After many starts and stops, hype and disappointment, foreign protein expression in plants is now routine and biopharmaceuticals produced in green plants will soon be with us. Plants have been part of medicine for thousands of years and pharmaceuticals still rely heavily on plant-based materials: more than half of our pharmacopoeia is extracted from plants or related to plant compounds. 

Plants have been part of medicine for thousands of years, but bioengineering plants to produce therapeutic proteins is changing the shape of biotech. Plant-made biopharmaceuticals are cheaper to develop and cheaper to produce. Will they take over the field?	

Plant-made biopharmaceuticals (PMBs), however, break the traditional mold of plants and medicines in two ways: plants are bioengineered, and the resulting protein drugs are foreign to it - just as they are to recombinant yeast or bacteria or to transfected mammalian cells. 	

PMBs are preparing the way for the "next wave" in biotech which will parallel the evolution of biotechnology itself. According to a University of Florida study, more than 300 PMBs are under investigation worldwide.

 Most of this work goes on at academic or government institutions. Eventually, this undercurrent of technology will provide the foundation for PMB companies, just as it did for biotech 20 years ago.	

PMBs could have a big impact on markets for therapeutic recombinant proteins. The argument for PMBs is compelling. Datamonitor, a business information company, estimates the world market for therapeutic proteins at about $30 billion, and predicts demand will rise to $59 billion by 2010.

 Leading the way are monoclonal antibodies, with 2003 sales exceeding $3 billion and growth approaching 60% yearly. The market for therapeutic vaccines, although of much smaller volume, is also growing quickly, with 61% growth on 2002 sales of $31 million. Table 1, from Datamonitor's report, lists leading marketed therapeutic proteins.	

Moreover, what PMB companies lack in capital funding they more than make up for with innovation and vision. Larry Grill, Ph.D., chief scientific officer at Vacaville, CA-based Large Scale Biology Corp. (LSBC), predicts "at least a couple of dozen" approved plant-based biotech products by 2010. Most managers at PMB firms share his optimism.	

Optimistically, PMBs' lower capital costs and greater production flexibility could simply steal the show. Realistically, PMBs eventually may dominate specific therapeutic protein markets or monopolize biogenerics. Or they could fail.	

Figure 1. These rice plants, developed by Ventria Bioscience, will grow into mature plants and express therapeutic proteins.	

On paper at least, PMBs offer numerous advantages over traditional fermentation and cell culture. Among these are				

ability to express several genes simultaneously

inherent safety, especially of food crops

low proteolytic activity in crude product

Compared to traditional biomanufacturing, PMBs offer lower capital investment and lower ongoing manufacturing costs. Brick-and-mortar plants take from two to seven years to build and cost anywhere from $100 million to $500 million, and this only includes the cost of the plant, not ongoing manufacturing and other functions. The Dow Chemical Co. and Sigma Aldrich estimate capital reduction at 75% to 80%, with manufacturing costs slashed 50% to 60%. Neil Cowen, Ph.D., vice president for product development at Epicyte Pharmaceutical in San Diego believes ongoing cost savings for manufacturing might even reach 75%.

Figure 2. Ventria's rice-based PMBs illustrate the basic method of protein expression in plants.	

Cost advantages for downstream PMB operations depend on the final dosage form. As with all therapies, injectibles tend to be high-cost products, while edible or topical PMBs will be cheaper to develop. 	

Still, potentially significant cost savings are possible downstream. Because of the large biomass expected from PMB processes, batches will be large enough for developers to consider continuous or semi-continuous processing, which biotech companies traditionally avoid. A side benefit of large batches is lower quality assurance and quality control requirements since these activities are performed per batch, not per volume or weight of product.	

PMB manufacturing resembles food processing more than pharmaceutical production. So where do good agricultural practices (GAP) end and good manufacturing practices (GMP) begin? According to Andrew Baum, chief executive officer of SemBioSys Genetics and head of the Biotechnology Industry Organization's working group on PMBs, GAPs apply while the crop is in the field. GMPs, however, extend across the entire enterprise, overlapping with and supplementing GAPs. For example, the United States Department of Agriculture (USDA) governs isolation of engineered crops, whereas FDA regulates materials and purity, equipment, and manufacturing - encompassing everything from seed stock to packaging.

Figure 3. SemBioSys fuses proteins with safflower seed oil bodies.				

USDA and FDA, which jointly regulate PMB projects, state that the field is part of the manufacturing "facility" and require that PMB operations be treated like any other pharmaceutical process. Therefore, the appropriate safety and quality controls, dedicated equipment if necessary, contamination safeguards, and trained staff to assure safe, consistent product must be in place. "Companies involved in this work must apply the same types of regulatory and quality standards, from planting to harvesting, as they would for a fermentation," says Epicyte's Cowen. 	

LSBC claims to be the only "plant" company with large-scale GMP extraction capability. As a result, LSBC extracts proteins for other PMB firms on a contract basis. Says LSBC's Grill, "We can extract 6,000 pounds per hour of plant tissue, for 24 hours per day, under GMP."	

Because they come from plants, PMBs are believed to be inherently safer than recombinant proteins from micro-organisms or cells. All mammalian cells carry viral genomes that spawn potentially pathogenic viruses. By contrast, food plant viruses are not pathogenic to humans.	

Much has been written about post-translational modifications (PTMs) - glycosylations, phosphorylations, and others - the finishing steps in eukaryotic protein-making. No two organisms glycosylate in quite the same way, and some, like bacteria, don't glycosylate at all. PTM differences have not proven to be a source of undesirable immunogenecity in PMBs. Epicyte, for example, has failed to condition an immune response in mice with plant-derived mouse antibodies. Similarly, circulating half-life, clearance rate, and other pharmacokinetic properties do not differ between native and plant-derived proteins. "Worries over glycosylation differences were overrated," says Grill.	

Since plants cannot transmit prions, the current scourge of the beef industry, replacing animal-derived proteins with plant-derived copies seems promising. For example, LSBC makes the protease inhibitor aprotinin in a tobacco-like plant instead of extracting it from cow lungs, the traditional source. 	

