
The Integral online process analyzer platform from Dionex offers sampling and sample preparation modules, optional industrial enclosures, and the latest Dionex Reagent-Free ion chromatography (RFIC) or HPLC technologies integrated into an online IC or HPLC process analysis solution. The integral migration path concept enables online IC or HPLC to be applied in the process analytical laboratory, in the pilot plant, or on the factory floor-wherever and whenever on-line IC or HPLC is needed. Integral can be configured to meet the needs of most chromatography applications. Instrument options include single- and dual-channel IC or HPLC systems that can be configured to increase sample throughput or to perform complimentary analyses for increased information, RFIC systems for just-add-water convenience, or UHPLC systems for high-resolution HPLC analyses. 


Using carbon-resin technology, the arium 611 is capable of producing 18.2 M x cm with total organic carbon (TOC) levels as low as < 1 ppb at up to 2 L/min in a compact user friendly design that has eliminated the need for a service contract. Different cartridge packs are available to meet specific feed water quality (RO, Dl, distilled water, tap water) and application requirements. Every unit of the arium 611 family includes sterile end filtration with a Sartopore capsule with a 0.2 µm PESU membrane, validated in compliance with HIMA and ASTM F-838-83 guidelines. With four different types of systems to choose from, the arium 611 is able to meet the most stringent demands for ultrapure water in the laboratory. 

www.sartorius-stedim.com/index.php?id=7073


The Waters Xevo quadrupole time-of-flight (QTof) mass spectrometer (MS) from Waters Corporation is a highly sensitive exact mass MS/MS benchtop instrument. Xevo mass spectrometers are designed around the philosophy of engineered simplicity, which combines instrument performance with simplicity of operation to enable scientists to convert data into business-critical knowledge faster and with greater assurance. Paired with Waters Acquity UltraPerformance Liquid Chromatography (UPLC), the Xevo QTof MS is the only commercially available MS system to give scientists a method of data acquisition that captures all of the data all of the time at high speeds to maximize the amount of information collected from minimal sample amounts. 


GE Healthcare has launched ÄKTAready a liquid chromatography system designed for process scale-up and production for Phase 1–3 drug development and full scale production to GLP and cGMP standards. Simplified system handling and reduced downtime between products and batches improves cost efficiency and productivity by saving time and expenditure for start-up, labor, and consumables. ÄKTAready operates with ready-to-use, disposable flow paths, eliminating the risk of cross-contamination and the need for cleaning and validation of cleaning procedures. ÄKTAready comprises the chromatography unit, Unicorn software, and a disposable ReadyToProcess flow kit including sensors and detection flow cells. 


Millipore’s Viresolve Pro solution enables productivity gains from process development to full-scale production. The Viresolve Pro is based on a dual layer PES membrane that simultaneously delivers high parvovirus LRV, capacity, and flux. The device formats were designed to fit into the majority of processes and enable productivity gains from process development to full-scale production. Every device format is fully disposable and is shipped gamma irradiated. For the convenience of optional in situ sanitization, all Viresolve Pro devices are also caustic stable. 

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Pittcon 2009 Product Focus

America's new president has promised change on many fronts, including healthcare. Some of his planned measures will benefit the pharmaceutical industry. Others, however, will present challenges. 

One refreshing change is that we will once again have a Whitehouse that values science. For pharma, the most immediate benefit of this change will be lifting the restrictions on embryonic stem cell research. The new administration also believes in funding scientific research, though in today's recession, it is hard to imagine where new research funds will come from. 

Expanding healthcare coverage is also a priority for the new administration. As an advocate for universal healthcare (see my editorial from October) I welcome the new president's desire to reform the system, though I believe his plans are not comprehensive enough. Only by requiring that everyone be insured—particularly the young and healthy—will the system work. We also need coverage that is not linked to employment. In the current recession, many Americans will lose their jobs and their healthcare coverage at the same time. That is unconscionable. 

How expanded coverage will affect drug sales is difficult to predict, but Obama does have plans to reduce drug costs, through increased use of generics, allowing drug reimportation, and eliminating restrictions that prevent Medicare from negotiating drug prices. All of these measures  pose threats to pharma profits. These threats are far from new, however.  

Faced with major losses from drug safety problems, looming patent expirations, and generics competition, traditional pharma has increasingly turned to higher-margin biopharmaceuticals. But biologics are not a panacea for pharma's ailments. Safety concerns, for example, can affect any treatment; just think of the label changes to erythropoietin last year. And even though biologics are a growing segment of the overall market, there were still only 13 new biologics approved in the US last year, 10 of which were vaccines (including six flu vaccines), compared to 21 small-molecule NMEs. And the biggest threat to biotech profits, of course, is follow-ons. Europe has demonstrated that it is possible to have a robust system for approving biosimilars, and follow-on biologics legislation in the US is going to come sooner rather than later. Wise companies are preparing for that day. Merck's recent announcement of the formation of a biosimilars division is a sign of this change, although their choice was likely precipitated by a need to recoup their investment in GlycoFi. 

Meanwhile, the current lack of venture capital threatens the survival of small biotechs, which have become the lifeblood of Big Pharma discovery. Surely, some of these companies, and the drugs they are developing, will be saved from doom by being acquired at bargain-basement prices. But in today's tight credit market, even Big Pharma's access to credit is not a given. 

Ultimately, the success of the industry will depend on its ability to devise new strategies for a new world. This will require thoughtful long-term plans and perhaps some bold moves. As we went to press, one big move was heating up: a Pfizer takeover of Wyeth. Such a megameger doesn't strike me as bold, however; it sounds like more of the same. And given the mixed results of past megamergers, it is far from a guarantee of future profits. 

This industry is full of smart, innovative people, so I am optimistic that it will survive. In what form, however, remains to be seen. Ultimately, how change plays out will depend not only on the moves of a new Whitehouse, but on pharma leaders' ability to adapt to changing times. 

Columns and Departments

How change plays out will depend not only on the new Whitehouse, but on pharma leaders' ability to adapt to changing times.

How Change Plays Out

At the moment, single-use mixing systems are fast evolving. This is reflected by the extent of ongoing innovation, which is presenting potential users with difficult choices to make. In this article, we present an overview of the current offerings in this area, discuss end-user feedback, and provide the suppliers' perspective on the latest technologies.    

The situation for mixing systems is more complex compared with the use of bags for holding liquids because mixing systems are used for processing and the duty varies depending on the application. Table 1 summarizes the key applications and disposable technologies for cell-culture available in the market today. We are certain that this situation will gradually resolve itself over the next few years as leading technologies and companies gain acceptance and market share.  

