The world is full of hope for the bright future for biological therapies: monoclonal antibodies, protein receptors, gene therapy, natural bioactive peptides for human healthcare treatment. Advancement in recombinant DNA biotechnology, molecular biology and immunology, the number of biotech drugs, including engineered peptides, proteins and monoclonal antibodies, available for clinical use has dramatically increased. The rising need for new and safer therapeutic molecules combined with the potential of peptides as active pharmaceutical ingredients (APIs) for effective drug formulation has contributed to rapid market development. The development of large synthetic and biological peptide libraries in combination with high-throughput screening processes has enhanced the prospects of obtaining new versatile drug candidates.
Peptides are emerging as a new class of biopharmaceutics due to their physiological dexterity and unique intrinsic physico-chemical medicinal properties. The benefits conferred by these characteristics include low unspecific binding to molecular structures other than the desired target, minimization of drug-drug interactions and less accumulation and toxic effect in tissues reducing risks of complications due to intermediate metabolites (Pichereau and Allary, 2005 ). Compared to small molecules, peptides offer valuable chemical and biological diversity on which intellectual property is still widely available. As a result, even the big pharmaceutical companies, traditionally focused on small molecules, are increasingly considering peptides in their pipelines for example Pfizer, GlaxoSmithkline and Lilly have recently acquired peptide-based products (Parmar, 2005). Small molecules share significant market as drugs by virtue of its small size, low price, oral availability, ability to cross membranes, and straightforward synthesis. The merits of peptides are higher potency, chemical and biological diversity alongside high specificity, affinity, and molecular recognition and have few toxicology problems, no accumulation in organs or face drug-drug interaction (Sato et al., 2006).
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Peptides are heteropolymers composed by amino acid residues linked by peptidic bonds between the carboxyl group of one amino acid residue and the Î±-amino group of the next one in a defined order. There are 20 amino acids that are essential to the body and are most often chosen by nature to assemble peptides and proteins. Despite the limited number of building blocks, peptides show physiological prowess and versatility. Peptides are polymers of amino acids made up of two to hundreds of amino acids. A protein consists of one peptide folded into a three-dimensional shape, or several peptides folded together. Biological functions as diverse as learning and memory, enzyme inhibition, blood pressure regulation, glucose metabolism, thermal control, analgesia, reproduction, immunosuppressant, antibiotic, antifungi, antiinflammatory and antitumoral activities are regulated by peptides (Thermofischer)
Figure1: The market breakdown of biotherapeutic drugs
(Source: Parmar 2005)
As seen from Figure1 majority of marketed peptide products and homologous compounds (proteins and antibodies) are peptide hormones or peptide derivatives that simulate the action of hormones. Peptides that are agonists or antagonists for receptors implicated in oncology and inflammation, peptides as antibiotics, or peptides that act as enzyme inhibitors in a variety of therapeutic indications are increasingly being tested for efficacy at the discovery and preclinical stages, suggesting that this class of drugs might soon occupy a larger niche in the marketplace. Other therapeutic peptides, such as antimicrobial peptides, with broad-spectrum antimicrobial activity against bacteria, viruses and fungi, are promised a great future, especially in counteracting the loss of efficiency of conventional antibiotics (Rotem andMor 2008)
Figure 2: Key classes of commercialized therapeutic peptides
GNRH/LHRH : Gonadotropin-releasing hormone also known as Luteinizing-hormone-releasing hormone
According to market research by Drug & Market Development Publications, the potential of peptide therapeutics "has recently intensified." That development is due to manufacturing improvements--peptides can be manufactured through transgenic, recombinant, or synthetic methods--and techniques that promise to make peptides more stable. peptides still need to surmount hurdles such as their high R&D expenses and difficult scalability. Yet they are drawing attention such that the global market for peptide-based active pharmaceutical ingredients (APIs) is expected to expand at a "growth rate nearly double the growth rate for APIs overall
Advantages of peptides over other drug candidates
Many therapeutic proteins, including products isolated from human blood, recombinant human cytokines, and recombinant growth factors, have elicited immune responses when administered to patients. Administration of megakaryocyte growth and development factor (MGDF) and Johnson & Johnson's erythropoietin (EPO) product EprexÂ® (epoetin alfa), show formation of cross-reactive neutralizing antibodies to the corresponding endogenous protein resulting in thrombocytopenia and pure red cell aplasia, respectively. These cases, have brought the issue of unwanted clinical immunogenicity to the forefront. Although even native human proteins can elicit an immune response, the perceived risk of clinical immunogenicity in response to non-native protein sequences is especially high. Nonnative protein sequences, include fusion proteins, bacterially derived biotherapeutics engineered variants, and most monoclonal antibodies (mAbs) (Chirino et.al,2004)
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Peptides possess novel functions and superior clinical performance; it is safe and effective drug for human biotherapeutics. Compared with proteins and antibodies, peptides have the potential to penetrate further into tissues owing to their smaller size. Moreover, clinical peptides, even synthetic ones, are generally less immunogenic than recombinant proteins and antibodies (McGregor,2008)
