell line applications (microcarrier use). In this study, these performances were evaluated for the four bioreactor technologies that were identified. 

Three tools for mixing characterization were used: mixing time experiments, correlation software, and the particle image velocimetry (PIV). Typical results of the PIV technique are shown in Figure 2.  

Figure 2. Results generated by the particle image velocimetry technique  

Mixing characterization was performed on the four selected technologies; their mixing configurations are shown in Figure 3. Mixing performances were evaluated based on several criteria such as mixing time, maximum shear levels, etc. These performances were then compared to the application's specific requirements: minimizing shear stress while assuring good homogeneity levels and maintaining all the microcarriers in complete suspension. Some results of this study are depicted in Figure 4. 

Figure 3. Mixing configuration of the four single-use bioreactors (SUB) evaluated  

This figure shows the combination of maximum shear stress (represented by the tip speed) and mixing time in the minimum operating conditions necessary to maintain microcarriers in complete suspension. In this graph, two technologies seem to show better results in terms of acceptable mixing time combined with a low maximum shear stress (tip speed). 

Figure 4. Mixing performances comparison of the four disposable bioreactors  

Aeration performances also were compared based on gas transfer capacity measurements (k

a) in our end-of-cell-growth conditions for each bioreactor. 

Gas transfer capacities in our operating conditions (minimum agitation speed required to maintain microcarriers in complete suspension) were evaluated for the four bioreactor technologies. A high gas transfer capacity is useful to decrease the amount of oxygen needed to maintain the dissolved-oxygen concentration to its set point. With low sparging flowrates, shear induced by bubble break-up at the liquid surface will be decreased. These low flow rates also have a positive effect on foaming. 

Switching to single-use bioreactors can have financial and performance benefits.

Disposable Bioreactors for Viral Vaccine Production: Challenges and Opportunities

The demand for single-use bioprocess systems in biotech and pharmaceutical manufacturing has expanded significantly over the past few years. Applications for single-use systems in biomanufacturing range from upstream media preparation single-use bioreactors, buffer preparation, and intermediate processing, to storage of active pharmaceutical ingredients (APIs), bulk formulations, and filling of final dosage containers. Despite their increased acceptance and implementation, a primary concern with single-use disposable polymeric equipment continues to be that of extractables and leachables. In contrast with final dosage container and closure systems, the absence of specific regulatory guidance for process equipment extractables and leachables has many drug manufacturers unsure of what data must be submitted. 

In response to the interest in single-use disposable manufacturing and to address the challenges of associated problems affecting the industry, the Bio-Process Systems Alliance (BPSA), the single-use biomanufacturing trade association, has developed best practice educational guides on a range of topics including component quality tests, disposal issues, irradiation and sterilization, the economics of single-use technology, and extractables and leachables. These guides tie together the expertise and leadership of BPSA's supplier and end-user member companies to provide recommendations for industry best practices.    

In December 2007 and January 2008, BPSA published its first white papers on extractables and leachables testing of single-use process equipment.

  This was the first independent consensus guide that provided basic concepts of extractables and leachables from single-use equipment, summarized the existing regulatory requirements, and recommended a risk-based approach that could reduce the amount of testing required by users. As part of this initiative, BPSA also conducted a training seminar in February 2008, at the headquarters of the FDA and CBER near Washington, DC, where we differentiated single-use process equipment from final dosage container and closures, discussed the BPSA recommendations for extractables and leachables testing of single-use equipment, and the proposed risk-based approach. BPSA also has encouraged its supplier member companies to develop more generic and comparable extractables data that would reduce the burden on users for redundant testing. 

Regulatory Expectations for Extractables and Leachables Data

In the ensuing period, BPSA has gotten positive feedback from users and regulators about the risk-based approach. However, FDA 483 observations and warning letters continued to cite insufficiencies in extractables data submitted by biopharmaceutical manufacturers in drug product applications.  

For example, in an April 2008 prelicensing inspection 483, the FDA stated, 

"Besides the 0.22 µm sterilizing filters, there was no leachable and extractable testing performed for the equipment and (--redacted--) materials used in (--redacted--) purification process including purification of (--redacted--)." 

The drug manufacturer submitted a validation project plan defining requirements for the evaluation of extractables and potential leachables from the product contact components of the process equipment, filters, and chromatography media used to manufacture (the) drug substances and products, and the risk assessment. FDA's subsequent 483 response review memo stated that the draft protocols and proposed corrective actions were considered to be adequate.

