Human immunoglobulin preparation against cytomegalovirus for intravenous introduction and method of its obtaining

FIELD: medicine.

SUBSTANCE: as feedstock for fractioning used is anticytomegalovirus plasma with activity index not less than 70%. Process of production includes: ultrafiltration of immunoglobulin at ion force not higher than 0.02 and pH value 4.0-5.7 with further diafiltration against distilled water with pH value 3.5-6.0. Immunoglobulin is incubated with maltose of 2-11% concentration at temperature 32-40°C and pH value 4.0-4.8 for 10-36 hours, or with pepsin in dose 1:10-6-1:10-5 in presence of 2-11% maltose. After that inactivation/removal of viruses is carried out by immunoglobulin incubation at pH value 4.0-4.4 and/or with application of depth filtration, and/or nanofiltration. Afterpurification of immunoglobulin is performed by methods of: ultrafiltration and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography, and/or dialysis. Immunoglobulin stabilisation is carried out with application of sodium chloride and/or maltose, and/or proline, and/or glucose and/or glycerol, whose content provides preparation osmolarity within 270-400 mOcm/kg. In obtained immunoglobulin preparation pH value 5.0-7.5 and protein content 4.5-10.5% are installed.

EFFECT: invention allows to obtain highly avid immunoglobulin preparation against cytomegalovirus for intravenous introduction with improved and stable qualitative quaracteristics.

7 cl, 5 tbl, 4 ex

 

The invention relates to medical biotechnology, in particular to a method for the specific preparation of immunoglobulin (IG) man against cytomegalovirus (CMV) for intravenous injection (IV).

For the treatment and prevention of cytomegalovirus infection are intravenous immunoglobulins (IVIG) as non-specific (normal), and specific [1, 2]. The main indicators of the quality of IVIG is not only the efficiency of their application, i.e. the magnitude of the titles of specific antibodies (ATA), but also the safety of the drug, due to the low anticomplementary activity (AKA)no or minimal content of aggregates, dimers and fragments, as well as undesirable impurities, such as immunoglobulin A (IgA) and isohemagglutinins. The need to control IgA and isohemagglutinins in IVIG due to the possibility of patients adverse reactions when their high content in the product.

According to the recommendations of the world Health Organization and the requirements of the European Pharmacopoeia technology receiving immunoglobulin for intravenous administration should ensure the stability of quality characteristics of the drug during storage and transportation and to preserve intact the structure of the molecule and the distribution of subclasses of immunoglobulin G (IgG), are appropriate to eastwoodiae normal donor plasma [3]. Used stabilizers should not only ensure the stability of the drug IVIG, but not to make new biological activity. In accordance with the requirements of the European Pharmacopoeia, the technology of obtaining IVIG must contain the stage of viral inactivation. Used methods of inactivation/removal of viruses should not have an adverse effect on activity and biological properties of the drug IVIG: protein electrophoretic purity, impurity - IgA, IgM and IgE, molecular parameters, the distribution of IgG subclasses, the titre of specific antibodies and the ratio of their concentration. The specific activity of the IVIG depends on initial activity of antibodies in the source of raw materials - donor plasma, and on the technology of production of the drug. At some stage of the production process of IVIG have a negative effect on the functional activity of specific AT and cause the loss or reduction of certain subclasses of immunoglobulin G [4]. It is known that antibodies to the pathogen CMV infection mostly found in IgG1 and IgG3 the subclasses of immunoglobulin G [5, 6].

Drugs non-specific IVIG are characterized by variability and low content of title anticytomegalovirus AT that it is not possible to obtain positive results in the treatment process is s disease, due to cytomegalovirus [1]. Therefore for the prevention and treatment of CMV infection are mainly used specific IVIG, which differ consistently high titer anticytomegalovirus antibodies and speed of onset of therapeutic effect [2]. In addition, the use of specific IVIG reduces the amount of drug that increases the safety of its application.

Known unmodified anticytomegalovirus intravenous IG CytoGam® (Medimmune, UK), which is obtained by fractionation vysokotirazhnoj plasma by the Cohn method 6 and 9. For viral inactivation using solvent-detergent treatment, as stabilizers of 5% sucrose and 1% albumin. It is known that the viral inactivation solvent-detergent treatment causes denaturation of proteins and significantly reduces the yield of immunoglobulin [7]. Used for stabilization of albumin carries a new biological activity, the possibility of contamination IVIG prions [8] and is almost never used global manufacturers. Sucrose is in the drug IVIG can cause people acute renal failure [9].

Intravenous immunoglobulin against cytomegalovirus Ivegam-CMVR(Biotest, Germany) with an activity of at least 50 RE/ml receive ethanol fractionation Stake. Process pre who sees the virus removal processing of β-propiolactone and nanofiltration. However, processing of immunoglobulin β-propiolactone causes the destruction of the IgG3 subclass, which leads to a significant decrease in the content of immunoglobulin G3 subclass in drug IVIG and its antiviral activity [6]. In addition, β-propiolactone significantly reduces Fc function of immunoglobulin reduces antigen-binding and antigen-dependent complement-binding, which ultimately affects the activity of the immunoglobulin preparation [10].

Another anticytomegalovirus IVIG preparation HUMAGLOBIN-CMV (M/S Human Serums Production & Medicine MFG Co Ltd., Hungary) contains up to 0.5% of polyethylene glycol and undergoes the process of pasteurization. At the stage of pasteurization positive moment can be considered as a reduction in the frequency and severity of adverse reactions to immunoglobulin (pain, redness, irritation) in comparison with the same raw drugs. However, the method of pasteurization drug IVIG is in absolute contradiction with the requirement of the Federal register, States: "Never in the production process, the product should not be exposed to temperatures above 45°C, and after sterilization, the product should not be exhibited at temperatures above 32°C over 72 hours [11]. Probably pasteurization may adversely affect the parameters of the specific activity, stability and security of the IMM is noglobulins drugs. Contained in the drug IVIG polyethylene glycol is mometasone connection and can cause side effects because of the possibility of overloading the reticulo-endothelial system.

