Download questions for self-preparation GENERAL CHARACTERISTIC OF

questions for self-preparation
GENERAL CHARACTERISTIC OF PRODUCERS OF BIOLOGICALLY ACTIVE AGENTS AND
MEDICINES. ……………………………………………………………….……………….1
BASES OF THE ORGANIZATION OF BIOTECHNOLOGICAL PRODUCTION OF
MEDICINES…………………………………………………………….……………..…….12
METHODS OF CULTIVATION OF PRODUCERS ………………………………………………13
PRINCIPLE AND DESIGNS OF BIOREACTORS. ……………………………………...………16
AIRPREPARATION IN BIOTECHNOLOGICAL PRODUCTION ………………………………19
PREPARATION OF NUTRIENT MEDIUMS …………………………………………...………..20
RECEIVING THE SOWING MATERIAL ……………………………………………..………...…27
ALLOCATION, CONCOCTION AND PURIFYING OF BIOTECHNOLOGICAL PRODUCTS ..28
DRYING OF BIOTECHNOLOGICAL AND PHARMACEUTICAL PRODUCTION …………..31
STANDARDIZATION OF BIOLOGICAL PRODUCTS …………………………..……………..33
CONTROL AND MANAGEMENT OF BIOTECHNOLOGICAL PROCESSES ………………..35
QUESTIONS FOR SELF-CHECKING:
1 . Fill in the table
Producer type
bacteria
fungi
algas
General characteristic
Industrially significant
representatives
Examples of use /
acheived metabolites
1
2
…
1
2
…
1
2
…
2 . What method of cultivation of microbioobjects is the most technological?
3 . What type of hashing is used at cultivation of animal and plant cells, why?
3 . What method carry out sterilization of technological gas environments for a fermenter?
4 . In what the main complexity consists at sterilization of multicomponent nutrient mediums and
how it overcome?
GENERAL CHARACTERISTIC OF PRODUCERS OF MEDICINES AND
BIOLOGICALLY ACTIVE AGENTS.
From an enormous variety (more than 2 million types) of live organisms of our planet in
biotechnology are investigated for application as producers and are used directly only the 100-th
share of percent. For designation of a producer as well as in other biological disciplines, apply the
binary nomenclature, i.e. the Latin name of a sort and a species behind which specify number of the
strain received by selection, for example, by Aspergillus awamori 16.
Now in biotechnology monocelled and the metaphytes constructed of cells of one type
(bacteria, fungi, algas), and also cells and fabrics of the highest plants and animals are used as
producers. Objects of biotechnology are enzymes, nucleinic acids, prostaglandins, lectins,
neuropeptides and various BAA (biologically active agents). In industrial biotechnology 3 types of
strains are applied:
1) The natural strains improved by natural and artificial selection (by production of a microbic
biomass);
2) The strains achieved as a result of induced mutagenesis;
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3) Genetic engineered strains (possess the highest genetic instability).
Industrial strains have to meet the following requirements:
1 . Harmlessness for the consumer and the service personnel.
2 . High growth rate of a biomass and target product (biologically active substance - BAS) at
economic consumption of a nutrient medium.
3 . The directed biosynthetic activity at the minimum formation of by-products.
4 . Genetic uniformity and stability in the relation to substrate and cultivation conditions.
5 . Absence of toxic substances in a target product and industrial drains.
6 . Resistance to phages and other foreign micro flora.
7 . Ability to grow on cheap and available substrate, waste of the food and chemical industry at
the high density of cells.
Only on set of these and other properties it is possible to estimate usefulness and profitability of
a producer. Bacteria of the sort Clostridium, Thermoanaerobacter, Bacillus, Acetobacter,
Pseudomonas, Brevibacterium are the most studied and are more often applied in biotechnology.
Let's characterize their features as producers in biotechnology.
Bacteria have very high speed of reproduction; their cells divide every 30-60 minutes (some
types - 8-10 minutes). They can process per day the volume of a biomass exceeding mass of a cell at
30-40 times (the mass of 10-12 g, volume - 10-12 ml), are capable to form a biomass of 1010 cells in 24 days.
Actually it doesn't occur as various limiting factors work. But possibilities of bacteria to fast
reproduction much more surpass other types of organisms, and this their property is the major by
production of microbic protein and BAS. Bacteria are biochemically universal in the sense that they
can acquire the most various nutrients and even are capable to choose the best organic compounds
from a mix therefore can adapt to the most various living conditions. For example, Pseudomonas
multivorans as a source of carbon can use 90 substances, including carbohydrates and their
derivatives, fatty acids, alcohols, amino acids and even cyclic hydrocarbons (phenol).
Depending on the relation to O2 of bacteria it is accepted to subdivide them into obligate
aerobes, facultative anaerobes, aero-tolerant anaerobes. The majority of producers in microbic
biotechnology are obligate aerobes therefore their cultivation proceeds at continuous inflow of O2,
some producers grow at the low maintenance of O2 (2-10%), they are called microaerofills, and
microaerobic by conditions in which them cultivate. Some representatives of the sort Bacillus,
Escherichia, belong to facultative anaerobes, metan-synthesising bacteria belong to aero tolerant
anaerobes.
The majority of bacteria is cultivated on complex organic mediums containing factors of
growth (vitamins, amino acids, purins, pyrimidines). The producers needing growth factors are called
auxotrophes, the strains which don't find this requirement, are prototrophes. Lactic bacteria can be
carried to auxotrophes. Many producers can grow on the synthetic mediums containing only one
organic substance as a source of carbon. Some producers use methane, methanol, methyled amines as
a power source. In microbic biotechnology ability of a number of bacteria to carry out activity as a
result of oxidation of molecular hydrogen, hydrogen sulfide, ammonium, nitrates, salts of bivalent
iron and some other inorganic connections is widely used. For many producers the labile metabolism
is characteristic.
It is expressed in ability to use a large number of different compounds of carbon, nitrogen, etc.
elements, and also switching from one type of a food on another. The majority of bacteria have
peptidoglicans as a part of a cellular wall (consisting of N-acetilglucosamin and acid Nacetilmuramic).
Archaebacterias, ancient representatives of procariotes, have special value as producers. They
live in mediums with extreme conditions (high concentration of the inorganic substances, increased
temperatures). Among archaebacterias halobacteria represent a great interest for biotechnology. They
grow in the medium containing 20-30% for NaCl (the concentrated solution, the Dead Sea), live on
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dry salty fish, leather products, have the proteins which normal functioning happens only at high
concentration of NaCl. These are sticks, cocci, the squares containing photoactive pigments
bacteriorhodopsin and halorodopsyn. Halorodopsin is capable to turn electromagnetic energy of light
into chemical energy due to which there is a phosphorylation and ATP synthesis. If to immobilize
purple bacteria of Halobacterium on the carrier, when lighting it is possible to receive electricity, ATP
and to desalinate sea water.
In the future it is assumed to provide by means of such microconverters of energy with
electricity certain dwellings in the countries with high solar radiation. Methanogenic bacteria
(archaebacteria) are widely studied in biotechnology as a way of obtaining additional energy from a
renewable substratum. Biological methanogenesis was known in China in the II century BC. In 1911
in Birmingham the plant on anaerobic decomposition of sewage was constructed. The gas generator
turned methane into electricity, providing life of the whole city. Now in China more than 7 million
biogas installations work. The biomass of methanogenic bacteria can be applied as a B12 vitamin
concentrate to agricultural animals.
Sulfur-depending archaebacterias and thermoplasmas also cause a great interest of
biotechnologists. They live in hot and sour reservoirs, in volcanic crevices. Energy receive for the
account of oxidation of H2, S, Fe2 + sulfites of metals. For example, Thermoproteales live at 108 ºС,
(not lower than 80 ºС), anaerobes, and the enzymes synthesized in their cells, possess high
thermoresistance. The main advantage of thermophilic anaerobic archaebacterias consist in the
following:
1. reduction of terms of cultivation;
2. opportunity to do without aeration;
3. reduction of probability of infection.
Actinomyceta is a group of the Gramm-positive bacteria which cells are capable to branching.
External similarity to fungi found reflection in their name ("radiant fungi", actis - a beam, myces - a
mushroom). But actually, these prokaryotic organisms have no any relationship with the fungi being
eucariots. The threads forming a mycelium of Actinomyceta, have diameter of 0,3-1 microns (at fungi
- about 50 microns).
Colonies of many Actinomycetes are colored by various pigments. Many Actinomycetes form
the dense substrate mycelium growing into a nutrient medium. Various chemical compounds with a
wide range of biological action belong to the antibiotics produced by Actinomycetes:
aminoglicosides, tetracyclines, actinomycines, macroleads, acsamycines.
The most important producers of these groups of antibiotics are Streptomyces griseus,
Saccharopolispora hisuta, Micromonospora olivoasterospora, Nocardia mediterranea.
Some main options of use of bacteria for preparation of drugs are known. The most popular is
based on getting biomass and the subsequent its use as a semi-product or a required preparation.
Some vaccines, medical diagnostic bacteriophages are developed this way. Other option is based on
use of bioobjects which collect in the medium of cultivation. Production of amino acids, vitamins,
enzymes, anti-enzymes, antibiotics, polysaccharides is based on this principle.
