VACCINES DEFINATION AND GENERAL REQUIREMTS OF VACCINES

VACCINES DEFINATION AND GENERAL REQUIREMTS OF VACCINES
VACCINES DEFINATION AND GENERAL REQUIREMTS OF VACCINES

Vaccines for human use are preparations containing substances capable of inducing a specific and active immunity in man against an infecting agent or the toxin or the antigen elaborated by it. They shall have been shown to have acceptable immunogenic activity in man with the intended vaccination schedule. They may contain an adjuvant.

Vaccines for human use may contain: organisms inactivated by chemical or physical means that maintain adequate immunogenic properties; living organisms that are naturally avirulent or that have been treated to attenuate their virulence whilst retaining adequate immunogenic properties; antigens extracted from the organisms or secreted by them or produced by genetic engineering; the antigens may be used in their native state or may be detoxified by chemical or physical means and may be aggregated, polymerised or conjugated to a carrier to increase their immunogenicity.


Bacterial vaccines are suspensions of various degrees of opacity in colourless or almost colourless liquids, or may be freeze-dried. The concentration of living or inactivated bacteria is expressed in terms of International Units of opacity or, where appropriate, is determined by direct cell count or, for living bacteria, by viable count.

Bacterial toxoids are prepared from toxins by diminishing their toxicity to a non- detectable level or by completely eliminating it by physical or chemical procedures whilst retaining adequate immunogenic properties. The toxins are obtained from selected strains of micro-organisms. The method of production is such that the toxoid does not revert to toxin. Toxoids may be liquid or freeze-dried. They may be purified and adsorbed. Adsorbed toxoids are suspensions of white or grey particles dispersed in colourless or pale yellow liquids and may form a sediment at the bottom of the container.
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Viral vaccines are prepared from viruses grown in animals, in fertilised eggs, in suitable cell cultures or in suitable tissues or by culture of genetically engineered cells. They are liquids that vary in opacity according to the type of preparation or may be freeze-dried. Liquid preparations and freeze-dried preparations after reconstitution may be coloured if a pH indicator such as phenol red has been used in the culture medium.

The production method for a given product must have been shown to yield consistently batches comparable with the batch of proven clinical efficacy and safety in man. Where justified and authorised, certain tests may be omitted where it can be demonstrated, for example by validation studies, that the production process consistently ensures compliance with the test.

Unless otherwise justified and authorised, vaccines are produced using a seed-lot system. The methods of preparation are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agents.

Where vaccines for human use are manufactured using materials of human or animal origin, Chicken flocks free from specified pathogens for the production and quality control of vaccines, Cell substrates for the production of vaccines for human use and. Tests for extraneous agents in viral vaccines for human use,
Unless otherwise justified and authorised, in the production of a final lot of vaccine, the number of passages of a virus, or the number of subcultures of a bacterium, from the master seed lot shall not exceed that used for production of the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy.

Vaccines are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man. Suitable additives, including stabilisers and adjuvants may be incorporated. Penicillin and streptomycin are not used at any stage of production nor added to the final product; however, master seed lots prepared with media containing penicillin or streptomycin may, where justified and authorised, be used for production.

Consistency of production is an important feature of vaccine production. vaccines for human use give limits for various tests carried out during production and on the final lot. These limits may be in the form of maximum values, minimum values or minimum and maximum tolerances around a given value. While compliance with these limits is required, it is not necessarily sufficient to ensure consistency of production for a given vaccine. For relevant tests, the manufacturer must therefore define for each product a suitable action or release limit or limits to be applied in view of the results found for batches tested clinically and those used to demonstrate consistency of production. These limits may be subsequently refined on a statistical basis in the light of production data.

Substrates for propagation

Substrates for propagation comply with the relevant requirements of the Pharmacopoeia in the absence of such requirements with those of the competent authority. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions shall be shown to be free from extraneous agents.

