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CMO's Ratchet up Use of Single-Use Devices

BioPharm International, November 2011 Issue (PDF)

The outcry from physicians, pharmacists, and patients over disruptions in the supply of vital medicines to treat cancer, pain, and other serious conditions is drawing attention on Capitol Hill and throughout the healthcare system. Oncologists cannot obtain widely-used chemotherapies to treat seriously ill patients. Surgeons are postponing operations because they lack key anesthetics and pain medications. Doctors are struggling to obtain vital parenterals for premature babies and for patients requiring infusion. And dozens of clinical trials have been halted because investigators don't have adequate supplies of cancer drugs used as components of treatment arms or as controls. 

While there always have been periodic drug shortages, the problem has become much more acute in the past two years. The main culprit appears to be drug-manufacturing and supply-chain failings, particularly related to the production of generic sterile injectables. Decisions by firms to exit certain low-profit markets often leave only one or two producers for a widely-used parenteral or other product. Some manufacturers have had to halt production due to problems meeting GMPs. Such developments raise charges that too-strict FDA oversight aggravates the problem—or that industry negligence and business practices put the public health at risk. Another possibility is that policies and practices that drive down reimbursement for old-line generics discourages industry investment in certain therapeutic categories.  

These issues made headlines in September as Congressional committees held hearings on the drug shortage crisis and broader pharmaceutical supply-chain problems. An FDA public workshop on Sept. 26, 2011, provided a forum for health professionals, patient advocates, drug distributors and manufacturers to air concerns and propose remedies for short drug supplies. The resulting publicity has boosted Congressional interest in legislation to address pharmaceutical supply-chain issues in general, and the drug shortage situation in particular. FDA has prepared a report analyzing steps it can take to tackle shortages with its current limited authority, and the Government Accountability Office (GAO) is examining the causes and responses to the drug-shortage crisis.  

Policymakers may take action because, despite extensive FDA efforts, "drug shortages are getting worse," said Douglas Throckmorton, deputy director of FDA's Center for Drug Evaluation and Research (CDER), at the FDA workshop. The agency recorded 178 drug shortages in the US in 2010, up from 157 in 2009—and much more than the 50–60 per-year range of previous years, reported Edward Cox, coordinator of CDER's drug-shortage program. The problem is even more serious when looking at all drugs and biologics: the University of Utah Drug Information Service recorded 210 shortages this year as of mid-September, slated to surpass last year's total 211 short-supply problems. And because supply problems often last more than a year, some 260 active shortages are in the Utah tracking system.  

Moreover, 74% of the shortages reported to FDA last year involved sterile injectables—and almost all of them for "medically necessary" drugs, noted Howard Koh, assistant secretary for health in the US Department of Health and Human Services (HHS), at a September hearing before the House Energy and Commerce Health subcommittee.  

Shortages in parenterals have taken a toll on hospitals and clinics: a survey by the American Hospital Association in June 2011, found that virtually all hospitals have experienced at least one drug shortage this year, and half report 21 or more supply problems. This builds on a March survey by the Premiere Healthcare Alliance that documented soaring shortages along with exorbitant prices for these products (see the June 2011 Washington Report column, "Safety Concerns and Shortages Challenge Manufacturers"). 

Koh cited a long list of underlying factors driving the short-supply trend:  industry consolidation, limited raw materials, changes to inventory and distribution practices, production delays, increases in demand, and business decisions to close down a manufacturing site. When there are only one or two sources for a crucial drug, explained pharmacist Richard Paoletti of Lancaster (Pennsylvania) General Health, a small shift in production lines, plus just-in-time inventory controls at hospitals, make it hard to buffer the impact.  

One response is for manufacturers to inform FDA in advance of supply problems. The agency reports that early warnings helped it head off 38 potential shortages in 2010 and 99 so far this year. When FDA knows of a looming supply interruption, its staff can expedite the review and approval of supplements for new suppliers, alternative production sites and changes in specifications—actions responsible for most (84) shortage preventions this year. Sometimes, FDA can bend the rules to permit continued marketing of a violative product with some corrective action. And the agency has had some success in encouraging other firms to ramp up production or to enter a market through speedy regulatory actions.  

FDA also has approved temporary importation of unapproved sources to fill supply gaps, a strategy that helped alleviate shortages this year in several cancer drugs. APP Pharmaceuticals worked with FDA to reduce a shortage of the widely used anesthetic propofol by importing the product from Germany. 

Unfortunately, FDA often lacks information on looming shortages because, under current law, only sole-source manufacturers of critical medicines have to notify FDA of plans to discontinue production. Some companies voluntarily inform FDA of problems likely to lead to short supplies, but most do not, and often the information comes in too late for timely resolution. 

To remedy this limitation, Congress is looking to enact legislation that would require six-months advance notification of production changes for a broad range of prescription drugs. Rep. Diana DeGette (D-CO) has bipartisan support for legislation in the House, and Sens. Amy Klobuchar (D-MN), and Robert Casey (D-PA) lead the early-notification campaign in the Senate. They propose leeway for manufacturers who show that a manufacturing problem could not be anticipated, as well as penalties for failure to comply. A related proposal requires manufacturers to inform FDA of situations that make a product vulnerable to shortages, such as a single API source, to highlight the need for back-up emergency sources.  

Although advance notification may help FDA head off some shortages, the danger is that wider release of such information could lead to hoarding and price gouging. Gray-market profiteers are inundating health professionals with faxes and phone calls offering scarce drugs at huge markups. Rep. Elijah Cummings of Maryland, top Democrat on the House Oversight and Government Reform Committee, launched an investigation of secondary distributors last month, seeking information on sources and profits for certain drugs. His targets include high-priced offers of leukemia treatment cytarabine by Allied Medical Supply (Florida); Superior Medical Supply (Colorado) offers for paclitaxel; Premium Health Services (Maryland) sales of leucovorin; fluorouracil offers from PRN Pharmaceuticals (Maryland); and Reliance Wholesale (Florida) marketing of magnesium sulfate.  

To avoid profiteering, FDA wants to be informed by manufacturers of possible shortage situations, but doesn't necessarily want to make that information public right away, noted Sandra Kweder, deputy director of CDER's Office of New Drugs at the House hearing. The agency first wants to assess if the problem is real, its likely impact, and what actions can be taken to mitigate difficulties. "Early notification to FDA is a very useful tool," said Kweder, "but it's different from early publication." 

Advance notification is not always possible. FDA officials concede that many manufacturing problems cannot be anticipated (e.g., equipment breakdowns and plant fires, and earthquakes, volcanoes, and other natural disasters)can suddenly disrupt supplies and material transport.  

Manufacturers now support stronger early notification requirements, largely to reduce complaints about industry responsibility for most shortages. FDA reports that most supply problems arise from GMP and product quality failings, along with difficulties obtaining active ingredients or problems at a manufacturing site. Industry consolidation has reduced the number of generic-drug firms making sterile injectables, Cox observed at the FDA workshop, and it takes time for another manufacture to establish a facility to produce these more complex and costly medicines.  

Reliance on contract manufacturers also has led to shortages for both brand and generic drugs. Johnson & Johnson faces serious supply problems for cancer and AIDS treatment Doxil (liposomal doxorubicin) because its contract producer, Boehringer Ingelheim's Ben Venue Laboratories, decided to exit the CMO business.  

The strong link between manufacturing issues and shortages raises questions about pharma's commitment to quality production. FDA wants manufacturers to prevent shortages by developing continuity of supply plans, with backup suppliers and alternative production strategies for critical products. Industry has a responsibility, FDA officials assert, to proactively identify and promptly resolve manufacturing problems and to implement quality-by-design strategies to prevent failures. 

