ast month, we described oral delivery technology for delivery of therapeutic peptides.

 We now describe a recombinant technology for the efficient and cost-effective production of these peptides. The two technologies are complementary to each other for developing a useful therapy. We will describe a direct expression process that is scaleable from 1 to 1,000 L without significant loss of fermentation productivity or yield following downstream purification. This platform technology for recombinant production enables the development of orally delivered peptide hormone drugs for chronic administration in a variety of therapeutic areas such as osteoporosis and diabetes. 

 ISSUES WITH RECOMBINANT PRODUCTION OF PEPTIDES

For peptide hormones that are 25 amino acids or greater, recombinant production in bacteria or yeast offers the potential to be more cost-effective and environmentally acceptable than chemical synthesis, particulary at production scales of hundreds of kilograms per year. However, the relatively small size and lack of tertiary structure of most peptides make them susceptible to rapid degradation in the cytoplasm. Moreover, greater than 50% of the known peptide hormones and neurotransmitters are post-translationally modified by the addition of an amide group to the 

-terminus of the peptide. Under normal physiologic conditions, amidated peptides are expressed as glycine-extended hormone precursors, and subsequently processed to yield the mature amidated hormone. 			

Amidation of the peptide is often required for full biological activity of the hormone.

  The enzyme that performs this post-translational modification is peptidylglycine α-amidating monooxygenase, or PAM.

  Since PAM is not present in prokaryotes, peptide hormones that are produced in 

The degradation problem can be circumvented by expression of these peptides with a larger protein as a fusion partner, which allows the resulting fusion protein to accumulate relatively undegraded in a soluble or insoluble form in the cytoplasm of the host cell.

  Although large amounts of fusion protein can be made in this manner to give yields on the order of grams/liter, there are  several disadvantages. Liberation of the fusion partner from the peptide hormone by chemical or enzymatic cleavage can be difficult and expensive, and the resulting yield of the peptide is greatly diminished, since the peptide represents only a fraction of the entire fusion protein. Also, purification from the large number of cytoplasmic proteins that are released by cell lysis, as well as from the fusion partner itself, may require several steps, which can increase the cost of production and reduce the overall yield of the peptide hormone. 

 ADVANTAGES OF DIRECT EXPRESSION RECOMBINANT TECHNOLOGY

We developed a direct expression process for the efficient production of amidated peptide hormones that involves two recombinant cell lines (Figure 1). In the main path, a glycine-extended precursor of the peptide is produced in recombinant 

 cells using a proprietary direct expression technology.

  The glycine-extended peptide is produced with an upstream signal sequence that translocates the peptide from the cytoplasm to the periplasm. The signal sequence is cleaved in the periplasm, and the peptide is secreted from the 

Figure 1. Dual Recombinant Process for the Production of Amidated Peptide Hormones						

High levels of the peptide are obtained due to several desirable features of the system. These features include the use of a unique, high-expression plasmid vector, a protease-deficient host cell, and a fermentation protocol that allows for high-density cell mass and secretion of the peptide across the outer cell membrane into the growth medium. Since 

 secretes very few endogenous proteins, the glycine-extended peptide hormone can be recovered from the conditioned medium in a relatively enriched form, which reduces the number of downstream purification steps.

Figure 2 shows a high-density SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation of crude conditioned medium from 1 L fed-batch fermentations producing four different peptide hormones: glycine-extended forms of salmon calcitonin, two analogs of a glucose regulatory peptide, and a parathyroid hormone analog. In each lane, the major band corresponds to the secreted recombinant peptide, which represents the major protein species in the crude conditioned medium. The identities of the four peptides have been confirmed by Western blots with the corresponding specific antibodies, and the intactness of each peptide has been confirmed by separation by reversed phase high-performance liquid chromatography (RP-HPLC), followed by mass spectroscopy of the peptide peak (data not shown). This SDS-PAGE experiment confirms that the secreted peptides are intact and that there are few contaminating proteins that need to be removed during purification. 

Figure 2. High-Density SDS-PAGE of Crude Conditioned Medium from Four Different Direct Expression Fermentations 

The other path produces 75 kDa bi-functional PAM in recombinant Chinese hamster ovary (CHO) cells (Figure 1). A readily scalable batch-culture bioreactor-production protocol has been developed. The recombinant CHO cells are grown in a medium that is free of proteins and animal-sourced components, and soluble PAM is secreted into the growth medium. PAM is separately purified from the conditioned culture medium by two column-chromatography steps to yield a preparation that is free of contaminating proteases and shows a single major 75 kDa band on SDS-PAGE. 

 PURIFICATION AND IN VITRO AMIDATION OF PEPTIDES

Glycine-extended peptides are purified from the conditioned growth medium by a three-column purification procedure. Conversion of the peptide-glycine substrate to the peptide-amide by purified PAM is carried out in an 

 reaction with the appropriate buffer and co-factors.  			

PAM is able to quantitatively convert a variety of glycine-extended peptides to the amide form at a mass-ratio of enzyme to substrate as low as 1:1,000 or greater, depending on the penultimate amino acid of the peptide. Hence, the amount of PAM needed is a very small fraction relative to the amount of peptide to be produced, and the high cost of producing PAM in CHO cells does not add appreciably to the overall cost of the process. 

Following amidation, the bioactive peptide is separated from any remaining glycine-extended precursor and other minor contaminants by one or more chromatography steps.  The purified peptide is lyophilized and stored at -80Â°C until formulated into the final product. 

The fermentation protocol used in the direct expression process has been shown to be readily scalable from 1 to 1,000 L for several recombinant cell lines with no significant loss of productivity. The peptide yield from any fermentation varies depending on the peptide. Examples of peptides that have been successfully expressed with this direct expression technology are salmon calcitonin, analogs of parathyroid hormone, glucose regulatory peptide analogs, secretin, and growth hormone releasing factor. For some peptides, yields of approximately 1gram/liter of intact peptide have been achieved. 			