Seed lots

The strain of bacterium or virus used in a master seed lot is identified by historical records that include information on the origin of the strain and its subsequent manipulation. Suitable measures are taken to ensure that no micro-organism other than the seed strain is present in a seed lot.

Culture media

Culture media are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man; if inclusion of such ingredients is necessary, it shall be demonstrated that the amount present in the final lot is reduced to such a level as to render the product safe. Approved animal (but not human) serum may be used in the growth medium for cell cultures but the medium used for maintaining cell growth during virus multiplication shall not contain serum, unless otherwise stated. Cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration although it is preferable to have a medium free from antibiotics during production.

Propagation and harvest

The seed cultures are propagated and harvested under defined conditions. The purity of the harvest is verified by suitable tests.

Control cells

For vaccines produced in cell cultures, control cells are maintained and tested as prescribed. In order to provide a valid control, these cells must be maintained in conditions that are essentially equivalent to those used for the production cell cultures, including use of the same batches of media and media changes.

Control eggs

For live vaccines produced in eggs, control eggs are incubated and tested
Purification

validated purification procedures may be applied.

Inactivation

Inactivated vaccines are produced using a validated inactivation process whose effectiveness and consistency have been demonstrated. Where there are recognised potential contaminants of a harvest, for example in vaccines produced in eggs from healthy, non-SPF flocks, the inactivation process is also validated with respect to the potential contaminants. A test for effectiveness of the inactivation process is carried out as soon as possible after the inactivation process.

Stability of intermediates

During production of vaccines, intermediates are obtained at various stages and are stored, sometimes for long periods. Such intermediates include
seed lots;
live or inactivated harvests from bacterial or viral cultures;

purified harvests that may consist of toxins or toxoids, polysaccharides, bacterial or viral
suspensions;
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  purified antigens;

  adsorbed antigens;

  conjugated polysaccharides;

final bulk vaccine;

vaccine in the final closed container stored at a temperature lower than that used for stability studies and intended for release without re-assay.

Except where they are used within a short period of time, stability studies are carried out on the intermediates in the intended storage conditions to establish the expected extent of degradation. For final bulk vaccine, stability studies may be carried out on representative samples in conditions equivalent to those intended to be used for storage. For each intermediate (except for seed lots), a period of validity applicable for the intended storage conditions is established, where appropriate in the light of stability studies.


Final bulk

The final bulk is prepared by aseptically blending the ingredients of the vaccine.

Adsorbents
Vaccines may be adsorbed on aluminium hydroxide, aluminium phosphate, calcium phosphate or other suitable adsorbent; the adsorbents are prepared in special conditions which confer the appropriate physical form and adsorptive properties.

Antimicrobial preservatives
Antimicrobial preservatives are used to prevent spoilage or adverse effects caused by microbial contamination occurring during the use of a vaccine. Antimicrobial preservatives are not included in freeze-dried products. For single-dose liquid preparations, inclusion of antimicrobial preservatives is not normally acceptable. For multidose liquid preparations, the need for effective antimicrobial preservation is evaluated taking into account likely contamination during use and the maximum recommended period of use after broaching of the container. If an antimicrobial preservative is used, it shall be shown that it does not impair the safety or efficacy of the vaccine. Addition of antibiotics as antimicrobial preservatives is not normally acceptable.

During development studies, the effectiveness of the antimicrobial preservative throughout the period of validity shall be demonstrated to the satisfaction of the competent authority.

The efficacy of the antimicrobial preservative is evaluated as described , If neither the A criteria nor the B criteria can be met, then in justified cases the following criteria are applied to vaccines for human use: bacteria, no increase at 24 h and 7 days, 3 log reduction at 14 days, no increase at 28 days; fungi, no increase at 14 days and 28 days.

Final lot

For vaccines for parenteral administration, the final lot is prepared by aseptically distributing the final bulk into sterile tamper-proof containers which, after freeze- drying where applicable, are closed so as to exclude contamination. For vaccines for administration by a non-parenteral route, the final lot is prepared by distributing the final bulk under suitable conditions into sterile, tamper-proof containers.