 In a related inspection, the FDA similarly observed, 

"There were no leachable and extractable testing performed for (--redacted--) materials used in buffer preparation."

The drug manufacturer agreed to implement an extractables and leachables assessment policy that included risk assessment, safety assessment, and model solvent study design, along with generic family-approach studies for leachables and extractables for the storage of (--redacted--) used in buffer preparation activities.

  This is consistent with BPSA's published recommendations. 

At IBC's 7th International Single Use Applications for Biopharmaceutical Manufacturing Conference, held in La Jolla, CA, on June 14, 2010, Destry M. Sillivan of the FDA provided an update and overview of types of data to be reviewed and specific areas of concern to the FDA. 

 Sillivan stated that, "the responsibility for establishing that single-use materials selected for the manufacturing process do not adversely impact the product falls on the manufacturer of the drug product under review," and recommended that drug sponsors, "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."

Following closely the recommendations made in 

 and FDA CBER training seminar, Sillivan stated that, "CBER recommends a risk-based approach be taken in evaluating extractables and leachables where you take multiple aspects into account (e.g., indication, safety issues, product characteristics, dosage, formulation, and stability profile)." If there is 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," he added. 

According to Sillivan, "Where there is relevant risk, the drug sponsor may have to determine toxicity based on maximum dosage of potential leachables based on extractables data. If the maximum dosage of potential leachables presents a safety risk, leachable evaluation and testing may be necessary. Additionally, if product quality could be affected by potential leachables, studies may need to be performed to assess the effect on product quality, including efficacy." 

New products contacting single-use materials often are reported in the product annual report with no information regarding material composition, no extractables or qualification studies performed in support of use of the new material, and no written justification of why the studies that were submitted were appropriate to support suitability for use of the new materials with the drug product. The FDA and CBER consider this level of information to be insufficient to determine if the change was submitted appropriately.

The FDA and CBER generally recommend that either the drug sponsor or the material manufacturer demonstrate through sufficient testing that the material is suitable for the submitted processes and product.  

BPSA Introduces Updated Extractables Guide

Recognizing the need for further education on extractables testing, including extraction and analytical methods, BPSA began to develop a follow-up guide providing more extensive information on methods of extraction and extractables analysis in 2009. The resulting 

 white paper, "Recommendations for Testing and Evaluation of Extractables from Single-Use Process Equipment"

  was developed by an expert team representing suppliers, users, and independent testing laboratories. 

begins with a review of the key concepts introduced BPSA's 2008 white paper, then expands on the previously proposed risk-based approach, providing recommendations for extraction conditions, explanations of analytical methods, and suggestions for users on how best to evaluate and compare supplier-generated data. 

The first section covers three revisions made to the 2008 extractables white paper.  

Two levels of risk evaluation are now differentiated—the first, for materials, includes information on polymer chemical compatibility, generic extractables data, and suitability for use. The second risk evaluation considers drug product-related toxicity and quality risk, evaluating the extractables data as presumptive leachables by calculating worst-case levels in downstream process fluids and final drug product, and applying recognized toxicological assessment methods.

A change was made to the decision tree flow chart originally published in the 2008 white paper, which included an option to test only for leachables in intermediates or final products in the absence of adequate supplier extractables data. Subsequent discussions with CBER reviewers highlighted the need to more strongly address concerns that leachables could be masked in protein solutions. BPSA recognized that to analyze for potential leachables in biopharmaceuticals, knowing the extractables from process equipment was not an option. In the 

, the option to skip extractables testing if unavailable and go directly to leachables testing is omitted.

, to refer to potential leachables from process equipment. Several end users have noted that in FDA and EMA documents, the term 

 only appears in reference to final product containers and packaging, not to process equipment, and looked to BPSA to recognize this distinction. In response, BPSA has introduced the term 

 when referring to chemicals that leach from process equipment, but may or may not be detected as "leachables" in final product dosage containers.

