The question of what insulin is made from is of interest not only to doctors and pharmacists, but also to patients with diabetes, as well as their relatives and friends. Today, this hormone, unique and so important for human health, can be obtained from various raw materials using specially developed and carefully tested technologies. Depending on the method of production, the following types of insulin are distinguished:

• Porcine or bovine, also called a preparation of animal origin
• Biosynthetic, also known as modified pork
• Genetically engineered or recombinant
• Genetically engineered modified
• Synthetic

Porcine insulin has been used for the longest time to treat diabetes. Its use began in the 20s of the last century. It should be noted that pork or animal was the only drug until the 80s of the last century. Animal pancreas tissue is used to obtain it. However, this method can hardly be called optimal or simple: working with biological raw materials is not always convenient, and the raw materials themselves are not enough.

In addition, the composition of pork insulin does not exactly coincide with the composition of the hormone produced by the body of a healthy person: their structure contains different amino acid residues. It should be noted that the hormones produced by the pancreas of cattle have an even greater number of differences, which cannot be called a positive phenomenon.

In addition to the pure multicomponent substance, such a preparation invariably contains the so-called proinsulin, a substance that is almost impossible to separate using modern purification methods. It is this substance that often becomes the source of allergic reactions, which is especially dangerous for children and the elderly.

For this reason, scientists around the world have long been interested in the question of bringing the composition of the hormone produced by animals into full compliance with the hormones of the pancreas of a healthy person. A real breakthrough in pharmacology and the treatment of diabetes mellitus was the production of a semi-synthetic drug obtained by replacing the amino acid alanine in a drug of animal origin with threonine.

In this case, the semi-synthetic method of obtaining the hormone is based on the use of preparations of animal origin. In other words, they simply undergo modification and become identical to the hormones produced by humans. Among their advantages is compatibility with the human body and the absence of allergic reactions.

The disadvantages of this method include the shortage of raw materials and the complexity of working with biological materials, as well as the high cost of both the technology itself and the resulting drug.

In this regard, the best drug for the treatment of diabetes mellitus is recombinant insulin obtained through genetic engineering. By the way, it is often called genetically engineered insulin, thus indicating the method of its production, and the resulting product is called human insulin, thereby emphasizing its absolute identity with the hormones produced by the pancreas of a healthy person.

Among the advantages of genetically engineered insulin, one should also note its high degree of purity and the absence of proinsulin, as well as the fact that it does not cause any allergic reactions and has no contraindications.

The frequently asked question is quite understandable: what exactly is recombinant insulin made from? It turns out that this hormone is produced by yeast strains, as well as E. coli, placed in a special nutrient medium. Moreover, the amount of the substance obtained is so large that it is possible to completely abandon the use of drugs obtained from animal organs.

Of course, we are not talking about simple E. coli, but about a genetically modified one that is capable of producing soluble human genetically engineered insulin, the composition and properties of which are exactly the same as those of the hormone produced by the cells of the pancreas of a healthy person.

The advantages of genetically engineered insulin are not only its absolute similarity to the human hormone, but also ease of production, sufficient quantities of raw materials and affordable cost.

Scientists around the world call the production of recombinant insulin a real breakthrough in diabetes therapy. The significance of this discovery is so great and important that it is difficult to overestimate. It is enough to simply note that today almost 95% of the need for this hormone is met with the help of genetically engineered insulin. At the same time, thousands of people who previously suffered from allergies to drugs received a chance to live a normal life.

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I have type 2 diabetes - non-insulin dependent. A friend advised me to lower my blood sugar levels with

Insulin (from Latin insula - island) is a peptide hormone produced in the beta cells of the islets of Langerhans in the pancreas. It has a multifaceted effect on metabolism in almost all tissues.

The main function of insulin is to ensure the permeability of cell membranes to glucose molecules. In a simplified form, we can say that not only carbohydrates, but also any nutrients are ultimately broken down into glucose, which is used for the synthesis of other carbon-containing molecules, and is the only type of fuel for cellular energy plants - mitochondria. Without insulin, the permeability of the cell membrane to glucose drops 20 times, and the cells die of starvation, and excess sugar dissolved in the blood poisons the body.

Impaired insulin secretion due to the destruction of beta cells - absolute insulin deficiency - is a key element in the pathogenesis of type 1 diabetes mellitus. Impaired action of insulin on tissue - relative insulin deficiency - plays an important role in the development of type 2 diabetes mellitus.

The history of the discovery of insulin is associated with the name of the Russian doctor I.M. Sobolev (second half of the 19th century), who proved that the level of sugar in human blood is regulated by a special hormone of the pancreas.

In 1922, insulin isolated from the pancreas of an animal was first administered to a ten-year-old boy with diabetes. the result exceeded all expectations, and a year later the American company Eli Lilly released the first animal insulin preparation.

After receiving the first industrial batch of insulin, over the next few years a huge path was taken in its isolation and purification. As a result, the hormone became available to patients with type 1 diabetes.

In 1935, Danish researcher Hagedorn optimized the action of insulin in the body by proposing a long-acting drug.

The first crystals of insulin were obtained in 1952, and in 1954 the English biochemist G. Sanger deciphered the structure of insulin. The development of methods for purifying the hormone from other hormonal substances and insulin degradation products has made it possible to obtain homogeneous insulin, called single-component insulin.

In the early 70s Soviet scientists A. Yudaev and S. Shvachkin proposed the chemical synthesis of insulin, but the implementation of this synthesis on an industrial scale was expensive and unprofitable.

Subsequently, there was a progressive improvement in the purity of insulin, which reduced problems caused by insulin allergies, kidney disorders, visual impairment and immune resistance to insulin. The most effective hormone for replacement therapy for diabetes mellitus was needed - homologous insulin, that is, human insulin.

In the 80s, advances in molecular biology made it possible to synthesize both chains of human insulin using E.coli, which were then combined into a molecule of biologically active hormone, and at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences, recombinant insulin was obtained using genetically engineered E.coli strains.

