[Recombinant granulocyte-colony stimulating factor (filgrastim): optimization of conjugation with polyethylene glycol].

In order to create an active pharmaceutical substance of the drug with prolonged action the modification of recombinant human granulocyte colony-stimulating factor GCSF (filgrastim) with polyethylene glycol (PEG, M 21.5 kDa) was conducted. A method for preparation of PEG-filgrastim designed for the development and scaling-up of the technological process of production was described. Modification of proteins with PEG was performed by selective covalent attachment of the molecule alpha-methyl-PEG-propionaldehyde to the alpha-amino group of the N-terminal methionine amino acid residue of the recombinant GCSF. The conditions of the reaction, which provide the desired product yield at least 85% of the total protein, also high protein concentration in the reaction mixture (more than 9 mg/mL) and reduce consumption of PEG in terms of terminal alpha-amino group of the protein was chosen. The data of RP HPLC and MALDI-mass spectrometry showed that the produced drug modified by the N-terminal residue and contains no more than 10% of products with a high degree of modification.

[DNA assay for genetically engineered pharmaceutical substances using the real-time PCR technique].

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.

[Optimization of the industrial production of the recombinant precursor of human insulin].

Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.

[Nanocomplexes of recombinant proteins and polysialic acid: preparation, characteristics, and biological activity].

A preparation of nanocomplexes containing recombinant proteins (interferons alpha2b and beta1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10-20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.

[Modification of recombinant proteins by covalent polysialation illustrated with the example of human insulin].

Methods of selective and nonselective covalent immobilization of genetically engineered proteins on molecules of natural polysialic acid are described by the example of human insulin. Such modification increases insulin lifetime in vivo.

[Validation of a method for monitoring of genetically engineered human insulin manufacture].

A method for monitoring the manufacture of genetically engineered human insulin by HPLC was developed. The method was validated by the estimation of its linearity, correctness, accuracy, specificity, and stability; the limits of detection and quantitative assessment were also determined. It was proven that HPLC analysis enables reliable and reproducible results to be obtained and can be used for monitoring insulin manufacture.

[Prospects for designing Russian gene engineering agents for medicine. Rastan is the first Russian recombinant human growth hormone].

The development of modern pharmacology cannot be imagined without the use of genetic engineering methods (recombinant DNA technology). The success of medicine is increasingly based on the active use of protein preparations obtained using the technology of transferring hereditary information (genes) from one organism to another. The emergence of the ability to express foreign genes in the cells of various organisms (both eukaryotes and prokaryotes) has become one of the revolutionary events in the science of the last two decades of the 20th century and laid the foundations of modern biotechnology.

[A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations].

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.

[The histamine-liberating action of polymyxin B and its analogs]. / Gistaminvysvobozhdaiushchee deistvie polimiksina B i ego analogov.

Histamine-releasing effect of polymyxin B1 and its deacylated analogues has been studied on purified rat mast cells. The structure-activity analysis showed that cyclic peptide fragment and acyl residue of molecule of polymyxin plays an important role in histamine-releasing activity. Histamine release, induced by polymyxin B1 and its analogue was blocked by metabolic inhibitor antimycin A. Preincubation of polymyxin B1 with lipopolysaccharide inhibits in dose-dependent manner polymyxin-induced histamine secretion from rat mast cells.

[Structural and functional investigation of polymyxins. Structure and biological properties of polymyxin M analogs]. / Strukturno-funktsional'nye issledovaniia polimiksinov. Struktura i biologicheskie svoistva analogov polimiksina M.

The effect of proteinases of plant and microbial origin on polymyxin M was studied. It was shown that this antibiotic was absolutely stable to the effect of papain and ficin. On hydrolysis with subtilisin there formed polymyxin decyclized analogs not described earlier. Their isolation, purification and biological activity are described. The structure of these compounds was assessed by one- and two-dimensional 1H NMR spectroscopy. The role of various functional groups, their space orientation and impact on antimicrobial activity of the compounds are discussed.

[The action of combinations of the nonapeptide polymyxin B with antibiotics on gram-negative bacteria]. / Deistvie sochetanii nonapeptida polimiksina B s antibiotikami na gramotritsatel'nye bakterii.

Activity of polymyxin B nonapeptide alone and in combination with other antibiotics against clinical strains of Pseudomonas and enteric bacteria was studied. It was shown that nonapeptide was highly active against Pseudomonas and moderately active against enteric bacteria. In combination with rifampicin, fusidic acid or erythromycin the nonapeptide had a potentiating effect on the tested strains.

[Structural and functional study of polymyxins. Isolation and properties of individual components of polymyxin M]. / Strukturno-funktsional'nye issledovaniia polimiksinov. Vydelenie i svoistva individual'nykh komponentov polimiksina M.

LPCC and HPLC revealed that polymyxin M was a mixture of five components of the polymyxin nature: PM1, PM2, PMx, PMy and PMz. The individual compounds PM1, PM2 and PMz were isolated. Their physicochemical properties and data on antimicrobial activity are presented.

[Structural and functional research on polymyxins. 1H NMR analysis of the conformation of polymyxin M in water]. / Strukturno-funktsional'nye issledovaniia polimiksinov. 1H-IaMR-analiz konformatsii polimiksina M v vode.

Spatial structure of polypeptide antibiotic polymyxin M in water was studied by one-and two-dimensional (COSY, COSY-45, RELAY) H NMR spectroscopy. Analysis of the signal spectral parameters revealed two intramolecular hydrogen bonds in the cyclic part of the molecule which was analogous to the structure of polymyxin B. However, configuration of both the beta-turns in the polymyxin M structure differed from that of the detected earlier beta-turns in the structure of polymyxin B.

[Structural-functional research on polymyxins. 1H-NMR spectra of polymyxin B and its shortened analog]. / Strukturno-funktsional'nye issledovaniia polimiksinov. 1H-IaMR spektry polimiksina B i ego ukorochennogo analoga.

Polymyxin B and its shortened analog were studied comparatively by 1H-NMR spectroscopy. Analysis of the signal chemical shifts, constants of spin-spin interaction of 3J HN-C alpha H and temperature coefficients of the NH signal chemical shifts revealed absolute structural identity of both molecules cyclic parts. This proved that there was no conformative interaction between the cyclic and linear parts of the polymyxin B molecule. Comparison of the results with the data on the biological activity showed that the hydrophobic N-end moiety of the polymyxin B molecule played a specific role in its antibacterial effect and toxicity.