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.bjmicrobiol.com.br/ Biotechnology and Industry Microbiology Biopharmaceuticals from microorganisms: from production to purification a b b Angela Faustino Jozala , Danilo Costa Geraldes , Louise Lacalendola Tundisi , c d e Valker de Araújo Feitosa , Carlos Alexandre Breyer , Samuel Leite Cardoso , f f,g Priscila Gava Mazzola , Laura de Oliveira-Nascimento , c h Carlota de Oliveira Rangel-Yagui , Pérola de Oliveira Magalhães , d c,∗ Marcos Antonio de Oliveira , Adalberto Pessoa Jr a Universidade de Sorocaba (UNISO), Departamento de Tecnologia e Processo Ambiental, Sorocaba, SP, Brazil b Universidade de Campinas (UNICAMP), Instituto de Biologia, Programa de Pós-Graduac¸ão em Biociências e Tecnologia de produtos bioativos, Campinas, SP, Brazil c Universidade de São Paulo, Departamento de Bioquímica e Tecnologia Farmacêutica, São Paulo, SP, Brazil d Universidade Estadual de São Paulo (UNESP), Instituto de Biociências, Campus do Litoral Paulista, SP, Brazil e Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduac¸ão em Ciências Farmacêuticas, Brasília, DF, Brazil f Universidade Estadual de Campinas, Faculdade de Ciências Farmacêuticas, Campinas, SP, Brazil g Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Bioquímica e Biologia Tecidual, Campinas, SP, Brazil h Universidade de Brasília, Faculdade de Ciências da Saúde, Departamento de Farmácia, Brasília, DF, Brazil a r t i c l e i n f o a b s t r a c t Article history: The use of biopharmaceuticals dates from the 19th century and within 5–10 years, up to Received 6 September 2016 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biophar- Accepted 22 September 2016 maceutical industry experienced a significant growth in the production and approval of Available online 26 October 2016 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 Associate Editor: Nelson Durán 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 Keywords: biopharmaceuticals. Biopharmaceuticals © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is Fermentation process an open access article under the CC BY-NC-ND license (http://creativecommons.org/ Biotechnology licenses/by-nc-nd/4.0/). Upstream process Downstream process ∗ Corresponding author. E-mail: [email protected] (A. Pessoa Jr). http://dx.doi.org/10.1016/j.bjm.2016.10.007 1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 52 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 to differentiate the products, the first biopharmaceutical Introduction version of the same therapeutic protein is set as the refer- ence medicine, whereas the following ones are denominated Biopharmaceuticals are mostly therapeutic recombinant pro- biosimilars. Biosimilars may present differences because teins obtained by biotechnological processes. They are derived of post-translational modifications (phosphorylation, glyco- from biological sources such as organs and tissues, microor- sylation) and different manufacturing processes. The term ganisms, animal fluids, or genetically modified cells and biobetter, also named biosuperiors, was recently used to refer 1,2 organisms. Although several different expression systems to therapeutic macromolecules of the next generation, which may be employed including mammalian cell lines, insects, present more effective drug delivery system, are modified and plants, new technological advancements are contin- by chemical methods (e.g., PEGylation) and/or engineered uously being made to improve microorganism production by means of molecular biology techniques to present better of biopharmaceuticals. This investment is justified by the pharmacologic properties such as higher activity, enhanced well-characterized genomes, versatility of plasmid vectors, 12,13 stability, fewer side effects, and lower immunogenicity. availability of different host strains, cost-effectiveness as com- Therefore, while a biosimilar represents a generic version 2,3 pared with other expression systems. of the original biopharmaceutical, biobetters need original Bioprocessing is a crucial part of biotechnology. There is an research and development and the costs are significantly anticipation that within the next 5 to 10 years, up to 50% of all higher.14 drugs in development will be biopharmaceuticals. Examples Additionally, while the first biopharmaceuticals were pre- include recombinant proteins obtained through microbial fer- dominantly delivered by injections, biobetters adopt different 2,3 mentation process. Bioprocessing for biopharmaceuticals approaches to drug delivery administration as oral, derma- production involves a wide range of techniques. In this review, tological and inhaled formulations which are related with we describe the main advances in microbial fermentation and different encapsulation approaches aiming to minimize the purification process to obtain biopharmaceuticals. biologic instability caused by protein aggregation and dena- turation as consequence of physicochemical modifications Biopharmaceuticals and the pharmaceutical industry processes of the biodrug as deamination, hydrolysis, oxi- 15 dation, among others. Protein engineering and rational Drug development is an extremely complex and expensive modification is also a very promising area in new biopharma- process. According to the Tufts Center for the Study of ceuticals and some aspects will be discussed later. 4 Drug Development (http://www.csdd.tufts.edu), it may take The use of biopharmaceuticals has grown worldwide in the approximately 15 years of intense research from the ini- last few years. In 2016, the total number of products approved tial idea to the final product and development and costs by the Foods and Drugs Administration (FDA) and European usually exceed $2 billion. Low-molecular mass molecules Medicines Agency (EMA) for use in humans reached 1357, of are generically named as drugs while high-molecular mass which >130 have different formulations (reference products), drugs, which are represented by polymers of nucleotides (RNA 737 are biosimilars, and the remaining 482 are classified as or DNA) or amino acids (peptides and proteins), are called 16 biobetters (http://www.biopharma.com). From 2013 to 2016, 5 biopharmaceuticals. Biopharmaceuticals based in nucleic 73 biopharmaceuticals were approved for use in humans. acids, such as small interfering RNA (siRNA), DNA vaccines, Among them, high prominence was given to monoclonal and gene therapy, are very promising strategies. However, clin- antibodies (23 approvals) widely used in several diagnostic 6 ical protocols were approved only very recently and just a procedures, treatment of inflammatory diseases, and neoplas- few nucleic acids-based drugs have been therapeutically used 16 tic tumors (http://www.biopharma.com). 7 to date and recent reviews addressed the state of the art of In addition, the European Medicine Agency (EMA) licensed 8,9 nucleic acids in therapies. In this review, we focused on pep- two new products based on gene therapy (insertion of a tides and proteins because they represent the major class of corrective gene able to produce a normal protein in the biopharmaceuticals.10 patient’s genome to cure a genetic disease) for use in human The use of proteins as drugs has been highlighted mainly therapeutic protocols. These products were Glybera, devel- by the high versatility of these biomolecules, which have dif- oped by the German company UniQure for the treatment ferent physiological roles in the human body including as of lipoprotein lipase deficiency, and Strimvelis, developed by catalysts, receptors, membrane channels, macromolecule car- GlaxoSmithKline (GSK) for the treatment of adenosine deam- 10,11 riers, and cellular defense agents. Some protein therapies 17 inase deficiency. Although biopharmaceuticals can be very provide high specificity, such as replacement of a patient’s effective for disease control or cure, treatment costs can reach defective protein or even fulfill its absence due to genetic 18 up to $1 million per patient. 10 defects or immunological complications. Biobetters based in protein structure engineering Biopharmaceuticals: reference, biosimilars, and biobetters One of the most promising areas of the biobetters relies in It is worth emphasizing that the same gene product, which protein structure engineering aiming the development of bio- encodes the identical amino acid sequence, could be obtained drugs with better pharmacological properties including higher by extraction from an animal tissue or by recombinant DNA activity, fewer side effects, and lower immunogenicity. The techniques. However, the same protein produced by differ- breakthrough in the determination of protein structures and ent manufacturers present different characteristics. In order their use as medicines dates from 1980s as a consequence 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 53 of the advances in recombinant DNA technology. In turn, High amounts of target