Decree of the Ministry of Health No. 48 / 2001 Coll.
Decree of the Ministry of Health amending Decree No. 1 / 1998 Coll., laying down requirements for the quality, procedure for the preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended by Decree No. 296 / 1999 Coll.
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48
DECLARATION
Ministry of Health
of 4 January 2001
amending Decree No. 1 / 1998 of the Ministry of Health Coll., laying down quality requirements, procedure for the preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended by Decree No. 296 / 1999 Coll.
The Ministry of Health, after consulting the Ministry of Agriculture and the Ministry of Industry and Trade, provides, pursuant to § 75 (4) of Act No. 79 / 1997 Coll., on Medicines and on amendments and additions to certain related laws, as amended by Act No. 149 / 2000 Coll.:
Decree No. 1 / 1998 Coll., laying down quality requirements, procedure for the preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended by Decree No. 296 / 1999 Coll., is hereby amended as follows:
1. In Part 2 of the Annex to the Test Method, Chapter 2.2. Physical and physico-chemical methods, Chapters 2.2.24 and 2.2.25 read:
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2.2.24 Intra-red absorption spectrophotometry
Infrared spectrophotometers are used to record spectra in the area from 4000 cm-1 to 670 cm-1 (2,5 μm to 15 μm) or in some cases up to 200 cm-1 (50 μm). Spectrophotometers with Fourier transformation use polychromatic radiation and Fourier transformation converts spectrum from original data to frequency representation. Spectrophotometers equipped with an optical system capable of producing monochrome radiation in the measured area may also be used. Normally the spectrum is given as a transmittance function, the ratio of the intensity of the released and the incident radiation.
Absorbance A is defined as the decadic log of the inverted value of T and is expressed by:
A = log10-1T = log10-I0I,
in which it indicates:
T - I / I0,
I0 - intensity of incident monochromatic radiation,
I- intensity of expired monochromatic radiation.
Preparation of the sample
For recording using permeability or absorption. The substance shall be modified by one of the following procedures.
Liquid. The liquid shall be tested either as a film between two plates permeable for infrared radiation, or in a bouquet of suitable thickness also permeable for infrared radiation.
Liquid or solid in solution. Prepare the solution in an appropriate solvent. The concentration and thickness of the bouquet shall be selected to obtain the appropriate spectrum. Good results are generally obtained with concentrations from 10 g / l to 100 g / l for a thickness of 0,5 mm to 0,1 mm. The absorption caused by the solvent should be compensated by inserting a similar cyvette with the solvent used into the reference beam.
Solid substances. The solids shall be tested dispersed in an appropriate liquid (suspension) or solid (halide tablet) according to what is preferable. If this is prescribed in the article, a melt film shall be prepared between the two plates permeable for infrared radiation.
(a) Suspension. A small amount of test substance with a minimum amount of liquid paraffin R or other suitable liquid shall be removed; In general, 5 mg to 10 mg of test substance is sufficient to prepare an appropriate suspension. The suspension shall be compressed between two plates permeable for infrared radiation.
(b) Tablet. Unless otherwise indicated, 1 mg to 2 mg of test substance with 300 mg to 400 mg of finely powdered and dried potassium bromide R or potassium chloride R shall be wiped. These amounts are generally sufficient to prepare a 13 mm diameter tablet and obtain a spectrum of appropriate intensity. The mixture is carefully spread, filled evenly with a suitable form and pressed in a vacuum of about 800 MPa (8 t.cm-2). Bad tablets may arise from various causes: e.g. insufficient or excessive smelting, moisture or other impurities in the dispersing environment and insufficient dilution of particles. The tablet does not apply if it shows a non-uniform permeability during visual inspection or if the transmittance at about 2000 cm-1 (5 μm) in the absence of a specific absorption belt is less than 75% without compensation.
Gases. Gases shall be tested in an infrared permeable pendulum having an optical path of about 100 mm. The valve shall be evacuated and filled to the required pressure by means of a tap or needle valve using an appropriate gas transfer tube between the valve and the container containing the test substance. If necessary, the pressure in the pendulum shall be adjusted by atmospheric application of gas permeable for infrared radiation (e.g. nitrogen R or argon R). To remove the interference of absorption caused by water, carbon dioxide or other atmospheric gases, the same cyvette shall be inserted into the reference beam which is either evacuated or filled with gas permeable for infrared radiation.
For recording using multiple reflection
If this entry is prescribed in the Article, the substance shall be adjusted by one of the following methods.
Solutions. The substance shall be dissolved in an appropriate solvent under the conditions laid down in the Article. The solution is evaporated on a thallium bromide iodide plate or on another suitable plate.
Solid substances. The substance is homogeneously spread out to thallium bromide iodide or other suitable plates.
Identification using reference substances
In the same way, both the test substance and the reference substance shall be modified and the spectrum from 4000 cm-1 to 670 cm-1 (2,5 μm to 15 μm) shall be recorded under the same conditions. The permeability minima (absorption maxima) of the test substance spectrum correspond both to the position and relative intensity of the reference substance (CRL).
If the spectrum recorded in solid state shows differences in permeability minima (absorption maxima) positions, the same test substance shall be processed in such a way as to crystallize or arise in the same form, or proceed as prescribed in the article, and the spectrum shall be recorded.
Identification using reference specter
Control of distinguishing ability. A spectrum of 0,04 mm thick polystyrene film shall be recorded. The difference x (see Fig. 2.2.24-1) between the transmittance percentages at the maximum transmittance A at 2870 cm-1 (3,48 μm) and the minimum transmittance B at 2849,5 cm-1 (3,51 μm) should be greater than 18. The difference between the transmittance percentage at a maximum transmittance C at 1589 cm-1 (6,29 μm) and a minimum transmittance D at 1583 cm-1 (6,32 μm) should be greater than 12.
