BIOPHARMACEUTICALS

he term Biopharmaceuticals refers to drugs that have unique qualities in the way that they are derived and manufactured as opposed to traditional drug products. Biopharmaceuticals are protein-based and may either be derived from genetically altered bacteria or fungi (also called biotech drugs), or may come from blood and blood plasma products (usually referred to as biologics).

Because of their unique status, biopharmaceuticals have not generally been able to be produced generically even though a number of these products may have patents that have expired. The official name for generics of these products (per the FDA) is followon biologics; however, they have commonly come to be called biogenerics. Some common examples of biopharmaceuticals include Enbrel, Aranesp, Epogen, Neupogen, Gaminex, recombinant human insulin, and human growth hormone, to name a few.

Fig: How Biopharmaceuticals are produced?

 ProspectsofBiopharmaceuticals
Biopharmaceutical products represent a diverse group of products that include proteins, peptides, nucleic acids, whole cells, viral particles and vaccines. The conformation of the macromolecule or cell must be maintained to retain biological activity, and animal models for biological activity and characterization assays are often developed in tandem with initial formulation studies. This presents the formulation scientist with a unique set of challenges when compared to those for small molecules. In the last 10 years, there has been an increase in the number of new biopharmaceutical applications, with 15 new approvals in 2003. Nine of these are new biopharmaceutical products, and six new indications or formulations of biopharmaceutical products already on the market. The product approvals were supported in 2003 by a considerable pipeline of 270 new biopharmaceuticals under clinical evaluation in the major indications. With 101 clinical studies, most of the biopharmaceuticals are designed for indications in oncology. This area, one with high therapeutic needs, is also a field where new tumour receptors have been discovered for which monoclonal antibodies have been developed as murine, chimeric, humanized or fully human antibodies. The production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. First, plant systems are more economical than industrial facilities using fermentation or bioreactor systems. Second, the technology is already available for harvesting and processing plants and plant products on a large scale. Third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as a food (edible vaccines). Fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). Fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. Last, health risks arising from contamination with potential human pathogens or toxins are minimized.

What are Edible Vaccines?

Edible vaccines are antigenic proteins that are genetically engineered into a consumable crop. The idea is that the crop food product contains the protein which is derived from some disease causing pathogen. People eat the crop, the food is digested, and some of the protein makes its way into the blood stream. Get enough of this protein into the blood stream and it causes an immune response. This immune response would now nuetralize the pathogen should the person ever encounter it in the future. The nice thing about edible vaccines is that it's a very cheap way to produce vaccines, and it's also a very convenient way to store, ship and distribute vaccines.

Proteins of microbial and viral pathogens were some of the earliest examples chosen to show the feasibility of transgenic plant expression systems. The rationale was that key immunogenic proteins of major pathogens could be synthesized in plant tissues and then fed as edible subunit vaccines to humans or commercially important animals. The proof of this concept has since been shown using several bacterial and viral proteins. The practical aspects of choosing particular foodstuffs in which to deliver defined doses of a vaccine are being explored, and efforts are under way to establish clear regulatory paths for the development of edible vaccines Oral delivery of vaccines is an attractive alternative to injection, largely for reasons of low cost and easy administration. The chances of acquiring mucosal immunity against infectious agents that enter the body across a mucosal surface are also increased with oral vaccines. However, a major concern with oral vaccines is the degradation of protein components in the stomach and gut before they can elicit an immune response. To guard against degradation, several delivery vehicles have been developed to ferry intact proteins to the gut. These include recombinant strains of attenuated microorganisms, bioencapsulation vehicles such as liposomes and transgenic plant tissues.

Plantderivedbiopharmaceuticals andhumanproteins
Generally, levels of pharmaceutical proteins produced in transgenic plants have been less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified. Plant-derived recombinant hepatitis-B surface antigen induced only a low level serum antibody response in a small human study, probably reflecting the low level of expression (15 ng g1 fresh weight) in transgenic lettuce. In spite of recent improvements in expression levels in potato with a view to clinical trials, expression levels should be increased further for practical purposes. Also, even though Norwalk virus capsid protein expressed in potatoes caus ed oral immunization when consumed as food, expression levels are too low for largescale oral administration (0.37% of total soluble protein). Expression of genes encoding other human proteins in transgenic plants has been disappointingly low: human serum albumin, 0.020% total soluble protein; human protein C, 0.001% total soluble protein; erythropoietin, 0.003% total soluble protein; and human interferon-, <0.001% fresh weight. A synthetic gene coding for the human epidermal growth factor was expressed only up to 0.001% of total soluble protein in transgenic tobacco. In spite of several successful reports of high-level expression of non-human proteins (e.g. phytase, glucanase) via the nuclear genome, there is a great need to increase expression levels

of human blood proteins to enable the commercial production of pharmacologically important proteins in plants.