Appearance

Each container (vial, syringe or ampoule) in each final lot is inspected visually or mechanically for acceptable appearance.

Degree of adsorption
During development of an adsorbed vaccine, the degree of adsorption is evaluated as part of the consistency testing. A release specification for the degree of adsorption is established in the light of results found for batches used in clinical testing. From the stability data generated for the vaccine it must be shown that at the end of the period of validity the degree of adsorption will not be less than for batches used in clinical testing.

Stability
During development studies, maintenance of potency of the final lot throughout the period of validity shall be demonstrated; the loss of potency in the recommended storage conditions is assessed and excessive loss even within the limits of acceptable potency may indicate that the vaccine is unacceptable.

Expiry
Unless otherwise stated, the expiry date is calculated from the beginning of the assay or from the beginning of the first assay for a combined vaccine. For vaccines stored at a temperature lower than that used for stability studies and intended for release without re-assay, the expiry date is calculated from the date of removal from cold storage. If, for a given vaccine, an assay is not carried out, the expiry date for the final lot is calculated from the date of an approved stability-indicating test or failing this from the date of freeze-drying or the date of filling into the final containers. For a combined vaccine where components are presented in separate containers, the expiry date is that of the component which expires first.

The expiry date applies to vaccines stored in the prescribed conditions.

Animal tests

In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, tests must be carried out in such a way as to use the minimum number of animals and to cause the least pain, suffering, distress or lasting harm. The criteria for judging tests must be applied in the light of this. For example, if it is indicated that an animal is considered to show positive, infected etc. when typical clinical signs or death occur then as soon as sufficient indication of a positive result is obtained the animal in question shall be either humanely destroyed or given suitable treatment to prevent unnecessary suffering. In accordance with the General Notices, alternative test methods may be used to demonstrate compliance with the . and the use of such tests is particularly encouraged when this leads to replacement or reduction of animal use or reduction of suffering.

TESTS

Vaccines comply with the tests prescribed in individual .s including, where applicable, the following:

pH

Liquid vaccines, after reconstitution where applicable, comply with the limits for pH approved for the particular preparation.

Adjuvant

If the vaccine contains an adjuvant, the amount is determined and shown to be within acceptable limits with respect to the expected amount (see also the tests for aluminium and calcium below).

Aluminium

Maximum 1.25 mg of aluminium (Al) per single human dose where an aluminium adsorbent has been used in the vaccine, unless otherwise stated.

Calcium

Maximum 1.3 mg of calcium (Ca) per single human dose where a calcium adsorbent has been used in the vaccine, unless otherwise stated.

Free formaldehyde

Maximum 0.2 g/l of free formaldehyde is present in the final product where formaldehyde has been used in the preparation of the vaccine, unless otherwise stated.

Phenol

Maximum 2.5 g/l is present in the final product where phenol has been used in the preparation of the vaccine, unless otherwise stated.

Water

Maximum 3.0 per cent m/m for freeze-dried vaccines, unless otherwise stated.

Extractable volume

Unless otherwise justified and authorised, it complies with the requirement for extractable volume.

STORAGE

Store protected from light. Unless otherwise stated, the storage temperature is 5 ± 3 °C; liquid adsorbed vaccines must not be allowed to freeze.

LABELLING

The label states:

 — the name of the preparation;

 — a reference identifying the final lot;

 — the recommended human dose and route of administration;

 — the storage conditions;

 — the expiry date;

 — the name and amount of any antimicrobial preservative;

 — the name of any antibiotic, adjuvant, flavour or stabiliser present in the vaccine;

 — the name of any constituent that may cause adverse reactions and any contra-indications to the use of the vaccine;

 — for freeze-dried vaccines:
  — the name or composition and the volume of the reconstituting liquid to be added;
  — the time within which the vaccine is to be used after reconstitution.