Validation of the wave scale. The wavy scale may be verified by using a polystyrene film that has a minimum permeability (absorption maxima) at the corneas in cm-1 listed in Table 2.2.24-1.
Tab. 2.2.24-1 Minimum permeability (and acceptable tolerance) of polystyrene film in cm-1.
| 3060,0 (±1,5) |
| 2849,5 (±1,5) |
| 1942,9 (±1,5) |
| 1601,2 (±1,0) |
| 1583,0 (±1,0) |
| 1154,5 (±1,0) |
| 1028,3 (±1,0) |
Working procedure. The test substance shall be prepared according to the instructions given in the reference spectrum. Under the working conditions used to check the resolution, the spectrum of the test substance shall be recorded and the absorption strips of polystyrene shall be superimposed at 2849,5 cm-1 (3,51 μm), 1601,2 cm-1 (6,25 μm) and 1028,3 cm-1 (9,72 μm). The two spectrum and the maximum polystyrene referred to above shall be compared. If the peak polystyrene positions are used as a reference, then the position of the significant peak spectrum of the test substance shall correspond to the significant reference spectrum maxima with a deviation of not more than 0,5% of the wavelength scale. The relative intensity of both spectra should be the same.
Impurities in gases
For the analysis of impurities in gases, a cyvette permeable for infrared radiation with an appropriate optical path (e.g. 1 m to 20 m) shall be used. It shall be completed as specified in paragraph Gases. The detection and quantification of impurities shall be carried out in accordance with the procedure laid down in Article 1.
Fig. 2.2.24-1 Typical spectrum of polystyrene used in controlling resolution
2.2.25 Absorption spectrophotometry in ultraviolet and visible areas
Absorbance A is defined as the decadic logarithm of the inverted transmittance T value for monochrome radiation and is expressed by:
A = log10-1T = log10-I0I,
in which it indicates:
T - I / I0,
I0 - intensity of incident monochromatic radiation,
I- intensity of expired monochromatic radiation.
In a homogeneous environment, measured by absorbance (A), the thickness of the layer (b) through which the radiation passes and the concentration of the absorbing substance in the solution (c) according to the relationship:
A = ε,
in which it indicates:
ε - molar absorption coefficient (absorption) when the concentration (c) is expressed in mol / l and the thickness of the layer (b) in cm.
The expression A1cm1% is a specific absorbance which expresses the absorption of a 10 g / l solution measured in a 1 cm layer at a specific wavelength:
A1cm1% = 10εMr,
Unless otherwise specified, the absorbance at the prescribed wavelength shall be measured in a 1 cm layer at (20 ± 1) ° C and measured against the solvent or mixture of solvents used. The absorbance of the solvent used, measured against air at the prescribed wavelength, should not exceed 0,4 and preferably be less than 0,2. The absorption spectrum shall be produced as an absorbance of the absorbance or its function (axis of the order) on the wavelength or its function (axis of the segments).
If a single wavelength value for maximum absorption is indicated in the cells, the value found may be within ± 2 nm.
Device. Spectrophotometers suitable for measurements in ultraviolet and visible spectrum areas consist of an optical system capable of providing monochrome radiation in the range of 200 nm to 800 nm and equipment suitable for absorbance measurement.
Wavelength check. The accuracy of the wavelength scale shall be verified by means of the absorption maxima of the Holmite R chloride solution, the positions of the lines for the hydrogen lamp or deuterium lamp or the positions of the mercury vapour lines as shown in Table 2.2.25-1. The tolerance allowed is ± 1 nm for the ultraviolet and ± 3 nm for the visible area.
Tab. 2.2.25-1 Absorption maxima for checking the wavelength scale.
| 241,15 nm (Ho) | 404,66 nm (Hg) |
| 253,7 nm (Hg) | 435,83 nm (Hg) |
| 287,15 nm (Ho) | 486,0 nm (Dβ) |
| 302,25 nm (Hg) | 486,1 nm (Hβ) |
| 313,16 nm (Hg) | 536,3 nm (Ho) |
| 334,15 nm (Hg) | 546,07 nm (Hg) |
| 361,5 nm (Ho) | 576,96 nm (Hg) |
| 365,48 nm (Hg) | 579,07 nm (Hg) |
Absorbance check. The accuracy of the absorbance scale shall be verified by a solution of potassium dichromate R at the wavelength specified in Table 2.2.25-2, in which the exact values of the specific absorbance and the permitted limits are given for each wavelength. The absorption tolerance is ± 0,01. For the control of the absorbance, use the potassium dichromate solution R prepared as follows: dissolve 57,0 mg to 63,0 mg potassium dichromate R previously dried at 130 ° C to constant weight in sulphuric acid 0,005 mol / l RS and dilute to 1000,0 ml.
Tab. 2.2.25-2
| Vlnová délka (nm) | Specifická absorbance | Maxima tolerance |
|---|---|---|
| 235 | 124,5 | 122,9 až 126,2 |
| 257 | 144,5 | 142,8 až 146,2 |
| 313 | 48,6 | 47,0 až 50,3 |
| 350 | 107,3 | 105,6 až 109,0 |
The limit of diffuse light may be monitored at given wavelength using appropriate solutions or filters. For example, the absorbance of potassium chloride solution R (12 g / l) measured in a 1 cm layer at 200 nm shall be greater than 2 compared to R water as a control fluid.
