If the next stages in the study conducted by molecular biologist, Dulce Elena Casarini, progress as planned, the first kit for the preventive diagnosis of hypertension taken from a urine exam is likely to be on the market next year. Completion of the non-invasive method is the crowning achievement of work that started more than 10 years ago when Dulce, a professor in the Nephrology Group of the Federal University of São Paulo (Unifesp), identified a new type of angiotensin-converting enzyme (ACE) that functions like a biological marker of high blood pressure. Her studies revealed that animals with hypertension, or those predisposed to developing hypertension, secrete ACE into the urine with molecular weight of 90 kDa (kilodaltons, the atomic mass unit). The kit is being jointly developed with Proteobras, a biotechnology company based in Paulínia, in inner-state São Paulo, headed by the engineer Paulo Roberto Pesquero and by his brothers who are molecular biologists on the scientific board, Professors João Bosco Pesquero, from Unifesp, and Jorge Luiz Pesquero, from the Federal University of Minas Gerais (UFMG).
Last year the company received funding of R$ 280,000 from a project in FAPESP’s Program for Innovative Research in Small Companies (Pipe) to develop three recombinant proteins, produced from cloned genes, and their respective antibodies for use in the kit. Pesquero expects that the kit will be suitable for use in public policy programs. “A person who has the hypertension marker can receive guidance on how to improve their quality of life, by adopting suitable eating habits, exercising and giving up smoking and drinking,” says Dulce. According to the biologist, a group of 1,150 volunteers with and without a history of hypertension in the family forms part of the first phase of the research and the results obtained in 2001 (see Pesquisa FAPESP nº 69) showed the efficiency of the kit in revealing the risk in people with a family history of the disease and who had ACE with a molecular weight of 90 kDa in their urine. “We are ending the second phase of this study in which the same patients were evaluated to learn which of those had the molecular marker and became hypertense.”
The kit will comprise a protein and its antibody, which can detect the presence of the biological marker of hypertension and can be used in the preventive diagnosis of renal lesions, particularly those in the renal tubules, microscopic structures that form part of the filter system of the kidneys. According to Dulce, this diagnosis is “extremely important” for the diabetic patient and will help doctors to treat the kidney early, to keep the person from developing what is often called diabetic nephropathy, which can lead to renal insufficiency. “This new function for our kit is the outcome of work that we completed recently, the results of which surprised us,” says the biologist. The patent on the kit has already been filed in the United States and in the main European countries and, since 2001, with the National Institute of Industrial Property (INPI).
In the laboratories of Proteobras, whose annual revenue is R$ 200,000 from producing reagents for the routines of laboratories that work with proteins and DNA, development of the diagnosis kit is at an advanced stage. “Our objective is to produce a molecule that recognizes this angiotensin-converting enzyme I, as if it were an antibody. But to develop this molecule, which is a recombinant protein, it’s necessary to have large amounts of the enzyme beforehand. This material is produced in the laboratory with cloning techniques,” says João Bosco. “We’ve already cloned the converter enzyme and now we’re in the expression and purification phase. Then we’ll start producing the antibodies for use in the kit.”
Development of the hypertension diagnosis kit is only one of the projects of Proteobras, a company created in 2004 in Mogi das Cruzes (SP) to supply the Brazilian market with recombinant proteins of socio-economic and commercial interest and which, until then, were imported. Seven years later, this market is still just beginning in the country and few Brazilian companies have mastered the production technology. Worldwide, the market for reagents, which include research with proteins, DNA and cell biology, is worth US$ 42 billion a year and is likely to reach US$ 81 billion by 2016, according to a study published in April of this year by an American market research company, BCC Research.
“Brazil is an enormous but repressed market. It’s difficult to establish its size, since all fields of knowledge, sectors of the economy and activities involving humans and other animals can use these products,” says João Bosco. Recombinant proteins have an application in health, biology, biochemistry, biotechnology, microbiology and agriculture. Clinical analysis laboratories use the material in various tests and for the treatment of certain genetic illnesses, such as diseases linked to the coagulation process of hemophiliacs and innate errors of the metabolism. “Another factor that limits the use of recombinants is that it is hard to obtain and to purify the protein. The process has various stages and is very difficult,” says João Bosco, for whom “the country today has a very small number of professionals with experience in techniques that involve proteins, which makes progress in the sector difficult.”