Liquid anticytomegalovirus IVIG preparation "CMV-Polyglobin" (Bayer, Germany) with a title anticytomegalovirus antibody 1:150000-1:1225000 obtained by fractionation according to the method of Kona, followed by alkylation and stabilization of the maltose [12]. The disadvantage of this method is that to reduce AKA drug use stage alkylation. Modification of IG using alkylation significantly reduces the titer anticytomegalovirus antibodies [13] and significantly increases unwanted rheumatoid activity of the drug [14]. In addition, in this case, the title anticytomegalovirus antibodies, in contrast to concentration is not constant and depends on the sensitivity and accuracy of the method of analysis.

Also known is a method of obtaining drug-specific hyperimmune anticytomegalovirus IG for intravenous injection [15]. The known method provides for the selection vysokotirazhnoj anticytomegalovirus plasma from native unvaccinated donors and ethanol fractionation method according to the method of Kona. To enable intravenous prefabricated maintained at neutral Zn is the significance of pH and temperature (4-30°C)within 2-30 days. In the stabilizer is used oligosaccharides in an amount of from 75 to 200 g/l, is selected from glucose, maltose and sucrose. The drug anticytomegalovirus of intravenous immunoglobulin has a specific activity of not less than 60 PE/ml and electrophoretic homogeneity - not less than 90%.

A method of obtaining a known drug involves incubation of the IG with carbohydrates under conditions that are suboptimal for reducing anticomplementary activity and stability anticytomegalovirus antibodies. So the incubation of the drug occurs when the value of pH from 6.4 to 7.0, which corresponds to isoelectrically point immunoglobulin (pH 5,8-7,3) and promotes spontaneous increase anticomplementary activity and aggregation. Keeping immunoglobulin at a temperature 4-30°C may cause (according to obtained experimental data) or to high AKA, or instability of the specific activity of the finished product - deviation titles anticytomegalovirus AT in the tests on the stability exceeds 20%. The duration of incubation of the immunoglobulin to 30 days significantly lengthens the manufacturing process.

The disadvantage of this drug is low electrophoretic purity of the immunoglobulin G, which does not match Tr is the requirements of the European Pharmacopoeia (2008) on human normal immunoglobulin for intravenous injection (not less than 95%) [16]. The disadvantages of the drug are quite high content of aggregates to 1.4% and dimers - to 14.3%, and relatively low specific activity - at least 60 PE/ml and the degree of concentration of specific antibodies (4-5 times), adopted for all previously released a native anticytomegalovirus immunoglobulins. In addition, the time of the dissolution of IVIG after the test for accelerated aging (according to the authors) has increased 3 times, which indicates the instability of the drug in terms of quality - solubility. High concentrations of oligosaccharides (200 g/l) in the composition of the drug give IVIG high osmolarity, which may be associated with serious adverse reactions. Sucrose and glucose, are used as stabilizers, and intravenous high-dose immunoglobulin can cause people acute renal failure.

The prototype of the present invention selected the most highly drug - Noticetext firm "Biotest Pharma (Germany), which represents 5% of the intravenous solution with a specific activity of at least 100 u/ml [17]. 1 ml solution contains: proteins of human plasma - 50 mg; immunoglobulin G is not less than 95%; antibodies against cytomegalovirus - not less than 100 RE/ml; immunoglobulin And not more than 2.5 mg/ml, the hemagglutinin: anti-A titer less than 1:64, anti-In-the engineers - less than 1:64 (after dilution to 30 g of protein/l); stabilizer glycine - 300 μmol/ml, the pH value of the preparation of 5.0 to 5.6. The distribution of subclasses of immunoglobulin G is: IgG1 - 59%, IgG2 - 36%, IgG3 - 3% and IgG4 - 2%. The production process includes fractionation anticytomegalovirus plasma ethanol in the cold to obtain fractions II and the processing of immunoglobulin octane acid. Inactivation/elimination of viruses carry out the processing of octanoic acid and solvent/detergent using a three-(n-butyl)-phosphate (TNBP) and Polysorbate 80 (tween 80). The product was then purified katakouzinos chromatography on SP-sportexe M-gel in the system liquid chromatography high-pressure and high-performance filtering [18, 19].

The known method provides for processing of immunoglobulin octanoic (Caprylic) acid. Intravenous immunoglobulin from human blood plasma obtained caprylate way, has a higher titer of anti-a and anti-b antibodies and consequently greater risk of hemolytic reactions [20]. So, in the normative-technical documentation on known drug maximum titer isohemagglutinins corresponds to the maximum permitted the title provided by the European Pharmacopoeia (2008). The use of IVIG with a high titer of isohemagglutinins can lead to intravascular hemolysis, the characteristic is karasuyama rapid decrease in hemoglobin, development hemoglobinemia hyperbilirubinemia, hemoglobinuria, there is need for red cell transfusions [21]. Therefore, M. Haase, and H. Mengel (1983) proposed to tighten the requirements for the preparations of IVIG on the content of hemagglutinin, reducing the critical titer anti-a and anti-b antibodies with 1:32 to 1:16 [22].

The disadvantage of this method is the use of solvent-detergent to inactivate viruses. With the stage solvent-detergent treatment in the process of obtaining IVIG associated instability of specific antibodies [23]. Thus, in known anticytomegalovirus drug IVIG content of the IgG3 subclass, neutralizing activity against CMV infection is much higher than other subclasses [5], is less than in normal blood plasma of man. Therefore, the drug disrupted the normal distribution of subclasses of immunoglobulin G.

In addition, the processing of the IG solvent-detergent leads to a higher content in the preparations of IVIG antibodies to human DNA (IgG) [23]. However, to prevent possible adverse reactions associated with high levels of antibodies to human DNA, a known drug is not controlled by the content of autoantibodies.

The high limit of the amount of immunoglobulin a in neocytotect (up to 2500 µg/ml) limiting the em its application for IgA-deficient patients whose frequency in the population ranges from 1/200 to 1/800 of cases [24].