A revised risk decision tree highlights materials risk evaluation, which incorporates a review of available generic extractables data and determining sufficiency, followed by application of that data to the drug product and performing a risk evaluation for toxicity and product quality. After the risk assessments are done, a decision can be made whether the available extractables data, when considered as potential migrants, are sufficient, or whether further testing for migrants from the process equipment into the actual process fluid, where they may be detected as leachables, is merited. In the latter case, both data on extractables and migrant (i.e., process-derived leachables) should be submitted in regulatory filings. 

 provides detailed considerations on extraction conditions, including selection and preconditioning of test articles such as autoclaving, irradiation, and flushing. BPSA recommends water and ethanol as primary worst-case solvents representing most bioprocess fluids, and also discusses options for other extraction fluids. Model extraction conditions are differentiated by component types and summarized in a detailed table covering filter capsules, tubing, sterile connectors and fittings, biocontainers, mixing bags, and bioreactors. 

This is followed by an extensive section on analytical methods, including detailed descriptions of applicable techniques—what they do, what they detect, and what their limits of detection are. This section on "what you need to know about analytical methods" is intended to guide suppliers in generating appropriate data, and enable users of single-use systems to better understand suitable extractables data and the implications to their process and product. 

Lastly, the guide provides recommendations for user actions when extractables data from suppliers are incomplete or inadequate. Suggestions also are provided for comparing data from different suppliers of comparable components where extractions may have been conducted under differing pretreatment or extraction conditions or different analytical methods. 

Availability of supplier-generated core (generic) extractables data generated according to consensus recommendations, as proposed by BPSA, can minimize duplicate testing by users for individual and multiple drug products. These conditions and analytical methods are intended to guide suppliers and users to develop more comparable extractables data that facilitate user and regulatory evaluations. Copies of the 

 is the senior vice president of global scientifi c affairs at Pall Life Sciences, Port Washington, NY, 516.801.9086, 

 He also is the chairman of the Board and Technology Committee at Bio-Process Systems Alliance. 

 1. BPSA Extractables and Leachables Subcommittee. Recommendations for extractables and leachables testing of single-use–part 1: Introduction, regulatory issues, and risk assessment. BioProcess Int. 2007 Dec;5(11):36–49.  

 2. BPSA Extractables and Leachables Subcommittee. Recommendations for extractables and leachables testing of single-use–Part 2: executing a program. BioProcess Int. 2008;6(1):44–53. BPSA. Available from: 

 3. FDA 483 response review memo. Available from: 

http://www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts/licensedproductsblas/fractionatedplasmaproducts/ucm161014.htm

 4. FDA 483 response review memo. Available from: 

http://www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts/licensedproductsblas/fractionatedplasmaproducts/ucm161016.htm

 5. Sillivan, DM. Review of single use processes and materials: overview of types of data to be reviewed and specific areas of concern. IBC's 7th International Single Use Applications for Biopharmaceutical Manufacturing, La Jolla, CA: 2010 Jun 14. 

6. BPSA Extractables Subcommittee. Recommendations for testing and evaluation of extractables from single-use process equipment. 2010: Washington, DC. Available from: 

BPSA eases confusion over extractables and leachables testing through guides.

Regulatory Expectations and Consensus Industry Recommendations for Extractables Testing of Single-Use Process Equipment

In this study, we tested the combination of a disposable bioreactor and a disposable dissolved oxygen sensor as a replacement for our standard bioreactors. The possibility to run a fed-batch cell culture process developed for the production of a monoclonal antibody in a 50-L single-use bioreactor was investigated. The single-use bioreactor was assessed both as a seed train and as a production bioreactor. Therefore, three configurations corresponding to different combinations of the 50-L disposable bioreactor and the reference 5-L glass bioreactor (fully scalable up to 300 L) were compared.

In the past decade, biopharmaceutical manufacturing processes have undergone multiple changes resulting in significant improvements in efficiency. In parallel with the development of high producing cell lines and robust chemically defined media for cell culture, the constant evolution of disposables has led to simpler operations. The use of disposables eliminates cleaning and sterilization steps, as well as cleaning validation, thus reducing costs and the time of operation per batch. Traditional disposable devices such as filters, tubing, bags, bottles, and syringes have commonly been used in biopharmaceutical manufacturing since the 1990s. The breakthrough in disposable bioreactor technology development in terms of larger capacities was the use of bag systems for cell culture. The 20-L rocker system was commercially available in 1998 and the technology became a success, especially in cell expansion operations. It allowed working with complete sterility, thereby securing the industrial cell culture processes. Further development of this system led to higher volumes and to the equipment available today on the market.    