Table 1.  Typical mixing applications and disposable technology options for cell culture 

It is, however, important in this current environment that you use a formal risk-assessment methodology to select the right mixing system on the basis of technology maturity, economics, supplier dependencies, supply chain, and validation. In our previous column published in the December 2008 issue of 

,  we described an approach that we have developed and successfully implemented for many projects that use disposable systems.  

Besides the technology selection methodology outlined in our last article, the following are a few key points to consider when evaluating disposable mixing systems:  

What are the benefits of moving to disposable technology as opposed to working with stainless steel mixing technologies for your given process? Can you quantify these benefits? Does moving to disposable mixing systems reduce the overall cost of goods of your process, improve operation flexibility, and product security?

Can a given disposable mixing technology be acquired from more than one vendor? If so, is it possible to do that for both hardware and consumables? 

Does the mixing technology have solid intellectual property (IP)? It is to be noted that there are numerous patent claims in this area—the IP situation is quite complex.

Is the level of particulate generation of a given disposable mixing technology within defined limits? 

How much floor and cleanroom height does the system require? How cleanable is the hardware?

The above list is not exhaustive but it illustrates the type of factors that need to be considered when selecting disposable mixing systems. 

In the current market, there are a large number of companies offering disposable mixing systems that evolve as companies introduce new developments and extend existing ranges. In this section, we provide an overview of the leading large-scale single-use products. 

ATMI offers the broadest range of mixing systems with a total of five different mixing technologies in their portfolio.  

The Newmix PAD drive overhead impeller mixer with the paddle enclosed in a film sleeve is suitable for a comprehensive range of applications covering all powder-to-liquid and liquid–liquid applications available in a range from 5–1,000 L.  

The LevTech Levmixer (a technology that ATMI recently acquired from LevTech) is a levitating radial impeller with two impeller sizes (4.95" and 6.35"). Standard bag sizes are available from 30–1,000 L, with the 2,000 L volume available as a custom item. Jacketed tanks are available and special bags can be customized with the Newsafe powder transfer system.  

The LevTech Magnetic Mixer system is a versatile mixing universal drive machine suitable for most powder-to-liquid and liquid–liquid applications. It is available in 30–1,000 L volumes. Magnetic coupling and agitation generates more particulates than the levitating technology. These particulates are within largely acceptable limits and are not an issue provided there is a subsequent sterilizing filtration step.  

The Wandmixer is suitable for small-scale mixing applications in the range of 5–200 L. 

The jet drive is suitable for totally contained liquid–liquid mixing applications in 50 and 200 L volumes. 

Newsafe contained powder transfer bags for media and buffer preparation applications are available in capacities from 5–100 L with triclamp port sizes available from 1–8 inches. These powder-transfer bags are available in antistatic format. 

The Millipore Mobius Mix is currently available in 100, 200, and 500 L sizes. A 1,000-L system is expected to be in the market later this year. The system includes a magnetically driven impeller with magnetic levitation technology. The system has no mechanical shaft and bearings. It doesn't have particulate generation and no recalibration is required.   

The Levmix system uses the LevTech levitated impeller licensed from ATMI (see description above) and Sartorius Stedim Biotech's Flexel 3D bag. Sartorius Stedim offers the system in both drum and  cubical Palletank format. The system is available from 50–650 L for Palletank and 50–1,000 L in drum format. Heating and cooling options are available. 

Flexel 3D Aseptic Mixing system uses a double recirculation loop system and peristaltic pumps for completely contained liquid–liquid mixing applications from 200–1,000 L. 

The Xcellerex XDM Quad Mixing system is equipped with a disposable bottom-mounted impeller. The engagement between motor and disposable impeller is by magnetic coupling, and agitation is driven through stainless-steel bearings in the impeller. The bags have an integrated three-inch triclamp port for powder addition. The system is available in 100–1,000 L sizes adapted to a square configuration container taking advantage of the natural baffling effect of this shape. Standard features also include probe sheath assemblies, floor scale or load cells, and heating and cooling options. 

The Hyclone Single-Use Mixer is currently available in 200, 500, and 1,000 L volumes. According to Jon Reid, Hyclone's EU Bioprocess market manager, Hyclone is expanding the volume range from 50 to 2,000 L and adding a docking station option, which will be available in April 2009. The Single-Use Mixer is based on the same concept as the Thermo Scientific Hyclone Single-Use bioreactor (SUB) with a single-use bioprocess container, an integrated overhead impeller, and reusable hardware. The system is available with or without a heating or cooling jacket, and has cutout sections to facilitate bag positioning and monitoring probe capability. The same hardware also can be used with open top liners for media and buffer preparation. The system sports a powder port for contained powder transfer up to 25 k in one shot into a closed bag system. 

Given the many mixing technology options available and the variety of applications, it is important to get feedback from end-users. Keeping this in mind, we interviewed several end-users to see if there are some general messages that need to be addressed by the suppliers.  

When considering mixing technologies, it is important to consider the application. We interviewed a German contract manufacturer who has been testing disposable mixers (5–200 L) for the last 18 months for ultra and diafiltration applications used in the GMP manufacture of a human therapeutic protein.  They tested the Millipore Mobius system, the LevTech system, and the ATMI Padmix system. The LevTech system was not retained because of the startup time. "If an operator accidentally switched the system off, we would lose an hour's production time," said the manufacturer. Although the ATMI Padmix system provided robust mixing, it was not retained because of the room height required to fit in the paddle for a 200-L system. The system that best fitted their requirements was the Millipore Mobius because it "works efficiently, is virtually particle free, is not cumbersome, and takes less than five minutes to set up." 

Another end-user selected the ATMI Padmix system for solid-liquid applications because it works efficiently for even the most difficult mixing applications. Contained powder transfer is also a nice feature of this system. According to this end-user, the drawbacks of the system are the ceiling height requirement because of the overhead impeller and certain manipulations of the paddle. 

The beauty of the LevTech technology as seen by end-users is that the levitating technology ensures there is virtually no particulate generation, which makes it the technology of choice for late-stage downstream processing applications. This also makes it useful for final formulation applications in vaccine manufacturing, when a final sterilizing filtration step may not be possible. The technology is suitable for a majority of powder-to-liquid applications, although the set up time for charging the superconductors (40 minutes) is not accommodating to the fast turnaround required in media and buffer preparation.  

A veterinary vaccine manufacturer tested both the Padmix and the Hyclone Single-Use Mixer for a viral inactivation step and found the ATMI Padmix very simple to use and liked the fact that it is a completely contained system. Another factor that contributed to this end-user's choice of the Padmix was that the mixer driver can be decoupled from the bag container allowing interchanging of containers. This end-user considers that disposable mixing systems have considerably matured over the last two years but durability and integrity of the bags can still be an issue. 

A major US contract manufacturer tested several mixing systems for blending chromatography column loads. In this case, they chose the Xcellerex XDM Quad Mixer working at 500-L scale. The selection was based on ease of use, mixing efficiency, and the fact that it is an all disposable impeller and that the system is reasonably priced.  

