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Facilitate Cell Culture Process Development

BioPharm International, August 2010 Issue (PDF)

One of the biggest trends in biopharmaceutical manufacturing over the past few years has been the increasing use of single-use systems for biomanufacturing. Nevertheless, although disposables are commonly used in upstream processes, their use in downstream operations is not so widespread. 

7th Annual Report and Survey of Biopharmaceutical Manufacturing

 shows that the growth in the use of disposable systems has steadily increased over the past five years. Significant improvements and product advances have taken place in 2007 to 2009. For example, three years ago, disposable tangential flow filtration (TFF) systems suitable for bench to commercial scale seemed unlikely, but we have them today. According to the survey, the highest average annual growth rate among single-use products was for membrane adsorbers (Figure 1).   

Figure 1. Average annual growth rate (2005â2009) for single-use products in all stages of R&D and manufacture (not growth in sales but in application first usage within a facility) 

Such incremental improvements in manufacturing are important, but the real industry performance change is likely to occur when all steps in biomanufacturing, including the costly and difficult downstream purification steps, can be made "in a box," using disposable, or modular devices. There are many reasons companies are seeking this kind of solution, including easier cleaning, faster turnaround times, easier validation, and better quality management.  

Contract manufacturing organizations (CMOs) are leading the charge, partly because of the need for flexibility in their manufacturing. By enabling flexibility and quick turnaround times between process runs, disposables enable CMOs to perform different processes more efficiently within a given facility. These efficiencies can reduce operating costs and capital investments. These savings then can be passed on to the client.  

"The transition of small- and mid-scale biopharmaceutical manufacturing processes to single-use systems is almost complete," says Geoff Hodge, VP of technology at Xcellerex, noting that depth filtration harvests, filtration steps including tangential flow and virus filtration can all be addressed with single-use solutions. Xcellerex now routinely uses disposable flow paths for its chromatography steps too. "These, coupled with pre-packed columns, offer an opportunity for us to dedicate columns to a single customer campaign and eliminate any product contact crossover from one campaign to the next," he says. "In most cases, single-use sensors are available off the shelf to support downstream operations."  

As the industry shifts its attention to purification capacity and cost, new technologies such as continuous processing, multi-column chromatography, and expanded bed chromatography are being developed to address downstream processing bottlenecks. In most new downstream technologies, vendors are incorporating single-use systems. 

Nevertheless, although single-use systems are widely accepted and used in upstream unit operations, their acceptance in downstream processes has been slow. In our survey, we also measured manufacturers' and CMOs' current use of single-use systems at all stages, from R&D through commercial production. Most respondents currently use disposable filter cartridges (94.2%), but products like membrane adsorbers (45.8%) remain at the bottom of the list again this year. In particular, the use of membrane adsorbers to replace Protein A remains a challenge for the industry, although data indicate it is slowly gaining ground.   

Companies such as Xcellerex, Millipore, Sartorius, and Hyclone, to name a few, are promoting the use of flexibility in their single-use systems. According to Alfred Dadson, senior director of manufacturing operations at Xoma, "Entirely disposable upstream processes that can support commercialization are a reality, and now companies are focusing on cost-effective downstream single-use innovations."    

Our survey identified the top five new technologies and equipment; they all relate to chromatography and methods to reduce its impact as a bottleneck on the process.  Another major area for anticipated improvement is filtration and other membrane processes. According to Scott M. Wheelwright, the founder of Strategic Manufacturing Worldwide, "These are historically the major unit operations in downstream processing ... there is work on precipitation and two-phase systems, both of which have the potential to make inroads into established methods for recovery and purification." Similarly, he says, conversion of large proteins to monoclonal antibody fragments or synthetic peptides also has the potential to impact the industry, but based on the [survey] responses, not many people are betting heavily on these alternatives. That means that the innovations in traditional downstream technologies of chromatography and filtration will continue to be important. 

The industry is demanding more flexibility across all unit operations and vendors have responded by developing technologies to improve downstream processes. Our annual study also continues to show downstream processing as the primary bottleneck today, and although no fully commercialized downstream alternative to current chromatography processes exist, a number of products are in development. Once such products are available, more companies will likely increase their overall use of single-use systems. This will make it possible to begin to realize the advantages expected from fully disposable production processes. 

In addition, as new regions such as China and India begin to produce biologics and biogenerics intended for Western markets, the need for consistent quality will likely dictate a greater use of disposables, because they can be implemented more efficiently and with fewer risks of quality problems. As new biomanufacturers enter the industry and new products enter the pipeline, the disposables option will become part of the decision process from the very start. This may help start-up organizations and those in less developed regions become more effective and competitive.  

 is president and managing partner at BioPlan Associates, Inc., Rockville, MD. He is also a member of 

 ediorial advisory board, 301.921.5979,  

Downstream Trends

Although single-use systems are widely used in upstream unit operations, their acceptance in downstream processes has been slow.

Downstream Disposables: The Missing Link in the Disposables Chain

At last month's CMC Strategy Forum on Quality by Design (QbD), the discussions, as always, were refreshingly candid. Those conversations highlighted some core differences of opinion between members of industry and regulators. This time, the particular manifestation of this disagreement was non-critical process parameters. Do companies need to provide ranges for them, even though they are not critical? Do they belong in the design space? And should they become part of the company's regulatory commitment?  

The underlying issue, of course, is the same one that has been at the heart of the QbD initiative since its inception. Can companies that demonstrate a high degree of process and product understanding gain "regulatory relief"—greater freedom to make manufacturing changes (improvements) without regulatory involvement? Company representatives, of course, argued for more flexibility. The regulators showed a willingness to hear the case for reduced oversight, but expressed concern about letting go too much. In particular, regulators pointed to the potential for unexpected consequences of manufacturing changes, particularly at the edges of a design space or if the interaction of critical quality attributes is not fully understood.  

In a hallway conversation, one member of industry shared his frustration over the FDA's reluctance to grant companies more freedom. "The agency should see that companies like ours have excellent scientific knowledge, and can manage our quality with greater flexibility," he said. "Yes," I said, "But not all companies are like yours. Some don't have as much experience, and others flout the rules." 