Peptides have other advantages over proteins and antibodies as drug candidates, including lower manufacturing costs as compared to recombinant production, higher activity per unit mass (15-60-fold, assuming 75Â kDa for one combining site of an antibody and 10-50 amino acids for a therapeutic peptide), lower royalty stack than antibodies because of a simpler intellectual property landscape during discovery and manufacturing, greater stability (lengthy storage at room temperature acceptable), reduced potential for interaction with the immune system (assuming the peptide contains no known immune-system signalling sequence) and better organ or tumour penetration (Ladner et.al,2004)
Therapeutic peptides also offer several advantages over small organic molecules that make up large percentage of traditional successful medicines. The first advantage is that often representing the smallest functional part of a protein, they offer greater efficacy, selectivity and specificity (limited non-specific binding to molecular structures other than the desired target) than small organic molecules. A second advantage is that the degradation products of peptides are amino acids, thus minimizing the risks of systemic toxicity (minimization of drug-drug interactions).Third, because of their short half-life, few peptides accumulate in tissues (reduction of risks of complications caused by their metabolites (Loffet, 2002)
Chemically modified peptides with improved bioavailability and metabolic stability may be directly used as drugs and many efforts have been made to develop peptide-based, pharmacologically active compounds. The most straightforward approach for peptide modification is to introduce changes into the side chains of single amino acids. The introduction of such functional groups that do not occur naturally in peptides restricts its conformational flexibility and enhances its metabolic stability. Affymax, an erythropoietin mimetic is a peptide drug to treat cancer and immune-related disorders. Erythropoiesis, the development of red blood cells, is stimulated when erythropoietin is secreted by the kidney. Myeloma patients and people with damaged kidneys, for example, do not produce enough erythropoietin and can become anemic.
The decreasing number of approved drugs produced by the pharmaceutical industry, which has been accompanied by increasing expenses for R&D, demands alternative approaches to increase pharmaceutical R&D productivity. This situation has contributed to a revival of interest in peptides as potential drug candidates. New synthetic strategies for limiting metabolism and alternative routes of administration have emerged in recent years and resulted in a large number of peptide-based drugs that are now being marketed (Vlieghe, 2010).
1.2 Emerging Market
Although peptide drugs have been on the market for decades - insulin being the most prominent example - it was not until 10 to 15 years ago that the pharmaceutical industry really started to work seriously on the development of a new generation of peptide-based therapeutics, prompted by advances in the understanding of the genetics of disease. Currently 60 per cent of the market comprises peptide-based therapeutics for the oncology segment followed by cardiovascular, infection and metabolic therapeutics. The approval of new peptide-based drug products such as Roche's HIV treatment Fuzeon (enfuvirtide) is stirring interest among many pharmaceutical companies. Globally, more than 40 peptide-based products are commercially available with six in the registration process.
The therapeutic peptides market emerged in the 1970s, when Novartis launched Lypressin, a vasopressin analogue. Since then, approximately 30 peptides have reached the market, representing a â‚¬5.3 billion opportunity in 2003 (over 1.5 per cent of the â‚¬325 billion global pharmaceutical market). Among the different classes of peptides, GNRH/LHRH agonists (leuprorelin,goserelin) account for almost 50 per cent of the market. Other key commercialised peptides include sandostatin (somatostatin analogue, Novartis), glatiramer (immunomodulator peptide, Teva) and salmon calcitonin (Miacalcin, Novartis). Several key therapeutic peptides were recently launched on the market, including: platelet aggregation inhibitors bivalirudin (Angiomax, The Medicines Company) and eptifibatide (Integrilin, Millennium); HIV cell entry inhibitor enfuvirtide (Fuzeon, Roche); and GNRH antagonist abarelix (Plenaxis, Praecis). Moreover, projects in Phase II/III have grown from 24 in September 2001 to 67 in January 2004. Peptides are expected to enjoy fairly attractive growth rates the market for therapeutic peptides rose from â‚¬5.3 billion in 2003 to â‚¬8 billion in 2005. It has been estimated that it will reach â‚¬11.5 billion in 2013 calculated at compounded annual growth rate (CAGR) of 8.1% (Pichereau and Allary, 2005 ). This excludes the prominent proteins and antibodies extracted from natural sources or produced by recombinant DNA technology, cell-free expression systems, transgenic animals and plants and enzyme technology.
Figure 3: Therapeutic Peptide Market Growth (03-13)
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More than 60 synthetic therapeutic peptides (comprising those used for medical diagnostics or imaging), with a size <50 amino acids, have reached the American, European and/or Japanese pharmaceutical markets through a marketing authorization as APIs, even if some of them are generics or discontinued (http://www.fda.gov/,http://www.fdaapproveddrugs.us,)
1.3 Sources of Peptides
Therapeutic peptides traditionally have been derived from three sources: (i) natural or bioactive peptides produced by plants, animal or human (derived from naturally occurring peptide hormones or from fragments of larger proteins); (ii) peptides isolated from genetic or recombinant libraries and (iii) peptides discovered from chemical libraries (Latham,1999)
Functional peptides are synthesized very rapidly within living cells, but until recently solution phase synthesis could artificially synthesize in vitro peptides at a slow rate and had poor impure yields. Recently a new technique known as solid phase peptide synthesis (SPPS) has been developed. SPPS results in high yields of pure products and works more quickly than classical synthesis, although still much more slowly in than living cells. Besides the classical synthesis in solution, solid-support synthesis is now the most widely used method to prepare synthetic peptides. The advantages of solid-support synthesis are its speed, versatility, ease of automation and low costs.