An alternative view is that FDA creates shortages through overly aggressive enforcement of manufacturing rules. Agency officials maintain that they don't halt production for minor violations, but only for significant problems with drug sterility and contamination. Before requesting a drug recall or seizure, they check to make sure such action won't precipitate a shortage. But strong action is needed when inspectors find glass and metal particles in vials and new impurities and degradants. In some cases, companies have been cited multiple times for violations and still fail to correct manufacturing deficiencies until threatened with total shutdown.  

FDA officials also emphasize that they can act fast to help bring online a new producer or new supplier when needed.  "We can turn things around in a matter of weeks," Kweder insisted at the House hearing, in response to manufacturer claims that FDA requires two-to-three years to approve a new manufacturing site. "This is not business as usual," she stated.  

But stakeholders feel there is more that FDA and other government agencies can do to prevent critical shortages. FDA should revise how it calculates risks and benefits from regulatory action to give greater weight to patient safety issues that arise with shortages. The federal government should establish stockpiles for medically necessary drugs, as done for treatments against bioterrorist attacks and pandemics. And antitrust officials should scrutinize proposed pharma company mergers to assess how the combination would affect limited drug supplies.  

At the same time, there's interest in providing more incentives for manufacturers to enter depleted markets. Tax credits or rebates could spur manufacturers to update facilities or launch production of low-profit drugs. Some kind of exclusivity could be offered for new production of a drug in short supply. Or, generic-drug makers might be eligible for reduced user fees on applications to produce hard-to-obtain medicines.  

Shortages in controlled substances, such as long-acting painkillers and drugs to treat children with attention deficit hyperactivity disorder, are generating calls for the Drug Enforcement Agency (DEA) to work more closely with FDA and industry to modify limits on active ingredients. Manufacturers receive DEA annual quotas on controlled drug substances, but would like a way to transfer allotments when one company ceases production. 

There's also support for more resources for FDA to deal effectively with shortages. FDA formed the drug-shortage program in 1999 to manage anticipated supply disruptions from the Y2K shift to the new millennium. But with only five staffers and limited legal authority, FDA is hamstrung in preventing supply disruptions.  

Parallel to the shortage crisis, FDA has been campaigning for more authority to control counterfeiters and manage an increasingly global pharma supply chain. A number of bills before Congress would empower FDA to deal more forcefully with illegal imports and to address drug manufacturing problems. The challenge is to ensure that efforts to strengthen FDA clout does not aggravate drug shortages.  

At a hearing Sept. 14, before the Senate Health, Education, Labor, and Pensions (HELP) Committee, Deborah Autor, recently named FDA deputy commissioner for global regulatory operations and policy, presented a long list of desired policy changes to promote drug safety and level the playing field between domestic and foreign manufacturers. Autor wants mandatory recall authority for drugs, power to detain and destroy violative imports at the border, and much stiffer penalties for noncompliance. Drug manufacturers and importers would have to register and list manufacturing facilities using identifier numbers, and importers would have to demonstrate that they meet quality standards, instead of FDA proving that they do not. Autor also seeks more authority to enforce track-and-trace standards, which would help hospitals and physicians know whether drugs from unknown or unusual sources are legitimate.  

Industry supports many of these changes, but is wary that continued shortages will spur calls for even greater government intervention in the market. The danger is that added rules could make low-profit drug markets less attractive to manufacturers. Yet, patients are waiting:  Short supplies of drugs to treat children with leukemia in the US is "shameful," lamented oncologist Len Lichtenfield at the FDA workshop. "Maybe we need more government intervention." 

Washington editor, Chevy Chase, MD, 301.656.4634, 

Regulatory Beat

Clamor mounts over compromised care and rising costs due to lack of crucial therapies.

Drug Shortages Create Crisis for Manufacturers, Regulators

Biosimilars manufacturers recognize that to compete and provide cost-savings relative to reference products, they must be cost-effective. Some improvements for biosimilar manufacturers will include: improved expression systems, higher titers to reduce capacity requirements, and better assay methods to permit process monitoring, and product characterization (1). 

Biosimilars manufacturers will need to increase expression-system yields to enable use of smaller bioreactors and lower cost facilities. New expression-system technologies, along with large single-use bioreactors for late-stage and commercial manufacturing, may begin to displace stainless steel for commercial manufacture. This shift is particularly true for recombinant monoclonal antibodies (mAbs), where a large number of biosimilar products are in development. mAbs have high repeated dosing requirements, and large amounts of protein in the magnitude of hundreds  of kilograms per year often are needed. 

New platforms (i.e., new host cells/organisms) and genetic-engineering advances applied to traditional platforms also can offer advantages in product yield, product quality, lower operating, purification, and infrastructure costs (2). Some newer technologies offer shorter times required to go from gene to transformed host cell line/organism to commercial-scale manufacture. Currently, there are just a few major players promoting new expression-system technologies, but there are many advances ready for adoption, adaptation,  and further development. 

Higher titers to reduce capacity requirements

The trend in increasing protein expression yields has had broad ramifications for biosimilars. From lowering construction requirements to increasing bottlenecks at the downstream end of production, the effects of upstream advances and the availability of more varied expression systems have created options and problems not previously seen. According to a recent BioPlan Associates analysis, the overall average yield reported for commercial mAb manufacture in 2011 is 2.18 g/L, up from 1.94 g/L in 2009 (see Figure 1) (1). It was not that long ago that mammalian-cell culture commercial production yields were lucky to achieve 1 g/L or greater (1). 

Figure 1: Annual change in titre, commercial, and clinical-scale production of monoclonal antibodies, 2008â2011 (data, reference 1). 

The next generation of commercial products will have an even greater average yield. For late-stage clinical manufacture, the overall average in 2011 is 2.68 g/L, up from 1.96 g/L in 2008. Although protein yields over 30 g/L are being reported by expression system developers and early adopters, these yields are the exception, and yields for commercial manufacture in the 5–10 g/L range are more likely in coming years (1). 

Better assay methods to permit process monitoring and product characterization will enable more biosimilar approvals. FDA and other regulatory agencies will largely rely on analytical data to support conclusions that biosimilar candidates are sufficiently similar to their marketed reference products to allow generic-like approvals. As such, regulators are likely to look for better analytic characterization through more combinations of attributes at greater sensitivities with multiple complementary methods. For drug developers, analytical method development and testing will be less expensive and quicker compared to reliance on clinical trials to support approvals. 

Figure 2: Select new product-development areas of interest (data, reference 1).  

Progress in the development of bioprocessing-related assays and analytical equipment, however, has not kept up with industry demands. Better analytical technologies are being sought by the industry for improving productivity, process monitoring, real-time product quality control, process optimization, and analytical characterization for biosimilars. These trends are reflected in a recent BioPlan Associates survey, where more than 32% of biopharmaceutical manufactures and 21% of CMOs expressed demands for improved analytical assays (see Figure 2). The survey showed that, after single-use/disposable bioprocessing equipment, more relevant and cost-effective assays and analytical equipment are strong needs. Areas of interest include better probes and sensors, in-house real-time analyses of process streams, use of biological assays to determine active agent-related quality (e.g., glycosylation variants), and outsourcing of specialized chemical analysis and biological assays.  

Figure 3: Percentage of all biopharmaceutical Industry vendors actively funding assay-related R&D (data, reference 1).  

More than one quarter (26.2%) of respondents cited analytical testing and drug-product releases a major factor likely to constrain their organization's production capacity during the next five years. Respondents identified 26 areas where new and improved testing technologies are required. More than 40%, cited glycosylation and comparability testing (primarily for demonstration of comparability between lots/batches of the "same" product, but also to show similarly for biosimilar products). Also, nearly 40% of all respondents cited the need for better host-cell protein assays, biophysical characterization during process development, and in-process testing and aggregation assays. Figure 3 shows the percentage of vendors funding assay-related R&D.    

 is president and managing partner at BioPlan Associates and a member of BioPharm International's editorial advisory board.  	

 1. BioPlan Associates, "8th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production: Survey of Biotherapeutic Developers and Contract Manufacturing Organizations" (Rockville, MD, April 2011).  

