EA017172B1 - Method for producing isoimmune blood plasma - Google Patents

Abstract

The invention relates to medicine, in particular to isoimmune donorship and provides to produce highly-active raw material of isoimmune plasm from blood of reimmunized donors for the production of immunoglobulin of human anti-Rh (anti-D) and anti-Rh diagnostic serums. The claimed invention is aimed at producing blood plasm with high titers of anti-Rh from isoimmune donors, shortening of reimmunization period and reducing intigen load on donor's organism. The essence of the invention is characterised in that the reimmunonization is carried out once Rh(D) by alloerythrocytes and additionally by Roncoleukin. At immunonization 3-5 ml Rh(D) of alloerythrocytes and Roncoleukin are intravenous or intramuscular injected in 1000 ME/kg dosage subcutaneous in the lower angle of left or right scapula 30 min after alloerythrocytes injection. After each two weeks of the first immunization blood is sampled with subsequent flow plasm therefrom.

Description

The invention relates to medicine, namely to isoimmune donation, and for the production of highly active raw materials - isoimmune blood plasma from reimmunized Bk ₀ (O) alloerythrocyte donors for the production of human anti-иммун immunoglobulin and diagnostic anti-Rhesus serum.

There is a method of obtaining biologically active blood plasma anti-Rhesus from individuals previously sensitized with antigens of the Rh system due to Rh-conflict pregnancies or the introduction of Rh incompatible blood [1]. The disadvantage of this method is, despite the presence of resusantibodies, a rapid decrease in their titers.

The closest in technical essence and the achieved result is a method of obtaining blood plasma from donors immunized with ΒΗ ₀ (Ό) alloerythrocytes 2-3 times 3-5 ml every 3-4 days every 4 months after an immunization or reimmunization cycle ΒΗο (Ό) by alloerythrocytes for the subsequent preparation of human anti-иммун immunoglobulin and diagnostic antiresus serum [2].

The disadvantages of this method are the following:

1) the need for frequent calls of donors during working hours for reimmunization with erythrocytes ΒΗ ₀ (Ό) (3-4 days every 4 months after the immunization or reimmunization cycle Β1ι ₀ (Ό) with alloerythrocytes);

2) a low and short-term immune response in donors, i.e. low titers of anti-Ό antibodies after reimmunization with donor alloerythrocytes Β1ι ₀ (Ό);

3) the complexity of obtaining and preparing each dose of washed thawed donated red blood cells (ΒΗ ₀ (Ό) alloerythrocytes) and the use of expensive routine testing methods for determining blood groups, antigens of the Rhesus system (Ό, С, Е, с, е and other Kell systems , Duffy, etc.), markers of hepatitis B and C viruses, human immunodeficiency virus (HIV 1 and 2). Compliance with the six-month quarantine of donor red blood cells with subsequent re-examination of donors for markers of the above viruses to exclude a seronegative window;

4) in 10% of isoimmune donors, there is a complete absence of an immune response to an antigenic stimulus after immunization and reimmunization with donor Β1ι ₀ (Ό) alloerythrocytes, which consists in the absence of anti-Ό antibody titers.

The objectives of the invention are to obtain blood plasma with high titers of Rh antibodies from isoimmune donors, reducing the time for re-immunization and, accordingly, reducing the antigenic load on the body of the donor.

The objectives are achieved due to the fact that in the method of producing isoimmune blood plasma, which includes the immunization of an isoimmune donor by introducing thawed cryopreserved ΒΗ ₀ (Ό) alloerythrocytes of active blood group 0 (I) or the same group with the blood of the immunized person, Ke11 negative into the body similar in antigens to the system ΜΝ8δ, ΌαΓΓί. in an amount of 3-5 ml intravenously or intramuscularly, after which, every two weeks after the start of reimmunization, blood is sampled until the titer of anti-Ό antibodies is reduced to 1:64 with the subsequent release of plasma from it, new is that re-immunization is performed once ΒΗ ₀ (Ό) donor alloerythrocytes and additionally Roncoleukin, with Roncoleukin at a dose of 1000 IU / kg administered subcutaneously in the lower corner of the left or right scapula 30 minutes after the introduction of Β1ι ₀ (Ό) alloerythrocytes.