The direct expression technology also offers advantages in purification.  No cell lysis is required to recover the peptide product, and, hence, the peptide does not need to be purified away from the large number of cytoplasmic and periplasmic proteins, nor from the high-molecular-weight DNA that would be liberated from lysed cells. 

 secretes few endogenous proteins, so there are relatively few contaminating proteins in the crude conditioned medium. Since no fusion partner is used, no chemical or enzymatic cleavage is required to liberate the peptide from the fusion partner. The potential for contamination of the final product by DNA and endotoxin is greatly reduced. The yield of peptide following purification and amidation is generally at least 50%, and the purity of the amidated peptide is typically over 98%. 

The direct expression fermentation technology, as well as the purification steps that utilize low-pressure chromatography, is readily scalable. Several proprietary peptides have been expressed and purified in 1,000 L batches at Unigene's manufacturing plant. Considering very large scale, for a peptide that yields 500 mg/L after purification, a single 20,000 L fermenter with the appropriate downstream purification suite, at one batch per week, would be large enough to supply about 500 kg per year. It should be pointed out that the direct expression technology can also be applied to peptides that are not 

-terminally amidated. For such non-amidated peptides,  CHO cell fermentation is unnecessary, and the peptide yield after purification may be higher since the amidation and downstream purification steps are eliminated. 

Vicki Ray and Christopher Meenan have been largely responsible for development of the direct expression cloning and fermentation technology. Angelo P. Consalvo developed the downstream purification and amidation steps. Duncan Miller contributed to the development of the PAM enzyme clone-and-fermentation protocol. Process scale-up and cGMP manufacture of peptides at the 1,000 L scale are conducted under the direction of Paul Shields. 			

1. Mehta NM. Oral delivery and recombinant production of peptide hormones. Part I: making oral delivery possible. 

2. Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide α-amidation.  

3. Mehta NM, et al. Purification of a peptidylglycine α-amidating enzyme from transplantable rat medullary thyroid carcinomas. 

4. Ray MVL, et al. Production of recombinant salmon calcitonin by 

5. Shuman HA and Silhavy TJ. The art and design of genetic screens: 

6. Ray MVL, et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in 

Escherichia coli. Protein Expression and Purification

7. Mehta NM, et al, inventors. Direct expression of peptides into culture media, 

Articles

Hormone Technology

This technology includes an efficient and scalable in vitro enzymatic amidation step for peptide hormones.

Oral Delivery and Recombinant Production of Peptide Hormones, Part II: Recombinant Production of Therapeutic Peptides

lthough biomedical researchers in both public and private organizations like to believe that government regulations and policies are based on scientific evidence, many aspects of the development and production of drugs and biologics are shaped by political concerns. The debate over drug importation is a prime example of politics trumping science as members of Congress reject the consensus of scientists and policy experts that opening US borders to therapies from abroad raises serious public health concerns. Similarly, FDA's rejection of non-prescription drug status for the morning-after pill, called Plan B, reflects political demands more than clinical evidence (see "Missed Opportunity"). 

Members of Congress often weigh in on FDA decisions to approve or reject new treatments for cancer and other life-threatening diseases, even when the science is weak. Pressure from policy makers to reduce spending on prescription drugs, moreover, is building support for more generic products, a trend that may shape the looming debate on "follow-on" biologics. This could have a direct effect on pharmaceutical and biotech manufacturers. 

Although the first embryonic stem cell was isolated in a US laboratory, future advances may occur elsewhere due to current administration policies. In August 2001, President Bush limited federal funding of embryonic stem cell research to already-established cell lines. Unfortunately, less than 20 of the 78 originally identified lines now appear to be available for research. To fill this widening gap, British officials have opened a national stem cell bank to store and distribute embryonic and adult stem cells to scientists around the world. And US states and research institutions are launching locally funded stem cell research initiatives.  			

However, pressure to liberalize federal policy may be having some impact. In April, 206 members of the US House of Representatives sent a letter to President Bush urging reconsideration of the current policy, and about 50 senators signed a similar plea for flexibility. Former First Lady Nancy Reagan made headlines recently by strongly supporting stem cell research as a way to find cures for Alzheimer's and other diseases.  

NIH Director Elias Zerhouni responded to the House's letter with a carefully crafted statement that offered research advocates some hope for future change. Although Zerhouni reiterated the Bush administration's policy that public funds should not encourage destruction of human embryos that have potential for life, he also acknowledged that access to additional stem cell lines could speed scientific research. Zerhouni subsequently explained that the current policy is based on the president's moral and ethical concerns and admittedly not on scientific evidence. The hope is that the White House might open the door for researchers to access embryos abandoned in fertility clinics and slated for destruction anyway.  

On another front, the worldwide clamor for low-cost, fixed-dose combinations (FDCs) of antiviral drugs for third-world countries produced a positive response from Washington. At the opening of the recent World Health Organization annual meeting in Geneva, Health and Human Services Secretary Tommy Thompson announced a program promising speedy approval of already-marketed AIDS drugs that manufacturers will co-package in easy-to-use blister packs or reformulate as single pills.  			

To encourage manufacturers to submit applications for these products, FDA issued a draft guidance for industry on May 14 that spells out what sponsors must do to gain speedy approval (see 

). The guidance lists drug regimens and components that FDA believes have sufficient clinical safety and efficacy data to support concomitant use and that fit agency criteria for two- and three-drug regimens. It notes that FDA has systems in place to handle priority review and fast-track designation of critical therapies. Plus, according to the guidance, FDA has the authority to refer to existing clinical safety and efficacy data, including published reports, to approve new combination drugs with little additional testing. FDA also expects to waive user fee payments and pediatric study requirements to speed these medicines to market. Manufacturers will need to propose systems for collecting and reporting adverse drug reactions in developing countries, an activity that local health agencies distributing the products are more likely to handle. 

