B B p A V P C M a b b c d e f g h a A R A A A K B F B U D h 1 B b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 ht tp : / /www.bjmicrobio l .com.br / iotechnology and Industry Microbiology iopharmaceuticals from microorganisms: from roduction to purification ngela Faustino Jozalaa, Danilo Costa Geraldesb, Louise Lacalendola Tundisib, alker de Araújo Feitosac, Carlos Alexandre Breyerd, Samuel Leite Cardosoe, riscila Gava Mazzola f, Laura de Oliveira-Nascimento f,g, arlota de Oliveira Rangel-Yagui c, Pérola de Oliveira Magalhãesh, arcos Antonio de Oliveirad, Adalberto Pessoa Jr c,∗ Universidade de Sorocaba (UNISO), Departamento de Tecnologia e Processo Ambiental, Sorocaba, SP, Brazil Universidade de Campinas (UNICAMP), Instituto de Biologia, Programa de Pós-Graduação em Biociências e Tecnologia de produtos ioativos, Campinas, SP, Brazil Universidade de São Paulo, Departamento de Bioquímica e Tecnologia Farmacêutica, São Paulo, SP, Brazil Universidade Estadual de São Paulo (UNESP), Instituto de Biociências, Campus do Litoral Paulista, SP, Brazil Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas, Brasília, DF, Brazil Universidade Estadual de Campinas, Faculdade de Ciências Farmacêuticas, Campinas, SP, Brazil Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Bioquímica e Biologia Tecidual, Campinas, SP, Brazil Universidade de Brasília, Faculdade de Ciências da Saúde, Departamento de Farmácia, Brasília, DF, Brazil r t i c l e i n f o rticle history: eceived 6 September 2016 ccepted 22 September 2016 vailable online 26 October 2016 ssociate Editor: Nelson Durán eywords: iopharmaceuticals ermentation process a b s t r a c t The use of biopharmaceuticals dates from the 19th century and within 5–10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biophar- maceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN �, �, and �) and growth hormones. The pro- duction of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals. © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is iotechnology pstream process ownstream process an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). ∗ Corresponding author. E-mail: pessoajr@usp.br (A. Pessoa Jr). ttp://dx.doi.org/10.1016/j.bjm.2016.10.007 517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by E Y-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) lsevier Editora Ltda. This is an open access article under the CC . dx.doi.org/10.1016/j.bjm.2016.10.007 http://www.bjmicrobiol.com.br/ http://crossmark.crossref.org/dialog/?doi=10.1016/j.bjm.2016.10.007&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:pessoajr@usp.br dx.doi.org/10.1016/j.bjm.2016.10.007 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ i c r o 52 b r a z i l i a n j o u r n a l o f m Introduction Biopharmaceuticals are mostly therapeutic recombinant pro- teins obtained by biotechnological processes. They are derived from biological sources such as organs and tissues, microor- ganisms, animal fluids, or genetically modified cells and organisms.1,2 Although several different expression systems may be employed including mammalian cell lines, insects, and plants, new technological advancements are contin- uously being made to improve microorganism production of biopharmaceuticals. This investment is justified by the well-characterized genomes, versatility of plasmid vectors, availability of different host strains, cost-effectiveness as com- pared with other expression systems.2,3 Bioprocessing is a crucial part of biotechnology. There is an anticipation that within the next 5 to 10 years, up to 50% of all drugs in development will be biopharmaceuticals. Examples include recombinant proteins obtained through microbial fer- mentation process.2,3 Bioprocessing for biopharmaceuticals production involves a wide range of techniques. In this review, we describe the main advances in microbial fermentation and purification process to obtain biopharmaceuticals. Biopharmaceuticals and the pharmaceutical industry Drug development is an extremely complex and expensive process. According to the Tufts Center for the Study of Drug Development4 (http://www.csdd.tufts.edu), it may take approximately 15 years of intense research from the ini- tial idea to the final product and development and costs usually exceed $2 billion. Low-molecular mass molecules are generically named as drugs while high-molecular mass drugs, which are represented by polymers of nucleotides (RNA or DNA) or amino acids (peptides and proteins), are called biopharmaceuticals.5 Biopharmaceuticals based in nucleic acids, such as small interfering RNA (siRNA), DNA vaccines, and gene therapy, are very promising strategies. However, clin- ical protocols were approved only very recently6 and just a few nucleic acids-based drugs have been therapeutically used to date7 and recent reviews addressed the state of the art of nucleic acids in therapies.8,9 In this review, we focused on pep- tides and proteins because they represent the major class of biopharmaceuticals.10 The use of proteins as drugs has been highlighted mainly by the high versatility of these biomolecules, which have dif- ferent physiological roles in the human body including as catalysts, receptors, membrane channels, macromolecule car- riers, and cellular defense agents.10,11 Some protein therapies provide high specificity, such as replacement of a patient’s defective protein or even fulfill its absence due to genetic defects or immunological complications.10 Biopharmaceuticals: reference, biosimilars, and biobetters It is worth emphasizing that the same gene product, which encodes the identical