The invention consists in the following.

Rhesus negative donors are injected once with 3-5 ml of thawed cryopreserved ΒΗ ₀ (Ό) erythrocytes of active donors of the blood group 0 (I) or the group of the same name with the blood of the immunized face, Ke11 negative, similar to the antigens of the ΜΝ8δ, ΌαΓΠ system, with a shelf life of at least 6 months, intravenously or intramuscularly. General and local reactions to administration should be absent for 30 minutes.

30 minutes after this, the Roncoleukin solution, diluted with sterile water for injection to 100,000 IU / ml, is injected subcutaneously in the lower corner of the left or right shoulder blade in an amount of 1000 IU / kg weight, i.e.

with a donor weight of up to 60 kg - 0.6 ml;

with a donor weight of 61 to 70 kg - 0.7 ml;

with a donor weight of 71 to 80 kg - 0.8 ml;

with a donor weight of 81 to 90 kg - 0.9 ml;

with a donor weight of 91 to 100 kg or more - 1.0 ml.

Roncoleukin is a preparation of recombinant human yeast IL-2, manufactured by Biotech LLC (St. Petersburg, Russia).

The practice of more than 8 years of use of Roncoleukin in clinical medicine has revealed its priority in many areas of cytokine immunocorrective therapy with recombinant IL-2 (rIL-2) compared with drugs used in foreign countries [3]. Roncoleukin was created on the basis of a producer of non-pathogenic strains of saccharomycetes yeast Baskagotusek segegyae. The resulting rIL-2 is identical in amino acid sequence to the peptide fragment of endogenous human

- 1 017172 fishing IL-2. The production technology of rIL-2 based on a yeast producer is more economical compared to a producer based on Ε.οοίί. therefore, the final cost of the drug is lower than that of the foreign analogues of RIL-2 (Prgoelixi, AIlieiks, Teseliks, Masgo1sh-2, Vyeliks). The foreign analogue of Roncoleukin, created in 2002 using a similar technology, is the drug RIL-2 A1b1eiksh (a1bt-t1e1eiksh-2, Nitap Seyote 8s1eise8, 1ps.) [4].

Due to the fact that Roncoleukin is a structural and functional analogue of endogenous human IL-2, the main mechanisms of the immune effects of endogenous IL-2 should be noted to assess the immunocorrective effectiveness of this drug.

IL-2 belongs to the family of hematopoietin cytokines and is a glycoprotein with a molecular weight of 15,000 Da (in the glycolized form of 17,000-22,000 Da). The main pharmacological effect is immunomodulatory [5, 6]. The immunological role of IL-2 is to regulate a specific (antigen-dependent) immune response by stimulating the proliferation and differentiation of immune cells involved in its implementation. Interacting with cell receptors, IL-2 induces the growth, differentiation and proliferation of T and B lymphocytes, monocytes, macrophages, oligo-dendroglial cells, epidermal Langerhans cells. IL-2 causes the formation of lymphokinactivated killers.

It also stimulates the cytolytic activity of natural killers and cytotoxic platelets, increases the resistance of cells to programmed cell death - apoptosis. The use of the drug IL-2 provides immune protection against tumor cells, as well as pathogens of viral, bacterial and fungal infections. Indications for its use are complex therapy of septic conditions of various etiologies, accompanied by immunosuppression in adults, in surgery, traumatology, gynecology, burn sepsis, severe pneumonia, treatment of kidney cancer in adults.

