RU2174408C2 - Preparation for prophylaxis and treatment of diphtheria and method of its preparing - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: invention proposes the preparation that is lyophilized mono-receptor fraction of F(ab)₂-fragments of equine IgG isolated from fermented equine hyperimmune serum using immunosorbent containing purified concentrated diphtheria anatoxin as ligand. Immunoaffine purification ensures to obtain the preparation with activity of antidiphtheria antibodies 2500 IU/ml, not less, and protein content 3-4%. For preparing the antidiphtheria preparation equine hyperimmune serum is used and for this purpose serum is fermented with pepsin followed by fractionation of proteins with salts and thermal denaturation of inert proteins. Isolation of the end product from fermented hyperimmune serum is performed by specific sorption method. EFFECT: preparing highly purified antidiphtheria preparation with decreased reactivity properties. 3 cl, 3 tbl, 2 dwg

Description

 The invention relates to medicine, in particular immunology, and can be used for immunoprophylaxis and immunotherapy of diphtheria.

 A traditional method for treating diphtheria is the intramuscular administration of antidiphtheria serum (PDS) obtained from the blood of horses hyperimmunized with diphtheria toxoid containing specific immunoglobulins that neutralize toxins of diphtheria bacteria. 1 ml of serum contains at least 1,500 international antitoxic units of activity (IU). Being an effective method of treating diphtheria, PDS has several disadvantages: the introduction of a significant amount of foreign protein makes it possible to develop anaphylactic reactions and serum sickness (Favorova L.A. et al. Diphtheria. - M .: Medicine, 1988. - P. 187-192) .

 Known drug immunoglobulin anti-diphtheria man, obtained from the plasma of donors vaccinated with diphtheria toxoid. In 1 ml of the drug contains from 5 to 50 IU of anti-diphtheria antibodies. The prophylactic dose of the drug is 300 IU with intramuscular injection, the therapeutic dose is from 1200 to 20,000 IU (Aleshin V.A., Zatsepin Yu.K., Bashlai A.G. et al. Obtaining anti-diphtheria immunoglobulin. // The role of immunobiological drugs in modern medicine. - Ufa, 1995. - Part 1. - S. 100-102).

 However, the release of anti-diphtheria homologous immunoglobulin is limited by the lack of raw materials and the imperfection of immunization schemes. The growth of “traditional” blood-borne infections (syphilis, hepatitis, HIV infection, new T- and B-lymphotropic viruses, the diagnosis of which is still difficult) is increasingly limiting the possibility of donor blood collection (L. Reicher, I. Reicher I. Affinity-purified xenogenic antibodies. Prospects for development, problems. // The role of immunobiological preparations in modern medicine. - Ufa, 1995. - Part 2. - P. 86-91).

 In addition, the preparation of one batch of an anti-diphtheria human immunoglobulin preparation requires blood from at least 1000 donors, therefore it is a mixture of molecules of different allotypes, the introduction of which can lead to the development of allergic reactions in patients (Pytsky V.I., Adrianova N.V. , Artomasova A.V. Allergic diseases. - M.: Medicine. - 1991. - S. 176).

 Closest to the proposed invention is a method for producing a heterogeneous serum preparation, comprising treating serum with rivanol, pepsin, aluminum hydroxide, followed by ultrafiltration and sterilizing filtration (patent N 2062617, class A 61 K 35/16, 39/00, 1996) .

However, this method does not allow to obtain preparations with a high degree of purification: the final product is a complex of F (ab) ₂ fragments of immunoglobulins of various specificities, moreover, antibodies (antitoxins) against the antigen by which the producer was immunized do not exceed 25% of the total protein content . As a result, antitoxic sera remain overloaded with ballast proteins and in some cases can cause anaphylactic reactions of varying severity on the part of the recipient. In addition, the drugs are contaminated with group blood substances (HVA), which can also cause complications with the introduction of sera.

 The technical result of the invention is to obtain a highly purified anti-diphtheria drug with reduced reactogenicity from heterogeneous raw materials. The drug for the prevention and treatment of diphtheria is a lyophilized form.

 The problem is solved by obtaining a drug for the prevention and treatment of diphtheria from fermented hyperimmune horse serum using immunoaffinity purification.

The invention consists in the following. The drug for the prevention and treatment of diphtheria is a lyophilized monoreceptor fraction of F (ab) ₂ horse IgG fragments isolated from fermented hyperimmune horse serum using an immunosorbent containing purified concentrated diphtheria toxoid as a ligand. Immunoaffinity purification allows to obtain a highly purified preparation for the prevention and treatment of diphtheria with anti-diphtheria antibody activity of at least 2500 IU / ml and a protein content of 3-4%.