  2. BioPlan Associates, "Biopharmaceutical Expression Systems and Genetic Engineering Technology: Current and Future Manufacturing Platforms" (Rockville, MD, 2008).  

Perspectives on Outsourcing

Biosimilar manufacturers need better expression systems and analytical tools to compete.

Biosimilars' Technology Needs

From an industrial R&D perspective, the design and development of protein therapeutics today appears somewhat akin to the rational design of small-molecule discovery back in the 1970s when lead compounds were generated from known physiological substrates or ligands. Facing a need to find novel and diverse small-molecule leads, attention in the 1980s centered on high-throughput screening (HTS) technologies and compound libraries. Those libraries, albeit large, were hardly diverse, with most therapeutic agents coming from a few target protein classes. Complementation of libraries with natural products, the development of combinatorial chemistry, and application of focused-library sets followed. This evolution, together with automated methods for content-rich assay systems and fast make-test cycles, enhanced discovery of novel, potent, and diverse lead series.  

Contrast this with the present analagous processes for protein therapeutics: the discovery and development of novel biologics is hardly diverse, efficient or rapid. State-of-the art protein discovery and development use multiple expression hosts (e.g., mouse, 

 Chinese hamster ovary (CHO), and NS0) and several reformatting steps between hosts are often necessary during testing, scale-up, and production. The process of developing cell-based protein expression systems that are efficient, consistent, and scalable often is difficult and sometimes impossible using currently available technology.  

To date, more than 150 protein drugs have been approved for clinical use, nearly all of which are produced in cell-based expression systems, such as 

). These cell-based systems have several limitations, and many biologics can't be developed in these systems. For example, these systems only allow the overexpression of proteins that don't affect the physiology of the host cells. For many expression systems, identifying cell lines that stably synthesize high protein titers of the desired product is a time-consuming and labor-intensive process. Ideally, the same production host for rapid variant discovery, production for animal testing, and manufacturing of a clinical candidate would be used. 

Ideally, one would want to emulate the huge leap made in iterative drug design seen in small-molecule discovery, namely, rapid make-test cycles and generation of multiple parallel libraries of drug candidates with diverse structural elements to optimize activity while maintaining feasibility for manufacture. An ideal system would do the following:  

Make fast make-test cycles a prerequisite for re-iterative design on the order of three to five days, similar to those for focused small-molecule libraries

Create efficient and rapid expression and purification that allows for libraries of hundreds to thousands of protein-sequence variants to be simultaneously tested per make-test cycle using standard off-the-shelf robotics equipment

Incorporate preferred sequences defined from selection technologies, such as ribosome or phage display, into whole protein therapeutics for testing

Enhance the diversity of chemical structures by expanding the library of available amino acids at specifically targeted points in the protein sequence from 20 natural to many hundreds of non-natural amino-acids

Optimize several properties (e.g., agonist or antagonist, affinity, stability, and predictive manufacturability) simultaneously through rapid high-throughput make-test cycles 

Create processes that are not only rapid but amenable to rapid scale-up and cGMP manufacturing once the desired therapeutic construct has been identified.

As ambitious as such a system would seem, several exciting technologies are emerging that improve expression systems and enhance diversity to enable modification of intrinsic properties of proteins, such as enzyme catalytic efficiency or binding. Others combine different properties in single therapeutics by conjugation chemistries. Further emerging technologies can lead to more rapid and parallel expression of many protein drug candidates. Getting all of these desirable technologies into a single amenable platform that has the flexibility to be scaled and support cGMP manufacturing is in sight. 

Early improvements in endogenous protein-based therapeutics produced new, commercially successful therapeutics with desirable properties by simply extending sequence incorporating fusion to proteins such as the constant fragment of antibodies (Fc) or by PEGylating to increase half-life. Beyond these early approaches, considerable effort to produce ever-more elegant constructs that combine two separate functions have been made. One promising approach, antibody drug conjugates (ADCs), involves using a targeting antibody to known tissue selective cell-surface antigens or receptors to target conjugated toxins or cytotoxic drugs and so enhance selectivity over normal tissue.  

Successful design of effective ADCS is complex and requires linking cytotoxic drug payloads to tumor-targeting antibody constructs. The selection of an ideal antigen target for optimal internalization and specificity for tumor tissues is critical. The design of linkers that are stable in circulation, but cleave when internalized in tumor cells to release the cytotoxic drug, adds to the complexity, but the other major technical hurdle has been to define how the cytotoxic payload with linker are conjugated to the targeting antibody. The biopharmaceutical companies Seattle Genetics and Immunogen have developed robust platforms that depend on conjugation of linkers and cytotoxic warheads to available cysteine or lysine residues, respectively, in the tumor targeting antibody sequence.  

Despite successes with ADCs, there are many examples where seemingly optimized functional components (i.e., antigen-binding motif, linker, and drug-conjugate) do not translate into a developable therapeutic candidate. ADCs produced using conjugation chemistries to endogenous cysteines and lysines inevitably lead to the production of multiple species of the ADC with the drug conjugated in varying payloads of between one and nine molecules per immunoglobulin G (IgG). Furthermore, all sites of conjugation are not equal. Some conjugations interfere with antigen-binding epitopes, thereby reducing binding affinity and/or drug half-life (1). All too often, poor efficacy is revealed in the clinic only after significant investment in cell-based expression systems and scale-up. 

Several technologies aim to provide chemically amenable sites within a protein sequence for the posttranslational chemical conjugation of small-molecule drugs, peptides, or other constructs to improve or add functionality. For example, sequence-specific conjugations producing homogeneous ADCs with fixed payloads are aimed at improving tumor-cell killing and increasing therapeutic index. Engineered ThioMabs (Roche/Genentech technology) that use natural cysteine residues that must be carefully unmasked during production for subsequent site-specific conjugation have shown preclinical proof-of-concept (2). These approaches await further clinical validation.  

Carlos Barbas' laboratory at The Scripps Research Institute exploited the use of exposed tyrosine residues within the complimentarity determining regions (CDRs) of IgG molecules as the basis for linking drug conjugates. CovX, acquired by Pfizer in 2008, was founded to develop this technology. These sorts of approaches are attractive in that posttranslational chemical coupling to a common IgG construct with resulting extended half-life represents a platform amenable to many different small-molecule or peptide agonists. 

The introduction of non-natural amino acids (nnAAs), or those amino acids not part of the 20 naturally incorporated ones into proteins, plays an important role in basic peptide and protein research. They are increasingly used to develop biologics with enhanced pharmacological properties beyond providing sites for drug conjugation. NnAAs can be introduced through chemical synthesis in peptides or biosynthetically in proteins. Currently, only peptides and very small protein drugs with nnAAs are on the market because they can be made synthetically and avoid the limitations of cell-based expression systems. A prominent example is the semisynthetic, broad-spectrum antibiotic, ampicillin, into which the nnAAs D-phenylglycine and D-4-hydroxyphenylglycine have been incorporated (3). 

The opportunities to broaden protein diversity and properties with nnAAs are enormous, as is the ability to incorporate chemical modifications in proteins that can endow current biopharmaceuticals with improved or new properties. These chemical modifications can change the characteristics of proteins, including ligand-binding properties, stability, spectroscopic properties, folding behavior, catalytic efficiency, and substrate specificity. These modifications provide possibilities to develop biobetters and biosuperiors that have superior pharmacological properties, including improved safety profiles, longer half-life, and enhanced activity (4). 

Much effort has been put into developing technologies that ensure a site-specific incorporation of the nnAAs with a high rate of yield. Various methods for site-specific introduction have been established, both semisynthetic and recombinant methods. Few methods, however, have made it from the bench at small-scale protein production to commercial scale.  