Plantderivedbiopharmaceuticals
Plant-derived biopharmaceuticals should meet the same standards of safety and performance as other production systems. However, many herbal medicines are now exempt from such close scrutiny and are not required to meet the same standards because of their classification as nutritional supplements. Because interest groups to confuse public perception have raised several environmental concerns, it is of paramount importance that regulating agencies distinguish between real and perceived public concerns (scientific versus non-scientific). If biopharmaceuticals that are potentially harmful are capable of persisting in the environment and might accumulate in non-target organisms, precautionary measures should be taken. Induction of biopharmaceutical production after harvesting (as was done in the case of glucocerebrosidase) might be one approach to minimize environmental exposure, provided that the use of viral vectors does not introduce additional environmental or regulatory concerns. Expression of potentially harmful proteins in a form that must be treated for activation might minimize the risk of exposure. For example, hirudin is produced as a fusion protein and is inactive in this form; it is activated only after it is purified from seeds. There are currently four methods of protein production from plants: (1) Stable nuclear transformation of a crop species that will be grown in the field or a greenhouse, (2) Stable plastid transformation of a crop species (3) Transient transformation of a crop species, and (4) Stable transformation of a plant species that is grown hydroponically such that the transprotein is secreted into the medium and recovered. Molecular Farming describes growing and harvesting genetically modified crops, with the object of producing not foodstuffs but pharmaceuticals. The idea is to use such crops as biological factories to generate drugs difficult or expensive to produce in any other way.

Genes from other sources, such as microorganisms, are sliced into the plants genetic apparatus, its genome. During normal growth these modified plants synthesise useful compounds, which are then extracted from the crop. The technique is already being used to produce vaccines for some animal diseases, such as mink enteritis virus. Many others are at the experimental stage, such as drugs to fight infant diabetes and Crohns disease. By one of lifes ironies, tobacco plants are especially suited to this purpose, so one day they may prove to be more valuable as a source of pharmaceuticals than of tobacco. Though the term has been around for a decade in the specialist literature, it is slowly becoming more widely known. A closely related term, pharming, seems more widely used for genetically modified animals than plants.

Classes of proteins within molecular farming

Proteins currently being produced in plants for molecular farming purposes can be categorized into four broad areas: (1) Parental therapeutics and pharmaceutical intermediates, (2) Industrial proteins (e.g., enzymes), (3) Monoclonal antibodies (MAbs), and (4) Antigens for edible vaccines

Productsinthemarket
Avidin
Avidin is a glycoprotein found in avian, reptilian and amphibian egg white. It is used primarily as a diagnostic reagent. The protein is composed of four identical subunits,

each 128 amino acids long. The usual source for commercial quantities of avidin is from chicken egg white but the resultant product is relatively expensive due to the cost of maintaining live animals.

b-Glucuronidase
GUS (b-glucuronidase; b-d-glucuronide glucuronosohydrolase) is a homotetrameric hydrolase (68 kDa/subunit) that cleaves b-linked terminal glucuronic acids in monoand oligo-saccharides and phenols. GUS is widely used as a visual marker in transgenic plant research.

Trypsin
Although both plant-derived GUS and avidin are protein products that have been commercially available for several years, their markets have been quite small and were designed to show proof-of-concept. Maize-derived trypsin, a more recent introduction, has a significant demand.

Productsclosetomarket
Many companies have been created to produce molecular farming products. Besides the three products described above, there are at least nine products thought to be close to reaching commercial market, i.e., in the next 5 years. Several of these proteins are normally derived from animal organs and, due to the possibility of animal pathogens being carried along with these proteins, there is a need for alternative low-cost supply.

Aprotinin
Aprotinin is a protein that inhibits serine proteases (including trypsin, chymotrypsin, kallikrein, and pepsin). This activity is known to modulate and lessen the systemic inflammatory response (SIR) associated with cardiopulmonary bypass surgery, which translates into a decreased need for blood transfusions, reduced bleeding, and decreased re-exploration for bleeding.

MarketpotentialofBiopharmaceuticalsproducts

Collagen is a structural protein currently rendered from hooves and connective tissue of animals. Vast quantities of collagen are consumed throughout the world each year in the form of gelatin. Based on their structural roles and compatibility within the body, collagen and gelatin are commonly used biomaterials in the medical, pharmaceutical and cosmetic industries. Collagen is used commercially in the areas of arterial sealants, bone grafts, corneal implants, drug delivery, incontinence, tissue engineering (artificial skin and cartilage), and as a viscoelastic supplement. Gelatin is used in the stabilization and delivery of vaccines and drugs, in capsules and soft gels, nutraceuticals, and as plasma expanders.