Distinguishing capacity (for qualitative analysis). If prescribed in the article, measurements shall be made of the resolution capability of the apparatus as follows: the spectrum of the toluene solution R 0,0 2% (V / V) in hexane R. The minimum absorbance ratio at 269 nm to absorption at 266 nm is recorded in the article.
Spectral slit width (for quantitative analysis). In order to avoid errors in measurement caused by the spectral width of the slit when using an instrument where the width of the slot at the selected wavelength can be changed, the width of the slot must be small compared to half the width of the absorption strip, but at the same time it must be as large as possible to obtain a high I0 value. In any case, the width of the slot of the apparatus should always be such that there are no changes in the absorbance reading when it is further reduced.
Flowers. The permitted tolerance of the inner distance of the opposite sides of the cell used is ± 0,005 cm. If filled with the same solvent, the pendants for the test solution and for the control fluid must have the same transmitant. Otherwise, an appropriate correction should be introduced.
The flowers should be cleaned and handled with care.
Derivative spectrophotometry
Derivative spectrophotometry means transformation of the absorption spectrum (zero order) in 1, 2 or higher spectrum derivative.
The first derivative of the spectrum (first order derivative spectrum) is the gradient's dependence of the absorption curve (change in the wavelength absorbance, dA / dλ) on the wavelength.
The second derivative of the spectrum (derivative spectrum of the 2nd order) is the dependence of curvature of the absorption spectrum on the wavelength (d2A / dλ2). The second derivative at any wavelength of λ is proportional to the concentration according to the following equations:
d2Adλ2 = d2A1cm1% dλλc 'b10 = d2Aεdλλcb10,
in which they indicate:
c '- concentration of absorbing substance in g / l.
Device. A spectrophotometer equipped with an analogue resistance-to-capacity differential module or digital differential, or, where appropriate, other device generating a derivative spectrum, shall be used. Some methods of creating the second derivative of spectrum give rise to a wave shift relative to a spectrum of zero order, and this must be considered in these cases.
Figure 2.2.25-1
Fig. 2.2.25-1
Distinguishing ability. If prescribed in the article, the second order derivative spectrum of toluene R (0,2 g / l) solution in methanol R using methanol R as control fluid shall be recorded. In the spectrum is a small negative extreme placed between two large negative extremes at 261 nm and 268 nm, see Figure 2.2.25-1. Unless otherwise stated, the ratio A / B (see Figure 2.2.25-1) is not less than 0,2.
Working procedure. Prepare the test substance solution, set the appropriate instrumental conditions according to the manufacturer's instructions, and the quantity of the substance determined shall be calculated as specified in the Article.
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2. In Part 2 of the Annex to the Test Method, Chapter 2.2. Physical and physico-chemical methods, Chapter 2.2.31 reads as follows:
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2.2.31. Electrophoresis
GENERAL PRINCIPLE
By applying an electric field of charged particles dissolved or dispersed in an electrolyte solution, migrate in the direction of the reverse polarity electrode. In gel electrophoresis, particle movement is slowed by interaction with the surrounding gel matrix, which acts as a molecular sieve. The opposite effect of electrical force and molecular sifting leads to different migration speeds according to particle size, shape and charge. Due to their different physicochemical properties, the various macromolecules migrate during electrophoresis at different speeds, dividing them into different fractions. Electrophoretic separation may be performed in systems without supporting phases (e.g. separation in free solution in capillary electrophoresis) and in stabilising media such as thin plate, film or gel.
FREE ELECTROPHOSIS DO NOT ABOLE ELECTROPHOUSE WITH MOVABLE EXTENSION
This method is mainly used to determine electrophoretic mobility, the experimental characteristics being directly measurable and reproducible. It is mainly used for substances with high relative molecular weights and low diffusion coefficients. The position of the cut-off shall be determined at the beginning by physical methods such as refractometry or conductometry. After application of the electric field, new interfaces and their relative position are observed after precisely measured time. The working conditions must be selected to determine as many interfaces as the components.
ZONE ELECTROPHOUSE IN THE SHEET
Only a small quantity of the sample is sufficient for this method.
The nature of the carrier, such as paper, agar gel, cellulose acetate, starch, agarosa, methylacrylamide, bulking gel, introduces a number of other factors affecting electrophoretic mobility:
(a) because of the porosity of the carrier, the measured migration distance is less than the actual migration path;
(b) some media are not electroneutral and may sometimes cause significant electroendosomatic flux;
(c) any heating due to Joule's effect may cause the liquid to evaporate from the carrier, causing the solution to move from the edges to the centre as a result of capillarity. Ion force therefore tends to grow gradually.
The migration rate then depends on four main factors, in particular the mobility of the charged particle, the electroendosomatic flow, the flow caused by evaporation and the intensity of the electric field. It is therefore necessary to work under fully defined experimental conditions and to use reference substances wherever possible.
The electrophoresis equipment consists of:
- DC power supplies whose voltage can be regulated and stabilised,
- electrophoretic chambers, usually rectangular, made of glass or solid plastic with two separate spaces, anodic and cathodic, containing an electrolyte solution. In each space is immersed electrode, e.g. platinum or graphite. The electrodes are connected by an appropriately isolated circuit with a direct current source whose positive pole is connected to the anode and negative pole to the cathode. The liquid level in both spaces shall be maintained at the same level to prevent the flow caused by the siphone effect.
The electrophoretic chamber is closed with an airtight lid, which maintains a saturated atmosphere during the operation and reduces the evaporation of the solvent. For safety reasons, a device is used to cut the electrical current when removing the lid. If the electrical power exceeds 10 W, cooling of the carrier is recommended.
