EDUARDO CESARPurification of monoclonal antibodies at EDUARDO CESAR
It was hard for biochemist Fernanda Alvarez Rojas to conduct experiments and conclude her doctoral program in medical sciences at the State University of Campinas (Unicamp) from 1998 and 2001. The researcher focused on the study of animal models for laboratory use in research projects on resistance to insulin. The study included experiments with the use of antibodies, essential components of diagnosis kits and new medical drugs. The antibodies are basically molecules produced by the immune system to battle diseases and have been used in scientific work to determine the characteristics of the antigens, the particles in the invading cells that induce the body’s immune response. The problem was that this product was not made in Brazil, and had to be – and still is – imported. This was the problem for Fernanda – and for many other Brazilian researchers. “We had to order the antibodies and it took a long time for them to get here; this delayed the research projects. In addition, upon arrival, many of them were outdated or damaged because of packaging problems during the transportation,” Fernanda recalls. “At one point, I conducted 50 experiments and only four were successful. It was a huge waste of time and money.” Episodes such as this led Fernanda, after she had concluded her graduate course, to consider producing the antibodies for her research projects and for those of her colleagues from the academic community.
In 2004, the biochemist partnered with two colleagues – nurse Eliana Araújo and pharmacist Alessandra Gasparetti – to set up Imuny Biotechnology, a company based in the city of Campinas, State of São Paulo, to produce and sell antibodies. The company was granted funds by FAPESP, through the Innovative Research in Small Enterprises Program (Pipe). Four years later, Fernanda and biologist Luís Antônio Peroni founded Rheabiotech, a spin-off of Imuny, to produce diagnosis kits; the parent company became responsible for the commercial operations of biotechnological inputs. Once again, the researchers obtained funds from Pipe, this time to develop kits for the agribusiness industry.
Soja Detecta was one of the first products to be produced by Rheabiotech. This product is used for the early detection of Asian soybean rust, a disease caused by the Phakopsora pachyrhizi fungus, which generates huge losses for soybean farmers. Soja Detecta was developed with the technical support of Bayer CropScience, the company that supplied infected soyplant leaves for lab tests. The product is undergoing field validation tests. The kit uses a serum with antibodies able to detect the fungus in the plant. To this end, it is necessary to macerate the soybean leaves and apply them to a special paper. This paper, with the samples of the leaves, is then placed in contact with reagents containing the antibodies. The paper turns red if the macerated leaves have been contaminated by the fungus.
Rheabiotech, which has already produced 300 different antibodies, earned R$30 thousand last year, even before implementing a commercial strategy. The company expects to increase its revenues to R$150 thousand this year. To this end, it is focusing strongly on new product development, including a kit that it is developing jointly with Empresa Brasileira de Pesquisa Agropecuária (Embrapa), to detect hog salmonellosis in slaughterhouses; two kits – one for the citrus fruit industry and the other for the diagnosis and prognosis of autoimmune and inflammatory diseases and some types of cancer – are also being developed by the company. This second kit has been given the financial support of the 2009 Subvention Program of Finep, the Studies and Projects Funding Agency of the Ministry of Science and Technology (MCT).
Two years after it was founded, Rheabiotech joined the small and select group of technology-based companies specializing in the production of antibodies, diagnosis kits and other biotechnological inputs for basic research, for the detection of diseases in humans, animals and plants. “The domestic market depends heavily on imported antibodies. I believe that 99% of these inputs – essential for the diagnosis of diseases and for scientific research, come from abroad,” says physician Fernando Kreutz, a partner in FK Biotecnologia, a pioneering company in the State of Rio Grande do Sul. He says that the domestic market for diagnosis reagents, which includes the antibodies used in research and in the detection of diseases, is a market worth about R$1.2 billion, while the market for therapeutic use antibodies totaled R$1.8 billion in 2009. “It is strategic for Brazil to have a sound industry for the production of these inputs and thus reduce its strong dependency on imported antibodies and diagnosis kits,” says Kreutz.
