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Article ; Online: Enhancing Human Glycoprotein Hormones Production in CHO Cells Using Heterologous Beta-Chain Signal Peptides.

Sinegubova, M V / Kolesov, D E / Dayanova, L K / Vorobiev, I I / Orlova, N A

2023 Volume 514, Issue 1, Page(s) 1–5

Abstract: We studied the influence of heterologous signal peptides in the β-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the β- ... ...

Abstract	We studied the influence of heterologous signal peptides in the β-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the β-chains were replaced with the heterologous signal peptide of human serum albumin, cell productivity was increased 2-2.5 times for human luteinizing hormone, human chorionic gonadotropin, and human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes.
MeSH term(s)	Cricetinae ; Animals ; Humans ; Cricetulus ; CHO Cells ; Protein Sorting Signals ; Glycoproteins ; Chorionic Gonadotropin/metabolism
Chemical Substances	Protein Sorting Signals ; Glycoproteins ; Chorionic Gonadotropin
Language	English
Publishing date	2023-12-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	2062390-2
ISSN	1608-3091 ; 1607-6729
ISSN (online)	1608-3091
ISSN	1607-6729
DOI	10.1134/S1607672923700576
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Article: Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur

Sinegubova, Maria / Vorobiev, Ivan / Klishin, Anatoly / Eremin, Dmitry / Orlova, Nadezhda / Orlova, Natalya / Polzikov, Mikhail

Pharmaceutics

2022 Volume 14, Issue 1

Abstract: Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese ... ...

Abstract	Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration operations. Studies on chromatography resin resource and ligand leakage showed that the immunoaffinity matrix employed was suitable for industrial use and stable for at least 40 full chromatography cycles, and the leaked single-domain antibody ligand was completely removed by subsequent purification steps. All chromatography resins employed withstood the same 40 cycles of use without significant changes in separation efficiency and product binding capacity. The resulting industrial purification process yielded batches of r-hFSH with consistent levels of purity and bioactivity.
Language	English
Publishing date	2022-01-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527217-2
ISSN	1999-4923
ISSN	1999-4923
DOI	10.3390/pharmaceutics14010096
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Article: Humoral Immune Responses in Patients with Severe COVID-19: A Comparative Pilot Study between Individuals Infected by SARS-CoV-2 during the Wild-Type and the Delta Periods.

Sukhova, Maria / Byazrova, Maria / Mikhailov, Artem / Yusubalieva, Gaukhar / Maslova, Irina / Belovezhets, Tatyana / Chikaev, Nikolay / Vorobiev, Ivan / Baklaushev, Vladimir / Filatov, Alexander

Microorganisms

2023 Volume 11, Issue 9

Abstract: Since the onset of the COVID-19 pandemic, humanity has experienced the spread and circulation of several SARS-CoV-2 variants that differed in transmissibility, contagiousness, and the ability to escape from vaccine-induced neutralizing antibodies. ... ...

Abstract	Since the onset of the COVID-19 pandemic, humanity has experienced the spread and circulation of several SARS-CoV-2 variants that differed in transmissibility, contagiousness, and the ability to escape from vaccine-induced neutralizing antibodies. However, issues related to the differences in the variant-specific immune responses remain insufficiently studied. The aim of this study was to compare the parameters of the humoral immune responses in two groups of patients with acute COVID-19 who were infected during the circulation period of the D614G and the Delta variants of SARS-CoV-2. Sera from 48 patients with acute COVID-19 were tested for SARS-CoV-2 binding and neutralizing antibodies using six assays. We found that serum samples from the D614G period demonstrated 3.9- and 1.6-fold increases in RBD- and spike-specific IgG binding with wild-type antigens compared with Delta variant antigens (
Language	English
Publishing date	2023-09-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720891-6
ISSN	2076-2607
ISSN	2076-2607
DOI	10.3390/microorganisms11092347
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Article ; Online: Targeted Knockout of the dhfr, glul, bak1, and bax Genes by the Multiplex Genome Editing in CHO Cells.

Orlova, N A / Dayanova, L K / Gayamova, E A / Sinegubova, M V / Kovnir, S V / Vorobiev, I I

Doklady. Biochemistry and biophysics

2022 Volume 502, Issue 1, Page(s) 40–44

Abstract: The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. ... ...

Abstract	The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr
MeSH term(s)	Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Editing ; Gene Knockout Techniques ; Glutamate Plasma Membrane Transport Proteins/genetics ; Tetrahydrofolate Dehydrogenase/genetics ; bcl-2 Homologous Antagonist-Killer Protein/genetics ; bcl-2-Associated X Protein/genetics
Chemical Substances	Glutamate Plasma Membrane Transport Proteins ; bcl-2 Homologous Antagonist-Killer Protein ; bcl-2-Associated X Protein ; Tetrahydrofolate Dehydrogenase (EC 1.5.1.3)
Language	English
Publishing date	2022-03-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	2062390-2
ISSN	1608-3091 ; 1607-6729
ISSN (online)	1608-3091
ISSN	1607-6729
DOI	10.1134/S1607672922010082
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Article ; Online: Manifestations of pathomorphosis at pregnant women with heart diseases

Kazachkova E.A. / Kazachkov E.L. / Vorobiev I.V.