Sweet hopes for diabetics Good news for diabetics.

Sweet hopes for diabetics Good news for diabetics.

A three-year study carried out by Universiti Teknologi Malaysia in Skudai has confirmed previous findings that cinnamon has the potential to lower sugar levels.

UTM research and development manager Prof Dr Mohammad Roji Sarmidi said their research showed that the spice, known as kayu manis locally, has positive effects on the disease, especially Type II diabetes.
Type II diabetes causes cells to lose their ability to respond to insulin, the hormone that tells the body to remove excess glucose from the bloodstream. This condition usually develops in people in their middle age and prematurely kills an estimated 100 million of the world’s population every year. Dr Mohammad Roji said herbalists all over the world had used cinnamon in the treatment of diarrhoea and arthritis, as cinnamon extract was found to improve blood circulation, heal wounds, reduce pain spasm and prevent ulcer and allergies.
“In the last decade, laboratory studies have also revealed that cinnamon extract mimicked insulin action in the cells,” he said. Insulin regulates glucose metabolism, helping body cells to convert glucose to energy and keep blood sugar levels normal. “Studies by the Agriculture Research Service in the United States have also found that certain substances in cinnamon helps cells become more responsive to insulin,” he added. Cinnamon is an ingredient used in cooking, and in cakes, pastries and beverages like coffee and tea.
Dr Mohammad Roji said UTM would conduct further studies next year, which would cover tests on animals and metabolic profiling for diabetic patients.

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Technorati Profile

Hybridoma - a technique for eternal production of monoclonal antibodies in cell cultures