- equipment for fixing the carrier:
Striped electrophoresis. The belt of the carrier, pre-moistened with the electrolyte solution used and immersed in both ends of the electrode compartment, is suitably strained and attached to the support of the carrier to prevent electrolyte diffusion. The support can be a horizontal frame, a stand in the shape of an inverted V or a plate with protruding tips located at appropriate distances.
Gel electrophoresis. The device consists of a glass plate (e.g. a microscopic slide), the entire surface of which is covered with a tight layer of gel of the same thickness. The electrical connection between the gel and the conductive solution is provided in different ways depending on the type of device used. Provisional measures should be taken to avoid condensation of moisture or drying of the solid layer.
- measuring or detection equipment.
Working procedure. The electrolyte solution is poured into the electrodes. The electrolyte-saturated carrier shall be placed in the chamber in a manner appropriate to the device. The start point shall be marked and the sample applied. The electrical current shall be connected for the prescribed time. When the source is switched off, the carrier is removed from the chamber, dried and the separated substances are visualized.
ELECTROPHOUSE IN POLYACRYLAMIDE GEL IN PULSES
At this type of electrophoresis is a stationary phase gel which is prepared from a mixture of acrylamide and N, N '-methylenebisacrylamide. Gels are prepared into tubes length 7,5 cm and with inside diameter 0,5 cm. One solution is applied to each tube.
Device. It consists of two containers above each other for a buffer solution made of suitable material such as polymethylmethacrylate. Each container is equipped with platinum electrode. The electrodes are connected to a source enabling work either at a constant current or at a constant voltage. The device has several holders at the bottom of the top container equally distant from the electrode.
Procedure for determination. Before the polymerisation of the gel, the solutions should be removed and the gel used immediately after preparation. Prepare the gel mixture according to the regulation and pour into suitable closed-bottom glass tubes to the same height about 1 cm from the upper edge. It is necessary to ensure that no air bubbles remain in the tube. The mixture of gel is overlaid with water R in order to avoid air access and be polymerised. Gel formation usually takes about 30 minutes and is terminated when a sharp interface between the gel and the layer of water appears. The water layer is removed. The bottom container should be filled with the prescribed silencing solution and the tube caps removed. The tubes shall be fixed to the holders in the top container so that the lower part of the tubes is submerged in the dampening solution of the lower container. The tubes should be carefully filled with the prescribed buffer solution. Prepare the test solution and control solution, both containing appropriate marker dye, thickening e.g. by dissolving sucrose R. These solutions are applied to the surface of the gel of individual tubes, using a different tube for each solution. The same buffer solution is poured into the top container. The electrodes shall be connected to the power source and the elecrophoresis shall be allowed at the prescribed temperature and at the prescribed constant voltage or current intensity. The energy source shall be switched off when the marking dye reaches almost the lower container. The tubes shall be removed immediately from the device and the gel released. The position of individual zones in the electrophoreogram shall be degraded in the prescribed manner.
ELECTROFORSIS SDS IN POLYACRYLAMIDE GEL
Scope of application. Gel electrophoresis in polyacrylamide is used for the qualitative characterisation of proteins in biological products, for the control of purity and for quantitative determination.
Purpose. Analytical gel electrophoresis is an appropriate method to demonstrate the identity and homogeneity of proteins in pharmaceutical products. This method is routinely used to determine the molecular weight of proteins and protein subunits and to determine the composition of the subunits of purified proteins.
By replacing the gels and probers described in this text, the commercially available gels and probers may be used, provided they provide adequate results and comply with the validation requirements set out in paragraph Validation of the test.
POLYAKRYLAMID GELS 'CHARACTERS
The networking properties of polyacrylamide gels are based on a three-dimensional network of fibres and pores, which consists of bifunctional bisacrylamide netting of polyacrylamide chains. Polymerisation is catalysed by the formation of free radicals from diammonium peroxodisulphate and tetramethylethylenediamine.
Increasing acrylamide concentration in gel reduces the effective pores. The effective size of gel pores is the working definition of its sizing properties, i.e. the resistance that places the movement of macromolecules. There are limits on acrylamide concentrations that can be used. At high concentrations of acrylamide, the gel breaks more easily and is more difficult to handle. The migration rate of protein passing through the gel is reduced as the gel pores decrease. By adjusting the pore size of the gel by changing the concentration of acrylamide, the differentiation ability of the method can be optimised for the protein product. Therefore, the gel is physically characterised by the corresponding composition of acrylamide and bisacrylamide.
The protein state is an important factor in electrophoretic mobility alongside the gel composition. For proteins, electrophoretic mobility depends on the value of pK of charged groups and the size of the molecule. It is influenced by the type, concentration and pH of the dampening solution, the temperature and strength of the field as well as the characteristics of the carrier material.
ELECTROFORSIS OF DENATURATION CONDITIONS IN POLYACRYLAMIDE GEL
The method quoted as an example is limited to analysis of monomer polypeptides with a mass range of 14,000 to 100,000 daltons. The mass range may be extended by different techniques (e.g. gradient gel, special dampening system), but these techniques are not covered by this chapter.
Electrophoresis under denaturing conditions in polyacrylamide gel using sodium dodecylsulphate (SDS-PAGE) is the most widely used method of electrophoresis to determine the pharmaceutical quality of protein products and will be subject to a model method. Typical analytical electrophoresis of proteins is carried out in polyacrylamide gel under conditions ensuring that proteins are split into their individual polypeptide subunits while minimising their clumping. The most commonly used is the strong anion detergent of sodium dodecylsulphate (SDS) in combination with heating, where proteins break down into gel before they are applied. Denatured polypeptides weigh SDS, become negatively charged and exhibit a constant charge / weight ratio, regardless of the type of protein. Because the amount of bound SDS is almost always proportional to the molecular weight of polypeptide and is independent of its sequence, SDS-polypeptide complexes migrate with a polyacrylamide mobility gel dependent on the size of polypeptide.