All of the aforementioned manufacturers to get specific drug IVIG against cytomegalovirus as a basic principle used only one criterion - selection of human blood plasma with high titers of total anticytomegalovirus antibodies using enzyme immunoassay systems, aimed at identifying all of the antibodies to the lysate cytomegalovirus. However, it is known that between the value of the total titer anticytomegalovirus antibodies and neutralizing titer of AT, i.e. protective activity, the correlation is weaker than that between neutralizing titer anticytomegalovirus of antibody avidity index, where there is a strong correlation [25]. Low avidity series of different immunoglobulin preparations against CMV can lead to variability in the clinical trials [26]. Therefore, the use of quality raw material Miscavige anticytomegalovirus plasma may lead to reduced neutralizing activity end anticytomegalovirus IVIG, in spite of a sufficient titer of specific antibodies in the product. Also known drugs IVIG against cytomegalovirus receive a standard technology normal intravenous immunoglobulin excluding the effect is basically the process stages (ionic strength, pH, conditions reducing AKA, the contents of auxiliary substances and others) on the title anticytomegalovirus AT when obtaining the drug and their stability during storage. That is, in the known methods is not provided optimal conditions for obtaining specific anticytomegalovirus of intravenous immunoglobulin. Therefore, not developed special saving titer anticytomegalovirus antibody technology. No data concerning the stability of the specific activity of the liquid preparation anticytomegalovirus IVIG in tests on accelerated aging and mixing, which is very important and necessary for specific immunoglobulin preparations in liquid form. In addition, there is evidence that elevated levels of immunoglobulin E in the IVIG preparations can cause adverse reactions of immediate type, including anaphylactic [27]. Therefore in IVIG preparations should determine the content of immunoglobulin E. especially in recent years around the world a deterioration of the General allergic status and increase the number of IgE-mediated allergic diseases [28].

Thus, the problem of obtaining high-performance, secure, and stable preparation of CMV immune globulin intravenous (IV) not once is Elena still.

The problem to which the invention is directed, is to obtain a highly effective and safe preparation of human immunoglobulin against cytomegalovirus intravenous and improved stability of the target product.

The technical result of the invention is to develop a method for preserving titles anticytomegalovirus antibody, and obtaining vysokoavidnyh drug cytomegalovirus immune globulin intravenous with superior and stable quality characteristics, content vysokoavidnyh anticytomegalovirus antibodies at least 70 RE/ml.

The invention consists in the following: as a source of raw materials for CMV immunoglobulin use vysokotirazhnyh anticytomegalovirus plasma of human blood, in which the avidity index anticytomegalovirus antibodies is not less than 70%. Selection vysokoavidnyh and vysokotirazhnoj anticytomegalovirus plasma carried out by titration of donor plasma immunoassay single-point method, and without confirmation data immunoassay from passported (certified) donors with known high titer anticytomegalovirus antibodies (at least 13 RE/ml) and the avidity index is not me is it 70%. The selection of immunoglobulin spend ethanol fractionation method for cold method Kona 6. The obtained filtrate B (III Stake) is subjected to ultrafiltration with an ionic strength of not more than 0.02 and pH value of 4.0 to 5.7, with subsequent diafiltration against distilled water with a pH of 3.5 to 6.0. Then in the semi anticytomegalovirus immunoglobulin injected maltose concentration 2-11% and spend incubation at a temperature of 32-40°C and pH 4,0-4,8 within 10-36 hours As one of the options of receiving IVIG against cytomegalovirus prefabricated treated with pepsin at a dose of 1:10-6-1:10-5in the presence of 2-11% maltose. To inactivate/remove viruses immunoglobulin incubated at acidic pH value of 4.0-4.4 for three weeks at a temperature of 21-27°C and/or subjected to depth filtration and/or nanofiltration. Purification of specific immunoglobulin exercise methods: ultrafiltration, and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography and/or dialysis. Then osmolarity IVIG is brought to a value 270-400 mOsm/kg with the use of auxiliary substances: 0.0 to 0.7% of sodium chloride, and/or maltose and/or Proline and/or glucose and/or glycine. In a final product set pH 5.0-7.5, the protein content of 4.5 to 10.5%. End the drug IVIG get in liquid or dry f is RME.

The claimed method allows to obtain vysokoavidnyh and safe preparation of CMV immune globulin intravenous, stable during storage and transportation, which is an immunoglobulin G with electrophoretic homogeneity of not less than 97%, the avidity index of not less than 70%, with a specific activity of not less than 100 RE/ml, in which the content vysokoavidnyh anticytomegalovirus antibodies is at least 70 RE/ml; protein concentration in a solution of 4,5-10,5%; titre anti-a and anti-hemagglutinin - not more than 1:16; the content of immunoglobulin a - no more than 250 µg/ml; immunoglobulin E is not more than 50 IU/ml; IgG to UN-denatured human DNA is no more than 40 AU/ml; the distribution of subclasses of IgG: IgG1 at least 60%, lgG2 - not less than 20%, IgG3-not less than 5%, IgG4 - not less than 2%; pH value of 5.0 to 7.5.

Unlike the prototype of the inventive method as a raw material for specific IVIG use vysokoavidnyh anticytomegalovirus plasma, in which the avidity index is not less than 70%. Carry out the screening and certification of permanent donors with high titers anticytomegalovirus antibodies and high avidity. Re-selection of specific plasma spend passported from donors without confirmation data immunoassay. The production process includes the t ultrafiltration anticytomegalovirus immunoglobulin when the ionic strength is not more than 0.02 and pH value of 4.0 to 5.7, with the subsequent diafiltration using distilled water with a pH of 3.5 to 6.0. To reduce anticomplementary activity and security of titles anticytomegalovirus antibody immunoglobulin incubated with 2-11% maltose at a temperature of 32-40°C and pH 4,0-4,8 within 10-36 hours As one of the variants of the drug IVIG against cytomegalovirus receive treatment with pepsin at a dose of 1:10-6-1:10-5in the presence of 2-10% maltose. For the inactivation/removal of viruses immunoglobulin incubated at acidic pH value of 4.0-4.4 and/or subjected to depth filtration, and/or nanofiltration. The method of producing drug IVIG against cytomegalovirus includes the purification of immunoglobulin methods: ultrafiltration, and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography and/or dialysis. Physiological osmolarity (270-400 mOsm/kg) IVIG sets with different combinations of excipients: 0.0 to 0.7% of sodium chloride, and/or maltose and/or Proline and/or glucose and glycine. End the drug IVIG can be obtained in dry form with a neutral pH value (6,0-7,5).

