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BIOTECHNOLOGICAL DRUGS INTRODUCTION We study biotechnological drugs because there is an unmet need in medicine: combining biotechnologies with medicine allowed to apply typical techniques of the biotechnological field to the medical one, in order to find new treat...

BIOTECHNOLOGICAL DRUGS INTRODUCTION We study biotechnological drugs because there is an unmet need in medicine: combining biotechnologies with medicine allowed to apply typical techniques of the biotechnological field to the medical one, in order to find new treatments and so in order to correct the defects that characterize a lot of pathologies (for example, the cloning techniques used in biotechnologies are used to produce proteins that can be used to treat some pathologies). The main problem with these drugs is the delivering into the body, because biotechnological drugs are usually macromolecules (mainly proteins, but also nucleic acids), hence they have a huge molecular weight. So this field of applications is in constant development. The field of biotechnology applied in medicine is usually called red biotechnology and it involves the production of new drugs based on the knowledge of the mechanisms that induce the development of a specific pathology, because thanks to this knowledge we can also find pharmacological targets. The main aim is in fact to correct the defects typically part of that disease, most of the times in order to reconstitute the function of specific molecules (especially proteins) that don’t work anymore in that pathology. In fact, for this reason, the field of red biotechnology is involved also in designing organisms to manufacture antibiotics and vaccines and in engineering genetic defects through genomic manipulation (gene therapy). So one of the main goals of red biotechnology is drug production, which is the process in which pharmaceutical products are produced through application of biotechnological techniques. These drugs are produced for diagnosis (in this case they are just tools, not treatments), cure treatments and prevention of diseases. It’s estimated that by 2030, almost 80% of pharmaceutical will be represented by biotechnological drugs (right now they represent the 20% of marketed drugs and 50% of drugs under development): this is because they are highly effective and they have great advantages. However, biotechnological drugs cannot fully replace chemical drugs because chemical drugs are easier to produce and they can be used against certain types of pathologies that it’s better not to treat with biotechnological drugs (for example, when we have a headache it is better to use chemical drugs like anti-inflammatories, instead of using biotechnological drugs that are harder to produce and that are usually also hard to deliver inside the body). Biotechnological drugs are for this reason really useful mainly for really important and massive diseases, while mild diseases should be treated with small drugs. Right now we have more than 400 new biotechnological drugs and biotechnological vaccines for more than 200 diseases are currently being tested. The disease against which a lot of biotechnological drugs are being developed is cancer, followed by infectious diseases, autoimmune disorders and cardiovascular diseases. Moreover, recent statistics have pointed out that the most researched category of biotechnological drugs are monoclonal antibodies. The most sold drug in the world right now is a biotechnological drug. The main application of biotechnological drugs, that firstly induced the production of these type of molecules, consists in the administration of biotech drugs in pathological situations of either lack or insufficiency of natural proteins. Traditional methods involved the purification from blood, urine, human tissues or animal organs; however these products didn’t have a good efficacy since, if we extract from blood for example, we could have the possible presence of pathogenic agents, or if we purify animal proteins, the products that we obtain could be highly immunogenic for our system. Also, all of these techniques required a lot of money and they eventually allowed us to obtain only a small amount of product that could be used for medical purposes. So there was the need to find a way to synthesize human molecules, and thanks to biotechnology this was possible. With the in vitro production of these proteins the safety of the drugs was improved, moreover we could be able to obtain a modified protein for particular purposes (for example we can engineer the gene to obtain a modified protein with modified characteristics) and in top of that we could produce proteins whose extraction was impossible, so the ones we weren’t able to use for medical purposes until that point (for example erythropoietin, interferon, chloroplasts by bombardment of leaves with chloroplast vectors using the gene gun. After confirmation of stable integration of foreign genes into all the chloroplast genomes in each plant cell and expression of the correct size protein and functionality, genetically modified lines are transferred to the greenhouse to increase biomass. Harvested leaves are lyophilized, powdered and stored in moisture free environment. Machines are commercially available for processing lyophilized leaf materials into desired particle size and packaging into capsules. Evaluation process includes microbial count in lyophilized materials, integrity of therapeutic proteins after prolonged storage (folding with disulfide bonds, pentameric or multimeric structures) and functionality in conferring immunity with vaccine antigens (protective immunoglobulins IgG1, IgA, cytokines, pathogen/toxin challenge) or developing tolerance with autoantigens (suppression of allergy, formation of IgE, inhibitory antibodies, destruction of pancreatic islets, etc.) or conferring desired functions (regulating blood glucose with insulin, exendin-4, etc.). It is an advantage because it reduces manufacturing costs, it allows a long-term stability and storage, a large-scale production, the elimination of the cold chain, of expensive fermentation, of purification steps, sterile injections and microbes and it presents comparable safety potency and efficacy. Another interesting delivery route could be the buccal delivery: we have a special formulation of the drug, in which the protein is embedded in a muco-acid film (used for a lot of chemical drugs), so that the protein will be directly absorbed in the blood stream from the oral mucosa. An attempt to produce a biotech drug with this formulation was made on insulin: the recombinant human insulin is bound to glycan-coated gold nanoparticles through non-covalent binding and embedded in a polymeric mucoadhesive film for delivery of insulin via the buccal mucosa. However, the trial updates for this formulation have interrupted so the trial was probably halted. Future developments in buccal mucoadhesive drug delivery system for biologics could be directed to vaccines, peptides or proteins. Pulmonary delivery could be favourable because the absorption appears to be inversely related to molecular mass (MM); moreover we have a large absorptive surface (the alveoli have more than 100m 2 of surface of absorption), and from the alveoli the protein could go directly to the bloodstream (because of the this diffusional layer and of the high vascularization), without any alteration in the protein structure (in fact in the lungs we have proteolytic inhibitors and there is a high tolerance to foreign substances). Moreover, through this route we can avoid the first-pass metabolism and we have reliable, metered nebulizer- based delivery systems that have been already created. In this route of delivery, it is easier for the protein to pass the epithelial layer either through transcytosis or through paracellular transport (passage between two cells), because the drug has to cross a monolayer of insoluble lipids (lung surfactants), epithelial cells, the interstitium (fibrosus material) and the vascular endothelium. For this passage, the formulation of the drug can also involve the formation of a liposome nanoparticle, which presents a lipidic bilayer containing the drug (which is water soluble), tagged with specific targeting compounds. So, the strategies that have been conceived for this route of administration are: - Intratracheal instillation: provides information about protein stability, systemic absorption and toxicity (it is still experimental). - Aerosol inhalation: it involves two-phases colloidal systems (very fine liquid droplets dispersed in a gaseous medium); aerosol particle