For all resulting detergent-polypeptide complexes, the same functional relationship between their electrophoretic mobility and molecular mass is assumed. The migration of SDS complexes towards anode occurs in a predictable way, with low molecular complexes moving faster than high molecular complexes. The molecular weight of proteins can therefore be determined from their relative mobility in calibrated SDS-PAGE and the occurrence of one strip in such a gel is a purity criterion.
However, the modification of a polypeptide skeleton such as N-or O-glycosylation has a significant effect on the apparent molecular weight of the protein, since SDS does not bind to the carbohydrate component in a similar manner to the polypeptide, thus maintaining a constant charge-to-weight ratio. The apparent molecular weight of proteins after post-translational modifications is therefore not a real reflection of the weight of the polypeptide chain.
Reduction conditions
Polypeptide subunits and three-dimensional structure are often maintained in proteins by the presence of disulfide bonds. The aim of SDS- PAGE analysis under reduction conditions is to violate this structure by reducing disulfide bonds. Complete denaturing and protein fission by the action of 2-mercaptoethanol or dithiothreitol (DTT) results in the development of polypeptide skeleton and its subsequent complexing with SDS. Under these conditions, the molecular weight of polypeptide subunits may be calculated by linear regression from the molecular weight of the appropriate standards present.
Non-educational conditions
For some analyses, complete protein splitting into peptide subunits is not desirable. If not operated by reducing probers such as 2-mercaptoethanol or DTT, disulfide covalent binding remains intact and the oligomeric form of protein is preserved. Oligomeric complex SDS- protein moves slower than SDS- polypeptide subunits. In addition, unreduced proteins cannot be completely saturated with SDS and therefore cannot bind the detergent in a constant weight ratio. This makes the determination of the molecular weight of these molecules using SDS@-@ PAGE analysis less accurate than the analysis of fully denatured polypeptides and it is therefore necessary that both the standards and unknown proteins are for valid comparisons in a similar configuration. However, the colour of a single belt in such a gel is a criterion of purity.
CARTRIDGS OF THE DISCOCONTINUAL TLUMAL SYSTEM OF GEL ELECTROFORSIS
The most popular electrophoretic method to characterise complex protein mixtures is the use of a discontinuous dampening system consisting of two contiguous but different gels: a distinguishing or separation gel (lower) and a focus (upper) gel. These two gels are made with different porosity and contain solutions of different pH and ion strength. In addition, ions with varying mobility are used in gel and electrode dampening solutions. The discontinuity of dampening solutions causes the concentration of large sample volumes in the focus gel and leads to improved resolution. When the current is switched on, the flow rate of the sample is created and the protein is applied to the focus gel. Glycine ions from the electrode buffer solution follow the proteins to the focus gel. A rapidly forming area of the moving interface with the highly moving chloride ions at the head and with relatively slow glycine ions at the back. The localized high-voltage gradient formed between the lead and end ion zones causes SDS- protein complexes to form narrow zones and migrate between chloride and glycine zones.
Within the wide limits, regardless of the height of the applied sample zone, all SDS- proteins are concentrated in a very narrow area and entered into the distinguishing gel as a thin, well-defined high protein density zone. Focus gel with large pores does not slow down the migration of most proteins and serves mainly as an anti-convection environment. At the interface of the focus and separation gel, proteins are strongly slowed down due to the limited pores of the separation gel. In the distinguishing gel, proteins are further slowed down due to matrix networking. Glycine ions overtake proteins that then move in the area of permanent pH formed by trometamol and glycine. Molecular sifting causes SDS-polypeptide complexes to be separated based on their molecular weights.
PREPARATION OF VERTICAL SDS OF POLYACRYLAMIDE GELLS WITH DISCOCONTULOUS TIMING SOLUTIONS
Assembly of gel casting cassette
The thin detergent is cleaned and thoroughly rinsed with water by two glass plates (e.g. 10 cm × 8 cm), a polytetrafluoroethylene comb, two delimitators and a silicone rubber hose (e.g. 0,6 mm × 35 cm diameter). All parts are dried with a paper napkin. Exhibitors and hoses shall be lubricated with non-silicone fat. The divisors shall be applied along each of the two short sides of the glass plate 2 mm from the edges and 2 mm from the long side corresponding to the bottom of the gel. The hose starts to be placed on a glass plate using one delimiter as a guide. The hose is carefully rolled at the bottom of the demarcator and follows the long side of the glass plate. While holding the hose with one finger along the long side, the hose is rotated again and placed on the other short side of the glass plate using the second delimiter as a guide. The second glass plate shall be placed exactly above the first plate and the form shall be pressed with the hand. Two clamps shall be placed on each of the two short sides of the form, four clamps shall be carefully placed on the longer side of the gel form and the bottom of the gel form shall be formed in this way. It shall be verified that the hose passes around the edge of the glass plate and has not been pushed out when the clamps are placed. The gel form is now ready to pour the gel.
Preparation of gel
In the discontinuous silencing SDS polyalkrilamide gel, it is recommended to first pour the separation gel, have it polymerized and then pour the focusing gel, since the composition of the two gels differs by the ratio of acrylamide bisacrylamide, dampening solutions and pH.