Environmental concerns with PMBs include altered plants contaminating wild strains and human exposure to plant material containing potent drugs. Government agencies are well aware of these issues and appear to have them under control. In August, USDA's Animal and Plant Health Inspection Service (APHIS) amended its regulations for genetically engineered plants that make drugs, requiring a standard permit for field testing. Previously, APHIS allowed transgenic plant work under an expedited permit.	

Figure 4. Minichromosome technology from Chomatin allows multiple gene insertion via a single operation.	

PMB companies generally operate in a manner that protects both workers and the environment. For example, Ventria Bioscience exclusively uses self-pollinating crops because their reproductive mechanism keeps genes within the plant, a plus for what Chief Executive Officer Scott Deeter calls "environmental stewardship." The USDA-mandated setback distances for barley and rice, which self-pollinate, are 50 and 100 feet, respectively, compared to one mile for wind-pollinated maize.	

PMBs are generally easier to express upstream - and about as difficult to purify downstream - as conventional biopharmaceuticals. For some dosage forms, such as oral and topical, PMB purity is primarily driven not by safety concerns, since food crops are already recognized as safe, but for purposes of standardizing formulations. Lower sterility requirements and the generally large quantities of PMBs available from food crops could improve downstream economics, allowing development of high-dose, non-sterile formulations that would be too expensive for proteins manufactured from cell culture or fermentation.				

Downstream innovations will almost certainly arise from the huge biomass volumes expected from manufacturing-scale PMB operations and the potential for continuous processing. Another source will be expression methods that engineer ease of purification into raw products. A third avenue, process naivete, stems from the fact that PMB developers must experiment with purification techniques in ways that traditional bioprocessors do not.	

As Chenming "Mike" Zhang, Ph.D., assistant professor of biological systems engineering at Virginia Tech, points out, "[The PMB] industry needs to develop unique purification processes for each product. Generic processes are not realistic for plant-derived proteins."	

Zhang's specialty, aqueous two-phase extraction (ATPE), is an example of a separation technique considered feasible for PMBs but which is generally untouchable for conventional biopharmaceuticals. A single ATPE operation produces 87% pure egg white lysozyme from ground tobacco leaves using two aqueous phases - one pure water and the other spiked with additives like poly(ethylene glycol), dextrin, and salts.

 The only drawback is that Zhang never knows which aqueous layer will dissolve the product (and therefore, where the protein will end up), a rather small disadvantage considering the complexity of typical downstream protein purifications and ATPE's ease of scale-up.		

Table 1. Classes and values of marketed therapeutic proteins in 2000 and 2001.	

SemBioSys' expression of proteins attached to oil bodies of safflower seeds is a good example of expression-level purification. After harvesting and grinding, company engineers use continuous centrifuges to isolate proteins attached to oil bodies. "By addressing the entire manufacturing issue, we offer the same expression benefits of other systems but change the economics of purification," says Chief Executive Officer Andrew Baum. SemBioSys calls its process the StratoSome Biologics Production System.	

Since StratoSome proteins are inexpensive, they could potentially be used orally or topically with minimal processing and without regard to unfavorable pharmacokinetics. "In cases where the oil-body assembly is the product," says Baum, "you're done." For injectibles, proteins are cleaved from the oil bodies and subjected to another centrifugation, followed by column chromatography.	

A related oil-body technology, StrataCapture Purification, uses affinity-modified oil bodies to purify proteins generated through more conventional expression systems.	

Articles

Product Development

After many starts and stops, hype and disappointment, foreign protein expression in plants is now routine and biopharmaceuticals produced in green plants will soon be with us.

Is a Green Plant in Your Manufacturing Future?

Chitosan and alginate hydrogels are high-molecular-weight carbohydrate polymers used in an extensive array of pharmaceutical applications. While many applications depend upon the ionized state of the polymer, other factors play an important role in the pharmacological activity of complexes that include these agents. This review focuses upon ionic versus nonionic interactions, and illustrates where modifications of these ionic sites dramatically alter their pharmacological activity and pharmaceutical applications.	

Both of these hydrogels are natural polysaccharides, and when hydrated, they dramatically increase the viscosity of aqueous solutions. (A hydrogel is a colloid in which the dispersion medium is water.) In addition to their swelling and dispersal properties, the ionic characteristics of these polymers are important. Additionally, the sequence of the monosaccharide units in these polymers affects their physical and pharmacological properties. Applications include encapsulating enzymes, formulating macromolecular complexes, and gene transfection.

Figure 1. A partial structure of chitosan.	

Chitosan is a polysaccharide hydrogel derived from acid extraction of chitin. The most common source of chitin is the shells of crabs and other crustaceans.

 Chitosan can be isolated in complexes with a molecular weight exceeding 10

 Upon acid extraction of chitin, the N-acetylglucosamine subunits undergo partial deacetylation to yield a chitosan polymer composed of poly D-glucosamine. A partial structure of chitosan is shown in Figure 1. The presence of aminosugars makes chitosan soluble in dilute solutions of organic acids. Chitosan has many applications based upon its ability to form complexes with anions and composite films with other polysaccharides.

 Hydration and dissolution readily occur at a pH below 5.5.

Alginate or alginic acid is a polysaccharide hydrogel derived from brown seaweed.

 Alginate polymers can be isolated with a molecular weight exceeding 100,000 daltons.

 The repeating units are composed of mannuronic and guluronic acid monosaccharide subunits as shown in Figure 2. These monosaccharides are arranged to facilitate the complexation of divalent cations between the carboxyl groups on adjacent alginate chains.

 A rigid chelate structure forms when an alginate mix is added to a divalent cation solution. This property is used to encapsulate enzymes, macromolecular complexes, and cells.

 Alginate also forms films that can be readily hydrated.	

In their ionized forms, chitosan is positively charged and alginate is negatively charged. Drug release and gene transfection appear to depend primarily upon the charge of the polymer.