Microbic cells are used as a protein source mainly in sterns for animals. Also they are used for
carrying out biological transformations. The microbic transformations called by also microbiological
transformations are possible to carry out by means of growing or not growing or even the dried-up
cells, and also dispute. The essence of such transformations is to turn one substance into another,
related on structure, by means of one or several enzymes formed by cells. Unlike the majority of nonbiological chemical reactions biological transformations happen at biological temperatures, and water
serves as solvent. The achieved products differ by high degree of purity, practically they don't contain
by-products. Biological transformation is strictly specific process, i.e. each enzyme catalyzes only
one type of reaction in a specific place of a molecule of a substratum. However at application of
bacteria as producers of pharmaceutical production the structure of their lipids has to be carefully
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studied as some of them (for example, mycobacterium) can contain toxic components. Besides
bacterial cells are small and process of concentration of a biomass is complicated because of that.
Using bacteria as producers of protein and vitamins by production of pharmaceutical
production has a number of priorities:
1 . Possibilities of use of waste of food and chemical productions for cultivation;
2 . Raised content of essential amino acids in bacterial cells in comparison with vegetable
proteins;
3 . High speed of reaction of biosynthesis of protein;
4 . Rather simple technology of commercial cultivation, independent of seasons and other
changing conditions of medium;
5 . Possibility of the directed influence by means of selection methods on a chemical
composition of cells for improvement of biological value of a target product.
However the pharmaceutical production achieved on the basis of bacterial cells has to be
exposed to careful medical and biological check for identification of cancerogenic, mutagen,
embriotropic action on human body.
It is known about 30 species of the bacteria being producers of various biotechnological
productions, including medicinal substances.
At cultivation of bacteria waste of different types of the industry, including natural and passing
gases (hydrogen), and also methanol, ethanol, propanol can be a carbon source. On gas nutrient
mediums sort Methylococcus, Pseudomonas, Methylophillus bacteria are cultivated. In Great Britain
production on methanol of a proteinaceous preparation prutin, protein content in which is 74% from
dry weight is organized. In Russia the technology of industrial production of meprin with use as a
nutrient medium methanol is developed.
In production of proteinaceous preparations it is possible to apply as producers and the
hydrogenium-oxydating bacteria accumulating in cells to 80% of protein, especially in the chemical
companies.
Fungi have similarity both to plants (top, or the apical growth, a strong cellular wall, existence
of a vacuole and cross partitions at many of them), and to animals (heterotrophic type of a feeding,
big or smaller need for vitamins, availability of chitin or chitosan, glycogen synthesis). At the same
time the micelial structure is inherent only in fungi and as a result absorbing way of nutrition
(osmotrophy); for them phenomena of dicariosis (separate location of two kernels in one cell,
capable to simultaneous division and imitating a diploidic kernel) and heterocariosis (location of
different kernels in one cell) are known.
Fungi have diameter of cells in 3-5 times more, than bacteria, and are steadier against phages.
Among fungi as producers of medicinal substances micromycetes (yeast, Penizillum, Aspergillum)
and macromycetes, forming fruit bodies during growth and development are applied.
Specific productivity of fermenter on a biomass at application of bacteria as producers is
higher, than at cultivation of fungi (7 g/kg • h for Candida, 22 g/kg • h for bacteria of Micrococcus
lactis). It is connected not only with the high growth rate of bacteria, but also with ability to
oxidation of wider range of carbohydrates.
In production of alcoholic drinks yeast represents the only industrially used strain of
microorganisms.
Besides production of beer and wine yeast are applied commercially to get technical alcohol
and glycerin, and also as additives to sterns for animals.
As producers are used Saccharomyces cerevisiae, Candida lipolytica. From compounds of
carbon yeast best of all uses hexoses, from polysaccharides utilizes inulin and starch, some can be
cultivated on methanol and ethanol, organic acids. As a nitrogen source by production of yeast are
applied ammonium salts (nitrates, nitrites). The majority of yeast grows in pH 3,0-8,0 borders, the
optimum temperature of cultivation is 28-30 ºС, and beer yeast has wider optimum of temperature.
Alcoholic fermentation at yeast differs from glycolysis at the highest plants only the by last stages
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(ethyl alcohol is formed) that is caused by availability of enzyme pyruvate decarboxylase catalyzing
transformation of pyruvate to ethyl aldehyde which is restored in ethanol then.
Strains of S. cerevisiae are subdivided into races of local and riding fermentation. To races of
local fermentation relate wine and beer yeast, to races of the riding – spirit, baking. Yeast of local
fermentation function at manufacturing at 6-10 ºС, riding – at 14-25 ºС. At the end of fermentation
local yeast settles on a bottom, and riding forms "cap".
At cultivation of yeast on ethanol the outlet on weight makes 30%, fermenter has productivity
2-3 times bigger, than at cultivation on n-alkanes. Ethanol application for manufacture a barmy
biomass doesn't meet psychological objections. In Czech Republic production of a biomass of C.
utilis on ethanol for addition in food of the person for the purpose of improvement of their
organoleptic properties is organized. Yeast are used in production of ergosterine and β-carothene.
From the point of view of production of pharmaceutical production producers of genuses Aspergillus,
Cephalosporium, Fusarium, Penicillum are most important. Antibiotics, organic acids and enzymes
belong to number of the main products of their metabolism.
For cultivation of yeast as a nutrient medium unbranched hydrocarbons from 10-30 by carbon
atoms in a molecule, i.e. liquid fractions of hydrocarbons of oil, and also whey are applied. 1 tonne of
serum contains 10 kg of protein and 50 kg of lactose. Now the effective industrial technology of
producing of protein from whey is developed by an ultrafiltration method which is applied in
production of dry skim milk.
Liquid waste from this production (perliate) is used further for cultivation of fodder yeast. Yeast
is cultivated on methanol and ethanol. At such technology the preparation contains 56-62% of
proteins and the smaller amount of harmful impurity (derivative benzene, amino acids, abnormal
lipids, toxins) is considerable, than at cultivation on oil n-paraffin.
Yeast according to the content of such amino acids as a lysine, threonine, valine and the leucine
considerably exceeds many vegetable proteins.
Proteinaceous preparations from a barmy biomass are applied as food additives. These are beer
and food yeast S. serevisiae, C. arborea, C. ufilis. In USA the compounding of preparation of
sausages from meat of turkey with addition of 25% of barmy protein is developed. In Great Britain in
production of sausages a proteinaceous preparation mucoprotein is applied. 14 types of yeast of
species of Candida are applied in utilization of whey and cultivation of a biomass rich in protein and
vitamins. Rhodotorula glutimis yeast is applied in production of food and medical appointment. Beer
contains S.carlsbergensis yeast not less than 48% of protein, 14 various vitamins and are
characterized by good balance on essential amino acids therefore are widely applied in medicine and
the food industry by production of sausages as casein substitute.
When processing yeast in food protein they are exposed to special transformation. At first walls
of food cells are destroyed (mechanical, alkaline, acid influence or by means of special enzymes),
than homogeneous barmy mass is processed by suitable organic solvent for the purpose of release
from low-molecular impurity and accompanying organic substances. The following stage is
processing by solutions of alkalis for dissolution of proteins and a dialysis. Proteins cleared by means
of such methodical receptions are sedimented, dried up and applied in food technology and medicine.
Micelial fungi form about 1200 antibiotic substances. Penicillin, cephalosporin, trychocetine,
phumaligin, griseofulvin represent the greatest interest for clinical practice. Penicillin is synthesized
by certain species of Penizillum (P. chrysogenium, P. brevicompactum, P. nigricans) and some
species of Aspergillus (A. flavus, A. nidulans). The main producer at industrial production of this
antibiotic is P. chrysogenium during vital activity of which various forms of the penicillin are formed.
They differ by structure of lateral part of a molecule of an antibiotic, biological activity and a range
of antimicrobic action. The most important producer of antibiotics of the cephalosporin’ group
applied in pharmaceutical industry is Cephalosporium acremonium and actinomycet Streptococcus
clavuligereus (cephalosporin C, cephamycin C, cefalexin, cephradin) is. In recent years new chemical
modifications of cephalosporins (cefaparol, cephartisin, cephamandol, cephaxytin) are produced.
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Production of antibiotics on the basis of micromycetes by a biomass in the form of a mycelium
represents big difficulties. It complicates designs of bioreactors and leads to change of hydrodynamic
properties of cultural liquid.
Now basidiomycetes, the fungi forming fruit bodies as foodstuff, are grown up artificially, by
more than 70 countries and world production them are exceeded by 1,5 million tones. It is counted
that fungi as alimentary product many times over surpass production of beef and fish breeding in a
protein exit and have no competitors. They are characterized by unusually fast growth in comparison
with other organisms of fruit bodies. Fungi are an additive to vegetable food poor of protein. At the
same time it is low-calorie food and suits inactive people. The chief producers of fungi are the USA,
France, Japan, Germany, Hungary. In the industry grow up 7-10 types of basidiomycetes: field
mushroom two-sporous, shiitake, wolvariella, oyster mushroom ordinary, black truffle, etc. For
example, sheetake is an export product of Japan. It is grown up in the closed rooms on sawdust with
various additives and on the rice straw moistened with extract from beans, at a temperature of 20 ºС.
From micoriz fungi only a black truffle is cultivated artificially in oak and beechen groves. There is a
firm supplying with micoriz saplings of economy.
