RU2770425C2 - Method of producing smallpox immunoglobulin from horse blood serum - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of producing anti-smallpox immunoglobulin from horse blood serum. Method of producing smallpox immunoglobulin from horse blood serum involves preparing an antigen from vaccine virus of B-51 and L-IVP strains, preparation of immune serum using calcium chloride, isolation of immunoglobulin by ammonium sulphate precipitation, purification in three stages, concentration, sterilizing filtration, packing and packaging under certain conditions.

EFFECT: method described above enables to obtain anti-smallpox immunoglobulin from horse blood serum with high content of monomer fraction of immunoglobulin, not less than 95 %, and low content of foreign fractions of dimers, polymers and aggregates, not more than 5 %, as well as absence of residual ethyl alcohol, which reduces anaphylactogenicity of immunoglobulin and brings the quality of the preparation in compliance with the requirements of the European Pharmacopoeia for heterologous preparations.

1 cl, 2 tbl, 1 ex

Description

The invention relates to the field of biotechnology, specifically to the development of technologies for the production of protective immunobiological drugs, in particular, to the development of a method for obtaining a specific means of emergency prevention and treatment of infectious diseases caused by orthopoxviruses pathogenic to humans.

The development of a specific liquid anti-small immunoglobulin from the blood serum of horses was carried out in the period from 1955 to 1965 by a team of authors led by A.T. Kravchenko and A.A. Vorobyov. The drug being developed was intended for the prevention and treatment of smallpox in humans, as well as the treatment of patients with complications after vaccination with smallpox vaccine (with the exception of children with complications after vaccination with smallpox vaccine). For anti-smallpox immunoglobulin from horse blood serum, obtained by the method of alcohol precipitation in the cold and the method of electrodecantation, were developed and in 1967 approved by the Ministry of Health of the USSR MRTU-42 No. 150-67 for the production of the drug.

With the termination of vaccination against smallpox in 1980, work on the creation of new therapeutic drugs, vaccines and other means of specific prevention of smallpox was curtailed. The cessation of smallpox vaccination led to the virtual disappearance of population immunity to smallpox. In the current situation, the smallpox virus is considered by many experts as one of the main damaging agents that can be used by terrorists [1-6]. The intensification of terrorism, in particular bioterrorism, makes the problem of protecting the Russian population from biological agents, especially smallpox, urgent [7, 8]. The existence of the problem of the resumption of smallpox vaccination is also recognized by experts from WHO, France, Germany, the USA and other countries [9, 10]. A number of countries are stockpiling smallpox vaccines and developing smallpox drugs.

In Russia, there are no safe means of biological protection against smallpox, suitable for use in conditions of the practical absence of population immunity. The release of metisazon and other chemotherapeutic anti-small drugs has been discontinued. The possible resumption of skin smallpox vaccination in modern conditions is associated with a high risk of developing a large number of post-vaccination complications [11, 12].

Previously, it was shown that specific donor immunoglobulin can be used to treat post-vaccination complications [13, 14].

At present, the serial production of anti-smallpox homologous immunoglobulin in the Russian Federation is problematic due to the practical absence of human donors with a high titer of anti-smallpox virus-neutralizing antibodies in their blood. Therefore, it is reasonable to obtain a heterologous anti-small immunoglobulin from the blood serum of horses, safe against HIV, viral hepatitis and prion infections.

A known method of obtaining, immunoglobulin anti-small from the blood serum of horses liquid, including the preparation of an antigen from the vaccine virus of strains B-51 and L-IVP, obtaining immune blood, obtaining immune serum using a 30% solution of calcium chloride, isolation and concentration of immunoglobulins by precipitation with ethyl alcohol in the cold according to Kohn, sterilizing filtration. This method is the only analogue (prototype) of the present invention.

EFFECT: method provides obtaining a non-toxic, pyrogen-free, sterile, specific means for emergency prevention and treatment of infectious diseases caused by orthopoxviruses pathogenic for humans with specific activity expressed by a virus-neutralizing antibody titer of at least 1:4096, a content of gamma globulin fraction of at least 80%, an impurity content of foreign proteins, not more than 20% and a residual amount of ethyl alcohol not more than 4%.