Generally, the size of the peptide determines the most suitable technology for its production: chemical synthesis, recombinant DNA technology, cell-free expression systems, transgenic animals and plants or enzymatic synthesis (Gill et.al,1996).
1.3.1 Chemical Synthesis
Chemical synthesis is a viable technology for the production of small and medium size peptides ranging from about 5 to 80 residues. The chemical route is often a better technological option than the biotechnological methods of recombinant DNA and biocatalysis for the synthesis of peptides. Since Emil Fischer's pioneering work in the early 1900's, synthesis methods have improved continually - especially with Merrifield's development of solid-phase syntheses. since the introduction of solid-phase synthesis byMerrifield (1986), this technology has gained more relevance and significant advances have been made in the development of polymeric carriers and linkers, reversible protective groups and methods for the activation of covalent bond formation (Albericio, 2004)
Solid-phase peptide synthesis (SPPS) consists in the elongation of a peptidic chain anchored to a solid matrix by successive additions of amino acids which are linked by amide (peptide) bond formation between the carboxyl group of the incoming amino acid and the amino group of the amino acid previously bound to the matrix, until the peptide of the desired sequence and length has been synthesized (Nilsson et al. 2005).
Solid-phase synthesis is usually carried out as follows:
1. loading of C-terminal amino acid to resin
2. deprotection: removal of N-terminal protecting group (PG) at amino residue
3. activation of next amino acid at carboxy residue
4. coupling reaction
5. start synthesis cycle 2-4 again or cleave fully-synthesized peptide off resin
Step 1 - Attaching an amino acid to the polymer
Peptide chains have two ends, known respectively as the N-terminus and the C-terminus, and which end is attached to the polymer depends on the polymer used. When polyamide beads are used the C-terminus of the peptide is attached to the polymer. The attachment is done by reacting the amino acid with a linkage agent and then reacting the other end of the linkage agent with the polymer. This means that a peptide- polyamide link can be formed that will not be hydrolysed during the subsequent peptide- forming reactions. Common linkage agents are di- and tri-substituted benzenes such as those shown below:
Step 2 - Protection
One of the critical steps in peptide synthesis is the necessity to block those functional groups that must not participate in peptide bond formation. In order to elongate the resulting dipeptide, one protecting group has to be removed. This has to be done under such conditions that the peptide bond itself is not harmed and transient protecting groups still stay on, too. Peptide chains have two reactive ends, known respectively as the N-terminus and the C-terminus. To ensure that only the desired dipeptide is formed the basic group of one amino acid and the acidic group of the other must both be made unable to react. This 'deactivation' is known as the protection of reactive groups, and a group that is unable to react is spoken of as a protected group. In classical organic sythesis the acids are protected, allowed to react and deprotected, then one end of the dipeptide is protected and reacted with a new protected acid and so on. There are four main groups used in this way: tBu (a tertiary butyl group), Trt (a triphenylmethyl group), tBOC (a tertiary butyloxycarbonyl group) and PMC (a 2,2,5,7,8-pentamethylchroman-6-sufonyl group) and FMOC (9-fluorenylmethoxy-carbonyl).
Fmoc amino acid protected with Fmoc Free COOH group reacts
with the NH2 group present on resin
Step 3 - Coupling
The FMOC protected amino acid is then reacted with the amino acid attached to the polymer to
begin building the peptide chain. The reaction is catalysed by DCC (1,3 dicyclohexylcarbodiimide), which is itself reduced to DCU (1,3-dicyclohexylurea). The next (NÎ± protected) amino acid is coupled to the already synthesized peptide chain bound to the polymeric matrix and, once coupled, its NÎ± amino group is deprotected.
Step 4 - Deprotection
Excess DCC is washed off the insoluble polymer with water, then the FMOC group removed with piperidine (a cyclic secondary amine). This is a trans amidification reaction. Steps 2 to 4 are repeated as each new amino acid is added onto the chain until the desired peptide has been formed.
Step 5 - Polymer removal
Once the peptide is complete it must be removed from the polyamide. This is done by cleaving
the polyamide - peptide bond with a 95% solution of trifluoro acetic acid (TFA). The side-chain protecting groups are also removed at this stage.
Step 5 - Polymer removal
This coupling-deprotection cycle is repeated until the desired amino acid sequence has been synthesized. Finally, the peptide-matrix complex is cleaved and side chain protecting groups are removed to yield the peptide with either a free acid or amide depending on the chemical nature of the functional group in the solid matrix. The cleavage reagent must remove the protecting groups of the side chains of the amino acids, which are stable at the conditions of NÎ± deprotection.Once the desired peptide has been made the bond between the first amino acid and the linkage agent is broken to give the free peptide (Bodanszky,1993).