size is one of the most important in determining drug deposition and distribution in the lung. The devices used for this type of delivery are the nebulizers (however they are  4. Human antibodies are instead complete human mAb, the first created was adalimumab (anti TNF- alpha antibody). Despite its nature as being completely human, there are reports describing the occurrence of production human anti-human antibodies (HAHAs, same name as for the humanized ones) in patients. This is due to the immune system of the receiving subject, because even if the immunogenicity is really low we could still have the activation of the immune system. Also this type is produced with recombinant technology. We have also different types of mAb that are modified from the normal mAb to make them less immunogenic or more effective: for example the primatized antibodies are genetically engineered from cynomolgus macaque monkey, they are structurally indistinguishable from human antibodies and so they may be less likely to cause adverse reactions and they are potentially suitable for long-term treatments (an example is lumiliximab, an anti-CD23 antibody). However, the production of antibodies in monkeys is not so easy, moreover it is unethical and they are not approved, at least extensively. We can mention also another type of antibody, the bi-specific antibody, which is particularly useful against tumoral cells: a characteristic of cancer cells is their ability to perform immune-escape, so they won’t be attacked by T cells anymore. If we use bi-specific antibodies, which are structured in order to have one Fab portion directed against a tumoral antigen and the other directed against an antigen of T cells (like CD3, especially in CD8+ cells), we can make T cells become closer to tumoral cells and in this way the probability that they are going to activate against them is higher. If we then consider the fact that the Fc portion of the mAb is going to recruit also NK cells (or macrophages), the final action against the tumor is going to be amplified by the contemporary activation of the T cell. This is really revolutionary because usually infiltrating T cells, once they get activated, become anergic so they cannot get activated against the tumor anymore, while circulating T cells are still able to act against the tumor but they cannot reach it and infiltrate it because the tumor can escape their action. So by bringing T cells and tumoral cells very close we can induce the activation of CD8+ cells against the tumor. Moreover, in this type of antibody, the two binding sites can significantly increase affinity or internalization rates of particular antigens on a cell's surface by binding to two different epitopes on an antigen, and they usually have a higher cytotoxic potential to bind to antigens with low expression level. With the development of antibody engineering, many types of bispecific antibodies have been designed to overcome short half-life, immunogenicity and side-effects caused by cytokine liberation. They include trifunctional antibodies, chemically linked Fabs, various types of bivalent and trivalent single chain variable fragments (scFvs), and fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bispecific T cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region. Why are scFvs so innovative? The part of the antibody which recognizes the antigen is the variable portion, which is made of light chains and heavy chains. The heavy chains have one variable domain (VH) and three constant domains (Ch1, Ch2, Ch3), while the light chains have one variable domain (VL) and one constant domain (CL). If we link together the variable domains of these two chains by a short peptide linker, we can create a molecule that only contains the light and heavy chains of the variable portion and that can recognize the antigen. This is an advantage because the resulting molecule is very small, much smaller than a whole antibody, and so these scFvs could possibly enter the cell and target intracellular antigens, like viral ones (something that an antibody cannot do because of its size; called intrabodies). So the sequence of the gene of the intrabody can be inserted in a vector (very difficult thing to do with a whole antibody, which is encoded by very long and complex sequences), which is going to be transfected inside our cell of interest, then the intrabody will be expressed inside it and it is going to target antigens found inside it (for example in the ER, which is pretty simple to induce because antibodies are normally produced there so you don’t need any particular folding or stability characteristic, or in the cytoplasm or in the nucleus if the target is located there, and in that case a correct folding of the intrabody is required).  Drosha-DGC8, inside the nucleus. Then the pre-miRNA is exported in the cytoplasm (thanks to exportin 5), where the DICER is going to transform it into a double stranded miRNA molecule (miRNA duplex or miRNA-miRNA). Even if the process is quite the same of the siRNA one, we have a difference between these two molecules: siRNA have a complete complementarity with the target mRNA, so also the duplex will be a linear double strand molecule, while miRNA do not have a precise complementarity, and we can understand it from the fact that their duplex contains a sort of “hole” inside it (region in which the nucleotides do not pair with each other). Again, only one strand will be the one used for the gene silencing (the other one is digested), and, incorporated in the RISC-AGO2 complex, it will bind either to the regulatory regions of the mRNA molecule (5’-UTR and 3’-UTR) or to the ORF (open reading frame, so the encoding region). This is because the miRNA doesn’t have a specific complementary to the coding region of the mRNA, so they can target a lot of parts of the mRNA molecule. Finally, the miRNA could also go back inside the nucleus and, always bound to the RISC-AGO2 complex, bind to the promoter region of genes (so of DNA). More than 500 miRNAs are encoded in the human genome, so they constitute the largest gene family; it has been estimated that more than 30% of protein-coding genes can be regulated by these miRNAs. Both miRNA and siRNA are molecules that are normally produced by the cell but they could also be injected inside a cell (or they could be expressed by it), like ASO molecules. What is the seed region? It is a short sequence present on the target mRNA that is usually perfectly complementary to the siRNA, while we don’t have the same level of complementarity between this region and miRNAs. So, if the molecule that binds to the seed region is perfectly complementary (usually siRNA, full match), the mRNA will be degraded through the action of the RNase H; if the molecule that binds the seed region is not perfectly complementary (usually miRNA, partial match) the mRNA will not be degraded, but there will just be a physical block of the translation. In both cases, the result is the repression of the translation and the mRNA will not be translated. However, these different levels of complementarity are index of the fact that siRNA will eventually have a single target (they are complementary to only one mRNA because they have the same sequence), while miRNA have multiple targets (because they are not fully complementary to any of them, so they could potentially target a lot of different mRNAs). We already said that these RNAi are physiologically produced by our cells, in particular miRNAs are the most represented ones. They have a lot of physiological roles, that they carry out through their regulatory action, for example they are involved in organ and tissues development, in stem cell differentiation and maturation, in cell growth and survival, in metabolic homeostasis, oncogenic malignancies and tumor formation (because it can regulate the expression of genes involved in the growth of the cell, so they are unfortunately also involved in these pathologic activities), viral infection and epigenetic modification. But we can use them also as therapeutics. In this sense we can consider them as both New Chemical Entities (NCEs), because they can be chemically synthetized, and New Biological Entities (NBEs), because they have a highly specific target and they have a complex modality of action. So they are different from the other biotech drugs that we talked about, which are proteins and cannot be chemically synthetized. Another big difference is that one molecule of RNAi can target one mRNA but then it can be recycled and target another mRNA molecule, so this means that RNAi based compounds can be used for several rounds of