Although skeptics complained that the FDA fast-track FDC approval initiative merely repeats the World Health Organization program to "prequalify" FDCs and will delay access to needed therapies, the policy is generating a positive response from manufacturers. Right after Thompson's announcement, California-based Gilead Sciences announced FDA granted priority review to its previously filed application for a fixed dose co-formulation of two approved antiviral medications: Viread (tenofovir) and Emtriva (emtricitabine). Both products have been on the market for more than a year and should receive an approval decision in the fall or sooner. 

Similarly, GlaxoSmithKline is hoping for speedy FDA approval for a combination of its Epivir (lamivudine) and Ziagen (abacavir). GSK also may develop a broader FDC with Boehringer Ingelheim's Viramune (nevirapine), while Gilead is working on a combination pill with Merck and Bristol-Myers Squibb. These manufacturers may first develop co-packaged versions of these therapies while testing and seeking FDA approval of new combo treatments.  

The fast-track approval strategy addresses the fact that the President's Emergency Plan for AIDS Relief (PEPFAR) is required to purchase only those drugs that demonstrate safety and effectiveness to a "stringent regulatory authority" such as FDA. Using taxpayer dollars to purchase drugs not approved for use at home threatened to open the door to charges that the US was foisting lower-quality treatments on poor nations, which can lead to drug resistance.  

By establishing an FDA review system for these products, the State Department will be able to purchase them for use in foreign markets. FDCs produced by generic drug makers that include patented products may be distributed by US health programs in areas where the manufacturer does not enforce its patents, but not in the US. Thus, the program carefully addresses political demands while accommodating scientific requirements. 

If there was a scientific rationale for rejecting the over-the-counter drug application for the morning-after pill, called Plan B, FDA officials failed to articulate it to the medical community or the public. Last December, two FDA advisory committees voted convincingly (23 to 4) to approve an OTC switch application from Barr Pharmaceuticals, a recommendation that FDA staffers usually follow. But in this case, agency officials responded to high-level demands to find a legitimate rationale for rejecting the submission. 			

Steven Galson, acting director of the Center for CDER, sent a non-approval letter to Barr in May. Because it is unusual for the CDER chief to sign such a letter, it suggested that Galson was overruling the pro-approval sentiment of his staff. FDA based its decision on a lack of data on appropriate use of the therapyby young women (under 16). In response, Barr is seeking dual status for Plan B: OTC access for adults (entails ID checks) and prescription status for patients under 16. Such a dual labeling system raises a host of novel legal issues that could take some time for FDA to resolve.  

FDA's action rekindled charges that its regulatory policy often stymies access to new treatments. More than 40 members of Congress sent a letter to acting FDA Commissioner Lester Crawford urging reversal of the Plan B decision. Manufacturers and political observers wonder if FDA is becoming more risk-averse in bringing new products to market, and the agency missed an opportunity with Plan B to lay such fears to rest. 

Columns and Departments

The debate over drug importation is a prime example of politics trumping science as members of Congress reject the consensus of scientists and policy experts that opening US borders to therapies from abroad raises serious public health concerns.

Regulatory Beat: Politics vs. Science in Biomedical R&D

wo quarterly meetings of the BioPharma Operations Excellence Consortium were held over the first three months of 2004. On February 3, the US West Coast chapter of the consortium had its first meeting of the year at Abgenix in Fremont, CA, and on March 25 in the Netherlands, the European chapter kicked off its first meeting, which was hosted by Centocor Leiden.  

The Abgenix meeting hosted 40 professionals from over 15 companies and focused on contract manufacturing, a hot topic in an industry periodically beset by capacity hortages. In addition to the presentations and group discussions, participants were given a facility tour of Abgenix's large-scale manufacturing plant. 			

One of the more intriguing sessions of the day was a presentation by Joe Dillon, vice president, supply chain, of Silterra, a semiconductor contract manufacturing organization (CMO) based in Malaysia. Dillon discussed the industry's background and how the consortium discussions reminded him of the semiconductor industry 20 years earlier. He also shared that since the '80s, the semiconductor industry has made major advancements that biopharmaceuticals companies might want to emulate. 

As an example, Dillon noted that advanced manufacturing execution systems allow customers of semiconductor CMOs to have full, real-time visibility of their products in the CMO plants. He also mentioned that there are a large number of "fabless" companies that focus on product development and outsource all their manufacturing to CMOs, mainly located in Asia. Plus, according to Dillon, manufacturing technology drives improvement in semiconductors, and, as a result, a semiconductor plant can expect to turn over 33% of its manufacturing equipment every three years. He added that labor only constitutes 5% of the product cost, while the tax advantage is the real driver to building CMOs in Asia. The semiconductor industry can thoroughly inspect each product made, Dillon said. He concluded by noting that although there are many differences between the industries, especially related to regulatory requirements, there are enough similarities that the future of biopharmaceuticals with respect to contract manufacturing might look much like the semiconductor industry. 

The other presentations of the day were given by Steve Chamow, vice president, process sciences at Abgenix; Karen Walker, director of quality at Abgenix; Patrick Hanley, Sr., manager of packaging at Genentech; Sid Advant, director of technology transfer and clinical operations at Diosynth; and Allan Harmon, plant manager for Baxter BioScience.  

The main focus of the presentations given by Walker, representing the development side, and Advant, standing for the CMOs, was the need to develop frequent communication between the organizations as well as a clear, focused set of expectations from the technology transfer to the development of the quality agreement. Harmon's presentation focused on Baxter BioScience's transition of its Hayward, CA facility to transition from supporting internal customers to opening its doors to external customers. For Baxter BioScience, becoming a contract manufacturer required the establishment of a customer-support organization, as well as improvements to the physical plant to provide added transparency to customers and offer visitors a better understanding of the current manufacturing processes.  

