RU2302427C2 - Method for producing intravenous injection immunoglobulin preparation and preparation produced in this way - Google Patents

Abstract

FIELD: biochemical engineering; medicine.

SUBSTANCE: method involves producing immunoglobulin preparation by concentrating III Kohn spirit method fractions by means of ultrafiltration under pH 3.5-5.0 condition, washing from ethanol and carrying out final solution concentration process. The subsequent restoration hydrate immunoglobulin envelopes is carried out by means of molecular filtration method by creating concentration gradient by introducing low-molecular substance in concentration of 0.1-2.5% by weight into immunoglobulin solution, with water of pH 3.8-4.5 being used, cleared from dialyzing solution. Then stabilizing agent is added and pH value is fixed at 3.5-5.0. Sterilizing filtration is carried out and the produced preparation is dried by subliming when needed at the final stage of refinement process. The so produced immunoglobulin preparation for making intravenous injections has solution transparency index less than 0.01, and contains monomer immunoglobulin IgG more than 97% under full absence of polymeric forms, proteins 4.5-11% and sodium chloride no more than 0.3%.

EFFECT: reduced risk of side reactions when administering the immunoglobulin preparation; mechanically aided and automated process of preparation production; prolonged storage period.

5 cl, 1 dwg, 2 tbl

Description

The invention relates to medicine, namely to the production of an immunoglobulin drug suitable for intravenous administration.

Immunoglobulin preparations for intravenous administration are widely and successfully used to treat a wide range of various diseases. For therapeutic use, a large set of drugs is proposed that differ in their quality, frequency of adverse reactions, form, physical parameters, duration of circulation in the body and effectiveness / 1, 2 /.

The domestic immunoglobulin preparation for intravenous administration is produced using enzymatic treatment, which leads to damage to the Fc fragment of the immunoglobulin G molecule (IgG - class G immunoglobulins, make up 80% of all blood serum antibodies) and its rapid elimination from the body within a few days / 3 /. This is due to the fact that during the production process, pepsin is added to the semi-finished product of the immunoglobulin, an enzyme that breaks down the mentioned Fc fragment, which provides excellent tolerance upon intravenous administration, but leads to a violation of most functions of the immunoglobulin molecule / 4, 5 /.

One of the main indicators of the quality of intravenous immunoglobulin preparations is anticomplementary activity - the ability, after intravenous administration, to spontaneously bind and activate complement. (The complement is a system of whey proteins, one of the most important factors of natural immunity, which functions in human blood, is found in lymph and tissue fluids).

Low anticomplementary activity leads to the absence of adverse reactions during intravenous administration of the drug, therefore, drugs with anticomplementary activity below 10 mg of protein, not activating two 50% units of complement (2 CH ₅₀ ), cannot be used in clinical practice / 6 /. The ability of an immunoglobulin preparation after intravenous administration to spontaneously bind and activate complement (anti-complementary activity) is explained by damage to IgG molecules during fractionation with subsequent formation of aggregates. Clinically, this is manifested by shortness of breath, trembling, nausea, and in some cases collapse / 7 /.

In this regard, one of the most important stages of the technological process of obtaining an immunoglobulin preparation is a decrease in anticomplementary activity.

The decrease in the anti-complementary activity of an immunoglobulin preparation can be achieved in various ways: by treatment with proteolytic enzymes / 5 /, by modifying immunoglobulin by acetylation / 8 / or sulfonation / 9 /, keeping at 37 ° C for 18 hours / 10 /, incubation at a temperature of 30-45 ° C / 11 /, gel treatment with aluminum hydroxide followed by heat treatment / 12 /. However, a similar effect on immunoglobulin can contribute to an increase in the number of aggregated forms of immunoglobulin / 13 /. A method for producing an intravenous immunoglobulin with a high content of monomers / 14 /, a method for producing an unmodified liquid immunoglobulin with a low ionic strength / 15 / is described. Known methods for producing an immunoglobulin preparation using polyethylene glycol / 16, 17, 18 /, ion exchange chromatography / 19 /, a combination of ion exchange chromatography, concentration by ultrafiltration and incubation at a temperature of 30-45 ° C / 20 /.