The use of affinity chromatography significantly reduced the content of contaminating proteins in the preparation with a higher mw than that of insulin. These proteins include proinsulin and partially cleaved proinsulins, which are capable of inducing the production of anti-insulin antibodies.

Using human insulin from the very beginning of therapy minimizes the occurrence of allergic reactions. Human insulin is absorbed more quickly and, regardless of the formulation, has a shorter duration of action than animal insulins. Human insulins are less immunogenic than porcine insulins, especially mixed bovine and porcine insulins.

1. Types of insulin

Insulin preparations differ from each other in the degree of purification; source of production (bovine, pork, human); substances added to the insulin solution (prolonging its action, bacteriostatics, etc.); concentrations; pH value; the possibility of mixing ICD with IPD.

Insulin preparations vary according to their source. Pig and bovine insulin differs from human insulin in amino acid composition: bovine insulin has three amino acids, and pork insulin has one amino acid each. It is not surprising that when treated with bovine insulin, adverse reactions develop much more often than when treated with porcine or human insulin. These reactions are expressed in immunological insulin resistance, insulin allergies, lipodystrophies (changes in subcutaneous fat at the injection site).

Despite the obvious disadvantages of bovine insulin, it is still widely used throughout the world. And yet, the disadvantages of bovine insulin in immunological terms are obvious: it is under no circumstances recommended to be prescribed to patients with newly diagnosed diabetes mellitus, pregnant women, or for short-term insulin therapy, for example, in the perioperative period. The negative qualities of bovine insulin remain when used in a mixture with pork, so mixed (pork + bovine) insulins should also not be used for the treatment of these categories of patients.

Human insulin preparations are completely identical in chemical structure to human insulin.

The main problem of the biosynthetic method for producing human insulin is the complete purification of the final product from the slightest impurities of used microorganisms and their metabolic products. New quality control methods ensure that biosynthetic human insulins from the above manufacturers are free from any harmful impurities; Thus, their degree of purification and glucose-lowering effectiveness meet the highest requirements and are almost identical. These insulin preparations do not have any undesirable side effects depending on impurities.

Currently, three types of insulin are used in medical practice:

Short-acting with a quick onset of effect;

Medium duration of action;

Long-acting with slow onset of effect.

Table 1. Characteristics of commercial insulin preparations

Short-acting insulin (RAI) - regular insulin - is a short-acting crystalline zinc insulin, soluble at a neutral pH value, the effect of which develops within 15 minutes after subcutaneous administration and lasts 5-7 hours.

The first long-acting insulin (LAI) was created in the late 1930s so that patients could inject less frequently than with ICDs alone—once a day if possible. In order to increase the duration of action, all other insulin preparations are modified and, when dissolved in a neutral medium, form a suspension. They contain protamine in a phosphate buffer - protamine-zinc-insulin and NPH (neutral protamine Hagedorn) - NPH-insulin or various concentrations of zinc in an acetate buffer - ultralente, lente, semilente insulins.

Intermediate-acting insulin preparations contain protamine, which is a protein with an average molecular weight. 4400, rich in arginine and obtained from rainbow trout milt. To form the complex, a protamine to insulin ratio of 1:10 is required. After subcutaneous administration, proteolytic enzymes destroy protamine, allowing insulin to be absorbed.

NPH insulin does not change the pharmacokinetic profile of regulatory insulin mixed with it. NPH insulin is preferred over insulin lente as an intermediate-acting component in therapeutic mixtures containing regular insulin.

In phosphate buffer, all insulins readily form crystals with zinc, but only bovine insulin crystals are sufficiently hydrophobic to provide the slow and sustained release of insulin characteristic of ultralente. Zinc crystals of pork insulin dissolve faster, the effect occurs earlier, and the duration of action is shorter. Therefore, there is no Ultralente preparation containing only pork insulin. Monocomponent pork insulin is produced under the names insulin suspension, neutral insulin, isophane insulin, and aminoquinuride insulin.

2. Getting insulin

Human insulin can be produced in four ways:

1) complete chemical synthesis;

2) extraction from human pancreas (both of these methods are not suitable due to inefficiency: insufficient development of the first method and lack of raw materials for mass production by the second method);

3) by a semi-synthetic method using an enzyme-chemical replacement at position 30 of the B-chain of the amino acid alanine in pork insulin with threonine;

4) biosynthetically using genetic engineering technology. The last two methods make it possible to obtain highly purified human insulin.

Currently, human insulin is mainly produced in two ways: by modifying porcine insulin using a synthetic-enzymatic method and by genetic engineering.

Insulin was the first protein produced commercially using recombinant DNA technology. There are two main approaches to obtain genetically engineered human insulin.

In the first case, separate (different producer strains) production of both chains is carried out, followed by folding of the molecule (formation of disulfide bridges) and separation of isoforms.

In the second, it is obtained in the form of a precursor (proinsulin) followed by enzymatic cleavage by trypsin and carboxypeptidase B to the active form of the hormone. The most preferred method at present is to obtain insulin in the form of a precursor, ensuring the correct closure of disulfide bridges (in the case of separate production of chains, successive cycles of denaturation, isoform separation and renaturation are carried out).

With both approaches, it is possible to obtain the initial components (A- and B-chains or proinsulin) individually or as part of hybrid proteins. In addition to the A- and B-chain or proinsulin, the hybrid proteins may contain:

A carrier protein that ensures the transport of the hybrid protein into the periplasmic space of the cell or culture medium;

An affinity component that significantly facilitates the isolation of the hybrid protein.

Moreover, both of these components can be simultaneously present in the hybrid protein. In addition, when creating hybrid proteins, the principle of multimerism can be used (that is, several copies of the target polypeptide are present in the hybrid protein), which can significantly increase the yield of the target product.

In the UK, using E. coli, both chains of human insulin were synthesized, which were then combined into a molecule of a biologically active hormone. In order for a single-celled organism to synthesize insulin molecules on its ribosomes, it is necessary to provide it with the necessary program, that is, to introduce a hormone gene into it.

A gene that programs the biosynthesis of the insulin precursor or two genes that separately program the biosynthesis of the A and B chains of insulin are obtained chemically.