The next step was the development of stirred-tank bioreactors with a configuration similar to conventional stainless steel bioreactors. Compared to traditional wave bioreactors, stirred-tank bioreactors offer the benefits of sparging, stirring of the suspension, and a higher utilization rate of the bag size as cultivation space. Such bioreactors are considered for use in the industry in cell amplification processes, to reach higher cell densities, or as production bioreactors. The single-use disposable bioreactor (SUB) from HyClone (Thermo Fisher Scientific) entered the market in 2006. Currently, disposable bioreactors up to 2,000 L (SUB, Hyclone and XDR, Xcellerex) culture volume are commercially available and plans are to develop 3,000 L bioreactor systems over the next few years.

In addition to disposable bioreactors, innovative single-use sensors are currently being developed, which will allow a cell culture process to run long-term in a fully disposable system. Single-use sensors also will solve technical problems sometimes raised by using plastic bags in single-use bioreactors, which can interfere with the functioning of stainless steel sensors because of static electricity problems, and therefore create drifts especially for pH measurement.

In this study, we tested the combination of a disposable bioreactor and a disposable dissolved oxygen (DO) sensor as a replacement of a standard bioreactors to run a fed-batch cell culture process developed for the production of a monoclonal antibody (MAb). The disposable equipment selected was the 50-L stirred-tank SUB by HyClone coupled to the TruLogic RDPD controller based on DeltaV technology and the disposable TruFluor DO probe by Finesse Solutions.  

The SUB was assessed both as a seed train and as a production bioreactor. Therefore, three configurations corresponding to different combinations of the 50-L disposable bioreactor and the reference 5-L glass bioreactor (fully scalable up to 300 L) were compared (Figure 1): seed train and production in a 5-L glass bioreactor (named 5L/5L); seed train in the 50-L SUB; production in 5-L glass bioreactor (named SUB/5L); and seed train and production in the 50-L SUB (named SUB/SUB).

The cell line used in this study was a Chinese hamster ovary (CHO) cell line developed for the production of a MAb. The cell culture process was performed using chemically defined cell culture media and feeds. A typical seed train was used for the production runs (Figure 1). After vial thawing, cells were grown in T-flasks and shake flasks for six days at 37°C, 5% CO

, and then transferred into a 2-L Cultibag RM Optical (Sartorius Stedim Biotech GbmH), followed by a 50-L wave bag. The two last steps of the seed train were performed in bioreactors (N-2 and N-1 steps). To properly assess the performance of the SUB as a seed train bioreactor and a production bioreactor, the cell suspension from the 50-L wave bag was split in a 50-L SUB (Hyclone, Thermo Fisher Scientific Inc.) and a 5-L benchtop-scale glass bioreactor (BIOSTAT B-DCU Quad, Sartorius-Stedim Biotech GbmH) for the N-2 step. The N-1 step consisted of the passage of the N-2 bioreactor, removing part of the suspension, and adding fresh media. Then, the seed train in the 5-L glass bioreactor was used to inoculate two 5-L glass bioreactors for the production phase. The seed train in the SUB was used to inoculate, in parallel, one SUB and two 5-L glass bioreactors for the production phase. The experimental plan is described in Figure 1 and was performed twice.    

Figure 1. Description of the process and experimental scheme. The cell amplification was performed in T-flasks and shake-flasks, followed by a passage in a 2-L bag rocker and a 50-L bag rocker. The inoculum was splitted to inoculate the N-2 bioreactors (SUB and 5-L glass vessel bioreactor). The N-1 SUB seed train bioreactor was used to inoculate two 5-L glass vessel bioreactors and itself as production bioreactor with the remaining inoculum. The 5-L glass vessel seed train bioreactor was used to inoculate one other 5-L bioreactor and itself as production bioreactor. 

The process developed in the 5-L glass bioreactors was adapted for the SUB as follows: pH, temperature, DO setpoints, and feeding strategy were unchanged compared to the 5-L bioreactor. Values for gas flow rates and agitation setpoint were scaled up by keeping the headspace renewal rate and the power per volume constant, respectively. The pH regulation was performed using CO

 in overlay and sodium hydroxide for the 5-L glass bioreactors.  