Therapeutic use
Established in 1999, FK Biotecnologia, whose main place of business is in Porto Alegre, the Rio Grande do Sul state capital, has already placed more than 150 monoclonal antibodies in the market, for the purpose of academic research. The company invests in the research and development of antibodies of therapeutic use for cancer treatment. These are specifically developed antibodies for the treatment of diseases. This market is still immature, and approximately 20 antibodies have already been approved for therapeutic use by the Food and Drug Administration (FDA), the United States regulatory agency for the medication sector. The industry’s revenues amount to roughly US$15 billion. Almost half of this comes from the sale of eight antibodies for cancer treatment. According to the Ministry of Health, Brazil spent R$389.8 million in 2009 on only four such antibodies. São Paulo’s Recepta Biopharma (see Pesquisa FAPESP issue 137) is another Brazilian company involved in this field of business.
EDUARDO CESARBioreactor for the production of monoclonal antibodiesEDUARDO CESAR
FK also invests in kits for diagnosis, another segment that also uses antibodies as its raw material. At the beginning of 2010, FK, in partnership with Lifemed, a company based in São Paulo, registered four new products with Anvisa, the National Sanitary Surveillance Agency, which is the Brazilian pharmaceutical industry’s regulatory body. The company intends to launch three HCG diagnosis kits in the first half of this year. HCG stands for human chronic gonadotropin, a hormone used in pregnancy tests. The kits contain monoclonal antibodies that detect the HCG secreted in great quantities at the start of pregnancy. With these new kits, the company expects to increase its revenue to R$10 million, a five-fold increase versus 2009. “Until last year, our production was limited to antibodies for research purposes. From 2010 on, we plan to move forward significantly in terms of the diagnosis kits. We are now ready to compete with the major global suppliers,” says Kreutz. The new kits will be sold in drugstores as well as exported to Africa and the Middle East.
Biologist and immunologist Sandro Gomes Soares, executive director of Invent Biotecnologia, a company installed at Supera, an incubator for technological companies on the Ribeirão Preto campus of the University of São Paulo (USP) in São Paulo State, agrees with Kreutz. He says that Brazil must learn to produce antibodies on a large scale and invest in research and development, in order to create a new generation of antibodies with the application of recombinant DNA techniques. In this technique, a gene that produces a given antibody is inserted or coded in the genome of an organism by means of biotechnological processes. “We need to move towards innovative products faster than the Brazilian pharmaceutical companies have,” says Soares.
Physiological response
Antibodies are also used as conventional biotechnological tools in Elisa and Western Blot tests, to quantify and locate proteins of interest to researchers. From the physiological point of view, antibodies are protein molecules produced in response to the entry of foreign substances into the organism, such as viruses, bacteria or tumor cells. Produced and secreted by lymphocyte B cells (the cells of our immune system), antibodies can recognize a specific target “the antigen” that exists in the cells of the invading substance. Each antibody contains two paratopes, which bind to the epitope, a specific part of the antigen. In figurative language, the antibody is the key and the antigen is the lock. When the antibody “plugs” into the antigen, an immunological action begins to neutralize the invading body. The testing of these mechanisms is fundamental for biotechnological research.
Antibodies can be divided into monoclonal and polyclonal antibodies. Monoclonal antibodies are produced in laboratories from a cell population derived from a single B lymphocyte, the cell of the immunological system that makes all antibodies have a single specificity. They can be produced continuously and react only against a single epitope of the antigen. Polyclonal antibodies are produced in mammals – rabbits, mice, horses, cattle, sheep – or in birds – usually, hens. When an animal is immunized, it produces a wide variety of antibodies that act against different epitopes of the antigen, because these antibodies originated from different lymphocyte B cells. The process to produce polyclonal cells is relatively simple and low cost – one milliliter of polyclonal antibodies costs around R$800, and 150 micrograms cost R$1,500. On the other hand, the use of polyclonal antibodies is limited to some procedures only, as in diagnosis kits and as an academic research tool.
Monoclonal antibodies are made using more refined techniques, require a longer period of time for conclusion and are expensive to produce. They have broader applications, and can also be used for therapeutic purposes. “Because they are more specific, monoclonal antibodies are used preferably in research projects for diagnoses and therapies that are efficient for certain pathologies,” explains Fernanda. “However, because of their ability to recognize and bind with specific antigens – protein, glycoprotein, lipoprotein, carbohydrates, nucleic acids and lipids – they can also be used efficiently in research studies on proteins by means of immunological assays.”