The next step is the inclusion of the insulin precursor gene (or individual chain genes) into the genome of E. coli, a special strain of Escherichia coli grown in the laboratory. This task is performed by genetic engineering.

The plasmid is isolated from E. coli using the appropriate restriction enzyme. the synthetic gene is inserted into a plasmid (by cloning with the functionally active C-terminal part of E. coli β-galactosidase). As a result, E. coli acquires the ability to synthesize a protein chain consisting of galactosidase and insulin. The synthesized polypeptides are cleaved from the enzyme chemically, and then purified. In bacteria, about 100,000 insulin molecules are synthesized per bacterial cell.

The nature of the hormonal substance produced by E.coli is determined by which gene is inserted into the genome of a single-celled organism. If the insulin precursor gene is cloned, the bacterium synthesizes the insulin precursor, which is then treated with restriction enzymes to cleave the prepeptide to isolate the C-peptide, resulting in biologically active insulin.

To obtain purified human insulin, the hybrid protein isolated from biomass is subjected to chemical-enzymatic transformation and appropriate chromatographic purification (prental, gel permeation, anion exchange).

At the Institute of the Russian Academy of Sciences, recombinant insulin was obtained using genetically engineered E. coli strains. from the grown biomass, a precursor is released, a hybrid protein expressed in the amount of 40% of the total cellular protein, containing preproinsulin. Its conversion into insulin in vitro is carried out in the same sequence as in vivo - the leading polypeptide is cleaved off, preproinsulin is converted into insulin through the stages of oxidative sulphitolysis, followed by the reductive closure of three disulfide bonds and enzymatic isolation of the binding C-peptide. After a series of chromatographic purifications, including ion exchange, gel and HPLC, human insulin of high purity and natural potency is obtained.

You can use a strain with a nucleotide sequence built into the plasmid that expresses a hybrid protein that consists of linear proinsulin and a fragment of Staphylococcus aureus protein A attached to its N-terminus through a methionine residue.

Cultivation of a saturated biomass of cells of a recombinant strain ensures the beginning of the production of a hybrid protein, the isolation and sequential transformation of which in tube leads to insulin.

Another way is also possible: a recombinant fusion protein is obtained in a bacterial expression system, consisting of human proinsulin and a polyhistidine “tail” attached to it through a methionine residue. It is isolated using chelate chromatography on Ni-agarose columns from inclusion bodies and digested with cyanogen bromide.

The isolated protein is S-sulfonated. Mapping and mass spectrometric analysis of the resulting proinsulin, purified by anion exchange chromatography and RP (reverse phase) HPLC (high performance liquid chromatography), show the presence of disulfide bridges corresponding to the disulfide bridges of native human proinsulin.

Recently, close attention has been paid to simplifying the procedure for obtaining recombinant insulin using genetic engineering methods. For example, it is possible to obtain a fusion protein consisting of an interleukin 2 leader peptide attached to the N-terminus of proinsulin via a lysine residue. The protein is efficiently expressed and localized to inclusion bodies. Once isolated, the protein is digested by trypsin to produce insulin and C-peptide.

The resulting insulin and C-peptide were purified by RP HPLC. When creating fusion constructs, the mass ratio of the carrier protein and the target polypeptide is very important. C-peptides are connected in a head-to-tail manner using amino acid spacers carrying the Sfi I restriction site and two arginine residues at the beginning and end of the spacer for subsequent protein cleavage by trypsin. HPLC of the cleavage products shows that the C-peptide cleavage is quantitative, and this allows the use of the multimeric synthetic gene method for the production of target polypeptides on an industrial scale.

1. Biotechnology: Textbook for Universities / Ed. N.S. Egorova, V.D. Samuilova.- M.: Higher School, 1987, pp. 15-25.

2. Genetically engineered human insulin. Increasing the efficiency of chromatographic separation using the principle of bifunctionality. / Romanchikov A.B., Yakimov S.A., Klyushnichenko V.E., Arutunyan A.M., Vulfson A.N. // Bioboundary Chemistry, 1997 - 23, No. 2

3. Glick B., Pasternak J. Molecular biotechnology. Principles and Application. M.: Mir, 2002.

4. Egorov N. S., Samuilov V. D. Modern methods for creating industrial strains of microorganisms // Biotechnology. Book 2. M.: Higher School, 1988. 208 p.

5. Immobilization of trypsin and carboxypeptidase B on modified silicas and their use in the conversion of recombinant human proinsulin into insulin. / Kudryavtseva N.E., Zhigis L.S., Zubov V.P., Vulfson A.I., Maltsev K.V., Rumsh L.D. // Chem.-pharmac. zh., 1995 - 29, No. 1 pp. 61 - 64.

6. Molecular biology. Structure and functions of proteins./ Stepanov V. M.// Moscow, Higher School, 1996.

7. Fundamentals of pharmaceutical biotechnology: Textbook / T.P. Prishchep, V.S. Chuchalin, K.L. Zaikov, L.K. Mikhaleva. – Rostov-on-Don: Phoenix; Tomsk: NTL Publishing House, 2006.

8. Synthesis of insulin fragments and study of their physicochemical and immunological properties. / Panin L.E., Tuzikov F.V., Poteryayeva O.N., Maksyutov A.Z., Tuzikova N.A., Sabirov A.N. // Bioorganic Chemistry, 1997 - 23, No. 12 pp. 953 - 960.

Insulin is a life-saving drug that has revolutionized the lives of many people with diabetes.

In the entire history of medicine and pharmacy of the 20th century, it is possible to single out, perhaps, only one group of medications that are of the same importance - antibiotics. They, like insulin, very quickly entered medicine and helped save many human lives.

Diabetes Day is celebrated at the initiative of the World Health Organization every year since 1991 on the birthday of the Canadian physiologist F. Banting, who discovered the hormone insulin together with J. J. McLeod. Let's look at how this hormone is obtained and made.

How do insulin preparations differ from each other?