As it turns out, just as we were wondering if the FDA could grant companies more freedom, only a few miles away in Washington, moves were being made to strengthen the FDA's oversight: US Representative Ed Towns (D-NY) introduced a bill giving the FDA mandatory recall authority. 

The bill was prompted by recent recalls of several over-the-counter drugs made by McNeil, Johnson & Johnson's consumer division. None of the defects posed a serious health risk, but the incident raised concern because of apparent gaps in the company's quality systems, such as a failure to properly investigate consumer complaints about foreign particles. What's worse, the company is accused of having delayed reporting problems to regulators, and of having quietly removed products from shelves without formally recalling them, to reduce scrutiny. The issue was particularly unsettling precisely because it happened at a Big Pharma company that you assume will resolve such matters openly, swiftly, and fully.  

As I watch this QbD debate, I find myself rooting for regulatory relief. We all want to see the continuous improvement that can come from it. Yet the McNeil case reminds us that product quality derives not just from the process and product understanding that can come out of process development, but from proper execution of the full gamut of actions required by cGMPs. Thus, regulatory relief requires that regulators trust companies to know what they are doing, 

Will the industry ever earn that trust from regulators? It's hard to say. At best, it will happen only slowly. As the FDA's Steven Kozlowski put it, "It's a bit like when your child goes off to college. At first, you want to have a lot of phone calls. Eventually, over time, you may become comfortable that he or she is acting responsibly and needs less attention." 

So far, it looks like we are in for a long freshman year. 

From the Editor

Regulatory relief requires that regulators trust companies to know what they are doing, and to do it-consistently.

Tug of War

The European Union (EU) debt crisis may have started in Greece, but it has spread throughout Europe. The immediate nature of the crisis was monetary, but the underlying cause of the problem is excessive government spending on social programs financed by uncontrolled government borrowing. Now, nearly all European governments are focused on reining in their budgets. 

As European governments begin to focus on their fiscal profligacy, spending on healthcare and bio/pharmaceuticals will get close attention. Healthcare accounts for 15% of public spending in most European countries and has been growing faster than most budget items. Government-run programs account for 75% of all healthcare expenditures in Europe and at least 70% of pharmaceuticals purchased there, according to data from the Organization for Economic Co-operation and Development and 

. As European governments make drastic cuts in public expenditures to lower deficits and reduce sovereign debt, spending on healthcare and bio/pharmaceuticals will likely be a major target.  

One key way European governments will cut spending is through their control of prices. Government agencies set prices for bio/pharmaceuticals in most European countries, and they are aggressively cutting prices. Spain and Greece already have announced plans to reduce prices for generic drugs by 25%, and Germany has announced 10% cuts.  

Another government tool will be driving more use of generics. The countries with some of the biggest problems, e.g., Spain and Italy, also have some of the lowest generic-drug penetration. IMS Health estimates that increased use of generic drugs by the 27 EU member states could generate €30 billion ($38 billion) in savings annually. Government agencies also could make it more difficult for new drugs to get coverage eligibility, especially expensive new biopharmaceuticals.  

Contract manufacturing organizations (CMOs) will feel the effects of reduced healthcare expenditures in the form of fewer or smaller batches, and pressure from clients to reduce prices. This situation will be especially difficult for European CMOs, which already must compete in a market with far too much capacity and widespread price-cutting.  

CMOs and CROs in China usually have been thought of as having labor cost advantages relative to US and European providers. However, a variety of factors appear to be pushing labor costs up in China, including industrial actions by Chinese workers; moves to increase the minimum wage and enforce tougher labor, worker safety, and environmental laws; the high cost of living in major cities like Beijing and Shanghai; and labor shortages across the country.  

The rising wages are being accompanied by an appreciation of China's currency, the renminbi, as the Chinese government looks to drive production growth for domestic consumption and control inflation.  

European and North American service and materials providers will be inclined to welcome the rising costs in China, because it will improve their cost position relative to their competitors in China. A 25% increase in labor costs, combined with a 10% appreciation in the renminbi, would raise the cost of sourcing in China by 25–35%.  European providers may get an even bigger gain in competitiveness in light of the rapid devaluation of the Euro and British Pound. After adding in the additional costs of managing long-distance sourcing relationships, bio/pharmaceutical companies will see the cost advantages of sourcing from China narrow and may stick with more local providers. 

The second order effects of the rising wage costs, however, may cause service providers in mature markets even more pain than they are facing today. That's because Chinese service providers and manufacturers will respond to their eroding competitive cost position in starting materials and simple chemistry by moving to higher value-added products and services such as formulation, advanced chemical intermediates, and biologics. This move to higher value-added services is exactly the strategy that North American and European companies have used to stay in business in the face of low-cost competition from the BRIC countries (Brazil, Russia, India and China). They will now be looking at lower-price competition in more sophisticated services and technologies that have been their refuge in recent years. 

The rising costs aren't likely to diminish global bio/pharmaceutical companies' interest in China either. China remains the world's fastest growing major economy, and rising living standards are expected to increase demand for high-value pharmaceuticals. IMSHealth has projected that spending on drugs in China will grow 20% annually over the next five years.  Bio/pharmaceutical companies will continue to be drawn to the opportunities of that rapidly-growing market. 

From a business perspective, one of the defining characteristics of the contract manufacturing industry is how fragmented it is. When an industry is fragmented, it means that there are many companies competing in the industry, and each has a relatively small market share. Fragmentation often is an indication of industry immaturity, i.e., the industry is relatively young and the sources of long-term competitive advantage have not yet been established. Fragmentation also suggests that barriers to entry for new competitors are low, and that there can be a lot of instability as companies fight for enough business to stay afloat. 

That degree of fragmentation in the dose contract manufacturing industry was recently documented in an analysis of FDA approvals by PharmSource. In our analysis, we found that 200 of the 487 products receiving FDA approval (NDA and BLA) during the period 2005–2010 involved a contract dose manufacturer. Of those 200, we were able to successfully identify the CMO for 185 (92%) of them. 

In total, 72 different CMOs had at least one of the 200 outsourced FDA approvals granted during the 2005–2010 period. Of those 72 CMOs, 44 (60%) received only one FDA approval over the five years covered by this analysis. Nearly half of the CMOs getting approvals (34 of the 72) are selling excess capacity and are not dedicated to the CMO business. 