amino acid sequence, could be obtained by extraction from an animal tissue or by recombinant DNA techniques. However, the same protein produced by differ- ent manufacturers present different characteristics. In order b i o l o g y 4 7 S (2 0 1 6) 51–63 to differentiate the products, the first biopharmaceutical version of the same therapeutic protein is set as the refer- ence medicine, whereas the following ones are denominated biosimilars. Biosimilars may present differences because of post-translational modifications (phosphorylation, glyco- sylation) and different manufacturing processes. The term biobetter, also named biosuperiors, was recently used to refer to therapeutic macromolecules of the next generation, which present more effective drug delivery system, are modified by chemical methods (e.g., PEGylation) and/or engineered by means of molecular biology techniques to present better pharmacologic properties such as higher activity, enhanced stability, fewer side effects, and lower immunogenicity.12,13 Therefore, while a biosimilar represents a generic version of the original biopharmaceutical, biobetters need original research and development and the costs are significantly higher.14 Additionally, while the first biopharmaceuticals were pre- dominantly delivered by injections, biobetters adopt different approaches to drug delivery administration as oral, derma- tological and inhaled formulations which are related with different encapsulation approaches aiming to minimize the biologic instability caused by protein aggregation and dena- turation as consequence of physicochemical modifications processes of the biodrug as deamination, hydrolysis, oxi- dation, among others.15 Protein engineering and rational modification is also a very promising area in new biopharma- ceuticals and some aspects will be discussed later. The use of biopharmaceuticals has grown worldwide in the last few years. In 2016, the total number of products approved by the Foods and Drugs Administration (FDA) and European Medicines Agency (EMA) for use in humans reached 1357, of which >130 have different formulations (reference products), 737 are biosimilars, and the remaining 482 are classified as biobetters16 (http://www.biopharma.com). From 2013 to 2016, 73 biopharmaceuticals were approved for use in humans. Among them, high prominence was given to monoclonal antibodies (23 approvals) widely used in several diagnostic procedures, treatment of inflammatory diseases, and neoplas- tic tumors16 (http://www.biopharma.com). In addition, the European Medicine Agency (EMA) licensed two new products based on gene therapy (insertion of a corrective gene able to produce a normal protein in the patient’s genome to cure a genetic disease) for use in human therapeutic protocols. These products were Glybera, devel- oped by the German company UniQure for the treatment of lipoprotein lipase deficiency, and Strimvelis, developed by GlaxoSmithKline (GSK) for the treatment of adenosine deam- inase deficiency.17 Although biopharmaceuticals can be very effective for disease control or cure, treatment costs can reach up to $1 million per patient.18 Biobetters based in protein structure engineering One of the most promising areas of the biobetters relies in protein structure engineering aiming the development of bio- drugs with better pharmacological properties including higher activity, fewer side effects, and lower immunogenicity. The breakthrough in the determination of protein structures and their use as medicines dates from 1980s as a consequence http://www.csdd.tufts.edu/ http://www.biopharma.com/ http://www.biopharma.com/ c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 53 o s i e m m i s f s i r c m t t t M e a b A w a d o E c e a g e c r a l a a r U p T b U t i l m b p m a s b High amounts of target by heterologous expression 3D structure determination Structural analysis at atomic level (binding sites, aggregation paths, Rational modification by SDM Protein with superior characteristics (Biobetter) Negatively charged path Uncharged path Fig. 1 – Pipeline of protein engineering to obtain biobetters. The protein is represented by molecular surface and colorized by coulombic forces (blue = positive, red = negative, and white = neutral). b r a z i l i a n j o u r n a l o f m i f the advances in recombinant DNA technology. In turn, tructural biochemistry has revolutionized our understand- ng of protein biology and afforded the beginning of protein ngineering processes that can create protein drugs that are ore effective than wild type proteins. Protein engineering ay increase catalytic activity, stability, lower immunogenic- ty, and susceptibility to proteolytic processes.11,19–21 Protein engineering involves manipulating the protein equence at the molecular level in order to change its unction. The most common manipulations in the protein equence are base pair cuts and exchanges. However, changes n protein structure caused by oxidation or irreversible eduction of disulfides are also considered. One factor that ontributed decisively to protein engineering was the develop- ent of techniques that allow the determination of proteins hree-dimensional structure at the atomic level. Among hese techniques, more emphasis is given to X-ray crys- allography because of its high resolution (reaching < 1 Å). ore recently, nuclear magnetic resonance (NMR) and Cryo- lectron microscopy (cryo-EM) have also gained space as lternative techniques for solving structures.22 Gene manipulation (e.g., codon replacement) by molecular iology is able to modify protein structure in a specific