The last interviewee, a veterinary vaccine manufacturer in France, indicated that they had tested several disposable mixing systems since 2003 for final bulk formulation working with aluminium gel hydroxide. This was a difficult mixing application, in which the product had to be maintained in a sterile condition because there was no downstream sterilization before filling. They tested the Wave Mixer, LevTech Mixer, Stedim Flexel 3D recirculation system, Millipore's Mobius, and ATMI's Padmix. The Padmix provided robust mixing but was apparently difficult to set up and the LevTech technology was efficient but the length of start-up time was a major drawback. The company selected the Millipore Mobius Mixer because of the plug-and-play nature of the technology along with the particle-free nature of the mixing system. 

As disposable technologies start to play an active role in the process and become more integrated into manufacturing, factors such as cost, testing, and supply-chain security become as important as the technologies' capabilities. Below are some specific comments made during the interviews. 

 In our work with users evaluating mixing technologies, we have seen situations where the proposed mixing technologies are more expensive in terms of upfront capital costs and operating costs. This is linked to the application and the actual technology being considered. In this situation, operational and other benefits have to be identified and it may be that the stainless-steel option is the most sensible.  

 Testing the integrity of a system before use in critical application is an issue for both stainless-steel and disposable systems. Some companies also expressed reticence to use disposable mixing technology from any supplier for later-stage downstream processing applications such as bulk product formulation because of the current unavailability of a post-integrity test system on the disposable-bag systems themselves. 

How to choose a disposable mixing system that fits your particular needs.

Evaluating Disposable Mixing Systems

Recent initiatives by industry and regulators around Quality by Design (QbD) included the integration of analytical methods into the overall QbD program. The benefits of QbD such as regulatory relief, increased operational flexibility, better product quality, and lower overall manufacturing costs, are well recognized. Many small companies find the implementation of QbD to be a challenge. A two-step process can facilitate the implementation of QbD. The gradual process to QbD may be more manageable for firms and regulators. 

STEP 1:  Method Performance Space Through Validation

The first step will expand the use of validation results to define a method performance space. Changes in this space can be implemented without regulatory filing just like QbD intends to do.  

A more effective use of development, validation, and post-validation data must be achieved to obtain immediate regulatory relief and increased operational flexibility. The intent of this is to set a method performance space, similar to the current vision for the design space. The required method development and validation elements—robustness and intermediate precision—can be used to generate this data. Potential method changes should be simulated during the development and validation studies. If these changes do not cause significant shifts in the overall test results, they are within the method performance space and should not require regulatory notification. Two out of the four aforementioned benefits of QbD can be immediately obtained. This step will get us ready for a method design space.  

STEP 2:   Method Design Space Through QbD

Executing QbD for methods to the fullest extent should grant many benefits. The advantage of generating a method design space before validation is that it allows for consideration of risks early in the development process. Because the intent is to push the method performance into a fixed set of design space limits during the product development phases well before validation, QbD will demand rigorous selection, development, and optimization. This will improve actual method performance. As a contributor to overall process variation, it will result in overall improved product quality. 

Even with the use of the two-step process to QbD, small companies may still be faced with other challenges. The validation of the design space for methods through validation protocol limits can be challenging. Critical method performance characteristics may be difficult to capture at the time when the design space limits are set. The design space limits need to be exact. 

QbD limits should take into account process variability. Typically, only method variation and process variation are considered. Actual imperfections in sample or sampling consistency, for example, may require tighter QbD limits for the process or method to comply with the overall QbD limits. Specific to QbD for methods, analytical equipment and reference standard qualification should be included. 

In addition to designing the space, risk-based recovery options for failed design space limits should exist that are similar to investigation instructions for out-of-specification results for production. Further, method control charts should be connected to process control charts to allow for adjustments in design space limits when needed. 

Undoubtedly, optimized QbD for methods and processes and products will bring substantial benefits, at minimum the reduction of regulatory burden for low-risk CMC changes.  

is the director of quality at Mpex Pharmaceuticals, Inc., San Diego, CA, 858.875.6664, 

QbD for Analytical Methods

The number of biotechnology-based human therapeutic products in the late-stage pipeline along with the average cost to commercialize a biotech product has been steadily increasing over time. In addition, the biotech industry is facing unprecedented challenges in the form of a sagging global economy and rising regulatory expectations. Companies have to continue to evolve their approaches to be more efficient with respect to time, resources, and cost. This article describes some of the technologies that can help optimize time and cost of biopharmaecutical manufacturing. 

Creating processes that are not only optimal and robust but also economical in a time and resource efficient manner is the Holy Grail for the biotech industry today. Regulatory initiatives such as Quality by Design (QbD) and process analytical technology (PAT) require an  improved process understanding earlier in the product lifecycle.

Recent advances in fermentation and cell culture processes have increased cell densities and protein titers in process fluids, requiring reliable upstream technologies to handle growing production demands.

  Moreover, within the bioprocessing industry there is a strong trend toward the application of single-use technologies, eliminating the expense of cleaning and cleaning validation, a major cost factor involved in operating reusable systems. One of the commonly used technologies for primary separation after fermentation is tangential flow filtration (TFF).

  TFF processes have traditionally used cassettes or hollow fiber formats. Typically, TFF cassettes are installed in stainless steel housings that require post-use cleaning and validation of cleaning. The installation and preconditioning steps before process use are time consuming and require a reasonable level of skill to avoid potential malfunction. Hollow fiber filter systems provide an alternative primary separation technology. However, this open channel format tends to require relatively high pumping rates for optimal performance. Achieving these high rates with fully disposable equipment can be difficult. 

Membrane chromatography has already proven to be a powerful alternative to traditional packed-bed chromatography in flow-through operations, such as polishing for the removal of viruses and contaminants in biologics manufacturing.

  Case studies have been described for the purification of monoclonal antibodies (MAbs) with a high isoelectric point and have demonstrated the popularity of their implementation. As flow-through utilization has expanded, membrane chromatography applications have also included the capturing of large molecules.

  Such bind-and-elute applications imply the demand for higher capacity and larger surface membrane area compared with flow-through applications.  

Manufacturers of biopharmaceuticals are required to characterize the ability of key process steps to clear viruses. Typically, the entire manufacturing process is qualified to attain a cumulative virus reduction factor, which significantly contributes to the documentation of virus safety. In many of these manufacturing processes, a virus retentive filter is used to achieve a robust and effective virus clearance step. The key to implementing virus filtration is the assurance of virus retention and device integrity while maintaining high productivity and ease of use.      

