RU2577703C1 - Method for producing positive control standard sample of human immunoglobulin to determine anticomplementary activity of preparations of human immunoglobulin, and standard sample of human immunoglobulin to determine anticomplementary activity of preparations of human immunoglobulin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, specifically to human immunoglobulin standard samples used to determine anticomplementary activity of human immunoglobulins, and methods for their preparation. Group of inventions discloses a method of producing a positive control standard sample of human immunoglobulin comprising thermal treatment of sample in a water bath, cooling thermally treated sample at a temperature of (22±3) °C, anticomplementary activity determination of positive control sample; and a standard sample of human immunoglobulin containing a positive control, produced by disclosed method, and negative control with anticomplementary activity in range of 10-45 %.

EFFECT: group of inventions can be used to assess quality of human immunoglobulin preparations and enables to determine anticomplementary activity to improve reliability of results.

2 cl, 4 dwg, 11 ex

Description

The invention relates to biotechnology, in particular to methods for obtaining controls of a standard sample of human immunoglobulin to determine the anti-complementary activity of preparations of human immunoglobulins; as well as standard samples of human immunoglobulin, designed to determine the anti-complementary activity of preparations of human immunoglobulins.

The invention can be used to determine the anti-complementary activity of preparations of human immunoglobulins in the production process to assess the quality of both the release and in the course of their research in order to confirm compliance with regulatory requirements (certification tests).

Human immunoglobulins are drugs containing a concentrated solution of an immunologically active protein fraction isolated by fractionation with ethyl alcohol at a temperature below 0 ° C from the blood plasma of healthy donors. One of the manifestations of the side effect of immunoglobulin preparations during intravenous administration is the spontaneous activation of the complement system in the Fc region of immunoglobulin molecules aggregated and damaged during fractionation, which in turn leads to the activation of macrophages and the release of vasoactive substances and the corresponding clinical manifestations [1]. The quality indicator characterizing the indicated property is “Anti-complementary activity”.

The critical parameters that affect the final result of determining the anti-complementary activity of human immunoglobulin preparations are:

- the source of the complement of guinea pigs;

- the activity of the used hemolytic serum;

- a batch of sheep erythrocytes and their sample preparation;

- the composition of the used buffer solution, its temperature and pH.

The variability of these parameters indicates the need to use a standard sample in determining the anti-complementary activity of human immunoglobulin preparations. The positive and negative controls of the standard sample confirm the reliability of the results in various ranges of anticomplementary activity values.

Analogues developed by the authors of the method for obtaining positive control of a standard sample of human immunoglobulin to determine the anti-complementary activity of human immunoglobulin preparations at the prior art have not been identified.

The closest to the claimed standard sample is “Human immunoglobulin standard (anti-complementary activity and molecular parameters), biological reference drug, series 1” [“Human immunoglobulin (ACA and molecular size), BRP batch 1”], approved by the European Directorate for the Quality of Medicines and health care (European Directorate for the Quality of Medicines, EDQM) in 2011. It is currently used to determine the anti-complementary activity of human immunoglobulin preparations [2]. Before use, this standard sample is dissolved in a predetermined volume (105.4 ml) of water for injection. The result is a solution with an immunoglobulin content of about 50 mg / ml, and there is a need to confirm the protein content in the resulting solution. Negative and positive controls are formed by changing the amount of the initial solution of a standard immunoglobulin sample: 0.2 ml and 0.6 ml for the negative and positive controls, respectively. A well-known standard sample is characterized by instability of certified characteristics, confirmed by a significant coefficient of variation: for negative control - 33%, for positive control - 15% [3]. Its use cannot be rational, since when the lyophilisate is dissolved, more than 105.4 ml of a solution with a limited shelf life (no more than 14 days) is obtained, and its consumption per study does not exceed 0.8 ml. When storing the resulting standard sample solution, it is possible to be contaminated with microorganisms and change the values of the certified characteristics.

The objective of the invention is to increase the stability of the certified characteristics of a standard sample, as well as increasing the efficiency of its use, simplifying sample preparation and improving the reproducibility of the results of determining the anti-complementary activity of human immunoglobulin.