manner. mong the several techniques used for gene manipulation, e highlight site-directed mutagenesis (SDM). This technique llows rational protein engineering based on its three- imensional structure.23,24 Using SDM, one can replace, delete, r insert one or more amino acids in the sequence of a protein. xamples include the insertion of post-translational modifi- ation sites (glycosylation, acetylation, phosphorylation, etc.), nhancement of kinetic characteristics by modification of the ctive site environment, and modification of protein aggre- ation paths.25–28 (Fig. 1) Biobetters generated by protein ngineering and gene manipulation may present superior haracteristics over the reference biopharmaceutical and rep- esents the major growing class among biopharmaceuticals. The reference recombinant protein is expressed in high mounts and the molecular structure is determined at atomic evels (crystallography or NMR). Afterwards, the protein is nalyzed using bioinformatic tools and regions of interest re identified. After gene manipulation by SDM, the modified ecombinant protein (biobetter) is obtained. pstream processing on biopharmaceuticals roduction he manufacturing technology for biopharmaceuticals can e divided into up- and downstream processes (Fig. 2). pstream process is defined as the microbial growth required o produce biopharmaceuticals or other biomolecules and nvolves a series of events including the selection of cell ine, culture media, growth parameters, and process opti- ization to achieve optimal conditions for cell growth and iopharmaceutical production. The main goal of the upstream rocess is the transformation of substrates into the desired etabolic products.29 This requires well-controlled conditions nd involves the use of large-scale bioreactors. Several factors hould be considered such as the type of process (batch, fed- atch, continuous, etc.) temperature, pH, and oxygen supply control, sterilization of materials and equipment employed, and maintenance of the environment to ensure it is free of contaminating microorganisms.30 Biopharmaceuticals produced by microorganisms Bacteria The use of protein biopharmaceuticals in human health dates from the 19th century with the use of diphtheria anti- toxin therapy.31 The antidote consists of immunoglobulins extracted from the serum of immunized animals that rec- ognize and neutralize the toxin (e.g., horse or sheep).31,32 In fact, several antitoxins are available to treat envenomation by snakes, scorpions, and wasps, or infections. However, the use of non-human animal antibodies can cause hypersensitivity of the patient to the animal serum, which is known as serum sickness.33 54 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 Culture at –80ºC Plate or Stock Flask Inoculum preparation Production bioreactor Centrifugation or filtration (cell harvesting) Precipitation and/or liquid liquid extraction Low resolution purification steps Final biophameceutical Quality control and packagingLiofilization Formulation: (Protein + Buffer + Salt + Protectants) High resolution purification steps DiafiltrationViral filtration Polishing cromatography Viral inactivationCromatography Fig. 2 – The biopharmaceutical manufacturing technology flowchart exemplifying the upstream and the downstream bioprocess. The 20th century experienced the use of several molecules coming from animal sources such as insulin, growth hormone (GH), glucagon, and asparaginase.34–36 However, the discov- ery of the prion diseases related to the administration of hGH revealed another potential risk associated with non-human animal proteins. This reinforced the need for the produc- tion of protein pharmaceuticals from other sources.37 At this time, the biopharmaceutical industry looked at heterologous expression of protein drugs by means of recombinant DNA techniques in microorganisms.38 With the advances of molecular biology and recombinant DNA, human proteins could be obtained by heterologous expression using Escherichia coli, as well as other bacteria. The classic example is human insulin, which is used to treat dia- betes mellitus types I and II (DMI and DMII). Initially, insulin was purified from the extracts of bovine and porcine pan- creas. However, the process was expensive and many cases of immune responses caused by animal insulin in patients were reported10,39 The human insulin gene was then isolated and the human protein could be obtained by heterologous expres- sion using E. coli (Fig. 3). Filamentous fungi The great diversity of molecules produced by filamentous fungi justifies the exploitation of these organisms. In par- ticular, the isolation and identification of taxol-producing endophytic fungi is a new and feasible approach to the pro- duction of this antineoplastic drug. The development and use of taxol-producing fungi have made significant progress worldwide.40 Taxol was produced by Fusarium oxysporum grown in potato dextrose broth. In addition, the filamen- tous fungus Aspergillus niger isolated from Taxus cuspidate was found to produce taxol.41 Extracellular enzymes produced by filamentous fungi have also been explored. �-d-galactosidase (lactase – EC. 3.2.1 23) is the enzyme responsible for the catalysis of lactose to glucose and galactose. Global market for lactase has been increasing significantly due to its importance in lactose intolerance treat- ment. Lactase is marketed in tablet or capsules to be used as a food supplement for individuals intolerant to lactose before the intake of milk or dairy products.42,43 Lactase also par- ticipates in the galactooligosaccharides (GOS) synthesis