While developments in chromatography media (resins) have brought the technique much closer to practical and theoretical limits of binding capacity, future advances are likely to focus on new surface chemistries and a more efficient use of chromatography as a unit operation. One way to gain productivity improvements is to use smaller equipment or the similar size equipment in a more time-efficient manner.  

 series and will present some of the most promising technologies that are being introduced in biotech manufacturing by some of the major biopharmaceutical vendors.   

TFF MICROFILTRATION CAPSULE FOR CELL CLARIFICATION AND HARVEST

The Kleenpak tangential flow filtration microfiltration (MF) capsule from Pall Life Sciences (East Hills, NY) uses an Ultipleat pleated filter cartridge and Kleenpak Nova capsule with their operation based on TFF principles. The capsule is configured similarly to hollow fibers or cassette formats with feed, retentate, and permeate ports. As Figure 1 shows, the pleated construction is used inside the capsule to create multiple parallel flow channels while increasing the effective membrane area in a reduced footprint. It is simple to install and requires limited preconditioning before exposure to process fluid.  

This technology has been tested with representative industrial strains such as Chinese hamster ovary (CHO) cells, bacteria such as 

, and yeast. Feed volumes of 50–100 L have been processed per single full-scale capsule (0.5 m

), depending on cell type and characteristics. The capsules can be manifolded together in series or in parallel to accommodate larger process requirements. For example, a six capsule assembly can process up to 1,000 L. Stable performance has been observed for monoclonal antibody (MAb) clarification from CHO cell feed with starting cell counts up to 1x10

  cells/mL and step yields of greater than 98% have been obtained. For clarification of hybridoma cells expressing monoclonal antibodies, filtrate flux rates of 70 L/m

/h (LMH) to a volumetric throughput (VT) of 180 L/m

 have been obtained with a protein transmission of 100%. Protein expression for these cells was approximately 0.1 mg/mL.  

A comparison of the Kleenpak TFF technology and two different types of cassettes for concentrating 

 with all three devices incorporating membranes rated at 0.2 µm (nominal) is presented in Figure 2. All three devices were operated at a crossflow flux rate (CFF) of 1.5 L/min/ft

. A five-fold volumetric concentration followed by a three-fold diafiltration buffer exchange was performed. The data shows that all three devices can be operated at a flux rate of 30 LMH throughout both concentration and diafiltration stages. Although the start and finish transmembrane pressure (TMP) values are different for the Kleenpak TFF filter compared to the cassette formats, the TMP gradient is similar for all three technologies and suggests stable operation. The flux and throughput performance of all three technologies was similar during these trials. Figure 3 presents a comparison of a Kleenpak TFF MF capsule with a Pall Microza hollow fiber module for clarification of IgG from a CHO cell culture with a cell density of approximately 2x10

/mL. Each format was operated at a pumping capacity or crossflow flux rate (CFF) of 20 L/min/m

. It is seen that the Kleenpak TFF outperforms the hollow fiber module by processing more than three times the filtrate volume for a similar terminal TMP.  

Experiments were conducted with a recombinant 

 solution at a cell density of approximately 4% w/w for production of a plasmid used for gene therapy treatment. The primary objective of the application was to concentrate the 

 feed by a minimum concentration factor of 10-fold using a single-use technology. Initial trials were conducted with the small-scale device and the filtrate flux rate for concentration was selected to be 22.5 LMH. It was estimated that six full-scale modules (membrane area of 3 m

) will be required to process 500 L of feed at a feed flow rate of approximately 21 L/min (CFF of 7 L/min/m

). To demonstrate scalability to a series and parallel configuration, a trial was conducted to compare the performance of a single full-scale module against the performance of three full-scale modules operated in series. The filtrate flux rate was maintained at 22.5 LMH for each module throughout the trial. Pressure gauges were installed throughout the system to enable accurate TMP determination across each module. The results shown in Figure 4 indicate that each of the three capsules manifolded together in series provided a VT versus TMP curve very similar to a single module operated in isolation.  

In summary, the Kleenpak TFF MF capsules' single-use TFF technology offers major benefits for cell harvesting and clarification. The self-contained format eliminates the need to purchase and install holders or housings and also reduces exposure to biologically active solutions. This is a critical consideration for manufacturers of vaccines and other products that present potential workplace hazards. The optimized feed channel screen provides good performance at low flows and the technology is scalable and reproducible. 

HYDROPHOBIC INTERACTION MEMBRANE CHROMATOGRAPHY

Sartobind Phenyl membrane adsorber from Sartorius Stedim Biotech (Goettingen, Germany) uses hydrophobic interaction chromatography (HIC) principles and assembled into a 30-layer radial flow process capsule (Sartobind). It is based on hydrophilic regenerated stabilized cellulose with the hydrophobic phenyl groups covalently attached to the cellulose matrix. The novel macroporous membrane structure with 1–3 µm pores has been designed for high flow rates and high binding capacities. As seen in Table 1, the binding capacity for a MAb on HIC membrane is found to be at par with conventional HIC resins even at significantly higher flow rates. Two MAbs were loaded onto a membrane device and on a column at specified flow rates. The bed height for the column was 30 mm and 8 mm for the membrane. The dynamic binding capacity as illustrated in Figure 5A was similar for the two formats. Combining the advantages of membrane chromatography with virtually no diffusion limitation, shorter processing time because of convective flow, and a ready-to-use disposable capsule format, the Sartobind HIC membrane adsorber represents a new membrane-based tool applicable for flow-through as well as bind-and-elute applications. It provides a robust alternative capturing large and unstable molecules that need to be processed rapidly.     

At least two subsequent polishing steps are typically implemented in MAb downstream processes. The Sartobind anion exchange membrane chromatography in combination with a hydrophobic interaction membrane chromatography offers different selectivities and could reduce host cell proteins, high molecular weight aggregates, DNA, and leached protein A to acceptably low levels that assure safety of the product. This may enable the downstream process to be just a one-column-step purification process.  

Figure 5B shows the effect of concentration of ammonium sulphate in 50 mM potassium phosphate buffer pH 7.0 on the static protein binding capacity of the phenyl membrane compared to the base cellulose membrane (without phenyl functional groups). Data shown on binding of human polyclonal antibody to the membrane indicates negligible nonspecific interaction from the cellulose base matrix. Scalability of the hydrophobic membrane adsorbers is further shown in Figure 6. The study involved loading of human polyclonal IgG in 0.9 M (NH

, 50 mM potassium phosphate, pH 7.0 on Sartobind phenyl membrane adsorber at 10 mL/min (Nano, 120 cm

/3 mL) and 500 mL/min (5-inch capsule, 5,000 cm

/125 mL), respectively. The amount of polyclonal IgG was normalized to membrane area. The normalized breakthrough curves of Sartobind phenyl Nano and 5" capsules in Figure 6 represent a successful 42-fold scale up.  