A recently formed biotechnology company, Redwood Biosciences, is using an approach based on the work of Carolyn Bertozzi's laboratory at the University of California, Berkeley. Her work focuses on genetically encoded aldehyde tags and aims to exploit a specific sequence (originally found within the sequence of sulphatases) that is posttranslationally recognized and modified by a formyl glycine-generating enzyme to produce a so-called aldehyde chemical handle (5). The incorporation of the CxPxR sequence at specific positions in candidate protein therapeutics provides a means to produce a site-specific nnAA with a reactive aldehyde amenable to drug conjugation. 

One of the oldest methods for nnAA incorporation into proteins uses auxotrophic strains from 

that cannot synthesize a specific natural amino acid and thus have to uptake it from the growth medium. A structurally similar nnAA can be supplied within the growth medium in place of the natural amino acid and will be alternatively incorporated into the protein. A major downside is that the specific nnAA will be incorporated at every site coding for the natural amino acid and can lead to misfolding and impaired function of the target protein, or the nnAA can be incorporated in the host-cell's proteins, which can have toxic effects (6). Allozyne has pioneered this type of cell-based expression system that incorporates nnAAs into protein sequences, but this approach requires extensive re-engineering of the target protein sequence used, due to the region-specific nature of the nnAA incorporation using this method. 

Ambrx has developed cell-based nnAA incorporation systems where 

or CHO cells are engineered with orthogonal pairs of transfer (tRNA) and tRNA synthetases to charge and incorporate nnAAs at selected codons at specific points in the coding sequence of the expressed protein. This approach is a significant advance and provides answers to at least some of the questions raised about nonspecific sites of conjugations in ADCs. For truly expanding the number and variety of nnAA that can be incorporated to determine the effect on function, even at a single amino-acid position, the approach demands significant investment to engineer further orthogonal pairs of tRNA synthetases and tRNAs that can recognize a library of nnAAs. A further complication is that these pairs should be exquisitely selective over natural amino acids to avoid their incorporation over the desired nnAA, which can be challenging in a drug-manufacturing context with strict regulatory requirements. When this challenge is taken into account, along with the variability in efficiency with which nnAAs are absorbed into the cell, these systems will not likely be amenable to fast reiterative make-test cycles with libraries of nnAAs at multiple sites of incorporation.  

All of these considerations suggest a clear need to move away from the conventional cell-based protein expression systems to address the critical requirement for a rapid make-test system that is amenable to many parallel re-iterations of site-specific incorporations of defined natural amino-acid sequences or multiple nnAAs at multiple sites. The answer may not lie with cell-based systems at all, but with completely 

 biochemical protein synthesis based on novel cell-free expression systems. 

CELL-BASED EXPRESSION SYSTEMS: PARALLEL REITERATIVE DESIGN AND SCALABLE PLATFORMS

Adnexus Therapeutics (acquired by Bristol-Myers Squibb in 2007) has developed an 

-based platform for producing adnectins. Adnectins are derived from human fibronectin and many trillions of adnectin variants can be generated to represent a screenable library for desirable therapeutic properties. Scale-up from a selected lead is rapid, albeit with the requirement for PEGylation for clinical candidate manufacture (7). 

Fabrus has recently addressed the rapid make-test cycle approach for biologics using arrays of predefined Fab antibody sequences produced in a high throughput expression system based on production of proteins in 

, cell-lysis, and subsequent protein purification (8). The large volumes of cell culture required for high-yield parallel production of Fabs requiring a significant investment in specialized robotics equipment. The method allows production of hundreds of variant Fab proteins over a one-week production cycle to begin variant testing in high-throughput biochemical-binding assays.  

Although cell-free protein synthesis has been practiced for decades as a research tool, only recently have advances suggested its feasibility for commercial biologics drug development and production as an alternative to conventional cell-based expression systems (9). An ideal cell-free protein production platform would produce fully soluble and correctly folded proteins at high volumetric productivities at any scale. The platform would be rapidly and predictably optimized by systems-level process design and control without the demanding requirements for maintaining cell viability and be readily adapted to high-throughput methods, including 

evolution of proteins to allow incorporation of nnAA into polypeptides. The platform would be based on simple batch systems using standard bioreactors that are known to scale to thousands of liters for both cell fermentation and subsequent cell-free protein production (10, 11). 

Early efforts at developing such a system focused on projected costs that were much too high, as well as on proteins with disulfide bonds that could not be folded effectively. By focusing on basic biochemical reactions and controlling cell-free metabolism, these limitations have been methodically addressed (12). Amino-acid supply has been stabilized, and metabolism activated to dramatically reduce substrate costs by requiring only the addition of nucleotide monophosphates to drive energy production. Commercially available 

, wheat germ, rabbit reticulocytes, and insect-cell extracts do not offer this advantage and are suitable only for research exploration at small scale. Control of the sulfhydryl redox potential has been gained and a robust disulfide isomerase added to facilitate oxidative protein-folding (13). These advances not only suggest production feasibility for pharmaceutical proteins containing the 20 natural amino acids, but they also provide enabling technology for incorporation of nnAAs at commercial scale. 

A recent publication demonstrates that this open cell-free system (OCFS) developed by Swartz and collaborators can be optimized for high-level production of proteins to allow for scale-up to commercial levels once the target protein is identified (14). The authors expressed a multidisulfide-bonded protein, biologically active granulocyte-macrophage colony-stimulating factor (rhGM-CSF), at titers of 700 mg/L in 10 h. Importantly, they could show that the product was linearly scalable from starting materials in 96-well plates up to 100-L culture volume (14). The open nature of the system allows mass spectrometry-based profiling of the cell-free metabolome and proteome. Rapid testing of the effects of addition and subtraction of various components for system optimization can be modeled without the requirements for tuning more complex cellular networks required for maintaining cell viability commonly encountered in mammalian cell-line development. 

The lack of a membrane-barrier in the OCFS provides the opportunity to express and study proteins that are difficult to express in cell-based systems. Many proteins that can't be readily expressed by 

 expression systems due to poor folding, inclusion body accumulation, or due to toxicity can be expressed in an

The advancements in genomic research and increased numbers of sequenced genomes require expression systems that allow fast production of the proteins under investigation. Cell-free expression systems can provide a useful tool for rapid screening and analysis of protein function, which is important for protein-drug discovery and development. DNA molecules can be amplified, transcribed, and translated in microplate wells and the expressed protein can be assayed immediately (15). Recently, HTS in a cell-free wheat germ system led to the discovery of a novel malaria vaccine candidate (16). Finally, the linear scalability allows proteins identified in display-based selections and HTS to be immediately scaled for production of multiple gram quantities, thus avoiding the delays and challenges of conventional mammalian cell line development. 

Currently, cell-based expression technologies exhibit several limitations with respect to protein production at all phases of the drug discovery and development pipeline. Rapid production of proteins with novel chemical modifications, such as ADCs, are particularly challenging. 

-based cell-free protein synthesis systems, however, provide robust, rapid,and scalable protein production. The 

-based OCFS system, in particular, allows rapid and multiplexed production of various difficult to express proteins and opens the unprecedented ability to explore therapeutics beyond the 20 amino acids that define today's proteins (17). The OCFS, combined with rational protein design and the focused use of libraries of nnAAs, allows for rapid exploration and identification of protein therapeutics, moving from the exploratory stage to clinical scale-up on an unprecedented, rapid timescale. 

Growing demand for new and better biopharmaceuticals has led to sophisticated advances in protein synthesis that now allow for:  

Rapid production of target proteins, including those that are difficult to express in cell-based expression systems

Straightforward scalability of protein expression from HTS to commercial levels

Combinatorial screening of many proteins to identify and optimize drug candidates

Introduction of site-specific chemical modifications, including nnAAs into proteins to improve pharmacological properties

These new approaches to protein expression will revolutionize the development of biopharmaceuticals, and open up the possibility to create drugs that were previously inaccessible, and even unimaginable until now. 

 is  vice-president of research, both at  Sutro Biopharma, 310 Utah Ave,  Suite 150, South San Francisco, CA 94080, 

* To whom all correspondence should be addressed

Features

Industrializing design, development, and manufacturing of therapeutic proteins.