Figure : Basic steps in solid phase peptide synthesis using Fmoc chemistry
The strategy of synthesis (Fmoc or t-Boc), the nature of the solid carrier, the coupling reagents and the procedure of cleavage of the peptide from the solid matrix are theimportant parameters during synthesis.
Pros and Cons
The chemical synthesis of peptides has been developed and automated to a high degree in the last decades. Protocols have been well established, which are amenable for scale-up to match the production levels required by the market. A key advantage of the chemical synthesis in solid phase is that the peptide product can be easily separated from impurities and side products. Major drawbacks refer to the racemization during peptide bond formation, the requirement of protection of the side chains of the amino acids that increases the cost of the substrates and reduces the yield of product recovery during deprotection, the difficulty of recycling the coupling reagent and the acyl donor used in excess to achieve rapid and complete acylation of the nucleophile, the time consumed in protection and deprotection reactions that reduce the productivity of the process and the toxic nature of solvents and coupling reagents that may lead to health and environmental concerns (Gill et al. 1996). Despite these restrictions, chemical synthesis of peptides can be considered the most mature technology available, being especially suited for medium size peptides up to 100 amino acid residues, which comprises most of the peptides of therapeutic relevance. Moreover, a key advantage of the chemical synthesis by SPPS is that the peptide product can be easily separated from impurities and side products. In addition, synthetic therapeutic peptides are now less expensive to produce than peptides or proteins obtained by recombinant technology (applicable only for peptide sequences including natural amino acids) or enzymatic route. As an example, even if it takes six to eight months and 106 steps to chemically synthesize at a multi-tonne per year scale the 36-mer anti-HIV peptide FuzeonÂ® (enfuvirtide, the first fusion inhibitor on the market for HIV treatment), its development has helped reduce costs of large-scale good manufacturing practices peptide synthesis to less than US$ 1 per gram per amino acid residue.
1.3.2 Recombinant Technology
The development of recombinant DNA technology in the mid 1970s marked the beginning of the modern biotechnology era. Recombinant DNA technology is particularly suitable for the synthesis of large peptides and proteins, as illustrated by the case of insulin and other hormones (Walsh, 2005). The manufacture of therapeutic proteins represented the first true industrial application of recombinant DNA technology. Production systems for recombinant peptides or proteins include bacteria, yeast, insect cells, mammalian cells and transgenic animals and plants. Genetic engineering enables the post translation modifications of the peptide sequence for example, glycosylation, phosphorylation or proteolytic cleavage for optimum biological activity these processing are not feasible during small molecule production (Rathore, 2009).
Recombinant DNA methodology has been employed for the generation of two monospecific anti-isotype antisera expressed as fusion peptides with GST (glutathione S-transferase) offers the possibility of large scale peptide production as an alternative to chemical peptide synthesis (Natalia et.al, 2006)
Compared with synthetic therapeutic peptides, the quality and/or purity of recombinant molecules is not always optimal and peptides produced by enzymatic routes have low productivity. Inability to incorporate unnatural amino acids or C-terminal amidation is another drawback of genetic engineering during the synthesis of therapeutic peptides. Moreover, the application of recombinant DNA technology typically requires a long and expensive R&D phase. The product can be obtained from very inexpensive starting materials via fermentation. This technique surpasses the use of toxic solvents and coupling reagents which are detrimental for human health and environment and hence can be applied for food grade applications. Despite significant advances, the synthesis of amino acid sequences by this method remains impractical due to low expression efficiencies and complexity encountered in product extraction and recovery (Gill et.al, 1996). several recombinant gonadotrophins (follicle stimulating hormone, FSH; luteinizing hormone, LH; and human chorionic gonadotrophin, hCG) have been approved for the treatment of various forms of subfertility/infertility. Cytokines approved include a range of recombinant haematopoietic factors, including multiple erythropoietinbased products used for the treatment of anaemia as well as a colony stimulating factor aimed at treating neutropenia.
Additional approved cytokines include a range of recombinant interferon-based products, mainly used to treat cancer and various viral infections, most notably hepatitis B and C, and a recombinant tumour necrosis factor (TNF) used as an adjunct therapy in the treatment of some soft tissue cancers. Blood-related approved therapeutic proteins include a range of recombinant blood coagulation factors used to treat haemophilia, recombinant thrombolytics and recombinant anticoagulants (Walsh,2003).
1.3.3 Enzymatic Synthesis of Peptides
Enzyme-catalysed chemical transformations are now widely recognized as practical alternatives to traditional organic synthesis, ans as convenient solutions to certain intractable synthetic problems (Koller and Wong, 2001).Enzymatic synthesis has been applied for the synthesis of peptides exceeding 10 residues. Its potential relies on the synthesis of very small peptides and most of the cases reported correspond to dipeptides and tripeptides. In recent years, proteases have been widely used to form peptide bonds. Proteolytic enzymes comprise a group of hydrolases that is the most relevant in technological terms, sharing about one half of the world market of enzymes, with annual sales of about US$ 3 billion (Chellapan et al. 2006).
