RU2799637C1 - Method of producing biologically active peptide-amino acid preparation based on human blood plasma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method of producing a biologically active peptide-amino acid preparation based on human blood plasma. The method of obtaining a biologically active peptide-amino acid preparation based on human blood plasma is characterized by the following: the supernatant obtained in the process of native proteins isolation from the blood plasma of donors is filtered through membrane filters with a cut-off threshold for molecular weight in the range of 10÷30 kDa and concentrated to suspension, then the resulting suspension is subjected to vacuum drying in an inert gas atmosphere at a temperature of 55°C to obtain a dry residue, which is then dispersed to visual uniformity and redissolved in water for injection using an ultrasonic disperser, while controlling the pH in the range of 5.0–6.0, the resulting solution of the peptide-amino acid preparation is subjected to triple ultrafiltration using membrane filters first with a porosity of 5 and 0.45 µm, after which it is sterilized using filters with a porosity of 0.22 µm.

EFFECT: invention makes it possible to obtain a biologically active peptide-amino acid preparation devoid of xenogeneity and having increased neuroprotective activity, to achieve a more complete processing of human blood plasma and to increase the yield of the final product compared to the known methods.

4 cl, 4 dwg, 2 tbl

Description

The invention relates to the field of medicine, namely to biological preparations consisting of molecules present in normal physiological conditions in the human body.

Blood plasma, which is one of the numerous components of donor blood, can be used as an independent therapeutic drug (fresh frozen plasma), as well as as a raw material for obtaining numerous pharmaceutical products of its fractionation. In the process of processing, a mixture of plasma from several hundred healthy donors is first checked for the absence of markers of bloodborne infections by enzyme immunoassay and polymerase chain reaction [1], then from one to five stages of low-temperature fractionation using alcohol precipitators in accordance with the Kohn method, resulting in native blood plasma proteins, which include fibrinogen, immunoglobulins, which make up the bulk of proteins, alpha and beta globulins, and albumin [2]. At the stage of albumin isolation, a by-product is also obtained - supernatant, which is a water-alcohol solution containing organic and mineral compounds that can be used to create a wide range of drugs, biological additives and nutrient media components. The resulting valuable supernatant has not previously been subjected to deeper processing to obtain additional plasma fractionation products, in addition to those listed above. The biologically active peptide-amino acid preparation described in the present invention is made from this by-product, the supernatant.

A known method for producing acidic alpha-1-glycoprotein, in which the ballast sediment of human blood serum is homogenized in water and dissolved in sodium hydroxide. The resulting solution is cooled to 3-15°C and ethanol is added to it. After centrifuging the mixture, the supernatant is simultaneously concentrated and subjected to diafiltration on hollow fibers, followed by chromatographic isolation of the target product [3].

The disadvantage of this method is the low yield of the final product - 0.3 grams per liter of initial plasma, which makes it impossible in conditions of a constant shortage of donor blood to organize mass production of the resulting drug.

A known method of isolating natural antimicrobial peptides (AMP) from leukocyte-erythocyte-platelet mass of blood, previously exposed to hemolysis with trypsin [4]. The selection of AMP is carried out by liquid chromatography on a separating column with a triple use of Sephadex G-25. The method allows to optimize the technology for isolating the most complete fraction of natural low molecular weight peptides containing AMP, to use Sephadex G-25 several times after washing, regeneration and drying, to increase the biological value of AMP.

The disadvantage of this method is the need to use a significant amount of hydrogen peroxide, as well as the use of erythrocytes, one of the most important blood components, as a feedstock.

A method is known for obtaining a biologically active preparation from the blood of cow embryos, which is a mixture of an unidentified composition that contains highly active low molecular weight peptides, amino acids, nucleic acid derivatives and other components. The method consists in that the blood of bovine embryos pre-frozen at a temperature of -18±2°C is thawed, separated and subjected to ultrafiltration using two sequentially connected membranes with retention limits of 100 and 10 kDa, respectively. EFFECT: invention makes it possible to improve the specific properties of a drug isolated from the blood of cattle, and a pharmaceutical composition obtained on its basis, used in the treatment of cerebral circulation disorders, metabolism, peripheral circulation disorders, thermal and radiation damage to tissues, as well as in the treatment of wounds, ulcers and inflammatory processes without the formation of keloid scars [5].

The disadvantages of this method include the xenogeneity of the resulting biologically active drug in relation to the human body, as well as only partial use of blood components of pharmaceutical interest isolated at the stage of separation of raw materials.

