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As the scientific director for a start-up Biotech company I have been asked to develop a system for the production of a protein therapeutic to treat a human autoimmune condition. I have thus studied about the various types of human autoimmune diseases and I have decided to treat Diabetes mellitus type 1 disease. In my following report I will be outlining the approaches and technologies that I would be using to produce a commercially viable product.
Human Autoimmune Diseases:
The immune system is the body's means of protection against microorganisms and other "foreign" substances. The immune system is very essential to survival and even a small decrease in the immune function can leave a person susceptible to infection. But the immune system itself can also cause disease by inappropriately attacking the body's own organs, tissues and cells causing prolonged inflammation and subsequent tissue destruction. Thus Autoimmunity is the failure of an organism to recognise its own parts and which in turn allows an immune response against its own cells and tissues. Any disease that results from such an inappropriate immune response is termed as autoimmune disease.
Autoimmune diseases can be broadly classified into 2 general types
Systemic autoimmune disease ( Those that damage many organisms)
Localized autoimmune disease ( Those where only a single tissue or organ is damaged)
There are about 80-100 autoimmune diseases identified so far and these diseases are said to be chronic and life-threatening. The National Institute of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune diseases and the prevalence is rising. Autoimmune diseases is also one of the top 10 leading causes of death in female children and women in all age groups up to 64 years of age.
Diabetes (medically known as diabetes mellitus) is the autoimmune disorder in which the body has trouble regulating its blood glucose levels. There are 3 main types of diabetes:
Type1: This is also called as Insulin dependent or Juvenile diabetes and this makes up to 10% of the diabetes cases. Most cases of Type 1 Diabetes are diagnosed in those under the age of 30. Type 1 Diabetes make very little or no insulin.
Type 2: This is also called as Non-insulin dependent or adult onset diabetes and this makes about 90% of the diabetes cases. Most cases of this kind are diagnosed in those over the age of 45 years. Those people with type 2 diabetes do make their own insulin but it is either not in sufficient amount to meet their body needs or their body has become resistant to its effects.
Gestational Diabetes: It is a form of hyperglycemia that is seen in some pregnant women, usually late in their pregnancy. The hyperglycemia associated with gestational diabetes usually goes away after the baby's birth, but both the women diagnosed with gestational diabetes and their babies are at an increased risk of eventually developing type 2 diabetes
Diabetes mellitus type 1:
Diabetes mellitus type 1, more commonly known as type 1 diabetes, is a disease in which the pancreas produces too little insulin to meet the body's needs. People with this type of Diabetes must take insulin injections to live. Insulin is a hormone that regulates the amount of glucose (sugar) in the blood and is required for the body to function normally. Insulin is produced by cells in the pancreas, called the islets of Langerhans. These cells are responsible for continuously releasing a small amount of insulin into the body. Every time a person eats, the glucose levels in the blood rises. This rise in glucose levels triggers the cells in the islets of Langerhans to release necessary amount of insulin. Insulin allows the blood glucose to be transported from blood into cells. Without insulin the blood glucose builds up in the blood and the cells starve of their energy source. The net effect is persistent high levels of blood glucose, poor protein synthesis and other metabolic derangements.
Figure 1: Statistics of treatment with insulin, tablets and diet medication among adults with diagnosed diabetes
[ Source: National Health Interview Survey 2006-2008]
Recombinant production of Human Insulin:
The initial source of insulin for clinical use came from bovine (cow) and porcine (pig). The insulin from these were purified, bottled and sold. When this was administered to human beings some people found that this foreign protein caused allergy or other types of reactions and this led to a steady decline in the production of animal derived insulin. Biosynthetic "human" insulin came as a breakthrough and insulin is now manufactured for widespread clinical use using recombinant DNA technology. Eli Lilly marketed the first such insulin, Humulin in 1982. Humulin was first produced by inserting an actual human DNA into a host cell (E.Coli).
Figure 2: Recombinant production of Human insulin
[Source: Understanding the Immune system with information provided by the National Cancer Institute and the National Institute of Allergy and Infectious diseases.]
Structure of Human Insulin:
Insulin is a polypeptide hormone and is produced in the cells of the pancreas called the islets of Langerhans. Insulin consists of two polypeptide chains A and B. Insulin is one of the smallest proteins in the body. Chain A consists of 21 amino acids and chain B consists of 30 amino acids [Insulin 1992]. These two chains A and B are linked by two inter-chain and one intra-chain disulfide bridge. In the human pancreas, insulin is produced as a single polypeptide chain, proinsulin, with the A- chain and the B-chain joined by the connecting peptide (C-peptide) [Mackin 1998].
. Figure 3: Human Insulin structure
[Source: Robert, J.F. Biochemistry and Physiology.]
Development of a system for the production of Human Insulin:
Mature insulin is so far not producible in prokaryotic host cells in its native conformation by recombinant techniques. This is because the correct disulfide bond formation occurs only at the proinsulin. Several strategies have been developed for the production of proinsulin in E.Coli and these are commercially being used. [Tang and Hu, 1993; Kang and Yoon, 1991; Sung et al., 1986]. However, the complex purification processes and the formation of correct disulfide bonds during folding are however the critical cost factors for the above methods.