There does seem to be market leadership emerging in the CMO industry. The 12 companies receiving the most approvals accounted for 105 (52%) of all approvals involving a dose CMO.  

 The analysis suggests that we should see more industry consolidation in the coming years. Consider that over 60% of CMOs listed in the PharmSource ADVANTAGE database that claim FDA compliance received no new FDA approvals in the period, while most CMOs that did receive an approval, received only one.  

It's hard to see how a dedicated CMO can survive long-term on one approval every five years, let alone none. Legacy products and technology transfer products (previously approved transferred from another manufacturing site to a CMO) may cover costs for a significant period, but new products are needed to replace the older products and drive the growth necessary to attract further investment.  

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

Outsourcing Insights

This month, we catch up on major developments around the world and their implications for the contract services industry.

Spanning the Globe

There has been a rapid uptake of disposable technologies in the biopharmaceutical industry over the last decade, including disposable modules for chromatography. Disposable membrane chromatography has proven to be a powerful alternative to polishing columns, particularly for process-scale monoclonal antibody purification, because of advantages such as high throughput, faster processing time, reduced buffer consumption, and elimination of column packing and packing testing activities. Disposable technologies also allow facilities to run multiple products in a single plant without the risk of cross-contamination between batches. This article demonstrates through, a case study, the practical and economic benefits gained from replacing reusable column chromatography with disposable membrane chromatography. The study looks at the whole process and evaluates the production costs for the stainless steel option, in which all unit operations are performed using stainless steel equipment items, and the disposable option, in which column chromatography is replaced by disposable membrane chromatography and all other unit operations are based on stainless steel equipment items.  

Significant upstream process advancements in the biomanufacturing industry have led to high cell culture titers producing a large mass of proteins. But we haven't seen the same improvement downstream, which now has to handle the elevated protein concentration and contaminant level. It has been much discussed in the literature that specific unit operations, such as traditional chromatography, can represent technological bottlenecks caused by dimension limitations.

  The improvement in titer affects production and material costs as the buffer, cleaning, and sanitization solutions required for downstream operations increase in proportion to the mass of product being purified.

  Therefore, large upstream protein production levels combined with the large scale demand of antibody-based therapeutics have driven the biopharmaceutical industry to search for alternate technologies to surmount the bottleneck downstream and fulfill the requirements for high-throughput production while delivering a product with high purity.

Manufacturers are exploring multiple ways of streamlining product recovery and purification processes. Strategies include standardized platform approach to downstream processes,

  and the evaluation of alternatives to chromatography separations to overcome the physical limitations of traditional chromatography.

  Among the novel technologies developed, membrane chromatography has already proven to be a robust alternative to columns, especially anion exchange chromatography (AEX) for polishing.  

AEX chromatography is perhaps the most powerful tool to remove a variety of viruses, DNA, and endotoxins.

  In most cases, AEX chromatography is carried out using flow through (FT) mode, with the impurities binding to the chromatographic support and the product and the product of interest flowing through.  

Replacing AEX column chromatography with AEX membrane chromatography has resulted in similar viral clearance at a much higher flow rate and load density compared with columns. Arunakumari, et al., recently reported >4 logs of minute mouse virus (MMV) and murine leukemia virus (MuLV) removal using Sartobind Q membrane adsorbers (Sartorius Stedim Biotech) in monoclonal antibody (MAb) processes at a load density of up to 20 kg MAb/L of membrane.

  The diffusion dependence and pressure limitation associated with column chromatography significantly limit throughput at large scales and lead to column oversizing in traditional polishing scale up. Because of the hydrodynamic benefits, membrane chromatography involves much smaller devices than columns with a similar output and no bottlenecks.

  Such disposable technology offers the added advantages of reducing buffer volume, processing time, and floor space requirement. Membrane chromatography is a disposable operation system; there is no need for column hardware, packing-related studies, resins life studies, carry-over studies, cleaning-in-place (CIP) and steaming-in-place (SIP) procedures, and validation. Membrane chromatography offers easy solutions to biomanufacturing, increases flexibility, and lowers capital requirements—all of which are benefits derived from disposable technology.  

Whereas the disposable approach has been accepted for many years for operating certain unit operations in upstream and downstream processes such as cell harvest, filtration, and buffer and media storage, an increased trend toward disposable technologies only has been observed in the last five years in response to the needs of biopharmaceutical companies. In an effort to increase capacity without exposure to excessive investment risk, biopharmaceutical companies are considering disposable manufacturing.

  The economic benefits of single-use membrane chromatography for polishing as a single unit operation has been demonstrated.

  In this paper, we analyze the cost impact of replacing reusable chromatography technology with single-use membrane chromatography technology in the FT mode for the AEX polishing step, looking at the overall cost of goods (CoG) and the breakdown of the CoG into different categories (i.e., capital charges, materials, and consumables). The FT AEX polishing step was implemented as a unit operation in the whole process at a production scale of 2,000 L. The study evaluates the production costs for the whole process based on all stainless steel equipment and compares it to the process in which the Q column is replaced by the Q membrane capsule. 

DISPOSABLE FT-AEX MEMBRANE CHROMATOGRAPHY FOR POLISHING

AEX resins typically are used as a polishing step in a FT mode for contaminant removal. Because the AEX resins remove only trace level of impurities, in principle, a small column should be significant to meet the impurity clearance requirements. However, the columns usually are over-sized at the manufacturing scale to meet process throughput requirements. AEX membrane chromatography provides an attractive alternative because of its convective mode of mass transport, which allows operation at a significantly higher linear flow-rate or smaller residence time than columns. The convective mode of mass transport coupled with the need for trace levels of impurity clearance enables the use of a membrane chromatography adsorber that is significantly smaller in size than a conventional column, which reduces buffer requirements.  

Zhou and his collaborators at Amgen have extensively studied the membrane chromatography technology using Sartobind membrane capsules.

  The authors estimated water and buffer consumption comparing a stainless steel column to disposable membrane chromatography during polishing steps for contaminant removal and virus clearance in MAb purification processes. The authors observed a 95% buffer savings with the FT membrane chromatography as compared to traditional column chromatography, regardless of the production scale.

