Читать книгу Biologics, Biosimilars, and Biobetters - Группа авторов - Страница 113

rDNA is as the name implies, taking DNA from two sources and recombining DNA into one. This allows for modification of DNA and is the basis for much of what is considered genetic engineering. The therapeutic applications have been discussed throughout this chapter; after a protein, messenger system, receptor, or biologic process in the body is understood well, scientists can attempt to augment that system using human‐made drug products.

In most cases, to do so specifically, requires the introduction of biologic products, biopharmaceuticals. For example, as history has demonstrated, it is possible to develop such biologic products from inoculating horses with diphtheria bacteria or producing insulin extracts from the pancreas of cows. While scientifically important as well as lifesaving at the time of discovery, these processes are fraught with several limitations, including antibody development following the introduction of agents of animal origin, scalability challenges with mass production from animal stock, lack of uniformity in the end product, and numerous opportunities for potential contamination.

Being able to produce a therapeutic, human protein such as insulin in a controlled environment mitigates many of these challenges. Being able to develop a gene in a laboratory and introduce it into a vector (e.g. bacterium or Chinese hamster ovary [CHO] cells) that will produce millions of copies of a modified, therapeutic protein is a game‐changer for drug development and the treatment of human disease.

As genes are the cornerstone of most molecular biology, it is helpful to be able to isolate and amplify specific gene fragments. Through rDNA technology, a gene of interest can be cloned by combining it with another DNA molecule (known as a vector) and inserting this into living cells where replication takes place. To obtain the genes required for this process, DNA needs to be “cut” or “spliced” into smaller fragments. The cutting is mediated by restriction endonucleases, which are enzymes found in bacteria that cut DNA. Through purposeful development, these enzymes are selected for their ability to cut DNA at specific sites. Once DNA fragments are cut as desired, they are then rejoined by ligase, which is an enzyme that catalyzes the joining of two large pieces or molecules.²⁴

Consider in this scenario, the specified genes to be cloned produce a useful protein such as insulin. To make many copies of that gene, it needs to be carried in a living cell (a cell that replicates). E. coli plasmid vectors and bacteriophage lambda are two of the most commonly used vectors for this purpose. The key difference among the two is that plasmid often lives symbiotically with the host cell and replicates each time the host cell replicates, whereas bacteriophage lambda acts as a virus and kills the host cell, leaving their packaged DNA intact.²⁵

Plasmid is a circular, double‐stranded DNA molecule. When an important section of DNA (such as the string of DNA that codes for insulin production) is isolated, it can be inserted into a plasmid, which then gets inserted to a bacterial cell such as E. coli. As E. coli bacteria reproduces, so too does the DNA molecule of interest (the insulin‐coding DNA strand in this example). Attaching an antibiotic‐resistance gene to the plasmid and exposing the E. coli cells to antibiotics ensures that only E. coli cells with rDNA inside reproduces. This makes the process more efficient. As the science progressed, it became possible to develop synthetic DNA fragments. These can be useful for making plasmids better suited for cloning or incorporation to study the impact of mutations, for example.²⁵

Several more examples beyond insulin can further demonstrate the importance of rDNA technology. The human somatostatin hormone consists of 14 amino acids and inhibits the secretion of somatotropin (growth hormone). It can be used therapeutically to treat acromegaly (excessive somatropin production) and analogs of somatostatin can be used to treat cancer. Somatostatin is produced using plasmid vectors and incubated in E. coli. Darbopoetin alfa, a 165‐amino acid protein, is a synthetic form of erythropoietin used to increase red blood cell levels. It is produced using rDNA technology, but unlike insulin or somatostatin produced in E. coli, darbopoeitin alfa is produced using CHO cells.²⁴

CHO cells are used to make nearly 70% of recombinant protein therapeutics today. The first product manufactured using CHO cells was a plasminogen activator called Activase^® (r‐tPA) in 1987. Rationale for the popularity of CHO cells in biopharmaceutical endeavors include accommodating complex protein folding and post‐translational modifications (shaping a protein after it has been made to a desirable structure), adaptive ability to study G‐protein coupled receptors in a stable environment, and structural characteristics related to cytoskeletal and microtubule structure, adhesion, and motility. Furthermore, there are logistical benefits to using CHO for protein synthesis; the cells can grow to very high densities in bioreactors, which benefits scalability and CHO cells have been found not to replicate human viruses such as HIV, influenza, polio, herpes, or measles.²⁶