The disadvantages of the known methods for the preparation and preparations of immunoglobulin are the complexity of the process, the high cost, the damaging effect of fractionating agents on the immunoglobulin molecule, the insufficiently high content of monomeric IgG and low anticomplementary activity.

The closest technical solutions are a method for producing an immunoglobulin preparation for intravenous administration and an immunoglobulin preparation for intravenous administration (Gabriglobin) / 21, prototype /.

This known method for preparing an immunoglobulin preparation for intravenous administration involves concentration of fraction III of the Cohn alcohol method by ultrafiltration at pH 3.5-5.0, washing from ethanol, final concentration of the solution, restoration of hydration shells of the immunoglobulin by molecular filtration by creating a concentration gradient of low molecular weight substances on opposite sides of a semipermeable membrane using distilled water (or, equivalently, purified water) for dialysis solution create, add the stabilizer to establish the immunoglobulin content of the solution is 5% and the pH 3.5-5.0, and sterilizing filtration.

A well-known immunoglobulin preparation for intravenous administration is obtained by a method including concentration of fraction III of the Cohn alcohol method by ultrafiltration at pH 3.5-5.0, washing from ethanol, final concentration of the solution, restoration of hydration shells of immunoglobulin by molecular filtration by creating a concentration gradient of low molecular weight substances on opposite sides of a semipermeable membrane using distilled water to prepare a dialysis solution, adding the stabilizer with the establishment of the immunoglobulin content of the solution is 5% and the pH 3.5-5.0, and sterilizing filtration.

When implementing this known method, the stage of restoration of the hydration shells of the immunoglobulin molecules by molecular filtration is carried out for 18-20 hours in manual mode. To do this, cut the viscose dialysis sheath into pieces, let it get wet, then seal one end of the sheath, fill the resulting container with a semi-finished product of immunoglobulin, seal the upper end of the sheath, check its tightness and conduct dialysis for 12-14 hours with a periodic change of the dialysis solution manually.

Alternatively, an ultrafiltration apparatus (for example, the AR-1.0 hollow fiber apparatus manufactured by the Design Bureau of Design Bureau of the City of Kirishi) can be used in diafiltration mode. At the same time, an immunoglobulin solution is fed into the primary circuit of the ultrafiltration apparatus, and the dialysis solution is circulated in the secondary circuit of the apparatus using a pump.

Distilled or purified water used as a dialysis solution has a pH value of 5.5 to 6.5. The use of such water without adjusting the pH causes a wave-like increase in the pH of the immunoglobulin solution during diafiltration (during restoration of the hydration shells of the immunoglobulin) from 3.8 to 5.4. A stepwise increase in the concentration of hydrogen ions in the immunoglobulin solution apparently leads to uncontrolled conformational changes in the immunoglobulin molecules and fluctuations in anticomplementary activity. Thus, in the prototype, the dialysis solution has non-optimal acidity, and in the immunoglobulin solution there are no substances that form an effective concentration gradient, as a result of which water molecules are embedded in the damaged or dehydrated by ethanol sections of the hydration shell of the immunoglobulin is not active enough to implement an effective process of restoring the hydration shells of immunoglobulin molecules and corresponding decrease in anticomplementary activity. The resulting preparation has an insufficiently high amount of monomeric immunoglobulin and an unnormalized amount of sodium chloride. (The content of sodium chloride in an immunoglobulin preparation reflects the efficiency of the process of removing alcohol and other low molecular weight substances used in the process of fractionation of immunoglobulin. The lower the content of sodium chloride, the more effective the diafiltration process).

The drawing shows a characteristic change in the pH of an immunoglobulin solution from the volume of water during diafiltration.

