Laboratory of Mammalian Cell Bioengineering

Ivan I. Vorobev
Dr Sci. (Biology)
Head of Laboratory
INB, room 512
Телефон +7 (495) 308-99-96
E-Mail ptichman@gmail.com

The laboratory was founded in 2013, aiming to bridge fundamental and applied research in the field of pharmaceutical biotechnology, to accelerate biopharmaceutical research and development from bench to industry.

 

Research Focus

To gain new insights into fundamental mechanisms that control the heterologous proteins biosynthesis and secretion and cultured mammalian cell fate.

To develop key technologies for biological pharmaceutics production, including producer cells bioengineering, purification methods of pharmaceutical grade proteins and pDNA, pharmaceutical preparations and analytical technologies.

Basic studies of cell genome organization and regulation of gene expression on a model of cultured Chinese hamster ovary cells.
Practical applications:

  •  To identify and overcome the factors limiting the super-production of secreted heterologous proteins by CHO cells.
  • To identify and overcome the factors limiting the industrial maintenance of dense culture of CHO cells, including the pathway of programmed cell death and autophagy.

Basic studies on protein chemistry and development of methods for mathematical planning of experiments
Practical applications:

  • To develop low-cost and fully scalable technologies for the production of pharmaceutical-grade therapeutic proteins.
  • To obtain conjugates of the therapeutic and vaccine proteins with desired properties.

 

Main Scientific Achievements
Contribution to the fight against the COVID-19 pandemic:

  • Development of the production technologies of the SARS-CoV-2 S protein RBD in the CHO cells, and SARS-CoV-2 NP – in bacterial cells.
  • Development of the surrogate Virus Neutralization Test (sVNT) based on antibody-mediated blocking of the interaction of ACE2 and SARS-CoV-2 Spike protein RBD.

Gonadotropic hormones for assisted reproductive technologies

  • Development of recombinant follicle stimulating hormone production technology in CHO cells.

Coagulation factor production for the treatment of hemophilia

  • Development of highly efficient cell lines, producing factor VIII as a full-length and B-domain deleted protein. Development of original methods for affinity purification of recombinant factor VIII.
  • Development of recombinant coagulation factor IX production technology in CHO cells.

Fibrinolysis: Producer line development for the recombinant human tissue activator of human plasminogen.

Fight against obesity, diabetes and their complications

  • Study of bioconjugate drug candidates of human oxyntomodulin and polysialic acid.
  • Process development for pharmaceutical plasmid DNA production.

 