Cultivation of mycelium of a field mushroom (etc. champignons) is specialized sterile
microbiological production and is carried out at the plants equipped with modern biotechnological
devices and devices. In our country more than 80 farms are engaged in cultivation of basidiomycetes
as food. Tomorrow of the mushroom industry is their use for the pharmaceutical purposes. For
example, cepe possesses natural antiholesterol and anti-virus action, the mushroom sheetake is
effective at hypertension treatment.
Thus, the problem of artificial cultivation of fungi is the actual biotechnological problem which
decision will allow to reduce the price and simplify utilization of household and industrial wastes, to
give a number of the necessary substances for medicine, without serious expenses to increase amount
of proteinaceous foodstuff for people and forages for cattle.
Algas are also used as producers in biotechnology. They grow more slowly, than fungi. The
general protein content in them can reach 40-70%, and proteins are full on aminoacide structure. At
cultivation of algas it is possible to receive at 2-10 times more solid, than at cultivation of the highest
plants.
Algas as fungi easily separate from a substratum, contain less nucleinic acids in biomass. These
are photosynthesizing organisms; they can be grown up both in photobioreactors, and on
carboniferous substrate. On protein of an alga have advantage in comparison with the highest plants
at 6-30 time. Not less than 100 types of macrofyt algas are used in food in all countries. From them
many dietary dishes are manufactured: salads, seasonings, candies, jam, jelly. Laminaria and
chlorella are the most popular edible and fodder algas.
To macrophyts, applied in human food belong to ulva, alaria, porphyry, rodimenia, chondrus,
undaria, furtsellaria, spirulina.
In Japan cultivation porphyries occupies 60 000 g of the water area.
In our country from the Black Sea algas get fillofor for production of iodine, an agar-agar make
from an anfeltion. The general production of algas is about 3 million t (People's Republic of China,
Japan), from them 2,2 million tons are cultivated. Spirulina is got and cultivated in reservoirs, it is a
traditional food product in the territory of Mexico and the Central Africa. In Russia from it receive
flavoring and proteinaceous and vitamin additives to vegetables, canned food, sauces (contains 9
irreplaceable amino acids). Biomass of chlorell and spyruline apply for replacement of food raw
materials for preparation of nutrient mediums at cultivation of microorganisms, cells of plants and
animals. Chlorella is of interest to creation of artificial ecological systems for life support of crews of
spaceships. It should be noted that use of algas as components of foodstuff are connected with
numerous technological problems:
1 . need of removal of a cellular wall for reduction of quantity of indigestible components;
2 . degreasing as some components of lipid fraction impact to a product unpleasant relish;
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3 . detoxication of pigmented proteins.
Potential producer of biotechnology of a kingdom of animals is the simplest and various groups
of soil invertebrates (earthworms, etc.).
Though protozoa aren't used now in industrial scale neither for production of a biomass, nor
for synthesis of biologically active agents, they along with other microorganisms play large role in
biological sewage treatment of the pharmaceutical enterprises. These processes which are being
widely applyed around the world, from the point of view of the microbiologist, are very difficult.
Sewage is a multicomponent mix of various nutrients and microorganisms. Therefore processing of
drains requires also large number of various representatives of the protozoa competing in
consumption of nutrients.
Protozoa are among nonconventional objects of biotechnology. Now they only win to
themselves a place in research work and the microbiological industry as BAS producers. It is thus
more rational to use free-living protozoa. They are an important component of geological breeds,
soils, the fresh and sea waters, some produce a cellulose multi-enzimatic complex.
The trypanosome became the first producer of an antineoplastic preparation Crucin (Russia,
France), possessing cytotoxic effect at direct contact with a tumor.
Free living flagellates Astasia longa is cultivated to produce Astaliside, possessing
antineoplastic action not through cytotoxic effect, and at impact on a cellular link of immunity.
Evglenic can be considered as perspective producers of glycans and other heteropolysaccharides.
Main producers of medicinal production of animal kingdom
#
Producer
Usage in biotechnology
#
Species
Drug
Organ
1
Human
Eritrocitary mass for hemotransfusion,
Blood
leucocitary mass
Horse, donkey, mule Anti-staphylococcal plasma, heterological
Blood
anti-toxic serums, antidiphtherial, antitetanic,
serums
Maral (Manchurian Pantokrin
Unossified antlers
deer, spotty deer)
Cow, yak
Insulin
Tissues of a pancreas,
Pancreatinum
parathyroid gland,
Paratireoidin
hypophysis, seed plants,
Tireotropin
cartilages
Gialuronidaza
Rumalon
Sheep
Normal erythrocytes for IFA-test
Blood
Goat
Heterological anti-serum to a virus of tickBlood
borne encephalitis
Pig
Pepsin
Stomach mucous
Insulin
Rabbit
Diagnostic serums
Blood
Newborn rabbit
Rage vaccine
Medium for virus
reproduction
Chicken
Lizotsim
Proteinaceous fraction,
Lecithin
yolk of eggs
Snake
Anti-toxic serums (anti-epha, anti-lebetina
Anti-genes for
viper)
immunization
Bees
Bee sting
Fabric of belly glands
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Scorpions
Anti-toxic serum
Poison
The majority of producers used in biotechnology in relation to temperature are mezophils: their
growth and development happens at a temperature 25-37 °C. Psihrophilic microorganisms grow at a
temperature 0-15°C, and thermophylls – at a temperature 60-80°C. All listed groups have
intermediate forms. For biosynthetic processes in industrial production it is desirable to use
thermophyllic microorganisms. Some thermophylls grow at 110ºС, and the microorganisms
developing at t 300 °C and under pressure are found in underwater emissions of superhot springs of
big oceanic depths.
Valuable producers of alcohols, amino acids, enzymes, molecular hydrogen are found among
thermophylls. Growth rate and metabolic activity at them is higher, than at mezophylls. The enzymes
synthesized by thermophylls (protease), have high resistance to heating, action of oxidizers, cleaners
and others to adverse conditions. Application of thermophylls in microbic biotechnology (producers
of Thermus aquaticus and T. caldophilus proteases) allows to lower costs of sterilization of the
industrial equipment and of cooling systems of bioreactors. It will allow to apply fermenter without
bulky heat-exchange devices and to simplify their design.
In microbiological synthesis for each culture of microorganisms there is an optimum, a
minimum and pH maximum. The majority of microorganisms best of all develops at pH 7,0 (neutral).
To acidophyllic microorganisms (some yeast, a mold) it is necessary to have a hydrogen indicator of
the medium 1,5-4,5, basophyllic - pH 8,5-9,5.
Acidophyllic forms don't grow at pH higher than 5,0-5,5, Thiobacillus ferrooxidans meets in
mine waters of fields of sulphidic minerals (pH sometimes less than 1,0). Alkalophylic bacteria grow
at pH more than 10 (some bacteria of the genus Bacillus), decomposing urea to ammonia. In the
industry it is more preferable to apply acidophyllic strains as the foreign microflora in such substrata
perishes and the means applied to sterilization decrease.
High osmotic pressure of the medium is one of the factors limiting growth of microorganisms.
Some yeast (Xeromyces bisporus) and micelial fungi belong to osmophyllic types, they can grow on
the substrata containing 20% of sugar and more.
Now the special importance for production of pharmaceutical production is gained by
researches of processes of reorganization of genetic programs of cells of producers in the direction of
increase in speed of biosynthesis of target products and conversion of a nutrient medium by methods
of modern genetics.
BASES OF THE ORGANIZATION OF BIOTECHNOLOGICAL PRODUCTION OF
MEDICINES
Manufacturing efficiency of pharmaceutical production using biotechnological methods
depends on all composed and the general material and power balance. Thus it is necessary to
remember that in biotechnology there are two active representatives of means of production and
between them there is interference. Really, than rate of functioning of bioobject is higher, especially
high demands are made to hardware registration of processes with its use. Optimization as bioobject,
and processes and devices of biotechnological productions is necessary.
The general technological scheme of production includes:
 preparation of a sowing material or inoculate where a number of stages enters, beginning from
seed test tubes and rocking chairs of flasks before carrying out cultivation in a seeding fermenter;
• preparation of a nutrient medium for a production fermenter which includes a choice and
realization of a compounding of the medium, and also the sterilization guaranteeing safety of all
plastic and power components in initial quantity and quality;
• preparation of the fermentative equipment guaranteeing safety against hit in process of
contaminating flora;
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• biosynthesis stage where possibilities of bioobject are used to the maximum for receiving the
medicinal beginning which is collected in a cell or secreted onto cultural medium;
• the stage of the concoction which has been at the same time intended and for removal of a
ballast;
• the stage of purifying realized at the expense of repetition of a number of the same operations
or at the expense of a set of various preparative receptions of increase of specific activity of the
medicinal generation (ultrafiltration, extraction, sorption, crystallization);
• stage of getting a final substance or medicinal form with the subsequent operations of
packaging and packing;
METHODS OF CULTIVATION OF PRODUCERS
The central stage of pharmaceutical production is fermentation. Fermentation is all set of
consecutive operations from entering into a nutrient medium of a sowing material (inoculate) before
completion of process of growth or biosynthesis of biologically active agents. At the heart of process
of fermentation cultivation of producers, i.e. cultivation of culture of microorganisms, cells of the
highest plants or mold fungi lies.
The culture of microorganisms is the population of the microorganisms which grown up in a
nutrient medium and being in a stage of reproduction or has finished it. At production of drugs and
dietary supplement the following methods of cultivation are applied.