The invention and the positive results obtained by the application of the proposed method are illustrated by graphic materials, which show:

in FIG. 1 - indicators of the general analysis of blood of donors, immunized according to the claimed method (donor C.) and the prototype (donor X.);

in FIG. 2 - indicators of a biochemical analysis of blood of donors re-immunized by the present method (donor C.) and the prototype (donor X.);

in FIG. 3 - the content of IL-2 in the blood serum of donors re-immunized by the present method (donor C.) and the prototype (donor X.);

in FIG. 4 - the content of IFN-α in the blood serum of donors re-immunized by the present method (donor C.) and the prototype (donor X.);

in FIG. 5 - titers of Rh antibodies from donors immunized by the present method and the prototype;

in FIG. 6 - analysis of the main data during the immunization of donors according to the claimed scheme and the prototype;

in FIG. 7 - indicators of the hemogram of donors re-immunized by the present method and the prototype;

in FIG. 8 - indicators of a biochemical analysis of blood donors, immunized according to the present method and the prototype;

in FIG. 9 - the content of interleukin-2 in the blood serum of donors re-immunized by the present method and the prototype;

in FIG. 10 - the content of interferon-alpha (IFN-α) in the blood serum of donors re-immunized by the present method and the prototype.

The invention is illustrated by the following examples.

Example 1

Donor S. was injected intramuscularly with 3 ml of thrice washed thawed cryopreserved Ρ1ι ₀ (Ό) active donor alloerythrocytes with blood group 0 (I), Ke11 negative, similar in antigens to the ΜΝ8δ, ΌαΓΓί system. After 30 min, Roncoleukin was administered at a dose of 1000 IU / kg subcutaneously into the region of the lower corner of the left shoulder blade. Before and every 14 days after the start of immunization, before plasmapheresis for 4 months of observation, the following titers of anti-Ό antibodies were determined: before immunization, 1:64, after the start of immunization after 3, 5, 7, 9, 11 weeks - 1: 1024, then a break of 1 month after 5 plasmapheresis and after 16 weeks - 1: 512.

Example 2

Donor K. was injected intramuscularly with 5 ml of thawed thawed cryopreserved Ρ1ι ₀ (Ό) alloerythrocytes of the active donor with blood group 0 (I), Ke11 negative, similar in antigens to the системы8δ, ,αΓΓί system. After 30 min, Roncoleukin was administered at a dose of 1000 IU / kg subcutaneously into the region of the lower corner of the left shoulder blade. Before and every 14 days after the start of immunization, before plasmapheresis for 4 months of observation, the following titers of anti-Ό antibodies were determined: before re-immunization 1:64, after the start of re-immunization after 3, 5, 7, 9, 11 weeks - 1: 1024, then break 1 month after 5

- 2 017172 plasmapheresis and after 16 weeks - 1: 1024.

Example 3 (prototype).

Donor X .., immunized according to the standard regimen of immunization 3 times in 3 ml intramuscularly after 3 days with thrice washed thawed cryopreserved Ρ1ι ₀ (Ό) alloerythrocytes of the active donor with blood group 0 (I), Ке11 negative, similar to antigens of the ΜΝ8δ system, ΌαΓΓί, had the following titers: before reimmunization - 1:64, after 3, 5, 1.9, 11 weeks - 1: 128, then a break 1 month after 5 plasmapheresis and 16 weeks after the start of immunization - 1: 128.

Example 4 (prototype).

Donor III., Immunized according to the standard scheme 2 times in 5 ml intramuscularly after 4 days with thawed thawed thawed cryopreserved Ρ1ι ₀ (Ό) active donor alloerythrocytes with blood group 0 (I), Ke11 negative, similar in antigens of the ΜΝ8δ, ΌαΓΓί system. had the following titers: before re-immunization - 1:64, after 3, 5, 7, 9, 11 weeks - 1: 256, then a break 1 month after 5 plasmapheresis and 16 weeks after the start of re-immunization - 1: 128.

The data of indicators of a general blood test, a biochemical blood test, the content of IL-2 in serum, as well as the content of IFN-α in the blood serum of donor C. and donor X. are shown in FIG. 1-4. All changes in the studied clinical and laboratory parameters were within the standard values.