  The buffer and water consumption did not include buffers for column packing and validation incurred with resin reuse. At the 2,000-L scale, a 35-L column was necessary to accommodate the throughput, whereas a 0.18-L membrane volume was sufficient to clear contaminants and pathogens and achieve the required product purity. The smaller membrane chromatography device provides product of the same quality while reducing floor space requirements, hardware equipment, processing time, and labor use. 

Figure 1 shows BioSolve (Biopharm Services, Ltd.), the Excel-based model that was used in this study. Models are useful tools to define process performance and gain insight into process fit in the confines of an existing plant. Of course, they only provide an approximation—a model of the real situation limited by the information available for input. The use of the model provides an estimation of CoG and CoG breakdown by cost categories. In the model, the CoG takes into consideration the fixed overheads of the facility and the variable operations costs of the process. The fixed overheads include capital charges, taxes, and insurance, whereas the variable costs include materials, consumables, labor, and waste management.

Figure 1. BioSolve model used to estimate total CoG and CoG breakdown by cost categories. Data provided by Biopharm Services.  

Figure 2 depicts the framework of BioSolve, which comprises the user interface, process definition, productivity levels, cost calculations, and outputs. The interface provides a list of predefined key input parameters (e.g., process scale, expression level, solution preparation basis, single-user options) that enable the user to perform quick what-if analyses for different scenarios. The process information is defined in the cost model, which consists of the sequence of unit operations, mass and volume balances, equipment sizing, process operating conditions, and resource allocation. The model computes the facility throughput, equipment list, materials and consumables usage, and labor requirements. The model outputs include plant productivity, bill of materials and consumables, capital expenditure, and CoG. 

Figure 2. Framework of the BioSolve model. The model comprises the user interface, process definition, productivity, cost calculations, and output. 

In this study, we evaluate the total CoG when the reusable AEX column chromatography was replaced by disposable membrane chromatography in a production process. The production process included upstream, recovery, and purification, and all unit operations were based on stainless steel equipment with the exception of the disposable Q membrane step in the disposable option (Figure 3). The process sequence was based on the manufacture of a mammalian-based MAb using generic production steps, derived from commercially relevant operations commonly practiced in the industry. 

Figure 3. Sequence of unit operations for a typical MAb production process  

The mammalian production cell culture is performed in fed-batch mode with two batch seed cell culture steps. The growth and production medium is DMEM medium with additives. The feeds are glucose and glutamine solutions. Removal of the cells is achieved by centrifugation and depth filtration. The downstream purification steps are standard industrial unit operations for the production of an antibody-based product, using routine solutions and cycle times. 

The study assumes a 2,000 L process scale with 2 g/L MAb expression titer. Table 1 shows the feed mass (3,027 g) into the AEX chromatography unit operation. Load densities of 10 kg/L for the membrane adsorbers and up to 100 g/L for the Q-column were selected. Both devices were used in the FT mode. With these assumptions, 3,027 g of product would require a 20-inch Sartobind Q capsule with 360 mL membrane volume against a 30-L column volume (20 cm bed height) of Q sepharose FF (GE Healthcare Bio-Sciences Corp.)	

The basis for the case study comparison is the steps used to define the chromatography operation (flush, equilibration, and wash). The buffers used for the disposable membrane operation are similar to that in the conventional resin-based chromatography case. Table 2 shows the subunit operations and the solution requirements for (a) the reusable column chromatography and (b) the disposable membrane chromatography. The stainless steel chromatography column requires cleaning and storage steps. The disposable nature of the membrane technology has eliminated the need for cleaning and washing, thereby reducing the amount of solutions required. 

Table 2. Subunit operation steps for a) reusable resin-based chromatography and b) disposable membrane chromatography  

COG WITH DISPOSABLE MEMBRANE CHROMATOGRAPHY

When the process is based on stainless steel equipment including reusable resin chromatography column, the overall CoG is $192/g of MAb. The CoG decreases to $184/g when reusable resin is replaced by disposable membrane chromatography (Figure 4). The need for capital requirement is reduced in the membrane chromatography option with 3% capital savings. Disposable membrane chromatography simplifies hardware installation, adding flexibility to the process. Despite a 0.3% increase in consumable cost, the overall CoG savings with disposable membrane chromatography is 4% because of reduced capital requirements, labor, and buffer and water consumption. The disposable chromatography option has the advantage of switching some of the capital costs to consumables costs that are only relevant when the plant is operational. 

Figure 4. CoG results for a typical 2,000 L scale production process based on stainless steel equipment using reusable resin and when resin is replaced by disposable membrane chromatography.   

Figure 5 represents the water and cleaning material usage with the Q membrane and Q resin. As we looked at the whole process, the buffer savings from the membrane is minimized because of the elimination of the CIP and SIP requirements that must be performed on the stainless steel-based equipment. A 3% water usage savings is observed with the membrane technology. 

Figure 5. Water usage for a 2,000 L scale production process based on stainless equipment using reusable resin and when resin is replaced with disposable membrane chromatography.  

ENVIRONMENTAL EFFECT OF DISPOSABLE TECHNOLOGIES

The increasing interest in disposable technology naturally has been followed by a growing concern about the environmental impact because of the solid plastic waste being generated from their use. Sinclair, et al., have compared the environmental footprint of a traditional biopharmaceutical manufacturing facility using fixed-in-place stainless steel equipment and a facility implementing disposable technologies.

  This paper demonstrated that the overall waste streams were reduced with disposable technologies, specifically water load and people requirements. A 25.5% reduction in CO

 emissions was estimated for the facility using disposables relative to the traditional stainless steel-equipped facility. The reduction in CO

 emission mainly is derived from the reduced usage of water, which more than compensates for the emission of CO

 associated with the use of plastics (i.e., between polymerization, transportation, and incineration).  

Disposable membrane chromatography significantly reduces the need for water load. Water and buffer usage is 95% less for disposable membrane chromatography because there are no CIP or SIP procedures. Because of its hydrodynamic benefits, membrane adsorbers can operate at much higher flow rate or lower residence time than columns, thereby shortening the overall process time and reducing labor requirements and energy consumption. The smaller membrane device sufficient to process the required throughput reduced the plant footprint and operating costs. 