Furthermore, the carbohydrate backbone confers some selective characteristics to the polymers. For instance, the degree of deacetylation affects chitosan's binding characteristics.

 The sequence of mannuronic and guluronic acid residues play an important role in the application and activity of alginate polymers.

 These residues determine the orientation of the carboxyl group for complexation with cations.	

Figure 2. Monosaccharide subunits of alginate.	

While studies have highlighted the importance of charge density and orientation, the presence of a polysaccharide chain is also very important. Chitosan has been reported to be an effective gene transfection agent,

  and has been reported to be selective for HeLa cells and related cell lines.

 Binding of chitosan to DNA results in condensation and interaction with sulfate groups in the extracellular matrix, internalization of the gene, and transcription.

 This mechanism does not account for the selectivity of chitosan-mediated transfection in certain cell types, since sulfate groups appear to be ubiquitous in the extracellular matrix of most cell types. The ionic binding of sulfate with the amino group of chitosan is the basis of this interaction.	

Studies at other labs indicate that cellular binding to chitosan could be disrupted with methyl α-D-mannopyranoside, a non-ionized, non-reducing monosaccharide.

 These findings suggest that cell surface interactions with chitosan are due in part to the interaction of the carbohydrate chains of chitosan (the backbone) and the extracellular matrix. This may explain, in part, the selectivity of transfection in certain cell types independent of the cell's ability to transcribe the DNA introduced in the transfer process. 	

Studies of alginate-mediated transfection reveal the importance of the carbohydrate backbone. Unmodified alginate is a poor transfection agent in most systems, presumably due to the lack of positively charged groups necessary to complex with DNA. Modification of the carboxyl groups of alginate with aromatic and heterocyclic amines containing two or more amino groups did not consistently yield an effective transfection agent.

 Among these derivatives, the alginate and basic fuchsin conjugate was the only effective transfection agent. Modification of alginate with basic fuchsin results in the incorporation of two widely-separated amino groups into the carbohydrate backbone, facilitating transfection. The results from these studies strongly suggest that the carbohydrate backbone of these hydrogels must affect the orientation of the amino groups and their interaction with certain cell types.	

Chitosan and alginate are both excellent film-formers with opposite ionic charges. These characteristics provide applications for composites of these polymers in the same system.

 Their excellent film forming abilities are currently under investigation in the design of a bilayer complex for selective blotting and removal of nucleic acids and other macromolecules. In this bilayer system, an external chitosan layer can be introduced onto cellulose fibers or other substrate. DNA will readily bind to the chitosan film. A calcium alginate layer can be formed over the chitosan, resulting in a bilayer film (Figure 3). The calcium alginate layer - as well as any inclusion complexes in the alginate - can be readily removed with any dilute citrate without significantly affecting the position of the DNA-chitosan blot. Potential applications include molecular watermarking in forensics as well as forming a substrate and a matrix for gene transfection. Other derivatives of these hydrogels are currently under development. These natural polymers and their broad applications will be a fertile area for innovation.		

Figure 3. Alginate and chitosan bilayer film.				

(1)		Synowiecki J, Al-Khateeb NA. Production, properties, and some new applications of chitin and its derivatives. 

Critical Reviews in Food Science and Nutrition 2003

(2)		Wu AC, Bough WA, Conrad EC, Alden KE. Determination of molecular-weight distribution of chitosan by high-performance liquid chromatography. 

(3)		Borchard G, Junginger HE. Modern drug delivery applications of chitosan. 

(4)		Tapia C, et al. Study of the influence of the pH media dissolution, degree of polymerization, and degree of swelling of the polymers on the mechanism of release of diltiazem from matrices based on mixtures of chitosan/alginate. 

(5)		Boylan HJ. The commercial exploitation of seaweeds, with special reference to alginate manufacture. 

(6)		Horton JC, Harding SE, Mitchell JR. Gel permeation chromatography - multi-angle laser light scattering characterization of the molecular mass distribution of 'pronova' sodium alginate. 

(7)		Seely GR, Hart RL. The binding of alkaline earth metal ions to alginate. 

(8)		Tonnesen HH, Karlsen J. Alginate in drug delivery systems. 

(9)		Gotoh T, et al. A new type porous carrier and its application to culture of suspension cells. 

(10)		Shu XZ, Zhu KJ, Song W. Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release. 

(11)		Venkatesh S, Smith TJ. Chitosan-mediated transfection of HeLa cells. 

(12)		Borchard G. Chitosans for gene delivery. 

(13)		Singla AK, Chawla M. Chitosan: some pharmaceutical and biological aspects - an update. 

(14)		Stabler C, et al. The effects of alginate composition on encapsulated betaTC3 cells. 

(15)		Erbacher P, et al. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. 

(16)		Venkatesh S, Smith TJ. Chitosan-membrane interactions and their probable role in chitosan-mediated transfection. 

(17)		Padmanabhan K, Smith TJ. A preliminary investigation of modified alginates as a matrix for gene transfection in a HeLa cell model. 

(18)		Wang L, Khor E, Lim LY. Chitosan-alginate-CaCl(2) system for membrane coat application. 

(19)		Coppi G, et al. Chitosan-alginate microparticles as a protein carrier. 

(20)		Serp D, et al. Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization. 

Chitosan and alginate hydrogels are high-molecular-weight carbohydrate polymers used in an extensive array of pharmaceutical applications. While many applications depend upon the ionized state of the polymer, other factors play an important role in the pharmacological activity of complexes that include these agents.

An Overview of the Biopharmaceutical Applications of Chitosan and Alginate

One goal of FDA's initiative to overhaul how it ensures high-quality pharmaceutical production is to apply more oversight and stricter rules to higher-risk products and processes (Inside Washington, October 2003). This approach involves revising field inspection programs and policies to use the agency's limited resources more effectively, particularly as FDA increasingly deals with more complex and diverse manufacturing operations. FDA officials are looking to link inspection priorities to risk factors: new proposals may omit preapproval inspections (PAIs) for low-risk products and select inspection sites based on manufacturing complexity and company compliance history. 	