Closest to the claimed invention in terms of essential features, is a method for obtaining deproteinized blood homoderivation of calves under the trade name "Actovegin" [6]. This drug is a deproteinized, endotoxin- and antigen-free calf blood ultrafiltrate containing more than 200 bioactive components. The process of its production consists in the fact that the blood of calves is subjected to preliminary preparation, the technological stages of which are not given by the manufacturers and are classified as "know-how", and deproteinization by two-stage ultrafiltration using sieves with a cut-off by molecular weight of 5 kDa. Next, the raw material is subjected to vacuum distillation, filtering the remaining precipitate through a filter with a porosity of 0.45 μm and titration to pH 6.4, followed by sterilizing filtration through filters with a porosity of 0.45 μm and 0.2 μm.

The disadvantage of this method is the xenogeneity of the resulting drug, which increases the risk of negative consequences in patients receiving therapy.

The problem to be solved by the claimed invention is to develop a biologically active peptide-amino acid drug with neuroprotective properties and used in the treatment of patients with acute and chronic cerebrovascular pathology, post-stroke cognitive impairment and peripheral ischemia.

The technical result of the present invention is to obtain a biologically active peptide-amino acid preparation, devoid of xenogeneity and having increased neuroprotective activity, as well as achieving a more complete processing of human blood plasma and increasing the yield of the final product compared to known methods.

The specified technical result in the method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma is achieved due to the fact that the supernatant obtained in the process of isolating native proteins from human blood plasma is filtered through membrane filters with a cut-off threshold for molecular weight within 10 ÷ 30 kDa and concentrated to a suspension state by vacuum distillation on a rotary evaporator or using chromatographic methods, after which the resulting suspension is subjected to vacuum drying in an inert gas atmosphere at a temperature of 55 ° C or the supernatant is subjected to convection, freeze-drying or spray drying immediately until a dry residue is obtained, which is then dispersed to visual uniformity and redissolved in water for injection using an ultrasonic disperser, while controlling the pH in the range from 5.0 to 6.0. The resulting solution of the peptide-amino acid preparation is subjected to triple ultrafiltration using membrane filters, first with a porosity of 5 and 0.45 μm, after which it is sterilized using filters with a porosity of 0.22 μm.

The novelty of the method lies in the fact that for the manufacture of a biologically active peptide-amino acid preparation, a supernatant is used, obtained at the fifth stage of isolation by the method of Conative proteins from human blood plasma.

The production of a biologically active peptide-amino acid preparation is based on the use of a supernatant, a by-product of the isolation of native proteins from human blood plasma [2], which is an aqueous-alcoholic solution rich in amino acids, peptides, proteins, nutrients, mineral compounds, and other organic compounds. and inorganic substances. The plasma used for fractionation is a mixture of plasma from several hundred healthy donors, which has passed the stage of control for the absence of markers of bloodborne infections and is devoid of group and Rh affiliation.

In practice, the method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma is carried out as follows.

The supernatant obtained after isolation of native proteins from the blood plasma of healthy donors is filtered through membrane filters with a molecular weight cut-off in the range of 10–30 kDa and concentrated using vacuum distillation on a rotary evaporator or using various chromatographic methods (ion-exchange chromatography, hydrophobic chromatography on the Sepharose sorbent, gel filtration chromatography on the Sephadex G-10 sorbent) to a suspension state. Next, the suspension is subjected to vacuum drying in an inert gas atmosphere at a temperature not exceeding 55°C to avoid oxidation of amino acids and denaturation of peptides. There are also options for concentrating the supernatant to dryness, which consists in the fact that the solution is subjected to convection drying in an oven at a temperature of 55°C or freeze-drying at a temperature of -100°C, or spray drying in a nitrogen stream. After that, the dry residue is dispersed to visual uniformity and redissolved in water for injection using an ultrasonic disperser to prevent the particles of the dry residue from conglomerating. In the process of re-dissolution, the level of acidity of the medium is controlled to maintain the stability of the chemical composition: pH should be in the range from 5.0 to 6.0. The resulting solution of the peptide-amino acid preparation is subjected to triple ultrafiltration: in order to achieve visual transparency and uniformity, the solution is first passed through membrane filters with a porosity of 5 μm and 0.45 μm, then the solution is sterilized using filters with a porosity of 0.22 μm. As a result, a biologically active peptide-amino acid preparation is obtained from a by-product of fractionation of human blood plasma. The yield of dry residue from 1 liter of the initial supernatant solution is presented in Table 1 and averages 6.6 g.

Table 1

Sample No. Date of receipt of the supernatant at the blood transfusion station Mass of dry residue, g 1 07/02/2021 7.7 2 07/19/2021 6.0 3 07/19/2021 6.2

The choice of the cut-off threshold, which ensures the production of an exclusively peptide-amino acid fraction with a molecular weight of up to 30 kDa, is confirmed by the literature data and is due to the need for deproteinization of the initial solution to obtain the drug in order to exclude the possibility of developing an immune response of the body.