with applications in functional foods such as low-calorie foods and as an additive in fermented dairy products, breads, and drinks. GOS, a group of oligosaccharides, are not digestible and are beneficial to the human or animal body. The benefits of GOS ingestion arise from a population of bifidobacteria in the colon that suppress the activity of putrefactive bacteria and reduce the formation of toxic fermentation products, avoiding intesti- nal constipation and increasing the production of vitamins B complex.44,45 Another biological drug of importance in fungi is the asparaginase enzyme. This enzyme is used for the treatment of selected types of hematopoietic diseases such as acute lym- phoblastic leukemia and non-Hodgkin lymphoma. As tumor cells are dependent on the exogenous supply of asparagine for their proliferation, the presence of the drug, which depletes the bloodstream from asparagine, causes its selective death. However, the drug, which is obtained from E. coli (ELSPARTM) and Erwinia chrysanthemi, causes severe immunological reac- tions. Thus, the fungi enzyme could provide an alternative to the bacterial enzymes as an anti-tumoral agent as it presents stability and optimum pH near physiological conditions. Li et al. (2015)46 demonstrated the production of a molecule with antifungal activity against a strain of Cytospora chrysosperma by submerged fermentation in a shaker. The active compound was obtained by extraction in organic sol- vents, liquid chromatography, and thin-layer chromatography. 47 Svahn et al. (2015) produced and isolated amphotericin B by using a strain of Penicillium nalgiovense isolated from Antarc- tica. It was the first time that amphotericin B was isolated from a different organism as it is usually isolated from Streptomyces b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 55 Human insulin gene Bacterial plasmid Recombinant plasmid Bacterial transformation and heterologous expression Protein purification (recombinant insulin) Fig. 3 – Recombinant protein production. Using recombinant DNA techniques, the target human gene can be isolated and ligated to a vector (plasmid). The plasmid containing the human gene is used to transform bacterial cells, which are able to produce high amounts of the r n c d s a c l e a p u t Table 1 – Biopharmaceuticals obtained from filamentous fungi. Compound Organism Taxol Taxomyces andrenae Beta-galactosidase A. foetidus Lovastatin Monascus rubber, A. terreus l-asparaginase A. terreus Ergot alkaloids Claviceps purpurea Griseofulvin P. griseofulvum Proteases Aspergillus sp ecombinant protein. odosus. Amphotericin B also showed a minimum inhibitory oncentration of 0.125 mg/mL against Candida albicans. Collagenolytic proteases (KollagenaseTM) have been irectly used in clinical therapy, including wound healing, ciatica in herniated intervertebral discs, retained placenta, nd as a pretreatment for enhancing adenovirus-mediated ancer gene therapy.48 Another alkaline protease with col- agenolytic activity was produced by A. niger LCF9 and the nzyme hydrolyzed various collagen types without amino cid release and liberated low molecular weight peptides of 49 otential therapeutic use. Carrez et al. (1990)50 detected the presence of interleukin-6 p to 25 ng/mL in a modified strain of A. nidulans expressing he human interleukin-6. Years later, Yadwad and colleagues Penicillium sp Amphotericin B Penicillium nalgiovense (1996)51 produced approximately 54 mg/L of interleukin-6 in an air-lift fermenter with a recombinant strain of A. nidulans and a medium supplemented with salts, fructose, and threon- ine. The production of biopharmaceuticals by filamentous fungi is well studied, but the applicability of biomolecules pro- duced by such organisms is still restricted by the high cost of purification of some molecules and by difficulty in filamentous fungal cultivation (Table 1).52 Nonetheless, the use of filamen- tous fungi for the production of compounds of interest is still an interesting strategy. Downstream process: Isolation and purification of Biophamaceuticals Downstream processing includes all steps required to purify a biological product from cell culture broth to final puri- fied product. It involves multiple steps to capture the target biomolecule and to remove host cell related impurities (e.g., host cell proteins, DNA, etc.), process related impurities (e.g., buffers, leached ligands, antifoam, etc.) and product related impurities (e.g., aggregates, fragments, clipped species, etc.). Each purification step is capable of removing one or more classes of impurities.53,54 Downstream processing usually encompasses three main stages, namely (i) initial recovery (extraction or isolation), (ii) purification (removal of most contaminants), and (iii) polishing (removal of specified con- taminants and unwanted forms of the target biomolecule that may have formed during isolation and purification).53,55,56 Initial recovery involves the separation between cell and supernatant (broth clarification). For this purpose, the main operations employed are centrifugation, filtration, sedimen- tation, and flotation. If the target biomolecule is produced extracellularly, the clarified broth is submitted to concentra- tion (e.g., ultrafiltration) followed by purification. For example, secreted and soluble proteins in the culture media of P. pas- toris can be directly recovered by centrifugation. Samples can then be concentrated and the target protein purified from the supernatant by processes such as ultrafiltration, precipitation, and/or chromatography.57 For intracellular biomolecules, the