The disadvantages of the existing method include the fact that the final product contains a large amount of extraneous blood serum proteins (up to 20% albumin, α - and β - globulins) and a residual amount (up to 4%) of ethyl alcohol, which increase the reactogenicity of the drug. When analyzed by gel filtration HPLC, about 88% monomers, 6% dimers, 3% polymers and 3% aggregates are detected in the preparation, which indicates a low purity of the immunoglobulin fraction. As a result, the drug has anaphylactogenicity, characterized by the Weigl anaphylaxis index of 2.5±0.5.

The aim of the present invention is to develop a method for the production of smallpox immunoglobulin, which provides the final product with a higher degree of purification, which will significantly reduce the anaphylactogenicity of the drug.

The technical result of the invention lies in the fact that the developed method provides for the production of smallpox immunoglobulin from the blood serum of horses with a high content of the immunoglobulin monomer fraction (not less than 95%) and a low content of foreign fractions of dimers, polymers and aggregates (not more than 5%), and also the absence of residual ethyl alcohol, which reduces the anaphylactogenicity of immunoglobulin (anaphylaxis index according to Weigl 1.5 ± 0.5) and brings the quality of the drug in line with the requirements of the European Pharmacopoeia for heterologous drugs [15].

This technical result is achieved due to the fact that the proposed method for producing anti-small immunoglobulin includes the method of precipitation of immunoglobulin with ammonium sulfate, as well as additional stages of chromatographic purification and concentration, consisting of three stages: gel filtration chromatography on a column with a Sephadex G-25 sorbent pre-equilibrated with a solution of 20 mm Tris, with a flow rate of 100 ml/min. After applying the solution, the separation is carried out in isocratic elution mode, feeding a solution of 20 mm Tris at the same flow rate; ion-exchange chromatography on a column with a Q Sepharose FF sorbent at a temperature of 20°C and a linear sample feed rate of 150 cm/h, followed by a gradient elution from 0 to 100% with a solution of 20 mM Tris 1 Μ NaCl (pH 8.0) at the same linear velocity within 80 minutes; gel filtration chromatography on a column with a Superdex 200 pg sorbent equilibrated with a phosphate-glycine buffer solution (pH 6.9) at a linear flow rate of 50 cm/h in an isocratic elution mode at the same speed; as well as concentration of the immunoglobulin solution on an automatic filtration and concentration system in a tangential flow through cassettes with an exclusion limit of 50 kDa at a flow rate of 500 ml/min, an inlet pressure of no more than 3 bar and an outlet pressure of no more than 2 bar.

The essence of the invention lies in the fact that in the method of obtaining a heterologous anti-small immunoglobulin, the isolation by the method of alcohol precipitation in the cold according to Kohn is replaced by precipitation with ammonium sulfate, and additional stages of purification and concentration of the preparation are included using the methods of ion-exchange and gel filtration chromatography, as well as the concentration of the immunoglobulin solution in a tangential flow through cassettes with an exclusion limit of 50 kDa. The advantage of isolation by precipitation with ammonium sulfate is the high solubility of albumin and other lighter proteins in it, the absence of a denaturing effect on proteins, as well as the need to create a special temperature regime and the availability of high-tech equipment. The ammonium salt was removed by gel filtration chromatography on a Sephadex G-25 sorbent. The next stage of ion-exchange chromatography on a Q Sepharose FF sorbent makes it possible to isolate IgG fractions and remove albumin residues, as well as a pseudoglobulin fraction, which contains α -, β - globulins and a minimum amount of specific antibodies. At the next stage of purification, using gel filtration chromatography on a Superdex 200 pg sorbent, high molecular weight fractions of dimers and polymers, low molecular weight fragmentary fractions, which reduce the quality of immunoglobulins, are separated.