Determination of the separation boundary of the components by molecular weight and control of the purity of the peptide-amino acid preparation was carried out by capillary gel electrophoresis using the Kapel-105M system by separating SDS-protein complexes according to the method PU 49-2016 of the 2018 edition of the company Lumex-Marketing LLC ". The results of the electrophoretic analysis of peptide-amino acid preparations made from the supernatant that had previously passed the stage of ultrafiltration through membranes with a cut-off threshold for molecular weight in the range of 10–30 kDa are shown in Fig. 1A and 1B, respectively. Figures 1A, 1B and 1C show the complete absence of proteins with a molecular weight above 10 kDa both in the reference drug Actovegin and in the peptide-amino acid drug obtained by the claimed method using membrane filters with a selected cut-off threshold. Based on the data obtained in the course of electrophoretic analysis, supported by scientific and technical literature, we can conclude that the selected cut-off threshold of at least 10 and not more than 30 kDa is sufficient.

The identity and purity of the peptide-amino acid preparation was determined by high performance liquid chromatography (HPLC), biuret reaction, capillary electrophoresis, spectrophotometry, and inductively coupled plasma spectral analysis.

Table 2 presents a comparative characteristic of the average quality indicators of three series of a peptide-amino acid preparation based on human blood plasma, obtained by the claimed method, and a reference preparation.

table 2

Index Method Indicator values Peptide-amino acid preparation The reference drug "Actovegin" Authenticity 1. Peptides Qualitative biuret reaction,
blue-violet coloration A blue-violet color was observed in the solution. A blue-violet color was observed in the solution. 2. Uric acid riboside (retention time), min HPLC 7 5 3. Amino acids HPLC Contains asparagine,
glycine,
arginine,
serine,
alanine,
tyrosine,
lysine Contains aspartic acid, glutamine, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, valine, methionone, phenylalanine, leucine, lysine, proline Transparency Visual comparison with reference solution and water Transparency like water Transparency like water pH Potentiometric in accordance with the State Pharmacopoeia of the Russian Federation XIV 5.01±1.2 6.5 Density, g / cm ³ Pycnometric in accordance with the State Pharmacopoeia of the Russian Federation XIV 1.009±0.0005 1.106 Purity 1. Absorption (optical density) Spectrophotometric at 420 nm 0.990±0.001 0.987±0.001 2. Protein Capillary gel electrophoresis Negative reaction Negative reaction 3. Heavy metals, mcg/ml Spectral using an inductively coupled plasma spectrometer 0 0.07 4. Sodium, mg/ml 4.5 9.2 5. Potassium, mg/ml 0.2 0.9

On FIG. Figure 2 shows a chromatogram of a peptide-amino acid preparation obtained by HPLC under isocratic elution conditions with a mobile phase flow rate (phosphate buffer with methanol, 82:18) of 0.5 ml/min. The results of the analysis show the presence of uric acid riboside in the preparation.

The results of the chromatographic qualitative analysis of the peptide-amino acid preparation for amino acid content are shown in FIG. 3. The chromatogram (Fig. 3) was obtained by HPLC under gradient elution conditions using an acetate buffer and an organic solvent with the addition of a modifier. The studied samples of the drug were preliminarily sample prepared using phenylisothiocyanate. Thus, the composition of the peptide-amino acid preparation includes about seven essential amino acids.

To assess the neuroprotective properties and effectiveness of the peptide-amino acid preparation obtained by the proposed method, experimental studies were carried out to study the survival of laboratory animals on a model of cerebral ischemia.

In one series, the experiment was carried out on 60 male white laboratory rats weighing 250-300 g, aged 3-4 months. The number of animals (n = 12 in each group) was sufficient to assess the nature and frequency of the recorded effects, and to conduct proper statistical processing. In addition, the number of animals was minimally sufficient in terms of ethical principles. The experiments were performed in five series of experiments.

On the first day of the study, pathology modeling was performed using the method of irreversible simultaneous occlusion of the common carotid arteries. Then, for seven days, the test drug was administered to animals from the experimental group intramuscularly at doses of 200 or 400 mg/kg/day; Animals from the comparison group were administered the reference drug "Actovegin" at a dose of 200 mg/kg/day. Rats from the control group were similarly injected with equivalent volumes of saline.

Methods widely used in preclinical studies of the pharmacodynamic properties of cerebroprotective drugs were used in the work. In the course of the study, such indicators as the psychoneurological state of the animals, disturbances in sensorimotor functions, the level of local cerebral blood flow, and cognitive functions of rats were controlled. Information about the survival of animals after irreversible ligation of the common carotid arteries, presented in the form of a curve constructed by the Kaplan-Meier method, is presented in Fig. 4.