1. Degree of purification.
2. The source of production is pork, bovine, or human insulin.
3. Additional components included in the drug solution are preservatives, action prolongers and others.
4. Concentration.
5. pH of the solution.
6. Possibility of mixing short-acting and long-acting drugs.

Insulin is a hormone produced by special cells in the pancreas. It is a double-chain protein containing 51 amino acids.

About 6 billion units of insulin are consumed annually in the world (1 unit is 42 mcg of the substance). The production of insulin is high-tech and is carried out only by industrial methods.

Sources of insulin

Currently, depending on the source of production, pork insulin and human insulin preparations are isolated.

Porcine insulin now has a very high degree of purification, has a good hypoglycemic effect, and there are practically no allergic reactions to it.

Human insulin preparations fully correspond in chemical structure to the human hormone. They are usually produced by biosynthesis using genetic engineering technologies.

Large manufacturing companies use production methods that ensure that their products meet all quality standards. No major differences in the action of human and porcine monocomponent insulin (that is, highly purified) have been identified; in relation to the immune system, according to many studies, the difference is minimal.

Auxiliary components used in the production of insulin

The bottle with the drug contains a solution containing not only the hormone insulin itself, but also other compounds. Each of them plays its own specific role:

• prolongation of the effect of the drug;
• disinfection of the solution;
• the presence of buffer properties of the solution and maintaining a neutral pH (acid-base balance).

Prolonging the action of insulin

To create long-acting insulin, one of two compounds is added to a solution of regular insulin: zinc or protamine. Depending on this, all insulins can be divided into two groups:

• protamine insulins – protafan, insulin basal, NPH, humulin N;
• zinc insulins – insulin-zinc suspension mono-tard, lente, humulin-zinc.

Protamine is a protein, but adverse reactions such as allergies to it are very rare.

To create a neutral solution environment, a phosphate buffer is added to it. It must be remembered that insulin containing phosphates is strictly forbidden to be combined with insulin-zinc suspension (IZS), since zinc phosphate precipitates and the effect of zinc insulin is shortened in the most unpredictable way.

Disinfectant components

Some of the compounds that, according to pharmacotechnological criteria, should already be included in the drug have a disinfecting effect. These include cresol and phenol (both of them have a specific odor), as well as methyl parabenzoate (methylparaben), which has no odor.

The introduction of any of these preservatives causes the specific odor of some insulin preparations. All preservatives in the quantities in which they are found in insulin preparations do not have any negative effect.

Protamine insulins usually contain cresol or phenol. Phenol cannot be added to ICS solutions, because it changes the physical properties of the hormone particles. These drugs include methylparaben. Zinc ions in solution also have an antimicrobial effect.

Thanks to this multi-stage antibacterial protection with the help of preservatives, the development of possible complications that could be caused by bacterial contamination when a needle is repeatedly inserted into a bottle with a solution is prevented.

Due to the presence of such a protection mechanism, the patient can use the same syringe for subcutaneous injections of the drug for 5 to 7 days (provided that he is the only one using the syringe). Moreover, preservatives make it possible not to use alcohol to treat the skin before injection, but again only if the patient injects himself with a syringe with a thin needle (insulin).

Calibration of insulin syringes

In the first insulin preparations, one ml of solution contained only one unit of the hormone. Later the concentration was increased. Most insulin preparations in vials used in Russia contain 40 units per 1 ml of solution. The bottles are usually marked with the symbol U-40 or 40 units/ml.

They are intended for widespread use precisely for such insulin and are calibrated according to the following principle: when a person draws 0.5 ml of a solution with a syringe, he gains 20 units, 0.35 ml corresponds to 10 units, and so on.

Each mark on the syringe is equal to a certain volume, and the patient already knows how many units this volume contains. Thus, the calibration of syringes is a calibration of the volume of the drug, designed for the use of U-40 insulin. 4 units of insulin are contained in 0.1 ml, 6 units in 0.15 ml of the drug, and so on up to 40 units, which correspond to 1 ml of solution.

In some countries, insulin is used, 1 ml of which contains 100 units (U-100). For such drugs, special insulin syringes are produced, which are similar to those discussed above, but they have a different calibration.

It takes into account exactly this concentration (it is 2.5 times higher than the standard). In this case, the dose of insulin for the patient naturally remains the same, since it satisfies the body’s need for a specific amount of insulin.

That is, if the patient previously used the drug U-40 and injected 40 units of the hormone per day, then he should receive the same 40 units when injecting insulin U-100, but administer it in an amount 2.5 times less. That is, the same 40 units will be contained in 0.4 ml of solution.

Unfortunately, not all doctors, and especially those with diabetes, know about this. The first difficulties began when some of the patients switched to using insulin injectors (pen-syringes), which use penfills (special cartridges) containing U-40 insulin.

If you fill such a syringe with a solution labeled U-100, for example, to the level of 20 units (that is, 0.5 ml), then this volume will contain as many as 50 units of the drug.

Each time, filling regular syringes with insulin U-100 and looking at the unit cutoffs, a person will take a dose 2.5 times greater than the one shown at this mark. If neither the doctor nor the patient notices this error in a timely manner, then there is a high probability of developing severe hypoglycemia due to a constant overdose of the drug, which often happens in practice.

On the other hand, sometimes there are insulin syringes calibrated specifically for the U-100 drug. If such a syringe is mistakenly filled with the usual U-40 solution, then the dose of insulin in the syringe will be 2.5 times less than the one written near the corresponding mark on the syringe.

As a result, a seemingly inexplicable increase in blood glucose may occur. In fact, of course, everything is quite logical - for each concentration of the drug you need to use a suitable syringe.

In some countries, such as Switzerland, there was a carefully thought out plan according to which a competent transition to insulin preparations labeled U-100 was carried out. But this requires close contact of all interested parties: doctors of many specialties, patients, nurses from any departments, pharmacists, manufacturers, authorities.

In our country, it is very difficult to switch all patients to using only U-100 insulin, because this will most likely lead to an increase in the number of errors in determining the dose.

Combined use of short-acting and long-acting insulins

In modern medicine, diabetes mellitus, especially type 1, is usually treated using a combination of two types of insulin - short-acting and long-acting.