Clinical trials according to the claimed method for producing blood plasma were carried out on 10 male donors aged 20 to 50 years. At the same time, as a comparison, reimmunization of 10 donors of the prototype was carried out.

Statistical analysis of the results was carried out using the software package M1stoyoy Ex, 1, ZhiMuya ν. 6.0. The studied data were presented as Μ ± 5 (where M is the arithmetic mean and 5 is the standard deviation). The quality control of the studies was carried out by statistical processing and analysis of the research data independently in 2 groups of the sample of donors immunized according to the claimed method and the prototype.

The significance of differences between the compared values was determined by one-way analysis of variance followed by pairwise comparisons according to the Newman-Cales criterion in identifying differences between the studied groups. Differences were considered significant at a probability of P <0.05.

As can be seen from the data shown in FIG. 5, the initial rhesus antibody titers did not significantly differ in the studied groups of donors and amounted to 58 ± 46 (stimulation index 0.8 ± 0.4) for donors immunized by the present method (reimmunization with alloerythrocytes + Roncoleukin), and for donors, reimmunized according to the prototype (reimmunization with alloerythrocytes), 53 ± 38 (stimulation index 0.9 ± 0.3).

It was found that Roncoleukin increased the titers of anti-antibodies in 2.5-3.1 times in the period from 3 to 16 weeks after reinimmunization of donors by the present method in comparison with the prototype. A single injection of Roncoleukin according to the claimed method increases the titers of anti-Ό antibodies in comparison with the prototype as follows: 3 weeks after the start of immunization - 2.5 times, after 5 weeks - 2.3 times, after 7 weeks - 2.4 times, after 9 weeks - 2.9 times, after 11 weeks - 2.5 times, after 16 weeks - 3.1 times. It should be noted that in donors immunized according to the claimed method, anti-Ό antibody titers were not less than 1:64 before immunization in 7 out of 10 donors, after 3 weeks in 8 out of 9 donors, after 5 weeks in 9 out of 10 donors after 7 weeks in 9 out of 10 donors, after 9 weeks in 9 out of 10 donors, after 11 weeks in 9 out of 10 donors, after 16 weeks in 9 out of 10 donors and at least 1: 128 before reimmunization in 1 out of 10 donors, in 3 weeks - in 8 out of 9 donors, in 5 weeks - in 9 out of 10 donors, in 7 weeks - in 8 out of 10 donors, in 9 weeks - in 8 out of 10 donors, in 11 weeks - in 8 out of 10 donors, after 16 not del - in 8 out of 10 donors (Fig. 6). Donors immunized according to the prototype have anti-Ό antibody titers of at least 1:64 before immunization in 7 out of 10 donors, in 3 weeks in 8 out of 10 donors, in 5 weeks in 8 out of 10 donors, in 7 weeks in 8 out of 10 donors, in 9 weeks - in 8 out of 10 donors, in 11 weeks - in 7 out of 9 donors, in 16 weeks - in 8 out of 10 donors and at least 1: 128 before re-immunization in 2 out of 10 donors, through 3 weeks - in 8 out of 10 donors, in 5 weeks - in 8 out of 10 donors, in 7 weeks - in 8 out of 10 donors, in 9 weeks - in 8 out of 10 donors, in 11 weeks - in 7 out of 9 donors, through 16 weeks - in 7 out of 10 donors.

As a result, from donors immunized according to the claimed method for the entire observation period after the start of immunization, received isoimmune plasma with rhesus antibody titers greater than 1:64 - 36.0 l (with an average titer of rhesus antibodies 1: 608 in 1 ml), s with titers of more than 1: 128–30.0 L (average titer of resusantibodies 1: 717 in 1 ml), and from donors immunized according to the prototype, isoimmune plasma with titers of more than 1:64–32.4 L was obtained (average titer of Rhesus antibodies 1 : 275 in 1 ml) and more than 1: 128 - 28.8 l (average Rh antibody titer 1: 301 in 1 ml).