The novelty of the invention lies in the fact that from the technologies for obtaining heterologous immunoglobulins from the blood serum of horses, it is not known to use a combination of isolation by precipitation with ammonium sulfate and additional operations of affinity-free purification and concentration of the drug by ion-exchange and gel filtration chromatography to obtain anti-smallpox immunoglobulin.

The inventive level of the proposed technical solution lies in the fact that it does not explicitly follow from the prior art that the use of the method of precipitation with ammonium sulfate, additional stages of purification and concentration in the technology for obtaining anti-smallpox immunoglobulin using the methods of ion-exchange and gel filtration chromatography, as well as the concentration of the immunoglobulin solution in a tangential flow through cassettes with an exclusion limit of 50 kDa provides a safer immunoglobulin preparation.

Industrial applicability lies in the possibility of using typical biotechnological equipment and materials available in the microbiological and pharmaceutical industry of the Russian Federation for the implementation of the claimed method for obtaining a specific immunoglobulin.

The proposed technical solution can be used to obtain a better means of emergency prevention and treatment of infectious diseases caused by pathogenic orthopoxviruses for humans for the needs of the Armed Forces and the population of the Russian Federation.

Execution example

According to the developed technology, experimental production batches of anti-small immunoglobulin from horse blood serum were prepared.

The preparation included the following steps:

1. Antigen preparation

For the preparation of antigens, uterine cultures of the vaccine virus (strains B-51 and L-IVV) were used, which are 1 ml of sterile lyophilized virus-containing homogenate XAO EC with a stabilizer (5% peptone), with a biological activity of at least 1⋅10 ⁷ OFU/ml, stored under vacuum in ampoules at a temperature not exceeding minus 18°C.

Antigens for immunization of animal producers were prepared by infecting a three-day monolayer of Vero cells (B) in rollers at the rate of 1 to 5 OFU per cell. Infected cells were incubated for 48 hours at a rotation speed of 0.125 rpm in a thermostated room at a temperature of 35.0 to 36.0°C.

Two days later, the roller with the contents was placed in a refrigerator and frozen at minus 20°C. It is allowed to store the roller at the specified temperature for no more than 6 months.

The roller with the contents is thawed, the exfoliated monolayer of cells is washed off with rotational movements and re-frozen-thawed. The virus-containing suspension is centrifuged at 3500 rpm for 20 minutes. The supernatant is carefully dispensed into 100 ml vials.

The contents of each roller are controlled for sterility by direct inoculation in liquid thioglycol medium. The biological activity of each antigen should be at least 1⋅10 ⁷ OFU/ml. Antigens retain their activity at temperatures from minus 18 to minus 22°C for 6 months.

2. Obtaining immune blood and blood serum of horses Immunization of horses was carried out after keeping them in quarantine for 45 days in a quarantine isolated room. Horses selected for immunization should have a normal body temperature and be in good general condition. In the absence of contraindications, horses are transferred to the production facility for immunization, which is built on the principle of isolated sections and the same number of walking areas for regular exercise of animals.

Immunization of animals is carried out in the form of cycles consisting of a single injection of antigen followed by bloodletting:

- the first cycle - subcutaneous injection of 20 ml of a suspension of the vaccine virus strain B-51 with a sterile syringe into the neck;

- the second and subsequent cycles - intramuscular or subcutaneous injection of a suspension of the vaccine virus strain L-IVP in a volume of 80-100 ml into the neck.

The interval between immunization cycles should be at least 1 month.

In the process of hyperimmunization, the temperature of the animals was measured daily. With its increase above the norm, further manipulations with animals were not carried out until the disappearance of these symptoms. In the process of immunization, small amounts of blood are taken from horses (sample size 20 ml) to monitor the dynamics of the increase in virus-neutralizing antibodies.

The main volume of blood is taken in several doses on the 14-35th day after the last immunization. Blood is taken sterile from the upper third of the jugular vein using a blood sampling rig with 10% sodium citrate solution at the rate of 35 ml of solution per 1 liter of blood. At the first bloodletting, the amount of blood taken is 1 liter for every 50 kg of animal weight, at the second - 30% less than the volume of blood taken at the first bloodletting.