cells harvested must be submitted to lysis (e.g., high-pressure homogenizer, sonication, passing through mills, etc.) followed by clarification to remove cell debris. The target biomolecule is purified from the clarified cell homogenate (usually by pre- cipitation and/or chromatography). In cases where proteins i c r o 56 b r a z i l i a n j o u r n a l o f m are expressed as inclusion bodies (as some recombinants pro- duced by E. coli), an extra step of protein refolding (buffer exchange) is required. These additional steps significantly contribute to increases in production time and costs for intra- cellular biomolecules.58 Efficient recovery and purification of biopharmaceuticals have been referred as a critical part of the production process. Purification process must be robust, reliable, easily scaled-up, and capable of removing both processes- and product-related impurities to ensure product safety. The achieved purity, the speed of process development, overall recovery yield, and throughput are some of the main key parameters that must be taken into consideration during downstream process development.55 To reach the stringency of purity required in the biopharmaceutical industry, sometimes exceeding 99%, chromatography steps are usually required. Chromatography allows for high resolution and has traditionally been the workhorse for protein purification and polishing.53,56 How- ever, chromatography has also been the major cost center in purification processes, mainly due to media cost and relatively long cycle times. In addition, the biopharmaceutical indus- try still faces practical limitations in terms of throughput and scalability.55 Chromatography Different strategies based on sequences of classical chro- matography have been described for nucleic acids, peptides, and proteins purification. In fact, chromatography is a very effective purification technique with a wide range of indus- trial applications and currently represents the favorite choice due to its high resolution capacity.56 The separation principle in chromatography is based on the differences in the affinity of the species carried by a fluid mobile phase toward a solid sta- tionary phase. When a sample is introduced and transported by the eluent along the column, some of its components will have more powerful interactions with the stationary phase than others, generating concentration profiles that will percolate the chromatographic column at different speeds. The less retained species will elute earlier from the column than the most retained ones, eventually allowing the collec- tion of the products of interest with a high purity degree.59 Based on the interaction between the solid stationary phase and biomolecules, chromatographic techniques can be sum- marized into five classes: (i) affinity, (ii) ion-exchange, (iii) hydrophobic interactions, (iv) size exclusion, and (v) mixed- mode chromatography.60 Affinity chromatography simulates and exploits natural biological processes such as molecular recognition for the selective purification of target proteins.61 This class of chro- matography is probably the only technique currently available that is capable of addressing key issues in high-throughput proteomics and scale-up.62 The most common example of an affinity process is protein-A chromatography, which has been applied for over a decade in industrial and academic settings for the capture and purification of antibodies.60 Sim- ilarly, protein-L may possibly come to play a role in antibody fragments purification.59 Another affinity-based strategy well established for recombinant proteins purification is the use of fusion tags, which are amino acid sequences attached to b i o l o g y 4 7 S (2 0 1 6) 51–63 recombinant proteins with selective and high affinities for a chemical or biological ligand immobilized on a chromato- graphic column. In particular, the polyhistidine (xHis) tag has been frequently used to purify recombinant proteins due to its binding capacity toward divalent metal cations.60 Despite the fact that affinity methods usually eliminate purification steps, increase yields, and downsize capital equipment, they do present some drawbacks, particularly regulatory ones since complete withdrawal of leached ligands is a requirement.61 Traditional choices in chromatographic set ups include particle-based resins, batch mode operation, and packed columns. In order to address the drawbacks from these standard parameters, some process alternatives are attracting the pharmaceutical industry, especially the chromatographic separations based on simulated moving bed (SMB), expanded bed adsorption (EBA), and single block monolith columns. SMB chromatography is the preferred choice for enan- tiomer separation of synthetic drugs in pharmaceutical industry. However, just recently, its use made a significant rise in biotechnology companies, especially for protein refolding and continuous downstream process.63 The system presents multiple small chromatographic columns sequentially con- nected and operated with countercurrent flow of fluids. Simulated moving comes from the periodical switch of mul- tiport inlets/outlets from column to column, in the direction of fluid flow, which gives the impression that the column bed is moving. These inlet/outlet valves (feed, desorbent, raffinate, and extract) are positioned in a way that minimize dead zones, allow desorbent recycling, optimize product recovery, and function as semi-continuous mode.64 Especially for refolding, SMB together with the recycling of aggregates lead to a the- oretical yield of 100%, excluding the folding