Саратовский научно-медицинский журнал, Vol 13, Iss 2, Pp 233-

2017 Volume 238

Abstract: Purpose: to study the structure of heart defects in pregnant women, the features of the medico-social portrait, the course of pregnancy and perinatal outcomes in patients with heart defects in the light of pathomorphosis. Material and Methods. A ... ...

Abstract	Purpose: to study the structure of heart defects in pregnant women, the features of the medico-social portrait, the course of pregnancy and perinatal outcomes in patients with heart defects in the light of pathomorphosis. Material and Methods. A retrospective clinical and anamnestic analysis of the medical documentation of 165 patients delivered in the maternity hospital of the Municipal Health Care Institution of the City Clinical Hospital No. 6 in Chelyabinsk in 1991-1994 was conducted, (group 1)and a prospective cohort study of 168 patients who were delivered to this hospital between 2011 and 2014 (group 2) in accordance with a modified WHO classification for assessing the risk of cardiovascular complications for the mother and offspring, the characteristics of the medico-social portrait, The course of pregnancy and perinatal outcomes in patients with heart defects in the light of the doctrine of pathomorphosis. Results. It is established that over the past 20 years, there have been significant changes in the structure of heart diseases, the medical and social characteristics of patients with this disease, complications of gestation, which must be taken into account when choosing the optimal tactics for pregravid preparation, pregnancy in women with heart defects. Conclusion. The significant changes in the structure and frequency of heart defects observed in modern pregnant women with heart defects, in the features of the medical and social portrait of these patients, and also during pregnancy, childbirth and perinatal outcomes, can be treated as pathomorphosis in the clinical (narrow) sense.
Keywords	heart disease ; pathomorphosis ; pregnancy ; Medicine (General) ; R5-920
Subject code	610
Language	Russian
Publishing date	2017-06-01T00:00:00Z
Publisher	Saratov State Medical University
Document type	Article ; Online
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Article: A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX.

Kovnir, S V / Orlova, N A / Shakhparonov, M I / Skryabin, K G / Gabibov, A G / Vorobiev, I I

Acta naturae

2018 Volume 10, Issue 1, Page(s) 51–65

Abstract: Hemophilia B patients suffer from an inherited blood-clotting defect and require regular administration of blood-clotting factor IX replacement therapy. Recombinant human factor IX produced in cultured CHO cells is nearly identical to natural, plasma- ... ...

Abstract	Hemophilia B patients suffer from an inherited blood-clotting defect and require regular administration of blood-clotting factor IX replacement therapy. Recombinant human factor IX produced in cultured CHO cells is nearly identical to natural, plasma-derived factor IX and is widely used in clinical practice. Development of a biosimilar recombinant human factor IX for medical applications requires the generation of a clonal cell line with the highest specific productivity possible and a high level of specific procoagulant activity of the secreted factor IX. We previously developed plasmid vectors, p1.1 and p1.2, based on the untranslated regions of the translation elongation factor 1 alpha gene from Chinese hamster. These vectors allow one to perform the methotrexate- driven amplification of the genome-integrated target genes and co-transfect auxiliary genes linked to various resistance markers. The natural open reading frame region of the factor IX gene was cloned in the p1.1 vector plasmid and transfected to CHO DG44 cells. Three consecutive amplification rounds and subsequent cell cloning yielded a producer cell line with a specific productivity of 10.7 ± 0.4 pg/cell/day. The procoagulant activity of the secreted factor IX was restored nearly completely by co-transfection of the producer cells by p1.2 plasmids bearing genes of the soluble truncated variant of human PACE/furin signal protease and vitamin K oxidoreductase from Chinese hamster. The resulting clonal cell line 3B12-86 was able to secrete factor IX in a protein-free medium up to a 6 IU/ml titer under plain batch culturing conditions. The copy number of the genome- integrated factor IX gene for the 3B12-86 cell line was only 20 copies/genome; the copy numbers of the genome-integrated genes of PACE/furin and vitamin K oxidoreductase were 3 and 2 copies/genome, respectively. Factor IX protein secreted by the 3B12-86 cell line was purified by three consecutive chromatography rounds to a specific activity of up to 230 IU/mg, with the overall yield > 30%. The developed clonal producer cell line and the purification process employed in this work allow for economically sound industrial-scale production of biosimilar factor IX for hemophilia B therapy.
Language	English
Publishing date	2018-03-11
Publishing country	Russia (Federation)
Document type	Journal Article
ISSN	2075-8251
ISSN	2075-8251
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Article: COVID-19 in Russia: Clinical and Immunological Features of the First-Wave Patients.