When using the manual mode stage in the technological process of preparation of the drug, production costs increase, mechanization of the process is impossible, the volume of release of the preparation of immunoglobulin is reduced, and it is not possible to stabilize the content of monomeric IgG and anticomplementary activity when releasing the next series of the drug.

Of the five tested series of the drug obtained using the prototype at the Ivanovo blood transfusion station, only two series met the requirements of the pharmacopeia article of the enterprise / 22 /, on anti-complementary activity. (Pharmacopoeia article of the enterprise is a document that determines the quality indicators of the drug, approved by the Ministry of Health of the Russian Federation). Due to the fact that the preparation is available in dry form, all analyzes are carried out after dissolving the dry mass in the supplied solvent (p.5 FSP No. 42-0265-16-21-01 / 22 /).

The technical task of this group of inventions, united by a single technical concept, is to create an effective way to obtain an immunoglobulin preparation for intravenous administration, as well as to create an immunoglobulin preparation for intravenous administration and to expand the arsenal of these methods and drugs.

The technical result that provides a solution to the problem of this group of inventions is to reduce the risk of adverse reactions due to a decrease and stabilization of the anti-complementary activity of the immunoglobulin preparation and an increase in the number of monomers while reducing the number of dimeric forms, providing the possibility of mechanization and automation of the process of obtaining the immunoglobulin G preparation, increasing the time storage of an immunoglobulin preparation.

Anti-complementary activity, as already mentioned, is one of the main indicators of the quality of intravenous immunoglobulin preparations. Low anticomplementary activity leads to the absence of adverse reactions with intravenous administration of the drug. Therefore, significant fluctuations of this indicator from series to series are unacceptable.

It should be borne in mind that during fractionation, during the isolation of immunoglobulin from blood plasma, hydration membranes are removed by introducing ethanol into the protein solution, which collects hydrated water, thereby reducing the charge of protein globules and leads to the deposition of immunoglobulins / 27 /.

In turn, this leads to disruption of the quaternary (spatial) structure of the immunoglobulin molecule, unmasking of the sites where binding and further activation of complement occurs, i.e. to increase anticomplementary activity, which contributes to the occurrence of adverse reactions. At the same time, dimeric and polymeric forms of immunoglobulin appear.

The need to restore the "hydration shells" of immunoglobulin to reduce and stabilize anticomplementary activity is explained by the fact that all protein molecules exist in solutions, and their spatial configuration depends on the interaction of amino acid radicals that form the protein chain with water molecules. In this case, nonpolar hydrophobic radicals of amino acids are directed deep into the molecule, and polar radicals are oriented towards water.

It is the formation of bulk hydrated or solvate shells around an immunoglobulin molecule that contributes to the most stable conformation of protein globules. Hydration membranes play a decisive role in the formation of the tertiary and quaternary structure of the immunoglobulin molecule and in its stability / 29 /. Violation of the spatial quaternary structure of the immunoglobulin molecule leads to the "opening" of the IgG molecule, on which the complement activation process is initiated. The protein conformation in aqueous solutions is affected by pH, temperature and the presence of low molecular weight compounds / 28 /.

The direction of the creation of the drug and the method of its preparation was chosen taking into account the results of a study of the causes of a significant fluctuation in the anti-complementary activity of the production series of the known immunoglobulin preparation. For this, a special study was carried out, as a result of which it was established that the most significant parameters for this stage are the mechanism for creating a concentration gradient and the pH of the solution used in ultra- and diafiltration.

The essence of the invention in terms of the method lies in the fact that the method of producing an immunoglobulin preparation for intravenous administration, comprising concentration of fraction III of the alcohol method of Kohn by ultrafiltration at pH 3.5-5.0, washing from ethanol, final concentration of the solution, restoration of hydrated shells of immunoglobulin by the method molecular filtration by creating a concentration gradient of low molecular weight substances on opposite sides of a semipermeable membrane using purified water for dialysis solution, the addition of a stabilizer, the establishment of a pH value of 3.5-5.0, as well as sterilizing filtration, characterized in that during ultrafiltration and during the restoration of hydrated membranes, water for the dialysis solution is used with a pH value of from 3.8 to 4.5 and the concentration gradient is created by introducing a low molecular weight substance into the immunoglobulin solution at a concentration of 0.1-2.5 wt.%, and, if necessary, freeze-drying is carried out after sterilizing filtration.