These policies are being applied to traditional drugs now, with a plan to phase in changes for biotech therapeutics transferred from the Center for Biologics Evaluation and Research (CBER) to the Center for Drug Evaluation and Research (CDER). FDA is continuing current oversight programs for biologics, with CDER staff now conducting PAIs and Team Biologics continuing to handle GMP audits (see "Assessing Team Biologics" sidebar). Meanwhile, certain CBER compliance practices are influencing new proposals for overseeing traditional drugs.	

One innovation announced in September is to create a pharmaceutical inspectorate (PI). Similar to Team Biologics, PI will raise the expertise of field officers who inspect drug production facilities (GMP implementation documents available at 

). FDA's Office of Regional Affairs (ORA), which oversees the agency's field force, plans to train some 50 investigators to form the PI over the next four years. A memorandum of understanding between ORA and CDER describes how PI members will be selected, trained, and assigned, noting that PI members will focus on facilities that use more complex production technologies. Unlike Team Biologics, these individuals will still be based in FDA district offices and will not travel the country. 	

FDA also plans to include more review specialists in plant inspections to better coordinate application reviews with GMP inspections. CBER scientists and reviewers have regularly participated in preapproval and GMP inspections for biotech products. Now this approach is being extended to drugs. Specialists - from FDA centers or its other offices - will likely play a larger role in conducting high-risk inspections, resolving technical issues, reviewing draft warning letters, and developing approaches for regulating and encouraging new manufacturing technologies. 	

In adopting a risk-based approach to compliance, FDA announced in September that it no longer will mandate PAIs for certain low-risk drugs, including some narrow therapeutic index drugs on FDA's list of the top 200 prescribed drugs. The agency still can conduct PAIs for these products, but district field office managers will gain more flexibility in deciding whether inspections are needed. 		

For biologics, CDER will continue the CBER practice of assigning staff to conduct PAIs, but compliance officials believe that current policy provides flexibility to omit a PAI for a less complex biotech product when warranted. 	

The new policy is expected to reduce the number of PAIs overall (particularly for generic drugs and postapproval changes) and to allow inspectors to focus on more high-risk products and processes. PAIs still will be conducted regularly for new molecular entities, priority applications, treatment INDs, manufacturers' first applications, companies experiencing GMP problems, and various problem situations. 	

Because scientific and technical manufacturing disagreements arise during GMP inspections, FDA launched a program for drugs and biotech products to resolve these issues efficiently, hoping to remove roadblocks to manufacturers adopting new technologies. A draft guidance issued in September describes a formal dispute resolution program. As a first step, FDA urges manufacturers to clarify disputes with the plant inspection team during the inspection process. Such discussions may occur as issues emerge or after the inspection when investigators usually discuss their observations with company management. 		

If the disagreement is not resolved and FDA issues a form 483 citation, a manufacturer can request the new, two-tiered dispute resolution process. The first tier involves referring the scientific or technical issue to the appropriate ORA district office within 10 days of the inspection's completion. If ORA disagrees with the manufacturer, it will write up a review of the issue, usually consulting with the center staffs involved. 	

If a manufacturer disputes ORA's position, it can move to tier two and appeal the issue to an FDA dispute resolution panel. The panel of FDA officials will review the case and issue a report. 	

FDA anticipates the program will address broad scientific issues, such as the adequacy of a manufacturer's process control design or the appropriateness of a particular lot testing or sampling scheme. FDA will launch a 12-month pilot in January and aims to refine the proposal and issue final guidance by January 2005. 	

With the oversight of biologics moved to CDER, the center's Office of Compliance (OC) is monitoring the manufacturing quality of biotech therapeutics. The 208 staffers transferred from CBER to CDER include several compliance officers who joined the new Therapeutic Facilities Review Branch in OC's Division of Manufacturing and Product Quality (see "Consolidation Update" sidebar). This division, headed by Joe Famulare, is responsible for overseeing foreign and domestic drug inspections, drug recalls, and GMP policy development, particularly as it relates to electronic records and electronic signatures. OC's Division of New Drugs and Labeling Compliance monitors labeling requirements, including oversight of Internet claims and health fraud.				

As part of a reorganization last January, the OC formed a Division of Compliance Risk Management and Surveillance to help implement new risk management initiatives. Among other responsibilities, this division will monitor reports of drug quality problems and other data to focus GMP inspections on facilities that make higher risk products. OC director David Horowitz plans to target more inspections this year at new establishments and plants that make prescription drugs and sterile products. For the future, FDA is developing more advanced risk-based models to select sites for inspection based on the complexity of manufacturing process, product type, and compliance history. The aim is to focus additional surveillance where quality problems could have a serious impact on patients. Horowitz says that over time, the model for prioritizing sites for inspection will also include the biotech products for which CDER has recently been assigned responsibility.  

Columns and Departments

One goal of FDA's initiative to overhaul how it ensures high-quality pharmaceutical production is to apply more oversight and stricter rules to higher-risk products and processes (Inside Washington, October 2003).

Inside Washington: FDA Revises Inspections and Compliance Policies for Drugs and Biologics

UK science expertise, backed by increased government funding, has led to a thriving bioscience industry, distinguished by the energy and creativity of its entrepreneurs." These words are not mine but those of Lord David Sainsbury, the British government's science and innovation minister. The facts more than confirm the accuracy of Lord Sainsbury's assertion. 	

The United Kingdom is home to the largest biotechnology industry in the world outside of the United States. The biotechnology market and related industries are currently estimated at $13 billion and include almost 300 dedicated biotechnology companies and more than 450 companies involved in bio-related activities. The British government spends nearly $1 billion annually on bioscience research, and charities such as the Wellcome Trust also provide significant funding. The pharmaceutical sector spends more than $2.7 billion per year on research and development (R&D), making it the largest user of modern biotechnology.	