The availability of membrane chromatography as prepacked, disposable, and portable capsules eliminates the need for packing and packing testing, cleaning, and cleaning validation, and adds safety to the product by preventing cross-contamination.  

Interest in membrane chromatography has grown because it can be used as a disposable device while maintaining product with the required purity; and it has been much discussed in the literature that single-use system has lower an environmental impact.

, is senior scientist, purification technologies, at Sartorius Stedim North America, Bohemia, NY, 626.241.2171 

 is the managing director, both at Biopharm Services Ltd, Chesham Buckinghamshire, UK.  

 is vice president of purification technologies at Sartorius Stedim Biotech GmbH, Göttingen, Germany.  Gottschalk is also a member of 

 1. Gottschalk U. Bioseparation in antibody manufacturing: the good, the bad, and the ugly. Biotechnol Prog. 2008;24:496–503. 

 2. Davis J. A purification platform for the production of mAbs from fermenters with titers of 5 g/L and beyond. BioPharm Int. 2009 Oct Supplement:20–3. 

 3. Davis J. Economics of Monoclonal Antibody Production; the relationship between upstream title and downstream costs. IBC San Diego 2008. 

 4. Low D, O'Leary R, Pujar NS. Future of antibody purification. J Chromatogr B. 2007;848:48–63. 

 5. Schukla A, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies—application of platform approaches. J Chromatogr B. 2007;848:28–39. 

 6. Thoemmes J, Etzel M. Alternatives to chromatographic separations. Biotechnol Prog. 2007;23:42–5. 

 7. Przybycien TM, Pujar NS, Steele LM. Alternative bioseparation operations: life beyond packed-bed chromatography. Curr Opin Biotechnol. 2004;15:469–78. 

 8. Fraud N. Membrane chromatography: an alternative to polishing column chromatography. Bioproc J. 2008;7:34–7. 

 9. Arunakumari A, Wang J, Ferreira G. Advances in non-Protein A purification processes for human monoclonal antibodies. BioPharm Int. 2009 Mar Supplement:22–6. 

Features

An analysis of flow rate, load density, viral clearance, and cost.

Technical and Economic Benefits of Membrane Chromatography During Polishing Steps

The meningitis vaccine (NeisVac-C) comprises the de-O-acetylated form of group C meningococcal polysaccharide conjugated to tetanus toxoid. The polysaccharide purification process includes base treatment and subsequent diafiltration to remove hydrolyzed cell impurities. During development, a 100 kilodalton (kDa) nominal molecular weight cut-off (NMWCO) ultrafiltration membrane was used for the diafiltration, and process yields ranged from 87 to 135%. Unexpectedly, the yield from the first 12 production lots varied from 18 to 100%. Investigation revealed that ultrafiltration membrane porosity varied with different manufacturers' lots. The corrective action included changing from a 100 kDa to a 50 kDa NMWCO membrane; this change occurred just before license approval. To support the change, a process stream sample spiked with >200% of cell impurities was purified with the tightest 50 kDa membrane available. Even under this worst-case condition, the impurities were removed to meet the original specifications achieved by the 100 kDa membrane. The change was implemented and >100 lots have since been produced with satisfactory yield and purity.

NeisVac-C was introduced in the UK in the year 2000. Currently, it is licensed in 39 countries. The vaccine is used to prevent the invasive disease caused by 

 serogroup C. The active ingredient of the vaccine is a polysaccharide–protein conjugate. Each dose of the vaccine contains 10 µg of de-O-acetylated group C meningococcal polysaccharide (GCMP) conjugated to 10–20 µg of tetanus toxoid protein (TT) and adsorbed to 0.5 mg of aluminum as aluminum hydroxide in saline.     

NeisVac-C is the only meningitis conjugate vaccine on the market that uses the de-O-acetylated form of GCMP. Clinical data collected over the past 10 years demonstrate that the de-O-acetylated form is more immunogenic than the conjugate vaccine prepared from the O-acetylated form of GCMP.

During polysaccharide purification development, we encountered a unique challenge with the ultrafiltration (UF) membrane used in the purification process. This article describes the problem and the solution.  

The antigen component of the vaccine, de-O-acetylated GCMP, was purified from the culture supernatant of 

 serogroup C, strain C11. The carrier protein, TT, was obtained from Statens Serum Institut, Denmark. The conjugation of GCMP to TT used reductive amination technology.

The GCMP content was determined by a resorcinol-HCl method. This method measured GCMP monomer (sialic acid) by a colorimetric assay using N-acetyl neuraminic acid (sialic acid) as a standard.

The protein impurity content in the process was determined by the Bradford method using BSA as a standard.

The nucleic acid impurity content was determined by absorbance at 260 nm, assuming an absorbance of 1 (in a 1-cm wide cell) for 50 µg/mL of nucleic acid.

The molecular weight (MW) of GCMP 100 kDa retentate was determined by a laser-light-scattering method (Wyatt Technology Corp) after fractionation on an HPLC with Superose-6 column (GE Healthcare).

 production strain, C11, is an O-acetyl positive strain. It produces GCMP with O-acetyl groups at either the C7 or C8 position, but not both.

  De-O-acetylation is accomplished by incubating native GCMP with NaOH at high temperature. Under these conditions, the acetyl groups at the O-positions and at most of the N-positions are removed (Figure 1). The N-acetyl groups are believed to be an important epitope of the vaccine, and therefore, are restored with acetic anhydride in a subsequent chemical reaction. The NaOH treatment also hydrolyzes the cell impurities, which must be removed. Diafiltration with a plate and frame system (Pellicon 2, Millipore) containing multiple 5 ft

  cassettes of polyethersulfone-based membrane (Biomax) was used to remove these impurities. The UF step used 10 process volumes of diafiltration with water for injection (WFI), followed by concentration of GCMP to a targeted volume. Diafiltration with a 100 kDa UF membrane was the method of choice during process development because the MW of the native GCMP was believed to be at least 300 kDa and the cell impurities had been degraded into low MW substances.   