Preparation of separation gel
In the conical flask, prepare an appropriate volume of solution containing the required acrylamide concentration for the separation gel using the values given in Table 2.2.31.1. The ingredients shall be mixed in the prescribed order. If necessary, the solution should be filtered prior to the addition of the solution of diammonium peroxodisulphonate and tetramethylethylenediamine (TEMED), if necessary using vacuum via an acetate cellulose membrane of 0,45 μm pore size; the solution should be kept under vacuum, the filtration unit should be rotated until no bubbles are formed in the solution. Add the appropriate amount of solution of diammonium peroxodisulphate and tetramethylethylenediamine according to Table 2.2.31-1, mix with a circular motion and pour immediately into the gap between the two glass plates of the form. Leave sufficient space for the focusing gel (length of comb teeth plus 1 cm extra). Using a narrowed glass pipette, the solution is carefully overturned with isobutanol with saturated water. The gel shall be held vertically at room temperature.
Preparation of focus gel
After complete polymerisation (about 30 min), isobutanol is poured out and the top part of the gel rinsed with water several times to remove the rest of isobutanol and unpolymerised acrylamide. The remaining water is removed from the top of the gel as much liquid as possible and then removed by the edge of a paper napkin.
In the conical flask, prepare an appropriate volume of solution containing the required acrylamide concentration using the values given in Table 2.2.31-2. The ingredients shall be mixed in the prescribed order. If appropriate, the solution should be filtered prior to the addition of the solution of diammonium peroxodisulphonate and tetramethylethylenediamine, if necessary using vacuum via an acetatcellulose membrane of 0,45 μm pore size; the solution should be kept under vacuum, under occasional swirling, by circling the filtration unit until additional bubbles are formed in the solution. Add the appropriate amount of solution of diammonium peroxodisulphate and tetramethylethylenediamine according to Table 2.2.31-2, mix with a circular motion and pour immediately into the gap between the two glass plates of the form directly onto the surface of the polymerised distinguishing gel. A clean polytetrafluoroethylene comb is immediately inserted into the focus gel, making sure that no air bubbles remain below the comb. Add another focus gel solution to fill the ridge space completely. Allow the gel to be polymerised in a vertical position at room temperature.
Tab. 2.2.31-1 Preparation of separation gel
| Složky roztoku | Objemy složek (ml) na objem gelové formy | |||||||
|---|---|---|---|---|---|---|---|---|
| 5 ml | 10 ml | 15 ml | 20 ml | 25 ml | 30 ml | 40 ml | 50 ml | |
| 6% akrylamid | ||||||||
| voda R | 2,6 | 5,3 | 7,9 | 10,6 | 13,2 | 15,9 | 21,2 | 26,5 |
| roztok akrylamidu(1) | 1,0 | 2,0 | 3,0 | 4,0 | 5,0 | 6,0 | 8,0 | 10,0 |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,3 | 2,5 | 3,8 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100 g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,004 | 0,008 | 0,012 | 0,016 | 0,02 | 0,024 | 0,032 | 0,04 |
| 8% akrylamid | ||||||||
| voda R | 2,3 | 4,6 | 6,9 | 9,3 | 11,5 | 13,9 | 18,5 | 23,2 |
| roztok akrylamidu(1) | 1,3 | 2,7 | 4,0 | 5,3 | 6,7 | 8,0 | 10,7 | 13,3 |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,3 | 2,5 | 3,8 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100 g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,003 | 0,006 | 0,009 | 0,012 | 0,015 | 0,018 | 0,024 | 0,03 |
| 10% akrylamid | ||||||||
| voda R | 1,9 | 4,0 | 5,9 | 7,9 | 9,9 | 11,9 | 15,9 | 19,8 |
| roztok akrylamidu(1) | 1,7 | 3,3 | 5,0 | 6,7 | 8,3 | 10,0 | 13,3 | 16,7 |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,3 | 2,5 | 3,8 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100 g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,002 | 0,004 | 0,006 | 0,008 | 0,01 | 0,012 | 0,016 | 0,02 |
| 12% akrylamid | ||||||||
| voda R | 1,6 | 3,3 | 4,9 | 6,6 | 8,2 | 9,9 | 13,2 | 16,5 |
| roztok akrylamidu(1) | 2,0 | 4,0 | 6,0 | 8,0 | 10,0 | 12,0 | 16,0 | 20,0 |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,3 | 2,5 | 3,8 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100 g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,002 | 0,004 | 0,006 | 0,008 | 0,01 | 0,012 | 0,016 | 0,02 |
| 14% akrylamid | ||||||||
| voda R | 1,4 | 2,7 | 3,9 | 5,3 | 6,6 | 8,0 | 10,6 | 13,8 |
| roztok akrylamidu(1) | 2,3 | 4,6 | 7,0 | 9,3 | 11,6 | 13,9 | 18,6 | 23,2 |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,2 | 2,5 | 3,6 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100 g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,002 | 0,004 | 0,006 | 0,008 | 0,01 | 0,012 | 0,016 | 0,02 |
| 15% akrylamid | ||||||||
| voda R | 1,1 | 2,3 | 3,4 | 4,6 | 5,7 | 6,9 | 9,2 | 11,5 |
| roztok akrylamidu(1) | 5,0 | 7,5 | 10,0 | 12,5 | 15,0 | 20,0 | 25,0 | |
| Tris 1,5 mol/l (pH 8,8)(2) | 1,3 | 2,5 | 3,8 | 5,0 | 6,3 | 7,5 | 10,0 | 12,5 |
| 100 g/l SDS(3) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| 100g/l APS(4) | 0,05 | 0,1 | 0,15 | 0,2 | 0,25 | 0,3 | 0,4 | 0,5 |
| TEMED(5) | 0,002 | 0,004 | 0,006 | 0,008 | 0,01 | 0,012 | 0,016 | 0,02 |
(1) Solution of 30% acrylamide: acrylamide-bisacrylamide (29: 1) RS
(2) Tris 1,5 mol / l (pH 8,8): Trometamol buffer solution at pH 8,8 (1,5 mol / l)
(3) 100 g / l SDS: sodium dodecylsulphate solution R (100 g / l)
(4) 100 g / l APS: solution of diammonium peroxodisulphate R (100 g / l). Diammonium peroxodisulphate provides free radicals that control polymerisation of acrylamide and bisacrylamide. As the solution of diammonium peroxodisulphate decomposes slowly, a fresh solution is prepared each week.