Immune serum was obtained by defibrinating blood plasma with a 30% solution of calcium chloride (1.8 ml of solution per 1 liter of plasma) by vigorous shaking for 15-30 minutes. The defibrinated plasma was left for 18 to 24 hours at room temperature. After the specified period, fibrin filaments and lumps were collected in the upper, and serum - in the lower layer of the liquid; a small amount of erythrocytes settled at the bottom of the bottle. Serum is taken into sterile glass bottles by decanting and stored at 4°C.

Sterile fresh sera were used for fractionation. Isolation and concentration of immunoglobulins were carried out only at the end of the control of specific blood activity after the main bloodletting.

3. Isolation of immunoglobulin G by ammonium sulfate precipitation

In order to get rid of the protein sediment that occurs during storage, the immune serum of horses is filtered through a Sartoplus P20 filter, feeding the protein solution using a peristaltic pump at a flow rate of not more than 100 ml/min. The volume of serum should not be less than 500 ml.

The original horse serum was diluted two times with a solution of 20 mm Tris, pH 8.0. A saturated (4.32 M) solution of ammonium sulfate was added dropwise to the resulting solution with constant stirring over 360 minutes until 50% saturation of the protein solution was reached. In this case, high molecular weight fractions of immunoglobulins precipitate, while albumin and other lighter proteins remain in solution. The precipitation of immunoglobulin is carried out by centrifugation at a rotor speed of 10000g for 20 minutes. The resulting precipitate was dissolved in a 20 mM Tris solution.

Analysis of series of anti-smallpox immunoglobulin, isolated by precipitation with ammonium sulfate, by high-performance liquid chromatography (HPLC) showed that the content of the immunoglobulin G fraction is not less than 80% and impurities - not more than 20%.

4. Chromatographic purification of immunoglobulin on Sephadex G-25 sorbent

The immunoglobulin solution obtained after precipitation contained a large residual amount of ammonium sulfate for removal, which was applied by the method of gel filtration chromatography.

The immunoglobulin solution from the precipitation stage was fed to the Sephadex G-25 sorbent column preliminarily equilibrated with a 20 mM Tris solution at a flow rate of 100 ml/min. After applying the solution, the separation was carried out in isocratic elution mode, feeding a solution of 20 mm Tris at the same flow rate. Fractions were collected in polypropylene centrifuge tubes from the beginning of the release of the chromatographic peak until it disappeared. For further purification, the concentration of total protein was adjusted to 1-1.5 mg/ml. For this, the required amount of a 20 mM Tris solution was added to the solution after the stage of gel filtration on the Sephadex G-25 sorbent.

5. Chromatographic purification of immunoglobulin on Sepharose FF O sorbent

Ion exchange chromatography of the immunoglobulin solution on a Q-Sepharose FF column was performed at 20°C. For this, the protein working solution was pumped at a rate of 100 ml/min to the chromatographic column using a sample feed pump. After applying the solution, the sorbent was washed with a 20 mM Tris solution at a rate of 100 ml/min. Immunoglobulin was eluted from the column with a solution of 20 mM Tris 1 Μ NaCl, under gradient elution conditions from 0 to 100%. Fractions of the target product were collected in bottles.

6. Concentration of the working solution of immunoglobulin

The concentration of the immunoglobulin solution after the stage of ion-exchange chromatography was carried out on a laboratory installation for tangential filtration.

Two Sartocon Slice 200 cassettes with an exclusion limit of 50 kDa were placed in the cassette holder, and the lid was tightened using a torque wrench with a force of 18 Nm. The flow rate was set such that the inlet and outlet pressures were 3 bar and 2 bar, respectively. 400 ml of purified water was poured into the working container, the filtrate and permeate line was opened and the process of washing the cassettes from the preservative ethyl alcohol was started, the filtrate and permeate tubes were placed in an empty container. The procedure was repeated twice. At the same time, the holder was visually assessed for tightness. Next, the filtrate tube was connected to the working container, 400 ml of a 20 mM Tris solution was poured in, and the procedure for washing the cassettes with the working buffer solution was started. After 10 minutes, the flow was stopped, the device was ready for operation.