Protein Engineering

diagnostic (IVD) tests are complementing targeted therapeutics to reduce side effects, improve efficacy, and help control healthcare costs. Analysts are projecting billion-dollar revenues for some new drugs linked to companion diagnostics.  

As the pharmaceutical industry develops more targeted therapeutics, and their interdependence with companion diagnostics grows, companies are considering ways to access diagnostics technology to complement their evolving product portfolios. The main strategy has been to seek companion diagnostic solutions by forming partnerships with diagnostic companies. Yet, the allocation of the overall financial value of the drug-diagnostic combination has made deal-making a challenge. 

Companion diagnostics partnerships in the industry more than tripled in 2010 compared with 2008, and the pace of deal activity has continued during the first half of 2011. The rising number of partnerships reflects the increasing seriousness with which bio/pharmaceutical companies view biomarker and diagnostic programs designed to accompany their drug-development efforts. They are making more systematic use of companion diagnostic programs to increase drug response rates and reduce side effects. Diagnostic companies, particularly those with strong molecular and tissue diagnostic capabilities, have been active in developing tools to respond to industry's specific needs.  

Driving the momentum in companion diagnostics partnership activity is the increasing role of diagnostics in the regulatory approval, reimbursement, and performance optimization of new drugs. Increasingly, regulatory agencies insist on validated diagnostics prior to considering marketing clearance. In addition, the growing use of tests that identify patients who would 

 benefit from certain therapies has raised the bar for obtaining reimbursement for new drugs. Increasingly, payers see companion diagnostics as useful tools to allocate healthcare funds more effectively and to control costs. Many healthcare professionals insist on specialist testing before prescribing and reimbursing treatment regimens that are expensive and not efficacious in certain patient subpopulations. 

In the US, some pharmacy benefit managers are adapting their business models by forming partnerships with, or acquiring, specialist clinical laboratories. Payers' preference for drugs that come with a companion test, particularly when these drugs are expensive and may lead to severe side effects, will likely increase over time with the rising pressure on healthcare budgets and greater availability of appropriate diagnostic tools. 

Bio/pharmaceutical companies have achieved some success in seeking improvements in drug-response profiles through better patient targeting. For example, drug-response rates of up to 80% have been reported for targeted subpopulations for cancers that generally have a 20% response rate. 

The prospect of repeating such technological wins is encouraging industry to accept changes that appear increasingly inevitable, including the decline of the mass-market blockbuster drug model, the emergence of smaller targeted markets, and the need for high-performance diagnostic tools to dominate well-defined smaller market segments. 

On the supply side, the technological feasibility of companion diagnostic programs is increasing. Companies continue to develop relevant expertise in molecular and tissue diagnostics, which will enable the development of better tools to guide treatment decisions.  

The diagnostics industry is providing new tools that could improve product offerings to physicians and patients and create value for shareholders. Unfortunately, many diagnostics industry players believe the economics of innovation are undermined by low pricing and reimbursement of tests, and the diagnostics partner's low share of prescription–diagnostic (Rx–Dx) partnership values. In this context, it is important that the government and its agencies support required changes. According to research by PwC, diagnostics companies are seeking action in three main areas, as outlined below. 

Pricing should reflect the value of the test rather than its cost. The price should reflect a reasonable proportion of the test benefits or the cost savings. In the US, the concept of value-based pricing is making gradual progress.  Europe, however, has yet to see value-based pricing applied to a personalized medicine test. The diagnostics industry fears, according to PwC research, that unless pricing is adapted to value creation, it will fail to achieve sufficient economic return to stimulate continued investment and innovation.  

The process to gain reimbursement for diagnostics should be accelerated and harmonized across countries. In many countries, reimbursement for a new test can take four to seven years following marketing clearance. Industry participants believe that health technology assessment (HTA) models need to be adapted to allow for faster reimbursement decisions. One practical solution to address reimbursement delays has been companion test subsidies from the bio/pharmaceutical partner.  This is not ideal; but in cases where the test determines drug eligibility; the alternative would be that severe limitations would be placed on drug availability. This arrangement may not be acceptable to industry. 

Diversity of health technology assessment procedures across countries is another issue in multi-country product launches. This problem has been recognized by the European Commission, which has sponsored the European network for Health Technology Assessment to work on greater cooperation. The US is also represented in this initiative through the Center for Medical Technology Policy.  

The share of value going to the diagnostic in Rx–Dx partnerships should be revisited. Diagnostics companies are concerned about not getting a fair share of the overall value of Rx–Dx combinations when negotiating deal terms with bio/pharmaceutical partners, and that they suffer from historically low recognition of the value of diagnostics. Traditionally, diagnostics represent less than 2% of healthcare spend despite influencing more than 60% of crucial healthcare decisions. Diagnostics partners are focused on trying to rebalance their share of the financial value in Rx–Dx combinations.  

One avenue that diagnostics companies are pursuing is to obtain a royalty on sales of the companion product. Companies have resisted such a move because they believe the Dx partner has not shared the risk or investment associated with drug development. Diagnostics players insist that this move should happen, arguing that where Rx–Dx combinations are relevant, the companion drug would not be able to make it through clinical trials or be reimbursed and commercialized without the companion diagnostic. Thus, the value of the drug is critically dependent on the contribution of the companion diagnostic.  

In the near-term, these challenges are not expected to undermine the pace of Rx–Dx deal activity. However, they may affect long-term diagnostics innovation if they are not addressed. At best, not addressing these economic issues could result in an undervaluing of diagnostic innovation by pharma. At worst, these issues could eventually discourage continued investment into diagnostics ventures and delay patient access to important new health technology. 

, a principal at Pricewaterhouse Coopers (PwC), and Loïc Kubitza, a director in PwC's Pharmaceutical & Life Sciences Advisory Services group, 

Final Word

With the rise in therapeutics comes more complex partnerships.

The Economic Challenge of Prescription–Diagnostic Combinations

It's almost inevitable that when people speak of pharmaceutical innovation that someone doesn't ask how pharmaceutical companies can become more like Apple. It's a question that sprung up again with the death last month of Steve Jobs, Apple's iconic founder and innovator-in-chief. What was it about Jobs and the company he founded that nurtured so much innovation and creativity, and can it really be brought into pharmaceutical innovation?  

Jobs outlined his personal trajectory in a tale of passion and loss in his 2005 commencement address at Stanford University. His path to Apple started when he dropped out of Reed College in order to be free to take any course that interested him rather than follow a prescribed curriculum leading to a specific major. This in turn led him to a calligraphy course that, he says, informed his ethic of blending aesthetics and technology to create Apple products. But as with many great relationships, what started out with so much generative promise ended in heartbreak—Jobs's specifically, when at the age of 30 he was asked to leave the company he'd founded.  

"I'd been rejected," he said, "but I was still in love." That first loss, painful as it may have been also relieved Jobs of the "heaviness of being successful," and replaced it "with the lightness of being a beginner again." Jobs the beginner embarked on yet another creative period that resulted in the formation of Next and Pixar, which ironically, brought him back to Apple and it to him for yet another creative period that yielded iPods, iPhones, and iPads and revolutionized both the computer and the communication industries.  

The other great focusing event in Jobs's life, as we all now know, was the imminence of his death from pancreatic cancer. In the face of death said Jobs, "we are already naked," and because of which, he admonished Stanford's class of 2005 not to be "trapped by dogma, which is living with the results of other people's thinking." Rather, Jobs urged the graduates to follow their individual passions. 

Jobs died the same week the winners of the 2011 Nobel Prizes were announced, and among those winners was Ralph Steinman, a Rockefeller University professor, who elucidated the biology of dendritic cells. Steinman died the same week as Jobs, just days before the Nobel announcement was made. But in an interview on National Public Radio on Oct. 3, 2011, Adam Steinman, the Nobelist's son talked about his father's passion for his work, and his desire to "instill a sense of excitement and discovery in the next generation."  