cleavage (obviously it also depends on the concentration because the lower will it be, the less frequently this phenomenon will happen, but at the same time the lower the dosed the less the side effects). So which type of RNAi have been used in the therapeutic field? Systemic administration: this type of administration is generally preferred for any type of drug because in this way it can easily reach every tissue. However, nucleic drugs should be modified prior to a systemic administration because their stability inside our body is very low. The first example is the one of Patisiran: it is a siRNA that targets the mRNA of a protein called transthyretin, that, when aberrantly produced, causes the accumulation of amyloid fibrils in different organs, determining the appearance of a pathology called Familial Amyloidotic enzymes; the presence of more than 20 proteases in a bacterial cell makes this task difficult, however there are some mutant E. coli species with defective proteases, like ompT; this strategy is used to increase the stability of the protein when we cannot change the sequence of the aminoacidic chain of the protein otherwise we will alter its function). However, expression vectors also need to be controlled: a continuous production of a protein could kill bacterial cells, causing inhibition of cell functions, loss of energy, loss of plasmid. To avoid this problem we can use specific vectors in which we induce the transcription of the protein just in a specific moment. These vectors are called inducible vectors and they work associated to substances which are able to induce their expression only in the case in which they are present. In this way, the bacterial system doesn’t overload and we are able to achieve an optimal production in a limited period of time, called induction phase. If we don’t use these strategy and we, for example, leave the bacteria grow in two days, it is possible that our protein will be produced only for the first two hours and then it won’t be produced anymore, either because the protein that we want the bacterium to express can be toxic for the host cell, or because, under some conditions, the recombinant protein can account for up to 30-40% of the total proteins, replacing those that are important for the metabolism of the bacterium. Moreover, when the protein is “induced”, proteolytic degradations are limited and the yield of production of the protein is improved. Another strategy of modification is to produce the protein in a specific region of the bacterial cell or directly outside of it. This could be performed to avoid the degradation of the heterologous protein inside the cell and to facilitate the purification process. The localizations could be: intracellular, periplasmatic, cell wall, extracellular. Except for the intracellular one, all the other localizations could induce a secretion of the protein so that we have the final product directly in the medium and it will be easier to recollect it. The eventual disulphide bonds of the protein occur during secretion, the N-terminal region will be identical to the original product and the correct folding is also guaranteed. All of these can be achieved by producing fusion proteins: for example, we can insert in the vector the gene of our protein of interest fused with the gene for another protein (for example a maltose binding protein), the product will be a fusion protein that can be used to achieve a different localization of the protein in the host cell or to achieve a better purification step (for example, in this case we exploit maltose bound to a column and we make our fusion protein bind to it). Then, the protein used for the purification will be removed and we will simply obtain our therapeutic protein. So in general fusion proteins can be used to increase the stability of the protein during the expression in a different localization of the cell, or to improve the purification steps. For this last purpose, the GST is one of the most used gene in laboratory practice (check table). BIOTECH DRUGS: PROCESSING AND PRODUCTION Which host cells can be used for the production of biotech drugs? 1. E. COLI: it is very easy to grow, it is cheap, we know all the media and all the techniques that are needed to grow this bacterium, so we have a lot of proteic drug that have been and are still produced inside these cells (insulin, INF-alpha, INF-gamma, IL-2, G-CSF, hGH and tPA). Moreover, the level of expression in E. coli is below 30% of the total proteins of the cell, we cannot obtain a higher percentage of protein, but it is still very high. E. coli cells grow rapidly on simple and inexpensive media, their molecular biology is well characterized and their appropriate fermentation technology is well established, so they are really easy to control for us. In E. coli we can produce potentially any type of protein that doesn’t need to be glycosylated. This is the only main problem of this type of cell: glycosylation cannot happen in bacterial cells, it’s a step that can only happen in eukaryotic cells. Another drawback is that, being bacterial cells, they present the LPS on their cell  protein (a viable embryo with the inserted gene must be brought to term and this gestation period ranges from 1 month for rabbits to 9 months for cows, after breeding they must bring their offspring to term before they begin to lactate); after some generations of transgenic offspring the recombinant gene is lost, so the animals will stop producing our protein of interest. So the microinjection technique is time-consuming and does not guarantee success. For all these reasons sometimes instead of using this technique we prefer the cloning technique (nuclear transfer): in this case the donor is an adult cell (any tissue cell from an adult goat), you remove the nucleus and you genetically engineer it to harbour the gene of interest, then you substitute it with the nucleus of an egg cells taken from an unfertilized animal. The “reconstructed” embryo is then grown for 7 days, then it is implanted in a surrogate mother and in this way we give birth to a cloned animal with the exact DNA as the tissue cell donor animal. This is the technique that has also been used for the Dolly sheep cloning. The problem with this technique is that the cloned animal could have a short life and health problems. So the microinjection is still preferred in the majority of the cases. In 2014, the first recombinant drug purified from the milk of rabbits was produced: ruconest (International Non-proprietary Name: conestat alfa) is a recombinant human C1 esterase inhibitor (rhC1iNH) developed and approved for the treatment of acute angioedema attacks in patients with hereditary angioedema. HAE is a rare, serious, autosomal-dominant genetic disorder with an estimated prevalence of one in 50,000. Clinically, patients with HAE experience recurrent acute attacks of soft tissue swelling that can affect multiple anatomic regions, including the gastrointestinal tract, facial tissues, vocal cords and larynx, oropharynx, urogenital region, and/or the arms and legs. The transgenic rabbits have been modified to produce the recombinant protein in their milk, however the quantity of milk that we can produce with these animals is not that much. So why do we use rabbits? This disease is very rare, so the amount of protein that we have to produce is not that much, so rabbits are perfect to provide the correct amount of this protein. We only saw the production of biotech drugs in milk, but are there any other biological fluid that could produce recombinant proteins? Potentially, we could use blood, but this means that to purify it we have to make the animal bleed, and this means that the animal could die, so only a low volume of blood can be harvested from animals at any given time, and this is not enough for a pharmaceutical protein production. Moreover, producing proteins in blood means that we could potentially induce side effects, mainly related to immunogenicity, in the producer animal. Another disadvantage is that serum contains a variety of native proteins, so the purification of the recombinant protein could be really complex; in addition, many proteins are poorly stable in serum. So, this production is not suitable for industry. Other fluids where we could think of producing recombinant proteins in transgenic animals are urine and seminal fluid. But these fluids are really difficult to collect, and they are not appropriate for the production of protein in an industrial scale (urine contains toxic molecules and seminal fluid is not produced in high quantities). 