The set of distinctive features of the proposed method allows to obtain highly effective for CMV immunoglobulin for intravenous administration with superior and stable quality features and advantages of the kami. In the claimed drug IVIG content of immunoglobulin G is at least 97%, vysokoavidnyh anticytomegalovirus AT least 70 RE/ml, the concentration of protein in solution up to 10.5%. In the developed drug is determined by the avidity index, which is not less than 70%. In addition, the claimed specific IVIG is controlled on the content of IgE and IgG to human DNA. Compared with the prototype in the drug reduced the limit of the amount of immunoglobulin a and isohemagglutinins. Moreover, it increased the content anticytomegalovirus antibodies of the IgG3 subclass.

The set of distinctive features of the claimed method provides the following advantages.

Stability vysokoavidnyh titles anticytomegalovirus antibodies from donors vysokotirazhnoj plasma over time (regardless of cyclicity and seasonality deviation anticytomegalovirus titles AT was no more than 15% during the observation period of 3 years) allows not only the specific screening plasma, but the selection of donors and implement certification (certification) regular donors with high titers anticytomegalovirus antibodies with high avidity. Screening of donors and the screening of plasma in passported donors without confirmation data immunoassay reduce the need to t the St-systems, to reduce the complexity of production and the cost vysokoavidnyh drug cytomegalovirus immune globulin intravenous.

Ultrafiltration semi-finished when the ionic strength is not more than 0.02 and pH value of 4.0 to 5.7 with subsequent diafiltration against distilled water with a pH of 3.5 to 6.0 promotes maximum safety of titles anticytomegalovirus antibodies and the output of the immunoglobulin. When you increase the specified pH causes a decrease in the specific activity of the drug and exit anticytomegalovirus immunoglobulin. The decrease in pH and/or increasing the ionic strength leads to reactivity (autoreactivity) IVIG to the test antigen (diploid fibroblasts of the human embryo) according to enzyme immunoassay test system "CMV-screen" ( " , Moscow) - the increase in the titer of approximately 2.1-fold (p<0,000; n=10), and increase in the titer of antikink antibodies, the content of which is an indicator of the denaturation of immunoglobulin in the process of obtaining the drug and reflects the preservation of natively antibodies [29].

Incubation of antibody with maltose concentration 2-11% at pH 4,0-4,8 within 10-36 h reduces anticomplementary activity of the drug and to provide an opportunity for intravenous injection. It should be noted that the temperature of incubation ocas which covers a significant impact on the stability of the title anticytomegalovirus antibodies. Comparison of titers anticytomegalovirus AT different temperatures keeping immunoglobulin with maltose showed that after the test for accelerated aging in the preparation incubated at 23°C, the reduction of the titer of specific antibodies 53.6%. In addition, after playing conditions storage for 2 years (4 weeks at 37°C) this product contains the maximum number of units. It was noted a strong correlation between the level of reduction credits anticytomegalovirus antibodies and content units in IVIG during storage (correlation coefficient µ=0,832; P<0,05; n=10). The most stable in respect of titles anticytomegalovirus AT were the preparations incubated with maltose at a temperature of 4 and 37°C (table 1). However, incubation of antibody with maltose at 4°C anticomplementary activity of the drug exceeded IN/1 mg protein and did not meet the requirements of the European Pharmacopoeia (2008). Thus, in the production of specific IVIG against cytomegalovirus incubation of antibody with maltose should be carried out at a temperature of 32-40°C and pH 4,0-4,8 within 10-36 h, as these conditions contribute to a better stability of the titles anticytomegalovirus antibodies, low anticomplementary activity and minimalist guest is gnome content units in the preparation of IVIG during storage.

The possibility of using the trace amounts of pepsin in doses of 1:10-6-1:10-5in the presence of 2-11% maltose helps prevent: increase anticomplementary activity of the drug, the turbidity of the solution IVIG, the appearance of aggregates and the increase in the content of dimers. It should be noted that this concentration of pepsin provides stability titer anticytomegalovirus antibody during storage and transportation.

The inactivation/removal of viruses by keeping the prefabricated IG at acidic pH values (of 4.0-4.4), with the subsequent nano - and/or depth filtration with high efficiency and allows you to get virologically safe intravenous immunoglobulin. This method of inactivation/removal of viruses is easy to perform, does not require the introduction of additional reagents and control their residual content in the finished product. A comparative analysis of the experimental series IVIG received from one of the material with and without the use of data stages of inactivation and removal of viruses, did not reveal significant differences in their qualitative characteristics.

The purification of specific immunoglobulin methods ultrafiltration, and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography and/or dialysis allows you to get asociaciones and virusopodobnyh drug IVIG against cytomegalovirus with improved quality characteristics, stable during storage and transportation. The combination of methods used cleaning reduces the amount of immunoglobulin a in the product to remove the residual content of pepsin, fragments, dimers and aggregated forms of the antibody.

Form of application of the claimed drug IVIG against cytomegalovirus may be different from a standard solution with a protein concentration of 4.5% to more concentrated to 10.5%. Increasing concentration of immunologically active fraction is achieved by improving the efficiency and reliability of IVIG, with significantly reduced time and volume of injection. To prevent dangerous for patients of high osmolarity and viscosity of the solution associated with a high concentration of protein in the IVIG, the drug is subjected to nano - and depth filtration. In addition, were selected special combination and concentration of excipients and pH of the drug.