(5) TEMED: tetramethylethylenediamine R
Tab. 2.2.31-2 Preparation of focus gel
| Složky roztoku | Objemy složek (ml) na objem gelové formy | |||||||
|---|---|---|---|---|---|---|---|---|
| 1 ml | 2 ml | 3 ml | 4 ml | 5 ml | 6 ml | 8 ml | 10 ml | |
| voda R | 0,68 | 1,4 | 2,1 | 2,7 | 3,4 | 4,1 | 5,5 | 6,8 |
| roztok akrylamidu(1) | 0,17 | 0,33 | 0,5 | 0,67 | 0,83 | 1,0 | 1,3 | 1,7 |
| Tris 1,0 mol/l (pH 6,8)(2) | 0,13 | 0,25 | 0,38 | 0,5 | 0,63 | 0,75 | 1,0 | 1,25 |
| 100 g/l SDS(3) | 0,01 | 0,02 | 0,03 | 0,04 | 0,05 | 0,06 | 0,08 | 0,1 |
| 100 g/l APS(4) | 0,01 | 0,02 | 0,03 | 0,04 | 0,05 | 0,06 | 0,08 | 0,1 |
| TEMED(5) | 0,001 | 0,002 | 0,003 | 0,004 | 0,005 | 0,006 | 0,008 | 0,01 |
(1) acrylamide solution: acrylamide (29: 1) 30% RS
(2) Tris 1,0 mol / l (pH 6,8): Trometamol buffer solution at pH 6,8 (1 mol / l)
(3) 100 g / l SDS: sodium dodecylsulphate solution R (100 g / l)
(4) 100 g / l APS: solution of diammonium peroxodisulphate R (100 g / l). Diammonium peroxodisulphate provides free radicals that control polymerisation of acrylamide and bisacrylamide. As the solution of diammonium peroxodisulphate decomposes slowly, a fresh solution is prepared each week.
(5) TEMED: tetramethylethylenediamine R
Location of gel in electrophoretic equipment and electrophoretic separation
After complete polymerisation (about 30 min), remove the polytetrafluoroethylene comb carefully. Flasks should be rinsed immediately with water or SDS- PAGE with electrode solution R to remove unpolymerised acrylamide. If necessary, the teeth of the sharpening gel shall be examined using a blunt hypodermic needle attached to the syringe. The clamps are removed on the short side, the hoses are carefully removed and the clamps are put back on. On the other hand, the short side is similar. Remove the hose from the bottom of the gel. The gel cassette shall be placed in an electrophoretic device. The top and bottom container shall be filled with electrophobic dampening solutions. Remove any bubbles caught at the bottom of the gel between the glass plates, preferably with a hypodermic needle attached to the syringe. Preelectrophoresis is never performed, i.e. the connection of voltage prior to application of the sample, as this violates the discontinuity of dampening systems.
Before applying the sample, the slot between the glass is carefully rinsed with an electrode solution RS. Prepare the test and control solution in the recommended sample buffer solution and adjust as specified in the individual article. An appropriate volume of individual samples is applied to the focus gel wells. Electrophoresis shall be started according to the conditions recommended by the device manufacturer. SDS- PAGE manufacturers can supply gels of different sizes of different thickness. The time the electrophoresis takes place and the applied current or voltage for optimal division may vary according to the device manufacturer. It shall be verified that the coloured forehead moves towards the separation gel. If the dye approaches the gel bottom, the electrophoresis will stop. The gel cassette is removed from the device and the glass plate is separated. Removes the demarcators and focus gel and immediately continues to dye.
_
Coomassie coloring is the most common method of protein coloring with a detection level ranging from 1 μg to 10 μg protein per belt. Silver dye is the most sensitive method of protein dye in gel and can be degraded belt containing 10 to 100 ng.
All gel dye steps are carried out at room temperature by gently shaking (e.g. in an orbital shaker) in a suitable container. Gels must be coloured with gloves to avoid dyeing of fingerprints.
Coomassie coloring
The gel is immersed in a large excess of the colouring solution of blue-acidic 83 RS and left to stand for at least 1 hour. Then remove the colouring solution.
The gel is discoloured with a large excess of RS discoloration solution, which changes several times until the coloured strips of protein are clearly distinguishable on a clean background. The more thoroughly the gel is discoloured, the less protein can be degraded. Discoloration may be accelerated by adding several grams of anexus or small mushrooms to the RS discoloration solution.
NOTE: Acid solutions used in this process do not fix protein in gel. It can completely lead to the loss of some low molecular proteins during coloring and decoloration of thin gels. The fixed fixation is achieved by allowing the gel to stand in a mixture of volume parts of trichloroacetic acid R, methanol R and water R (1 + 4 + 5) for 1 hour before immersion in the colouring solution blue acid RS.
Silver dyeing
The gel is immersed in a large excess of the fixation solution R and left to stand for 1 h. Remove the fixation solution R, add a fresh fixation solution R and incubate at least 1 h or, if appropriate, overnight.