Cultivation methods
Superficial;
Liquid medium
solid medium
(Depth of layer is
about 2 – 3 cm)
interior
continuous
recurrent
With feeding
With dialysis
semi-continuous
detachable-fill-up
The solid-phase superficial fermentation is carried out on the humidified, loose or paste-like
medium. Growth of a producer happens on a surface of firm particles, and also in a time filled with
water or air. Hashing isn't allowed if micromycetes are cultivated. Typical example is silo or compost
preparation in heaps. Operated process of a solid-phase fermentation takes place by production of
enzymes by means of micromycetes.
For superficial cultivation on firm mediums a beet or grape press, a grain peel, wheat or rice
bran with various added nutrients are applied. Optimum humidity of a substratum is 40-70%.
Sterilization is carried out by direct introduction of steam on medium when hashing. If moistening is
carried out by acidified solutions, sterilization conditions are 15-20 min. at 95 ºС. Providing O2 is at a
loss with increase in a layer of a substratum, therefore for each strain or production a peculiar
thickness of a layer of a nutrient medium. At cultivation of micelial fungi hashing isn't allowed. At a
solid-phase superficial fermentation maintenance of constant temperature in all volume of a nutrient
medium is a very big problem. For example, temperature of composts increases from a surface to
deep layers.
The kinetics of growth of population on a surface (film) differs from deep conditions. At solidphase cultivation at the beginning of the culture growth when in the medium there is no gradient of
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concentration of the substratum, all cells can grow in a colony with maximum for this medium
specific growth rate, i.e. under the exponential law. In the central part of a substratum growth of cells
is limited by diffusion and, thus, the biomass grows with the maximum specific speed only on a
surface of a nutrient medium.
Operated process of solid-phase superficial cultivation is applied by production of enzimatic
medicines.
Example: production of amilolitic enzymes. As a nutrient medium wheat bran is used. The sterilized nutrient
medium after seeding of the corresponding strain of a micromycete distribute on ditches and transport in rastilny
cameras where a certain temperature and humidity of air is maintained, and also ventilation along a substratum surface
for the purpose of aeration is provided.
For Aspergillus awamori as producer of amylases distinguish 3 stages of cultivation of culture: 1) Growth prior
to germination of conidia (t - 32º - 38ºС, 100% humidity of air); 2) Stage of an intensive gain of a biomass at t 27°С; 3)
At the third stage stop giving the conditioned air into the camera. The general duration of process is 42 hours. Essential
lack of this method is existence of big floor spaces, difficulty of automation of technological process. Now types of the
mechanized vegetable installations which to some extent eliminate the specified defects of a cuvette way are developed.
Cultivation of biomass of micromycetes is applied on the special trays established on the moving conveyor, and each tray
has special air duct and agitator.
The deep fermentation is characterized by presence of cells in suspension. In the conditions of
laboratory in flasks, of 50-250 ml a liquid nutrient medium in which sow pure culture (from
ampoules or a flask) is poured, then it is placed for 24 hours into a thermostat with a certain
temperature where it grows and breeds. Aerobic producers grow up in special flasks on rocking
chairs in a heat chamber. Here this example is periodic deep cultivation, i.e. growth of a producer is
carried out in the closed system (an exchange of gases, heat), but the nutrient medium and a sowing
material aren't entered in the course of producer growth.
At periodic cultivation some phases in cultural development are marked out.
1 . Latent phase. The culture of microorganisms masters the nutrient medium, the noticeable
increase in number of cells doesn't occur. During this period the cell metabolism is reconstructed, the
enzymes necessary for use of new substrata are synthesized, protein biosynthesis is activated.
2 . The phase of exponential growth is characterized by fast accumulation of a biomass and
metabolism products. Thus a stock of nutrients in the medium in optimum concentration, increase in
number of cells proportionally time, i.e. the linear growth of culture.
3 . The phase of delay of growth is a short period during which the growth rate of culture
decreases that is connected with accumulation of toxic products of a metabolism and an expenditure
of nutrients of the medium.
4 . The stationary phase is a speed of a gain of a biomass is completely compensated by the
speed of death and lysis of cells.
5 . The phase of dying off of culture – is characterized by a substratum starvation,
accumulation of the substances inhibiting growth, the speed of a gain of a biomass is equally to zero.
By production of medicines an important role is played by any phase. So, at production of
primary metabolites reduction to a minimum of a latent phase and increase in an exponential phase
are important. Duration of a latent phase depends on structure of a nutrient medium, age and weight
of inoculate. Transfer of cells from one medium in another, quic change of conditions when scaling
can make versatile impact on a producer. The monitoring system and regulation of fermentative
activity turn on also adaptation the mechanism, i.e., facing new nutrients, cells start acquiring them
only after synthesis of enzymes.
Biotechnologically valuable products are synthesized in an exponential phase (nucleotides,
enzymes, vitamins - primary metabolites). In a stationary phase and a phase of dying off secondary
metabolites - antibiotics, dyes are synthesized.
At continuous cultivation process constantly proceeds in an exponential phase, there is no
change of phases of cultural development, as at periodic cultivation (it is possible to compare to a
greenhouse where vegetation and fructification of plants goes during the whole year). It is possible to
tell that at continuous cultivation activity of culture lasts at the expense of continuous giving of a
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nutrient medium and selection of cultural liquid together with which toxic metabolites are also
removed of the reactor.
Continuous cultivation can be carried out in the conditions of large-capacity production in the
cascade of reactors. For the first time this method was applied in 1915 by production of alcohol in
1940 - to receiving acetone.
Continuous process can be used in production of BAS if the culture at long cultivation doesn't
lose ability to synthesis, i.e. it is genetically steady and uniform.
For Brechibacterium flavium 22 strain (production of lysine) economic efficiency at a
continuous mode is 6 times higher, than at periodic cultivation.
Characterizing continuous cultivation, it should be noted that high efficiency of process can be
reached at bigger value of D (dilution speed), and it can be connected with carrying out of an
unutilized substratum. Therefore, this method cannot always be applied, for example, in production
of antibiotics and other preparations of medical appointment.
PRINCIPLE AND DESIGNS OF BIOREACTORS.
In industrial manufacturing of pharmaceutical production the strain producer is the main
productive force. That’s why the cultivation stage in special bioreactors or fermenters is the central
stage of industrial production.
The principle of deep cultivation of populations of microorganisms in aerobic conditions
consists in continuous inflow to the fermentative medium of a source of O2 - air - at intensive hashing
of a nutrient medium. At design of fermenter kinetic features of a population, instead of separate cells
are considered.
The design of bioreactors in industrial biotechnology has to consider mass transfer processes,
i.e. a metabolism between various phases (between a cell and a liquid nutrient medium). Therefore,
an important component of the reactor is the system of hashing serving for providing uniform
conditions in the device.
Hashing is one of the major factors defining a hydrodynamic situation in a fermenter. With
increase in its intensity speeds a mass exchange increase and there is a uniform distribution in all
volume of nutrients, the speed of biochemical reactions increases in cells of microorganisms owing to
lack of limiting factors. However, the excessive increase in intensity of hashing can be connected
with mechanical impact on a cell.
By production of drugs by biotechnological methods aerobic cultures are used. Therefore the
design of reactors provides existence of knots of aeration to provide O2 transfer from gas to liquid
medium and further to cells in quantities, optimum for this strain in this growth phase.
For aeration of the cultural medium air, or the air enriched with O2 is used, more rare oxygen is
used. During metabolic processes CO2 which is subject to removal is formed.
Device of a fermenter
Fermenter represents hermetic cylindrical capacity with a spherical cover and the bottom. The
volume of the device can be from 0,01 to 100 m3. The best material for production of a fermenter is
stainless steel. Fermenter is supplied with thermostatic, mixing and regulating pH of the medium
devices, system of giving of a nutrient medium, defoamer of water and couple, a coil for sterilization.
Production cultivation is carried out in sterile conditions. Before filling of a fermenter with a
nutrient medium it is washed, checked on tightness, sterilized. At the same time all pipeline is
sterilized. Fermenter is filled with a nutrient medium for 70%, рН of medium and temperature are
brougth to optimum parameters, characteristic for a microorganism producer.
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The stage of cultivation lasts 48-72 hours. There are the bacterial cultures which stage of
cultivation makes 24 hours. For fungi cultivation of 12 days is characteristic. On continuation of all
cycle the growing deep culture needs to be supplied with oxygen. Bubbling devices are usually used
for these purposes. At aeration foam which disturbs cultivation process is formed. For its repayment
defoamer - vegetable and animal oils, silicone solutions or hashing of the cultivated medium in the
top layer is used.
Fig. 1. Fermenter for cultivation of
microorganisms on gaseous hydrocarbons: 1 - case of
a fermenter; 2 - cooling shirt; 3 - mixer; 4 - mixer
drive; 5 - supply of gaseous hydrocarbons; 6 - supply
of oxygen-containing gas; 7 - giving of a liquid,
nutritious mix; 8 - giving of sowing culture; 9 - exit of
barmy suspension upon termination of a fermentation;
10 - production of gas from a fermenter; 11 - exit of a
gas mix to recirculation; 12 - the gas analyzer giving a
signal of a pas the control device of the valve; 13 pressure in a fermenter; 14 - catcher of carbon dioxide
On a way of hashing and aeration
bioreactors are subdivided into devices with
mechanical, pneumatic and circulating
hashing. Devices with mechanical hashing
have the mechanical mixer consisting of the
central shaft and blades of a various form.