equilibrium as a limiting factor for productivity.65 Nevertheless, SMB is more complex to implement and requires a higher investment cost. EBA chromatography is a 3-in-1 process intended to cap- ture the product directly from the cell suspension, combining clarification, concentration, and initial purification. The bot- tom feed from the EBA system creates a flow that gradually expands the resin and form a stable particle gradient.66 This gradient consists of particles of different size ranges and dif- ferent densities, which requires a narrow range of calculated flow rates. All adsorbents in direct contact with the feed- stock may bind to cells/cell debris, disrupting the gradient and reducing recovery. This issue is addressed with studies on adsorption pH to identify conditions with maximum prod- uct adsorption and minimum cell adhesion.67 Several studies have shown the value of EBA. It efficiently removes precip- itates and captures target proteins from refold pools of E. coli-based production68 and it promotes enhanced recovery of Human Epidermal Growth Factor from E. coli homogenate and Pichia pastoris culture medium.67 Particle-based resins rely on mass transfer mainly through diffusion, requiring long times for large biomolecules. On the other hand, the single block monolith column has interconnected channels that transfer mass mainly through convection, which allows for high flow velocity. In addi- tion, monolith does not have the packing step and tolerates the passage of air, reducing costs, and time with packing validation and repacking/replacing solid phase due to air inter- ruption. Other significant advantages are easy scale-up due to b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 57 Table 2 – Unit operations for continuous downstream process. Centrifugation Filtration Precipitation/crystallization Chromatography Split-bowl centrifuge Single pass tangential flow Tubular reactor Expanded bed Disk nozzle centrifuge Batch topped off Simulated moving bed fl s d w u o I i A W s i i b h g a e a r T w i S c t e t h fl Membrane cascades Diafiltration ow independent of dynamic binding and compatibility with everal organic, polymer-based, and inorganic media.69 The isadvantage of higher buffer consumption can be decreased ith the SMB set up, which can also be combined with single se technology. Monoliths are widely applied to the recovery f proteins such as coagulation factor IX (ion exchange)70 and gG (affinity chromatography)29 from a variety of cell culture ncluding P. pastoris71 and E. coli.69 lternative separation techniques ith a burgeoning biotechnology market, there is an ongoing earch for new and improved alternatives to chromatography n an effort to lower costs and improve yields, while maintain- ng high product purity.56 Several promising alternatives have een described in literature including affinity precipitation, igh-performance tangential flow filtration, filtration strate- ies based on thiophilic and affinity interactions, two-phase queous systems, high-gradient magnetic fishing, preparative lectrophoresis, and isoelectric focusing.53,55,56,58 Magnetic separation with immunocapture supports stands mong the techniques used in purification kits, but just ecently its application to industrial scale showed viable paths. he initial high costs of the beads from the kits were surpassed ith new materials and broader size dispersion and bind- ng capacity, without decreasing batch-to-batch consistency. ub-micron superparamagnetic particles of coated magnetite rystals can be functionalized according to the desired selec- ivity. These particles have been used for the purification of nzymes and inclusion bodies.29 Filtration with ion-exchange membranes substitutes flow- hrough chromatography for polishing steps. They remove ost-cell proteins, nucleic acids, and viruses with increased ow rates, reduce buffer consumption and time, when Table 3 – Examples of therapeutic native proteins obtained by p Biopharmaceutical Commercial name Botulinum toxin type A Botox Botulinum toxin type B Myoblock Collagenase Collagenase, santyl l-Asparaginase Elspar PEG-l-asparaginase Oncaspar * Adapted from Leader et al., 2008. Annular compared to traditional polishing. Hydrophobic interac- tion membranes can remove dimers and aggregates from monoclonal antibody production and substitute more chro- matography steps.72 General trends The aim of downstream process should be to deliver the highest yield of the purest product at the shortest time/cost. However, traditional processes and quality control does not bring the efficiency needed to keep pace with current upstream production. To address current issues, some general trends emerge as most relevant including single use mod- ules, continuous production, process analytical technology, and quality by design.73 The disposable units are compatible with continuous mode and bring faster routine operation because no cleaning or cleaning/validation has to be performed.73 Continuous pro- cesses generally result in higher productivity, less buffer consumption, and smaller footprint. A general end-to-end continuous process can be accomplished by perfusion cell reactors coupled with a continuous capture step, inte- grated with some of the downstream technologies described in Table 2. A recent and extensive review on continuous downstream processing of biopharmaceuticals describes and discusses each set up option in detail.64 Process consistency over time can be assured with the aid of process analytical technology (PAT) and Quality by Design (QbD) concepts described in the International Council for