In this study we investigate the periplasmic production of proinsulin in E.Coli as a C-terminal fusion to DsbA. The bacterial periplasm is the most favourable compartment for the production of proinsulin. DsbA is the most important catalyst of disulfide bond formation and it is the most important oxidase of free sulfhydryl groups in the periplasm.
The E.Coli strains that were used are XL1 blue (Stratagene) and this was used as the host strain. The expression strains are C600 and RB791 (both E.Coli Genetic Stock Centre, New Haven). The cloning vector was pDsbA3 (Jonda et al., 1999). The vector pRK5-PI (Mather and Ullrich, 1987) was the source of the proinsulin gene.
The plasmid preparation was carried out according to Sambrook et al. (1989) and the vector pDsbA3-PI was constructed by inserting the human proinsulin gene into pDsbA3. Additionally a 18 nucleotide sequence was introduced as shown in the figure.
Figure 4: Map of the vector pDsbA3-PI. The restriction sites used for cloning are indicated. The linker sequence between DsbA (residues 1-189) and proinsulin (residues 196-281) are shown in italics.
[Source: Winter, J. et al. (2000). Journal of Biotechnology.84.]
Now the E.Coli harbouring the pDsbA3-PI was grown in LB medium. After the cells reached an optical density of 1, periplasmic fractions were prepared by Osmotic shock. [Kang and Yoon, 1994]. The soluble periplasmic proteins and the residual insoluble proteins were analysed by SDS-PAGE. This human insulin contains an additional arginine residue at the C-terminus of the B-chain. Cleavage by using trypsin is carried out and an optimal release of insulin from the fusion protein was obtained by incubation with 100Âµg trypsin for 10 min. The released insulin was then analysed by sandwich-ELISA. Using a standard curve created with native insulin, the amount of proinsulin produced n E.Coli was calculated.
Figure 5: Trypsin digestion products of purified DsbA-proinsulin were analysed on SDS gels. DsbA-proinsulin (20 mg) was incubated with trypsin in a mass ratio of 10:1 for 0, 0.17, 1, 10, and 50 min (lanes 2-6); lanes 7 and 8, human insulin standard; molecular weight markers (M) are indicated in lane 1.
[Source: Winter, J. et al. (2000). Journal of Biotechnology.84.]
In order to achieve efficient production of the fusion protein in soluble form with a native proinsulin part the expression was performed in E.Coli C600 and RB791 strains. The cells grew much faster in E.Coli RB791 than C600 resulting in higher cell density.
The yield of native proinsulin obtained from DsbA-proinsulin was 1000-fold higher than the secretory expression yield described for isolated proinsulin so far [Chan et al, 1981]. This indicates that DsbA is a very effective fusion partner for obtaining high amounts of soluble and correctly disulfide bridged proinsulin. In conclusion the data clearly indicates that the yield of human native proinsulin produced in E.Coli can be significantly increased by fusion of the proinsulin gene to DsbA. The great advantage of the fusion protein is that the proinsulin part can be cleaved off from the DsbA part by the addition of trypsin. Trypsin is commonly used for the conversion of proinsulin into insulin. By this proteolytic conversion it can be clearly shown that the proinsulin part of the fusion protein was folded correctly.
There are specific aspects that present potential limitations to the feasibility of the project and these are
Human Insulin may not be well accepted by some people and they may prefer to use insulin produced from animals.
The cost for producing Human insulin is almost double when compared to others so the cost may be a potential limitation.
During the development of Human Insulin there might be some problems with the post translational modifications.
Instability of the plasmid is also another limitation.
There might be problems in the folding of the disulfide bridge.
Biological activity and immunogenicity of recombinant protein may differ from the natural protein.
Secretes proteins only in the periplasm
But the greatest advantage of this system is that it gives us high quantities of the protein. Proteolytic cleavage events are a characteristic modification of many therapeutic proteins. Typically specific proteolytic enzymes cleave off the pro-peptide, yielding the mature protein. Proteolysis thus can also serve as a mechanism for releasing biologically active protein from an inactive precursor form. Proteolytic processing of proinsulin yields mature insulin product.
Figure 6: Proteolysis of Proinsulin into insulin.
[Source: Rhodes CJ et al. Insulin biosynthesis, processing, and chemistry. In: Kahn CR, Weir GC, King GL, et al, eds. Joslin's Diabetes Mellitus. 14th ed. Philadelphia, Pa: Lea & Febiger; 2005:65-82.]
Thus the proteolytic processing of proinsulin yielded mature insulin and this insulin can be given to patients who are suffering with Diabetes mellitus type 1 autoimmune disease. The system thus developed is mainly based on the recombinant DNA technology and this system gives us increased amounts of human proinsulin in the periplasmic space of E.Coli by fusion to DsbA. As the scientific director for a start-up Biotech company I would thus develop the above system for the production of Human Insulin for treating Diabetes Mellitus Type 1 autoimmune disease.