And blades have to be located in some stages
(for effective transport of components of a nutrient medium directly to cells). Aerating devices of
bioreactors of bubbling type have in the lower part of the reactor a horizontal pipe with openings
through which spray (bubble) air in the form of small bubbles. The bubbler is supplied with the
mechanical vibrator.
Fermenter with mechanical hashing of bubbling type
It is the vertical device of the cylindrical form about 63 m3 made of high-alloyed X18H10 I
steel with elliptic covers and the bottom. On covers drives of the mixing device, unions for loading of
a nutrient medium and a sowing material, the union of giving and an air conclusion, observation
ports, hatches for immersion of a washing mechanical head, unions for kip devices are located. In the
device there are shaft with the mixing device, the bubbler for air supply. The device is supplied with a
shirt from 6-8 circles sections, the surface area of cooling of 60 sq. m, temperature of sterilization is
130-140 ºС, a consumption of sterile air to 1 m3/min. Height of a column of liquid of 5-6 m, at pH 8.
Fermenters with pneumatic hashing and medium aeration
In fermenter of pneumatic type the mechanical mixer is absent. Hashing of a liquid nutrient
medium is carried out by vials of gas. The mixing device is the diffusor executed in the form of the
cylinder, built in in a fermenter. Air under pressure arrives through openings in liquid. And in a place
of input the rarefaction is created, and air rising from below up, mixes cultural liquid. The coefficient
of filling of fermenter with pneumatic hashing is 10-15% lower, than with the mechanical. Devices
are calculated for work under excessive pressure. Mass transfer speed between gas and liquid in such
devices is below, than with mechanical hashing and for overcoming of this shortcoming various
modifications are entered. The air-lift type of a fermenter is supplied with a diffuzer, and through it
passes a column of a liquid nutrient medium which is parted forcibly by vials of air that promotes
increase in its volume and liquid mixes up through the upper edge of a diffuzer down and it leads to
hashing and liquid aeration in other volume of the device, i.e. out of a diffuer. In such devices
practically there is no threat of excess foaming and coefficient of filling them above.
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Pneumatic fermenters are characterized by planned hashing and they can be applied at
cultivation of cells of the highest plants and animals. For example, the culture of cells of Digitalis
lanata forms in such reactor P-methyldigoxyns (means for treatment of cardiovascular ischemia).
Fermenters with pneumatic hashing attract also with simplicity of a design and small energy
consumption, i.e. hashing and aeration is combined in one knot. However they can't be applied in
biotechnological processes to cultivation of fungi and actinomycetes, grown up on nutrient mediums
with high viscosity.
Fermenters with circulating hashing
These equipment contains the special devices (pumps) creating the directed stream of liquid on
a vicious circle. Liquid entrains vials of gas. The special fermenter with circulating hashing and
diffusor existence, i.e. a combination of circulating hashing and pneumatic was created.
AIRPREPARATION IN BIOTECHNOLOGICAL PRODUCTION
Purification of air can be carried out essentially different methods based on destruction of
organisms or removal of them. One of the most effective ways of sterilization of air is radiation by
ultra-violet beams. This method is used for air disinfecting in boxes.
By our and foreign experience it is proved that technologically and in the industry the way of
purification of air by means of fibrous and porous materials is economically justified. By such way it
is possible to receive air with degree of purity of 99,9999%.
For sterilization of air membranes with a diameter of time of 0,45 microns are also
recommended. Porosity of membranes reaches 80%. Removal of microorganisms by means of a
membrane is based on sieving effect. Generally filtering materials are delivered by Millipor (USA),
in our country (Vladimir) membrane release "Vladipor" is adjusted. For membranes high pressure
differences aren't required, but their reliable work requires exact performance of conditions of
sterilization. It is possible to sterilize membranes only by saturated water vapor, from superheated
steam in membranes there are cracks, and membranes fail.
PREPARATION OF NUTRIENT MEDIUMS
Biotechnological production has to have a producer, raw materials for preparation of nutrient
mediums, the equipment for all stages of technological process. Typical elementary structure of
microorganisms the following: (in % to the weight of a dry biomass) carbon - 50%, nitrogen - 7-12%
and microelements. All cells contain also oxygen and hydrogen. The medium for cultivation of
microorganisms has to include elements which are a part of cells, and to keep their proper correlation.
Choice of structure of nutrient mediums
Many of producers applied in the industry are chemogeterotrophes which needs for energy and
carbon are satisfied with simple sugars. In the industry as sources of energy and carbon crude sugar,
and also these or those semi-products, for example, beet sugar or corn molasses (50-70% of
fermented sugars), serum or waste of the canning industry are often applied. Yeasts are grown on
sulfate-alcohol the bard, a by-product of paper production.
For providing various types of a metabolism of microorganisms nutrient mediums have to
conform to the following requirements:
1 . To contain all elements which are necessary for the cell: macroelements (carbon, nitrogen,
oxygen, sulfur, phosphorus, potassium, calcium, magnesium, iron) and microelements (manganese,
molybdenum, zinc, cobalt, nickel, vanadium, chlorine, sodium, silicon, etc.). All elements have to be
presented in slightly-assimilated by concrete microorganism substances. Various organic compounds
can be a source of carbon: carbohydrates, polyatomic alcohols, organic acids, amino acids, proteins,
etc. Ammonia solts, amino acids, peptides, proteins are a source of nitrogen. Source of other
macroelements are inorganic substances - phosphoric salts and other acids. Microcells arrive into a
nutrient medium with organic substrate, salts and water. Vitamins (especially groups B) and other
factors of growth are brought into medium as a part of organic substrata or in the form of pure
substances.
2 . To have sufficient humidity (not less than 20% of water).
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3 . Concentration of salts in the medium has to be made isotonic, i.e. correspond to
concentration of salts in a microbic cell (for the majority of microorganisms - 0,5%; the halophilic 3%).
4 . Concentration of hydrogen ions (pH) of the medium has to be optimum for a grown-up
microorganism (pH 4,5-8,5 range).
5 . Oxidation-reduction potential (Eh of the medium has to correspond to requirements of a
microorganism: for anaerobes – 0,120-0,60, for aerobes – more than 0,80.
6 . Nutrient medium has to be sterile.
Natural, semi-synthetic and synthetic nutrient mediums are applied for production of
pharmaceutical production. Natural mediums are received from animal fabrics, microorganisms,
plants, vegetables, fruit, grain and other products, and also production wastes. They contain all
complex of necessary components, but are a little suitable for large-capacity productions because of
inconstancy of structure. The complex natural mediums consisting of biomeal, wheat bran are widely
used for receiving enzymes.
Originally microorganisms and cells of tissues are cultivated on the habitats, representing
extracts of a plant and animal material (grape juice, milk, peptone, serum). Similar mediums are
convenient as contain all sources necessary for growth and reproduction, but they possess one
shortcoming - uncertainty of the contents macro - and microelements.
Semi-synthetic mediums consist of connections of the known chemical nature and a complex of
uncertain substances. These are liquid paraffin, wood hydrolysates, molasses, bran, corn extract and
other waste of food and not food productions (antibiotics, amino acids, yeast, enzymes). Synthetic
mediums consist of compounds of the known chemical nature: methanol, ethanol, natural gas and
methane.
Carboniferous raw materials. The most characteristic carboniferous raw materials are
carbohydrates. Carbohydrates are one of the main parts of a nutrient medium for cultivation of
microorganisms. They are used for synthesis of cellular structures and at the same time they are
power sources. For industrial biosynthesis glucose and starch, hydrolyzates of various waste of
processing of agricultural raw materials (sunflower pod, corn cabbage stumps, wood spill, etc.) are
used most often.
The choice of a source of carbon in biotechnology of albumens, lipids and amino acids has very
great value. It not only presents ways of a metabolism of this microorganism, but also often assumes
structure of other components of the medium and even technology and hardware registration of
production of a target product, in particular, if use of the substratum remote for microorganisms
needing preliminary processing is supposed.
Nitrogen-containing raw materials. Biosynthesis of many biologically active agents is carried
out on nutrient mediums of complex and often a changeable chemical composition. Various sources
of nitrogen can be presented in them by proteins, peptides or free amino acids. At an industrial
fermentation corn extract, a soy flour or a hydrolyzate of yeast are used. Corn extract contains all
complex of amino acids, but their quantitative structure can change considerably from party to party.
Soy flour is rich with albumens. It also, as well as corn extract, contains all amino acids,
however generally they are connected in idea of proteins. At an assessment of natural substances (a
soy flour, corn extract, etc.) it is necessary to take into account that their impact on the directed
process of a metabolism of microorganisms is caused not only availability of proteins and amino
acids, but also presence along with them of carbohydrates, nucleinic acids, microcells, organic acids
and other ss.
From mineral nitrogen-containing substances ammonia salts of sulfuric, hydrochloric or nitric
acids are applied most often. Sulfate of ammonium is suitable for biosynthesis of many substances.
The need for these or those nitrogen-containing connections is defined by physiological
opportunities of microorganisms. Part of microorganisms are capable to synthesize amino acids on
the basis of medium components with use of nitrogen of inorganic connections, others demand
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maintaining in structure of medium of ready forms of amino acids or other organic sources of
nitrogen.
The content of nitrogen in the nutritious medium has to be rather high as the biomass, for
example, of yeast, consists approximately for 45-55% of dry weight of protein, in which there is
about 6% of nitrogen. Therefore cultivation of yeast requires about 100 mg of nitrogen per 1 l of a
nutrient medium.