It would be much more convenient for patients if drugs with different durations of action could be combined in one syringe and administered simultaneously to avoid double puncture of the skin.

Many doctors do not know what determines the possibility of mixing different insulins. This is based on the chemical and galenic (determined by composition) compatibility of long-acting and short-acting insulins.

It is very important that when mixing two types of drugs, the rapid onset of action of short-acting insulin is not prolonged or disappears.

It has been proven that a short-acting drug can be combined in one injection with protamine insulin, and the onset of short-acting insulin is not delayed because soluble insulin does not bind to protamine.

In this case, the manufacturer of the drug does not matter. For example, it can be combined with humulin N or protaphan. Moreover, mixtures of these drugs can be stored.

Regarding zinc-insulin preparations, it has long been established that insulin-zinc suspension (crystalline) cannot be combined with short-acting insulin, as it binds to excess zinc ions and is transformed into long-acting insulin, sometimes partially.

Some patients first inject a short-acting drug, then, without removing the needle from under the skin, slightly change its direction and inject zinc insulin through it.

Quite a few scientific studies have been carried out on this method of administration, so we cannot exclude the fact that in some cases, with this method of injection, a complex of zinc-insulin and a short-acting drug may form under the skin, which leads to impaired absorption of the latter.

Therefore, it is better to administer short-acting insulin completely separately from zinc insulin, to make two separate injections into areas of the skin located at a distance of at least 1 cm from each other. This is not convenient, which cannot be said about the standard dose.

Combined insulins

Now the pharmaceutical industry produces combination drugs containing short-acting insulin together with protamine insulin in a strictly defined percentage. Such drugs include:

• mixtard,
• actrafan,
• insuman com.

The most effective combinations are those in which the ratio of short- and long-acting insulin is 30:70 or 25:75. This ratio is always indicated in the instructions for use of each specific drug.

Such drugs are best suited for people who maintain a constant diet and have regular physical activity. For example, they are often used by elderly patients with type 2 diabetes.

Combined insulins are not suitable for so-called “flexible” insulin therapy, when there is a need to constantly change the dosage of short-acting insulin.

For example, this should be done when changing the amount of carbohydrates in food, decreasing or increasing physical activity, etc. In this case, the dose of basal insulin (long-acting) remains practically unchanged.

Currently, according to WHO (World Health Organization), there are about 110 million people in the world who suffer from diabetes. And this figure may double in the next 25 years. Diabetes is a terrible disease that is caused by a malfunction of the pancreas, which produces the hormone insulin, which is necessary for the normal utilization of carbohydrates contained in food. In the initial stages of the disease, it is enough to use preventive measures, regularly monitor blood sugar levels, and consume less sweets. However, insulin therapy is indicated for 10 million patients; they inject drugs of this hormone into the blood. Since the twenties of the last century, insulin isolated from the pancreas of pigs and calves has been used for these purposes. Animal insulin is similar to human insulin, the difference is that in the pig insulin molecule, unlike human insulin, in one of the chains the amino acid threonine is replaced by alanine. It is believed that these minor differences can cause serious problems in the functioning of the kidneys, visual disturbances, and allergies in patients). In addition, despite the high degree of purification, the possibility of transmission of viruses from animals to humans cannot be ruled out. And finally, the number of people with diabetes is growing so quickly that it is no longer possible to provide all those in need with animal insulin. And this is a very expensive medicine.

Insulin was first isolated from the bovine pancreas in 1921 by F. Banting and C. Best. It consists of two polypeptide chains connected by two disulfide bonds. Polypeptide chain A contains 21 amino acid residues, and chain B contains 30 amino acid residues, the molecular weight of insulin is 5.7 kDa. Below is the amino acid sequence of human insulin:

Gli-Ile-Val-Glu-Gli-Cis-Tre-Ser-Ile-Cis-S-Lei-Tir-Gli-Lei-Gli-Lei-Glu-Asn-

Fen-Val-Asn-Gli-Gis-Lei-Cis-Glu-Ser-Gis-Lei-Val-Glu-Ala-Lei-Tir-Lei-Val-Cis-Glu-Glu-

Fen-Val-Asn-Gli-Gis-Gis-Lei-Cis-Glu-Ser-Gis-Lei-Val-Glu-Ala-Lei-Tir-Lei-Val-Cis-Glu-Glu

Trp-Lis-Pro-Trp-Tyr-Fen-Fen-Glu-Ark

The structure of insulin is quite conservative. The amino acid sequence of human insulin and many animals differs by only 1-2 amino acids. In fish, compared to animals, the B-chain is larger and contains 32 amino acid residues.

Its cost was very high. To obtain 100 g of crystalline insulin, 800-1000 kg of pancreas is required, and one gland of a cow weighs 200 - 250 grams. This made insulin expensive and difficult to access for a wide range of diabetics.

Genetic engineering, born in the early 70s, has made great strides today. Genetic engineering techniques transform bacterial, yeast and mammalian cells into “factories” for the large-scale production of any protein. This makes it possible to analyze in detail the structure and functions of proteins and use them as medicines. Currently, Escherichia coli (E. coli) has become a supplier of such important hormones as insulin and somatotropin.

In 1978, researchers from Genentech first produced insulin in a specially engineered strain of Escherichia coli. Insulin consists of two polypeptide chains A and B, 20 and 30 amino acids long. When they are connected by disulfide bonds, native double-chain insulin is formed. It has been shown that it does not contain E. coli proteins, endotoxins and other impurities, does not produce side effects like animal insulin, and is no different from it in biological activity. Subsequently, proinsulin was synthesized in E. coli cells, for which a DNA copy was synthesized on an RNA template using reverse transcriptase. After purifying the resulting proinsulin, it was split into native insulin, while the stages of extraction and isolation of the hormone were minimized. From 1000 liters of culture fluid, up to 200 grams of the hormone can be obtained, which is equivalent to the amount of insulin secreted from 1600 kg of the pancreas of a pig or cow.