To summarize, the newly designed membrane structure in combination with appropriate hydrophobic surface functionalization provides a new scalable and disposable tool for the large-scale purification and separation of biomolecules as well as for polishing applications in the biopharmaceutical industry. The unique membrane-based technology addresses the requirements for high throughput production while reducing process time and complexity (no packing and packing testing) and adding flexibility.  

Parvovirus filters are commonly used as means of effective removal of viruses from biological products by manufacturers as required by applicable industry regulations.

  The Viresolve Pro Solution from Millipore (Billerica, MA) is based on a dual layer polyethersulfone (PES) membrane and is designed to simultaneously deliver high parvovirus retention, capacity, and flux. It is also supported by an extensive safety assurance package. The formats are fully disposable, shipped gamma irradiated, and are caustic stable. The binary gas test (BGT) has been developed as a manufacturing release test and provides an increased sensitivity over air-water diffusion in its ability to detect small defects that could negatively impact virus retention. The expectation is that high capacity, flux, and fast set-up, combined with easy integrity testing, would translate into productivity gains from process development to full-scale production, thus reducing the cost of ownership for the virus filtration unit operation.  

Table 2 shows the results of a mice minute virus (MMV) retention study where grab samples were collected from two MAb solutions at various flow decay points. These samples were collected to determine if virus passage would occur through the Viresolve Pro Micro device as the membrane became increasingly fouled. Challenging the membrane at high flow decay tests the robustness. All feed and filtrate samples were assayed using validated TCID

 procedures and large volume assays were performed for filtrate samples with a total volume of 4 mL assayed for each sample. The log reduction value (LRV) was determined as the log

 of the ratio of input virus load and the output virus load as shown in the calculation below:  

 are input volume (mL) and virus titer  (TCID

Three Viresolve Pro Micro devices were tested for each MAb. The MAb 1 solution was approximately 6.0 g/L protein concentration. The MAb 2 solution was approximately 4.5 g/L protein concentration. After adding a 1% v/v MMV virus spike, feeds were filtered through a 0.22 µm filter. As can be seen from the results obtained with MAb 2, even at 90% flow decay, no passage of MMV was observed. With MAb 1, no passage of MMV was observed even after 13 kg/m

  of the protein was challenged. It was not possible to run to such high flow decays with MAb 1, because adequate feed material was not available. The results presented in Table 2 demonstrate robust virus clearance performance across a range of volumetric loadings, mass loadings, and flow decay points.  

Figure 7 shows capacity performance of Viresolve Pro Micro devices on 16 proteins tested. Most feed streams were tested at 30 psig (2.1 bars) using constant pressure operation. No prefiltration was used except the industry standard practice of a sterilizing grade filter. The achieved protein mass capacity averaged 6,900 g/m

  when limited to 4 h processing time and 75% flow decay. The results demonstrate achievement of high mass capacities and short processing times with Viresolve Pro devices. 

Further, the BGT has been developed as a new, proprietary release test that uses a two- component gas mixture, in which there is a large difference in permeability between the two gases across a wetted membrane and concentration is measured on the downstream side. A deviation from the expected concentration is an indication of the presence of a defect that could negatively affect the virus-retention capabilities of the device. Data presented in Table 3 demonstrates the superior sensitivity of this test in comparison to the traditional air-water flux test. Three devices, constructed from the same membrane lot, were tested with air-water diffusion, BGT, and virus retention testing. Although the BGT could discriminate among devices with small differences in retention, the air-water diffusion test could not. 

In summary, the Viresolve Pro Solution can provide high capacities and high viral retention under a range of processing conditions. This performance allows for increased productivity in manufacturing operations, often reduces the system size required for manufacturing. The flexibility of the fully disposable devices allows for ease of use and speed in process development and manufacturing. Enhanced release testing delivers a higher degree of virus-retention assurance. 

INCREASING PRODUCTIVITY IN BIOLOGICS MANUFACTURING

As discussed above, single-use technologies offer higher productivity in terms of process time (cleaning is not required) as well as floor space (no cleaning equipment). However, to maximize the benefit, the technology should provide the whole unit operation. For example, in the case of chromatography step, using a single-use column alone yields only partial benefit as the chromatography skid still requires cleaning post-use. The ReadyToProcess program at GE Healthcare Bio-Sciences AB (Uppsala, Sweden) aims to provide a more integrated solution that includes the prepacked, presanitized, and prequalified columns and a chromatography system with a disposable flow path. The flow path is also presanitized and contains an air-trap, UV and conductivity meters, and a flow meter. The flow path can be assembled on the system in less than 10 minutes. As seen in Figure 8, implementation of this technology could lead to reduction in changeover time by up to three days, from seven to four days. This reduction would give a 20% to 30% increase in the number of batches produced per year for a 49-week production schedule based on seven or 14-day production campaigns, respectively. Furthermore, if changeover time is reduced to no more than one day per production line, without disrupting any of the operation ongoing in the production suite (the parallel production line concept), then the increase in the number of extra batches made per year could be between 40 to 160%. The exact efficiency depends on the changeover time and the length of a single campaign. While a similar increase in number of batches could be achieved by using duplicate columns and skids such that the cleaning is performed when the column and skid are offline, this will require a significantly higher capital expenditure and floor space for the duplicate columns and skids. Another possibility is to use a single system for all chromatography steps during manufacturing of a single batch. This will result in reduction of the cost of equipment by 66% as well as lower floor space requirement.  

Figure 9 presents a comparison of break-even plot for conventional and ReadyToProcess based operations. The analysis assumes that: 1) each batch produced generates the same level of revenues, either current or future; 2) the fixed cost for the conventional operation is three times higher than for the ReadyToProcess one; and, 3) the operating cost per batch for the conventional technology is 50% lower. The two arrows in the figure show the level of net income after tax for the reference (conventional) case and the case when 40% more batches can be produced per year, respectively. The increase in the number of batches is related to the decrease in changeover time. It is seen that the higher operating cost associated with the use of single-use technologies is outweighed by significantly lower capital cost contribution. Thus, an introduction of such technologies results in a higher net income as long as number of batches produced using these ready-to-use technologies is larger than the number of batches that could be produced using conventional techniques. A real revenue curve for an average biologic is expected to be steeper than the revenue curve shown in Figure 9.  

Technologies enabling quick changeover times, and at the same time providing high manufacturing flexibility allowing quick responses to market demands, will become more popular because they offer higher productivity as well as better economics.  

 is senior R&D engineer, applications, both at Pall Life Sciences, East Hills, NY; 

 is senior scientist, purification process development at Sartorius Stedim North America, Edgewood, NY; 

 is director, virus safety solutions, both at Millipore Corporation, Billerica, MA; 

 is R&D staff scientist at GE Healthcare Bio-Sciences AB, Uppsala, Sweden; and 

 is the director of process development at Amgen, Inc., 

Features

Choosing the right tools to enhance the process.