Furthermore, the UK is characterized by an abundance of technically qualified and highly skilled personnel in its academic institutions, industries, and hospitals, as well as excellent education and training structures to ensure that this supply of talent is ongoing. In the past 50 years, British scientists have earned more than 46 Nobel prizes, two-thirds of which were for advances in medicine, chemistry, and physiology. The UK has discovered 15 of the world's current 75 best-selling drugs, and British companies account for 62% of the products in late-stage clinical trials in Europe. The UK is home to a significant number of world-leading research institutes including the Sanger Center and the Roslin Institute; international research establishments such as the European Bioinformatics Institute; and regulatory bodies such as the European Agency for the Evaluation of Medicinal Products and the Medicines Control Agency.	

The UK presently funds approximately 4.5% of the world's science, produces 8% of all scientific papers, and receives 9% of the citations - all with only 1% of the world's population. In short, British academic institutions and UK-based companies remain firmly on the frontlines of biotechnology research. They do not simply compete on a global scale; they lead the way. 	

The UK's biotechnology industry is unrivalled in Europe and continues to spawn companies that rival those of the US. The biotechnology communities in the UK and US have been inextricably linked since the UK's Francis Crick and James Watson of the US demystified the double helix 50 years ago. 				

For example, Eli Lilly plans to spend more than $350 million over the next four years to expand its UK operations. The investment includes $112 million to improve manufacturing operations in Southeast England. Eighty million dollars will support overall UK operations, including the Erl Wood research site in southeast England, where a further $64 million is committed to a neuroscience research center. An additional $72 million is dedicated to adding capacity at its Merseyside manufacturing facility, and $24 million is set aside for UK business support.	

Another American biotechnology company investing in the UK is Genzyme, one of the top five biotechnology companies in the world. Genzyme has recently completed a plan that included $88 million to expand its facilities in East Anglia. Two large-scale, state-of-the-art plants were officially opened in August 2002, enabling a tenfold increase in production capacity of the active ingredients for the kidney disease drug Renagel and doubling staffing levels to more than 200.	

The idea of biotechnology clusters may be valid in a country as vast as the US, but the UK is so geographically compact that, especially from the perspective of US business, it can be considered one "cluster" - one that can act as a transatlantic stepping stone into continental Europe. Within the UK cluster, there are hubs of special expertise, often growing out of the particular strengths of local universities.				

Expert advice is abundantly available to help individual investors identify the right location for their business proposition. For many investors, the first port of call has been UK Trade & Investment, which provides free and confidential advice to both potential and current investors. UKT&I works closely with the government's science and technology team that has representatives based in consulates around the US. It works hand-in-glove with the 12 development agencies directly responsible for England, Northern Ireland, Scotland, and Wales.	

Companies established in the UK support all aspects of biopharmaceutical production, from development and manufacturing of active ingredients, to formulation and delivery. The UK biomanufacturing marketplace is diverse and deep and includes specialist third-party contract manufacturers such as Avecia (Billingham), CAMR (Salisbury), Cobra Therapeutics (Keele), Excell Biotech (Edinburgh), and Lonza Biologics (Slough); contract testing and research organizations such as Q-One (Glasgow); and manufacturing facilities (such as PowderJect, Eli Lilly, Avecia) for vaccines and biologics. Also present are the in-house process development organizations of "big pharma" and biotechnology firms such as AstraZeneca, Celltech, Cambridge Antibody Technology, Delta Biotechnology, GlaxoSmithKline, Merck, Pfizer, and Protherics. Leading academic institutions such as University College London's Advanced Center for Biochemical Engineering, Aston Academy of Life Sciences, and the University of Cambridge support the entire sector.				

An excellent example of the UK's commitment to biomanufacturing is the recent announcement of the creation of a $50 million National Biomanufacturing Center at Speke, Merseyside. The center is the first of its kind in the UK and is expected to open in 2005. It will focus on biotechnology business development and the manufacture of innovative biological medicines, including vaccines. Also, the center will provide advice, services, and trained personnel to biotechnology businesses throughout the region. Its program is designed to support start-up companies spinning out of area universities and should provide a crucial bridge between academic innovation and commercial biomanufacturing development.	

In addition to the Eli Lilly and Genzyme expansions, other recent developments on the biomanufacturing front include the following:	

Abbott Laboratories is investing approximately $50 million to create 125 new, highly skilled positions in biomanufacturing, taking the total employed at its Dartford facility to more than 400. The investment will be for the production of advanced biological reagents for use in diagnostic testing equipment.

PowderJect, which was recently acquired by Chiron, is investing a further $140 million at its Merseyside site. 

A $166 million biomanufacturing center, BioCampus, is to be created in Edinburgh. This fully serviced 25-acre site will be home to specialist biotechnology companies engaged in the manufacture of their own products.

The dynamism of the UK biotechnology industry is founded on Britain's outstanding universities and research institutions, which remain at the forefront of global research. "The past decade has seen a dramatic change in UK universities with the recognition that our research base is developing knowledge that has a value beyond its scholarship and additional to its traditional output in publication," says Sir Richard Sykes, Rector of Imperial College London. "Today, more and more universities are looking to exploit their intellectual assets in ways that more directly bring income and rewards to fund their work. Investors have faith in technologies based on high quality research and UK universities have a strong and credible track record in supplying it. Markets may swing up and down but there will always be a place for inventions that address healthcare, quality of life, or quality of environment needs and concerns."				

In the UK, as in the US, technology transfer is often overseen by university-owned companies such as ISIS Innovation, which is wholly owned by the University of Oxford and provides the university's researchers with commercial advice; funds patent applications and legal costs; negotiates exploitation and spinout company agreements; and identifies and manages consultancy opportunities for university researchers. ISIS is just one of many similar UK organizations spearheading technology transfer.	

According to Maire Smith, chief executive officer of the technology transfer group Manchester Innovation, "The key to technology transfer is having a body that understands the bridge between the university world and the commercial world. It is essential to have staff with commercial management experience who can visualize a market requirement for a technology or area of research." Another primary focus is nurturing entrepreneurialism. "The incubator helps, as it shifts a researcher's mentality from academia to business," Smith says.	