Har- monisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. QbD preconizes that the product is the process. Therefore, it is essential to know the critical process parameters and link them with critical mate- rial attributes to predict and adjust their impact on critical urification from natural sources. Host organism Clinical use Clostridium botulinum Several kinds of dystonia; cosmetic procedures C. botulinum Several kinds of dystonia; cosmetic procedures Clostridium histolyticum Treatment of the chronic dermal ulcers and burned areas E. coli Acute lymphocytic leukemia (ALL) E. coli Chemically modified asparaginase (PEGylated) to the ALL treatment 58 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 Table 4 – Examples of therapeutic recombinant proteins obtained by heterologous expression in E. coli. Biopharmaceutical Other commercial names Clinical use Aldesleukin (interleukin-2) Proleukin Melanoma and renal cancer treatment Anakinra (interleukin 1 (IL1) receptor antagonist) Antril, Kineret Rheumatoid arthritis treatment Calcitonin (salmon calcitonin) Fortical Post menopausal osteoporosis treatment Denileukin diftitox (interleukin-2 and Diphtheria toxin fusioned) Ontak T-cell lymphoma treatment Filgrastim (analog to the granulocyte colony-stimulating factor) Neupogen Neutropenia treatment (as consequence of AIDS, chemotherapy, bone-among others) Filgrastim pegylated Neulasta Neutropenia treatment (as consequence of AIDS, chemotherapy, bone- marrow transplantation, among others) Growth hormone (GH) Genotropin, Humatrope, Norditropin, Norivitropin, Nutropin, Omnitrope, Protropin, Siazen, Serostim, Valtropin Prader-Willi and Turner syndromes Glucagon Glucagon Hypoglycemia Glucarpidase (bacterial carboxypeptidase G2) Voraxaze Control of methotrexate conc. in patients with deficient renal function Insulin (inhalation) Exubera Diabetes mellitus treatment Insulin (fast-acting) Lispro Diabetes Insulin (zinc extended) Lente, Ultralente Diabetes mellitus treatment Interferon-�2a Roferon-A Chronic hepatitis C. chronic myelogenous leukemia, hairy cell leukemia, Kaposi’s sarcoma Interferon-�2b Intron A Chronic hepatitis C. chronic myelogenous leukemia, hairy cell leukemia, Kaposi’s sarcoma Interferon-�2b pegylated Peg-intron Chronic hepatitis C. chronic myelogenous leukemia, hairy cell leukemia, Kaposi’s sarcoma Interferon-�1b Betaseron Multiple sclerosis Interferon-�1b Actimmune Chronic granulomatous disease, severe osteopetrosis Mecasermin (insulin-like growth factor 1) Increlex GH and IGF1 deficiencies Mecasermin rinfabate (insulin-like growth factor I and its binding protein IGFBP-3) iPlex GH and IGF1 deficiencies Nesiritide (B-type natriuretic peptide) Natrecor Acute decompensated heart failure (ADHF) treatment Oprelvekin (interleukin 11) Neumega Prevention of severe thrombocytopenia (patients in chemotherapy) OspA (Outer surface protein A fragment from Borrelia burgdorferi) LYMerix Lyme disease vaccine Palifermin (truncade keratinocyte growth factor) Kepivance Treatment of oral mucositis in (patients undergoing chemotherapy) Parathyroid hormone Preos, Preotact Treatment of osteoporosis and hypoparathyroidism Pegvisomant, modified GH (prevent GH binfing to receptor) Somavert Acromegaly treatment Ranibizumab (Mab fragment) Lucentis Age related macular degeneration Reteplase (plasminogen activator) Rapilysi Acute myocardial infarction treatment Somatropin, tasonermin Humatrope hGH deficiency treatment Tasonermin (cytokine) Beromun Soft sarcoma treatment Urate oxidase, PEGylated Krystexxal Gout Teriparatide. Parathyroid hormone Forteo Severe osteoporosis treatment The data were obtained from manufacturer pages and from16 http://www.biopharma.com. http://www.biopharma.com/ c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 59 q u g d t e t m o a t G b I t m i a ( m e r c ( n o 30 25 20 15 10 5 0 2005 2006 2007 To ta l n um be r of p ro du ct s 2008 2009 2010 2011 2012 2013 2014 2015 Monoclonal antibody related products Recombinant proteins Fig. 4 – Commercial biopharmaceutical products approved from 2005 to 2015. Dark green bars represent monoclonal antibody related products and non-related total recombinant proteins are represented in red. The data used concerning the number of biopharmaceutical approvals are available at biopharma biopharmaceutical products16 (http://www.biopharma.com/approvals). b r a z i l i a n j o u r n a l o f m i uality attributes of the final product. Processes developed nder QbD knowledge contain design spaces instead of sin- le value or extremely narrow parameters; values inside the esign space results in good product performance and brings he necessary flexibility to continuous processing.74 How- ver, knowledge of the process requires process analytical echnology (PAT) tools that include analytical chemistry and athematical and statistical modeling/analysis. Among the ptions, near infrared spectroscopy and principal component nalysis are trending choices for analytical and mathematical ools, which can be applied to several steps.75 lobal consumer market of microbial iopharmaceuticals n 1982, human insulin was the first recombinant protein hat was FDA approved for use in humans as a biophar- aceutical product.10,39 In the 1980s, the biopharmaceutical ndustry experienced a significant growth in the production nd approval of recombinant proteins including interferons IFN �, �, and �) and growth hormones. In the 1990s, the first onoclonal antibodies (MAb) and related products experi- nced an extraordinary growth, and in 2015, these products epresented two-thirds of the products approved for commer- ial