Abgenix's Chamow discussed the challenges of increasing efficiency in biologics production as well as the key factors to increasing titer. Most of the newer, large-scale production facilities coming on-line are focused on batch production. A discussion developed about perfusion vs. batch fermentation.  From a manufacturing perspective, perfusion production theoretically can be more efficient, requiring smaller vessels and fewer changeovers compared to batch processing. This is especially true for processes where large quantities of material are required and suites can be dedicated to products. Many limitations of perfusion were discussed as well. Current technology and equipment constraints have made large-scale production challenging. Also, perfusion requires a long-term, up-front investment for validation. Instead of running three batches over two months to validate a batch process, one may need three runs over at least six months to validate the perfusion process. Because the biopharmaceutical industry is still young, it is too early to tell which technology will become the standard in the end — but batch processing has a healthy lead.  

Hanley kicked off an interesting discussion of Genentech's validation of automated vial inspection for liquid-filled vials. Genentech has been exploring the use of automation, due to the ergonomic risks associated with vial inspection and the significant labor costs and space requirements needed to meet growing capacity demands. The discussion focused on the difficulty manufacturers face in validating current manual inspection processes and identifying liquid particles in products. Hanley went on to describe his company's newly proposed inspection process and how it is being validated against current manual processes. Although the process is not fully validated, Hanley said Genentech expects that in the future, it will be able to significantly limit the growth of the inspection group, be more consistent in its inspection of vials, and, most importantly, dramatically minimize the injury rate. 

The Centocor session, facilitated by Tefen's European office, was the European chapter's first meeting, hosting over 30 professionals from 18 companies. The large number of participating companies, all European-based biopharmaceutical manufacturers, demonstrated the need for such a forum and the willingness of organizations to share challenges and improvement initiatives. As it was the group's  first meeting, a large portion of the day was dedicated to introductions, development of clear expectations, charter statements, and working procedures.  			

Pedro Tetteroo, vice president and general manager of Centocor Leiden,  delivered the day's keynote address. He presented Centocor's vision and manufacturing strategy as well as the company's successful growth story over the past four years. Tetteroo concluded by emphasizing the importance of operations and process excellence to Centecor's success.  

Other presentations were given by Eric Goossens, director of production operations, and Johan Telen, director of process excellence at Centocor Leiden; Jo Dalle, director of manufacturing and Wim Croonen, director of human resources at Genzyme (Belgium); Dave Sherwood, director of global quality and compliance at Celltech (UK), and Martin Wrankmore, manufacturing continuous improvement leader at Lonza Biologics (UK). The presentations provided overviews of the companies' current operations excellence programs. The speakers mainly focused on lean operations and Six-Sigma methodologies.  

Tefen presented  a case study of a successful one-year deviation reduction program it led in partnership with a large, multi-site biopharmaceutical organization, using a Six-Sigma framework. It not only significantly reduced the backlog of open nonconformances (NCs),  but it also allowed for the implementation of required infrastructure for continued reduction in NC rates and closure cycle time, including a mechanism for visibility enhancement and elevation of NC reduction ideas from the staff. The program resulted in more than a 35% reduction in the average number of NCs per lot and more than a 50% reduction in average closure time per reported deviation. 

The East Coast chapter's meeting was held on May 11 at Centocor, Malvern, PA. Meeting highlights will be summarized in a future column.  			

At press time, the European chapter's next meeting was set for July 7 and it was to be hosted by Serono in its Vevey, Switzerland facility. The next West Coast meeting has not been announced. For more information about the consortium and details on future meetings, please e-mail us at 

Although there are many differences between the industries, especially related to regulatory requirements, there are enough similarities that the future of biopharmaceuticals with respect to contract manufacturing might look much like the semiconductor industry.

Operations Excellence: BioPharma Consortium Meets in California and the Netherlands

herapies based on living cells hold great promise as potential cures for life-threatening diseases. These therapies can repair, restore, replace, or regenerate the affected body system in the patient. One example is the use of 

 manipulated peripheral-blood or bone marrow progenitor stem cells as immunotherapies. Researchers are trying to create engineered tissues or biohybrid organs by combining cells with various medical-device biomaterials and other biologically active molecules.

A lingering concern is always the very simple question: "Is it safe?" However, answering that question for therapies based on living cells is not simple because biological safety (biosafety) is largely relative; for cellular-based therapies (CBTs) it is perhaps best discussed in terms of biosafety risk. Such risks can be assessed only after considering numerous factors, such as the source of the cells or tissue, preparation methods, the intended recipient, where in the body the cell-based therapy will be implanted, and the method of cell delivery. This article provides an overview of biosafety issues facing cell- and tissue-based products and discusses a systematic, practical approach for addressing them.  

CBTs are prepared from living sources, which raises clear biosafety concerns regarding the potential presence of infectious disease agents transmitted from the donors. Highly specialized manufacturing processes are often used, commonly necessitating the use of non-FDA approved ancillary materials (for example, media, cytokines, or growth factors) of uncertain safety and quality. Frequently, the final cell-containing product is intended for implantation or delivery into a specific site in the body and interacts with the cellular milieu in a specialized manner to yield the therapeutic effect. This is why biosafety concerns cover the delivery systems used and the post-implantation fate of the cells (Table 1). 

Table 1. Types of Biosafety Risks for Cell and Tissue-Based Therapies  

Manufacturing procedures for therapies based on living cells or tissues are unique (Table 2). This is important since many physiochemical techniques such as viral inactivation, filtration, and terminal sterilization are incompatible with therapies dependent upon living cells since such methods would render the cells nonviable. Using such techniques to make a CBT safe would eliminate the possibility of it also being efficacious.

Table 2. Unique Aspects of Cell and Tissue-Based Products  

Given the inability to completely assess risks that may be lurking in a product derived from living sources and the inability to inactivate or eliminate unknown threats inherent in living cells, it is obvious that providing an absolute assurance of biosafety is not possible. Consequently, a systematic, risk-based approach to biosafety must be employed that considers the unique characteristics of the product in relation to the condition of the recipient.

  Such an approach requires an ongoing biosafety risk assessment throughout all stages of development — before, during, and after administration of the product. 