4. HEN AS A BIORECTOR: an egg can be easily engineered with retroviruses containing the gene for our biotech protein, to obtain a recombinant egg. Retrovirus particles bearing a transgene, or Barred Rock cESs transformed with an oviduct-specific expression vector, are injected into the sub- germinal cavity of freshly laid, stage X embryos. In the case of retroviruses, the egg is sealed with a plug of hot glue or with eggshell membrane and cement and incubated to hatch, yielding G0 founders chimeric for the transgene. In the case of cESs, the egg is sealed and incubated for 3 days before transfer to a surrogate eggshell and incubated to hatch, yielding high-grade black feathered chimeric chicks. The egg white can contain 4g of proteins, of which 50% is ovalbumin, so eventually just 25% is represented by the recombinant protein. However, once we produce a transgenic rooster, its offspring can produce annually up to 300 eggs, so eventually 300g of recombinant protein can be produced. The main problem consists in the pattern of glycosylation: glycosylation is very different compared to the native glycosylation of human proteins, in fact a lot of attempts have been made with the production of antibodies, but the egg derived mAbs are really immunogenic because of this different glycosylation (reduced serum half-life, of 100 or 200 hours, because the different glycosylation enhances the ADCC activation). For this reason, no biotech drug has been produced with this method yet. disulphide bonds form between two different chains (interchain) we have the aggregation of different proteins. This phenomena are generally called disulphide exchange. Each one of these modifications can alter the structure, and thus the function, of our protein of interest, which must be kept in its native form. They could occur at any step of purification, so both during chromatography and after chromatography (for example oxidization could happen because we have potent oxidizers in solution but also just by contact with air, so it could happen also at the end of the process). To avoid it we perform all the steps at low temperatures or by adding chemical reagents that prevent these modifications. Once we have purified our protein and once we are sure that it was kept at its native form, we have to stabilize the protein. This is performed by adding stabilizing excipients, like human serum albumin. The human serum albumin does not alter the activity of the protein (obviously we have to use a recombinant one because if we used the one extracted from blood directly there would be the risk of transmitting infections from the blood of the donor). It is stable at low pH or at elevated temperature and it is very useful because it could work as an alternative target for proteases that are eventually still present in traces. It is also an effective cryoprotectant and once injected in blood it doesn’t cause anything wrong because we already have it in our blood (if we inject little quantities of it nothing happens in our body). Human serum albumin is the most used stabilizing agent, but we could also use amino acids, like glycine (used for IFN, EPO, factor VIII, …), which reduces the surface absorption of products, inhibits aggregated formation, stabilizes the conformation of the protein and prevents heat denaturation. We could also use polyols (like glycerol, mannitol, sorbitol, PEG and carbohydrates) or surfactants (protein denaturants which reduce the surface tension and increase the solubility of the protein, they avoid the mis-folding of the protein and they reduce the rate of protein denaturation at interfaces). The final step before the lyophilization is the sterilization: we pass the protein solution in a filter with pores of 0.22 micrometres which can get rid of bacterial cells. In this way we are 100% sure that we don’t have any bacteria in the solution. However, we could also have other types of microorganisms, like viruses, which can infect mammalian cell cultures. The filters that we use do not get rid of viruses so we have to change filters and use the ones that have 0.1 micrometres pores. Our sterilized protein then is ready to be lyophilized: lyophilization is the process through which we remove the solvent directly from a solution while in the frozen state, obtaining a powdered product (3% water). It is extremely useful to preserve the biological properties of the product, however it can promote the inactivation of some proteins, so specific excipients are added (e.g. HSA), along with amino acids (e.g. lysine, glycine). The whole process is performed while decreasing the temperature and, while it lowers, it induces the formation of ice crystals, instead the protein will aggregate and separate from the water molecules. The crystalized water is removed, while the temperature is still lowered until molecular mobility ceases (the lowering of the temperature is responsible for an increase in the viscosity of the solution, the mobility ceases around -60° of temperature). Finally, we apply a vacuum which is going to remove all water crystals (through sublimation), other solutes and salts, so that eventually only the protein will remain. After this process, a label is applied on the final product, which contains information on the name, the strength, the batch number, the date of manufacturing, the expiry date and the storage conditions of the drug. First, though, we have to perform a quality control of potency, sterility, final volume, absence of endotoxins or other toxic substances. In this phase, some mis-labelling errors could happen, so we have to pay attention. This process is performed for any lyophilized Once we are sure of what we are dealing with as our final product we wait for the FDA approval; if the FDA approves it the product is licenced, marketed and sold. This in the picture is a recap of all the steps. Remember: different process run at different times equals to different product, even if it is performed by the same pharma company. This doesn’t mean that the protein will be different, but that the total product will be slightly diverse (because maybe the DNA vector that we use for the cloning is a little different, or the host cell has different expression levels, or the fermentation process occurs differently, or the downstream protocol is run in a different way, or the final formulation is different). This is something typical of biotech drugs, chemical drugs are always the same even if the processes are different and run at different times. The paradigm of biopharmaceutical is that the process is the product. THERAPEUTIC HORMONES 1. Insulin: insulin has a lot of functions in the body, the most important is the reduction of glucose in the blood stream. It is a very small protein (6 kDa in weight), it is composed by two chains, which are the A chain (21 amino acids in length) and the B chain (30 amino acids in length), that are kept together by disulphide bonds, which are both intrachain and interchain. The structure and the length of the protein are really important to know because through the different composition we can obtain different types of insulin. Physiologically, it is produced as a pre-proinsulin, which is a single polypeptide molecule which contains the two chains (A and B) bound together by a linking peptide called C chain. Then the disulphide bonds are produced and the C chain is digested, so that the protein is only made by the A and B chains. This physiological process can be reproduced in vitro to produce recombinant insulin. Moreover, we know that native insulin molecules are kept together by zinc atoms, forming multimers, in particular hexamers, which determine how quickly the insulin is going to be absorbed into the tissues, and this is really important for a pharmacological application. In the past, insulin was obtained by pigs, and about 70 pigs per patient per year were needed for the production of this protein for diabetic patients. Porcine insulin differs of only 1 amino acid from the human one, however it was immunogenic (the bovine insulin differs of 3 amino acids so it’s even more immunogenic than the porcine one). So in order to facilitate the treatment of diabetic patients, the recombinant insulin was first produced and marketed in 1982, becoming the first marketed biotechnological drug. The main advantages is that we can produce huge amounts of the protein in this way, with a low grade of immunogenicity, however the disadvantage is that E. Coli is not able modify the eukaryotic pre-mRNAs nor it can operate post translational modifications of the protein. Anyway, with biotechnological approaches and with recombinant DNA techniques, it is possible to produce