 The resulting preparation is monofractional, while the prototype preparation has pronounced impurities of an inactive protein (Fig. 1). The drug for the prevention and treatment of diphtheria is monoreceptor: in the reaction of double immunodiffusion with diphtheria toxoid forms one line of precipitation, while serum (prototype) - 2-3 lines (Fig. 2).

 The characteristics of the preparations obtained by known methods and proposed are presented in table. 1-3.

A high degree of purification and the absence of HVA reduce the reactogenicity of the drug for the prevention and treatment of diphtheria obtained from heterogeneous raw materials (Table 1, 2), while it has high protective properties: the minimum dose of antitoxin in blood serum is 0.03-0.125 IU / ml , corresponding to the protective titer for diphtheria, protects 100% of the animals from the administration of 1.8-5.6 LD ₅₀ diphtheria toxin (table. 3). Freeze drying allows you to get highly active dry dosage formulations suitable for long-term storage.

 The implementation of the method.

 To prepare the drug for the prevention and treatment of diphtheria, hyperimmune horse serum is used, for which it is fermented with pepsin, salt fractionation of proteins and thermal denaturation of the ballast protein are carried out. Isolation of the target product from fermented hyperimmune serum is carried out using a specific immunosorbent containing purified concentrated diphtheria toxoid as a ligand. The drug is concentrated by ultrafiltration on membranes with a threshold for retention of substances with a molecular weight of 100 kD. Sterilizing filtration is carried out on membranes with a pore diameter of 0.22 μm.

Example
1. Fermentolysis of hyperimmune serum.

 30 l of hyperimmune horse serum is loaded into the reactor and diluted with 60 l of pyrogen-free distilled water to a content of 3% protein. With stirring, 243 g of pepsin dissolved in a small amount of acidified distilled water are added. Proteolysis is carried out at a pH of 3.2 at room temperature for 1 h and 1 h at a pH of 4.2. (pH is adjusted to the desired value with 2.5 mol / L hydrochloric acid solution or 2.5 mol / L sodium hydroxide solution).

At the end of the 2nd hour of proteolysis, 14.5% ammonium sulfate (13 kg) was added to the reactor through the loading hatch, the pH was adjusted to a value of 4.3-4.33. The mixture is heated to 56 ^o C and maintained at this temperature for 45 minutes. After this, the enzyme is freed from the ballast protein by filtration through calico cassette filters. The ballast protein precipitate is discarded, and a clear filtrate containing proteins with antitoxic activity is collected and adjusted to pH 7.0-7.2.

 2. Preparation of monospecific immunosorbent.

To obtain the immunosorbent, a standard preparation of cyanogen bromide sepharose 4B (Pharmacia, Sweden) is used, and a purified concentrated diphtheria toxoid with an activity of 450 Lf / ml is used as a specific ligand. 200 g of dry Sepharose is poured with two liters of a 0.001 mol / L hydrochloric acid solution for 30 minutes and washed with preservatives in the same solution. 1.2 l of diphtheria toxoid is combined with a precipitate of swollen Sepharose (900 ml), mixed well and kept at a temperature of 4 ^o C for 24 hours. Then determine the volume of the supernatant (V ₁ ) and the titer of toxoid (T ₁ ) in the flocculation reaction. If the titer does not exceed 10 Lf / ml, the decantate is discarded; otherwise, sorption is continued. The formed Sepharose-toxoid complex is washed from an unbound protein with a sterile pyrogen-free carbonate-bicarbonate buffer pH 9.0 under the control of spectrophotometry. After establishing a neutral pH, the last portion of the washings is checked for pyrogen according to the generally accepted method.

 In the absence of pyrogens in the washings, the free active groups of cyanogen bromide sepharose are blocked with two liters of a 1 mol / L ethanolamine solution pH 9.0 for two hours in a darkened place with constant stirring.

где A - титр иммуносорбента;
V, T - объем и титр анатоксина;
V₁, T₁ - объем и титр надосадочной жидкости;
V₂, T₂ - объем и титр промывных вод;
C - объем иммуносорбента.The immunosorbent is washed three times with alternating sterile pyrogen-free buffer solutions: 2 l of acetate pH 4.0; 2 l of boric borate pH 8.0. Wash water (V ₁ ) is monitored for the presence of diphtheria toxoid in the flocculation reaction (T ₂ ). The immunosorbent titer is calculated by the formula

where A is the titer of the immunosorbent;
V, T - volume and titer of toxoid;
V ₁ , T ₁ - volume and titer of the supernatant;
V ₂ , T ₂ - volume and titer of wash water;
C is the volume of immunosorbent.

Таким образом, получают 900 мл суспензии иммуносорбента с титром 588 Lf/мл.

Thus, 900 ml of an immunosorbent suspension with a titer of 588 Lf / ml are obtained.