Figure 1. The de-acetylation of group C meningococcal polysaccharide (GCMP) 

During the early phase of this process development, we encountered a serious transmembrane plugging problem. When the process stream was cooled to ambient temperature after base treatment at elevated temperature, a large amount of insoluble material soon blocked the UF membrane. Attempts to neutralize or to filter the polysaccharide before diafiltration were not successful. The final UF protocol involves maintaining the process stream at 50 °C (the highest temperature the membrane can tolerate) while performing the diafiltration. 

After use, the UF membranes were cleaned according to the manufacturer's recommendation: recirculation of 0.1 N NaOH at 40–50 °C for 45 min and 0.1 N phosphoric acid at 40–50 °C for 45 min.

  The membranes were rinsed with WFI and tested for integrity and normalized water permeability (NWP).

  An NWP specification is established before the first use of a membrane; and after cleaning, the NWP must be at least 60% of the NWP of the new membrane for it to be reused.  

Figure 2. Ultrafiltration step yield for development and production lots 

During development, the yield for the 100 kDa UF step was excellent and ranged from 87 to 135% (Figure 2). There are two possible reasons for the yield being higher than 100% in many lots: the crude starting material contains a high level of impurities and some of them may inhibit the sialic acid assay, or the de-O-acetylated polysaccharide may have a higher light absorbance than native polysaccharide in the sialic acid assay. 

When the process was transferred from development to manufacturing, the production yield dropped drastically after the 5th lot (Figure 2). At that point, production was halted and an investigation started.  

The diafiltration step is modeled with the following equation:

 is the volume concentration factor (the amount that the feed stream has been reduced in volume from the initial volume). For constant volume diafiltration, 

 is a measure of the extent of washing that has been performed during diafiltration and is based on the volume of diafiltration buffer introduced into the unit operation compared to the retentate volume. Ten volumes of WFI were used and 

 is the retention coefficient (the fraction of polysaccharide that was retained by the membrane). 

 = 1 – (GCMP concentration in the permeate/GCMP concentration in the feed). 

The ideal membrane would retain 100% GCMP with 

 = 1 and the theoretical yield would be 100%. If the UF membrane allowed GCMP to leak through, then the yield would drop drastically. The expected recovery can be calculated from the above equation using a hypothetical retention coefficient (Table 1). For example, a 10% leak of GCMP into the permeate would lower the recovery to 37%. Based on this analysis, it seemed likely that the 100 kDa UF membrane retention coefficient had changed; causing the low yield in the production lots 6–12.  

Table 1. Retention coefficient and recovery of desired component 

The retention behavior of a UF membrane can be characterized by its R90 value. R90 is not included in the certificate of analysis provided by the vendor for each lot of UF membrane but can be obtained by request. For 100 kDa NMWCO membranes, the vendor reported an R90 range of 160–485 kDa. 

R90 was determined by the vendor as part of the quality control procedure using the method described by Tkacik and Michaels.

  A mixture of dextran fractions was allowed to equilibrate in a UF system. An HPLC sizing column was used to measure the concentration of dextran in the permeate and retentate. In a plot of the rejection coefficient versus the MW of the dextran, R90 is the MW of that dextran with 90% retention. A higher R90 implies a membrane with a higher porosity. UF membranes are produced in large rolls, which are cut and assembled into UF cassettes. When new membrane rolls are produced, R90 is tested in two locations on the roll and the average is reported. When cassettes are produced, the R90 reported is the R90 of the membrane rolls used to make the cassette.  

Table 2.  R90, yield, and reuse for ultrafiltration membranes used in development and production  

After the R90 values obtained from the vendor for the membrane lots used in the NeisVac-C development were compared to those used in early production lots, the reason for the yield problem became clear (Table 2). From development through the 5th production lot, low value R90 membranes were used. Starting with production lot 6, the membranes used had much higher R90 and these lots of membrane could no longer retain GCMP completely and a low yield was observed. The membrane reuse also was tracked. There was no significant impact of membrane reuse on the yield. 

The corrective action from the investigation was to change from a 100 kDa NMWCO membrane to 50 kDa NMWCO membrane for this UF processing step. The vendor reported R90 values of 95–160 kDa for a 50 kDa UF membrane. With the change, GCMP yield returned to levels seen during development (consistently close to 100%) but purity had to be evaluated.  

To evaluate purity, the production process was scaled down 400 fold. A representative process stream was spiked with impurities (autoclaved whole cells), treated with NaOH, and then the tightest 50 kDa membrane obtainable (R90 of 93 kDa) was used to diafilter GCMP. As shown in Table 3, the removal of impurities was satisfactory at the small scale using typical starting material and spiked starting material. For the spiked starting material, the protein level dropped from 260% to <1% and the nucleic acid level dropped from 226% to 3% after NaOH treatment and 50 kDa diafiltration.  

Table 3. Small-scale purity data before and after NaOH treatment and filtration with a 50 kDa membrane 

At the time of the yield investigation, the marketing authorization application had been submitted for approval to the Medicines Control Agency (MCA) of the UK. There were communications between MCA and Baxter to finalize the product specifications. The 100 kDa membrane yield problem and the 50 kDa membrane change proposal were communicated to the MCA and the change proposal was accepted.  

The 50 kDa membrane change was implemented in production. For the five subsequent lots, the yield returned to the expected range and the purity was well within the specifications established by the 100 kDa membrane (Table 4).  

Table 4. Results from production lots after 50 kDa membrane implementation  

The size of the GCMP after 100 kDa processing was evaluated as part of the low yield investigation. Based on absolute MW determination by laser light scattering, the MW of GCMP was determined to be 200–300 kDa. A rule of thumb in UF is that the membrane of choice should have a NMWCO one-third to one-fifth of the MW of a product that is to be retained.

  According to this rule, if GCMP sizing data had been available during process development, the 50 kDa membrane would have been a better choice than the 100 kDa membrane. 

Since the UF membrane was changed from 100 kDa to 50 kDa, over 100 batches of commercial vaccine have been produced with satisfactory yield and purity.  

Throughout the production history, the 50 kDa UF membranes have been reused up to 11 times. Other UF steps have reused UF membranes for up to 26 cycles. Most of the time, UF membranes are retired because they exceed our internal specification of 24-h dirty membrane hold time. Most of the NWP values following cleaning appear to be stable. Because meningitis vaccine purification involves multiple UF steps and each UF step uses multiple UF membranes cassettes, the ability to reuse UF membrane represents a substantial savings in production cost.  