From the experimental results shown in Fig. It follows from Table 4 that the highest percentage of survival, which is 100%, corresponds to the group of animals that were injected with a peptide-amino acid preparation at doses of 200 mg/kg/day, the smallest (66.67%) - to the group that was injected with placebo. The percentage of survival in the group of animals treated with Actovegin is 91.67%. Based on the data obtained, it can be concluded that there is a positive effect in rats that have undergone irreversible ligation of the common carotid arteries from the administration of a peptide-amino acid preparation and Actovegin, in contrast to placebo.

The obtained results of studies on the possibility of using a biologically active peptide-amino acid drug based on human blood plasma, manufactured by the method proposed by the authors, in case of cerebral ischemia in rats caused by irreversible ligation of the common carotid arteries, suggest that this drug has a significant neuroprotective effect and does not inferior in effectiveness to the reference drug Actovegin in terms of the effect on the survival of animals and the timing of the restoration of their motor and neurological functions.

The proposed method ensures the production of a biologically active peptide-amino acid preparation with a stable composition due to the removal of the blood plasma protein fraction with a molecular weight of more than 30 kDa during the filtration process, which eliminates the risk of side effects, primarily autoimmunization.

The advantage of the present invention is the exclusion of the xenogeneity of the resulting final biologically active peptide-amino acid preparation based on human blood plasma, which also has increased neuroprotective activity, as well as providing more efficient processing of the feedstock and a high yield of the peptide-amino acid product, averaging 6.6 g with 1 l of supernatant, compared with known methods.

Information sources

1. National standard of the Russian Federation GOST R 52249-2009 "Rules for the production and quality control of medicines" (approved and put into effect by order of the Federal Agency for Technical Regulation and Metrology dated May 20, 2009 N 159-st).

2. Tkhai S. V., Rusanov V. M., Uiba V. V. Production of blood products: features of technology, organization and technical support / S. V. Tkhai, V. M. Rusanov, V. V. Uiba. - Moscow: Medpraktika-M, 2014. - 151 p.

3. Copyright certificate No. 1662039, Method for obtaining acid alpha-1-glycoprotein, IPC A61K39 / 00, / Lyutov A. G., Enikeeva S. A., Aleshkin V. A., Novikova L. I., Khakimova F. Z .; patent holder Ufa Research Institute of Vaccines and Serums named after I.I. Mechnikov, publ. 02/10/1996.

4. Patent RU No. 2729016, Method for isolating natural antimicrobial peptides from leukocyte-erythocyte-platelet blood mass, IPC C07K1/12, C12P21/06, G01N30/02. / Bazikov I. A., Maltsev A. N., Baturin V. A., Ramesh K. Goyal, A. Najirul Amin, Efremenko A. A., patent holders Bazikov I. A., Federal State Budgetary Educational Institution of Higher Education " Stavropol State Medical University" of the Ministry of Health of the Russian Federation (StGMU of the Ministry of Health of Russia, publ.

5. Patent EA No. 000633B1, Method for obtaining a biologically active drug from the blood of cow embryos and a pharmaceutical composition based on it with radioprotective, antihypoxic, immunomodulating, wound healing and antiherpetic activity, / Plenina L. V., Khlyustov S. V., Fedorova N. I., Babuk L. V., Fedulov A. S., Marchenko L. N., patentee Enterprise of Diagnostic and Medicinal Products “Dialek”, publ. 12/29/1999.

6. Method for obtaining deproteinized calf blood homoderivate under the trade name Actovegin, Machicao, F. Pleiotropic neuroprotective and metabolic effects of Actovegin's mode of action / F. Machicao, D. F. Muresanu, H. Hundsberger [et al.] // Journal of the Neurological Sciences. - 2012. - V. 322. - P. 222-227.

Claims (4)

1. A method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma, which consists in the fact that the supernatant obtained in the process of isolating native proteins from the blood plasma of donors is filtered through membrane filters with a cut-off threshold for molecular weight in the range of 10÷30 kDa and concentrated to a suspension state, after which the resulting suspension is subjected to vacuum drying in an inert gas atmosphere at a temperature of 55°C until a dry residue is obtained, which is then dispersed to visual uniformity and redissolved in water for injection using an ultrasonic disperser, while controlling the pH in range from 5.0 to 6.0, the resulting solution of the peptide-amino acid preparation is subjected to triple ultrafiltration using membrane filters, first with a porosity of 5 and 0.45 μm, after which it is sterilized using filters with a porosity of 0.22 μm. 2. A method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma according to claim 1, which consists in the fact that the supernatant is concentrated to a suspension state by vacuum distillation on a rotary evaporator. 3. A method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma according to claim 1, which consists in the fact that the supernatant is concentrated to a suspension state using chromatographic methods. 4. A method for obtaining a biologically active peptide-amino acid preparation based on human blood plasma according to claim 1, which consists in the fact that the suspension is subjected to convection, freeze-drying or spray drying.

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