Reading these stories, one might suggest, as Jobs did in his Stanford address, that it's the threat of loss that should make one insist on following their passions to discovery. But I keep thinking of one my professor's observation that children are the true scholars, because they want to learn everything and are unfettered by dogma. Jobs and Steinman and other great innovators are the ones that, in spite of prevailing dogma, never lose their childlike passion for learning and exploring. So the pharmaceutical industry may be misguided in looking for ways to inspire innovative thinking in its scientists. The real secret might be in not wringing it out of them. 

From the Editor

At a time when the industry is struggling with innovation, it might do well to learn a lesson from a few great innovators.

Passion, Innovation, and Loss

In the biopharmaceutical industry, flexible containers such as plastic bags or liners are often used for bulk intermediate storage, cell culture resuspension, viral inactivation, final formulation, final fill, or as bioreactors. In such applications, the bag is hermetically sealed and sterilized. Sterility of the bag must be maintained to avoid contamination of the product. Any breach of the sterile condition is considered a serious risk and often results in disposal of valuable product, sometimes after significant cost and effort has been expended in the course of making it.  

At the moment, disparities exist between defect sizes that are readily detectable using current on-line technology, and the speculated value for the critical defect size (e.g., the defect size at which sterility of a package is lost). Lampi  established, and Chen and Keller independantly substantiated, that the critical defect dimension for bacterial penetration for flexible bags is 11 μm or less, while Gilchrist determined that the dimension was nearly twice that:  22 μm (1–4). Blakistone later established that critical defect size was 7 μm (5). Discrepancies in the critical defect size could be attributed to differences in the bio-test methodology, concentrations of test microbes, test times, or positive/negative pressure in the test bag. Therefore, one of the objectives of this article is to determine the critical defect dimension at which sterility breach occurs in biopharmaceutical containers. Such information would provide a foundationfor avoiding inherently problematic conditions, as well as for empirically evaluating and tailoring leak detection equipment for their specific needs. 

The package's integrity controls microbial ingress into the package and thereby preserves product sterility. The current technologies employed in the biopharmaceutical packaging industry (e.g., vacuum bubble test, dye-penetration test, pressure-decay test, constrained-plate pressure-decay test) are only capable of reliably detecting leak rates in the range of 10

  cc/s, which is equivalent to detecting defect sizes in the scale of 90–500 μm. Thus, industry needs a method of testing the integrity of a flexible container that detects defects corresponding to the minimum size needed to block water-borne microbes. This article describes a new test methodology, called helium integrity testing (HIT), that can detect such defects.  

A defect can be defined as an unintended crack, hole, or porosity in an enveloping wall or joint, which must contain or exclude different fluids and gases allowing the escape of closed medium. The shapes of defects (e.g., cracks, fissures, porosity, damages, etc.) are very different, unknown, and nonuniform. Therefore, it is impossible to measure their sizes with any precision except in the case of an ideal or artificial leak as used for calibration. There are many methods for detecting defects, a list of which along with their sensitivities can be found in the literature (6–11). Figure 1 summarizes the sensitivity limits of each of them.  

Figure 1: Leak detection sensitivity chart. (ALL FIGURES ARE COURTESY OF THE AUTHORS) 

The most practiced method in the industry involves observation of gas or fluid flow through a test bag under defined conditions of temperature and pressure. Consequently, defect sizes are measured by the pressure decay method or by the tracer gas leak testing method. The pressure decay method is discussed below to illustrate some of the limitations observed in ensuring integrity of test bags, and varying adaptations of the tracer gas leak testing method are discussed along with their limitations.  

The pressure-decay test consists of pressurizing the system with a high pressure gas, usually dry air or nitrogen. The test part is isolated from the gas supply and, after a stabilizing period, its internal pressure is monitored over time. The pressure drop (Δp) is measured over the interval (Δt) (see Figure 2). If the pressure in the system drops quickly, there is a large leak present in that component or section of the system. If the system's pressure drops slowly, there is a small leak present. If the pressure remains the same, that component is leak-free. The leak rate, Q, can easily be computed considering the volume V of the component by using the  following equation:   

Leak-detection sensitivity is related to the test time, the pressure transducer resolution, and the test bag volume. Several external factors, such as temperature variations and mechanical deformations, affect this test. The internal pressure depends on temperature, and therefore thermal fluctuations may cause changes in pressure, altering the results. Longer test times allow for a more sensitive check, but make the test very time-consuming because smaller defects may require holding periods of several hours. The higher the pressure, the faster leak determination can be made. However, operator safety concerns along with the risk of damaging the test product limit the maximum admissible pressure value. 

The difficulties increase when using this technique for checking the defects in flexible bags. Calibrating the system when the test product being measured is extremely pliant is challenging. Therefore, reliable test sensitivity is reduced for flexible bags. This issue can be addressed by the use of constraining plates to constrain bag stretching or internal volume (11, 12). For products with a more complex shape it may be necessary to make a tool that conforms closely to the product outline. However,  it is not always possible to fit the final product into the test assembly, leading to products being tested without final assembly elements such as tubing connections or filters, because tubing port connections and manifolds are sensitive to over stretching or bending caused by constraining plates. In the case of large test bags, such as 1000-L or 2000-L, it may require hours to fill the test bag with pressurized gas and to empty the bag. Even after such long test times, one can only detect defects as small as 500 μm while smaller defects go unchecked. Thus, the inability to test fully assembled product risks assuring integrity of a finished product. It is therefore necessary to have a method that is easy to implement (e.g., not bulky or cumbersome), reliable, and that does not contaminate the interior compartment of the container undergoing testing. 

The term "tracer gas leak testing" describes a group of test methods to detect and measure a tracer gas flowing through a leak. These techniques differ for the tracer gas used and for the realization technology. The most commonly used tracer gases are halogen gases (e.g., CFC, HCFC, and HFC refrigerant), helium, and a mixture of nitrogen and hydrogen (95% / 5%). Helium has been used successfully as a tracer gas for a long time because of its physical properties (12, 13). It is neither toxic nor flammable and is does not react with other compounds. Helium has low viscosity and relatively low molecular mass, so it easily passes through cracks. In the same environmental conditions, it flows through orifices 2.7 times faster than air. Since its concentration in air is low (i.e., 5 ppm), it is easy to detect an increment of helium concentration. It is important to remember that background concentration in air is a limiting factor for any tracer gas detector. 

There are two ways to carry out leak testing with tracer gas: internal detection of tracer gas entering from leaks (i.e., outside-in method) and external detection of tracer gas escaping from leaks of a filled unit (i.e., inside-out method). The inside-out method can be executed with atmospheric sniffing or with vacuum chamber detection, while the outside-in method is generally implemented by putting the unit to be tested in a room containing the tracer gas or, very rarely, spraying the tracer gas on the unit surface. 

In the outside-in leak testing technique, the unit to be tested is put into an enclosure containing the tracer gas. The part is connected to a vacuum pump and evacuated. A tracer gas detector (i.e. helium mass spectrometer) is placed in the vacuum line to detect the tracer gas pulled in by the pump. The sensitivity, depending on tracer gas and test time, can reach 10

  mbar ∙ l/s. This method can be fully automatic, so it is not operator dependent. The gas containment hood can be configured to prevent dispersion, which reduces working area pollution and tracer gas consumption, and saves money by avoiding the need for a recovery system. The drawbacks include a large amount of tracer gas use because, in the case of a big leak in the part under test, a large amount of tracer gas escapes and is lost. In addition to the loss of tracer gas, a long pumping time could be required to lower the tracer gas in the detector to an acceptable level compatible with system function. The system is unusable during this time. Another disadvantage is that this method does not identify where the leak is, it only determines if a leak is or is not present. This technique only works for rigid test parts. For flexible parts, the test bag may collapse, blocking any trace gas from leaking. 