Selected publications

  1. Sinegubova MV, Orlova NA, Vorobiev II. 2023. Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins. PeerJ 11:e16287 https://doi.org/10.7717/peerj.16287
  2. Kolesov DE, Gaiamova EA, Orlova NA, Vorobiev II. Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test. Biochemistry (Mosc). 2023 Sep;88(9):1274-1283. doi: 10.1134/S0006297923090079. PMID: 37770394
  3. Khodak YA, Shaifutdinov RR, Khasanov DS, Orlova NA, Vorobiev II. Location and Orientation of the Genetic Toxin-Antitoxin Element hok/sok in the Plasmid Affect Expression of Pharmaceutically Significant Proteins in Bacterial Cells. Biochemistry (Mosc). 2023 Sep;88(9):1326-1337. doi: 10.1134/S0006297923090122. PMID: 37770399
  4. Sukhova M, Byazrova M, Mikhailov A, Yusubalieva G, Maslova I, Belovezhets T, Chikaev N, Vorobiev I, Baklaushev V, Filatov A. 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. Microorganisms. 2023 Sep 20;11(9):2347. doi: 10.3390/microorganisms11092347. PMID: 37764191
  5. Khodak YA. Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines. Biochemistry (Mosc). 2023 Sep;88(9):1248-1266. doi: 10.1134/S0006297923090055. PMID: 37770392
  6. Khodak, Y.A.; Ryazanova, A.Y.; Vorobiev, I.I.; Kovalchuk, A.L.; Ovechko, N.N.; Aparin, P.G. High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure. BioTech 2023, 12, 9.  DOI: 10.3390/biotech12010009 PMID: 36648835
  7. Kovnir S.V., Dayanova L.K., Gayamova E.A., Dybovsky L.N., Vorobiev I.I., Orlova N.A. Knockout of Bax, Bak1 Genes and Overexpression of Bcl2, Becn1 Genes Increase Lifespan and Maximum Density of CHO-S Cell Culture. Biotekhnologiya. 2022; 38(4): стр. 16-22 DOI:10.56304/S0234275822040081
  8. Ryazanova A.Yu., Khodak Yu.A., Orlova N.A., Sinegubova M.V., Dayanova L.K., Kovnir S.V., Korobova S.V., Ledov V.A., Kovalchuk A.l., Alkhazova B.I., Golovina M.E., Vorobiev I.I., Aparin P.G. Receptor-binding Domain of Sars-cov-2 Spike Protein Fused to a Non-glycosylated Crystallizable Fragment of Human Igg1: Obtaining and Assessment of Immunogenicity. Biotekhnologiya. 2022; 38(6): – С. 12-19. – DOI 10.56304/S0234275822060102.
  9. Orlova NA, Dayanova LK, Gayamova EA, Sinegubova MV, Kovnir SV, Vorobiev II. Targeted Knockout of the dhfr, glul, bak1, and bax Genes by the Multiplex Genome Editing in CHO Cells. Dokl Biochem Biophys. 2022 Feb;502(1):40-44. doi: 10.1134/S1607672922010082. Epub 2022 Mar 11. PubMed PMID: 35275305.
  10. Kolesov DE, Sinegubova MV, Dayanova LK, Dolzhikova IV, Vorobiev II, Orlova NA. Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD. Diagnostics (Basel). 2022 Feb 3;12(2). doi: 10.3390/diagnostics12020393. PubMed PMID: 35204485.
  11. Kolesov DE, Sinegubova MV, Safenkova IV, Vorobiev II, Orlova NA. Antigenic properties of the SARS-CoV-2 nucleoprotein are altered by the RNA admixture. PeerJ. 2022;10:e12751. doi: 10.7717/peerj.12751. eCollection 2022. PubMed PMID: 35036106.
  12. Sinegubova M, Vorobiev I, Klishin A, Eremin D, Orlova N, Orlova N, Polzikov M. Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur®) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency. Pharmaceutics. 2022 Jan 1;14(1). doi: 10.3390/pharmaceutics14010096. PubMed PMID: 35056992.
  13. Sinegubova MV, Orlova NA, Kovnir SV, Dayanova LK, Vorobiev II. High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector. PLoS One. 2021;16(2):e0242890. doi: 10.1371/journal.pone.0242890. eCollection 2021. PubMed PMID: 33529230.
  14. Orlova NA, Kovnir SV, Khodak YA, Polzikov MA, Nikitina VA, Skryabin KG, Vorobiev II. High-level expression of biologically active human follicle stimulating hormone in the chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors. PLoS ONE, 14(7):e0219434, 2019. DOI: 10.1371/journal.pone.0219434.
  15. Kovnir SV, Orlova NA, Shakhparonov MI, Skryabin KG, Gabibov AG, Vorobiev II. A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX. Acta Naturae. 2018 Jan-Mar;10(1):51-65.
  16. Kovnir SV, Orlova NA, Khodak YА, Kondrashova MP, Gabibov AG, Skryabin KG, Vorobiev AI, Vorobiev II. 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. Bull Exp Biol Med. 2017 Jun;163(2):245-249. doi: 10.1007/s10517-017-3776-0. Epub 2017 Jul 18.
  17. rlova NA, Kovnir SV, Gabibov AG, Vorobiev II. Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system. BMC Biotechnol. 2017 Mar 24;17(1):33. doi: 10.1186/s12896-017-0353-6.
  18. Mokrushina YA, Pipiya SO, Stepanova AV, Shamborant OG, Knorre VD, Smirnov IV, Gabibov AG, Vorobiev II. [Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates]. Mol Biol (Mosk). 2017 Nov-Dec;51(6):958-968. doi: 10.7868/S0026898417060088.
  19. Smirnov IV, Vorobiev II, Belogurov AA, Genkin DD, Deyev SM, Gabibov AG. Chemical Polysialylation of Recombinant Human Proteins. // Methods Mol Biol. 2015;1321:389-404.
  20. Kolyadko VN, Lushchekina SV, Vuimo TA, Surov SS, Ovsepyan RA, Korneeva VA, Vorobiev II, Orlova NA, Minakhin L, Kuznedelov K, Severinov KV, Ataullakhanov FI, Panteleev MA. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa. PLoS One. 2015 Dec 15;10(12):e0144940. doi: 10.1371/journal.pone.0144940. eCollection 2015.
  21. Orlova NA, Kovnir SV, Vorobiev II, Gabibov AG, Vorobiev AI. Blood Clotting Factor VIII: From Evolution to Therapy. Acta Naturae. 2013 Apr;5(2):19-39.
  22.  Orlova NA, Kovnir SV, Hodak JA, Vorobiev II, Gabibov AG, Skryabin KG. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells. BMC Biotechnol. 2014 Jun 14;14:56. doi: 10.1186/1472-6750-14-56.