An immunoglobulin solution was added to the instrument's working vessel, and the permeate line for collecting fractions below 50 kDa was placed in a drain vessel. They started the flow, controlling the inlet and outlet pressure indicators, they should not exceed 3 Bar and 2 Bar, respectively. When the concentrate volume reached 40 ml, 20 ml of a 20 mM Tris solution was fed through the sample line to flush the line. The concentrated product was collected by opening the drain valve of the instrument's working container. Sanitization of the device was carried out with 0.5 Μ sodium hydroxide solution for 30 min using a method similar to preservative rinsing.

7. Gel filtration of immunoglobulin on Superdex 200 prep grade sorbent

The immunoglobulin solution from the concentration stage was fed to a column with a Superdex 200 pg sorbent, previously equilibrated with a phosphate-glycine buffer solution, at a flow rate of 13 ml/min. After applying the solution, the separation was carried out in an isocratic elution mode, feeding a phosphate-glycine buffer solution at the same flow rate. Fractions of 8 ml were collected in tubes for collection of fractions from the beginning of the release of the chromatographic peak until the disappearance of the peak. Fractions containing immunoglobulin monomers were pooled, the protein concentration was 30 mg/ml. The procedure of precipitation, concentration and purification of the immunoglobulin was carried out at least three times.

8. Sterilizing filtration

The immunoglobulin solution was sterilized in a boxing device in compliance with asepsis rules. The resulting 3.0% immunoglobulin solution was sterilized in a disposable vacuum filtration system with a membrane filter with a pore diameter of 0.22 μm.

After sterilizing filtration, a 35 ml immunoglobulin sample was taken to control identity, specific activity, sterility, protein content, molecular parameters, pH value, transparency and color.

9. Packing a series of immunoglobulin into vials

The operation of packing drugs into vials was carried out in a box. Immunoglobulin was poured manually using a dispenser in compliance with asepsis rules.

At the first stage, the preparation for intradermal tests was packed in 8.45 ml, in the second stage - the main preparation of immunoglobulin - 6.35 ml in vials with a capacity of 10.0 ml.

In the process of filling vials from a series of the main drug and the drug for the intradermal test, 2 vials were selected for analysis (1 vial of each drug at the beginning and end of filling) to control the nominal volume.

The vials were sealed using a special device for manual capping with aluminum caps.

Upon completion of the bottling, both preparations were tested for sterility. To do this, the contents of 5 vials of immunoglobulin and 5 vials of immunoglobulin for intradermal tests were combined.

Table 1 presents the characteristics of the obtained batches of the drug.

Comparative characteristics of smallpox immunoglobulin preparations obtained by the proposed method in comparison with the prototype are presented in table 2.

Findings. The proposed method allows to obtain experimental production series of non-toxic, pyrogen-free, sterile means for the prevention and treatment of infectious diseases caused by orthopoxviruses pathogenic for humans, containing a larger amount of the active Ig G monomer fraction, a smaller amount of dimers, polymers and aggregates, not containing ethyl alcohol. alcohol. The drug has protective efficacy and less anaphylactogenicity, which reduces the risk of anaphylactic reactions when administered to humans.

Bibliographic data

1. Vorobyov A.A. Evaluation of the probability of using bioagents as biological weapons // Epidemiology and infectious diseases. - 2001. - No. 6. - S. 54-56.

2. Problems of bioterrorism in modern conditions /A.A. Vorobyov, B.V. Boev, V.M. Bondarenko, A.L. Gunzburg // Zhurn. microbiol. - 2002. - No. 3. - S. 3-12.

3. Onishchenko G.G. Features of the organization of prevention of controlled infections // Epidemiology and infectious diseases. - 2003. - No. 4. - S. 4-7.

4. Smallpox as a biological weapon: medical and public health response / D.A. Henderson, T.V. Inglesby, J.G. Bartlett et. al. // JAMA - 1999. - Vol.281. - P. 2127-2137.