The problem is solved due to the fact that to determine the anti-complementary activity of preparations of human immunoglobulins, a positive control of a standard sample of human immunoglobulin is used, obtained by a method comprising the following stages:

- heat treatment of a sample of human immunoglobulin in a water bath at a temperature of (56 ± 1) ° C;

- cooling a heat-treated sample of human immunoglobulin at a temperature of (22 ± 3) ° C;

- determination of the anti-complementary activity of the positive control sample, while the values of the determined activity should be in the range from 60 to 95%.

A standard sample of human immunoglobulin for determining anti-complementary activity contains a positive control obtained by the method described above, and a negative control with anti-complementary activity in the range of 10-45%.

The inventive standard sample of human immunoglobulin used to determine the anti-complementary activity of preparations of human immunoglobulins, includes two ampoules of transparent neutral glass containing a solution of human immunoglobulin, marked on the outer surface, reflecting information about the name of the standard sample, the name of the control indicating the value of anti-complementary activity, manufacturer , date of manufacture, shelf life, storage conditions, instructions for use, and packaged in containers for storage and transportation.

There is a causal relationship between the set of essential features of the claimed standard sample and the technical result achieved, namely, the use of two components with certified protein content: a human immunoglobulin solution with anti-complementary activity in the range of 60-95% for a positive control, and a human immunoglobulin solution with anti-complementary activity in the range of 10-45% for negative control, allows to increase the stability of certified characteristics, exclude additional final stages of sample preparation that affect the error in determining the target value; while a more convenient form of release (ampoule) allows you to more rationally store and use the inventive standard sample.

For the stage of heat treatment and cooling in the method of obtaining a positive control, the duration of exposure to temperature is determined by the volume of the human immunoglobulin solution placed in the container, and can be 5 minutes or more.

For negative control of a standard sample of human immunoglobulin, a human immunoglobulin sample that has not undergone heat treatment is used to determine anti-complementary activity.

Additionally, a standard sample of human immunoglobulin for determining anticomplementary activity is certified for protein content. The amount of protein is determined by the technology for producing human immunoglobulin preparations.

Due to the solution of such problems as increasing the stability of the certified characteristics of a standard sample of human immunoglobulin for determining anticomplementary activity and its more rational use, the invention improves the reliability of the results of determining the anticomplementary activity of human immunoglobulin preparations in various ranges.

Brief Description of Drawings and Drawings

FIG. 1. Preparation of samples with positive and negative control of the inventive standard sample of human immunoglobulin to determine anticomplementary activity and complement control.

FIG. 2. The procedure for preparing samples to determine the residual activity of complement.

FIG. 3. The average value of the certified characteristics of anticomplementary activity in a standard sample of human immunoglobulin.

FIG. 4. The study of the stability of a standard sample of human immunoglobulin to determine anticomplementary activity.

The possibility of carrying out the claimed invention is shown by the following examples.

Example 1. Preparation of gelatin-saline / gelatin-barbital buffer solution

1. Gelatin-saline solution (JSR)

1 g of gelatin is placed in a 250 ml volumetric flask, approximately 200 ml of purified water is added and left to swell. The mixture is heated at a temperature of (45 ± 3) ° C until complete dissolution. The resulting clear solution was cooled to room temperature.

8.5 g of sodium chloride, 0.1 g of anhydrous calcium chloride, 0.04 g of 6-aqueous magnesium chloride are placed in a 1000 ml volumetric flask, dissolved in a small amount of purified water and the prepared gelatin solution is added. The total volume of the solution was adjusted to the mark with purified water and mixed. The pH of the solution is adjusted to 7.2-7.3 with 0.1 M sodium hydroxide solution. The solution is used freshly prepared.

2. Gelatin-barbital buffer solution

2.1. Preparation of an initial solution of calcium and magnesium

4.412 g of calcium chloride dihydrate and 20.332 g of magnesium chloride 6-aqueous are dissolved in a small amount of purified water in a 100 ml volumetric flask, the solution volume is adjusted to the mark with purified water and mixed. The solution is stored at a temperature of (5 ± 3) ° C for 1 month.