Unlike Rheabiotech, which intends to become a large-scale producer of antibodies, Célula B is an extension service of the Federal University of Rio Grande do Sul (UFRGS). Célula B specializes in producing monoclonal antibodies and polyclonal serums in rabbits, mice and hens ordered by its clients, mainly researchers from universities and research centers, such as Unicamp, Embrapa and the Heart Institute of the Hospital das Clínicas teaching hospital of the University of São Paulo Medical School. “Our objective is not to produce antibodies on an industrial scale, but rather to develop small quantities of commercially unavailable serums, needed by researchers working in the characterization of a new protein that has no antibodies. The user provides us with the protein and we produce the antibody on a small scale to meet the researcher’s needs,” says veterinarian Itabajara da Silva Vaz Júnior, coordinator of Célula B.
The antibodies produced by UFRGS have been used to conduct research in oncology, biochemistry, immunology, molecular genetics, molecular biology, cytology, histology, and physiology. “We can apply an antibody that we know will react against a given protein, in cell culture, animal tissue or even on the entire animal, and observe its effect on physiology. Does the antibody block the function? Does it block growth? In addition, our antibodies can also be used to test the existence of a given protein in the different tissues of an animal, and thus show in which tissues or organs this protein is expressed,” he emphasizes. The service provided by Célula B began in 2003, following a Finep public notice, the aim of which was to fund research projects and the production of monoclonal and polyclonal antibodies. The service also evaluates reagents and immunological components and provides a training program for technicians from research institutes and companies.
The Bio-Manguinhos monoclonal antibodies laboratory of the Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro was one of the first government institutions to produce antibodies. “Since 1983, the lab has developed monoclonal antibodies for research into and diagnosis of specific antigens that are of interest to public health authorities, such as hepatitis A, B and C, leptospirosis and yellow fever,” says Márcia Arissawa, the laboratory’s manager. She says that a new wing was recently built to house the newly-equipped lab, because of the increasing demand for monoclonal antibodies and the search for new areas in which these components can be used. The new facility includes special rooms with a very low concentration of particles in suspension that can be used to produce batches of monoclonal antibodies.
PROTEIMAXAntibodies – Packaging for the domestic and foreign markets PROTEIMAX
Essential purification
Invent Biotecnologica, which also enjoys funding provided by FAPESP’s Pipe Program, focuses on strategies for the production and development of molecules and vectors for therapeutic purposes and for the production of antibodies. “The creation of a new strategy for the purification of polyclonal antibodies in birds is at an advanced stage of development at the company,” says Sandro Soares. The advantage of using birds instead of mammals for the production of antibodies is well known. It is possible to extract antibodies from eggs in much higher quantities than from serum. The extraction is non-invasive and barely affects the animal. According to Soares, a bird can produce 30 grams of antibodies a year, whereas a rabbit produces only 2.5 grams.
However, the production of antibodies in birds is hampered by the existence of lipid residues (fats) that come from the egg yolks, where the antibodies are concentrated, when conventional purification techniques are applied. “Invent has focused its efforts on developing a purification strategy with a high recovery rate and purity. As a result, we developed a low-cost methodology and a scalable process for production in industry.” The company is now working on the development of a commercial kit for the purification of antibodies and on the creation of a services sector to meet the needs of the academic community. “We are focusing on the scaling of antibody purification and protein production processes. We will not be as competitive as the international companies unless we learn to conduct the production process on an industrial scale,” says Soares.
Bioaptus, a company based in the State of Minas Gerais, was created in 2009. Despite such a short existence, it is already enjoying the results of the launching of Anfitech, the commercial name of a synthetic antibody, comprised of an organic molecule that the company developed without using cells or immunizing animals. This synthetic antibody, developed for the diagnosis and treatment of diseases, is already being widely used by many clients from the agriculture, animal husbandry, human and veterinary pharmaceutical products, and laboratory medicine sectors. “Anfitech was developed to have high affinity with and specificity for a given molecule. The product is an innovation for bioscience research. A researcher can order a specific Anfitech antibody for a given protein of his line of research,” says Luiz Augusto Pinto, one of the partners in the company, whose client list includes the Federal University of Minas Gerais (UFMG), the Nuclear Technology Center (CDTN) of the Ministry of Science and Technology, and the Oswaldo Cruz Foundation.