Part of this shift comes through introducing the entrepreneurial researcher to experienced management, venture capitalists, and business angels. These groups frequently take on both a financial interest and a business mentoring role in any new company. UKT&I recently organized its second annual Biotech VC mission in which nearly 30 US venture capitalists met with British biotechnology companies that were looking to raise R&D funding.	

One established venture capital group is Global Life Science Ventures (GLSV). It closed its first $110 million biotech fund in 1996. Although GLSV had a global investment focus - the bulk covered Germany and Switzerland, with a third going to the US - it has expanded its focus to include the UK. From new offices in London, the VC group closed a second fund of $237 million in autumn 2002. Although the group will consider any European or US investment, it will only take a lead investor role where it has a base. "In the rest of Europe there are good deals, but we need direct dialogue and frequent interaction to mentor a company properly," explains Philip Morgan, managing director of GLSV. "It made sense for us to come to the UK. It's significant in terms of the number of life science companies and there are managers who have been through the biotech process before," he says. "The UK has a substantial interest in biotech and a large unquoted sector. There is also a good group of VCs in the UK that we are interested to syndicate with."	

Even while the global economy remains soft and difficulties abound for companies seeking funding, the British biotechnology sector is growing and managing to attract significant investment. Within Europe, the UK is still attracting the lion's share with around $528 million in public and private investments in 2002, approximately half of the European total for the year.				

More than half of the investment funds raised came from private venture sources, making 2002 the second best year ever for private equity infusions into British biotechnology. According to Keith Binding, director of Critical 1, a life sciences intelligence group, "This demonstrates that VCs are prepared to back UK biotech with large sums of money."	

Why do VCs find these companies so attractive? "The UK has sailed a fairly steady course, as its companies are more mature and product focused. UK companies are therefore more attractive to venture capital than other European companies as venture capitalists are looking for products," explains Binding.	

The UK's public markets are also offering biotech companies increasing opportunities. "Last year there were only nine biotech IPOs in the world. Three of those were UK companies, and one was Canadian but listed on the London Stock Exchange. In the public markets, it's clear that London has re-emerged as the European market best able to support bioscience companies," Binding says. Today, some 56 British technology companies are publicly traded, the largest number of any European nation.	

In addition, the British government serves as a significant source of funding for the biotechnology industry. In partnership with the Wellcome Trust, the government has committed $1.5 billion for the renewal and modernization of the science base infrastructure in the UK. Furthermore, the UK National Health Service (NHS) spends in excess of $17 billion per year on goods and services and is one of the few healthcare systems in Europe currently increasing its expenditure. The government estimates health-related expenditure growth of 7.5% annually for the next five years.	

The UK has created a climate for biotechnology in which regulatory requirements aim to be				

flexible and responsive to advances in scientific knowledge

sympathetic to innovation while maintaining appropriate safeguards.

The UK has practical and effective arrangements in place for the clearance of products, such as novel foods, through the Advisory Committee on Novel Foods and Processes (ACNFP), and diagnostics, through the Medical Devices Agency (MDA). The Gene Therapy Advisory Committee (GTAC) has taken a pragmatic and sensible approach to controls on gene therapy. The Advisory Committee on Releases to the Environment (ACRE) provides advice on genetically modified organism (GMO) consents.	

The UK established the Agriculture and Environment Biotechnology Commission (AEBC) to consult widely on biotechnology issues and the environment. The Human Genetics Commission (HGC) has been set up to look at genetic technologies and their impact on humans, providing strategic advice to the government. The Food Standards Agency (FSA) has taken responsibility for the assessment of genetically modified (GM) foods and the development of new policies.	

The European Medicines Evaluation Agency (EMEA) is responsible for fast-track approval of biotechnology-derived pharmaceutical and veterinary products throughout Europe. It provides a single EU-wide license valid in all member states. The UK Medicines Control Agency (MCA) provides approval of other medicinal products and has a strong international reputation.	

In 1999, the National Institute for Clinical Excellence (NICE) was established as a Special Health Authority for England and Wales. Part of the National Health Service, NICE provides health professionals and the general public with authoritative, robust, and reliable guidance on current best practices. NICE produces specific guidance on the use of new and existing medicines, clinical guidelines for specific disease treatment, and the safety of interventional procedures. It also funds studies on improving the quality of patient care.	

One particular area in which the UK has been successful in adapting legislation and regulations to changing circumstances is in the use of embryonic stem cells, where the UK is now in a leading position internationally and attracting top scientists from around the world.	

Against this backdrop it is hardly surprising that more and more leading biotechnology companies are committing to the UK. Researchers and businesses alike are assured not only of a world-class environment today but also into the future. To close - as I opened - with words from David Sainsbury: "The UK government sees bioscience as a key industry for the future and is determined to create the best possible environment for the industry to grow and flourish." 		

For UK Trade and Inward Investment Opportunities, contact:

Hannah Morris, Life Sciences and Healthcare Analyst

International BioMarket

UK science expertise, backed by increased government funding, has led to a thriving bioscience industry, distinguished by the energy and creativity of its entrepreneurs." These words are not mine but those of Lord David Sainsbury, the British government's science and innovation minister.

The United Kingdom: Europe's Biotechnology Hub

Biopharmaceutical products in storage change as they age, but they are considered to be stable as long as their characteristics remain within the manufacturer's specifications. The number of days that the product remains stable at the recommended storage conditions is referred to as the shelf life. The experimental protocols commonly used for data collection that serve as the basis for estimation of shelf life are called stability tests. 	

Shelf life is commonly estimated using two types of stability testing: real-time stability tests and accelerated stability tests. In real-time stability testing, a product is stored at recommended storage conditions and monitored until it fails the specification. In accelerated stability tests, a product is stored at elevated stress conditions (such as temperature, humidity, and pH). Degradation at the recommended storage conditions can be predicted using known relationships between the acceleration factor and the degradation rate.	