An explanation of the diversity of the immune system
The most important task for the immune system is to defend the body against bacteria, virus and other microorganisms. The specific defense is exerted by a subgroup of white blood cells (lymphocytes). The immune system needs to recognize and react specifically with a large number of foreign substances (antigens). How the lymphocytes develop these vital properties and how they build up the highly specialized recognition system of the immune apparatus has long been an area of intensive research.
Niels K. Jerne is the leading theoretician in immunology during the last 30 years. In three main theories he has elucidated central issues concerning specificity, development and regulation of the immune system in a comprehensive and convincing way. By his theories Jerne has outlined the development of modern immunology.
Theory 1: Specificity is predeterminedIn his Natural-Selection
Theory of Antibody Formation from 1955 Jerne explains the development of a specific antibody response in the following way. Each individual has a large number of natural antibodies with specificities for all antigens towards which the individual can respond. These antibodies develop already during fetal life in the absence of external antigens. The foreign antigen then selects the antibody molecule which has the best fit. The antigen-antibody binding stimulates the production of this particular antibody specificity.Jerne's natural-selection theory contrasted to the dogmatic views of the antibody response as formulated in the instruction theories which were prevailing at that time. According to these theories the antigen serves a template for the production of antibodies.In Jerne's natural selection theory it is implied that the generation of the enormous number of antibody specificities is independent of exogenous antigens. This view on the nature of the immune system constitutes the basis for modern immunology.
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Theory 2: Reactivity against self-antigens creates diversity
The natural-selection theory is mainly concerned with the maturation of the immune system after it has acquired the ability to react with antigen. In the second theory on the Somatic Generation of Immune Recognition set forth in 1971 Jerne explains how the immune system develops from stem cells to mature lymphocytes which can react with antigen. He presupposes that every individual possesses all genes needed for the production of antibodies, and antibody-like molecules, which can bind all strong transplantation antigens of the species. Jerne suggests that lymphocytes mature in the thymus gland and in other lymphoid organs where they are exposed to the transplantation antigens of the individual. Cells which recognize the antigens are stimulated and enter cell division. As mutations accumulate in rapidly dividing cells new immunological specificities may develop. At the same time the specificities of the lymphocytes for self transplantation antigens are weakened. The mature lymphocytes will recognize foreign antigen associated with transplantation antigens. The theory explains how the immune system normally matures through the influence of self antigens. It also offers an explanation for the regulation of immunological specificity by genes belonging to the transplantation system.
Theory 3. Antibodies, anti-anti-bodies ...In his third main theory, the Network Theory from 1974, Jerne explains how the specific immune response is regulated. The theory has greatly stimulated research and led to new insights into the immune system. Recently its principles have been applied to diagnosis and treatment of disease.
A basis for the network theory was the observation that antibodies can elicit anti-antibodies directed against antigen binding structures on the first antibody .Moreover, anti-antibodies can stimulate the production of still another generation of antibodies, anti-anti-antibodies. Essentially, this antibody cascade is endless successively adding new specific properties to the immune system. The various antibody generations either stimulate or suppress the production of one another. Under normal conditions the network is balanced. When an antigen is introduced the equilibrium is disturbed. The immune system tries to restore balance which leads to an immune response against the antigen.1. Infectious diseases. Anti-antibodies have been used in animals as a kind of vaccine against parasitic infections (trypanosomiasis), urinary tract infections, hepatitis and other infectious diseases.
2. Allergy. Anti-pollen antibodies may elicit allergic symptoms when an allergic person is exposed to pollen. The production of anti-pollen antibodies has been prevented in animals by anti-antibodies.
3. Autoimmune disease. Autoimmune disease may be caused by antibodies directed against the body's own tissues. Experimental autoimmune disease has been successfully treated with anti-antibodies.
4. Transplantation. Anti-antiimmunity may be important in organ transplantation by contributing to immunological tolerance against antigen on the foreign graft.
5. Endocrinology. Anti-antibodies against hormones and hormone receptors may prevent binding of the hormone to the receptors. This has been described for insulin and its receptor.
6. Tumours. Anti-antibodies have been attempted as treatment of certain tumours of the human immune system.
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Hybridoma - a technique for eternal production of monoclonal antibodies in cell cultures
Besides gene technology, which has already been honoured by several Nobel Prizes, the hybridoma technique represents the most important methodological advance within the field of biomedicine during the 1970s. The development of this technique is based on several observations concerning basic biological phenomena.
There are cells in the body - immune lymphocytes - which can produce millions of different antibodies. However, each single cell can only produce antibodies with a certain predetermined specificity. A prerequisite for the formation of a multitude of antibodies is, therefore, the existence of an excess of lymphocytes. If the body is exposed to a certain foreign antigen there may be stimulation of a lymphocyte which fortuitously has been endowed with the capacity to identify this particular antigen. This lymphocyte then starts to divide and forms a clone of cells which produces identical - monoclonal - antibodies.
The development of a clone of cells in connection with a normal immune response occurs under carefully controlled conditions. In rare cases, however, the body loses control over a clone of antibody producing cells. This may lead to formation of a special type of tumour (myeloma). Myeloma cells usually retain their capacity to produce a certain antibody, but because of the accidental emergence of the tumour one normally does not know with which antigen this antibody reacts.
White blood cells responsible for producing antibodies are highly specialized cells. As a consequence they lack capacity to survive for a longer time if they are removed from the body and incubated in a tissue culture medium. In contrast, myeloma cells can occasionally be cultivated continuously. Since long, biomedical research workers have nourished the dream to be able to propagate clones of cells which produce antibodies with predetermined specificity. This dream materialized when Georges J.F. Köhler and César Milstein in 1975 introduced the so-called hybridoma technology for production of monclonal antibodies. The principle features of the hybridoma technology is as follows
Figure 2. Principle steps in the production of a hybridoma. Spleen cells are prepared from animals, usually mice, which have been immunized with a selected antigen. These cells are then fused with myeloma cells maintained in culture in the laboratory. The product of this fusion is referred to as a hybridoma. Surprisingly, a hybrid of two cells can survive and also continue to divide. In this particular hybrid the myeloma cells contribute the capacity for survival, whereas the spleen cells direct the synthesis of antibodies with the preselected specificity. By special arrangements it is possible to achieve a multiplication of hybridoma cells but not of isolated myeloma cells. The hybrids obtained are propagated in a highly diluted state so that colonies deriving from single hybrid cells can be isolated. By use of a sensitive method the clones which produce the specific antibodies are identified. A particular hybridoma can then be used for future, unlimited production of a highly specific antibody.
The availability of monoclonal antibodies has opened completely new possibilities for basic as well as applied biomedical research. The following examples of the use of monoclonal antibodies can be given.
1. Detailed studies of the distribution of different functions in different parts of antigen molecules. These studies may concern building elements of infectious agents; cell products such as enzymes and hormones; surface structures of cells etc. The mapping of variations in the surface components of influenza virus which explain the occurrence of repeated infections is one example.
2. High degree purification of substances, e.g. interferon, by taking advantage of the unique capacity displayed by a particular monoclonal antibody to bind to a certain antigen. In this case one uses a technique referred to as affinity chromatography.
3. Diagnostic characterization of diseases by identification of special structures on the surface or on the inside of cells. Hereby it is possible to distinguish between different forms of tumours and follow the development of tumours. Furthermore, it is possible to distinguish between different kinds of normal white blood cells. This is of importance for the characterization of certain immune deficiency conditions as seen e.g. in connection with the disease AIDS (acquired immune deficiency syndrome).Diseases caused by infectious agents can also be diagnosed by use of monoclonal antibodies. Thus, virus infected cells and bacteria or parasites inside or outside cells can be identified with a unique degree of specificity.
4. Treatment of AIDS diseases. Monoclonal antibodies against specialized white blood cells have been used with some success in connection with transplantation. There may also be possibilities to use monoclonal antibodies for treatment of tumours.