Proteobras has already mastered the production process, which starts with determining the protein that is to be produced. The first step is to isolate the DNA to be used which can be extracted from the blood or saliva of a person who has the protein. Then it is necessary to multiply the number of copies of this DNA – a process scientists call amplification – using a polymerase chain reaction (PCR) technique. Once this has been accomplished, DNA cloning can begin; the DNA is inserted into a cloning or expression vector, which gives rise to a recombinant vector. The vector is a type of molecular matrix that serves as a support for the DNA. This recombinant vector is inserted into an organism (a bacteria, yeast or mammal, insect or plant cell) that will act as a bioreactor, where the protein will be produced or expressed, in scientific parlance. The choice of organism depends on factors such as quantity, cost, the type of molecule to be cloned and its application. The organism is then placed on a plate with an antibiotic that will kill those organisms that have not incorporated the DNA, leaving just the positive organisms that are resistant to the antibiotic. This is the selection stage. The positive clones are isolated and multiplied in a culture medium. At the end of the process the organism expels the protein and the scientists then purify the substance. “This is the most important part of the process, which can be carried out by separating the protein by size, by electric charge, or by other methods,” explains the professor from Unifesp.
Besides the molecules still under development, Proteobras will begin selling two new products to the scientific community in November: a DNA polymerase and a DNA molecular weight standard. The polymerase is an enzyme of biotechnological interest with applications in diagnoses and used both in the DNA amplification process (during the process for producing recombinant proteins), for example, and in paternity tests and other molecular examinations carried out in clinical analysis and hospital laboratories. The molecular weight standard is used in developing recombinant proteins and serves to show whether the size of the DNA fragment to be cloned and selected by the researcher is correct; it has therefore many applications in molecular biology. According to João Bosco, this input has not been produced in Brazil so far.
To make the development of new molecules feasible, Proteobras has relied on funds from research development agencies ever since it was founded. The first public funding, obtained in 2005, was came from a PIPE [Program for Innovative Research in Small Companies] grant for R$ 75,000, whose aim was to establish a production protocol for the bovine follicle-stimulating hormone (FSH) for application in superovulation, a technique for increasing and improving herds. In 2009, the company received funding of R$ 390,000 from the Economic Subsidy Program of the Studies and Projects Funding Agency (Finep) to create a protocol for producing recombinant proteins and developing studies into protein characterization.
With his strong entrepreneurial spirit, João Bosco Pesquero is also helping to put together Exxtend, which was created in October this year in a partnership between Proteobras and a German group, K&A. The objective of the new enterprise is to offer a basic reagent, called an oligonucleotide, used by researchers in molecular biology and for diagnoses in the health area. Its main users are research laboratories, medical clinics, hospitals, research institutes and private laboratories. The German company, which is active in 80 countries, is the second largest producer of this material in the world. It started with the technology and supplied some of the equipment used in the synthesis of oligonucleotides. “At present we don’t have any company producing oligos in Brazil, just representatives of foreign groups. These reagents are imported from Europe, the United States and Asia and take on average 15 to 20 days to reach the hands of researchers.” Exxtend is going to make the product available to its customers in small flasks shipped by mail in about two days. According to João Bosco, there are no accurate data about the oligonucleotide market in Brazil, but estimates indicate that demand for the reagent is around 200,000 to 300,000 units annually. If all this material is sold, the company will achieve revenues of R$ 7 million (each oligo unit costs around R$25). “With Proteobras and Exxtend, we intend producing various research materials in the country which are currently being imported,” says João Bosco.
Development of a hypertension diagnosis kit using markers involving the angiotensin-converting enzyme and isoforms (nº 2009/51574-1); Modality Innovative Research in Small Companies (Pipe); Coordinator Paulo Roberto Pesquero – Proteobras; Investment R$ 280,000.00 (FAPESP)