Upcoming Technologies to Facilitate More Efficient Biologics Manufacturing

Venture capital is the lifeblood of early-stage biopharmaceutical companies. Start-ups and companies with promising preclinical drug candidates rely on venture funding to get them to proof-of-concept—that point, usually after completing a successful Phase 2A clinical study, where companies can expect to get a big licensing deal or be acquired outright by a major biopharmaceutical company. 

In the wake of the current global financial crisis, venture capital has become increasingly hard to come by, however, and the situation could get worse in 2009. The problem has been especially difficult for biopharmaceutical companies looking for new investors: venture capitalists (VCs) are holding on to their cash to make sure their current portfolio companies have adequate funding to advance their most promising compounds. Further, the VCs are reluctant to allow new investors into their current portfolio companies because at the low prices at which early-stage companies are being valued these days, their current equity positions would be diluted too much. 

Still, the biopharmaceutical industry could fare better than some other sectors in 2009, according to the National Venture Capital Association (NVCA). In a survey of its members conducted in late November and early December of 2008, the NVCA found that 92% of VCs expect total venture investment to decrease in 2009 versus 2008. However, 58% of VCs expect investment in biotech to either grow or remain stable next year, while 42% expect it to decrease.  

VCs will face problems raising funds and liquidating investments, according to the NVCA survey. Most expect institutional investors to reduce their commitments to venture capital in the coming year, and there is concern that some limited partners will not be able to meet current commitments. An overwhelming majority of respondents (72%) expect the IPO market to remain closed in 2009 and 87% expect transaction values to decline. Most expect returns on VC investing to decline over the next 10 years. 

Because early-stage companies are dependent on contract research organizations (CROs) for most of their development activity, and because they typically take compounds only through proof-of-concept, the venture capital downturn will hurt service providers that focus on preclinical and Phase 1 services, including toxicology testing, process development, and small-scale manufacturing. This is especially true for the many small- and mid-size CROs whose customer base is comprised primarily of the venture-backed companies. Even market leaders in early development are feeling the pinch. In late December, Covance reported that its fourth quarter 2008 toxicology and clinical pharmacology revenues were down from what was expected. 

Covance blames two developments for its disappointing outlook. One is a slowdown in new projects coming from smaller biopharmaceutical companies. Projects from smaller biopharmaceutical companies are usually one-off engagements, and they depend on the flow of new projects to replace completed projects. With funding down, those replacement projects aren't forthcoming. Those engagements only account for 10–20% of Covance's volume, but the slowdown is enough to hurt the company's overall performance.  

The bigger concern for Covance are the project delays coming from what the company termed "strategic clients," i.e., the major biopharmaceutical companies, some of which have dedicated space contracts with Covance. Although the company expects many of the delayed projects to proceed in 2009, it does not expect its dedicated space clients to exceed their contract minimums in 2009 as they have in past years.  

Despite the near-term uncertainty, large CROs and contract manufacturing organizations (CMOs) like Covance and Lonza are well positioned in the long-term. That's because their core customers are increasing the share of their development activity that is outsourced. In 2008, for example, Eli Lilly and Company effectively outsourced all of its toxicology testing to Covance while Novartis outsourced much of its biopharmaceutical process development and clinical manufacturing to Lonza under long-term contracts. 

Most major pharmaceutical companies have established global sourcing units that are pursuing strategies to reduce outsourcing costs by standardizing processes and reducing the number of vendors that must be qualified, audited, and managed. These programs started five years ago and resulted in companies reducing the number of CROs they work with by as much as 80%. The major CROs and CMOs are well-positioned to be selected as preferred providers thanks to their large-scale, abundant capacity, and financial strength. 

Things don't look so good for the small- and mid-size CROs and CMOs, however. With the major biopharmaceutical companies consolidating their vendor base and the early-stage companies having trouble raising cash, the small service providers are facing a rapidly shrinking market. Many entered the market only recently, attracted by the growing pipeline and ample funding for early-stage companies. They have minimal business development and marketing experience, haven't created brand equity and long-term relationships with better-funded companies, and are not well-capitalized. We expect a number of them to disappear in the downturn. 

What is normal in the foreseeable future is likely to be very different from what was normal in the recent past. The global financial crisis has changed investors' perception of risk and undermined their confidence that they can manage it. Major institutional investors are indicating that they plan to reduce their allocation venture capital in coming years. VCs will have a tougher time getting an attractive return on their investments so long as IPOs remain scarce and valuations are low.  

The new normal will consist of somewhat fewer opportunities than the industry has been used to, but it will lead to a smaller but healthier and more capable CRO and CMO industry. 

 is president of PharmSource Information Services, Inc. , Springfield, VA, 703.383.4903, 

What the current lack of venture capital means for the CRO market.

Less Venture Capital Means Less Work for CROs

To reform the nation's healthcare system to make quality care more available and affordable, new strategies are needed to tackle the ever-rising cost of health services and products. The Congressional Budget Office (CBO) and the Centers for Medicare and Medicaid Services (CMS) estimate that the nation will spend $2.6 trillion for healthcare in 2009, 17% of the gross domestic product (GDP). Without any policy changes, that amount will rise to 20% of GDP by 2017 and nearly 50% in 2082, according to a CBO report on "Key Issues in Analyzing Major Health Insurance Proposals".

Annual spending will increase from $8,300 per person today to $13,000 by 2017, and federal outlays for Medicare and Medicaid will grow from $720 billion in 2009 to about $1.4 trillion by 2019. The number of uninsured, moreover, will rise from 45 million today to about 54 million in 10 years because health insurance premiums will increase much faster than income, making coverage more difficult to afford. 

One bright spot in the health spending picture is a slowdown in expenditures on prescription drugs to a 45-year low in 2007, according to the CMS Office of the Actuary. Outlays for drugs rose only 4.9% to $227.5 billion, the slowest rate of growth since 1963. And prescription drug prices increased a paltry 1.4% in 2007, even less than the 3.5% price rise in 2006.

Still, the portion of the economy devoted to healthcare continued to expand as outlays for hospitals, physicians, and other healthcare services grew faster than inflation. Spending on prescription drugs is projected to total $264 billion this year, with about $100 billion coming from government coffers. The Medicare drug budget is slated to exceed $50 billion this year, an amount that attracts attention from budget cutters on all sides.  

One prime target of reformers is to promote generics competition for costly biotech therapies. The CBO estimates that the federal government could save $9.2 billion over 10 years by establishing an abbreviated pathway for the Food and Drug Administration (FDA) to approve follow-on biotech therapies (FOBs) for market. For its analysis, the CBO assumes a 12-year exclusivity period for brand-name products plus limits on requirements for duplicating innovator clinical trails. The resulting lower-priced FOBs would save Medicare and Medicaid about $8.1 billion over the 2010–2019 period, while other government health programs would see costs drop by another $1 billion during that time.  