As a low molecular weight substance, it is preferable to use maltose or sodium chloride.

The essence of the invention in terms of the preparation lies in the fact that an immunoglobulin preparation for intravenous administration was obtained by a method involving concentration of fraction III of the Cohn alcohol method by ultrafiltration at pH 3.5-5.0, washing from zanol, final concentration of the solution, restoration of hydrated shells of immunoglobulin by the method molecular filtration by creating a concentration gradient of low molecular weight substances on opposite sides of a semipermeable membrane using purified water for dialysis of the insulating solution, adding a stabilizer, setting the pH to 3.5-5.0, as well as sterilizing filtration, and during ultrafiltration and during the restoration of hydration membranes, water for the dialysis solution is used with a pH from 3.8 to 4.5, and the gradient concentrations are created by introducing into the immunoglobulin solution a low molecular weight substance in a concentration of 0.1-2.5 wt.%, while the preparation in liquid form has a solution transparency of less than 0.01, contains monomeric immunoglobulin G more than 97% in the complete absence of gender dimensional forms of protein of from 4.5 to 11% sodium chloride and no more than 0.3%.

If necessary, the immunoglobulin preparation can be released in dry form, and prior to freeze drying it has the following indicators: solution transparency - less than 0.01, monomeric immunoglobulin G content more than 97% in the complete absence of polymer forms, protein - from 4.5 to 11% and sodium chloride - not more than 0.3%.

The process of complete restoration of the hydrated shells of immunoglobulin in the technological scheme for producing immunoglobulin suitable for intravenous administration is extremely important, since it allows increasing the stability of the dosage form, preventing the formation of polymers and aggregates and eliminating spontaneous activation of complement.

A laboratory indicator of the effectiveness of this process is the indicator of the transparency of the solution, the content of monomeric IgG and the anticomplementary activity of immunoglobulin.

Marked quality indicators of immunoglobulin preparations prepared in accordance with the prototype and the claimed method are shown in table 1.

Table 1 Laboratory quality indicators of immunoglobulin preparations prepared according to the prototype and the claimed method Immunoglobulin preparation The transparency of the solution (optical density, dimensionless value) The content of monomeric IgG (%) Anti-complementary activity (mg protein, not activating 2 CH ₅₀ ) Obtained by the prototype 0.0080 ± 0.00068 92.44 ± 1.372 7.0 ± 2.4 Obtained by the claimed method 0.0033 ± 0.0013 97.166 ± 0.298 10.0 ± 0.0

If the average value of the anti-complementary activity of five series of immunoglobulin prepared according to the prototype was 7.0 ± 2.4, then the anti-complementary activity of 10 series of the immunoglobulin preparation Gabrilobin prepared by the proposed method was (10 ± 0) mg of protein that did not activate 2 CH ₅₀ . In the preparation prepared by the claimed method, the content of monomeric IgG was higher, and the solution was significantly more transparent.

The stability of the main parameters of the drug from batch to batch is illustrated in table 2, which summarizes the most significant quality indicators of ten production runs of the immunoglobulin preparation obtained by the claimed method.

table 2 Quality indicators of ten production runs of an immunoglobulin preparation prepared by the claimed method The value of indicators Quality indicators Minimum and maximum value Average value Deviation Transparency 0.001-0.006 0.0033 0.0013 Color 0.009-0.013 0.0107 0,00104 pH 4.25-4.38 4.33 0,0276 Protein,% 4.61-5.21 4.89 0.153 Sodium chloride, % 0,1-0,12 0.102 0.0036 Electrophoretic homogeneity,% 98-98 98 0 The polymer content,% 0-0 0 0 The content of dimers,% 1.99-3.50 2,777 0.345 The content of monomers,% 96.50-97.58 97,166 0.2988 The content of fragments,% 0-0.45 0,088 0.1408 Anti-complementary activity, mg protein 10-10 10 0 Antibodies to alfastaphylolysin, IU / ml 2-2 2 0 Antibodies to measles virus, IU / ml 25-25 25 0