) were controlled offline on ABL5 (Radiometer Medical). Viable cell density (VCD) and viability were measured using a Vi-Cell automatic cell counter (Beckman Coulter). Metabolites and electrolytes were controlled offline using a Nova Bioprofile 100+ (Nova Biomedical). Protein production was determined using a Protein A–based assay on the Gyrolab platform (Gyros).

The 50-L SUB was operated using a TruLogic RDPD (R&D and process development bioprocess) controller (Finesse Solutions). The disposable TruFluor DO probe (Finesse Solutions) was compared to the InPro6800 polarographic probe (Mettler Toledo) at a setpoint of 40%. The disposable DO probe was inserted in a sleeve manufactured with the SUB 50-L bag. The sleeve contained the disposable sensor, and the probe contained a non-invasive reader connected to the transmitter. The standard DO probe was inserted with a Kleenpack connector (Pall Corporation) after sterilization. After insertion, the standard DO probe was calibrated as usual. The DO was controlled using the InPro6800 probe, but signals from both probes were recorded on the controller.  

The seed trains performed in the 50-L SUB and in the 5-L glass bioreactors resulted in comparable cell density and viability (Figure 2). For all configurations, viable cell densities at the end of the growth phase ranged from 2.7 to 3.2 × 10

  cells/mL, and viability ranged from 95.5 to 98.7%.  

Figure 2. Profiles of viable cell density (SUB VCD and 5-L Bio VCD) and viability (SUB Viab and 5-L Bio Viab, in %) of the seed train bioreactors. The passage of the wave bag into the N-2 bioreactors was performed at working day 20, and the passage from N-2 to N-1 bioreactor was performed at working day 23. VCD and Viab were determined using the Vi-cell cell counter (Beckman Coulter). The graphs show mean values with errors bars of two different runs. The variability of the viability is so low that the error bars are not visible. 

The production phase performed in the 50-L SUB and in the 5-L glass bioreactors also gave similar results for the maximum VCD obtained at production day 7 (working day 33) and integral viable cells (IVC) (Figure 3 and 4). The IVC curves (Figure 4) show comparable cell growth throughout the process between different scales and configurations, whether the seed train material was coming from the 50-L SUB or the 5-L bioreactor. The metabolites profiles such as glucose, lactate, glutamine, and glutamate were all comparable between different scales and configurations (data not shown).   

Figure 3. Viable cell density bar graph at production day 7 (working day 33) of the production bioreactors. The viable cell density reached a maximum value at production day 7 (working day 33) and started decreasing on the next day. The values shown here are means of two or three values with error bars. 

The MAb titers obtained at production day 7 (corresponding to the highest point of viable cell concentration) were comparable for all configurations (Figure 5), again demonstrating the similar performance of the SUB and the 5-L bioreactor in seed train and production. The titer was plotted against the IVC (slopes: 5L/5L 22.62, SUB/SUB 21.81, SUB/5L 19.23) and the relationship was found to be linear, as expected.

  The slopes were very close for the three configurations, demonstrating that the same specific productivity is reached in different configurations.    

Figure 4. Integral viable cell density of the three configurations (SUB/SUB, SUB/5L, and 5L/5L). The graphs are means of two or three runs with error bars. 

The pH in the bioreactor was regulated using CO

 in overlay in the 5-L glass bioreactors and in the 50-L SUB. The pCO

 levels were compared between the SUB and the 5-L runs to assess the CO

 stripping performance of the SUB (Figure 6). As shown in Figure 6, the 50-L SUB cultures or the 5-L bioreactor coming from the SUB seed train have appropriate pCO

 levels below 60 mmHg. In addition, to assess the impact of pH regulation using CO

 data were compared to historical data from a 5-L acid-regulated run and showed that the 50-L SUB production run with the 50-L SUB seed train have even lower pCO

 levels than the acid-regulated runs (40–54 mmHg).    

Figure 5. Product titer bar graph at production day 7 (working day 33). The viable cell density reached a maximum value at production day 7 (working day 33) and started decreasing on the next day. The values showed here are means with error bars of two or three values. 

A drawback of single-use bioreactors is the possible impairment of sensors based on electric potential differences because of the static electricity phenomena.

  However, disposable sensors now are available based on fluorescence, which may not cause the same problems. One of these probes was tested in the single-use bioreactor to monitor the density of oxygen, in comparison to a reference stainless steel polarographic probe. The trends of the two probes are identical, even if a small constant difference (<2%) is noted between the two curves, probably because of calibration (data not shown). Thus, in this specific configuration, no impairment of the stainless steel DO probe was observed in the environment of the SUB. The disposable fluorescence DO probe showed a comparable performance, and could probably be used to replace the reference stainless steel probe. 