The savings would be even greater—about $12 billion over 10 years—if the government also revised billing codes for biologics dispensed by physicians under Medicare Part B. Placing an FOB in the same billing code as a brand-name counterpart would provide a strong incentive for physicians to prescribe the biosimilar because CMS would reimburse physicians based on a weighted average of the prices paid to manufacturers for all drugs in the same billing code. Consequently, a physician dispensing the less expensive FOB would be able to retain the difference between the product cost and Medicare payment, while those providers dispensing the brand-name product would face a financial penalty.  

Manufacturers object that such incentives risk dispensing biosimilars that raise concerns about patient safety and therapeutic efficacy. And the CBO acknowledges that authorizing FOBs could cut revenues to biotech companies and reduce R&D in new therapies. But the analysts point out that these losses would be offset by larger gains for private health insurance plans, which would translate into lower insurance premiums, higher wages, and a parallel boost in federal tax revenues.  

Medicare drug plans already are lowering outlays for expensive drugs covered under Part D by revising formularies to have four or more tiers, including a top tier for specialty drugs that cost more than $600 a month. Specialty tier drugs now routinely carry coinsurance rates up to 33%, compared to a median 25% three years ago, according to an analysis presented to the Medicare Payment Advisory Committee (MedPAC) in December, requiring patients to shoulder a larger portion of the cost. 

Another promising strategy for cutting drug expenditures, according to the CBO, is to mandate rebates on medicines purchased for Medicare beneficiaries, an idea championed by Rep. Henry Waxman (D-CA), newly named chairman of the House Energy and Commerce Committee. Waxman believes that manufacturers are raking in big profits from Part D caused by the elimination of rebates previously paid to state Medicaid programs for drugs delivered to low-income seniors now covered by the Medicare drug benefit.  

The CBO says that a mandatory 15% rebate of the average manufacturer price beginning in 2011 would save the government $33 billion over five years and $110 billion over 10 years (2011–2019). Manufacturers would have to pay the rebates or lose sales to Medicare Parts B and D, as well as to Medicaid, the Veterans Health Administration, and other government health programs. Although the envisioned policy is similar to the Medicaid drug rebate program, it would drop the best price provision to avoid affecting prices negotiated by private purchasers. 

The downside is that manufacturers would try to offset the added rebates by charging higher prices initially and developing new strengths and dosage forms for existing drugs that could command higher prices. If manufacturers cannot recoup some of the lost revenues, the policy could curb industry investment in R&D.  

Other strategies for cutting expenditures are less promising, says CBO, despite all the rosy predictions last year about how certain innovations would save billions and improve quality of care. Wider adoption of health information technology, for example, would require considerable up-front investment and thus would reduce overall spending only modestly.  

Prevention and disease management programs may moderate the need for expensive care for some patients, but such initiatives have costs, especially if provided for large populations. Anti-obesity and anti-smoking campaigns that enable people to live longer, moreover, may increase demand over the long run for more care for the elderly. Modifying the system for determining medical malpractice similarly would only have a modest impact on total healthcare expenditures. And funding research that compares the effectiveness of different treatment options may have long-term benefits, but high front-end costs.  

Similarly, the CBO predicts that permitting the federal government to negotiate lower Medicare drug prices with pharmaceutical companies is likely to produce small savings. The Secretary of Health and Human Services (HHS) might negotiate small savings by persuading manufacturers to reduce prices for select single-source products, but the agency would not have sufficient leverage to secure significant discounts.  

Moreover, the high cost of filling in the dreaded Part D coverage gap rules out that strategy. Such a move, says the CBO, would cost $42 billion during 2010–2014 and $134 billion over 10 years.  

These issues will be examined closely in coming months as Congress tackles critical health policies. The legislators moved quickly to reauthorize the State Children's Health Insurance Program (SCHIP). Now they are working to gain agreement on a 2010 budget, tax changes, and additional economic stimulus proposals.  

Looming ahead is the Medicare physician pay fix. Last summer, Congress enacted a limited Medicare reform bill that put off hard decisions on revising Medicare's outmoded payment system for doctors and set the stage for another heated battle over Medicare rates. A lot of skeptics maintain that there's no time to address broad health issues this year, but reform advocates insist that American industry cannot be competitive globally unless the nation develops a more efficient and cost-effective healthcare system. 

Washington editor, Chevy Chase, MD, 301.656.4634, 

 1. Congressional Budget Office [homepage on the Internet]. Key Issues in Analyzing Major Health Insurance Proposals. 2008 Dec [cited 2009 Jan 2]. Available from: 

 2. CMS Office of the Actuary, Health Affairs. National Health Spending in 2007. 2009 Jan/Feb;28(1). 

With cuts in healthcare, biotech drugs are under scrutiny.

Biotech Drug Prices Under Scrutiny

Using one unified single-use cell culture platform for facilities that require culture of different types of cells can significantly reduce equipment costs, the risk of cross-contamination, and the requirement for training, cleaning, sterilization, and validation. Here, a bioreactor platform consisting of a presterilized, flexible, disposable plastic bag on a rocking platform and filled with media and a gas mixture is shown to support the growth of

, Chinese hamster ovary (CHO) cells, and hybridoma cells as measured by the daily cell density, nutrient consumption, and production of metabolites. 

Single-use disposable components offer many advantages in biologics manufacturing, and they collectively provide a platform to culture diverse cell types in a facility. This is particularly important for multi-use contract manufacturing facilities such as PacificGMP that service the needs of diverse clientele.    

Disposable components include bioprocess bags, tubing, capsule filters, tangential flow capsules, bioreactors, chromatography capsules, and mixing systems. They are supplied clean and ready to use, which obviates the need for sterilization and decreases the requirement for services such as water for injection (WFI) water systems, and steam generators. Disposable components are not used for subsequent operations, eliminating the chance of cross contamination between process runs. Because the need for stainless-steel equipment is reduced or eliminated, long lead times for equipment installation can be avoided. Single-use ystems are less complex, reducing engineering requirements. There is no need for clean-in-place (CIP) or steam-in-place (SIP) operations, or any of the associated piping, valves, controls, or pressure rating of vessels. Moreover, the use of disposable components reduces the complexity of validation. Because there are fewer reusable components, fewer items need to be tracked, and extensive validation studies for sterilization and cleaning can be eliminated. Finally, by removing the limitations of hard piping and stationary tanks, disposable components allow for operations to be more rapidly reconfigured for a new process run. 