Human gastric lipase

Human gastric lipase or lack thereof, is a protein involved in the condition known as exocrine pancreatic insufficiency (EPI). The absence of lipase results in the inability to digest food lipids.

Human lactoferrin
Human lactoferrin is a natural defense iron-binding milk protein that purportedly possesses anti-bacterial, antifungal, anti-viral, anti-neoplastic and anti-inflammatory properties

Public acceptance
Sales are a good measure of the publics perceived benefits of specific products. However, todays public also wants to know that not only is there a benefit for the direct end user, but that there are otherwise no significant risks to the general public. This is illustrated by the recent concerns and debates over the use of GMO products produced in plants. While the initial concern involved GMO food products, this now encompasses non-food products as well. The fear is that the non-food products may inadvertently enter the food chain and present an unintentional risk. The following issues are to be considered for public acceptance: Adulteration Changes (variations) Wide distribution Complex production technology Potency Stability Environmental impact Quality management concepts and good manufacturing practice The standard operating procedure Documentation and document control Validation Validating biopharmaceutical production Facilities Equipment Downstream Analytical procedures Automated systems Regulatory agencies and mutual recognition Pitfalls to avoid Quality management burden or asset?

Trendsinnewbiopharmaceuticalsandvaccinesapprovedinthe USA
A total of 65 new biopharmaceuticals were approved during 19962005, with 32 approved during 19962000 and 33 approved during 20012005. Mean clinical development and approval times for new biopharmaceuticals increased during the two five-year periods. Mean clinical development and approval times were 68.0 and 15.9 months, respectively, for the 19962000 cohort. Mean clinical development and approval times were 83.0 and 18.5 months, respectively, for the 20012005 cohort. Increases in mean phase lengths were largest for the oncology therapeutics. A total of 15 new vaccines were approved during 19962005, comprising fewer than one-quarter of the number of new biopharmaceuticals approved in the same period. Only 4 of the 15 vaccines were indicated for a previously unmet medical need. The remaining 11 vaccines were approved for prevention of influenza and childhood illnesses. Mean clinical development time for innovative vaccines was similar to that for new biopharmaceuticals approved during 20012005.

Table1:NewproteintherapeuticsapprovedintheUSA
Companya Aventis Pharmaceutical, Inc. Serono, Inc. Amgen, Inc. BioMarin Pharmaceutical, Inc. Novo Nordisk, Inc. Unigene Laboratories, Inc. Tercica, Inc. Halozyme Therapeutics, Inc. Insmed, Inc. Bristol-Myers Squibb Application submission 18/06/2003 30/04/2001 15/06/2004 29/11/2004 05/12/2002 06/03/2003 28/02/2005 23/03/2005 03/01/2005 17/11/2004 31/03/2005 14/08/2003 08/01/2003 26/09/2003 24/05/2004 04/08/2003 07/07/2003 21/05/2004

Trade name

Generic name

Recombinant proteins Apidra Luveris Kepivance NAGLAZYME Levemir FORTICAL INCRELEX HYLENEX (r) IPLEX ORENCIA Insulin glulisine Lutropin alfa Palifermin Galsulfase Insulin determir Calcitonin (salmon) Mecasermin Hyaluronidase (human) Mescaserim rinfabate Abatacept

Monoclonal antibodies ERBITUX Avastin TYSABRI Vitrase Amphadase VIGIV Cetuximab Bevacizumab ImClone Systems, Inc. Genentech, Inc.

Natalizumab Biogen Idec, Inc. Non-recombinant proteins ISTA Pharmaceuticals, Hyaluronidase (ovine) Inc. Hyaluronidase (bovine) Vaccinia immune globin intravenous (human) Amphastar Pharmaceuticals, Inc. DynPort Vaccine Company LLC

Company name listed on FDA approval letter; bmonths; cTysabri voluntarily withdrawn from US market on 28/02/2005. Abbreviations: AA, accelerated approval; FT, fast-track designation; O, US orphan designation; P, priority review; r, recombinant; R, rolling application; S, standard review. Note: product first administration to humans and filing dates are confidential.

Futureresearch
One of the keys to success in the future will undoubtedly be the level of expression of the recombinant protein in plants. This is the one of the most important aspects with regard to economics. The expression level affects the cost of growing, processing, extraction, purification and waste disposal. Clearly there will be a drive towards higher levels of expression and there is much more room for improvement compared to other established systems.

Dr. P. E. Rajasekharan Senior scientist Division of Plant Genetic Resources, Indian Institute of Horticultural Research, Hessaraghatta Lake P.O.Bangalore 560 089, Email: [email protected]