A comprehensive program for assessing, minimizing, and monitoring risks to biosafety includes the following elements, which are also illustrated in Figure 1: 

1. Qualification of materials used in product manufacturing 

Biosafety and qualification of cells and tissues 

Biosafety and qualification of key ancillary materials

3. Preclinical pharmacology and toxicology 

Figure 1: Biosafety Must Be Assessed At All Stages 

 Qualification of materials used in product manufacturing

Federal regulations mandate establishment of a quality control unit as part of good manufacturing practices (GMP).

  This is particularly important for cell- and tissue-based products, since the manufacturing process entails the use of source and ancillary materials of differing complexity, variability, and risk for introduction of adventitious agents. FDA has made it clear via conference presentations, mass mailings to IND sponsors and guidance documents that developers of gene therapies and cell- or tissue-based products must have a qualification program in place prior to initiating clinical studies.

A critical function of a manufacturing quality-assurance and quality-control program (QA/QC) is the development of a qualification program for the source of cells and tissues that will be used as part of the final product, and all critical reagents, including ancillary materials such as cytokines and growth factors needed to prepare the final product but are not intended to be part of the final product. 

Table 3. QC/QA Biosafety Program for Product Manufacturing  

QA/QC is your first line of defense in evaluating and, where possible, eliminating known risks to biosafety presented by a source material or key ancillary materials and at key stages in manufacturing as outlined in Table 3. There may be unforeseen risks to biosafety that only emerge during preclinical safety testing in relevant animal models. Some risks can only be evaluated when this stage has been reached. 

Biosafety and qualification of cells and tissues.

 A core set of documents available from FDA regarding donor screening as well as direct testing of cells and tissues for pathogens of concern, is applicable to the diversity of sources of cells and tissues used to create cellular- and tissue-based products (autologous, allogeneic, xenogeneic, banked vs. non-banked).

  For developers of therapies based on human cells and tissues, FDA's draft reviewers' guidance contains helpful information on testing autologous and allogeneic cell and tissue sources as well as testing of cell banks.

  The guidance also cites key documents, such as those from the International Conference on Harmonization (ICH).  

Similarly, for therapies based on animal-derived cells and tissues (xenotransplantation), or cells and tissues that are co-cultured with animal cells (for example, murine feeder cells), valuable information can be found in FDA's guidance on xenotransplantation.

  It is important to recognize that these guidance documents describe only general expectations for biosafety assessments and in the case of ICH guidance documents, often specifically exclude CBTs from their scope. However, in the absence of more specific guidance for cell containing products, the principles described in these documents are applicable. 

There are additional biosafety concerns to be addressed for cell-based therapies involving 

 genetic modification of cells (regulated as gene therapy). Specific biosafety testing or information to be gathered depends on the vector system used and can include product testing and long-term patient follow-up.

Biosafety and qualification of key ancillary materials.

 Often, in order for a cell- or tissue-based therapy to possess the desired therapeutic characteristics, it must undergo a variety of manufacturing steps involving enzymes, cell selection or depletion with antibodies, and 

 culture in serum-containing media with a cocktail of cytokines and growth factors. Typically, these processing materials are only transient and are not intended to be part of the final product. The biosafety of these ancillary materials must be assessed since they come in contact with cells that will be subsequently administered to patients. The degree of biosafety necessary to satisfy regulatory authorities depends largely on its origin (synthetic vs. animal- or human-derived) and whether the vendor supplied the reagent for "

  research only" versus a product previously approved by FDA for human use.

Regardless of the regulatory status of an ancillary material, the manufacturer of the cell- or tissue-based product is ultimately responsible for developing procedures to evaluate risks to biosafety and performing additional testing. Demonstrating removal or absence of an ancillary material from the final product is one example where additional testing is needed regardless of whether or not the ancillary material is FDA-approved.  

 21 CFR 610 requires that the final product is tested prior to release for distribution and administration in patients.

 These release tests include a number of specifications that must be met for microbiological safety, including bacterial and fungal sterility, pyrogenicity (endotoxin), and mycoplasma (for cultured products). Also included are a number of release specifications relating to product quality and characterization, such as identity, purity, potency, and cellular viability. There are many legitimate issues regarding the need for some types of testing, or the length of time it takes to obtain test results in relation to the limited stability of many CBTs. 

Fortunately mechanisms exist for alternative test methods.

  Overall, confirming that the final product meets specifications for release is a key component of ensuring product quality and biosafety for biological products. For example, a variety of cell-based therapies are being investigated for various types of cardiac disease. One approach involves the use of bone marrow to stimulate angiogenesis in ischemic heart tissue. Unfractionated bone marrow contains a complex mixture of cells including T cells, B cells, NK cells, monocytes, macrophages, stromal cells, and neutrophils, which makes it difficult to determine which cells may contain therapeutic vs. non-therapeutic contaminating elements. This raises biosafety concerns since these cells produce a variety of cytokines that could lead to deleterious effects such as inflammation, fever, acute phase response, lymphocyte activation, and proliferation. 

  studies are needed to determine the significance of these concerns.

 Once information becomes available regarding these issues, it may be necessary to establish release specifications for the purity of the final product to avoid the possibility of adverse clinical events. Thus, the ability to characterize the final product by the purity-impurity profile can have important biosafety implications. 

Undesirable pharmacological activity or unrecognized toxicities are unlikely to be detected through 

 testing, and generally must be evaluated in a relevant animal model. This is, of course, due to the unique aspects of CBTs. 

 Because of the diversity of cell sources and disease targets for CBTs, it is clear that no single type of preclinical study design will be appropriate for all products. The information gained from preclinical studies will be used to design clinical studies. The cellular construct, dose, site of implantation, and duration of treatment should mimic those planned for clinical studies. 

Design preclinical animal safety studies to address issues specific to the cellular construct being used, including issues related to the site of implantation, and the post-implantation fate of the cells. For example, if the cells being implanted have been genetically modified, studies evaluating the level and persistence of gene expression, as well as the potential for inappropriate expression of the gene product, will likely be necessary. Similarly, if the cells are implanted as part of a construct containing a biomaterial or seeded within a device component, additional preclinical safety assessments will be needed (Table 4). 