insulins with altered pharmacokinetic properties (faster- or slower-acting) and to select insulin with higher receptor affinities (ensuring economic benefits from using smaller quantities of insulin per therapeutic dose). How do we produce insulin? The first method is the one of insulin crb: we have two cell lines of production (of E. coli), in one we clone the gene of the A chain, in the other one we clone the gene of the B chain. We make these cells express the two chains, we purify them through chromatographic steps and then we incubate both chains under appropriate oxidizing conditions to promote the formation of interchain disulphide bonds. In the second method, on the other hand, we produce the whole molecule in a single cells line (insulin prb): we insert the gene encoding for human pro-insulin in recombinant E. coli, we purify it from the molecules of the bacteria and then we remove  recombinant glucagon has been produced by Novo Nordisk in Saccharomyces cerevisiae and by Eli Lilly in E. coli. 3. Growth hormone: it is used to treat dwarfism. It was the second recombinant protein produced and marketed after insulin (1985). Before the production of its recombinant counterpart, growth hormone was purified from the pituitary glands of corpses because this protein, in order to be used as a therapy, must be human. However, extracting it from corpses had a lot of disadvantages: first of all we didn’t have a lot of corpses to purify it from, so the amount of hormone that we could obtain was vey low, moreover we extracted the protein from neuronal tissue, and this could cause the insurgence of infections in the patients, such as the Creutzfeldt-Jakob disease (mad cow disease). So the production of a recombinant growth hormone in E. coli was a really great innovation, because the native and recombinant forms of the molecule are identical. The growth hormone is used for the treatment of dwarfism but also of short stature in general, for the induction of lactation, for the counteraction of ageing, the treatment of obesity, body building and induction of ovulation. Other than the recombinant growth hormone, also an analogue that acts as an antagonist has been produced: it is called Somavert and it is used for the treatment of selected patients suffering from acromegaly. This molecule, commercially called Pegvisomant (Peg stands for pegylated), is able to bind to the receptor for the growth hormone but it does not induce receptor dimerization and thus the JAK/STAT pathway, preventing the expression of the genes regulated by the activation of this receptor (like IGF-1). The main adverse effect of this drug is that it may raise endogenous GH circulating levels because we don’t have any negative feedback regulating its synthesis in the pituitary gland or hypothalamus. The main difference between this analogue and the growth hormone molecule is that it has a substitution of 9 amino acids and it is pegylated, so that we lower the immunogenicity and we raise the half-life of the molecule. The aminoacidic substitutions include the G120K mutation, which is able to increase the affinity of the molecule with the GH receptor, but at the same time it prevents its activation. 4. Gonadotrophins (or gonadotropins): they are really important also in the veterinary field. They are a group of hormones constituted by the gonadotropin releasing hormone, released by the hypothalamus, the follicle stimulating hormone (FSH) and luteinizing hormone (LH), produced by the pituitary gland, they have the gonads as target organs (ovaries and testis), and human chorionic gonadotrophins, released in the placenta. They bind to different receptors but these receptors all share the same subunit. They must be glycosylated, otherwise they cannot function, so they cannot be produced by E. coli. Human gonadotropin is excreted in the urine during pregnancy (used for pregnancy tests), so before being reconstituted in vitro (recombinant gonadotropin), it was extracted from the urine of pregnant women through ion exchange chromatography and gel filtration. This process had a lot of disadvantages, because dealing with urine is very complicated (urine is toxic) and also the quantity of hormone that was extracted was really low. However, even nowadays it’s still available as an extracted protein because the process is cheaper. Menotrophin (human menopausal gonadotrophin) is the name given to FSH-enriched extracts from human urine (2,5 l/dose). Gonadotropins could also be extracted from pituitary glands (especially FSH and LH which are produced from this gland). However, in 1985 the extraction of gonadotrophins from the human pituitary was abandoned because of the risk of the Creutzfeldt-Jacob disease. Now the human chorionic gonadotropin is produced as a recombinant protein, it has the name of Ovitrelle and it was approved in 2001; it is used for the treatment of female infertility due to anovulation and for patients undergoing assistive reproductive technology, in fact it is used to trigger final follicle maturation and luteinization after follicle stimulation. It is produced in CHO cell lines because it has to be glycosylated, it is identical to the human protein and it is purified by multistep chromatography. The number of oocytes retrieved with recombinant hCG was similar to the number retrieved when urinary-derived hCG was used. Sometimes it is used also for the treatment of male infertility in assisted reproductive techniques. For veterinary use, the use of gonadotropins is pretty similar: we use it to obtain an identical offspring from surrogate dams (animals that receive multiple embryos coming from the same donor). So a cow is inseminated from the sperm of a superior bull (selected species of bull, with some particular characteristics), then we recover the embryos produced, we split them and we reimplant them in other cows, which are surrogate mothers, for differentiation, growth and birth. This technique is used to amplify the offspring of a genetically selected sperm donor and it is called embryo transfer; the aims various cell lines were found to secrete substantial amounts of IFNs (for example Namalwa cell line was able to produce 8 subtypes of IFN-). However, it was still produced in mixtures, so we still weren’t able to obtain single proteins. DNA recombinant technology led to the production of a single IFN subtype in the 1980s. Most interferons have been produced in E. coli, yeasts, and some mammalian cell lines. Most IFNs currently in medical use are produced in E. coli because the majority of human IFNs are not normally glycosylated (mostly IFN-a and IFN-b). IFN-g is glycosylated but the non-glycosylated form displays a biological activity similar to the native protein, so the glycosylation is not mandatory for this molecule to be use in therapeutical applications. However, the production of IFN in recombinant microbial systems means that any final product contaminants will be microbial in nature. So a high degree of purification is required: up to 99% of purity can be achieved with extensive chromatographic purification. The first recombinant INF was produced in 1980 (INF-alpha2a), in 1986 it was approved for the treatment of hairy cell leukemia (Intron A), but it can be used also against viral infections like in hepatitis. PEGylated interferons are now available: the PEG molecule is able to prevent the degradation of the molecule, shielding it from enzymatic degradation, so we have a prolonged half-life (13 hours to 25 hours compared to the 4 hours of the non-pegylated protein) with the same intrinsic biological activity as non-PEGylated INFs. Moreover, the PEGylation lowers the immunogenicity of the protein, allowing multiple and frequent dosing and achieving a higher and sustained interferon concentration in blood. If we administer the interferon with an antiviral chemical drug, we can see that the combination of this molecule with the non-PEGylated molecule reaches the 44% of remission of the HCV infection, while the combination with the PEGylated molecule allows us to reach a remission of 56%, so we obtain better results (both these combination work even better than immunotherapy). We can see that another main advantage is that, with better results, the dosage can be decreased, and this is very important for the adverse reaction profile (the less drug we use the less side effects we could have). We could have two different types of PEGylated interferon: PEG interferon alfa-2b (linear molecule, with a weight of 12 kDa) and PEG interferon alpha-2a (larger and branched molecule, with a weight of 40 kDa). This changes their pharmacokinetics: the volume of