In animals and humans, insulin is synthesized in β-cells of the islets of Largehans. The genes encoding this protein in humans are localized in the short arm of chromosome 11. Mature insulin mRNA consists of 330 nucleotides, which corresponds to 110 amino acid residues. This is the amount of them that contains the insulin precursor - preproinsulin. It consists of one polypeptide chain, at the N-end of which there is a signal peptide (24 amino acids), and between the A- and B-chains there is a C-peptide containing 35 amino acid residues.

The process of insulin maturation begins in the ciscerns of the endoplasmic reticulum, where the signal peptide is cleaved from the N-terminus under the action of the signalase enzyme. Next, in the Golgi apparatus, under the action of endopeptidases, C-peptide is excised and mature insulin is formed. On the trans side of the Golgi apparatus, the newly synthesized hormone combines with zinc, forming supramolecular structures (tri-, tetra-, penta- and hexamers), which then move into secretory granules.

The latter are separated from the Golgi apparatus, move to the cytoplasmic membrane, associate with it, and insulin is secreted into the bloodstream. The rate of hormone secretion is determined by the concentration of glucose and Ca 2+ ions in the blood. Adrenaline suppresses the release of insulin, and hormones such as TSH and ACTH, on the contrary, promote its secretion. In the blood, insulin is found in two forms: free and bound to proteins, mainly with transferrin and α 2 - globulin. The half-life of insulin is about five minutes, and decay begins in the blood, because Red blood cells contain insulin receptors and a fairly active insulin-degrading system. Erythrocyte insulinase is a Ca-dependent, thiol proteinase that functions in conjunction with glutathione-insulin-irans hydrogenase, which cleaves disulfide bonds between the two polypeptide chains of insulin.

Insulin fragmentation and breakdown occur primarily in the liver, kidneys and placenta.

Insulin fragments have biological activity and are involved in a number of metabolic processes. One of the main functions of insulin is the regulation of the transport of glucose, amino acids, ions and other metabolites into the cells of the liver, kidneys, adipose tissue of other organs. The mechanism of action of this hormone differs from that of other peptide hormones and is unique in the regulation of metabolic processes. The insulin receptor is a tetramer consisting of two α- and two β-subunits, one of which has thyroxinase activity. Insulin, when interacting with α-subunits located on the surface of the cytoplasmic membrane, forms a hormone-receptor complex. Conformational changes in the tetramer lead to activation of the transmembrane β-subunit of the receptor, which has tyrosine kinase activity. Active tyrosine kinase is capable of phosphorylating membrane proteins. Membrane channels are formed through which glucose and other metabolites penetrate into cells. Free insulin under the action of tissue insulinase breaks down into seven fractions, five of which have biological activity.

In addition, insulin stimulates a number of biosynthetic processes: the synthesis of nucleotides, nucleic acids, enzymes of glycolysis and the pentose phosphate cycle, and glycogen. In adipose tissue, insulin activates the formation of acetyl Co A and fatty acids. It is one of the inducers of the synthesis of cholesterol, glycerol and glycerate kinase.

Mutations in the structure of the insulin gene, disruption of post-transcriptional and post-translational processing mechanisms lead to the formation of defective insulin molecules and, as a consequence, disruption of metabolic processes regulated by this hormone. As a result, a serious disease develops – diabetes mellitus.

The development of technology for the production of artificial insulin is truly a triumph for geneticists. First, using special methods, the structure of the molecule of this hormone, the composition and sequence of amino acids in it were determined. In 1963, the insulin molecule was synthesized using biochemical methods. However, it turned out to be difficult to carry out such an expensive and complex synthesis, including 170 chemical reactions, on an industrial scale.

Therefore, in further research, the emphasis was placed on developing technology for the biological synthesis of the hormone in microbial cells, for which the entire arsenal of genetic engineering methods was used. Knowing the sequence of amino acids in the insulin molecule, scientists calculated what the sequence of nucleotides in the gene encoding this protein should be in order to obtain the desired sequence of amino acids. They “assembled” a DNA molecule from individual nucleotides in accordance with a certain sequence, “added” to it the regulatory elements necessary for gene expression in the prokaryotic organism E. coli, and integrated this construct into the genetic material of the microbe. As a result, the bacterium was able to produce two chains of the insulin molecule, which could later be combined using a chemical reaction to produce a complete insulin molecule.

Finally, scientists managed to carry out the biosynthesis of the proinsulin molecule, and not just its individual chains, in E. coli cells. After biosynthesis, the proinsulin molecule is capable of being transformed accordingly (disulfide bonds are formed between chains A and B), turning into an insulin molecule. This technology has significant advantages, since the various stages of extraction and release of the hormone are reduced to a minimum. During the development of this technology, proinsulin messenger RNA was isolated. Using it as a template, a DNA molecule complementary to it was synthesized using the enzyme reverse transcriptase, which was an almost exact copy of the natural insulin gene. After stitching the necessary regulatory elements to the gene and transferring the construct into the genetic material of E. coli

It became possible to produce insulin in a microbiological factory in unlimited quantities. Its tests showed almost complete identity with natural human insulin. It is much cheaper than animal insulin and does not cause complications.

Somatotropin is a human growth hormone secreted by the pituitary gland. A deficiency of this hormone leads to pituitary dwarfism. If somatotropin is administered in doses of 10 mg per kg of body weight three times a week, then in a year a child suffering from its deficiency can grow 6 cm. Previously, it was obtained from cadaveric material, from one corpse: 4 - 6 mg of somatotropin in terms of final pharmaceutical product. Thus, the available amounts of the hormone were limited, in addition, the hormone obtained by this method was heterogeneous and could contain slow-growing viruses. In 1980, the company "Genentec" developed a technology for the production of somatotropin using bacteria, which was devoid of these disadvantages. In 1982, human growth hormone was obtained in culture of E. coli and animal cells at the Pasteur Institute in France, and in 1984, industrial production of insulin began in the USSR. In the production of interferon, both E. coli, S. cerevisae (yeast), and a culture of fibroblasts or transformed leukocytes are used. Safe and cheap vaccines are also obtained using similar methods.