The key lesson learned from the UF yield investigation is that in designing a UF process and validation program, one should consider the sensitivity of the desired separation to variation in the retention profile of different lots of membrane.

  In this case, the UF step was used for concentration and purification of GCMP. The impact of membrane variability on product yield was significant. The impact of this variability on product yield and purity should be evaluated as part of process development and confirmed during process validation.  

, is a senior research manager, all at Baxter Healthcare,  Beltsville, MD, 301.419.8587,  

 1. Borrow R, Findlow J. Prevention of meningococcal serogroup C disease by NeisVac-C. Expert Rev Vaccines. 2009;8:265–79. 

 2. Jennings HJ, Lugowski C. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J Immunol. 1981;127:1011–8. 

 3. Svennerholm L. Quantitative estimation of sialic acids II. A colorimetric resorcinol-hydrochloric acid method. Biochimica et Biophysica Acta. 1957;24:604–11. 

 4. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. 

 5. Frasch CE. Production and control of Neisseria meningitidis vaccine. Bacterial Vaccines. 1990;123–145. 

 6. D'Ambra AJ, Baugher JE, Concannon PE, Pon RA, Michon, F. Direct and indirect methods for molar-mass analysis of fragments of the capsular polysaccharide of Haemophilus influenzae type b. Anal Biochem. 1997;250:228–36. 

How to balance porosity, plugging, and lot-to-lot variability in filters.

Meningitis Vaccine Purification: Quality Control of Ultrafiltration Membrane Porosity

The US District Court for the Southern District of New York shocked the patent community recently when, in the face of conventional wisdom, it ruled that isolated DNA sequences are not patentable. A diverse group of plaintiffs won the case attacking patents controlled by Myriad Genetics on summary judgment because of the District Court judge's application of the century-old "product of nature" doctrine. Regardless of the outcome on appeal, the 

 case will have a broad and lasting significance for the life sciences industry. 

 The crux of the case is the patentability of isolated DNA. The US Patent and Trademark Office, courts, and most commentators have long accepted that DNA, when isolated and distinguished from its natural form, can be patented. The suit challenged the validity of several Myriad patents relating to the BRCA1 and BRCA2 genes, which are strongly correlated to the occurrence of breast, ovarian, and prostate cancer, and methods for evaluating cancer risk by comparing a patient's DNA with the BRCA½ sequences.  

 Resurrecting the "product of nature" doctrine articulated by the Supreme Court more than a century ago, District Court Judge Sweet ruled that isolated DNA sequences are not patentable.  The Court applied a test requiring compositions derived from nature to possess "markedly different characteristics" from the natural material, and "possess a new or distinctive form, quality or property." According to the Court, purification of a composition, "without more," cannot make it patentable. The court opined that isolated DNA is not markedly different from native DNA sequences, and rejected the historically accepted position that distinctions between the two are sufficient to meet the "markedly different" test.   

 The court also held that certain method claims involving analyzing and comparing patient DNA with BRCA½ DNA sequences were unpatentable as mental processes. Although the DNA used in the method must be isolated from patients, that step was not recited in the claims. The court used similar reasoning to invalidate claims for screening candidate cancer drugs by measuring their effect on cells containing the BRCA1 cancer gene.   

 decision, the Supreme Court held that certain method claims were unpatentable as abstract ideas. Although the 

 decision is not directly on point, comments in the Supreme Court's opinion regarding abstract ideas and mental processes, coupled with a vigorous public-policy critique of gene patents, suggest that 

 decision is not binding on other courts. However, the Federal Circuit Court of Appeals or the US Supreme Court may adopt some or all of the 

 Court's reasoning. Because of this uncertainty, organizations involved with gene patents should proactively position their patents to meet the legal standards set forth in 

 Patent owners should use strategic patent management practices such as tailoring claims and method recitations to withstand a 

-like test, amending pending claims, and using alternative methods of IP protection. Even if 

 is ultimately reversed, these steps will strengthen the patent holder's patent portfolio. At the same time, patent licensees may want to re-evaluate the strength of the licensed patents in light of 

 and consider whether to continue paying license fees or challenge the validity of the patents. 

 case is to drive down the cost of cancer diagnostic tests and expand research efforts. Reducing healthcare costs is a crucial public priority, but many fear that the wholesale invalidation of gene patents will stifle investment in the development of new tests and drugs, preventing future advances in medicine. This landmark case, however, will continue to provoke widespread debate and concern.   

 is an attorney, both at Venable LLP, Washington, DC,  202.344.4000, 

Final Word

In light of the new ruling, patent licensees may want to re-evaluate the strength of their licensed patents.

Myriad Decision Places Biotech at a Crossroads

A number of high-profile drug and biotech manufacturing breakdowns in recent months is raising questions about industry investment in, and commitment to, modern production systems able to meet US Food and Drug Administration standards. For the past decade, the FDA has campaigned for a more efficient, risk-based approach to monitoring biopharmaceutical manufacturing activities that encourages consistent production of high-quality products. 

Yet, the massive recall in April of children's cold medicines produced by the McNeil unit of Johnson & Johnson (J&J, New Brunswick, NJ) reflects repeated failures by the company in meeting regulatory requirements. Genzyme Corp. (Cambridge, MA) continues its struggle to restore production capacity after contamination problems prompted closure of its Allston Landing, MA facility. And several vaccine makers are working hard to eliminate extraneous viruses from cell banks and to correct manufacturing issues identified by FDA inspectors. These and other situations raise questions about whether drug and biotech manufacturers are cutting corners on maintaining the quality systems needed to comply fully with current good manufacturing practices (cGMPs). 

At a hearing before the House Oversight & Government Reform Committee in May, FDA Principal Deputy Commissioner Joshua Sharfstein said that he wanted to "send the message" to manufacturers that the FDA is significantly strengthening its oversight and criminal enforcement of manufacturing activities. In a seminal speech last August (2009), FDA Commissioner Margaret Hamburg emphasized the importance of industry adherence to regulations [see BioPharm, Regulatory Beat, October 2009]. Hamburg outlined initiatives to accelerate the correction of manufacturing deficiencies identified during plant inspections and to streamline the process for issuing warning letters to those companies that fail to address violations expeditiously. That policy appears to be taking root, as the FDA steps up the volume and timeliness of untitled and warning letters, and moves aggressively to compel industry action. 