Physiological osmolarity (270-400 mOsm/kg) in anticytomegalovirus IVIG reach when several excipients: sodium chloride, maltose, Proline, glucose and glycine, with a minimum content in the product of each of the stabilizers. It is known that preparations of IVIG with a minimum content of each of the excipients the most beneficial you Lagat when the index of conditional latitude pharmacological security stabilizers LD50/MDS. Index conditional latitude pharmacological security stabilizers determined the risk of intravenous immunoglobulins. The sum of the concentrations used combinations of excipients provide the osmolarity of the claimed preparation, corresponding to the standards of the European Pharmacopoeia (2008) - not less than 240 mOsm/kg, and the stability of the qualitative characteristics of IVIG (molecular parameters, AKA, the specific activity) during storage and transportation (table 2). In addition, during freeze-drying using stabilizers maltose and glucose promotes stability AKA drug and prevents the formation of aggregated forms of the antibody. Used stabilizers have good tolerance when the intravenous route of administration due to the ability of renal cells to metabolize these substances. On the other hand, the presence of a single stabilizer in the composition of the drug IVIG can be recommended only if intolerance of other auxiliary substances.

The liquid form IVIG significantly reduces the cost of the drug and is more convenient for the consumer. The pH value of 5.0-6.0 in liquid form specific immunoglobulin for intravenous contributes to the stability of the quality characteristics of the drug and a greatest extent and stability of title anticytomegalovirus AT i.e. its specific activity during storage and transport.

Freeze-dried form of the drug allows you to receive IVIG with a neutral pH value (6,5-7,5), provides more stable performance during storage and transportation, increases the shelf life of the target product and safety applications due to neutral pH values.

The claimed method allows to obtain vysokoavidnyh, safe and stable preparation of cytomegalovirus immune globulin intravenous (IV) with improved quality characteristics (table 3). Use as raw material vysokoavidnyh plasma of donors with high titres of antibodies to CMV allows you to get anticytomegalovirus drug IVIG with high avidity (not less than 70%), in which the content vysokoavidnyh anticytomegalovirus antibodies is at least 70 RE/ml. it is Known that vysokoavidnyh antibodies significantly more active discouvery in a number of biological reactions, such as binding of complement and opsonization, and have a higher neutralizing activity [30].

In the claimed drug entered the control of the concentration of IgE and IgG, which are indicators of the safety of intravenous immunoglobulins. Determination of immunoglobulin E makes the drug IVIG more baie is dangerous in the treatment of patients prone to IgE-mediated allergic diseases. Control of IVIG on autoamtically activity helps to prevent possible adverse reactions [14].

Developed the drug belongs to the group of intravenous immunoglobulins with low content of immunoglobulin a (no more than 250 µg/ml) and can be used with less risk for the treatment of IgA-deficient patients.

The drug has a distribution of IgG subclasses, similar to the plasma of human blood (table 4). In comparison with the prototype content anticytomegalovirus antibodies of the IgG3 subclass increased by more than 1.7 times.

In accordance with the requirements of the European Pharmacopoeia on IVIG (2008) claimed the drug control on the content of all substances added to it and remaining in it after processing.

The preparation of cytomegalovirus immune globulin for intravenous administration was controlled by the following methods.

Electrophoretic homogeneity by electrophoresis on films of cellulose acetate (FS 42-3874-99, p.20). Molecular parameters by the gel filtration method (FS 42-3874-99, p.28). Anticomplementary activity of 1 mg of protein immunoglobulin should not consume more than 1 unit (SN) complement (MUK 3.3.2.1063-010). Title anticytomegalovirus antibodies to CMV were determined relative to the industry standard sample against the cytomegalovirus antibodies person CCA IgG anti-CMV No. 42-28-371-03 and was expressed in activity units of the Institute, Paul-Ehrlich - RE/ml. as calibrator cytomegalovirus antibodies were used international standard CMV IgG+ with an activity of 300 U/ml. The avidity anticytomegalovirus antibodies were determined by enzyme immunoassay system for the detection discouvery immunoglobulin G to cytomegalovirus infection CMV Diagnostician" (LLP Biotech company bioservice), DS-EIA-anti-CMV-G-Avidity (LLC "Scientific production Association "Diagnostic system"), CMV-IgG-Avidity-DS (CJSC "MBS"). Title vysokoavidnyh antibodies (TWAT) was calculated by the formula: TAT=TAT×IA/100, where TAT - titer antibody, IA - the avidity index. To determine the concentration of immunoglobulin E (IgE) used an enzyme immunoassay "IgE-ELISA-best-strip" (JSC "Vector-best, Novosibirsk, Russia). The content of anti-a and anti-immune isohemagglutinins was determined by the modified method of indirect haemagglutination (European Pharmacopoeia) using the test red blood cells a and b, prepared according to the "Instructions for the preparation and preservation of erythrocyte mass", approved by the chief of preventive and curative management of health Ministry 30.09.1968, the Content of IgA was determined by enzyme-linked immunosorbent assay test system "IgA-ELISA-BEST-strip" (JSC "Vector-best, Novosibirsk, Russia). The determination of concentrations of subclasses of IgG: IgG1, IgG2, IgG and IgG4 was carried out using enzyme immunoassay test system "Subclasses of IgG-ELISA-BEST" (JSC "Vector-best", , Novosibirsk). For the determination of autoantibodies was used the test system Vecton-IgG-strip for the detection of autoimmune antibodies of class G to UN-denatured double-stranded DNA. Proteolytic activity was determined according to the European Pharmacopoeia (Ph. Eu.r monograph 0682). Sodium chloride on FS 42-3874-99, p.59. Glycine by MUK 4.1/4.2.588-96, p.114. The quantitative content of maltose and glucose were determined by specific enzyme sets: Maltose/Sucrose/D-Glucose (BOEHRINGER MANNHEIM/R-BIOPHARM AG, Germany) or ENZYTEC™ D-Glucose, Enzytec™ Maltose ("SCIL Diagnostics GmbH, Germany). Residual ethanol by MUK 4.1/4.2.588-96, s. Definition of progenote were performed according to the requirements of the European Pharmacopoeia (2008, 6.0, monograph 0918). Osmolarity was determined by freezing point depression method on millionometer-cryoscope thermoelectric MT-4. The study of the stability of the preparation was carried out tests on mixing and accelerated aging. Test mixing, which is similar to the conditions of transportation of the drug was carried out by stirring for 2 h at a frequency of 80 horizontal oscillations per minute at a temperature of 20°C. Test for accelerated aging was carried out by maintaining the drug in thermostat at 37°C for 4 weeks and is analogous to the storage conditions within 2 years [European Pharmacopoeia. Ed. III-2000., V.2.6.20, monograph No. 0918]. Statistical treatment the processing of the results was performed using the computer programs Excel 4.0" and "Statistica 5.7".