Recombinant DNA technology is based on the production of highly specific DNA probes, which are used to study the expression of genes in tissues, the localization of genes on chromosomes, and identify genes with related functions (for example, in humans and chicken). DNA probes are also used in the diagnosis of various diseases.

Recombinant DNA technology has made possible an unconventional protein-gene approach called reverse genetics. In this approach, a protein is isolated from a cell, the gene for this protein is cloned, and it is modified, creating a mutant gene encoding an altered form of the protein. The resulting gene is introduced into the cell. If it is expressed, the cell carrying it and its descendants will synthesize the altered protein. In this way, defective genes can be corrected and hereditary diseases can be treated.

If the hybrid DNA is introduced into a fertilized egg, transgenic organisms can be produced that express the mutant gene and pass it on to their offspring. Genetic transformation of animals makes it possible to establish the role of individual genes and their protein products both in the regulation of the activity of other genes and in various pathological processes. With the help of genetic engineering, lines of animals resistant to viral diseases have been created, as well as breeds of animals with traits beneficial to humans. For example, microinjection of recombinant DNA containing the bovine somatotropin gene into a rabbit zygote made it possible to obtain a transgenic animal with hyperproduction of this hormone. The resulting animals had pronounced acromegaly.

Now it is even difficult to predict all the possibilities that will be realized in the next few decades.

Lecture 5. Integrated processing of biological raw materials

Integrated processing of biological raw materials is understood as a set of technological processes (technologies) aimed at obtaining products of various natures from one source. Such a source can be the biomass of industrial microorganisms, algae, plant and animal cells and agricultural waste.

At the same time, it is important that the cost of all products of complex processing of raw materials is lower than the sum of the costs of each type of commercial product obtained in production, taking into account the costs of environmental measures. This is of particular importance when processing biological raw materials, which includes natural biopolymers of protein, carbohydrate, lipid and nucleotide nature. Cells containing them in significant quantities are of interest for complex processing, since they make it possible to isolate valuable products from them, primarily for food and medical purposes.

Differences in the physicochemical properties of natural biopolymers predetermine the choice of technological methods for their isolation and purification. For example, the depth of complex processing of microbiological raw materials may be different. The technologies used in it must be flexible, and the volume of products must meet the needs of the market. When processing microbial mass to obtain lipid products, bacteria, yeast, microscopic fungi and algae are used. Polynucleotide and protein products are obtained from the biomass of bacteria and yeast.

In the biotechnological production of products, the basis is equipment, especially equipment associated with the fermentation stage, as it determines the composition and properties of bioproducts and culture fluid. In addition, in most cases, it is at the fermentation stage that the main economic indicators of biotechnological production and the competitiveness of the resulting bioproducts are laid down.

There are various biotechnological methods for intensifying fermentation: the use of a more active producer strain, hardware improvement, optimization of the composition of the nutrient medium and cultivation conditions, the use of biostimulants, emulsifiers, etc. All of them are capable of ensuring maximum productivity of the biotechnological process and increasing the yield of the final product.

At the same time, the equipment has the most significant influence on the nature of the fermentation process and its final technological indicators. Considering the variety of fermentation apparatuses currently used in biochemical production, we can conclude that in all reactors certain physical processes (hydrodynamic, thermal and mass transfer) occur, with the help of which optimal conditions are created for the actual biochemical transformation of the substance (biochemical reaction).

To carry out these physical processes, the biochemical reactor is equipped with standard structural elements, which are also widely used in chemical apparatus for carrying out physical processes themselves (stirrers, contact devices, heat exchangers, dispersants, etc.). A fermenter of any design must satisfy the basic requirements of the cell cultivation process: ensure the supply of nutrients to each cell, the removal of metabolic products, ensure the maintenance of optimal operating parameters, the required level of aeration, mixing, a high level of automation, etc.

The importance of biochemistry in biotechnology

Fundamental biochemistry is the basis for many biological sciences, such as genetics, physiology, immunology, microbiology. Advances in cell and genetic engineering in recent years have significantly brought biochemistry closer to zoology and botany. Biochemistry is of great importance for such sciences as pharmacology and pharmacy. Biological chemistry studies various structures at the cellular and organismal levels. The basis of life is a set of chemical reactions that ensure metabolism. Thus, biochemistry can be considered the basic language of all biological sciences. Currently, both biological structures and metabolic processes, thanks to the use of effective methods, have been studied quite well. Many branches of biochemistry have developed so intensively in recent years that they have grown into independent scientific directions and disciplines. First of all, we can note biotechnology, genetic engineering, biochemical genetics, environmental biochemistry, quantum and space biochemistry, etc. The role of biochemistry is great in understanding the essence of pathological processes and the molecular mechanisms of action of medicinal substances.

All living organisms consist of cells and their metabolic products. This was proven in 1838 by M. Schleiden and T. Schwann, who postulated that plant and animal organisms are built from cells arranged in a certain order. 20 years later, R. Virchow formulated the foundations of cell theory, pointing out that all living cells arise from previous living cells. Subsequently, the cell theory developed and was supplemented as methods of cognition improved. Each cell is a separate functional unit that has a number of specific features, depending on its nature. Microorganisms are represented by individual cells or their colonies, and multicellular organisms, such as animals or higher plants, consist of billions of cells connected to each other. The cell is a kind of factory in which diverse and coordinated chemical processes are carried out, just like in a real factory, the cell has a control center, areas for monitoring certain reactions, and regulatory mechanisms. The cell also receives raw materials, which are processed into finished products, and waste, which is thrown out of the cell.

Cells constantly synthesize substances necessary for their vital functions. These substances are increasingly used in industry and medicine. Some of them are unique and cannot be obtained by chemical synthesis.

Insulin is a pancreatic hormone that plays a vital role in the body. It is this substance that promotes adequate absorption of glucose, which in turn is the main source of energy and also nourishes brain tissue.

Diabetics who are forced to take the hormone by injection sooner or later think about what insulin is made from, how one drug differs from another, and how artificial analogues of the hormone affect a person’s well-being and the functional potential of organs and systems.