Temperature is the most common acceleration factor used for chemicals, pharmaceuticals, and biological products because its relationship with the degradation rate is characterized by the Arrhenius equation. Several methods of predicting shelf life based on accelerated stability testing are described in the article. Humidity and pH also have acceleration effects but, because they are complex, they will not be discussed in detail here. Also, details on statistical modeling and estimation are outside the scope of the article, but we provide references to computer routines. 	

Figure 1. A simulated set of stability results also showing the estimated degradation and 95% confidence limits.	

The assessment of shelf life has evolved from examining the data and making an educated guess, through plotting, to the application of rigorous physical-chemical laws and statistical techniques. Regulators now insist that adequate stability testing be conducted to provide evidence of the performance of a drug or a biopharmaceutical product at different environmental conditions and to establish the recommended storage conditions and shelf life.

 Recently, Tsong reviewed the latest approaches to statistical modeling of stability tests,

 and ICH has published some guidelines for advanced testing design and data analysis.

Modeling has become easier due to availability of standard statistical software that can perform the calculations. However, an understanding of the general principles of stability testing is necessary to apply these programs correctly and obtain appropriate results. Thus, the purpose of this paper is to provide an outline of the basic approaches to stability testing, as well as to create a foundation for advanced statistical modeling and shelf life prediction. 		

Table 1. Estimates of the degradation model and Table 2. Estimates of degradation rates, days of stability and 95% confidence limits.	

Since degradation is usually defined in terms of loss of activity or performance, a product is considered to be degrading when any characteristic of interest (for example potency or performance) decreases. Degradation usually follows a specific pattern depending on the kinetics of the chemical reaction. The degradation pattern can follow zero-, first-, and second-order reaction mechanisms.

 In zero-order reactions, degradation is independent of the concentration of remaining intact molecules; in first-order reactions, degradation is proportional to that concentration.

 Zero- and first-order reactions involve only one kind of molecule, and can be described with linear or exponential relationships. Second- and higher-order reactions involve multiple interactions of two or more kinds of molecules and are characteristic of most biological materials that consist of large and complex molecular structures. Although it is common to approximate these reactions with an exponential relationship, sometimes their degradation pattern needs to be modeled more precisely, and no shortcuts will suffice.		

The degradation rate depends on the activation energy for the chemical reaction and is product specific. We don't always have to deal with higher-order equations; in many cases, the observed responses of different orders of reactions are indistinguishable for products that degrade slowly. 	

The degradation rate depends on the conditions where the chemical reaction takes place. Products degrade faster when subjected to acceleration factors such as temperature, humidity, pH, and radiation. Modeling of the degradation pattern and estimation of the degradation rate are important for assessing shelf life. Experimental protocols used for data collection are called stability tests. In practice, evaluators use both real-time stability tests and accelerated stability tests. The real-time stability test is preferable to regulators. However, since it can take up to two years to complete, the accelerated tests are often used as temporary measures to expedite drug introduction.	

In real-time stability tests, a product is stored at recommended storage conditions and monitored for a period of time (t

). Product will degrade below its specification, at some time, denoted t

 can be obtained by modeling the degradation pattern. Good experimental design and practices are needed to minimize the risk of biases and reduce the amount of random error during data collection. Testing should be performed at time intervals that encompass the target shelf life and must be continued for a period after the product degrades below specification. It is also required that at least three lots of material be used in stability testing to capture lot-to-lot variation, an important source of product variability.

The true degradation pattern of a certain product, assuming that it degrades via a first-order reaction, can be described as follows:	

The observed result (Y) of each lot has a random component φ associated with that lot, as well as a random experimental error, ε.	

Both α and δ represent the fixed parameters of the model that need to be estimated from the data, while φ and ε are assumed to be normally distributed with mean = 0, and standard deviations of σ

 respectively. Equation 2 is a nonlinear mixed model. Details on the estimation process are outside the scope of this paper.

Let C represent a critical level where the essential performance characteristics of the product are within the specification. A product is considered to be stable when Y ≥  C. Product is not stable when Y < C, while Y < C occurs at t

. The manufacturer determines the value of C. The estimated time that the product is stable is calculated as	

Here, a and d are the estimated values of the intercept and the degredation rate. The standard error of the estimated time can be obtained from the Taylor series approximation method and is used to calculate confidence limits. The labeled shelf life of the product is the lower confidence limit of the estimated time.

 Public safety is paramount, that is why we use the lower confidence limit. Lots should be modeled separately when lot-to-lot variability is large. More details on this issue are found in references 9 and 10.	

We simulated data for three lots tested for a total period of 600 days (Table 1 and Figure 1). The product loses its activity as it ages, but it is considered to be performing within the specification until it reaches 80% of its activity (C = 0.8). The estimated lot-to-lot standard deviation is 0.000104, and the estimate of experimental error is 0.000262. Therefore, the shelf life of the product was determined to be 498 days. This represents the lower 95% confidence limit corresponding to the estimated time of 541 days.

Figure 2. A set of simulated data showing degradation of product at four different temperatures. 	

In accelerated stability testing, a product is stored at elevated stress conditions. Degradation at recommended storage conditions could be predicted based on the degradation at each stress condition and known relationships between the acceleration factor and the degradation rate. A product may be released based on accelerated stability data, but the real-time testing must be done in parallel to confirm the shelf-life prediction.

 Sometimes the amount of error of the predicted stability is so large that the prediction itself is not useful. Design your experiments carefully to reduce this error. It is recommended that several production lots should be stored at various acceleration levels to reduce prediction error. Increasing the number of levels is a good strategy for reducing error.

Table 3. Predictions of parameters at 25ÃÂ°C based on the Arrhenius equation. 		

Temperature is probably the most common acceleration factor used for chemicals, pharmaceuticals, and biological products since its relationship with the degradation rate is well characterized by the Arrhenius equation. This equation describes a relationship between temperature and the degradation rate as in Equation 4.	