distribution of the larger PEGylated interferon is lower than the smaller one (8L vs 20L), in fact, being a larger molecule, it is harder for this interferon to pass through tissues, in fact also the absorption half-life is longer, just like the mean elimination half-life. If we change and we modify the molecule by adding a different type of PEG molecules we can obtain different molecules with different pharmacokinetics characteristics (for example, if we want to have a longer half-life, we will use a bigger PEG molecule, but we have to remember that in this way also the time of absorption will be prolonged, while the volume of distribution will be lower, so some characteristics will have to be “sacrificed”). When talking about recombinant interferons we have to mention Infergen (interferon alphacon-1): it is not a real natural interferon molecule, it is “invented”. This protein is in fact an engineered INF approved for the treatment of hepatitis C, produced in E. coli, with a higher antiviral, anti-proliferative and cytokine-inducing activity than native type I interferons: the aminoacidic sequence presents the most frequently occurring aminoacidic residues in native IFNs-a. So it is not the copy of any other existing interferon, but it still works in a very good way. For what concerns INF beta, it is used for the treatment of multiple sclerosis, it inhibits the production of pro-inflammatory cytokines (like TNFalpha and INF-gamma) and it can be produced in either E. coli or CHO cell lines, because even if it is not glycosylated it yields the same therapeutic efficacy as the glycosylated protein. The recombinant INF-gamma is a particular interferon because it is used against the CGD (Chronic Granulomatous Disease), a rare genetic disorder where there is a defect in the granulocyte response to infections, so granulocytes do not activate properly. This pathology is responsible for the development of a lot of infections and granulomas in the patients and sometimes they could be lethal. It is produced in E. coli, hence it lacks the carbohydrate component, but it still displays the identical biological activity as the glycosylated protein. It is a potent activator or phagocytes, in particular of granulocytes, that’s why it is used against CGD. So interferons are really important for their therapeutic effect against infections, tumors and other types of disorders, however they could be toxic: INF elicit a number of unwanted effects, such as flu- L19-IL2 (darleukin) and the F16-IL2 (teleukin): in this way, IL-2 is bound to the variable portion of an antibody that is able to recognize a tumoral antigen in a specific way, so that the delivery of IL-2 will be specific (in this cases, these Abs are vascular targeting Abs) and it will induce an immune modulation on immune effector cells and stromal cells only in the site of the tumor. This expedient is much better than administering simple IL-2 without a specific target, which can cause really dangerous side effects, as we saw. Darleukin has already been tested in clinical studies in melanoma and pancreatic cancer. More recently, combination of Darleukin with other immune-cytokines or with chemotherapeutic drugs has shown potent synergistic activity in pre-clinical studies, and therefore the current clinical trial strategy is based on this concept. On the other hand, Teleukin has previously been tested in clinical studies in various solid cancers. More recently, Teleukin has shown the potential to enhance the therapeutic performance of other agents, including taxanes. We saw that this is the only way through which we can use IL-2 has a therapeutic agent without it causing multiple serious side effects. But if we change our point of view we could think of using IL-2 as a target, not as a therapy. In fact, when too much IL-2 causes damage, prevention of IL-2 is necessary. A high amount of IL-2 can be present in autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus,...), because we have an over-activation of the immune system so also IL- 2 will be produced in higher quantities, but we could also have a high level of IL-2 in organ transplantation, and we can block its production to avoid host vs graft complications. IL-2 can be blocked by administering soluble forms of the IL-2 receptor (IL-2R), we can administer a mAb towards the IL-2 receptor, or against IL-2 itself, or we could administer IL-2 variants that fail to initiate the signal transduction, or we could deliver IL-2 coupled to bacterial toxins. The last method that we mentioned can be explained through the example of Ontak (denileukin diftitox): it is a really effective drug against IL-2, it is a fusion protein produced in E. coli formed by A and T fragments of the diphtheria toxin and by IL-2. In this way, the toxin is brought in close proximity to the cell and it will bind to the cell-surface IL-2 receptor through the IL-2 portion; in this way it will be internalized through endocytosis and it will cause the death of the cell. In fact, the acidic pH in the endocytic compartment causes a conformational change that enables the translocation of the A chain of DT to the cytosol. Once in this compartment, DT modifies elongation factor 2 (EF2) by adenosine diphosphate (ADP)-ribosylation, which leads to inhibition of protein synthesis and cell death. Another important cytokine is IL-1, a family of proteins characterized by an array of 12 beta strands and by the absence of a classical secretory N-terminus peptide sequence. The two main components of this family are the IL-1alpha and the IL-1beta: the first one is generally associated with the plasma membrane of the producing cell and so it acts locally, it has a widespread production (in endothelial cells and keratinocytes), it is important in priming T cells during contact hypersensitivity and for the induction of high levels of serum IgE and its pro-domain has a nuclear localization sequence (nuclear IL- 1a has transcriptional transactivating activity); the second one is generally secreted and it circulates systemically, it is mainly produced by monocytes and macrophages and since it can circulate to the brain, it is important for the induction of fever. They are both non-glycosylated, so they could be potentially really easy to produce directly in E. coli, however therapeutically they cannot be used because they give the same problems of IL-2, so they are mainly used as targets. The two proteins signal through the same receptor complex and have identical biological activities in solution. Because of their potency and extensive functions, their activity is tightly regulated (through mRNA induction, regulation of processing and secretion, expression of a receptor antagonist and of a decoy receptor). IL- 1 proteins are usually produced as longer peptides and then they undergo maturation through digestion with caspases (caspase 1, also called ICE, IL-1-converting enzyme). Stimulation of cells for the production of IL-1 leads to the production of inactive ICE which, when activated, cleaves inactive pro-IL- 1, which is released across the cell membrane in an activated mature form. In the case of IL-1alpha, calpain cleavage of active pro-IL-1 stimulates release of mature IL-1 across the cell membrane. Some clinical studies demonstrated that no significant anti-tumoral response was observed in the treatment of various cancer with IL-1, so they are actually more harmful than useful. So instead of studying them as biotechnological drugs they have been identified as targets: there are a lot of diseases where we have a major production of IL-1 and so by reducing its amount we can ameliorate the clinical severity of these disease, which are typically acute/chronic inflammatory diseases.  