use in the world according to the Biotrack database76 Fig. 4). Currently, the total market sales from microbial recombi- ant products reached approximately $50 billion, representing ne-third of the total sales of biopharmaceuticals. The choice Table 5 – Examples of therapeutic recombinant proteins obtain Biopharmaceutical Comm Albumin Recombumin Hepatitis B surface antigen Engerix, Fendrix Recombivax HB Hepatitis B surface antigen and hepatitis A virus inactibated Ambirix, Twinrix Hirudine Refludan, Revas HPV vaccine Gardasil HPV surface antigens Silgard Glucagon like peptide 1, Liraglutide Victoza Insulin Humulin, Novol Mixtard, Insulat Actraphane, Insulin aspart; insulin glulisine; insulin lispro (fast-acting insulin analog) Novolog (aspart) Humalog (lispro Insulin detemir (long-acting insulin) Levemir Isophane insulin (intermediate -acting insulin) Humulin N Platelet Derived Growth Factor-BB Regranex Parathyroid hormone Preos, Preotact Rasburicase Ranibizumab, Fa Somatropin (GH) Valtropin Sargramostim Leukine The data were obtained from manufacturer pages and from16 http://www. of microorganism in the production of biopharmaceuticals relies on many factors including low cost production, easy manipulation, and propagation, and molecular biology meth- ods. Some of the most important biopharmaceuticals obtained by natural sources or by heterologous expression are shown in Tables 3–5. ed by heterologous expression in S. cerevisiae. ercial name Clinical use Manufacture of human therapeutics Hepatitis B vaccine Hepatitis A and B vaccine c Anticoagulant HPV vaccine HPV vaccine Diabetes mellitus treatment in, Protaphane, ard, Actrapid, Diabetes mellitus treatment , Apidra (glulisine), ) Diabetes mellitus Diabetes mellitus Diabetes mellitus Treatment of neuropathic, chronic, diabetic ulcer Treatment of osteoporosis and hypoparathyroidism sturtec Treatment of leukemia, lymphoma and tumor lysis syndrome GH deficiency treatment Neutropenia treatment (as consequence of AIDS, chemotherapy, bone- marrow transplantation, among others) biopharma.com. http://www.biopharma.com/approvals 60 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 S (2 0 1 6) 51–63 Table 6 – Top-10 biopharmaceuticals based on sales revenues in 2015. According to Gal77 and Igea78. Rank Product® Product type Production system Company Use 2015 sales (US$ million) Patent expiry U.S.A E.U. 01 Humira (Adalimumab) Anti-TNF� MAb CHO AbbVie (U.S.) Inflammatory diseases 14,021 2016 2018 02 Enbrel (Etanercept) Anti- TNF� MAb CHO Amgen (U.S.) Pfizer (U.S.) Takeda Pharm. (Japan) Inflammatory diseases 9027 2028a 2015 03 Remicade (Infliximab) Anti- TNF� MAb SP2/0 Johnson & Johnson (U.S.) Merck (U.S.) Mitsubishi T. (Japan) Inflammatory diseases 8957 2018 2014 04 Lantus (Insulin glargine) Insulin analog E. coli Sanofi (France) Diabetes 7209 2014 2014 05 Avastin (Bevacizumab) Anti-VEGF MAb CHO Roche (Switzerland) Cancer 6905 2019 2022 06 Herceptin (Trastuzumab) Anti-HER2 MAb CHO Roche (Switzerland) Cancer 6754 2019 2015 07 Prevnar family Polysaccharides conjugated to diphtheria protein Streptococcus pneumoniae and Corynebacterium diphtheriae Pfizer (U.S.) Pneumococcal vaccine 6245 2026 n.a. 08 MabThera/Rituxan (Rituximab) Anti-CD20 MAb CHO Roche (Switzerland) Cancer and autoimmune diseases 5827 2015 2013 09 Neulasta (PEGfilgrastim) Recombinant G-CSF E. coli Amgen (U.S.) Cancer- related infections 4715 2015 2017 10 Lucentis (Ranibizumab) Anti-VEGF FAb E. coli Novartis (Switzerland) Roche (Switzerland) Macular degeneration 3630 2020 2022 a The main patent on Enbrel (Etanercept) was originally expected to expire on October 2012, but owing to a filing loophole, Amgen secured an additional 16-year period of exclusivity. n.a., data not available. CHO, Chinese Hamster Ovary mammalian cell. SP2/0, Mouse myeloma cells. c r o b c r i T c E m t g e c t T c f n r i i m m d a C N i p i i o C T r b r a z i l i a n j o u r n a l o f m i Biopharmaceuticals are revolutionary in the pharmaceuti- al industry. According to global revenues, 10 biotechnological elated products figured among the top-25 best-selling drugs n 2015; 4 of them produced by microorganisms77,78 (Table 6). hese biopharmaceuticals are marketed by leading pharma- eutical companies primarily located in U.S.A, Japan, and urope and comprise a narrow scope of treatment profile, with ost drugs for the treatment and management of inflamma- ory diseases (e.g. rheumatoid arthritis) and cancer. Patents for cloning and production of several original- eneration (branded) biopharmaceuticals have expired or will xpire within the next years (Table 6). Similar to chemi- al drugs, once the patent of a biological product is expired he marketing of biosimilars and generics is possible.79 hese patent expirations, combined with rising healthcare osts and population aging worldwide are paving the way or the development of biosimilars and biobetters, opening ew commercial opportunities.80,81 Many biosimilars are cur- ently under development and these follow-on products will nevitably play substantial competition and an increasing role n healthcare in upcoming years.79,82 In Brazil the scenario is odest, but considering the global panel and recent govern- ent incentive for the national biopharmaceutical industry evelopment, we expect to see more patents in the near future nd also novel opportunities for biosimilars and biobetters. onclusion and future trends ew technological advancements are continuously made to mprove the discovery, rational modification, production, and urification of biopharmaceuticals. 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