The immunoglobulin preparation prepared by the proposed method had stable quality indicators in all series, the content of the monomer fraction was more than 97%, the polymers were absent in all series, and the sodium chloride content was less than 0.1%.

The clinical use of the claimed preparation of immunoglobulin showed that when administered intravenously, it is well tolerated, does not cause adverse reactions and is not inferior in therapeutic efficacy to the best foreign samples, which is confirmed by clinical trials in the Chelyabinsk City Clinical Hospital No. 1; Chelyabinsk inter-territorial burn center them. R.I. Lifshits on the basis of the city clinical hospital №6; Institute of Rheumatology RAMS; Department of Propaedeutics of Internal Diseases of the Yaroslavl Medical Academy, Izmailovsky Children's Clinical Hospital and the State Scientific Center "Institute of Immunology of the Ministry of Health of the Russian Federation."

During controlled clinical trials, it was shown that this immunoglobulin is effective in purulent-septic complications of the underlying disease in trauma and general surgical patients; sepsis; burns; systemic lupus erythematosus; systemic vasculitis; SARS, thrombocytopenic purpura in children and agammaglobulinemia / Aleshin V.A. et al., 2003, Donyush E.K. et al., 2003, Onishchenko G.G., 2002 /.

A method of obtaining an immunoglobulin preparation is implemented as follows.

Each batch of raw materials for the preparation of the drug is prepared from blood plasma obtained from a group of donors by fractionating its proteins with ethanol at a temperature below 0 ° C (Kohn's method).

In fraction III (filtrate “B”) of the Cohn alcohol method, a pH of 3.5–5.0 was established and concentrated in an ultrafiltration unit to reduce the solution volume by 10–12 times. Ultrafiltration is a type of membrane filtration for the formation of protein-free liquid. Ultrafiltration is characterized by the use of membranes with a pore size of 1-300 kD, removes all colloidal suspensions, organic and inorganic contaminants with a molecular size larger than the pores of the membranes. The transfer of substances through the membrane occurs under the influence of a difference in concentrations, a difference in electric potentials on both sides of the membrane or a pressure difference.

Then ethanol is removed using 5-6 volumes of purified water with a pH value of 3.8 to 4.5, and then the immunoglobulin solution is concentrated to a protein content of 6-7%. After that, hydration shells are restored, for which a low molecular weight substance, preferably maltose or NaCl in an amount of 0.1-2.5% is introduced into the immunoglobulin solution and molecular diafiltration is performed using purified water with a pH value of 3.8-4.5. Installation for diafiltration is collected in accordance with the scheme recommended by Brock / 26 /. An immunoglobulin solution with a low molecular weight substance is fed into the primary circuit of the installation, and a dialysis aqueous solution with a pH of 3.8-4.5 is circulated in the secondary circuit of the apparatus using a pump.

The use of a low molecular weight substance to create a concentration gradient and a dialysis solution with a controlled pH allows one to efficiently restore the immunoglobulin molecular shells using only an ultrafiltration unit, there is no need for manual dialysis, stable indicators of anticomplementary activity, monomer content and the absence of polymer forms from series to series are achieved .

Get the claimed preparation of immunoglobulin with anti-complementary activity of 10 mg of protein, not activating 2 CH ₅₀ with the content of monomeric IgG more than 97%, the complete absence of polymer forms, the concentration of sodium chloride less than 0.3%, transparency less than 0.01 and a protein content of 4.5 up to 11%.