Recombinant DNA Technology in the Synthesis of Human Insulin

Recombinant DNA Technology in the Synthesis of Human Insulin
Since Banting and Best discovered the hormone, insulin in 1921. diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.
Although bovine and porcine insulin are similar to human insulin, their composition is slightly different.Consequently, a number of patients' immune systems produce antibodies against it, thus neutralising its actions and resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance, as well as a projected decline in the production of animal derived insulin.These factors led researchers to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart.
This has been achieved using Recombinant DNA technology.
This method is a more reliable and sustainable method than extracting and purifying the abattoir by-product.Understanding the genetics involved.
The structure of insulin.
Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains are linked by a disulfide bond.
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Inside the Double Helix.
The genetic code for insulin is found in the DNA at the top of the short arm of the eleventh chromosome. It contains 153 nitrogen bases (63 in the A chain and 90 in the B chain).DNA Deoxyribolnucleic Acid), which makes up the chromosome, consists of two long intertwined helices, constructed from a chain of nucleotides, each composed of a sugar deoxyribose, a phosphate and nitrogen base. There are four different nitrogen bases, adenine, thymine, cytosine and guanine.The synthesis of a particular protein such as insulin is determined by the sequence in which these bases
are repeated .
Insulin synthesis from the genetic code.
The double strand of the eleventh chromosome of DNA divides in two, exposing unpaired nitrogen bases which are specific to insulin production .
The role of the mRNA strand, on which the nitrogen base thymine is replaced by uracil, is to carry genetic information, such as that pertaining to insulin,from the nucleus into the cytoplasm, where it attaches to a ribosome
The nitrogen bases on the mRNA are grouped into threes, known as codons. Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to a specific amino acid, collectively known as an anti-codon pair with complementary bases (the codons) on the mRNA. The Vector (Gram negative E. coli).
A weakened strain of the common bacterium, Escherrichia coli (E. coli), an inhabitant of the human digestive tract, is the 'factory' used in the genetic engineering of insulin. When the bacterium reproduces, the insulin gene is replicated along with the plasmid, a circular section of DNA . E. coli produces enzymes that rapidly degrade foreign proteins such as insulin. By using mutant strains that lack these enzymes, the problem is avoidedIn E. coli, B-galactosidase is the enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the insulin gene needs to be tied to this enzyme. Inside the genetic engineer's toolbox
Restriction enzymes, naturally produced by bacteria, act like biological scalpels, only recognising particular stretches of nucleotides, such as the one that codes for insulin.
This makes it possible to sever certain nitrogen base pairs and remove the section of insulin coding DNA from one organism's chromosome so that it can manufacture insulin . DNA ligase is an enzyme which serves as a genetic glue, welding the sticky ends of exposed nucleotides together.
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Manufacturing Human insulin.