3. Isolation of the monoreceptor fraction of F (ab) ₂ horse IgG fragments, drug concentration.

где V_ф, T_ф - объем и титр ферментата,
V_и.с., T_и.с. - объем и титр иммуносорбента.To isolate the F (ab) ₂ fragments of horse IgG, a columnless “Batch” method is used. In this case, all containers and solutions used must be sterile and pyrogen-free. The sorbent and fermented antitoxic serum with an activity of 110 IU / ml are combined in amounts equivalent to titer

where V _f , T _f - volume and titer of the enzyme,
V _I.S. , T _i.s. - volume and titer of immunosorbent.

Т.е. одной порции иммуносорбента достаточно для истощения 4,8 л ферментированной противодифтерийной антитоксической сыворотки.

Those. one serving of immunosorbent is sufficient to deplete 4.8 liters of fermented antidiphtheria antitoxic serum.

 Sorption of specific antibodies is carried out at room temperature for 1 h, pH 7.0. The sorbent is washed from unbound proteins with a sterile pyrogen-free buffered saline solution (PBS) pH 7.0 to zero according to the readings of a spectrophotometer. Sterile pyrogen-free PBS, acidified to a pH of 2.2 - 2.4 with 1 mol / L hydrochloric acid, is used as an eluent.

 The exposure of the immunosorbent in the eluent is 10-15 minutes. A specific protein is subsequently eluted with portions of PBS pH 2.4: 1.5-1.0-0.5 L. Select eluates with a protein content of at least 0.01%, combine and adjust the pH to 6.8-7.2. The combined eluates are tested for pyrogen by a conventional technique.

The immunosorbent is washed from the PBS protein (pH 7.0) and stored in a sterile pyrogen-free borate-borate buffer solution (pH 8.0) at a temperature of 4 ^° C. The immunosorbent is used repeatedly (up to 20 times).

 Get 90 l of eluates with a specific protein content of 0.095-0.1%. The concentration of eluates is carried out by ultrafiltration on an AP-2 installation with hollow fibers of the VPU-100 brand under a pressure of 0.06 ± 0.02 MPa. The concentration process is continued until the protein content in the concentrate is within 3-4%.

Get 3 l of concentrated affinity purified F (ab) ₂ fragments of horse IgG with an activity of 2500 IU / ml and a protein of 3%. The purified concentrated antitoxin is clarified by sequential filtration through membranes with a pore diameter of 0.8-0.65-0.45 μm. Sterilizing filtration is carried out on membranes with a pore diameter of 0.22 μm. A sterile preparation for the prevention and treatment of diphtheria is poured into ampoules of 1.0 ml.

 4. Lyophilization.

Ampoules with the drug for the prevention and treatment of diphtheria are placed in the receiving cassette and put on freeze in the low-temperature counter of the TG-50 apparatus, which provides a freezing temperature of at least minus 38 ^o C. When forced ventilation, the drug is frozen for at least 18 hours, without forced ventilation less than 20 hours. The product is loaded into the sublimator when the condenser reaches a temperature of no higher than minus 50 ^o C, shelves no higher than minus 15 ^o C. After which the sublimator is evacuated. The defrosting point of the drug is minus 33-36 ^o C. When the drug reaches a temperature of no higher than minus 38 ^o C and the vacuum stabilizes no more than 9 Pa, the shelves begin to heat up. One hour after loading, heating at 0 ^o C and then at 5 ^o C per hour. The final heating temperature of the shelves is 34-40 ^o C. After keeping the drug at positive temperature for 20 hours, the drying is considered complete.

 Thus, the proposed method allows to obtain a drug for the prevention and treatment of diphtheria with reduced reactogenicity, with high protective properties, in lyophilized form.

Claims (2)

1. The drug for the prevention and treatment of diphtheria, containing F (ab) ₂ IgG fragments of a horse, characterized in that it is a monoreceptor fraction of F (ab) ₂ IgG fragments of a horse with an anti-dephteric antibody activity of at least 2500 IU / ml and contains as an excipient, a 0.9% solution of sodium chloride with a pH of 6.8 - 7.2 in the following ratio of ingredients:
Monoreceptor fraction of F (ab) ₂ fragments of horse IgG with an activity of anti-diphtheria antibodies of at least 2500 IU / ml - 3 to 4 wt.%
0.9% sodium chloride solution with a pH of 6.8 - 7.2 - Up to 1 ml
2. The drug for the prevention and treatment of diphtheria according to claim 1, characterized in that it is a lyophilized form.  3. A method of obtaining a preparation for the prevention and treatment of diphtheria, including fermentolysis of hyperimmune horse serum with pepsin, characterized in that after fermentation, salt fractionation and thermal denaturation of whey proteins are carried out, the target product is isolated using a specific immunosorbent containing purified concentrated diphtheria toxoid as a ligand, followed by concentration by ultrafiltration on membranes with a threshold for the retention of substances by molecular weight 100 D, sterilizing by filtration on membranes with a pore diameter of 0.22 microns.

Images