Enzymes are remarkable catalysts: capable of accepting a wide array of complex molecules as substrates and highly selective catalyzing reactions with unparallel chiral (enantio-) and positional (regio-) selectivities. As a result biocatalyst can be used for simple and complex transformations without the need of tedious blocking and deblocking steps that are common in
chemical peptide synthesis and enantio- and regioselective organic synthesis. Such high selectivity also affords efficient reactions with few by-products, thereby making enzymatic transformations environmentally friendly alternative to conventional catalyst (Schmid et.al,2001; Kumar and Bhalla, 2005).The high specificity and high reactivity under mild operation conditions, which is characteristic of enzymatic processes, can have a strong impact on process economics, since it will reduce the number of operations required for the synthesis. This attributes have resulted in myriad applications in food and pharmaceutical sectors, speciality chemicals and polymers where precise reaction selectivity on complex substrates is critical. Some relevant examples are the synthesis of the leading non-caloric sweetener aspartame, the lysine sweet peptide, kyotorphin and enkephalin (Oyama et al. 1987, Kullman, 1979; Clapés et al. 1989), and some nutritional dipeptides and tripeptides. Telios Pharmaceutical Co. has explored the enzymatic synthesis of the tripeptide Arg-Gly-Asp as a new drug for the healing of heavy burns and dermal ulcer (Chen et al. 1998).
1.3.4 Natural sources
Natural products from environment have been the single most productive source of leads for the development of drugs and the most of the active ingredients of medicines (Harvey, 2008) Natural sources like plants, animals, microbes and marine life contain biologically active compounds, antibiotics, immunosuppressants and anticancer drugs etc some of which in future will prove to be valuable lead candidates for novel drugs. Historically, the most important natural sources for medicinal peptides and compounds have been plants. (Martin Tulp and Lars Bohlin, 2004) . Plants have been an integral part of the ancient culture of India, China and Egypt as medicine, and their importance even dates back to the Neanderthal period .In the modern world, the finding of cinchona in 17th century, followed by digitalis, morphine, and so on, and then introduction of synthetic aspirin, a derivative of a plant-based drug, compelled human beings to believe in the wonders of natural wealth. A large number of plants used in the traditional medicine have now become a part of the modern world health care system. Natural products offer large structural diversity , and modern techniques for separation, structure elucidation, screening and combinatorial synthesis have led to revitalization of plant products as sources of new drugs. The introduction of herbals in the form of nutraceuticals and dietary supplements are also changing the plant-based drug market (Saklani and Kutty,2008)
Enzymatic Biotransformation of Peptides
Proteases are among the best studied enzymes in terms of structure-function relationship. Proteases can be exquisitely specific for a particular peptide bond in a protein substrate or relentlessly nonspecific (Walsh, 2001). The proteases that are used for peptide synthesis are selected on the basis of their specificity against amino acid residues on either side of the splitting point and include the majority of the commercially available endo and exoproteases (Kumar and Bhalla, 2005). The broad specificity of proteases restricts their application in peptide synthesis, since the peptide product that accumulates during the reaction can be attacked by the proteases simultaneously with the reaction of synthesis (Schellenberger et al. 1991). There are five families of proteases in which serine, threonine, cysteine, aspartic or metallic groups play a primary catalytic role. In the first three groups, the nucleophile in the catalytic center is part of an amino acid residue, while in the second two groups the nucleophile is an activated water molecule. In cysteine proteases the nucleophile is a sulfhydryl group and the catalytic mechanism is similar to the serine proteases in which the proton donor is a histidine residue.
Proteases are active at mild conditions, with pH optima in the range of 6 to 8; they are robust and stable, do not require stoichiometric cofactors and are also highly stereo and regioselective (Bordusa, 2002). These properties are quite relevant to use them as chemical bench reagents in organic synthesis. This is possible because proteases can not only catalyze the cleavage of peptide bonds (hydrolytic activity) but also their formation synthetic activity (So et al. 2000). The other reactions of relevance for organic synthesis, proteases can catalyze are esterification and transesterification in the resolution of racemic alcohols and carboxylic acids and the stereoselective acylation of meso and prochiral diols the synthesis of glycoconjugates and the kinetic resolution of racemic mixtures, even though in this case lipases and esterases are more useful for the resolution of non amino acidic derivatives (Wong et al. 1993; Bordusa, 2002). Subtilisin, chymotrypsin, trypsin and papain have been widely used proteases in the enzymatic synthesis of peptides.
Bacteria, fungi, plant and animal produce proteases and are used for applied synthesis of small peptides. Microbial proteases are the most important in terms of market-share because of the advantages of their intensive production. However, plant (papain, bromelain etc) and some animal proteases(trypsin, pepsin etc) are still relevant for certain medical and industrial applications. Microbial and plant proteases have been traditionally used in degradation processes and are available at low cost; animals proteases are expensive and scarce. New sources of proteases are continuously being reported, especially from exotic organisms that thrive in extreme environments, being their proteases abnormally stable and/or active at such extreme conditions. As an example, the thermophilic and alkalophilic proteases from the salt tolerant marine fungus Engyodontum album has been recently reported and characterized (Chellapan et al. 2006). Compared with serine proteases, however, there have been few reports of the use of cysteine proteases in enzymatic peptide synthesis. Recombinant cathepsin L1 (rFheCL1) has been successfully expressed at high level in yeast systems, while cathepsin L1 extracted from liver flukes is notably stable (Deborah et.al, 2006)
1.4.1 Need to Engineer Proteases
In this sense, enzymatic synthesis of peptides is a less mature technology than chemical synthesis and no general protocols of synthesis are available, being each situation a particular case that has to be extensively studied and optimized to be technologically competitive. Despite the technological advances in peptide synthesis by biocatalysis, the low productivity, the low yield and the high cost of enzymes could hamper competitiveness in a broad spectrum of cases.