The safety of the use of Roncoleukin during reinimmunization of donors by the present method was determined by assessing their condition based on a study of a complex of clinical and laboratory parameters, including indicators of a general blood test with a leukocyte formula and a biochemical blood test, the content of endogenous IL-2 and IFN donors in blood serum -but.

- 3 017172

During re-immunization, no allergic and clinically detectable pathological reactions were noted in the studied groups of donors. After reimmunization according to the claimed method and the prototype, deviations from the norm in the state of donor health and indicators of a general blood test for the entire observation period were not detected (data are shown in Fig. 7).

Before re-immunization and 7 and 16 weeks after the start of re-immunization according to the claimed method, deviations from the range of normative values in the studied parameters of the biochemical analysis of blood in donor groups were not detected (Fig. 8).

Before re-immunization and 7 and 16 weeks after the start of re-immunization in the examined groups of donors re-immunized according to the present method and the prototype, there were no deviations from the range of physiological norm values in the content of endogenous IL-2 and IFN-α in blood serum (Fig. 9 and 10).

The inventive method leads to an increase in the titers of anti-ре antibodies in immunized donors, reduces the introduction of Ρ1ι ₀ (Ό) alloerythrocytes to once instead of two or three, resulting in a decrease in the antigenic load on the donor’s body and the time of immunization is reduced by 1 week, which leads to savings financial costs in obtaining isoimmune plasma.

The proposed method for the reinimmunization of donors will contribute to the production of highly active raw materials for the production of human anti-Rhesus immunoglobulin (anti-Ό) (used in obstetric practice to prevent the production of Rh antibodies in Rh-negative pregnant women, i.e. for passive immunization of Rh-negative mothers) as well as diagnostic anti-Rhesus serum, necessary for determination of Rh antigens.

Information sources.

1. Donskov, S.I. Blood groups of the Kiekik system. Theory and practice. M .: VINITI RAS, 2005, p. 151-154.

2. Instructions for use Donor immunization to obtain standard serum and immunoglobulin anti-Rhesus (registration number 119-1108 of November 13, 2008), Minsk, 2008, 8 pp.

3. The child, J. A. Roncoleukin - a new and effective treatment / Zh.A. Child // Honey. Knowledge, 1999, No. 4, p. 17-19.

4. Simbirtsev, A.S. Immunotropic drugs based on cytokines / A.C. Simbirtsev // [Electronic resource], 2006, Access mode: yyr: //^te^/a5uoteb.gi/yr/sop1ep1.ryr? 1b = 697. - Date of access: 11.24.2006.

5. Mikhailenko A.A., Konenkov V.I., Bazanov G.A., Pokrovsky V.I. Guidelines for Clinical Immunology, Allergology, Immunogenetics, and Immunopharmacology (for general practitioners) / Ed. IN AND. Pokrovsky, t. 2. Tver: LLC Publishing House Triad, 2005, p. 395.

6. Popovich A.M. Achievements and prospects for the clinical use of Roncoleukin in the treatment of immunodeficiencies of various etiologies // Materials of the VII All-Russian Forum with int. participation of the Days of Immunology in St. Petersburg. Acad. IN AND. Ioffe, 2003, p. 2-7.

Claims (1)

CLAIM A method of producing an isoimmune blood plasma, comprising the immunization of an isoimmune donor by introducing thawed cryopreserved Ρ1ι ₀ (Ό) alloerythrocytes of active blood group 0 (I) donors or the same group with the blood of the immunized donor, negative for Ke11 factor, similar to antigens of the system ΜΝ8 ΌαΓΓί. in an amount of 3-5 ml intravenously or intramuscularly, blood sampling every two weeks after the start of immunization to reduce the titer of anti-antibodies to 1:64, followed by the release of plasma from it, characterized in that the immunization is carried out once Ρ1ι ₀ (Ό) donor alloerythrocytes and additionally Roncoleukin, with Roncoleukin being injected subcutaneously into the region of the lower angle of the scapula at a dose of 1000 IU / kg 30 minutes after the introduction of Ρ1ι ₀ (Ό) alloerythrocytes.