Sometimes producers of biologically active agents need separate amino acids, more rarely –
they need all 20. Organic nitrogen-containing compounds and ammonia nitrogen are usually easily
acquired by microorganisms, nitrates are acquired more slowly as nitrogen of nitrates has to be
restored at first and only then is realized by a cell during metabolism.
The lack of nitrogen of a nutrient medium leads to so-called "obesity" of cells, i.e. to increase
of the maintenance of lipids in them at the expense of reduction of proteinaceous or amino acid
fractions.
Process of selection of a nutrient medium for cultivation of microorganisms and for
manifestation of the maximum biosynthetic activity of a target product by it is very labor-consuming
and difficult, demanding knowledge of physiology of a microorganism. Development or choice of the
medium can be carried out within several months or years depending on complexity of an objective
and degree of study of this microorganism.
Essentially the structure of each nutrient medium for each microorganism can be defined in two
ways: method of multistage empirical selection or with use of mathematical methods of planning of
experiment.
First way is the most popular. Usually on the basis of studying of physiological features of a
microorganism are defined qualitative structure of the medium, and quantity of components is
established experimentally when one component of the medium is changed in certain limits, and the
others are left at invariable level. This method is reliable, but quite long.
When using mathematical methods of planning of experiment it is possible to find much
quicker and scientifically to prove optimum structure of a nutrient medium. Mathematical planning
of experiments is acceptable when studying new producers of pharmaceutical preparations.
There is a concept of the "minimum" mediums containing only power supplies, necessary for
growth and "rich" mediums which contain additional substances in the form of amino acids, vitamins
(i.e. growth factors). Enrichment of mediums for cultivation leads to increase in growth rate and
change of fermental structure of a biomass.
If a main objective of biotechnological process is synthesis of medicines, certain substances are
added to medium - predecessors or its close analogs which join into a product molecule. So, in
production of penicillin and B12 vitamin phenylacetic acid is used as predecessor.
There are nutrient mediums of general purpose (universal) and special nutrient mediums.
Nutrient mediums of general purpose are suitable for cultivation of many species of microorganisms
and are applied as a basis for preparation of special nutrient mediums. For example broth, agar,
Hottinger's broth, Hottinger's agar, meat-pepton etc. Special nutrient mediums are intended for
selective cultivation of certain species of microorganisms, studying of their properties and storage.
Preparation and sterilization of nutrient mediums
One of important stages of microbic biosynthesis is preparation of nutrient mediums.
Department of preparation of a nutrient medium on modern biotechnological production is the shop
equipped with capacities for storage of solid and liquid substances, means of their transportation and
apparatuses with mixing devices for preparation of solutions, suspensions or emulsions.
For preparation of a production nutrient medium previously sugar and salt are dissolved, such
insoluble components, as a soy flour and chalk are carefully suspensed. Starch-containing raw
materials previously are pasted. For acceleration these processes are carried out in small devices with
mixers (reactors), and then solutions are mixed in the mixer reactor with the flat bottom supplied with
15
the bubbling device for input of steam. The concentrate of the medium making about one third of
necessary volume, for final dissolution and a suspension is heated by acute steam till 70-80 ºС. At
this temperature there is no decomposition of thermolabile components of the medium. Preparation
of more concentrated mediums gives a chance of use mixers of smaller capacity.
Necessary condition of successful sterilization of a nutrient medium is careful homogenization
of its firm components. At a sterilization temperature large particles slowly get warm, and in them the
constant microflora, capable to infect cultural liquid can remain.
For preparation of nutrient mediums in biotechnological production molasses (a by-product of
sugar plants), aceton-butyl bard (production wastes of acetone and butanol), serums (a by-product of
the dairy industry), wood hydrolyzates, sulphitic lye (pulp and paper industry waste) are used.
Aceton-butil bard contains about 1% of carbohydrates, it is used for producing B12 vitamin by a
microbiological way.
Spill, sawdust, the agricultural waste, the low-gone wrong peat and their hydrolyzates are used
in production of fodder yeast, ethanol. They contain 2,5-8,0% of monosaccharides (after hydrolysis),
a corn flour - 67-70%. Acetic acid is applied to preparation of nutrient mediums in production of a
lysine. Methyl alcohol is the product of catalytic synthesis from carbon and hydrogen oxides. It is a
source of carboniferous raw materials for production of microbic, fodder and food protein.
The nutrient medium before giving into a fermenter has to be disinfected. At this stage of
preparation of a substratum it is necessary to solve two problems: completely to destroy all
contaminational microflora which presents in the volume of liquid necessary for cultivation, and to
keep biological full value of a nutrient medium.
There are following methods of sterilization of the equipment, nutrient mediums and air:
thermal, chemical, filtrational, radiation. The thermal method is most often applied to sterilization of
the equipment and nutrient mediums and can be carried out as object heating untill all
micropopulation won't be lost yet.
Liquid nutrient medium after loading into a fermenter is heated to a certain temperature by
supply of steam into internal volume of a fermenter. By this way sterilization of pipes and fittings is
held.
Thermal sterilization leads to certain chemical changes in structure of a nutrient medium. Some
of them are reduced to decomposition of substances unstable to heating that leads to loss of
substances necessary for feeding of a microorganism. In the course of sterilization there can be an
interaction between various components of the medium and formation of the products inhibiting
growth of microorganisms. The majority of changes of chemical ingredients of the medium arises at
temperatures above than sterilization temperature.
Therefore, effective sterilization in combination with the minimum changes of the medium can
be reached by influence of more high temperature, and also fast heating and cooling.
If to carry out sterilization of carbohydrates separately, and then aseptically to add to other in
advance sterilized nutrient medium, it is possible to prevent reactions between carbohydrates and
other compound components of the medium. Those components which are extremely sensitive to
heating, also can be sterilized separately. Thus ionizing radiation or a filtration can be applied to
sterilization via special membrane filters.
For ensuring control of sterilization disputes the test of microorganisms of Bacillus
stearothermophilus of a strain 1518 are used. If after carrying out sterilization from an ampoule with
test culture seeding yields negative result, it is considered that destruction of all microorganisms,
contaminated the medium took place.
Solving a problem of the guaranteed sterility of a nutrient medium, it is necessary to remember
that the mode of disinfecting shouldn't reduce its biological full value. If the structure of a sterilized
phase includes thermolabile components, it is necessary to aspire to the increased temperature (more
140ºС), and also to reduction of time of processing. Lability of components can be changed at the
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expense of shift of pH of the sterilized medium. For example, for glucose pH=3,0, and for sucrose pH=8,0 are optimum.
The thermal way of sterilization is applied most often in the microbiological industry.
Sterilization is carried out within several minutes, thus physical and chemical properties of
components of the medium don't change.
The chemical way of sterilization is an application of disinfecting agents – β-propionates,
ethylene oxide, propylene oxide. Need of elimination of the sterilizing agent from a nutrient medium
after death of foreign microflora appears the main problem in this case. Therefore chemical
antiseptics have to decay easily at change of conditions after completion of sterilization. The choice
of such compounds is insignificant and while they can't be considered easily available. It is possible
to carry to number of best of them propiolactone, possessing strong bactericidal action and easily
hydrolyzed to nontoxical milk acid. Chemical sterilization of nutrient mediums didn't find industrial
application, however we use it in laboratory and laboratory equipment.
Filtrational method of sterilization is applied to air and the gases brought to reactors. From
filters of various types membrane filters made of teflon are most perspective.
The thermal way of sterilization is applied most often in the microbiological industry.
However for sterilization of firm nutrient mediums currents of high frequency are applied.
Sterilization is carried out within several minutes, thus physical and chemical properties of
components of the medium don't change.
The method is based on ability of semi-permeable membranes (like microfiltrational) to pass a
liquid phase and to detain cells of microorganisms.
The method of a sterilizing filtration is ideal means of sterilization of thermally unstable liquid
and gas mediums. Sterilization is carried out at a low temperature and demands only pressure
gradient on the different parties of a membrane. Membrane sterilization has prospects in development
of microbic biotechnology. The main difficulty is existence of the heat-resistant membranes, capable
to take out repeated thermal sterilization them in use. It is possible to claim that in process of creation
of perfect designs of membrane devices for the sterilization, calculated on long operation, this
method it will be widely applied in large-capacity productions.
RECEIVING THE SOWING MATERIAL
For receiving a sowing material initial culture of a producer which arrives in laboratory factory
from research institute or from sowing station is used.
As a rule, the sowing material containing young, growing cells of microorganisms at an initial
stage of a spore-growing (disputes, conidia) arrives in test tubes on slanted agar mediums or in the
form of pure cultures in ampoules.
Each production culture has the passport in which the producer and its collection number, series
and production date, average activity of a series and an expiration date are specified. The
characteristic of the medium for cultivation and culture storage is presented in the passport. The
received sowing material subject to careful microbiological and biochemical control as the further
production cycle depends on its activity and purity.
Depending on a type of a producer, its physiology-biochemical abilities preparation of a sowing
material (stage of production fermentation) takes place in some stages:
1. Initial culture
2. Slanted agar medium
3. Cultivation on rocking shelves in flasks on a liquid nutrient medium (one or two stages)
4. Sowing devices (one, some stages)
5. Stage of a production fermentation.
Initial culture at optimum temperatures is grown up in test tubes on a slanted agar nutrient
medium. For microscopic fungi, actinomycetes producers are cultivated before an optimum sporegrowing (72-120 hours), for bacterial cultures the phase is established experimentally.