This relationship can be used in accelerated stability studies when the following conditions are met:	

A zero- or first-order kinetics reaction takes place at each elevated temperature as well as at the recommended storage temperature.

The same model is used to fit the degradation patterns at each temperature.

These requirements do not fully guarantee that the Arrhenius equation can be used to predict the degradation rate at storage temperature, but they are a good start. Do not compromise the analytical accuracy during the course of the study to distinguish between the degradation rates at each temperature.	

Select temperature levels based on the nature of the product and the recommended storage temperature. The selected temperatures should stimulate relatively fast degradation and quick testing but not destroy the fundamental characteristics of the product. It is not reasonable to test at very high temperatures for a very short period of time, since the mechanisms of degradation at high temperatures may be very different than those at the recommended storage temperature. Choose the adjacent levels appropriately so that degradation trends are larger than experimental variability. Choosing levels depends on the nature of the product and analytical accuracy, but other practical implications may be considered. Testing should be performed at time intervals that encompass the target stability at each elevated temperature. Acquire some data below C so that the degradation trend can be determined. 	

Humidity and pH can be used along with temperature to accelerate degradation, but modeling of multi-factor degradation is very complex. A model for parameter estimation and prediction of shelf life when temperature and pH are used as acceleration factors is given by Some et al.

Quality Management

Biopharmaceutical products in storage change as they age, but they are considered to be stable as long as their characteristics remain within the manufacturer's specifications. The number of days that the product remains stable at the recommended storage conditions is referred to as the shelf life.

Assessing Shelf Life Using Real-Time and Accelerated Stability Tests

I'm a big basketball fan and, hailing from Boston, grew up watching Larry Bird roam the parquet floor of the old Boston Garden.

While I cheered Larry the basketball legend, my admiration of him went beyond mere hoops and hard courts. He had a folksy, common sense approach. When Bird would go three quarters without a basket, he was able to turn things around with the game on the line in the fourth quarter. He would invariably nail the game winner with seconds left.

Most players, having an off day, would have lost confidence and stopped shooting. Not Bird. As he so often said, "The only shots you know you'll miss are the ones you never take."

So it goes with the biopharmaceutical investment market, where an off year or two doesn’t break an industry that keeps firing away with new ideas, breakthroughs, patents, and life-saving drugs.

After losing roughly 60% of its value from January 2000 through the end of 2002, when terms like 

 fueled investor antipathy, the biopharmaceutical market, as measured by the Nasdaq Biotechnology Index was up 40% for the first half of 2003.That's manna from heaven for the biopharm industry, which has seen its coffers rise more than $2.6 billion (in equity financing) so far in 2003. That's a far cry from the $1.5 billion they raised for all of 2002.

The industry has benefited from a strengthening economy and a revitalized stock market which climbed 17% year-to-date through September 2003 and could post a return of 25% for the year, if current growth trends in the S&P 500 continue through the holidays. Those are numbers much higher than Wall Street forecasters predicted back in January. The sentiment then was that investors should take their 8% annual returns for 2003 and be thankful for that.

But the run-up in the stock market, just like the run-up in the Nasdaq's biotech index, grew legs faster and longer than most insiders had figured. Like freshmen at a college keg party, thirsty investors quickly quenched their thirst on double-digit market gains after three years of flat market brew.

Consequently, fear and timidity, it seems, are giving way to excitement and confidence on the part of investors who know that the last place to be during a market run-up is on the sideline. So, investors are stampeding the market, looking for the best places to park their money and make it work for them. For millions of investors, that means the biopharmaceutical market, where the numbers only begin to tell a rebirth story. Why biopharmaceuticals? Let's have a look:

Public and private enterprises in the United States poured nearly $70 billion into medical research last year; half will come from the biopharmaceutical sector. Investors, especially seasoned ones, like to follow the money because the money usually leads to significant returns on investment. But higher revenues aren't everything. Job creation in the industry, reduced health care costs due to revolutionary new drugs, and the number of lives saved by the research and products produced by the industry all count too. While you can't put a single price tag on all these elements, they certainly boost the image of an industry that only two years ago was hemorrhaging money on all fronts.

Investors also can relate to what biopharmaceutical companies are doing to cure their illnesses and ease their pain. Consider a cancer survivor who can point to a new biotech diagnostic that may have saved his life. Or the diabetic who benefits from a new drug that lengthens his. When I worked on Wall Street as a young bond trader I used to hear analysts, traders, and portfolio managers I worked with talk about "hands-on" investors — that is, investors who plowed money into Procter & Gamble because they swore by P&G's toothpastes and shampoos. Or those who loaded up on Gap shares because that's where they bought their blue jeans or sweaters. It's an investment mindset long championed by Wall Street legends like Peter Lynch, the first manager of Fidelity Investments' Magellan Fund. Lynch roamed the shopping malls of Boston and its suburbs examining which stores attracted the most shoppers regularly. Now, hands-on investing has come home to roost in the pharmaceutical sector, where millions of Americans have experienced first hand the lifestyle freedom provided by drugs like Prilosec or Viagra. Why, investors reason, shouldn't millions of other heartburn or erectile dysfunction sufferers enjoy their benefits too? Investors figure that millions benefit from such drugs and are quick to conclude that the companies that make these drugs are worth 100 shares or so in a financial portfolio.

That was the motto of fictional Faber College (from the movie classic 

) but it fits the biopharm market as well. Professional investors say that there is no substitute for knowledge when investing in a company and that hunches, bar charts, and timing mechanisms are akin to dialing 1-900-PSYCHIC when compared to knowing what a company does and why it does it well. That hurdle seems to have been cleared in the last year or so by a nation of health care consumers who happen to be investors (or, I'm tempted to say, a nation of investors who happen to be health care consumers). Investors have taken notice of the updraft in the economy, have decided to jump back into the market, and have picked out a few sectors — defense, consumer products, housing and construction, as well as biopharmaceuticals — where they feel their money has the best shot for growth.

In a tale of two markets, shares in biopharm companies bounce back.

BioPharm International-11-01-2003

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