For Amgen's purposes, this usually involves isolating a human gene with therapeutic potential, then introducing it into bacteria, yeast or an animal cell line. Without recombinant DNA technology, most of these proteins do not exist naturally in sufficient quantities. The recombinant systems, however, can be induced to produce the protein in high quantities under controlled conditions. In the end, we can produce large quantities of a highly purified protein for clinical use.
The technique of recombinant DNA, illustrated above, is fairly easy to grasp. Using proteins called restriction enzymes, individual genes from human DNA are isolated and inserted into small circular pieces of DNA cut with the same enzyme, known as plasmids. Once inserted into a plasmid, the gene can be glued in place using another enzyme called DNA ligase. Restriction enzymes and DNA ligase are the scissors and glue of recombinant DNA technology.
Once constructed in this way, the recombinant plasmid is inserted into a bacterial, yeast or cultured animal cell in a process called transformation. Transformed cells are separated from non-transformed cells in a selection procedure that takes advantage of drug-resistance genes also found on the plasmid.
A pure population of recombinant cells is then established through the process of cloning. In cloning, a single cell is selected, and it gives rise to a whole population of identical cells, or clones, by normal cell division. In this process, all of the resultant cells are expected to contain a copy of the plasmid carrying the inserted human gene.
Once the gene has been inserted and the cell line cloned, the cells are then coaxed to turn on, or "express," the human gene. Depending upon the cell system selected, the recombinant protein may be found inside the cells or outside in the surrounding medium. While this may sound straightforward, it is not; it took three years to clone the gene for EPOGEN®.
Amgen has developed a successful research focus on hematopoietic growth factors. Hematopoietic growth factors are protein hormones produced by the body to regulate the production and maturation of the various blood forming cells. Unspecialised precursor cells progress into specialized cell types, such as red blood cells (erythrocytes), white blood cells (leukocytes) and platelets. This progression is directed in part by various protein factors. Some of these factors appear to play a very specific role, while others seem to have more generalized functions.
Scientists at Amgen have used the capabilities of genetic engineering to isolate the genes for some of these protein factors. One such gene is erythropoietin (which stimulates progenitor cells found in the bone marrow to form mature erythrocytes, or red blood cells). It is a protein hormone produced by a specific cell type in the kidney.
Patients with chronic kidney disease are often unable to make adequate quantities of erythropoietin to maintain normal concentrations of erythrocytes in circulation. As a consequence, these patients are usually chronically and severely anaemic, that is, they have persistently low numbers of red blood cells in circulation. In some cases, in addition to dialysis, these patients require frequent blood transfusions to maintain adequate levels of red blood cells.
Presumably, the anaemia associated with kidney disease would be managed if an outside source of erythropoietin could be found. Unfortunately, the body produces erythropoietin in very small amounts, making it inconceivable to isolate enough natural erythropoietin to treat all patients with the disease. This is where recombinant DNA technology and Amgen enter the picture.
At Amgen, a research effort aimed at producing the human erythropoietin gene was led by Dr. Fu Kuen Lin. Dr. Lin's team initially obtained very small quantities of human erythropoietin from collaborators at the University of Chicago. The scientists used this material to determine the sequence of amino acids in part of the erythropoietin molecule.
Armed with the sequence information, Dr. Lin was able to design very short pieces of DNA, called oligonucleotides, that might match the human DNA sequence for erythropoietin. Simultaneously, pieces of human DNA that might contain the gene for erythropoietin were randomly cloned into bacteria. He then used the short pieces of DNA as tags to spot the erythropoietin gene in a technique called autoradiography. With this method, Dr. Lin was able to isolate the human gene for erythropoietin.