17
Grown-up culture (1-5% from volume) from a surface of the slanted agar medium is sterilely
washed away by water and transferred to Erlenmeyer's flasks at 750 ml containing 50-100 ml of a
liquid nutrient medium. The sowed flasks grow up on a rocking shelf (120-240 rpm) at a temperature
of 28-30-50 °C within 18-36 hours, i.e. deep way that increases culture growth rate. All stages of
growth of a producer supervise on morphological indicators of microorganisms. It is thus established
that the best results are yielded by culture which is in a stage of a physiological maturity.
Ready culture is sterilely transfered to the sowing device (a small inoculator) with previously
sterilized nutrient medium. The sowing device is equipped with a mixer, the aerating device, and also
instrumentation for regulation рН of medium, temperatures and extents of aeration.
When receiving inoculate it is desirable to carry out scaling when growth of population
happens to the maximum speed, i.e. in an exponential phase.
ALLOCATION, CONCOCTION AND PURIFYING OF BIOTECHNOLOGICAL
PRODUCTS
Process of allocation and purifying represents a number of the consecutive technological
operations which quantity increases with increase of desirable purity of the final product.
Cultural liquid is very sensitive to various influences therefore losses at allocation of the final
product are observed. Cultural liquid represents the difficult multiphase system containing from 1 to
5% and more of solid, separate microbic cells or a mycelium, products of biosynthesis and the
remains of a nutrient medium. As a rule, allocation of the final product is connected with certain
difficulties and generally depends on preliminary purification of native solution.
The first stage of preparation of cultural liquid for further processing is the division of the
weighed phase or microbic mass which structure includes microorganisms, products of their activity,
and also the remains of an unused nutrient medium. Division firm and liquid phases is carried out
with preliminary coagulation of high-molecular substances with the subsequent natural (upholding
and a filtration) or compulsory sedimentation centrifuges.
Effective method of coagulation of disperse systems is processing by their high-molecular
polyelectrolytes – floculants. Thus unlike usual coagulation floculants are formed or precipitation of
friable structure that considerably improves filtration process.
The main advantage of centrifugation in comparison with other methods of processing of nonuniform systems, for example, upholding and filtering consists in increase in productivity of division.
Productivity of modern centrifuges of continuous action reaches hundred m3/h. By means of
centrifugation it is possible to allocate from particle suspensions with the size to the 100-th shares of
a micrometer. Centrifugation is widely applied in biotechnological productions of medicinal
production, first of all for allocation from cultural liquid of a meristem of plants, biomass of yeast,
bacteria, fungi, segregation various products of microbiological synthesis (antibiotics, enzymes,
vitamins, etc.), transported previously into a firm phase, and also to division of the emulsions which
are forming at extraction.
For extraction of intracellular products of biosynthesis disintegration methods are widely used.
Disintegration, i.e. destruction of cellular covers of microorganisms, is carried out in several ways:
chemical, biological, physical (mechanical).
Chemical ways of disintegration are based on destruction of the ordered structures of a cellular
wall of a microorganism. The most known chemical ways are processing of cellular suspension
directly by alkali, urea, glycerin, ammonia, peroxide. The chemical way is intended for allocation of
total proteins of food appointment and didn't find broad application in chemical and pharmaceutical
industry owing to impossibility to receive a pure product.
Biological ways are carried out by means of lytic enzymes.
Physical ways. Physical (mechanical) disintegration is the process happening at high speeds
and being accompanied by fast hashing of the destroyed material in an area of coverage of
disintegrant forces. Physical disintegration can be carried out in a continuous mode with process
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automation. The most known ways of destruction of a biomaterial are freezing and thawing,
ultrasonic influence, and detrition of cells, extrusion.
If the target product represents a soluble metabolite or it is synthesized in cell and isn’t secreted
into cultural liquid, resort to the following methods of allocation: extractions, sorptions,
chromatography, and allocation by means of membranes.
Extraction from cells is carried out by organic solvents (for example, an antibiotic drug
griseofulvin by acetone, or bensylpenicillin at рН 2,0-3,0 – butilacetate). Extraction of enzymes is
carried out in two-phase systems, for example, a glucane-dextran and polyethyleneglycol
incompatible with it.
Method of adsorption is applied in microbiological productions generally when receiving
crystal amino acids and also the high purifying and immobilized enzymes. At allocation and an
immobilization of enzymes organic sorbents are used (starch, cellulose, synthetic and ion-exchange
pitches) or inorganic (zeolites, aluminum hydroxide, silica gels, etc.).
Processes of adsorption are carried out in devices of periodic or continuous action with a
motionless or mobile layer of adsorbent. Adsorbers with a boiling layer of adsorbent are perspective.
Vertical adsorbers (ion exchangers) are used for allocation from cultural liquids of amino acids, the
last when passing through a layer of an ionite are adsorbed, and accompanying impurity are taken
away from a column with the fulfilled cultural liquid. After pitch saturation by amino acid supply of
cultural liquid stop and pitch wash out water then pitch is ready to a repeated cycle.
Chromatographical separation of BAS use in various options: gel filtration, ion-changing
chromatography, affine chromatography.
In case of high specificity of such natural substances, as enzymes, antibodies and lectines an
affine chromatography is used. Enzymes form complexes with inhibitors, antibodies - with the
corresponding anti-genes (an immunosorption chromatography), lectins - with special receptors of
cellular walls.
The dialysis and electrodialysis, the return osmosis, ultrafiltration and microfiltration belong to
membrane methods of division. The community of all membrane methods of division is that a basic
element of their hardware registration are membranes.
Membrane methods of division possess a number of advantages:
- concoction and purifying happen without change of an aggregate state and phase
transformations;
- the processed product isn't exposed to thermal and chemical influences;
- mechanical and hydrodynamic impact on a biological material insignificantly;
- easily provide tightness and aseptic conditions.
The main restrictions in application of membrane methods of division are connected with that
some materials of which membranes are made, don't maintain very low and very high values рН and
high temperatures.
DRYING OF BIOTECHNOLOGICAL AND PHARMACEUTICAL PRODUCTION
Cultural liquid received at the end of a cycle contains from 0,1 to 5% of solids. At the
subsequent stages from useful products (a biomass, antibiotics, enzymes, amino acids and other
substances) are taken. Then turn into a final commodity form.
The majority of products of biological synthesis are issued in a dry form with residual humidity
no more than 5-12%.
Removal of moisture and thermal influence during drying of a biomass conduct to the essential
changes influencing quality of a ready-made product. The greatest difficulties arise if necessary to
keep such thermolabile objects, as live microorganisms (bacterial preparations) or enzymatic
preparations. The main reasons for their thermolability are the denaturation of protein and
inactivation of enzymes at thermal influence, increase in concentration of electrolytes and toxic
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substances during removal of moisture, and also structural and mechanical damages to drying
process.
On an initial aggregate state of moisture the following methods of drying are distinguished:
drying from a liquid state, evaporation from a firm condition, passing a liquid phase - sublimation.
Drying of cultural liquid or biomass is carried out in the contact, convective and radiation way.
At contact drying heat is transmitted to a dried-up material through heated surfaces and
evaporating moisture passes to air. For drying of products of biological synthesis are applied one and two-rolled cupboard dryers.
At convective drying warmly, necessary for drying process, it is delivered by the gaseous
drying agent which plays a role of heat carrier and medium role to which moisture from a material
passes. This method is widely applied to drying of products of biological synthesis and, first of all, in
pneumatic, aero gushing, spray dryers and dryers in a fluidized layer.
As the majority of products of biological synthesis are thermolabile substances, sparing
methods are the most applied for their drying. Thus, seek to reduce temperature and drying time. For
these purposes vacuum or drying of a fine material are used. Even more favorable conditions for
drying of thermolabile substances are created at sublimation.
The sublimation method of drying is based on removal of moisture from the frozen condition,
and, moisture passes to a gaseous phase, passing the liquid.
Characteristics of sublimation are minimum in comparison with other methods of drying
meaning change of structure of a dried-up material and lower temperatures of drying. Therefore
sublimation drying is applied to especially thermolabile products, for example live microorganisms,
the enzymes, some antibiotics.
Excessive dehydration in the course of sublimation drying can be a cause of death of cells. The
special protective mediums including glycerin, sucrose, kollidon and other substances which are
slowing down formation of intracellular ice are applied to protection of cells against death when
freezing and the subsequent drying, reducing concoction of electrolytes and protecting cells from
rough irreversible dehydration.
The received dry products of biosynthesis are usually used in the crushed condition.
Sometimes, at spray drying of additional crushing of the dried-up product it isn't required. In other
cases when the ready-made product has to be dissolved very quickly or mix up previously to a
homogeneous condition, enter crushing operation.
Crushing depends on the size of pieces of an initial material and respectively shares on some
classes:
• crushing - large, average, small;
• grinding - rough, average, thin, colloidal.
It is difficult to dose, pack and transport the dried-up powdery preparations. For increase in
average density, reduction of volume and decrease in dust formation preparations granulate. Various
ways of a granulation are known: extrusion with the subsequent centrifugal rolling, pseudoliquefaction and an irrigation powder liquid with drawing the glazed film on a granulate surface,
pressing and molding by means of forming machines in the dry or liquid way.
For volume pressure head dispensing of liquid mediums in exact and adjustable quantities
pumps and dosing units are used.