2.2. Preparation of the initial barbital buffer pH 7.3

83.0 g of sodium chloride and 10.192 g of sodium barbital are dissolved in 1700 ml of purified water in a volumetric flask with a capacity of 2000 ml, constantly stirring. The pH is adjusted to 7.3 with 1 M hydrochloric acid. Add 5 ml of the initial solution of calcium and magnesium, adjust the volume of the solution with purified water to the mark and mix. The solution is stored at a temperature of (5 ± 3) ° C for 1 month.

2.3. Preparation of gelatin solution

1.25 g of gelatin is placed in a 1000 ml volumetric flask, 500 ml of purified water is added and mixed thoroughly. The resulting mixture is heated at a temperature of (45 ± 3) ° C until the gelatin is completely dissolved. The resulting solution was cooled to room temperature, the volume of the solution was adjusted to the mark with purified water and stirred. Use only freshly prepared solution.

2.4. Preparation of gelatin-barbital buffer solution

4 parts of gelatin solution are added to 1 part of the initial barbital buffer solution, mixed thoroughly and the pH is adjusted to 7.2-7.3 with a 1 M sodium hydroxide solution or 1 M hydrochloric acid solution. The solution is used freshly prepared.

The turnover of barbital and its sodium salt in Russia is limited, as these substances are included in the List of psychotropic substances, the circulation of which in the Russian Federation is limited and with respect to which certain control measures may be excluded in accordance with the legislation of the Russian Federation and international treaties of the Russian Federation (list III); To use it, you must have a license. In view of the tightening of the requirements for the circulation of barbital and its sodium salt, it is necessary to use gelatin-saline solution as an alternative.

Example 2. Preparation of sheep red blood cells

Sheep blood can be used no earlier than 7 days after collection and used within 21 days.

Sheep blood stabilized in citrate solution: take one volume of blood in one volume of citrate solution and mix. Store at (5 ± 3) ° C.

Preparation of citrate solution:

20.5 g of glucose, 8.0 g of trisubstituted sodium citrate and 4.2 g of sodium chloride are dissolved in 750 ml of purified water, the pH of the solution is adjusted to 6.1 with citric acid solution (100 g / l), the volume of the solution is adjusted to 1000 ml purified water. Steam sterilized for 30 minutes at a temperature of 100 ° C. Store at a temperature of (5 ± 3) ° C for 1 month.

Example 3. Preparation of a hemolytic system (suspension of sensitized sheep erythrocytes)

Preliminarily titrated according to the method described in the European Pharmacopoeia [4], hemolytic serum is diluted to 2 MGE / ml and added to a 5% suspension of sheep erythrocytes prepared according to the method described in the European Pharmacopoeia [4], in a 1: 1 ratio. Incubated in a thermostat at a temperature of (37 ± 0.5) ° C for 15 minutes, used within 6 hours after preparation, stored at a temperature of (5 ± 3) ° C.

Example 4. Preparation of complement guinea pigs

Prepare a pool of serum from the blood of at least 10 guinea pigs. Serum is separated from the blood clot by centrifugation at a temperature of (5 ± 3) ° C. Serum is stored in small quantities at temperatures below minus 70 ° C.

Determination of the initial activity of complement is carried out according to the method described in the European Pharmacopoeia [4]. After determining the initial complement activity, a complement solution is prepared containing 100 CH ₅₀ / ml, i.e. 1 ml of the complement solution is diluted with JSR in N times.

Example 5. A method of obtaining a positive control of a standard sample of human immunoglobulin to determine anticomplementary activity

Applicable materials and equipment:

- water bath Julabo ED-19M (Julabo Labortechnik GmbH, Germany);

- technical glass thermometer ТТМ (measurement range from 0 to 100 ° C, OJSC "Termopribor", Russia);

- purified water.

The method is as follows. To obtain a positive control of a standard sample, 1.5 ml ampoules with a human immunoglobulin sample are subjected to heat treatment in a water bath with the following parameters:

- water temperature (56 ± 1) ° C;

- heat treatment time 5 minutes.

In this case, a change in the release form of a human immunoglobulin sample leads to a change in the duration of heating.

As a result, human immunoglobulin samples having high anticomplementary activity are obtained.