There are several options for disposable, scalable bioprocess bags for completing upstream processing steps. The design of a cell culture system will affect the shear forces experienced by cells as well as the mass transfer of gases, and different types of cells will have different sensitivities to these parameters. Generally, mammalian cells are more sensitive to shear forces than bacteria, but bacteria are more sensitive to oxygen limitation. The following studies demonstrate that the Wave Bioreactor (GE Healthcare Chalfont St. Giles, UK) effectively supports the growth and productivity of different types of cells, and the versatility of the platform is further demonstrated by the flexibility of feed strategies and scalability of the systems. 

Each bioreactor consists of a presterilized, flexible, disposable, plastic Cellbag that is placed on a rocking platform and filled with up to half of the bag volume with media. The bioreactors used in these studies were the System20/50EH, System200, and System1000 (GE Healthcare) with maximum culture volumes of 25, 100, and 500 L of media, respectively. The following naming convention will be used to identify the culture set up: the volume of media in a Cellbag of a certain volume, e.g., 100/200 L indicates 100 L of media in the System200 bioreactor. 

The remaining volume of the Cellbag is inflated with a process gas mixture, composed of carbon dioxide (CO

), which is mixed with air in a controlled manner. These gases are added using a sterile inlet filter that is pre-attached to the bag. Continuous airflow provides oxygenation and gas exchange for pH control and CO

removal. Exhaust air passes through another sterile filter and a backpressure control valve. The backpressure control valve ensures that the Cellbag is always fully inflated at any airflow and prevents overinflation.  

Liquid mixing and mass transfer of gases are achieved by rocking the Cellbag back and forth at a user-determined rocking angle and frequency. The waves generated by the rocking motion greatly increase surface area to enhance gas transfer. The wave motion also promotes bulk mixing and off-bottom suspension of cells and particles without any damage to the cells. Temperature is controlled by a heater located in the base plate of the unit that warms the underside of the bag. The heater is regulated by a noninvasive temperature sensor that is also in the base plate of the unit. 

Additional ports allow sterile additions and sample withdrawal without the need to place the bioreactor inside a laminar flow cabinet. A sterile tubing welder is used to attach media, buffer, and glucose stock solutions to the system. 

A hemacytometer was used for cell counts, and viability was established by trypan blue dye exclusion. Optical density (OD

) was measured with a spectrophotometer (Bio-Rad SmartSpec 3000). Cell culture biochemistry analysis was conducted on a Bioprofile 300A and 400 (Nova Biomedical, Waltham, MA). The pH was determined online using the Cellbag probe and offline using a PHM220 pH meter (Meter Lab, Lyon, France). Oxygen, CO

, temperature, rocker speed, and angle were determined and controlled through the Wave Bioreactor controllers. 

The 20/50-L bioreactor was seeded from an overnight 

 culture in 20 L of Terrific Broth with additives. The culture was maintained for 10 hours at 40–45 rocks per minute (rpm) and a rocking angle of 11.9–12.0°. A foam trap was attached in place of the outlet filter to avoid clogging from the high rpm and angle used for bacterial fermentation. 

 of 19.8 was reached after 9.5 hours (Figure 1) versus shake flasks cultures with maximum OD

 of 2.0 after overnight culture. This strong growth was supported by maintaining the dissolved oxygen at >75% (Figure 2). Initial glutamine and glutamate concentrations were approximately 6 and 8 mM, respectively, and were reduced to 4 and 3 mM (Figure 3). Variability (2–10 g/L) in the glucose concentration indicates spiking of the media followed by consumption. Even with the rapid growth of the bacteria, pH was maintained at an average of 6.75.  

Therefore, by using the Wave platform, an improvement in bacterial growth compared to shake flask culture of almost 10-fold was achieved without any optimization of growth media or feeds.  

Wave bioreactors were seeded at a cell density of 1–2x10

  cells/mL in HyQ ADCF-MAb media (HyClone, Logan, UT). The 25/50-L bioreactor ran at 22.5–24.4 rpm and an angle of 8.0°. The 100/200-L bioreactor ran at 20.1–21.1 rpm and 8.0° angle. The level of CO

 was adjusted to maintain the pH of the cultures at 6.9–7.1. The 25/50-L run required 6% CO

 with the exception of the last day at 4% CO

, whereas the 100/200-L run occurred in the presence of 7–8% CO

 was added to the 100/200-L run on the fourth day after perfusion was initiated.  

Perfusion was initiated one day after the bioreactors were at full capacity. In perfusion mode, feed solutions were fed to the bioreactor continuously, and spent media was constantly removed. Running a process in perfusion mode compared to in batch or fed-batch mode can increase the longevity and cell density of the culture, which in turn increases the overall productivity of the run. The perfusion rate (percent total culture volume per day) started at 5–12% and increased to >50% by the second or third day of perfusion. The maximum cell density in the 25/50-L and 100/200-L bioreactor was approximately 9x10

  cells/mL, respectively (Figure 4). These cell densities occurred 14 and 6 days after seeding and corresponded with a cell viability of 65–77%.   

The concentration of glucose was generally 3.0–3.5 g/L. The use of perfusion kept the glucose metabolism product, lactate, relatively low compared to cultures in shaker flasks. The maximum lactate for the 25/50-L and 100/200-L bioreactor was 1.15 and 1.25, respectively. The level of glutamine in the 25/50-L bioreactor was maintained between approximately 1–2 mM until day 13 of culture, and the product from glutamine metabolism, ammonium, was generally 3–4 mM (Figure 5). Ammonia production in the 100/200-L bioreactor was similar, but the glutamine was initially above 2 mM and fell to below 1 mM by day five of culture.  

Perfusate was collected for 11 and five days from the 25/50-L and 100/200-L bioreactors, respectively. The total collected media volume of 151 L from the 25/50-L bioreactor contained an average IgG concentration of 117 mg/L. From the 100/200-L bioreactor, 320 L of media that was collected yielded an average IgG concentration of 73 mg/L, which correlates to the reduced viable cell density in this bioreactor compared to the 25/50-L. 

The controlled addition of nutrients and resultant lower levels of metabolites allowed for maximum product concentrations of up to 124 mg/L. This is an improvement of approximately 80% compared to shake flask cultures, which typically reached a maximum of 22 mg/L.  

Suspension cells were seeded from spinner flasks into the 25/50-L, 100/200-L, and 500/1,000-L bioreactors at 1x10

  cells/mL in serum free IS CHO-V (Irvine Scientific, Santa Ana, CA) with additives. Seeding cells at lower densities (e.g., 6x10

  cells/mL) is also possible with no deleterious effects on the cell growth or production. In addition, the cells can reach much higher densities inside of the Cellbag than a spinner flask. This can most likely be attributed to the increased oxygenation inside the Cellbag, as well as the ability to control pH. In a spinner flask, the density was maintained between 2x10

Are disposable bioreactors effective for cell culture?

BioPharm International-02-01-2009