Table 4. Preclinical Biosafety Considerations for Engineered Cell-Biomaterial Constructs 

The principles involved in designing and developing appropriate preclinical testing to determine the safety of cell-based products are similar to those encountered for other biological products. Key components of protocol design for preclinical studies include: 

discerning activity and toxicity of the product (mechanism of action)

recommendation of an initial safe dose and dose escalation scheme in humans

identification of potential toxicity or activity target organs

identification of parameters that should be monitored clinically

identification of patient eligibility criteria.

When available, relevant animal species and animal models of disease should be utilized in preclinical studies. We all recognize that no single species will be representative or predictive for all patients. Ideally, the animal disease model chosen should have a similar pathophysiology and anatomy to humans, which should improve predictability of human risks as well as facilitate modeling of route of administration and dose exploration. Standard animal models of disease are frequently modified to generate the preclinical toxicity data. For example, immunological reactions to human cells in animals often necessitate that preclinical toxicology studies be performed with autologous animal cellular products (animal products that are analogous to the intended clinical product), rather than the actual human product.

  Follow good laboratory practices (GLP) for preclinical safety studies. However, given the unique design requirements of preclinical assessments of CBTs, regulatory authorities recognize that this is not always possible. In these cases the "spirit of GLP" should be followed. 

Performing preclinical safety studies in an animal model of the disease depends onthe existence of such a model. Where models do exist, recognize their limitations, such as whether the model is relevant to the pathophysiology of the disease in humans, the physical size of the animal, and potential technical limits on feasibility. Other important considerations are the availability of the animal model, including age, gender, and numbers needed for statistical considerations; the need for specialized housing; animal welfare concerns; costs; and the availability of historical data to support the animal model. 

In some cases, more than one animal model may be needed. For example, a small-animal model could provide important preclinical safety and proof of principle information. However, a study in larger animals may still be necessary if there are issues relating to the size of the cellular implant or if specialized tools are needed to deliver the therapy that cannot be effectively modeled in the small animal. Limit unnecessary use of animals, and discuss specific concerns with knowledgeable consultants or directly with regulatory authorities prior to initiating preclinical pharmacology and toxicology studies.  

 What do you do when no relevant preclinical animal model exists? In those situations, obtaining preclinical safety data in normal animals is necessary. At a minimum, treated animals should be monitored for general health status, serum biochemistry, and hematologic profiles. Microscopically examine target tissues for histopathological changes. Pharmacological studies in normal animals may also provide useful information regarding the 

 function, survival time, and appropriate trafficking of the modified cells.

One consequence of not being able to obtain relevant information concerning the pharmacological and toxicological effects of a CBT in an animal model is the need to design a resource-intensive clinical program. This includes frequent patient monitoring for adverse effects, since limited information is available as to which clinical safety parameters are important. Additional patients may be necessary, since information on the effects of the product in relation to the specific stage of disease may be unclear. It may be necessary to include long-term patient follow-up since information on long-term consequences of the therapy, such as patient morbidity or mortality, cannot be anticipated in the absence of animal studies. 

The goal of a scientifically sound preclinical study design is the ability to identify potential biosafety concerns that need to be examined in the clinical trial. The data from preclinical studies serve as the basis for risk-benefit decisions that are made in the context of the patient population, severity of disease, and the availability of alternative therapies. 

CBTs are delivered to patients in a number of ways, including infusion or injection via syringes or catheters and, in some cases, surgical implantation as part of construct-containing cells and medical-device biomaterials. Since there are no systems specifically approved for the delivery of CBTs, some preclinical assessment is usually necessary. For example,  catheters may present issues with clogging of lumens by the cell suspension, loss of viability of the cells, and uncertainty regarding the dose of cells delivered due to dead space in the catheter system. For catheters with needles there may be additional safety concerns regarding the ability to precisely deliver cells to a localized injection site versus inadvertent injection into the systemic circulation.

  Biosafety issues related to the post-implantation fate of the cells include a variety of risks that often overlap (Table 5). One example is a product derived from stem cells intended to be injected into a specific region of the brain. Ideally, safety studies in an animal model would address the ability of those cells to migrate away from the implantation site. Such migration might be acceptable if the cells are expected to integrate into appropriate anatomic structures within the brain that are distant from the implantation site. Conversely, this may be undesirable if the cells migrate to regions where they normally do not exist and aberrantly secrete excitotoxic substances. Similarly, it would be important to determine the influence of the local microenvironment at the implantation site on cellular differentiation and expression of the desired phenotype.

Table 5. Biosafety Assessment of Cellular Fate Post-Implantation  

It should go without saying that assessing biosafety is an essential element of investigational clinical studies. After all, a fundamental tenet of good clinical practice (GCP) is that a clinical trial of an investigational therapy should be initiated and continued only if the anticipated benefits justify the risks.

  GCP forms the basis of the worldwide clinical trials system. Initiation of a clinical study should be predicated on a risk-benefit assessment largely based on biosafety data accumulated from preclinical safety studies, qualification of components, and testing of the final product. For example, in the US, adequate information about chemistry, manufacturing and control of the product, as well as adequate pharmacological and toxicological data,  must be provided prior to initiating investigational studies.

  To ensure the safety of patients, incorporate the information collected regarding possible risks and side effects into the design of the clinical studies. 

Intense clinical monitoring of the human subject is expected after administration of the product. FDA requires sponsors to report serious and unexpected adverse events associated with use of a product as soon as possible and within 15 calendar days. Sponsors must report any unexpected fatal or life-threatening experience associated with the use of the product as soon as possible and within seven days. 

The need to have a well-defined clinical monitoring program is something FDA has emphasized for both cell-based and gene therapy products in recent years.

  In terms of gene therapy products, which include genetically modified cells, FDA recently announced the "Gene Therapy Patient Tracking System,"

 which includes a provision for the collection of short- and long-term patient safety and outcome data.