Cool heat-treated samples of human immunoglobulin with the following parameters:

- temperature (22 ± 3) ° C;

- cooling time 15 minutes.

Example 6. A method of obtaining a positive control of a standard sample of human immunoglobulin to determine anti-complementary activity

Analogously to example 5, it is possible to use 25 ml bottles of a human immunoglobulin sample to obtain a positive control of a standard sample, while the heat treatment time in a water bath is 15 minutes and the cooling time is 20 minutes.

Example 7. A method of obtaining a positive control of a standard sample of human immunoglobulin to determine anticomplementary activity

Analogously to example 5, it is possible to use 3 ml ampoules with a human immunoglobulin sample to obtain a positive control of a standard sample, while the heat treatment time in a water bath is 7 minutes and the cooling time is 15 minutes.

Example 8. Determination of the certified characteristics of anti-complementary activity

In the prepared positive control obtained according to example 5, for a standard sample of human immunoglobulin and a negative control, which is a human immunoglobulin solution that has not undergone heat treatment, certified characteristics are determined as follows.

Sample preparation with positive and negative control of the inventive standard sample and complement control is shown in FIG. one.

The preparation of samples with a positive control of the claimed standard sample according to examples 6, 7 and samples with a negative control, which is a human immunoglobulin solution that has not undergone heat treatment, is carried out depending on the certified value of the protein content in the human immunoglobulin solution indicated in the passport for the standard sample.

The prepared samples are incubated in a thermostat at a temperature of (37 ± 0.5) ° C for 60 minutes. After the incubation time has elapsed, a 1:50 dilution is prepared for the sample with complement control and a 1:20 dilution for negative and positive control samples. Then, the residual complement activity (in duplicate) is determined in accordance with FIG. 2.

To prepare control samples without hemolysis, 1.3 ml of BR are added to three tubes.

To prepare control samples with complete hemolysis, 1.3 ml of purified water are added to three test tubes.

Then, 0.2 ml of hemolytic system is added to each tube (tubes 1-12 and test tubes with control samples), mix thoroughly and incubate in a thermostat at a temperature of (37 ± 0.5) ° C for 60 minutes, then cool for 10 minutes at a temperature of (5 ± 3) ° C and centrifuged at 1000 g for 5 minutes

The optical density of the supernatant is measured on a spectrophotometer in a cuvette with a layer thickness of 10 mm at a wavelength of 541 nm. The comparison solution is JSR. The degree of hemolysis (Y) is calculated by the formula 1:

Where:

Ap is the average optical density of the test sample;

Ao is the average optical density of the control sample without hemolysis;

Ak - the average value of the optical density of the control sample with complete hemolysis;

On graph paper with a logarithmic scale on both axes or using software, a graph is plotted with Y / (1-Y) values on the abscissa axis and the amount of complement diluted in ml on the ordinate axis. An optimized curve is constructed (when plotting, the values of the degree of hemolysis are taken into account, which are in the range from 0.15 to 0.85; values outside the specified range are not taken into account), corresponding to the plotted points, and the ordinate is determined for the complement dose leading to 50% hemolysis, i.e. Y / (1-Y) = 1.

If centrifugation and / or reading of the results cannot be done immediately, the tubes are stored at a temperature of (5 ± 3) ° C for no more than 1 hour.

The complement activity is calculated in hemolytic units (CH ₅₀ / ml) according to the formula 2:

Where:

B is the reciprocal of the degree of dilution of complement;

C is the volume of diluted complement (ml), causing 50% hemolysis (Y / (1-Y) = 1);

5 - multiplier to account for the number of red blood cells.

The result of the titration of complement is considered reliable provided that the slope of the straight line is from 0.15 to 0.40, preferably in the range of 0.18-0.30.

The anti-complementary activity (in percent) in the negative and positive controls is calculated with respect to the complement control, taken as 100%, according to formula 3:

Where:

b - value of CH ₅₀ / ml for complement control, calculated by the formula 2;

and - the value of CH ₅₀ / ml for the negative, positive control and the test sample, calculated by the formula 2.