For the industrial sector, Bioaptus offers solutions for the development of analytical products and methods. The company’s main clients in this line of business include producers of in vitro diagnosis kits for lab medicine and vaccines for humans and animals. Installed at Inova, the company incubator at UFMG, Bioaptus’ revenues last year amounted to R$200 thousand. The company expects to increase its revenues by 300% in 2010. Next year, the goal is to achieve revenues of R$4 million from Anfitech sales. According to Luiz Augusto Pinto, Anfitech is an alternative to the antibodies currently being imported from abroad. The company has already begun negotiations to close business deals with interested parties in the United States and Europe.
The foreign market is a major source of revenue for Proteimax, a biotechnology company based in the town of Cotia, in the Greater São Paulo. Twenty two percent of its R$200 thousand revenue in 2009 came from the sale to the foreign market of polyclonal antibodies produced in rabbits, rats, mice and guinea pigs. “Our main line of research focuses on a class of proteins called receptors, coupled to the G protein, the acronym of which is GPCR. A total of 70 antibodies are the base of our platform for the identification of new compounds with therapeutic potential,” says molecular research Andrea Sterman Heimann, a director of the company, which is also involved in two projects funded by Pipe. Many medical drugs act by activating or blocking these receptors. The G proteins provide communication between the extracellular medium and the intracellular one, allowing a pharmaceutical product to produce – or not – an effect on a given organ of the body. At present, approximately 50% of the most widely used medical drugs in the world act directly or indirectly by activating or blocking GPCR receptors.
Andrea agrees with the idea of encouraging the rise of a strong Brazilian industry centered on the production of inputs for research purposes, but she feels that this industry should not aim at substituting imports, but rather on the development of innovative and competitive products in the global market. She says that the market for antibodies for research purposes is not big enough to support Brazilian companies in this line of business. Moreover, several of these inputs are produced by foreign companies in huge quantities to meet the needs of the global market, which prevents small companies from being competitive. “Proteimax was gaining the market share of a specific antibody in Brazil and the foreign competitor lowered the price of its products to block our progress. The competitor was able to do this because its products are sold to buyers throughout the world. This is why I think the best strategy is to beat competition by being innovative,” she adds.
The Projects
1. Production of polyclonal antibodies (nº 03/13387-9); Type Program of Innovative Research in Small Companies (Pipe); Coordinator Fernanda Alvarez Rojas – Rheabiotech; Investment R$ 386,454.14 and US$ 17,030.05 (FAPESP)
2. Development of diagnosis kits for phytopathogens that are important for agriculture (nº 08/53621-4); Type Program of Innovative Research in Small Companies (Pipe); Coordinator Luís Antônio Peroni – Rheabiotech; Investment R$ 233,358.98 and US$ 60,833.30 (FAPESP)
3. Pilot scale production and purification of polyclonal antibodies derived from chicken eggs (nº 05/00705-8); Type Program of Innovative Research in Small Companies (Pipe); Coordinator Sandro Gomes Soares – Invent; Investment R$ 145,713.69 and US$ 94,578.48 (FAPESP)
4. Specific formation antibodies: proposal for the generation of guided antibodies and protein G-coupled receptors (GPCRS) (nº 04/14258-0); Type Program of Innovative Research in Small Companies (Pipe); Coordinator Andrea Sterman Heimann – Proteimax; Investment R$ 186,485.31 and US$ 139,159.44 (FAPESP)
5. New molecular strategy for cancer diagnosis: specific formation antibodies (nº 08/01470-2); Type Program of Innovative Research in Small Companies (Pipe); Coordinator Andrea Sterman Heimann – Proteimax; Investment R$35,608.00 and US$12,800.00 (FAPESP)