  A similar system is under consideration for long-term patient follow-up for recipients of xenotransplantation products.

  Upon marketing approval, FDA expects ongoing postmarketing safety data collection and risk assessment based on observational data, as it does for all approved products, as part of pharmacovigilance.

Products based on living cells appear to hold significant therapeutic promise for addressing unmet medical needs. In light of the approximately 340 ongoing clinical trials involving cell therapies, perhaps this is an under- statement.

  However, such enthusiasm must be tempered by the potential biosafety risks these products present. Many of these risks, such as those presented by infectious agents, are common to all biological products, and well-established procedures such as donor screening and testing, as well as qualification of source and ancillary materials, should greatly minimize those risks.  

However, the very characteristics that many cells  possess, such as the ability to proliferate, differentiate, and integrate into a diseased or damaged body system, also elicit concerns regarding biosafety. Since there are a number of uncertainties regarding the ability of 

 systems to address these issues, we are left to ponder the notion that "absence of evidence is not evidence of absence."

 An ongoing biosafety vigilance program (before, during, and after administration of the product) should significantly minimize the actual biosafety risks inherent in CBTs. 

This article is dedicated to my former colleagues in the Office of Cellular, Tissues and Gene Therapies (OCTGT), CBER, FDA. The basis of the information presented in this article has been gleaned from conversations with many of you over the years, as well as guidance documents you have written and presentations you have made. Thanks for so freely sharing your knowledge and enthusiasm for these products with me. 

1. FDA. Code of Federal Regulations, Title 21 Part 600, Section 3(p): Definition of safety for biological products. 2003 April 1. 

2. FDA. Code of Federal Regulations, Title 21 Parts 210 and 211:  Good manufacturing practices. 2003 April 1. 

3. Frey-Vasconcells J. Presentation on GMPs. Biotechnology Industry Organization (BIO) Annual Meeting; 2000 Mar 26-30; Boston, MA. 

4.  FDA. Dear gene therapy IND or master file sponsor letter. 2000 Mar 3. Available at: 

5.  International Society for Cellular Therapy. Cel therapy regulatory requirements: recent developments. 

 www.celltherapy.org/imember/telegraft/Sept2002.pdf 

6.  FDA, CBER. Draft guidance for reviewers: instructions and template for chemistry, manufacturing, and control (CMC) reviewers of human somatic cell therapy investigational new drug applications (INDs). 2003 August 15. Available at: 

7.  FDA, CBER. Guidance for industry: source animal, product, preclinical, and clinical issues  concerning the use of xenotransplantation products in humans. 2003 April 3. Available at: 

8. FDA, CBER. Guidance for human somatic cell therapy and gene therapy. 1998 Mar 30. Available at: 

9. FDA, CBER. Eligibility determination for donors of human cells, tissues, and cellular and tissue-based products; final rule and notice. 2004 May 25. Available at: 

10  FDA, CBER. Guidance for industry: supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients in clinical trials using retroviral vectors. Available at: 

11. USP Expert Committee on Gene Therapy, Cell Therapy and Tissue Engineering. Ancillary materials for cell, gene, and tissue-engineered products; in-process revision, 

12.  FDA. Code of Federal Regulations, Title 21 Part 610. General biological product standards. 2003 April 1. 

13.  International Conference on Harmonisation.  Preclinical safety evaluation of biotechnology-derived pharmaceuticals, guideline S6. Available at: 

14.  FDA, Biologics Response Modifiers Advisory Committee. Cellular products for the treatment of cardiac disease, FDA briefing document. 2004 Mar 18. Available at: 

www.fda.gov/ohrms/dockets/ac/04/briefing/4018b1.htm 

15.  Jensen D. Cardiac catheters for delivery of cell suspensions. Presented at FDA Biologics Response Modifiers Advisory Committee Meeting; 2004 Mar 18; Silver Springs, MD. Available at: 

www.fda.gov/ohrms/dockets/ac/04/slides/4018s1.htm 

16.  International Conference on Harmonisation. Good clinical practice, consolidated guideline E6. Available at: 

17.  FDA. Code of Federal Regulations, Title 21 Part 312: investigational new drug applications. 2003 April 1. 

18.  FDA, CBER. Gene therapy patient tracking system. 2002 June 27. Available at: 

 www.fda.gov/cber/genetherapy/gttrack.htm 

Biosafety

Is it safe? Answering that question for therapies based on living cells is not simple.

Biosafety Considerations for Cell-Based Therapies

he biopharmaceutical industry features growth and job mobility. Competition for talented employees creates opportunities for them but headaches for management. 

The key employees are a company's greatest assets. Besides losing the talent when they quit, the second biggest loss could be confidential, proprietary, or trade secret information. Your strongest defense is requiring employees to sign non-competition and non-disclosure agreements.   

These agreements are legal documents and must be written broad enough to protect the company and narrow enough to be enforceable. We have prepared a series of three columns to describe the basic components of such agreements. If you are an employer or employee in the US, you want to know what you are signing.  

 THE PURPOSE OF NON-COMPETITION AND NON-DISCLOSURE AGREEMENTS

		Generally speaking, the purpose of non-competition and non-disclosure agreements is to protect the confidential, proprietary, and trade secret information of employers. The aim of these agreements is to ensure that competition in a given marketplace is conducted on an equal footing and that no company can inappropriately use another's secret information to gain an unfair competitive advantage. However, the courts view non-competition and non-disclosure agreements as, in part, restraints on trade and restraints on an individual's ability to earn a living. Therefore, courts scrutinize non-competition and non-disclosure agreements to ensure that they serve the limited purpose of protecting confidential, proprietary, or trade secret information. 			

The definition of confidential, proprietary, or trade secret information varies from state to state. Generally speaking, product formulations, manufacturing processes, client lists, customer lists, database information, and marketing and strategic plans fall within this definition. These are the appropriate topics for non-competition and non-disclosure agreements. Conversely, information that is generally regarded as public record and standard knowledge in a given industry is not properly subject to protection by non-competition and non-disclosure agreements. This is the first level of narrowing.  