5. Modeling potential responses to smallpox as a bioterrorist weapon / M.I. Meltzer, I.W. Leduc, J.D. Millor //Emerg. Infect. Dis. - 2001. - Vol. 7. - P. 959-969.

6. Flow cytometry and T-cell. Response monitoring after smallpox vaccination /F. Poccia, Cr. Giola, C. Montesano et. al. //Emerg. Infect. Dis. - 2003. - Vol.9, No. 11. - P. 1468-1470.

7. Bioterrorism as a national and global threat / G.G. Onishchenko, L.S. Sandakhchiev, S. V. Shchelkunov // Zhurn. microbiol. - 2000. - No. 6. - S. 83-85.

8. Infectious diseases at the end of the 20th century and sanitary and epidemiological well-being in Russia in the 21st century / V.I. Pokrovsky, G.G. Onishchenko, B.L. Cherkassky // Zhurn. microbiol. - 2002. - No. 3. - S. 16-23.

9Henderson D.A. Smallpox: clinical and epidemiological features //Emerg. Infect. Dis. - 1999. - Vol. 5, no. 4. - P. 537-539.

10. Tara'o Toole. Smallpox: an attack scenario // Emerg. Infect. Dis. - 1999. - Vol. 5, no. 4. - P. 540-546.

11. Non-parenteral methods of immunization against smallpox /A.A. Vorobyov, V. N. Podkuiko, V.V. Mikhailov, A.A. Makhlai //Journal. microbiol. - 1996. - No. 5. - C. 117-121.

12. Meltzer M.I. Risks and benefits of preexposure and postexposure smallpox vaccination//Emerg. Infect. Dis. - 2003. - Vol. 9, No. 11. - 1363-1370.

13. Marennikova C.S., Manevich G.R., Svet-Moldavskaya I.A. On the effectiveness of γ-globulin with a high content of anti-small antibodies in the treatment of post-vaccination encephalitis // Vopr. Virusol. - 1968. - No. 1. - S. 9-13.

14. Bondarev V.N., Voitinsky E.Ya. Prevention and treatment of post-vaccination complications in children. - L .: Medicine, 1972.

15. European Pharmacopoeia 7.0. Volume 1, 2010: 1273 p.

Claims (1)

A method for producing anti-smallpox immunoglobulin from horse blood serum, which includes the steps of preparing an antigen from a vaccine virus of strains B-51 with an activity of at least 1⋅10 ⁷ IU/ml and L-IVP with an activity of at least 1⋅10 ⁷ IU/ml, obtaining immune serum using a 30% solution of calcium chloride, isolation of immunoglobulin, purification and concentration, sterilizing filtration, packaging and packaging, characterized in that the isolation of antipox immunoglobulin from horse blood serum is carried out by precipitation by adding ammonium sulfate, purification of immunoglobulin is carried out in three stages, for the first purification stage, gel filtration chromatography is used on a column with a Sephadex G-25 sorbent, previously equilibrated with a 20 mM Tris solution, at a flow rate of 100 ml/min, in isocratic elution mode, feeding a 20 mM Tris solution at the same flow rate, for the second stage of immunoglobulin purification ion-exchange chromatography is used on a column with a sorbent Q Sepharose FF at 20°C and a linear sample feed rate of 150 cm/h followed by gradient elution of the Ig G fraction from 0 to 100% 20 mM Tris 1 Μ NaCl pH 8.0 at the same rate, for the third stage of purification is used gel filtration chromatography on a column with a Superdex 200 pg sorbent equilibrated with a phosphate-glycine buffer solution of 0.02 Μ sodium phosphate with 2% glycine, pH 6.9 at a linear flow rate of 50 cm / h in isocratic elution mode at the same flow rate, and for concentration of antipox immunoglobulin solution, an automatic tangential filtration system with cassettes with an exclusion limit of 50 kDa is used at a flow rate of 500 ml/min, an inlet pressure of no more than 3 bar, and an outlet pressure of no more than 2 bar.