Pneumatic batchers, automatic dosing scales, tape batchers, vibrobatchers are intended for
dispensing and packaging of bulks.
Substantially efficiency of pharmaceutical production depends on statement of microbiological
control. The museum culture of a microorganism producer, sowing material, nutrient mediums, air,
cultural liquid, and also finished goods are supervised. Depending on purpose of microbiological
production various demands are made to its contamination. The products used in the medical
industry, have to be almost free from microorganisms.
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One of the main tasks of biotechnological production is maintenance of a production strain in
an active condition. In the course of numerous transfers of microorganisms and also long influence of
conditions of cultivation eventually there can be spontaneous mutations which lead to decrease in
efficiency of production culture. In this regard periodically, two times a year, and carry in some cases
and more often out resowings of a production strain and selection of options with the highest
efficiency.
Biochemical control of quantity and specific activity of products of microbiological synthesis
allows to track an exit of intermediate and final products. This type of control provides release of the
pharmaceutical production which meets the requirements of standards.
STANDARDIZATION OF BIOLOGICAL PRODUCTS
In process of further development of biotechnology problem of quality control of the raw
materials used for drawing up nutrient mediums, and qualities of products of biosynthesis becomes
more and more actual. Now raw materials and nutritious substrata for microbic industrial
biosynthesis estimate on structure of these or those components connected with quality.
Quality control of products of the biosynthesis used in medicine is extremely important:
antibiotics, enzymes, other bioorganic compounds.
According to an official technique there is a classification of indicators for an assessment of
quality of microbiological production. Indicators of appointment characterize the properties of
production defining the main functions for which performance it is intended, and cause area of its
application. Two important subgroups belong to group of indicators of appointment: indicators of
structure and indicators of functional efficiency.
Indicators of the content of the main substance include:
•contents (%) of crude protein in yeast in terms of the solid is absolute (SIA);
•contents (%) of protein;
•contents (g) of antibiotics on 1 kg of a drug;
•content (mg) of vitamin on 1 kg of a drug;
•preparation caption;
•activity of enzymatic drugs;
•coefficient of biological activity;
•other indicators.
Indicators of the content of foreign impurity:
•contents (%) hydrocarbons in protein;
•content of metallomagnetic impurity in protein;
•contents in production of substances, excellent on structure from the main component and
reducing quality of production;
• contents (%) ashes in terms of ADS, etc.
Indicators of structure and physical and chemical characteristics:
• size of particles;
• humidity (%) ;
• bulk weight (g/l);
• dissolution speed;
• density (g/cm3), etc.
Biological product quality control on indicators of appointment is reduced to the analysis of its
structure and physical and chemical properties.
Qualitative characteristics of production (appearance, color, a smell, taste) are recommended to
be determined by an organoleptic method in comparison with a basic sample.
Also any biotechnological drug (for example, a vaccine) is exposed to standardization (control
check of a ready-made product) by means of the following tests:
1) test for sterility;
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2) test for preservative presence;
3) check on presence of adjuvants (for example, aluminum alum);
4) check on activity and identity.
Documentation, samples, marking and packing are subject to check. All above-mentioned tests
are necessary in order that the ready-made product possessed necessary properties: it was safe,
reliable and possessed necessary preventive or medical action. The international recommendations
(WHO recommendations) concerning such requirements, facilitate an exchange of biological
preparations between the certain countries and serve as the management for the workers responsible
for production of these preparations, and also for those who makes the decision on the questions
concerning methods of their analysis and control.
CONTROL AND MANAGEMENT OF BIOTECHNOLOGICAL PROCESSES
At the present stage of development of biotechnological production of medicinal, preventive
and diagnostic means high quality of production can be reached taking into account a number of the
general requirements to the organization and production control. Such requirements have to warn
possible mistakes when carrying out technological processes and a message to creation of
ecologically safe enterprises.
Their general name - GMP is deciphered as "Good Manufacturing Practice".
GMP should be understood as uniform system of requirements for the organization of
production and quality control of any medicines from the beginning of processing of raw materials
before production of ready-made products, including the general requirements to rooms, the
equipment and the personnel; they also completely extend on all biotechnological productions
occupied with production of medicines.
The rules of GMP contain only minimum practical instructions, but are the general
management regulating as production has to be organized and as control tests have to be organized.
There are national GMP (at more than 40 countries - the USA, Japan, Germany, India, etc.),
regional GMP (EEC), GMP WHO. The rules developed by WHO, make one of basic elements
"Systems of satisfaction of quality of pharmaceutical preparations in international trade which is
recommended to all member countries of WHO. It is a special type of the multilateral agreement for
the purpose of assistance to bodies of health care importing (including developing) the countries in an
assessment of the legal status and a technological level of medicines bought by them. It gives a
certain guarantee to the import countries.
It should be noted that requirements of GMP belong only to preparations of medical
application.
All national and other rules GMP are included in the collection "International drug GMP"
which is periodically republished (in the USA).
- national, regional, international, - the following main heads contain the rules GMP:
introduction, terminology, personnel, buildings and accommodation, equipment, process of
production and ecological safety, laboratory control, registration and reporting.
Without analyzing in detail all parts of the relevant official document (documents), we will note
separate requirements for some of sections.
Requirements to the personnel are connected with level of its qualification, state of health,
personal hygiene, order of use of technological clothes and footwear, materials for their production
(for example lint-free fabric), retraining and medical examinations of the personnel.
Requirements to buildings and rooms specify their arrangement, planning for the purpose of,
prevention of mixture of different types and series of initial raw materials, semi-products and ready
for using pharmaceutical products. Special rooms for carrying out concrete technological operations,
etc. are listed. Rather interestingly in this regard to note that in a number of the national guides to
GMP it is indicated, in particular, the technological operations connected with production, processing
and packing of antibiotics of penicillin and cephalosporins which are recommended to be carried out
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in the rooms isolated from where other medicines are made. This results from the fact that beta
lactams can show allergenic action in the extremely insignificant quantities getting to other medicines
in order that sensibilised patients had serious complications.
The considerable attention is paid in GMP to facing materials, bathroom equipment and a
question of maintenance of purity in rooms.
Under special control rooms where sterile medicines - concerning their isolation are made,
features of planning, ventilation, maintenance of a constant difference of pressure between certain
rooms get to 3-5 mm of water column, and, in rooms of higher classes of purity pressure has to be
higher. Unsterile medicines shouldn't be made in those rooms where sterile production is produced.
Requirements of GMP to a design, the sizes and arrangement of the equipment which is
especially used by production of sterile medicines are numerous. As one of examples it is possible to
note the instruction that by production of injection preparations it is necessary to avoid the filters
separating fibers. If they are forcedly applied (asbestos, etc.), after them it is necessary to use
additional membrane filters with a size of time no more than 0,45 microns.
In all GMP the detailed instructions which are intended for optimum carrying out all stages of
process of production are given. Rules concern:
1) sampling, raw materials quality control;
2) control directly behind processes of production, processing, packing. Rules for processing of
the rejected series are given.
The maintenance of microorganisms in water for purifying and washing of received medicines
is normalized.
Control of process of production of medicines has to be exercised according to technological
documentation. It should be noted that the rules GMP of the different countries don't coincide with
offered methods of sterilization of a ready-made product. As a whole advantage is given to thermal
methods of sterilization. GMP of some countries allow also radiation sterilization and sterilization by
an ethylene oxide.
Much attention in GMP is paid to ensuring accurate implementation of production instructions.
The list of documents, points of control, possible deviations from the accepted norms is in
detail regulated. Ways of fixing of results are regulated.
The set of requirements for registration and the reporting, containing in GMP, allows to track a
course of performance of production and precisely to establish in case of need all sequence of actions
of the personnel, brought to a deviation of quality of a product from the demanded parameter.
It’s needless to say that the rules of GMP provide a strict regulation of those emissions which
can arrive from the pharmaceutical and biotechnological enterprises in medium. Rules recommend
almost full prevention of receipt of strains producers of recombinant proteins in drains and gas - air
emissions, an exception of hit of allergen-active products of microbiological synthesis in the
atmosphere by means of various technical solutions, dumping prevention with sewage of the
biotechnological semi-products, capable to affect activity and structure of the biocenoses used on
installations of biological purification of polluted water.
Valid the law in a number of the countries of the rule GMP allow the enterprises to let out
competitive production. As showed the international experiment of introduction in medical practice
of the new medicines made in the biotechnological ways, the organization of their preliminary tests
also has to submit to certain rules. The official instances authorizing industrial release of a new
preparation, demand observance of these rules and the corresponding registration of documentation.
By all this reliability and an objectivity of experimental results and results of tests in clinic increases.
The rules GLP are deciphered as "Good Laboratory Practice" that means the correct
(appropriate) organization of carrying out laboratory work at preparation research. The rules GLP
cover all system of preclinical tests. As only one of examples we will note that rules make certain
demands to planning of rooms of a vivarium where experimental animals, to conditions of the
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maintenance of animals, their selection, etc. contain. The organization of preclinical laboratory
researches has to provide objectivity by comparison of data in control and experience.
For permission to industrial release of a new preparation the rules GCP "Good Clinical
Practice", i.e. the appropriate organization of work have very essential value when carrying out
clinical tests. Observance of the rules GCP allows to receive objective results and gives certain
guarantees of approach to the maximum safety in case of preparations for the first time given to the
person.
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