The term "biotechnology" often refers to the techniques of recombinant DNA. This latter term simply refers to the transfer of a gene from one organism into another organism: literally, the recombination of DNA from different sources.
For Amgen's purposes, this usually involves isolating a human gene with therapeutic potential, or genetically engineering a potential therapeutic, then introducing it into bacteria, yeast or an animal cell line. The recombinant systems are 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 and ultimately commercial therapeutic use.
The technique of recombinant DNA, illustrated above, is fairly easy to grasp. Using proteins called restriction enzymes, individual genes from human DNA or genetically engineered in the laboratory, 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 DNA sequence, designed to produce a potential therapeutic.
Once the gene has been inserted and the cell line cloned, the cells are then coaxed to turn on, or "express," the DNA sequence. Depending upon the cell system selected, the recombinant protein that is produced may be found inside the cells or outside in the surrounding medium.
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. 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. Epogen® provides an example of applying recombinant DNA technology to produce a human therapeutic.
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.
Producing Aranesp® (darbepoetin alfa) and Neulasta® (pegfilgrastim) 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 a standard interferon molecule, resulting in a larger molecule that stays in the body longer.
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