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. At Amgen, we use the bacteria Eschericia coli (E. coli), baker's yeast, and a number of mammalian cell lines, including the Chinese Hamster Ovary cell line (CHO cells) we use to produce EPOGEN® (Epoetin alfa).
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. Unspecialized precursor cells progress into specialized cell types, such as erythrocytes, 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. Two such genes are erythropoietin and granulocyte-colony stimulating factor, better known as the Amgen products EPOGEN® (Epoetin alfa) and NEUPOGEN® (Filgrastim). Since EPOGEN® was Amgen's first product, we will use it as an example of how genetic engineering works.
EPOGEN® is Amgen's trade name for Epoetin alfa, a recombinant human erythropoietin. It is a protein hormone produced by a specific cell type in the kidney. As outlined above, erythropoietin stimulates progenitor cells found in the bone marrow to form mature erythrocytes (red blood cells). 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 anemic, 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 anemia associated with kidney disease would be eliminated 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 cloning 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. Dr. Lin subsequently cloned the human gene into the Chinese Hamster Ovary cell line for production of the human protein. This cell line continues to be used today for the production of EPOGEN®.
Subsequent to the cloning of erythropoietin, many people have been responsible for making EPOGEN® a successful product. These efforts have included clinical development, the scale-up and implementation of a manufacturing process, the coordination of all of the elements necessary for regulatory submission, the protection of Amgen's patent position on EPOGEN®, and the successful marketing and launch of the product.
Amgen's newest products use principles of biotechnology as well. Producing Aranesp® and Neulasta® requires efforts beyond the cloning of a gene and purification of the resulting protein. In the case of Aranesp®, the process of glycosylation takes place, meaning that ogliosaccharide units are added to proteins, creating different types of bonds than would form otherwise. And producing Neulasta® requires a process called pegylation, in which a polyethylene glycol (PEG) molecule is attached to protein Neupogen® (Filgrastim), resulting in a larger molecule that stays in the body longer.