Enzymes are in general labile catalysts, so that process engineering of enzymatic reactions should be designed carefully. This implies the optimization of most relevant operational parameters: pH, temperature, organic solvent concentration, and the assessment of the activity and stability of the biocatalyst under operation conditions, the solubility of reactants, the stability of reactants and products (Blanco et al. 1991). The higher number of critical variables in an enzymatic process makes its optimization cumbersome. However, the limitations of chemical synthesis are generally overcome by protease synthesis. Enzymes are the biological catalysts responsible for cell metabolism. In cell they function well under the mild conditions of room temperature, atmospheric pressure, neutral pH and absence of functional group protection. To become process biocatalysts they must be robust enough to withstand the harsh conditions of an industrial process, which usually implies the modification of the enzyme to produce a stable biocatalyst.
1.4.2 Methods of Tailoring Proteases/Biocatalysis Engineering
Non aqueous enzymology has gained considerable attention in recent years as an efficient tool in the preparation of drug molecules, biopolymers, peptides and food ingredients. Few of proteases have been successfully applied to the synthesis of pharmaceutical and nutritional interest such as nutritional dipetides and tripeptides, artificial sweeteners and opioid growth factor (Krishna H, 2002). Enzymes in non-aqueous environments can catalyze backward reactions suppressed in aqueous solutions. Proteases in non aqueous medium shift the equilibrium of reaction catalyzed towards thermodynamically favorable synthesis of peptide bonds rather than hydrolysis (Clapes and Valencia, 1991). Despite their good catalytic properties, proteases are not ideal catalysts for the synthesis of peptides. Its specificity and selectivity might limit their potential, particularly in the case of rather large peptides where unwanted hydrolytic reactions will occur over the formed product and the substrates. Besides, the use of non-conventional reaction media and the conditions of temperature and pH required for synthesis can be detrimental both for protease activity and stability (Bordusa, 2002). There are numerous enzymes that remain catalytically active in non polar organic solvents as reviewd by Dordick. The polar solvent inflict stress on the native enzyme, severely perturbed from its optimum. Selective evolution was never applied to organisms to cause them to develop enzymes for biocatalysis in organic media. The protein solvent interaction shall influence the noncovalent interactions positively or negatively within the enzyme responsible for activity and stability. The activity of the biocatalyst will be preserved if the enzyme has attained balance in new environment. The loss of catalytic potential can be annulled by reengineering the extrinsic and intrinsic parameters of the reaction system, like reaction media, nature of solvents.
The term "medium engineering" originally stems from Klibanov and refers to the possibility of influencing enzyme properties by altering the nature of the solvent in which the reaction is carried out. Peptide bonds can be synthesized using proteases in either thermodynamicically controlled manner or kinetically controlled manner. This frequently implies the substitution of the usual aqueous medium for a non conventional medium in which water has been replaced partially or almost totally by another solvent (Hari Krishna, 2002). These reaction media that include organic solvents, eutectic mixtures, and more recently, ionic liquids offer other potential advantages as well: the possibility of using poorly water soluble substrates; the modification of the equilibrium of reaction as a consequence of the alteration of the partition coefficients of substrates and products in the case of biphasic systems; the reduction of inhibitory effects by substrates and products; the easiness of biocatalyst and product recovery,the increase in the thermo stability of the biocatalyst, variation in substrate specificity and the increase in the stereo and enantiospecificity in the resolution of racemic mixtures (Klibanov, 2001). There are basically two types of biocatalytic systems in non conventional medium: homogeneous systems which are mixtures of water and water miscible solvent and heterogeneous systems in which a second phase is produced by the presence of a water-immiscible solvent.
Selective catalysis is now becoming requirement for chemical industry and is on verge of significant growth. Enzyme's inherent catalytic prowess and specificity present in natural aqueous environment can be extended towards synthetic chemistries with novel reaction systems. Though reduced reaction rates and long term catalytic stability looms as barriers to the widespread implementation of nonaqueous biocatalysis on industrial scale. These result due to undesired effects of organic solvents: partial loss of tertiary and secondary structure, solvent penetration into active site and rigidification of enzyme conformations. The deactivation trends in organic solvents are not universal and can be mitigated by employing proper engineering approaches (Hudson, 2005). Biocatalyst engineering include approaches that range from chemical modification, immobilization to genetic and protein engineering. Protein engineering (site directed mutagenesis) entails the controlled alteration of a gene's nucleotide sequence, such that specific pre-determined alterations in the resultant polypeptide's amino acid sequence are introduced. Immobilization is an powerful tool designed to improve enzyme properties: stability, activity,specificity ans reduction of inhibition (Mateo et al.,2007). Adsorption to a solid surface increase the surface of contact on relatively large area facilitating the mass transfer of substrates and products (Adlercreutz). The conversion of enzymes into water -insoluble products possessing specific catalytic activity is of considerable industrial interest as they may be readily separated from the reaction mixture, thus reducing downstream processing cost and allowing its reuse. If the insoluble enzyme is catalytically stable it may be employed repeatedly for industrial bioconversions in large amount of substrate. This allows the complete utilization of expensive enzyme making the process economical. Reactions in packed bed column reactor make the process continuous with easy handling, less waste and enhanced stability towards stressful environmental conditions.