Differences between different types of insulin

Insulin is a vital drug. People suffering from diabetes cannot do without this remedy. The pharmacological range of medications for diabetics is relatively wide.

The drugs differ from each other in many aspects:

Every year in the world, leading pharmaceutical companies produce colossal amounts of “artificial” hormone. Insulin manufacturers in Russia also contributed to the development of this industry.

Every year, diabetics around the world consume over 6 billion units of insulin. Given the negative trends and the rapid increase in the number of patients with diabetes, the need for insulin will only increase.

Sources for obtaining the hormone

Not every person knows what insulin for diabetics is made from, but the origin of this most valuable drug is really interesting.

Modern insulin production technology uses two sources:

• Animals. The drug is obtained by treating the pancreas of cattle (less commonly), as well as pigs. Bovine insulin contains as many as three “extra” amino acids, which are foreign in their biological structure and origin to humans. This can cause the development of persistent allergic reactions. Porcine insulin is distinguishable from the human hormone by only one amino acid, which makes it much safer. Depending on how insulin is produced and how thoroughly the biological product is purified, the degree to which the drug is accepted by the human body will depend;
• Human analogues. Products in this category are produced using the most sophisticated technologies. Leading pharmaceutical companies have established the production of human insulin in bacteria for medicinal purposes. Enzymatic transformation techniques are widely used to obtain semi-synthetic hormonal products. Another technology involves the use of innovative genetic engineering techniques to obtain unique DNA recombinant insulin formulations.

How insulin was obtained: the first attempts of pharmacists

Drugs obtained from animal sources are considered drugs produced using old technology. Medicines are considered to be of relatively low quality due to insufficient purification of the final product. In the early 20s of the last century, insulin, even though it caused severe allergies, became a real “pharmacological miracle” that saved the lives of insulin-dependent people.

The first releases of the drugs were also difficult to tolerate due to the presence of proinsulin in the composition. Hormonal injections were particularly poorly tolerated by children and the elderly. Over time, this impurity (proinsulin) was removed by more thorough purification of the composition. They abandoned bovine insulin altogether, as it almost always caused side effects.

What is insulin made of: important nuances

In modern therapeutic regimens for patients, both types of insulin are used: both animal and human origin. The latest developments make it possible to produce products of the highest degree of purification.

Previously, insulin could contain a number of undesirable impurities:

Previously, such “supplements” could cause serious complications, especially in patients who were forced to take large doses of the drug.

Improved medicines are free of unwanted impurities. If we consider insulin of animal origin, the best product is the monopeak product, which is produced with the production of a “peak” of the hormonal substance.

Duration of pharmacological effect

The production of hormonal drugs has been established in several directions at once. Depending on how the insulin is made will determine how long it lasts.

The following types of drugs are distinguished:

Ultra-short-acting drugs

Ultra-short-acting insulins act literally in the first seconds after administration of the drug. The peak of action occurs after 30 – 45 minutes. The total time of exposure to the patient’s body does not exceed 3 hours.

Typical representatives of the group: Lizpro and Aspart. In the first version, insulin is produced by rearranging amino acid residues in the hormone (we are talking about lysine and proline). In this way, the risk of hexamers occurring during production is minimized. Due to the fact that such insulin quickly breaks down into monomers, the process of absorption of the drug is not accompanied by complications and side effects.

Aspart is produced in a similar way. The only difference is that the amino acid proline is replaced with aspartic acid. The drug quickly breaks down in the human body into a number of simple molecules and is instantly absorbed into the blood.

Short-acting drugs

Short-acting insulins are presented in buffer solutions. They are intended specifically for subcutaneous injections. In some cases, a different format of administration is allowed, but such decisions can only be made by a doctor.

The drug begins to “work” after 15 – 25 minutes. The maximum concentration of the substance in the body is observed 2 - 2.5 hours after injection.

In general, the drug affects the patient’s body for about 6 hours. Insulins of this category are created for the treatment of diabetics in a hospital setting. They allow you to quickly remove a person from a state of acute hyperglycemia, diabetic precoma or coma.

Intermediate-acting insulin

The drugs slowly enter the bloodstream. Insulin is produced according to a standard procedure, but the composition is improved at the final stages of production. To increase their hypoglycemic effect, special prolonging substances - zinc or protamine - are added to the composition. Most often, insulin is presented in the form of suspensions.

Long acting insulin

Long-acting insulins are the most modern pharmacological products today. The most popular drug is Glargine. The manufacturer has never hidden what human insulin for diabetics is made from. Using DNA recombinant technology, it is possible to create an exact analogue of the hormone that is synthesized by the pancreas of a healthy person.

To obtain the final product, an extremely complex modification of the hormone molecule is carried out. Replace asparagine with glycine, adding arginine residues. The drug is not used to treat comatose or precomatous conditions. It is prescribed only subcutaneously.

The role of excipients

It is impossible to imagine the production of any pharmacological product, in particular insulin, without the use of special additives.

Auxiliary components help improve the chemical qualities of the drug, as well as achieve the maximum degree of purity of the composition.

According to their classes, all additives for insulin-containing drugs can be divided into the following categories:

1. Substances that predetermine the prolongation of drugs;
2. Disinfecting components;
3. Acidity stabilizers.

Prolongators

In order to extend the time of exposure to the patient, prolonging drugs are added to the insulin solution.

Most often used:

Antimicrobial components

Antimicrobial components extend the shelf life of medications. The presence of disinfecting components helps prevent the proliferation of microbes. These substances, by their biochemical nature, are preservatives that do not affect the activity of the drug itself.

The most popular antimicrobial additives used in insulin production are:

Each specific drug uses its own special additives. Their interaction with each other is necessarily studied in detail at the preclinical stage. The main requirement is that the preservative should not interfere with the biological activity of the drug.

A high-quality and skillfully selected disinfectant allows you not only to maintain the sterility of the composition over a long period, but even to make intradermal or subcutaneous injections without first disinfecting the dermal tissue. This is extremely important in extreme situations when there is no time to treat the injection site.