			The employer's first step in considering whether to use non-competition and non-disclosure agreements is to assess, analyze, and understand the benefits of such agreements. The first step in the assessment process is identifying the types of information categorized as confidential, proprietary, or trade secret. Then identify particular employees or groups of employees who possess or will learn about that information. The best and most-enforceable non-competition and non-disclosure agreements are ones that are narrowly tailored to ensure that those employees do not disseminate the information following cessation of their employment with the company.   			

The simplest and most cost-effective way to address non-competition and non-disclosure issues is to do so at the drafting stage. Properly thought-out and drafted agreements will provide employers with sufficient protection and will likely preclude dissemination of its confidential, proprietary, and trade secret information. In contrast, "boilerplate" agreements, with no thought or legal guidance behind them, put employers and their most important information at risk. 

Companies should perform the above-referenced assessment as a matter of course when drafting non-competition and non-disclosure agreements. Courts are notorious for subjecting agreements to intense scrutiny. Advising the court of such an assessment at the litigation stage of any non-compete case will certainly benefit the company. It demonstrates to the court that the employer is, in good faith, simply trying to protect its most important information and not unduly attempting to restrain trade or an individual's right to earn a living as a trained specialist. 

Maw v. Advance Clinical Communications, Inc. 359 N.J. Super. 420 (App. Div. 2003)

 went to two levels of state court. The case underscores the importance of the assessment process and provides employers with a clear view of how strictly courts may assess non-competition and nondisclosure agreements. The New Jersey Appellate Court sided with an employee, a graphic designer, who refused to sign a non-competition and non-disclosure agreement and was terminated for her refusal to do so. The court's decision was that the employee could properly assert a cause of action for retaliation and wrongful discharge. Essentially, the court concluded that the employee's position with the company did not provide her with access to any confidential or proprietary business information. Thus, the court concluded that the employer had no legitimate, good-faith basis to compel her to execute a non-competition and non-disclosure agreement, as there was no need to restrain her future employment.   							

The Maw case was recently overturned by the New Jersey Supreme Court 

[Maw v. Advance Clinical Communications, Inc. 2004 WL 941640 (N.J. Supreme Ct., May 4, 2004)

]. While the New Jersey Supreme Court did, ultimately, dismiss the employee's retaliation claim, the Appellate Division's decision demonstrates the hesitancy of courts to provide blanket approval to non-competition and non-disclosure agreements and illustrates that it is imperative for employers to engage in an assessment process prior to requesting that employees sign non-competition and non-disclosure agreements. Executives, research and development employees, and sales people certainly possess information subject to protection. There are other classes of employees who do not possess sensitive information. So, in addition to assisting employers in non-compete litigation, a proper assessment process also limits an employer's potential liability for retaliation and wrongful discharge litigation. 

			Companies can't protect their employees from competitors forever. So, when drafting non-competition and non-disclosure agreements, address the scope and time of any non-competition clause. Courts teach that in order for a non-competition clause to be enforceable, the clause must be "reasonable" in both scope and time. 			

Similar to the definition of "confidential, proprietary, and trade secret information," the definition of "reasonable in scope and time" varies from state to state. Generally, non-competition clauses that restrict an individual's ability to work in a given industry across the United States for many years will be deemed to be unenforceable (unless there is some extraordinary reason for such a broad restriction). Conversely, the courts look at non-competition clauses that are narrowly drawn to prevent employees from causing substantial harm to a company more favorably, and they are more likely to enforce such clauses exactly as written. 

What if an employer is concerned about a high-level salesperson leaving its employ for one of its two chief competitors but is not concerned with the employee going to any of its other competitors? That issue should be dealt with at the drafting stage. Specifically, the non-competition clause should state, in clear and concise terms, that the employee is not permitted to work for either of the chief competitors for a certain period of time. Such a specific clause heads off any argument by the employee that the clause is too broad and restrictive and makes it likely that the court in any non-compete litigation will enforce the clause. 

			We have found, when faced with potential or actual non-compete litigation, that many employers do not appreciate the nuances and potential risks associated with such agreements. The scope of such risk, and an explanation of typical non-compete litigation, will be the subject of the next column in this series. 			     

The employer's first step in considering whether to use non-competition and non-disclosure agreements is to assess, analyze, and understand the benefits of such agreements.

Career Development: Keeping Your Secrets in Your Company

 few months ago, I wrote a column on the rapid advancements in human genome technology and how Wall Street was treating those advances like a luckless poker player treats a pair of aces on the deal and two more on the flop. In other words, very enthusiastically. It turns out that genomics is only half the story. In Wall Street brokerage firms, in financial advisor circles, and on mutual fund trading desks, the other shoe has dropped in the form of the biochip — those DNA-styled microchips that more and more industry companies are starting to use to examine the genomic templates of various diseases (and hopefully to help them find ways to cure them). In laymen's terms, biochips are sliver-sized microarray disks that harbor tens of thousands of DNA segments (or "probes" as laboratories call them) that represent human genes. 

Biochips are creating a great deal of interest in biopharmaceutical and investment circles. What can new chip technology offer investors? Apparently plenty. 

According to a 2002 report by the consulting firm BioPerspectives, the protein biochip market will reach $700 million in sales by 2006.

BioPharm International-07-01-2004

Oral Delivery and Recombinant Production of Peptide Hormones, Part II: Recombinant Production of Therapeutic Peptides

Regulatory Beat: Politics vs. Science in Biomedical R&D

Operations Excellence: BioPharma Consortium Meets in California and the Netherlands

Biosafety Considerations for Cell-Based Therapies

Career Development: Keeping Your Secrets in Your Company

StreetTalk: Hello, Mr. (Bio) Chips

Affinity Chromatography — from Textile Dyes to Synthetic Ligands by Design, Part I

BSE Offers Lessons in Risk Assessment

Final Word: Canada's Landmark Decision: Planting the Seed