Sniffing, an inside-out technique, involves a probe or wand being moved over the test part. The probe detects the leak as it passes over the leak. The speed, distance from the part, and the probe sensitivity determine the accuracy of leak detection. However, sniffing has the distinct advantage of being able to locate a leak on the test part, unlike the other methods described, and has the ability to sense leaks as small as 10

  mbar∙l/s, depending on the tracer gas. Sniffing is not recommended in a high volume production environment, other than for locating leaks for repair. Disadvantages include a high chance of missing leaks due to operator dependency, fragile equipment in rugged environments, and the rejection of good parts because of the inability to quantify the leak. The minimum leak rate measurable by a sniffer is the concentration of the tracer gas in the working area, a value known as background level. This level may change during the production cycle and increases because of leaking units. Relating to the tracer gas used, in case of a big leak in the part under test, a lot of tracer gas escapes from it and may remain for a long time in the working area, strongly affecting the subsequent tests causing rejection of good parts. 

Vacuum chamber inside-out leak testing is the most complex system of leak detection, but it is theoretically suitable for finding very small leaks, using the proper tracer gas. This method involves placing the test bag inside a vacuum chamber connected to the integrated helium leak detection system. The vacuum chamber encapsulates the entire test bag, ensuring that any helium  molecules escaping from the bag are captured and directed towards the sensor. 

Depending on the vacuum chamber dimensions, the evacuation group could call for a high pumping speed, which would introduce high level of particles in the test bag. In the case of a large leak in the part under test, a large amount of tracer gas escapes relative to the amount of tracer gas used. A long pumping time could be required to lower the tracer gas in the detector to an acceptable level compatible with system function, during which the system is unusable. 

Although use of helium gas as a tracer gas has been well documented in the literature, its use for detecting defects in flexible bags used in the biopharmaceutical industry has been limited because of the operational and functional issues listed below: 

Long down times due to inefficient process:

 Helium gas disperses slowly into the atmosphere, so, in the case of large leaks, its high concentration will contaminate the area for a long time, even hours, effectively making it inefficient for production purposes. 

Conventional belief that highly pressurized systems provide high sensitivity to method detection results in large quantities of helium being used and hence, high costs. The most suitable helium detector is based on a mass spectrometer, which is an expensive and delicate apparatus requiring much care and maintenance. There are high costs due to loss of tracer gas for the test. 

 To be able to detect smaller leaks, the test time can be long which affects production cycle time and hence overall cost of the product. 

 The testing method can impact product cleanliness performance and may increase bio-burden level of test bag. 

 Traditional hard vacuum leak testing is often difficult to perform on flexible wall parts. The pressures resulting from a full vacuum can damage the part. 

To address issues discussed above, ATMI developed the helium integrity test (HIT) method and apparatus which allows for fine leak testing of parts using a helium mass spectrometer (MS) leak detector. The technology is based on the inside-out testing principle, however, it addresses issues that prevented its use in leak testing of flexible bags. This method can achieve desired results at lower part differential test pressures and with faster cycle times compared with traditional helium accumulation methods. 

The HIT apparatus includes the following main components (see Figure 3):  

Vacuum pumps for bag evacuation and test chamber evacuation

Figure 3: Helium integrity test (HIT) apparatus.  

The HIT apparatus is composed of a test chamber with a closable lid that when closed, forms a tight seal such that no helium can enter into the test chamber from outside. The test unit is a flexible two-dimensional bag with connecting ports that enable fluid communication with the helium source and vacuum pump. The test chamber is large enough to house the unit to be tested. The test chamber is connected to a vacuum pumping group equipped with the tracer-gas detector for chamber evacuation and gas detection. A second vacuum group is used to evacuate the unit under test before filling it with gas. A tracer-gas filling device (i.e., helium supply) completes the testing apparatus. The unit to be tested is put into the vacuum chamber and connected to service hoses, then the vacuum chamber and the unit are evacuated. During chamber evacuation, the test unit is pressurized with the tracer gas. After a stabilization time, the detector is linked to the vacuum line to detect the tracer gas flow through a leak and drawn in by the pumping group. This method can be made fully automatic, so it depends little on an operator. Its sensitivity can reach < 10

 cc/s flow rates. To prevent long down times, the HIT system employs an in-line pressure-sensing test method as a preliminary leak test that detects gross leaks before the final automatic leak-test operation using a tracer gas is begun. This approach prevents large quantities of tracer gas from leaking into the atmosphere. A specialized pumping technique reduces the stress on the test unit by reducing the internal pressure of the test part along with the external (i.e., chamber) pressure. 

To improve test time per test unit, the test chamber was modified to include spacers that allow the simultaneous testing of four units. The spacers are held upright by connecting rods, and each spacer has enough cavities in it such that when a test bag inflates against it, it does not block the path of helium molecules flowing through the defect. The spacers constrain the test bag in the test chamber, further increasing the helium pressure in the bag and resulting in increased sensitivity of leak detection. The spacers also ensure that test bags experience minimal stress during chamber evacuation by limiting bag expansion. 

The test bags were prepared by welding two sheets made from ATMI's proprietary TK8 film. To create defective bags, one of the two sheets was modified to incorporate a defective patch. A defective patch is a piece of TK8 film (4 in. x 4 in.), with a 10 μm ± 1 μm hole drilled by a laser. The laser-drilled holes were validated for defect size by measuring flow rate through the defect area. Hereafter, "defective bags" implies a test bag with a 10 μm defect, and "good bags" implies bags with no defect. 

The size of the test bag depends on the size of the two sheets used in making one. The nominal volumes of test bags in this study were 1, 5, 10, 20, and 50 L. The helium leak rates through defective bags and good bags measured using HIT technology are discussed below. The test bags post-HIT tests were further characterized for product performance such as liquid particle count (

 <788>) to ensure that the HIT process did not affect product performance. 

In this study, test bags for microbial challenge were prepared by thermally welding TK8 film sheets having defects of specific sizes (2, 5, 10, 15, 20, and 50 μm). The test bags were aseptically filled with 50 mL microbial growth medium (trypticase soy broth). The outside of the test bag was sprayed with a 0.9% saline solution containing approximately 10

. The test bags were then transferred to an incubator maintained at 30–35 °C and monitored for 15 d. The growth medium inside the test bag was periodically checked for microbial growth, which indicates microbial ingress. 

A test bag was filled with ultrapure water (deionized water filtered using a 0.05 μm filter) and gently shaken to ensure that all the bag surfaces came into contact with the solution. A sample of the solution from the test bag was then passed through particle measuring equipment (PMS). The instrument reports the number of particles per mL of solution with particle sizes greater than 25 and 10 μm. 

Defects as small as 10 μm can be detected without compromising product cleanliness using helium integrity testing.

Integrity Testing of Flexible Containers

At the IBC Single-Use Applications meeting, held in Boston on Jun. 6–9, 2011, I gave a talk entitled "Standardization of Single-Use...Pros, Cons and Possibilities." The topic of standardization has appeared in many surveys as one of the top 10 needs for expanding single-use implementation. However, there is little definition of what is meant by "standardization," which could be applied to many areas.  

One particular area of discussion is the interchangeability of sterile connectors to enable single-use unit systems with sterile connectors from different suppliers to be linked. In preparing my talk, I also discovered that there is already an interchangeable "standard sterile connector" recognized by industry. If you search for this phrase in Google Images, you'll find a photo of a traditional two-way hosebarb fitting. 