The investigation into McNeil's manufacturing problems promises to affect manufacturers more widely. Deborah Autor, director of compliance at the Center for Drug Evaluation and Research (CDER), acknowledged at the May hearing that the FDA's criminal investigative unit is examining whether there is potential criminal liability for McNeil's "phantom" recall of Motrin products in 2008 and for the company's slow response to FDA inspection citations. Sharfstein commented that the FDA plans to consider corporate structure more specifically when enforcing the law, which involves applying its experience at one facility to other operations run by the same company. Manufacturers need strong compliance programs in place, he said, adding that the FDA much prefers to see the industry adhere to the rules than for the agency to take enforcement action. 

Manufacturers of older biomedical products in older facilities should take this advice to heart and invest in modernizing operations before serious difficulties arise. Quality control problems at Genzyme's outdated facility not only are proving costly to the company, but have precipitated shortages in important treatments for rare diseases. Genzyme CEO Henri Termeer nearly lost his job and had to add new members to his board of directors in June to placate investor Carl Icahn.  

Genzyme's sorry saga began several years ago when the company began to receive citations for GMP violations from the FDA. The situation deteriorated when Genzyme discovered a virus in bioreactors at its Allston Landing facility last year, just when it was trying to scale up production to meet growing demand for new and existing therapies [see Regulatory Beat, February 2010]. The plant, which is more than 15 years old, had to shut down for several months in 2009 for a $9 million decontamination process.  

In addition to losing millions in revenue, Genzyme has to finance a $150 million program to renovate and bring the facility into compliance. Moreover, the company has to pay $175 million in fines for GMP violations and to accept an FDA consent decree that requires third party monitoring of operations to ensure that new production complies with regulations. Potentially more damaging, the FDA speeded up review and approval of competing therapies from two other firms to alleviate product shortages, a move that may limit Genzyme's ability to regain market share.  

All signs point to continued FDA emphasis on speedy and efficient responses to manufacturing violations. In June, the FDA's Center for Biologics Evaluation and Research (CBER) admonished Australian vaccine maker CSL for failing to fully correct manufacturing deficiencies found during an April 2010 inspection. In an untitled letter, the FDA cited inadequate testing of containers and closures, plus CSL's failure to investigate batch discrepancies or to establish testing procedures to ensure conformity to standards. Even though this FDA communiqué was not an official warning letter, Mary Malarkey, director of CBER's Office of Compliance and Biologics Quality, called for a meeting with CSL CEO Brian McNamee and his senior management to discuss how the company will develop a corrective action plan to address its violations and to ensure production of safe, pure, and potent vaccines.  

Although some members of Congress initially chastised the FDA for failing to keep McNeil's adulterated children's medications off the market, the agency came out looking pretty tough from the May hearing. The FDA was able to document a history of regulatory violations, failed inspections, and product recalls going back several years, along with multiple efforts by the agency to compel McNeil to comply with quality standards. Sharfstein cited frequent inspections and numerous warning letters, plus a meeting with corporate executives when those efforts failed to produce results.  

Sharfstein also made the case for stronger FDA regulatory and recall authority. The J&J recall was voluntary because, under current policy, it's very long and difficult for the FDA to compel a company to pull products off the market. Even though most manufacturers comply with FDA recall requests, there often is a lag between when the company becomes aware of adverse events and quality problems, and when that information reaches the regulators. Several members of the House Oversight Committee indicated support for strengthening the FDA's powers, as did Rep. Rosa DeLauro (D-CT), chair of the House Appropriations subcommittee that oversees the FDA's annual budget. DeLauro sent a letter to Commissioner Hamburg complaining of McNeil's "reckless behavior" in disregarding GMPs at its facilities and suggested that the FDA might benefit from added authority to recall drugs, to require company monitoring of consumer complaints, and from more resources to conduct more frequent inspections.  

The need for multiple inspections of J&J plants over the last few years also has raised the issue of whether manufacturers that require repeat site audits should pay additional fees for the extra services. Reinspection user fees have been on the FDA's wish list for several years as a way to boost revenue from those firms that create more work for agency inspectors. Some policymakers have proposed that the FDA collect fees for all field inspections, as done in Europe and many other countries. That's not likely to happen, but asking violative companies to pay more for reinspections and re-reviews may gain support.  

Another outcome may be new rules from the FDA requiring more extensive pharmaceutical company monitoring of suppliers and contract manufacturers. The FDA holds the marketer of a finished drug responsible for ensuring that outside contractors comply with GMPs, but much of that oversight currently involves review of data and reports on quality control systems and product attributes. Now the agency is considering requiring biopharmaceutical companies to conduct on-site audits to ensure that contractors comply with rules and standards. 

There's more talk of FDA injunctions and consent orders in the wake of examining a number of legal issues as part of the process for investigating McNeil's actions. The pharmaceutical legal community is watching closely to see if the FDA seeks charges against J&J and McNeil executives for not doing more personally to prevent company violations of food and drug laws under the FDA's "responsible corporate officer" doctrine; that could involve individual misdemeanor charges, which can carry stiff fines and even prison terms. 

Washington editor, Chevy Chase, MD, 301.656.4634, 

Plant closures, product recalls prompt FDA re-evaluation of GMP enforcement efforts.

BioPharm International-08-01-2010

BioPharm International, August 2010 Issue (PDF)

Downstream Disposables: The Missing Link in the Disposables Chain

Tug of War

Spanning the Globe

Technical and Economic Benefits of Membrane Chromatography During Polishing Steps

Meningitis Vaccine Purification: Quality Control of Ultrafiltration Membrane Porosity

Myriad Decision Places Biotech at a Crossroads

Manufacturing Compliance in the Spotlight

Disposable Systems as a Platform Technology for R&D and Clinical Supply

USP <63> Mycoplasma Tests: A New Regulation for Mycoplasma Testing

Development of a Novel Rolling-Tube Cell Culture Platform and Demonstration of System Feasibility