example infliximab can be injected every 2 months while etanercept every month and adalimumab every week: this could be a problem for the patient, the lower is the frequency of injection per month the more compatible the therapy is for the life-style of the patients). Certolizumab pegol is a singular antibody: first of all it is PEGylated, to prolong its half-life (which, without PEGylation, would be much lower because it is a smaller antibody compared, for example, to adalimumab, so it would be digested in a faster way) and reduce its antigenicity, moreover recent studies have assessed that this mAb is not able to pass the placenta so it can also be used on pregnant women. In vitro, this antibody didn’t induce monocytes or lymphocytes apoptosis, complement activation or ADCC. In addition, along adalimumab, it is administered sub-cutaneously, which is a much easier administration route compared to the intravenous one of infliximab, because the patient can perform it even by himself without having to go to the hospital. Another particular aspect of mAb compared to the recombinant protein (Etanercept) is that antibodies (with the exception of certolizumab pegol) can form complexes because they have two Fab portions that can bind to two different molecules of TNF, meanwhile the recombinant protein only binds to a single molecule: the formation of immunocomplexes induces a higher immunogenicity, a higher clearance (through phagocytosis) and higher probability to have Fc-mediated effects. However, antibodies are able to bind to both the soluble and the membrane-bound TNF-alpha, while the recombinant proteins only binds to the soluble TNF. The ant-TNF drugs are used in inflammatory bowel diseases (like Chron’s disease), spondyloarthropathies (like psoriasis), juvenile rheumatoid arthritis and Adult Still’s disease (diseases against which they have a confirmed efficacy); we are now trying to use them also against vasculitis, scleroderma, graft vs host disease, inflammatory myositis, interstitial lung disease, Sjögren’s syndrome, inflammatory eye and ear disease, asthma, hepatitis C and sarcoidosis. Which are the disadvantages of these therapies? If TNF is removed from our body in pathogenic conditions, the pathology is reduced, but if we remove it in physiological conditions we could have some complications, like the reactivation of some infectious diseases (like Hepatitis B and tuberculosis), because TNF is produced in our body against infectious agents so if we have latent infections, we could make them reactivate if we deprive our body of TNF. Sometimes we could also have the reactivation not only of infectious diseases but also of tumors, like skin cancer. We could also have the development of autoimmune diseases (Lupus-like syndrome), demyelinating disorders, congestive heart failure and liver toxicity. Other side effects could be related to the immunogenicity of the drug that we use, especially with mAbs, and with the fact that if we interrupt the therapy, we could have the reoccurrence of the symptoms. Moreover, other disadvantages are related to the costs of the therapy (etanercept costs 15 000 euros per year per patient). [N.B. in this paragraph we talked about drugs used against autoimmune diseases but we have to remember that they cannot cure the disease, instead they are just able to ameliorate the symptoms, because autoimmune diseases are chronic: this is because they are due to genetic abnormalities which cannot be changed and so we cannot fully recover from an autoimmune disease] GROWTH FACTORS A lot of growth factors (GFs) are produced by our body (gamma-INF is also considered a growth factor because it promotes the clonal expansion of T cells), of these we have two specific haemopoietic growth factors which are erythropoietin and thrombopoietin, all the other growth factors have -GF as a suffix. We have a lot of growth factors that have been approved for medical use, of these the most important are G-CSF (granulocyte growth factors), GM-CSF (granulocyte-monocyte growth factor), platelet derived growth factor (PDGF); recently the epidermal growth factor (EGF) has been approved for the treatment of chronic wounds. Growth factors are really important for haemopoiesis, because they regulate the growth and the differentiation of the cell of the bone marrow. They are also used in vitro for the same process, so to obtain the growth of specific cell lineages.  Biosimilars of epoetins have been produced, since it was one of the first marketed recombinant protein and so its patent has expired. However, not every biosimilar of epoetins work: there have been studies where samples from Brazil, Colombia, India, Indonesia, Iran, Jordan, Korea, Lebanon, Philippines, Thailand, Venezuela, Vietnam, and Yemen have been tested against the European quality specifications for epoetin alpha, and they have pointed out how several biosimilar tested were found to be inconsistent in quality and potency and 26 out of 31 didn’t conform to all European specification for epoetin alpha. Some of them contained additional forms (basic isoforms), which reduce the clinical efficacy of the final product, moreover 2 samples contained endotoxins (safety concern) and 17 of them contained more than 2% of aggregates (concerns for immunogenicity). So, even if biosimilar versions of epoetin have been available in developing countries for many years and they are widely used for economic reasons, we have to be sure that the biosimilar that we produce is actually conform to all the safety rules of the European biosimilars, which are finely regulated. 3. Thrombopoietin (TPO): it is produced in the liver, it stimulates the production of platelets by megakaryocytes. There are some medical conditions in which platelets are not sufficiently produced and in general these conditions are all grouped under the term of thrombocytopenia (low platelet quantity); but we may need to produce platelets also for the transfusion (transfusion through blood is expensive and could lead to complications). TPO is able to induce the growth and the differentiation of the megakaryocyte progenitors and it is needed until we have the final production of platelets and their release in blood circulation from the megakaryocyte. We have two forms of recombinant TPO: one is the full molecule, it is a highly glycosylated protein, which for this reason is produced in mammalian cells, fully identical to the endogenous one; the other is a shorter PEGylated molecule made out of only the portion of the growth factor that binds to the receptor (PEGylated recombinant human megakaryocyte growth and development factor), which is produced in E. coli. The advantage of the second molecule is that it has a longer half-life because of the PEGylation. Another type of TPO for therapeutic use is the Romiplostin: it is a “peptibody”, which is made of 4 identical portions of 14 amino acids of the TPO protein bound to the Fc portion of an IgG4 molecule, kept together by disulphide bonds. It is able to bind easily to the receptor because it has a higher affinity to the receptor than the normal protein (the Fc portion is only used to create the molecule, it doesn’t have any therapeutical activity). It is used for treatment of thrombocytopenia in patients with chronic immune thrombocytopenia/idiopathic thrombocytopenic purpura (ITP) who have had an insufficient response to corticosteroids or immunoglobulins. Finally, we can say that thrombopoiesis can also be elicited by a chemical molecules, which is highly used, called Eltrombopag. Trials of a modified recombinant form, megakaryocyte growth and differentiation factor (MGDF), were stopped when healthy volunteers developed autoantibodies to endogenous TPO and then developed thrombocytopenia themselves. Romiplostim and Eltrombopag, structurally different compounds which stimulate the same pathway, are used instead. The potential clinical users of these biotech drugs are people that are affected by cancer and are undergoing chemotherapy (solid tumors or leukemia), have underwent a bone marrow transplantation, radiation therapy, anaplastic anemia or bone marrow failure states, immune thrombocytopenic purpura (ITP and thrombocytopenia of HIV, harvesting of peripheral blood progenitor cells and platelet apheresis. Side effects of thrombopoietin therapy imply thrombocytosis, thrombosis, marrow fibrosis, veno- occlusive disease, interaction with other growth factors. 4. Wound healing growth factors: a lot of growth factors generally cooperate to heal a wound alongside various types of cells (keratinocytes, fibroblast,...), but there are some pathological conditions in which this is not possible, like for example in diabetic patients, in patients affected by varicose ulcers, ulcerous cancers like the rodent ulcer, peptic ulcers and decubitus ulcers, so wounds due to continuous pressure on a particular area of the skin. The phases of wound healing involve coagulation, inflammation, migration and proliferation of the cellular component, angiogenesis, epithelization, contraction, fibroplasia and remodelling. The most important growth factors involved in this process are the FGF (fibroblast growth factor), TGF (transforming growth factor), PDGF (platelet-derived growth factor), IGF (insulin-like growth factor),  effective alternative to the recombinant factor because an antibody has a much longer half-life than the normal protein, so we can administer it once a month (or even more) and ameliorate the life-style of the patient. There are some differences between the action of the antibody and the recombinant factor, for example the recombinant factor is specifically able to bind only to the non-activated forms of factor IX and X, while the antibody is not able to distinguish between the zymogen and the enzyme. Moreover, factor VIII activated has an on-off mechanism (once it is activated it become inactive and does not perform its action until further required), instead the antibody doesn’t have an on-off mechanism. There is also a different in affinity, because factor VIII has a higher affinity to factor IX and X compared to the antibody. However, the antibody is still really used because the advantages are more than the disadvantages: if we had to choose between a protein and an antibody in terms of faster absorption, a protein should be preferred, but since we are talking about products that need to reach the blood stream and act there, we should favour a molecule with a longer half-life, and so in this case the antibody is the best option. 