Improvement of enzyme activity retention during immobilization procedure can be increased by several methodologies like presence of substrate, substrate analogues or reversible inhibitors that protect the active site and incorporation of spacer arms. The latter allows the adhesion to the polymeric support from a certain distance from the surface in order to keep biologically important sites accessible to substrate. It provides flexibility to molecule by shielding it from hydrophobicity of carrier. The carrier can be designed to include flexible side chain which act as spacer chains which shall ensure free movement of catalyst molecule in the reaction mixture. Long and flexible spacers with hydrolytic character can improve activity of enzyme when conventional small hydropbhopic "spacer arm" usually 6 to 12 carbon atoms r unsuitable . protein being large molecule need large surface area. A large research of work has been devoted to the polymeric carriers, especially to immobilization of the proteins onto carriers. Enzymes are often immobilized onto solid supports to increase their thermal and operational stability, and recovery (Altun and Cetinus, 2007)
Large Applications of immobilized enzymes
In 1967 immobilized enzymes were used for the first time in an industrial process. Immobilized Aspergillus oryzae aminoacylase was employed for the resolution of synthetic racemic DL-amino acids. In England immobilized penicillin acylase was used to prepare 6-aminopenicillanic acid from penicillin G or V and in Unites States immobilized glucose isomerase was used to convert glucose into fructose (Katchalski and Kraemer,2000)
The success of syntheses depends on several factors, while fundamental reaction parameters, such as temperature, ionic strength, reactant concentrations and pH play an important role. As a general rule, it can be considered that an increase of the nucleophile concentration (HN) increases the product yield. Due to the specificity of proteases to a particular amino acid, only those acyl donors that have a specific amino acid in the C-terminal position can be coupled to nucleophiles without side reactions. For instance, trypsin requires arginine or lysine residues as carboxylic terminal components in the structure of the acyl donor .The manipulation of the leaving group is generally useful to increase the specificity of the protease to a previously less specific amino acid, so increasing reaction rate. In this way, the manipulation of the leaving group affects the aminolysis/hydrolysis ratio of an acyl donor and therefore the conversion yield, since the substarate-enzyme intermediate formed is the same regardless of the change produced in the leaving group.
Natural Peptides of Pharmaceutical significance
The global market for plant-derived drugs was worth an estimated $18 billion in 2005 this figure shall grow more than $26 billion by 2011, at an average annual growth rate (AAGR) of 6.6% between 2006 and 2011. The U.S. accounts for 50% of the global plant-derived drug market and is expected to grow faster than other markets at an AAGR of 7.5% per year vs. 5.3% . A total of 26 plant-based drugs were approved/launched during 2000-2006, which also include novel molecule-based drugs like Galanthamine HBr (Reminyl1), Miglustat (Zavesca1) and Nitisinone (Orfadin1) (Saklani and Kutty,2008)
1.5.1 Combating Oncological disease
Cancer is a complex disease that involves uncontrolled multiplication and spread (metastasis) of abnormal form of body's own cells. As per WHO 13% of world deaths, that is, about 7.6 million deaths accounted in 2005 are because of cancer, and this percentage is expected to increase in coming years.Plant derived compounds have played an important role in treatment of cancers, and some of the most promising and better drugs have come up from plant sources like Taxol , Camptothecin , Combrestatin , Epipodophyllotoxin and Vinca alkaloids (vinblastine, vincristine). Another potential drug of plant origin, known for its anticancerous and chemopreventive action is bowman birk inhibitor (BBI) present in Glycine max soybeans (Sessa, 2001). Soy protein products became famous when US Food and Drug Administration approved health claim in 1999 indicating that food product containing at least 6.25g of soy protein per serving would reduce the risk of coronary diseases (Kumar, 2004). BBI is a double headed trypsin-chymotrypsin inhibitor having market as anti carcinogenic agent. In plants protease inhibitors may represent a form of storage protein or may be involved in plant defence mechanism against pest and diseases. Legume seeds contain various protease inhibitors classified into several families: Kunitz, Bowna birk, potato I&II, squash, cereal superfamily and thaumatin like inhibitors (Mello et.al, 2001)
Biological active peptides from natural source are present with myriad of proteins and undesirable entities. The prospect of acquiring array of important therapeutic biomolecules in pure form from nature has made commercial realization of biotechnology possible. There is need of to develop novel technologies like chromatographic and membrane separation techniques by means of which active peptide fractions can be produced and enriched.