Examples of specific performance.

Example 1. To obtain a preparation of human immunoglobulin against CMV for intravenous administration as a source of raw materials using plasma unvaccinated cytomegalovirus donor in the amount of 200 liters with high titer and avidity anticytomegalovirus antibodies. First carry out the screening and selection of donor human blood plasma, in which the title anticytomegalovirus antibodies is at least 13 RE/ml, the avidity index is not less than 70%. Determination of the titer and avidity index anticytomegalovirus antibodies carry out titration donor plasma immunoassay single-point method on the available enzyme immunoassay system. Then hold certification (certification) of regular donors with title anticytomegalovirus antibodies at least 13 RE/ml and the avidity index is not less than 70%. The selection of specific immunoglobulin perform classical ethanol method at low temperatures by the method of Cohn 6 to obtain a filtrate III. The obtained filtrate B (III Stake), with a pH of 5.1 and a protein content of 0.3%, ethanol - 17%, is subjected to concentration by ultrafiltration installation polyethersulfone membranes with a limit of cut-off molecular weight 10000-100000 Yes. The ultrafiltration immunoglobulin hold p and ionic strength of 0.02 and pH value of 4.0. Then the semi-finished product is subjected to diafiltration against distilled water with a pH value of 3.5. Then IG is injected maltose concentrations up to 2% in solution and incubated at pH 4.0 and temperature (33±1)°C for 36 h Later immunoglobulin subjected to purification by the methods of depth filtration, nanofiltration in a tangential flow at the facility Viresolve™ 180 (Millipore, USA) and anion-exchange chromatography using, for example, DEAE-sepharose. Then the content of maltose in the immunoglobulin is brought to a concentration of 2.0% and introduced an auxiliary substance to the concentration in solution: sodium chloride - 0.1%, Proline - to 1.2%, glucose 0.5% and glycine to 1.3%. The resulting osmolarity IVIG corresponds to a value of about 323 mOsm/kg (table 5, option 1). The protein content in the solution was adjusted to a concentration of 9.5-10.5 per cent, the value of pH to 6.5 and 7.5. After sterilizing filtration immunoglobulin subjected to freeze-drying, and the dry form. Preparation of immunoglobulin against cytomegalovirus for intravenous administration has electrophoretic homogeneity γ-globulin fraction of 99.9%; specific activity 160 PE/ml; the avidity index - 83,9%, the content of vysokoavidnyh anticytomegalovirus antibody - 134 D/ml; the concentration of protein in solution to 10.5%; the titer of anti-hemagglutinin - 1:2, the titer of anti-hemagglutinin - 1:1; content is the use of immunoglobulin a - 188 µg/ml; IgE - 38 IU/ml; IgG to UN-denatured human DNA - 29 AU/ml; the distribution of subclasses of IgG: IgG1 - 64%, IgG2 - 26%, IgG3 - 7%, IgG4 - 3%; molecular weight distribution: units - 0.1%, the monomers and dimers - 98,3%, fragments of 1.6%; AKA - 0,4 SN/mg; pH value of 6.5-7.5.

Example 2. Selection of raw materials - vysokotirazhnoj anticytomegalovirus human plasma with high avidity carried out without confirmation data immunoassay permanent passported (certified) donors with a known (previously established) Tyr anticytomegalovirus antibodies at least 13 RE/ml and the avidity index of not less than 70%. The selection of specific immunoglobulin spend ethanol fractionation method for cold method Kona 6. The obtained filtrate III is subjected to ultrafiltration at ionic strength of 0.015 and the pH value of 5.7, with subsequent diafiltration against distilled water with a pH of 6.0. Then in the semi-finished product is injected maltose concentrations up to 11%, set the pH value of 4.8 and incubated at a temperature of (39±1)°C for 10 h After which the immunoglobulin is treated with pepsin at a dose of 1:10-6when the pH value of 4.4 and the concentration of maltose 11%. To inactivate/remove viruses immunoglobulin is maintained at a pH value of 4.4 for three weeks at a temperature (26±1)°C and p will gorhaut deep virusderived filtering. To remove residual pepsin and low molecular weight products of the cleavage solution IG dosimat methods of ultrafiltration and anion exchange chromatography. After that, the protein content in the IG lead up to a concentration of 4,5-5,5%, maltose - up to 10%, the value of pH to 5.0 to 5.5. The osmolarity of the obtained product has a value of 300 mOsm/kg (table 5, option 3). End the drug IVIG is subjected to sterilizing filtration, and liquid form. The preparation of CMV immune globulin intravenous is an immunoglobulin G with electrophoretic homogeneity of 99.9%, the avidity index is 78%, with a specific activity of 120 RE/ml, in which the content vysokoavidnyh anticytomegalovirus antibodies 94 RE/ml; protein concentration in a solution of 4,5-5,5%; titre anti-a and anti-hemagglutinin - 1:1; the content of immunoglobulin a - 158 µg/ml; IgE - 33 IU/ml; IgG to UN-denatured human DNA - 25 AU/ml; the distribution of subclasses of immunoglobulin G: IgG1 - 66%, IgG2 - 26%, IgG3 - 6%, IgG4 - 2%; pH value of 5.0-6.0.