In my talk, I discussed how standardization of advanced sterile connectors must begin with the end-user's company. Different systems' assemblers and integrators can generally source whatever advanced sterile connector is specified, so the first effort must be for the end-user's company to decide what its preferred sterile connector will be. Since the introduction of the first advanced sterile connector (Pall's Kleenpak), there are now five additional designs on the market from other suppliers, but there is still no industry consensus on which design might become the "standard" of the future. Unlike the easily copied tri-clamp style hygienic flange connector, which is commonly used in stainless-steel and some single-use systems, or the aforementioned hosebarb fitting, many advanced single-use sterile connectors incorporate patented technology and designs. Users calling for the standardization of sterile connectors have yet to address which design should dominate or why the patent-holder should license their design to competitive suppliers. Once bioprocessers agree on a preferred design within their own facilities or companies, once there is consensus on an industry-preferred design (such as happened with tri-clamp style connectors), and once patents expire, options will appear to either "standardize" one of the original sterile connector designs. The alternative is that users will prefer an innovative proprietary design that is superior to current designs, but has yet to be seen. Until then, care has to be taken to avoid inhibiting innovation and it may not be realistic to expect meaningful voluntary standardization from the bioprocessing supply industry. 

One area where there is an industry initiative underway to standardize connectors, however, is for polymeric tri-clamp style hygienic flange seal connectors. In response to stimulus letter from the Bio-Process Systems Alliance (BPSA), the American Society of Mechanical Engineers bioprocessing equipment standards group (ASME–BPE) has formed a task force to review the requirements for single-use polymeric tri-clamp style connectors and how the ASME–BPE Standard for stainless steel hygienic flange seal connectors can be modified in response to accomodate their use. Unlike their stainless steel counterparts, single-use polymeric tri-clamp connectors do not require cleanable finishes, crevice-free cleanable seals, or thermal resistance and mechanical strength suitable for steaming in place. They also do not require tolerances that are readily achieved with stainless steel on a lathe, but are difficult to achieve with plastic molding, thus incurring unnecessary mold costs. Expansion and relaxation of the ASME–BPE standard to cover the specific requirements for single-use polymeric tri-clamp style connectors will be a benefit to both suppliers and users. 

The PDA held its first Single-Use Workshop in Bethesda on June 22–23, 2011. The main focus was to preview the draft PDA Technical Report on Single-use Manufacturing, which is currently in development, and to solicit attendee feedback. As a member of the PDA Single-use Task Force, I served on the planning committee and as a moderator and presenter on the report for this workshop.  

The purpose of the new report is to provide the reader with critical concepts and topics to consider when implementing a single-use manufacturing strategy for drug or vaccine production. The draft report discusses single-use systems that may be in direct or indirect contact with raw materials, intermediates, intermediate products, pharmaceutical drug substances or the drug product. The primary goals for developing a single-use manufacturing strategy are customer-based and focus on patient safety and product availability, as well as product and process understanding and control.  

The workshop program focused on QbD principles and other high-level topics to guide users on their initial decision, selection, validation, and implementation of single-use processes. The workshop included several opportunities for Q&A and open discussion, which gave the task force valuable feedback from attendees on the draft report. Key take away points included: 					

The report should aim to help end-users move away from 'gut-feeling' to fact-based decisions. 

Control of suppliers will also come under more scrutiny and supplier audits can be expected to increase. 

More detail on materials and manufacturing methods (e.g., films) may be required. 

Partnership between the supplier and end user was stressed; those companies that truly embrace partnership will be the ones most likely to achieve success. 

The industry will expect suppliers to have pharmaceutical standard quality systems in place, particularly with regard to having an appropriate materials change control and change notification program. 

Training in single-use will also be a key requirement/capability from suppliers. 

Clear communication strategies encompassing quality, trust, track record, openness, and security will be in focus.

The target date for the report's publication is by the end of this calendar year. Current activities entail completion of the manuscript incorporating the feedback from the June workshop and final technical review of completed sections. Follow-on workshops are planned for Uppsala, Sweden, on Nov. 28, 2011 and Phoenix, AZ, on Apr. 18–19 2012 (see 

REGULATORY GMP FOCUS EXPLAINED AT BPSA CONFERENCE 

The third important single-use conference of the summer was the BPSA International Single-use Summit, which was held in Washington, DC, on Jul. 27–29 2011. This inaugural conference for BPSA served as a forum for suppliers and users to highlight the business model for single-use. In addition to business leader speakers, the summit featured J. David Doleski, the consumer safety officer at FDA's Center for Drug Evaluation and Research. Doleski opened with a review of relevant FDA regulations that impact single-use manufacturing, including 21 

 211.65 on Equipment, 211.94 on Drug Containers, 600.3 on Biological Purity and 600.11 on Biological Control. Each of these has similar statements about assuring that process equipment and containers do not adversely affect the drug or biological product. Additional compendial standards noted were USP General Chapters <87> and <88> on Biological Reactivity, <661> on Plastics, and <381> on Elastomers, as well as relevant FDA guidances, including cGMP for Phase I Investigational Drugs (July 2008) and Container Closure Systems for Packaging Human Drugs and Biologics (May 1999). 

Advantages of single-use manufacturing were recognized as: 					

Reduced need for cleaning and sterilization systems and validation

Potentially greater control over aseptic operations (as facilitated with sterile connectors/disconnectors and tube welders/sealers). 

Doleski's talk continued with an excellent overview of topics that should be incorporated in process documentation and FDA filings. Initially highlighted were considerations for vendor partnerships and materials control, included establishment of manufacturing (quality) agreements, vendor audits, notifications of changes in product (materials or design), certificates of analyses, and flow path testing for endotoxin, particulates and bioburden (where necessary). With regard to sanitization (e.g., irradiation for microbial control) or sterilization, bioprocessers should note where sterilization is performed (i.e., contract irradiator) and provide documentation on the sterilization validation method, sterilization records, impact on materials (supplier validation data) and repeated sterilization where applied (note that single-use systems are generally not suitable for multiple irradiations at doses > 25 kGy). 

Extractables and leachables studies are performed to assess the potential impact of leachables on product quality, efficacy, and safety. This can begin with compatibility and extractables data from the supplier, testing with additional model solvents under manufacturing process parameters (e.g., temperature, pH, pressure, and time) where needed, considering the cumulative effect of all manufacturing equipment and conducting further risk assessments to determine if a leachable study is necessary (e.g., for final product formulation).  

Process-validation considerations should take into account the full range of the manufacturing process, and incorporate multiple unit operations and actual manufacturing parameters, such as mixing speed and duration of perfusion culture. Sterile-media simulations should be conducted for filling of sterile product (i.e., bulk or unit dosage). Where fluids are stored in single-use containers, validation should include the length of time and temperature range, with assessments of the impact of fluid on materials, and the impact of materials on product, buffers or media, and container integrity (i.e., leakage) after storage. Where bulk fluids are shipped, considerations should include the effect of pressure changes, such as altitude, effect of motion (i.e., acceleration or vibration) and the protection offered by external containers. Other environmental considerations can include light, chemicals, and other mechanical forces that may affect the contained fluid. 

Despite commonly cited concerns, leak integrity issues with today's improved biocontainer designs are rare. However, possible issues should be qualified and noted, such as movement or shipping of storage containers, operator error (handling training), improper operation parameters (i.e., tube welding), and exposure to extreme temperature.  

In this quarter's column, highlights from the IBC Single-use Applications meeting, the PDA Single-use Workshop, and the BioProcess systems Alliance International Single-Use Summit are presented.

BioPharm International-11-01-2011

BioPharm International, November 2011 Issue (PDF)

Drug Shortages Create Crisis for Manufacturers, Regulators

Biosimilars' Technology Needs

Protein Engineering

The Economic Challenge of Prescription–Diagnostic Combinations

Passion, Innovation, and Loss

Integrity Testing of Flexible Containers

Focus on Standardization, Quality by Design, and Regulatory GMP

CMOs Ratchet up use of Single-Use Devices to Improve Performance

Development and Commercialization of Biosimilars in India

Developing and Sustaining a Quality Culture

Biobetters and the Future Biologics Market