Bobik, T V / Kostin, N N / Skryabin, G A / Tsabai, P N / Simonova, M A / Knorre, V D / Stratienko, O N / Aleshenko, N L / Vorobiev, I I / Khurs, E N / Mokrushina, Yu A / Smirnov, I V / Alekhin, A I / Nikitin, A E / Gabibov, A G

Acta naturae

2021 Volume 13, Issue 1, Page(s) 102–115

Abstract: The coronavirus disease outbreak in 2019 (COVID-19) has now achieved the level of a global pandemic and affected more than 100 million people on all five continents and caused over 2 million deaths. Russia is, needless to say, among the countries ... ...

Abstract	The coronavirus disease outbreak in 2019 (COVID-19) has now achieved the level of a global pandemic and affected more than 100 million people on all five continents and caused over 2 million deaths. Russia is, needless to say, among the countries affected by SARS-CoV-2, and its health authorities have mobilized significant efforts and resources to fight the disease. The paper presents the result of a functional analysis of 155 patients in the Moscow Region who were examined at the Central Clinical Hospital of the Russian Academy of Sciences during the first wave of the pandemic (February-July, 2020). The inclusion criteria were a positive PCR test and typical, computed tomographic findings of viral pneumonia in the form of ground-glass opacities. A clinical correlation analysis was performed in four groups of patients: (1) those who were not on mechanical ventilation, (2) those who were on mechanical ventilation, and (3) those who subsequently recovered or (4) died. The correlation analysis also considered confounding comorbidities (diabetes, metabolic syndrome, hypertension, etc.). The immunological status of the patients was examined (levels of immunoglobulins of the M, A, G classes and their subclasses, as well as the total immunoglobulin level) using an original SARS-CoV-2 antibody ELISA kit. The ELISA kit was developed using linear S-protein RBD-SD1 and NTD fragments, as well as the N-protein, as antigens. These antigens were produced in the prokaryotic
Language	English
Publishing date	2021-05-07
Publishing country	Russia (Federation)
Document type	Journal Article
ISSN	2075-8251
ISSN	2075-8251
DOI	10.32607/actanaturae.11374
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Article ; Online: Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids.

Kovnir, S V / Orlova, N A / Khodak, Yu А / Kondrashova, M P / Gabibov, A G / Skryabin, K G / Vorobiev, A I / Vorobiev, I I

Bulletin of experimental biology and medicine

2017 Volume 163, Issue 2, Page(s) 245–249

Abstract: Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in ... ...

Abstract	Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies.
Language	English
Publishing date	2017-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	390407-6
ISSN	1573-8221 ; 0007-4888 ; 0365-9615
ISSN (online)	1573-8221
ISSN	0007-4888 ; 0365-9615
DOI	10.1007/s10517-017-3776-0
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Article: [Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates].

Mokrushina, Yu A / Pipiya, S O / Stepanova, A V / Shamborant, O G / Knorre, V D / Smirnov, I V / Gabibov, A G / Vorobiev, I I

Molekuliarnaia biologiia

2017 Volume 51, Issue 6, Page(s) 958–968

Abstract: Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the ... ...

Abstract	Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.
MeSH term(s)	Amino Acid Sequence ; Antibodies, Catalytic/chemistry ; Antibodies, Catalytic/genetics ; Antibodies, Catalytic/metabolism ; Antibody Affinity ; Antibody Specificity ; Biocatalysis ; Catalytic Domain ; Cloning, Molecular ; Gene Expression ; Genetic Vectors/chemistry ; Genetic Vectors/metabolism ; Hydrophobic and Hydrophilic Interactions ; Immunoglobulin Fab Fragments/chemistry ; Immunoglobulin Fab Fragments/genetics ; Immunoglobulin Fab Fragments/metabolism ; Kinetics ; Models, Molecular ; Organophosphorus Compounds/antagonists & inhibitors ; Organophosphorus Compounds/chemistry ; Organophosphorus Compounds/immunology ; Organophosphorus Compounds/metabolism ; Pichia/genetics ; Pichia/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Structure, Secondary ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/immunology ; Recombinant Proteins/metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Substrate Specificity
Chemical Substances	Antibodies, Catalytic ; Immunoglobulin Fab Fragments ; Organophosphorus Compounds ; Recombinant Proteins
Language	Russian
Publishing date	2017-11
Publishing country	Russia (Federation)
Document type	Journal Article
ZDB-ID	213542-5
ISSN	0026-8984
ISSN	0026-8984
DOI	10.7868/S0026898417060088
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Article: Coagulation Factor IX for Hemophilia B Therapy.

Orlova, N A / Kovnir, S V / Vorobiev, I I / Gabibov, A G

Acta naturae

2012 Volume 4, Issue 2, Page(s) 62–73

Abstract: Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in ... ...

Abstract	Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in the manufacturing of FIX, improved variants of the recombinant protein for therapy, transgenic organisms for obtaining FIX, and the advances in the gene therapy of hemophilia B.
Language	English
Publishing date	2012-08-07
Publishing country	Russia (Federation)
Document type	Journal Article
ISSN	2075-8251
ISSN	2075-8251
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