Example 3. As a source of raw materials for CMV immunoglobulin use vysokotirazhnyh anticytomegalovirus the blood plasma of a person with the avidity index anticytomegalovirus antibodies not less than 70%. Selection vysokoavidnyh and vysokotirazhnoj anticytomegalovirus plasma performance is given as the titration donor plasma immunoassay single-point method, and without confirmation data immunoassay passported from donors. The allocation of specific immunoglobulin fraction conduct alcohol method at low temperatures by the method of Cohn 6. The obtained filtrate III is subjected to ultrafiltration with an ionic strength of 0.005 and the pH value of 4.9, with subsequent diafiltration against distilled water with pH value of 4.8. Then in the semi-finished product is injected maltose concentrations up to 6% and spend incubation at a temperature (36±1)°C and a pH value of 4.4 for 24 hours and Then in the immunoglobulin add pepsin at a dose of 1:10-5. Removal of pepsin cake mix is poured into viscose dialysis tube and dialysis against distilled water. For the inactivation/removal of viruses immunoglobulin subjected to incubation at pH 4.0 and temperature (22±1)°C for three weeks, depth filtration and nanofiltration on the Planova filters (Asahi Chemical Industries, Japan). The purification is conducted by the methods of anion-exchange chromatography. Osmolarity IVIG is brought to a value of 333 mOsm/kg by adding sodium chloride to a concentration of 0.1% and glucose - 2,0%, the content of maltose in the immunoglobulin brought to a concentration of 6.0% (table 5, option 11). Immunoglobulin set the pH value of 5.7 to 6.8, the protein content of 4.5-5.5% and sterile-filtered. End the drug IVIG get in the soup form. The resulting preparation of CMV immune globulin intravenous is electrophoretic homogeneity of 99.9%, the avidity index - 90%, specific activity - 143 PE/ml, the content vysokoavidnyh anticytomegalovirus antibodies is 129 PE/ml; protein concentration in a solution of 4,5-5,5%; the titer of anti-hemagglutinin - 1:2, the titer of anti-hemagglutinin - 1:2; the content of immunoglobulin a - 173 µg/ml; IgE - 37 IU/ml; IgG to UN-denatured human DNA - 21 AE/ml; the distribution of subclasses of immunoglobulin G: IgG1 - 68%, IgG2 - 22%, IgG3 - 8%, IgG4 - 2%; pH value of 5.7 to 6.8.

Example 4. The selection of raw materials and the allocation of the γ-globulin fraction of immunoglobulin against CMV carried out according to examples 1, 2 and 3. The removal/inactivation of viruses and purification of immunoglobulin spend one or combination of following methods - incubation at pH value of 4.0-4.4, ultra-filtration, depth filtration, nano-filtration, anion exchange chromatography, dialysis. Various options for the possible formulation of the drug with different combinations of auxiliary substances are presented in table 5. Received options ready anticytomegalovirus IVIG fully meet the qualitative characteristics of the claimed drug.

Table 1
The effect of temperature of incubation of antibody with maltose on the title anticytomegalovirus antibodies and anticomplementary activity in immunoglobulin preparations against cytomegalovirus
Incubation temperature, °CTitle anticytomegalovirus antibody, PE/mlAKA drug SN/mg protein
sourceafter the test on mixingafter the test on accelerated agingsourceafter the test on mixingafter the test on accelerated aging
4109,0105,6 (-3,1%)85,9 (-21,2%)0,80,91,2
23138,4108,5 (-21,6%)64,2 (-53,6%)0,91,00,7
30129, 9mm991 (-23,7%) 73,1 (-43,0%)0,20,30,0
32119,3110,8 (-7,1%)109,5 (-8,2%)0,30,40,3
37to 108.2108,5 (+0,3%)100,0 (to-7.6%)0,30,50,5
40of 116.7124,6 (+6,8%)111,7 (-4,3)0,30,50,1
43128,1161,8 (+26,3%)147,8 (+15,4)0,80,91,1
Note: In parentheses are the change of title anticytomegalovirus antibodies relative to the original value, %. Change of specific activity - drop or increase in the activity of not more than 10% of installed domestic and foreign regulatory document is nami [31, 32, 33].

Table 3
Qualitative characteristics of the proposed drug immunoglobulin against cytomegalovirus intravenous and drug prototype Noticetext (Biotest, Germany)
The claimed preparation, M±SD (n=10)Noticetext (Biotest, Germany) M±SD (n=10)P
12
Specific activity, RE/ml143,2±27,3 (at least 100)141,3±13,6 (*not less than 100)P1,2>0,05
The avidity index, %to 89.9±6,8 (at least 70)57,2±5,7 (not rated)P1,2<0,05
Title vysokoavidnyh anticytomegalovirus antibody, PE/ml128,7±13,6 (at least 70)80,8±8,8 (not rated)P1,2<0,05
Title isohama-glutinin, reverse titleanti2,0±1,3 (16)13,0±3,9 (*less than 64)P1,2<0,05
anti2,0±0,9 (16)6,1±2,1 (*less than 64)P1,2>0,05
Immunoglobulin A, ug/ml173, 0mm±15,3 (no more than 250)1907,5±120,8 (*not more than 2500)P1,2<0,05
IgE, IU/ml37,2±4,6 (50)70,0±8,8 (not rated)P1,2<0,05
Autoantibodies to UN-denatured human DNA, AE/ml21,10±7,7 (40)97,00±21,70 (not rated)P1,2<0,05
Note: PE/ml titer anticytomegalovirus antibodies expressed in units of activity of the Institute, Paul-Ehrlich. Title vysokoavidnyh antibodies (TV AT) was calculated according to the formula of TAT=TAT × IA/100, where TAT - titer antibody, IA - the avidity index.
* These instructions for use n apart Noticetext [19].

Table 4

The distribution of subclasses of immunoglobulin G in the inventive preparation of human immunoglobulin against cytomegalovirus intravenous and drug the prototype Noticetext (Biotest, Germany)
X2The drug (number of episodes)The subclasses of immunoglobulin G, %
IgG1IgG2IgG3IgG4
1Declare (M±SD, n=10)66,5±2,524,2±1,27,0±0,92,3±0,1
2Noticetext* (prototype)593632
3The normal plasma donor**60,3 is 71.519,4-31,05,0 an 8.4of 0.7 to 4.2
R - reliability of differences P1,2>0.05 to P1,3>0,05P1,2<0.05 to P1,3>0,05P1,2<0.05 to P1,3>0,05P1,2>0.05 to P1,3>0,05
Note. *These instructions for use neocytotect [19].
**Information about the distribution of IgG subclasses is given by ..French (1986) [34].