Example 1

Fraction III in an amount of 300 L, obtained by fractionation of plasma by the Cohn alcohol method, is placed in a reactor (vessel) and the pH value is adjusted to 3.5 using a 1 M HCl solution. The filtrate is concentrated in an ultrafiltration unit. After reducing the solution to 30 L, ethanol is removed (washed) using 150 L (5 volumes) of purified water with a pH of 4.2 and the immunoglobulin is concentrated to 25 L (6.5% protein).

The hydration shells of the immunoglobulin are restored by molecular filtration. To do this, 30 g of sodium chloride (0.1% concentration) is introduced into the immunoglobulin solution and stirred until complete dissolution. Diafiltration is carried out using purified water with a pH of 3.8 in an amount of 150 l. After that, the pH is checked and, if necessary, adjusted, the stabilizer is introduced and the protein content (concentration) is set to 4.5%. Spend clarifying and sterilizing filtration, filling bottles. The immunoglobulin preparation before the freeze-drying started had the following parameters: the solution transparency value was 0.0030, the content of monomeric immunoglobulin G was 97.2%, no polymer forms, protein 4.5% and sodium chloride 0.25%. To obtain a dosage form, the drug is dried from a frozen state (lyophilized).

Example 2

In fraction III, obtained by fractionation of plasma with the Kohn alcohol method, the total volume of 100 L is adjusted to pH 4.2 and concentrated on an ultrafiltration unit for 2 hours, until the total volume decreases to 10 L. After that, ethanol and salts are removed using 50 l of purified water with a pH value of 4.5. To restore hydration membranes, 250 g of maltose, which is 2.5% (concentration), is added to the immunoglobulin solution (10 L). After dissolving the low molecular weight ion, diafiltration of the immunoglobulin concentrate is ensured in an ultrafiltration unit using purified water with a pH value of 4.5 in an amount of 300 l. At the final stage, the solution is concentrated to a protein content (concentration) of 11%, the pH value is set within the required limits, clarifying and sterilizing filtration is carried out, it is poured into bottles and 3.7 l of immunoglobulin are obtained in liquid form with anticomplementary activity of 10 mg of protein that do not activate 2 CH ₅₀ , a solution transparency of 0.0025, with a monomeric IgG content of 98.4%, a complete absence of polymers, a sodium chloride concentration of less than 0.2%.

Information sources:

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Claims (5)

1. A method for preparing an immunoglobulin preparation for intravenous administration, including concentration of fraction III of the Cohn alcohol method by ultrafiltration at pH 3.5-5.0, washing from ethanol, final concentration of the solution, restoration of hydration shells of immunoglobulin by molecular filtration by creating a concentration gradient of low molecular weight substances on different sides of a semipermeable membrane using purified water for dialysis solution, addition of a stabilizer, setting values pH 3.5-5.0, as well as sterilizing filtration, characterized in that during ultrafiltration and during the restoration of hydration membranes, water for the dialysis solution is used with a pH value of from 3.8 to 4.5, and a concentration gradient is created by introducing into the immunoglobulin solution a low molecular weight substance in a concentration of 0.1-2.5 wt.%, and, if necessary, after sterilizing filtration, freeze-drying is carried out. 2. The method according to claim 1, characterized in that maltose or sodium chloride is used as the low molecular weight substance. 3. The method according to any one of claims 1, 2, characterized in that after sterilizing filtration, freeze-drying is carried out. 4. The preparation of immunoglobulin for intravenous administration in liquid form, obtained in accordance with the method according to claim 1, having a solution transparency of less than 0.01, containing monomeric immunoglobulin G more than 97% in the complete absence of polymer forms, protein from 4.5 to 11 % and sodium chloride - not more than 0.3%. 5. Freeze-dried preparation of an immunoglobulin for intravenous administration, obtained in accordance with the method according to claim 1, having the following parameters before freeze-drying: the transparency of the solution is less than 0.01, the content of monomeric immunoglobulin G is more than 97% in the complete absence of polymer forms, protein from 4.5 to 11% and sodium chloride - not more than 0.3%.

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