The major blockage for developing a large scale purification process is development of efficient process to purify proteins. The functional activity of the peptide medicinal molecules is dependent on the labile architecture of amino acids which are sensitive to harsh conventional separation techniques such as distillation and liquid-liquid extraction. Therefore, mild separation techniques such as membrane filtration, precipitation, centrifugation, electrophoresis and chromatography needs to be employed to prevent irreversible denaturation. The distinct physicochemical characteristics of proteins are exploited for their purification with the above mentioned process. Based on these physicochemical characteristics of peptides many purification methods have been employed such as membrane filtration, precipitation, centrifugation, electrophoresis and chromatography.
Integration of process
The remarkable advances in biotechnology in last few decades can be attributed to combination of increased knowledge of functioning of biological systems and on development of process technology associated with the production and harvesting of products for biotransformation.
Isolation of biomolecules from processing waste and intermediate steps
In situ product
Figure: Next generation approach
The processing waste of food industry are rich in medicinal small and large molecules. The whey like rich waste can be processed further for extracting the bioactive entities making the process economically prudent and reducing the environmental pollution.
1.4.1 Scope of present work
The present work deals with production of therapeutic peptides by biocatalytic routes using proteases and isolation from natural sources. The research was carried out in following parts
Part I: Engineering Trypsin for Semisynthesis of Insulin
Part II: Engineering of Pepsin for hydrolysis of IgG
Part III: Purification of Bowman-Birk inhibitor from Soy flour
Part I: Engineering Trypsin for Semisynthesis of Insulin
Trypsin (EC 126.96.36.199) a serine proteases with narrow specificity was employed for for biotransformation of recombinant Single Chain Insulin into human Insulin Ester (hIE). the critical factor in most bioprocesses is biocatalyst stability i.e. the capacity to retain activity through time. Soluble proteases show high propensity towards autodegration and self proteolysis resulting in loss of activity, selectivity and specificity. Preservance of multiple non covalent forces within the enzyme is essential for its optimum functioning . Chapter 2, elucidates the forces which stabilizes enzymes and engineering strategies which can be employed for obtaining maximum output during biocatalysis. To achieve maximum stabilization of soluble trypsin screening studies for suitable adsorbent and efficient covalent and non covalent coupling chemistry was done. The protein structure of trypsin was analyzed by using molecular structure visualization software (Discovery Studio, Accelrys).The distribution of functional groups, charge and hydrophobic on the surface determined the chemistry used for enzyme adsorption. On the surface properties of trypsin solid adsorbents having diverse properties were used for trypsin immobilization. The nature of adsorption: single point and multiple point attachment influence on behavior of enzyme was investigated. The preparation having the best stability for maximum time was screened and all the physico chemico parameters were derived. Chapter 3, focuses on the bioconversion of rSCI into insulin hormone by the engineered trypsin. Covalent, hydrophobic and ionic modifications of trypsin on synthetic polymers having varied properties were employed under aqueous and non aqueous conditions. Solvent engineering and reaction engineering of the coupling reaction was done for amplifying the yield of Insulin ester. Role of water in the reaction media and coupling medium for promoting hydrolysis ns synthesis was explored for the kinetically controlled synthesis of peptide. For large scale synthesis continuous operation of the process is the primary requirement. The synthesis of Insulin Ester was performed in packed bed reactor and parameters providing better control over enzymatic bioconversion were investigated.
Part II: Engineering of Pepsin for hydrolysis of IgG
Pepsin when applied for large scale protein hydrolysis needs to be immobilized due to their inherent autocatalytic characteristics. Modification of pepsin that could enhance stability, operational parameters and reusability were attempted. Chapter 4 discusses the mild immobilization technique developed for pepsin to maintain and preserving the 3D conformation of the enzyme. Different parameters during covalent carbodiimide coupling of pepsin to amine support was optimized to prevent proteolysis of the biocatalyst. A simple adsorption chemistry for pepsin stabilization and reutilization was developed using positively charged Polyethyleneimine polymer of different molecular weight was coupled to Sepabeads support.
The electrostatic interaction was strengthened by addition of NaCl to prevent bleeding of the biocatalyst thereby increasing the yield per cycle. Optimization all the external paprmeters influencing pepsin activity was performed.The efficacy and specificity of the process was judged by successive digestion of human polyclonal antibody IgG into F(ab)2 fragments.
Part III: Purification of Bowman-Birk inhibitor from Soy flour
Production of protein isolate generating high volume wastes rich in isoflavones and potent enzyme-inhibitors, lectins, tannins oligosaccharides like staychose and raffinose and phytates in soya seeds which act as antinutritional factors (ANF). Recovery of BBI and isoflavones in pure form from soy processing waste will be a considerable value addition to soy processing industry. BBI has been isolated from various sources using salt precipitation followed by solvent extraction or multistep chromatography. Low yields due to multiple steps, low purity, loss of activity and long processing time are the major drawbacks. The complex nature and low concentration of desired molecules in effluent necessitated the need for a multistep process to achieve concentration and effective purification of BBI. For preserving functional activity of these medicinal molecules non denaturing techniques like chromatography and ultrafiltration were investigated. Isoflavones are the major contaminants in the effluent which were captured by reversed phase chromatography