1. A method of producing human immunoglobulin against cytomegalovirus intravenous, including the selection of donor plasma with a high titer anticytomegalovirus antibodies, specific fractionation of plasma ethanol method for cold method Kona 6 to obtain a filtrate III, the inactivation/removal of viruses, purification and stabilization of immunoglobulin glycine, characterized in that the selection of the plasma is conducted from a certified donor with a title anticytomegalovirus antibodies at least 13 RE/ml and the avidity index of not less than 70% without confirmation data immunoassay, as raw material for fractionation using anticytomegalovirus the plasma with the avidity index of not less than 70%, the production of the process includes: ultrafiltration immunoglobulin when the ionic strength is not more than 0.02 and pH value of 4.0 to 5.7 with subsequent diafiltration against distilled water with a pH of 3.5 to 6.0, incubation of antibody with maltose concentration 2-11% at a temperature of 32-40°C and pH 4,0-4,8 within 10-36 h, or with pepsin at a dose of 1:10-6-1:10-5in the presence of 2-11% maltose, the inactivation/removal of viruses by incubation of antibody with pH value of 4.0-4.4 and/or using depth filtration, and/or nanofiltration, purification of immunoglobulin methods: ultrafiltration, and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography and/or dialysis, the stabilization of the immunoglobulin with the use of auxiliary substances: 0.0 to 0.7% of sodium chloride, and/or maltose and/or Proline and/or glucose and/or glycine, the content of which provides the osmolarity of the drug within 270-400 mOsm/kg, obtained anticytomegalovirus immunoglobulin establish a pH value of 5.0-7.5 and the protein content of 4.5 to 10.5%, if necessary, the finished drug lyophilizer.

2. The method according to claim 1, characterized in that conduct the screening and certification of permanent donors with a title anticytomegalovirus antibodies at least 13 RE/ml and the avidity index of not less than 70%.

3. Vysokoavidnyh the preparation of human immunoglobulin against cytomegalovirus intravenous obtained according to claim 1, which is an immunoglobulin G with electrophoretic homogeneity of not less than 97%, the index even the STI - at least 70%, with a specific activity of not less than 100 RE/ml, in which the distribution of subclasses of immunoglobulin G is: IgGI not less than 60%, IgG2 not less than 20%, IgG3 not less than 5%, IgG4 not less than 2%, the content of vysokoavidnyh anticytomegalovirus antibodies is at least 70 RE/ml titer anti-a and anti-hemagglutinin not more than 1:16, the content of immunoglobulin And not more than 250 µg/ml of immunoglobulin E is not more than 50 IU/ml, autoantibodies to UN-denatured human DNA is not more 40 AU/ml

4. The preparation according to claim 3, characterized in that the immunoglobulin has a protein concentration in a solution of 4,5-10,5%.

5. The preparation according to claim 3, characterized in that the osmolarity of the preparation is in the range 270-400 mOsm/kg

6. The preparation according to claim 3, characterized in that the preparation has a pH value of 5.0 to 7.5.

7. The preparation according to claim 3, characterized in that the drug may be in liquid or dry form.



 

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3 cl, 6 tbl, 4 ex

FIELD: medicine, veterinary.

SUBSTANCE: invention relates to field of veterinary microbiology. As active substance vaccine contains mixture of inactivated virus suspension of strain of virus of infectious rhynotracheitis of cattle, SCV No 2333, of family Herpesviridae, subfamily Alphaherpesviridae, inactivated virus suspension of strain of virus of cattle paraflu-3 SCV No 2334 subfamily Paramixovirinae, genus Paramixovirus, inactivated virus suspension of strain of virus of cattle respiratory syncytial disease SCV No 2335, subfamily Pneumoviridae, genus Pneumovirus, inactivated virus suspension of strain of virus of cattle diarrhea SCV No 2336, family Flaviviridae, genus Pestivirus, with activity of each virus strain being not less than 106.5 TCD50 in 1 cm3 of vaccine, and inactivated suspension of cells of bacteria Pasteurella multocida serovar A strain 1231, inactivated suspension of cells of bacterium Pasteurella multocida serovar B strain 656, inactivated suspension of cells of bacterium Pasteurella multocida serovar D strain T-80, inactivated suspension of cells of bacterium Mannheimia haemolytica strain 169, in efficient quantity, taken in mixture in equal proportion with concentration not less than 3 billion microbial cells in vaccine dose.

EFFECT: vaccine possesses high specificity, stability in preservation of biological properties, creates expressed immunity, is harmless.

3 cl, 10 tbl, 6 ex

FIELD: medicine, veterinary.

SUBSTANCE: invention relates to field of veterinary immunology and virology. Method includes aerosol treatment of chicks with vaccine. Chicks are treated with vaccine, obtained by multiple infection of chicken embryos with material, containing Newcastle disease virus, selection of embryos, which survived to 19-day age, their killing by cooling, preparation of virus suspension from chorioallantoic fluid and liver and isolation from it of defective interfering virions.

EFFECT: method presents reliable and safe method of bird vaccination.

1 cl, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.

EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.

36 cl, 14 dwg, 6 tbl, 13 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to biotechnology. The vaccine contains an inactivated rabies antigen, a culture medium, a shielding desiccation medium of peptone, sucrose and gelatin, and also an immunity stimulator saponin. The desiccation environment in addition contains aluminium oxide hydrate in the following ratio in an end product (wt %): aluminium oxide hydrate with adsorbed inactivated rabies virus - 6.0-9.0, saponin - 0.75-1.5, peptone - 30.0-33.0, sucrose - 35.0-37,0, gelatin - 9.0-10.0, culture medium - the rest.

EFFECT: dry vaccine exhibits high level of immunogenic activity.

3 tbl

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

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