href="https://www.diabetes.co.uk/insulin/history-of-insulin.html">https://www.diabetes.co.uk/insulin/history‐of‐insulin.html.22
| 1869 – The Islets of Langerhans cells are discovered in the pancreas |
| 1901 – The Islets of Langerhans are identified as cells that produce insulin |
| 1916 – Pancreas extract is found to lower blood sugar when given to diabetic dogs |
| 1921 – Studies show that pancreas removal in dogs results in the symptoms of diabetes; cow pancreas extract demonstrated improvement in the dog's health when given and was termed “insulin” |
| 1922 – A young boy with type 1 diabetes lives for 13 years beyond typical mortality as the first recipient of medical insulin; Eli Lilly becomes the first insulin manufacturer |
| 1936 – The addition of protamine is found to prolong the action of insulin |
| 1950 – Intermediate acting insulin (NPH) is marketed by Novo Nordisk |
| 1955 – Insulin becomes the first protein to be fully genetically sequenced |
| 1963 – Insulin becomes the first human protein to be chemically synthesized |
| 1978 – Genentech makes the first synthetic human insulin via recombinant DNA techniques, which is the first human protein to be manufactured using biotechnology |
| 1982 – Synthetic insulin is renamed as human insulin to better differentiate from animal sources |
| 1996 – Eli Lilly develops the first insulin analogue, which is genetically modified to change pharmacokinetics |
3.2.10 Blood Factor Products
Like the scientific developments that improved the production of insulin, blood coagulation factor products for the treatment of hemophilia have also undergone dramatic and important advances. Hemophilia A and B are bleeding disorders caused by dysfunction or deficiencies in coagulation factors VIII and IX that result in impaired blood clot formation and potential hemorrhage. In the 1950s, the only treatment option available was the infusion of human whole blood or plasma. Then in the mid‐1960s, scientists discovered how to collect factor products that allowed for collection in a more concentrated form. Donors were now able to donate plasma for production of Factor VIII for replacement. Industries developed around sourcing the material, and patients with hemophilia benefited significantly, being able to treat themselves in their home for the first time.23
Unfortunately, the donated pool of plasma used in this process became a host for transmission of blood‐borne disease such as hepatitis and HIV. The development of recombinant factor was a way to avoid reliance on human‐produced product and to limit viral contamination. In 1984, factor VIII DNA was cloned and in 1992, the first recombinant FVIII product, Recombinate®, was licensed for marketing.23 Today, there are over a dozen recombinant products developed for hemophilia treatment, with subsequent iterations focusing on using less animal and human‐derived elements, limiting the development of inhibiting antibodies and transmission of blood‐borne pathogens. Recent drug development efforts have been aimed at extending the half‐life of factor products to allow for less frequent dosing, potentially resulting in greater compliance with prophylactic dosing and less reliance on acute bleeding treatment. Human cell lines have been used to produce the latest generation of factor products and genetic therapy is being studied, which could potentially require a single one‐time treatment.23
3.3 rDNA and Biologic Drug Manufacturing
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,