2. Anticoagulants: we mainly use two proteins, heparin and hirudin, however heparin is not an engineered anticoagulant but it is an extracted protein (from the pancreas of pigs or lungs of beef). Heparin is one of the most used anticoagulant protein: it is able to bind to antithrombin and enhance its activity by attaching to thrombin (factor IIa) or factor Xa, inhibiting their actions, thus preventing the coagulation cascade and stopping it (it can also inhibit factor XII, XI, IX, VIII and V). We have also a low molecular weight heparin available for the same purposes, except that it is only able to increase the action of antithrombin on factor X and not on thrombin. Heparin, although efficacious (especially in patients that underwent a surgery, to prevent the formation of thrombi), presents clinical disadvantages, including the need for a cofactor (antithrombin-AT III) and poorly predictable dose- response effects (it is unpredictable because its effect are not linked to the dose). That’s why recombinant anticoagulants have been developed: hirudin is the protein naturally produced by the leech as an anticoagulant, which is able to directly inhibit thrombin without binding to antithrombin first (it doesn’t require co-factors). Its effects depend on the dose (differently from heparin) and it is less likely to indue unintentional haemorrhages compared to the other anticoagulants that we have. Recombinant hirudin is slightly different from the natural one because the first two amino acids have been substituted (Val, Val -> Leu, Thr) and it is devoid of the sulphate groups normally present in tyrosine. It is safe and effective, it is administered intravenously and it is produced in Saccharomyces cerevisiae. It is available with two different names: in USA it is called lepirudin, while in Europe it is called desirudin. It has a short half-life but its action is more controllable than the one of heparin, so we know exactly which is the therapeutic window for each patient and we know that if we give too much hirudin we can cause bleeding (the dose is monitored and adjusted to give an aPTT ratio of 1.5-2.5 as above this range there is an increased risk of bleeding). Its clearance is mostly renal. Lately a new recombinant hirudin has been developed: it is called bivalirudin, it is a really short protein (20 amino acids), because it just contains the portion of the molecule that is able to bind to thrombin. It has the same effect as the whole molecule: its binding to thrombin (both fibrin-bound and circulating thrombin) prevents the formation of fibrin fibres so that the coagulation cascade is prevented; then the thrombin digests a piece of the recombinant protein and so, once the hirudin is removed, it can work again to form the clots and so the coagulation cascade becomes normal again (in this way, the use of the molecule is restricted for, for example, a period following a n intervention and it doesn’t last for too much time increasing the bleeding risk). Hirudin is used in cases of heparin induced thrombocytopenia (whether complicated by thrombosis or not, also in patients with cardiopulmonary bypass and vascular surgery), thrombosis prophylaxis after major orthopaedic surgery and in case of acute coronary syndrome and percutaneous coronary intervention. We could also produce recombinant antithrombin, which is a natural inhibitor of coagulation because it is physiologically able to bind to thrombin and to inhibit it, as well as to factors IXa and Xa. It is the only marketed recombinant protein produced in the milk of a goat. Its oligosaccharide composition usually varies. It is used for the treatment of hereditary and acquired anti-thrombin deficiency. Another factor that could lead to the formation of clots if absent is protein C: Xigiris is a recombinant human activated protein C produced in an engineered mammalian cell line and characterized by the presence of several gamma-carboxyglutamate and beta-hydroxylated residues. It is indicated for the DNase is an enzyme used for the treatment of cystic fibrosis (disease where we have an abnormal response of granulocytes against infections in the lungs; the lung become full of dead granulocytes that release their DNA, which is viscous and obstructs the airways). It induces the hydrolysis of long DNA chains into small fragments, it is produced in CHO cells and it is administered as an aerosol. Glucocerebrosidase is a protein approved for the treatment of Gaucher disease (lysosomal storage disease affecting lipid metabolism, specifically the degradation of glucocerebrosides), which is characterized by the lack of this enzyme. The recombinant form of glucocerebrosidase is produced in CHO cells, and an engineered form with mannose is used to facilitate the uptake by macrophages. It is the first approved biotech drug that was produced in carrots. THERAPEUTIC ANTIBODIES Antibodies are very useful as therapeutic agents, even if they have some disadvantages related to their big molecular mass, because they have multiple advantages. Antibodies were first produced in mice, by the immunization of the animal, the isolation of the antibody- forming cells from its spleen and, after the formation of hybridomas with tumoral cells and the expansion of these hybridomas in vitro, the collection of the antibodies from the cell culture. However, we already saw that mouse antibodies cannot be used because they are immunogenic. How can we produce fully human antibodies? One way is through the phage display technology. We start from the creation of a library, and it is a non-immune library: it is produced by using B lymphocytes obtained from non-immunized donors as a source of genes of antibodies. The difference with an immune library is that the immune library is obtained by cloning antibodies or antibody fragments-coding sequences derived from B lymphocytes of donors (from their spleen), previously immunized with the target antigen. We use non-immune libraries because since we have to produce fully human antibodies, we cannot immunize people to obtain our library, that’s too dangerous. So, we exploit antibodies that people already have produced in different occasions. Therefore, from the blood of donors, we isolate the B lymphocytes, we extract the RNA from these cells, and we isolate the RNA encoding for the light and heavy variable chains of the antibodies that could be produced by the lymphocyte. This isolated RNA is cloned into specific phage vectors in the form of cDNA, in frame with a surface protein. In this way, after the expression of the antibody, it will be expressed on the surface of the phage. Hence from this process we obtain a lot of different phage particles that display a different variable region of a different antibody on their surface. So eventually our library will me made out of all the variable portion of every antibody already produced by the B lymphocyte of our donors. Thanks to a technique called panning, I can isolate the phage particle carrying our variable fragment of interest simply by exposing our phage library to the antigen of interest (binding by affinity), immobilized on a surface. Then we elute the specific phage that we were able to isolate thanks to the panning technique by removing the phages that did not bind and the ones who bind un-specifically and we amplify it. Finally, we make these infect a E. coli cell line to produce a high quantity of this antibody for further analyses. The library that we produce at the beginning of this process usually comes from the antibodies of a lot of different donors, so it is very large, moreover we can add also mutations and rearrangements in vitro to increase the complexity of the library.