Test results are considered reliable if:

1. the values of the anti-complementary activity of the negative and positive control are in the range of 10-45% and 60-95%, respectively;

2. complement activity in complement control is in the range from 80 to 120 CH ₅₀ / ml.

Otherwise, re-analysis is carried out.

The results of determining anticomplementary activity in a standard sample of human immunoglobulin are presented in FIG. 3.

From the data presented in FIG. 1, it follows that there is no need to dissolve the standard sample and confirm the protein content, while the following positive aspects are observed:

- simplification of sample preparation.

From the data presented in FIG. 3, it follows that the certified value of the anti-complementary activity of the positive control of the standard sample prepared by the proposed method meets the stated requirements: the anti-complementary activity in the range of 60-95%, the certified value of the anti-complementary activity of the negative control falls within the range of 10-45%, also the standard sample has the coefficient of variation for negative control is 5.7%, for positive control - 3.6%, while the following positive aspects are observed:

- increased stability of certified characteristics of a standard sample.

Example 9. The study of the stability of a standard sample of human immunoglobulin to determine anticomplementary activity

To study stability, samples of the obtained standard sample of human immunoglobulin are stored at a temperature of (5 ± 3) ° C to determine anti-complementary activity and the controlled parameters are determined at the time of storage, then every month for the first three months, then with an interval of 3 months up to 15 months. Controlled parameters: anti-complementary activity and protein content. The results of stability studies are presented in FIG. 4. To establish the final shelf life, observations are ongoing.

Example 10. A kit containing a standard sample of human immunoglobulin for determining anti-complementary activity

The kit contains a positive control (1.5 ml ampoule) obtained in Example 5 and a negative control (1.5 ml ampoule), which is a human immunoglobulin solution that has not undergone heat treatment, certified according to the method described in Example 8. The kit is packaged in a cardboard box and apply instructions for use and a passport for a standard sample.

Example 11. Storage and transportation of the standard sample

Packaged reference kits are stored at a temperature of (5 ± 3) ° C in a dark place. Transportation is carried out by all types of covered transport according to SP 3.3.2.1248-03 at a temperature of (5 ± 3) ° C.

Similarly, taking into account the disclosure of the invention in the description by a specialist in this field can be obtained, other variants of the invention are made, which are covered by the claims below.

BIBLIOGRAPHY

1. Buchacher A., Schluga P., Mullner J., Schreiner M. Anticomplementary activity of IVIG concentrates - important assay parameters and impact of IgG polymers // Vox sanguinis. 2010. V. 98. N 3p1. P. e209-e218.

2. Leaflet to a standard sample of human immunoglobulin BRP. [Electronic resource] URL: http://crs.edqm.eu/db/4DCGI/db/4DCGI/ leaflet? Leaflet = Y0001504_1 (accessed: 03/21/2014).

3. Sandberg E., Costanzo A., Daas A., Buchheit K.-N. Calibration of the human immunoglobulin BRPs for ACA and molecular size (batch 1) and for Fc function and molecular size (batchs 1 & 2) // Pharmeuropa Bio & SN. 2012.P. 1-15. [Electronic resource] URL: http://pharmeuropa.edqm.eu/PharmeuropaBioSN/ (accessed: 03/21/2014).

4. European Pharmacopoeia 8.1, 01/2010: 20617 corrected 7.6 “Test for anticomplementary activity of immunoglobulin)). [Electronic resource] URL: http://online6.edqm.eu/ep801/# (accessed: 03.21.2014).

Claims (2)

1. The method of obtaining a positive control of a standard sample of human immunoglobulin for determining the anti-complementary activity of preparations of human immunoglobulins, comprising the following stages:
- heat treatment in a water bath of a sample of human immunoglobulin at a temperature of (56 ± 1) ° C;
- cooling a heat-treated sample of human immunoglobulin at a temperature of (22 ± 3) ° C;
- determination of the anti-complementary activity of the positive control sample, while the values of the determined activity should be in the range from 60 to 95%. 2. A standard sample of human immunoglobulin for determining the anti-complementary activity of preparations of human immunoglobulins, containing a positive control obtained by the method according to claim 1, and a negative control with anti-complementary activity in the range of 10-45%.

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