Figure 6. The pCO2 profiles for the different configurations (SUB/SUB, SUB/5 L, and 5 L/5 L), measured with ABL-5 gas analyzer (Radiometer). The values showed here are means with error bars of two or three values.

The aim of this study was to asses a disposable bioreactor in combination with a disposable probe for a fed-batch MAb production process. The equipment chosen was the HyClone SUB coupled to the TruLogic RDPD controller and the disposable TruFluor DO probe by Finesse Solutions. The single-use DO sensor showed comparable results to conventional ones. The equipment was readily implemented because the set-up time was only one day.  

The SUB gave results comparable to the 5-L glass vessel bioreactor (small-scale reference for the process) for the seed train and the production steps. This shows that despite different material, agitation, and aeration, the disposable bioreactor had a performance similar to standard bioreactors, at least for the fed-batch process tested here. The scale-up to 50-L also was straightforward. Given that all HyClone disposable bioreactors have the same overall reactor geometry ratio up to 2,000 L, it can be expected that the scale-up to larger volumes such as 300 L could be performed using the same principles. The scale-up to a higher volume such as 1,000 L could be more complex because process scale-up is rarely linear between such different scales.   

The single-use bioreactor showed the capacity to be used either as a seed train bioreactor or a production bioreactor, or both. If this double use is to be implemented, the bag aeration configuration should be carefully defined to be able to cope with different oxygen demand in cell expansion and production. In this case, the same bag was used for both phases, a limitation in oxygen flow rate appeared toward the end of the culture.  

The bioprocess container bag used for these experiments was equipped with a 20-mm sparger membrane. The bubbles released by this system were small enough to have sufficient oxygen transfer to the culture, but big enough to strip CO

. The bag is now available with a dual sparge system, consisting in a 20-mm porous frit for the oxygen transfer and an open pipe for CO

 stripping. Many different disposable bioreactors systems coexist on the market and new versions are frequently released, showing the high dynamism of single-use technology. Each system presents its own features and advantages. Some other disposable bioreactors currently are being assessed in our company.

This study enabled us to demonstrate the applicability of using a single-use bioreactor for producing a MAb at 50-L scale, and we can expect that further scale-up to at least 300-L can be achieved. In the future, it can be expected that disposable bioreactors will become far more common in biopharmaceutical manufacturing. Their use is of specific interest when producing material for early clinical trials to avoid a capital investment early, when the final production bioreactor volume, as well as the future of the molecule, are unknown. Some people claim that the use of disposable bioreactors also has big advantages when building a new facility, because the need for utilities might be reduced in a fully disposable environment, therefore reducing start up time, installation costs, and campaign turnaround.

  On the other hand, some concerns exist about the environmental impact of disposables, although assessing the latter is far from simple. The reduced use of purified water, clean and pure steam, and cleaning chemicals compared to stainless equipment has to be balanced with the increased plastic waste. One way to reduce the impact of such waste could be to convert back part of the 32.6 GJ/ton of energy stored in plastic in waste-to-energy incineration facilities, not necessarily solving the issue of carbon footprint.

  The ultimate solution might reside in recycling these disposable products, requiring further development on innovative transformation methods. Some other interesting future directions with respect to single-use bioreactors could be the development of systems for perfusion process applications, as well as more insights on leachables and extractables.

 is a biotech process sciences upstream specialist, 

 is a biotech process sciences upstream coordinator, 

 is a biotech process sciences manager, and 

Despite different material, agitation, and aeration, the performance of the disposable bioreactor is similar to that of stainless steel bioreactors.

BioPharm International-11-02-2010

BioPharm International, November 2010 Supplement (PDF)

Implementing Single-Use Technology in Tangential Flow Filtration Systems in Clinical Manufacturing

An Economic Analysis of Single-Use Tangential Flow Filtration for Biopharmaceutical Applications

Disposable Bioreactors for Viral Vaccine Production: Challenges and Opportunities

Regulatory Expectations and Consensus Industry Recommendations for Extractables Testing of Single-Use Process Equipment

Evaluation of a Single-Use Bioreactor for the Fed-Batch Production of a Monoclonal Antibody