Fixing solution R is removed and the gel is washed by a large excess of water R for 1 h. The gel is then allowed to soak 15 min in a solution of glutaraldehyde R 1% (V / V) and washed twice for 15 min in a large excess of R water, then soaked 15 min in the dark with a fresh silver nitrate test R. Finally, the gel is washed three times for 5 min in a large excess of water R and immersed to about 1 min in the R-development solution until satisfactory colouring occurs. The development is stopped by incubation in the R blocking solution for 15 min and the gel is washed with R water.
DRINKS OF POLYACRYLAMID GELLS
Depending on the colouring method used, gels are treated in a slightly different way. For Coomassia coloring, the gel should be left in a solution of glycerol R (100 g / l) at least 2 h (incubation is possible overnight) after discoloration steps. For silver colouring, add one step to the final rinsing - leaving a gel of 5 min in glycerol R solution (20 g / l).
Two foil porous cellulose film is immersed in R water and incubated 5 to 10 minutes. One of the films is placed on the drying frame. The gel is carefully grabbed and placed on a cellulose film. Remove all trapped air bubbles and pour several millilitres of R water around the edges of the gel. The second film is placed on the gel from the top and the air bubbles caught are removed again. A drying frame shall be completed and placed in the oven or kept at room temperature until dried.
DETERMINATION OF MOLECULAR WEIGHT
The molecular weight of proteins is determined by comparing their mobility with the mobility of several protein standards of known molecular masses. For calibration gels are available protein mixtures with well-known molecular weights mixed for homogeneous colour. They can be obtained in different molecular weight ranges. Concentrated protein stock solutions of known molecular weight are diluted in an appropriate sample buffer solution and applied to the same gel as the test protein sample.
Immediately after electromigration, the position of the branding dye in the gel shall be marked with bromophenol blue to identify the leading edge of the electrophoretic ion forehead. This can be done by cutting the cuts at the end of the gel or inserting a needle with Indian ink into the gel on the forehead of the dye. After dyeing, the migration distance of each protein strip (standards and unknown proteins) shall be measured from the beginning of the separation gel. The migration distance of each protein is divided by the distance travelled by the dye. The normalized migration distances thus obtained are called relative protein mobility (relative to the face of the dye) and are commonly designated as Rf. The log curve of the relative molecular weight (Mr) of protein standards is formed as a function of Rf. The graph is slightly sigmoid. Unknown molecular weights can be determined by linear regression analysis or interpolation from log Mr versus Rf curves if the values of unknown samples are found in the linear portion of the graph.
TEST VALIDATION
The test may be evaluated if the protein standards of the molecular weight are distributed along 80% of the length of the gel and include the required separation range (e.g. the range covering the product and its dimer or the product and its impurities) of separation obtained for significant protein strips, showing a linear relationship between the log molecular weight and Rf. Additional validation requirements with regard to the test solution may be specified in the relevant article.
_
Where the limit of impurities in the article is specified, a control solution corresponding to the level of impurities should be prepared by dilution of the test solution. For example, if the limit is 5%, the control solution is prepared by dilution of the test solution at a ratio of 1: 20. No impurity (no belt other than the main belt) on the electrophoreogram obtained with the test solution shall be more intense than the main belt obtained with the control solution.
In validation conditions, impurities should be quantified by normalisation to the main belt using the integration densitometer. In this case, the response shall be validated on linearity.
"
3. In Part 2 of the Annex to the Test Method, Chapter 2.2. Physical and physico-chemical methods, the following Chapters 2.2.42, 2.2.43 and 2.2.44 are inserted after Chapter 2.2.41:
"
2.2.42. Solid density
The density of solids corresponds to their average mass per unit of volume and is usually expressed in grams per cubic centimetre (g / cm3), although the international unit is a kilogram per cubic metre (g / cm3 = 1000 kg / m3).
Unlike gases and liquids whose density depends only on temperature and pressure, the density of the solid also depends on its molecular arrangement and therefore changes with the crystal structure and degree of crystals.
If solid particles are amorphous or partially amorphous, their density may also depend on the way they are prepared and processed.
In contrast to liquids, the density of two chemically equivalent solids may therefore be different, and this difference reflects the difference in the structure of the solid state. Particle density is an important physical characteristic for pharmaceutical use.
The density of a solid particle may reach different values depending on the method used to measure its volume. It is useful to distinguish between three levels of density expression:
- crystal density which only includes a solid fraction of the material; the density of the crystal is also called the right density,
- particle density, which also includes the volume of cavities inside the particle,
- total density, which also includes free volume between particles, produced in a powder layer; total density is also called apparent density.
I. Crystal density
The substance crystal density is the average mass per volume unit (volume unit weight), except for all cavities which are not essential parts of the molecular arrangement. It is an intrinsic property of the substance and should therefore be independent of the method of determination. The crystal density can be determined either by calculation or by simple measurement.
A. Calculated crystal density is obtained using crystalographic data (size and composition of the base cell) of the perfect crystal, e.g. from X-ray diffraction data, and the molecular weight of the substance.
B. Measured crystal density is the ratio of weight to volume after measuring these values for monocrystal.
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Regulation Information
| Citation | Decree of the Ministry of Health No. 48 / 2001 Coll., amending Decree of the Ministry of Health No. 1 / 1998 Coll., laying down requirements for the quality, procedure for the preparation, testing, storage and dosing of medicinal products (Czech Pharmacopoeia 1997), as amended by Decree No. 296 / 1999 Coll. |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 15.02.2001 |
|---|---|
| Effective from | 15.02.2001 |
| Effective until | - |
| Status | Valid |
The regulation text is for informational purposes only.
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