The first step is to chemically synthesise the DNA chains that carry the specific nucleotide sequences characterising the A and B polypeptide chains of insulin The required DNA sequence can be determined because the amino acid compositions of both chains have been charted. Sixty three nucleotides are required for synthesising the A chain and ninety for the B chain, plus a codon at the end of each chain,signalling the termination of protein synthesis. An anti-codon, incorporating the amino acid, methionine, is then placed at the beginning of each chain which allows the removal of the insulin protein from the bacterial cell's amino acids.
The synthetic A and B chain 'genes' are then separately inserted into the gene for a bacterial enzyme, B-galactosidase, which is carried in the vector's plasmid. At this stage, it is crucial to ensure that the codons of the synthetic gene are compatible with those of the B-galactosidase. The recombinant plasmids are then introduced into E. coli cells. Practical use of Recombinant DNA technology in the synthesis of human insulin requires millions of copies of the bacteria whose plasmid
has been combined with the insulin gene in order to yield insulin. The insulin gene is expressed as it replicates with the B-galactosidase in the cell undergoing mitosisThe protein which is formed, consists partly of B-galactosidase, joined to either the A or B chain of insulin . The A and B chains are then extracted from the B-galactosidase fragment and purified. Biological implications of genetically engineered Recombinant human insulin. Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely identical to that of the natural molecule. This reduces the possibility of complications resulting from antibody production. In chemical and pharmacological studies, commercially available Recombinant DNA human insulin has proven indistinguishable from pancreatic human insulin. Initially the major difficulty encountered was the contamination of the final product by the host cells, increasing the risk of contamination in the fermentation broth. This danger was eradicated by the introduction of purification processes. When the final insulin product is subjected to a battery of tests, including the finest radio-immuno assay techniques, no impurities can be detected. The entire procedure is now performed using yeast cells as a growth medium, as they secrete an almost complete human insulin molecule with perfect three dimensional structure.
This minimises the need for complex and costly purification procedures. The issue of hypoglycaemic complications in the administration of human insulin.
Since porcine insulin was phased out, and the majority of insulin dependent patients are now treated with genetically engineered recombinant human insulin, doctors and patients have become concerned about the increase in the number of hypoglycaemic episodes experienced. Although hypoglycaemia can be expected occasionally with any type of insulin, some people with diabetes claim that they are less cognisant of attacks of hypoglycaemia since switching from animal derived insulin to Recombinant DNA human insulin.(16) In a British study,
published in the 'Lancet", hypoglycaemia was induced in patients using either pork or human insulin, The researchers found "no significant difference in the frequency of signs of hypoglycaemia between users of the two different types of insulin."
An anecdotal report from a British patient who had been insulin dependent for thirty years, stated that she began experiencing recurring, unheralded hypoglycaemia only after substituting Recombinant DNA human insulin for animal derived insulin. After switching back to pork insulin to ease her mind, she hadn't experienced any unannounced hypoglycaemia. Eli Lilly and Co., a manufacturer of human insulin, noted that a third of people with diabetes, who have been insulin dependent for over ten years, "lose their hypoglycaemic warning signals, regardless of the type of insulin they are taking." Dr Simon P. Wolff of the University College of London said in an issue of Nature , "As far as I can make out, there's no fault (with the human insulin)." He concluded, "I do think we need to have a study to examine the possible risk.