Comparison of Aflibercept Producers Derived from СНО and СНО-GS Cell Lines
https://doi.org/10.33647/2074-5982-21-1-46-57
Abstract
When creating cell lines expressing recombinant proteins, the type of selection marker and selection conditions are of essential importance. In this work, we aim to obtain and compare cell lines based on CHO and CHO-GS as potential aflibercept producers to identify the most suitable cell line for obtain ing an aflibercept biosimilar. As a result, 10 monoclonal cell lines-producers of a potential aflibercept biosimilar were obtained based on СНО and CHO-GS cell platforms. In the two groups, the productivity on the 15th day of periodic cultivation with feeding achieved 2.5 g/L. The СНО-GS group showed an in crease in the specific cell productivity of aflibercept producers. The conclusion is made that the СНО-GS glutamine auxotroph line is a more preferable option. This cell line enables a comparable yield of target protein at lower concentrations of viable cells, which has a favorable effect on further stages of purification and isolation of target protein.
Keywords
Supporting agencies: this publication was prepared as part of the development of the drug aflibercept at GENERIUM JSC.
About the Authors
S. S. Timonova
GENERIUM
Russian Federation
Russian Federation
Sofya S. Timonova
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
I. A. Kirik
GENERIUM
Russian Federation
Russian Federation
Inessa A. Kirik
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
D. T. Zaripova
GENERIUM
Russian Federation
Russian Federation
Dolia T. Zaripova
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
R. I. Farsieva
GENERIUM
Russian Federation
Russian Federation
Regina I. Farsieva
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
I. A. Kargopolov
GENERIUM
Russian Federation
Russian Federation
Ivan A. Kargopolov
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
R. L. Anisimov
GENERIUM
Russian Federation
Russian Federation
Roman L. Anisimov
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
A. A. Piskunov
GENERIUM
Russian Federation
Russian Federation
Alexander A. Piskunov
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
R. A. Khamitov
GENERIUM
Russian Federation
Russian Federation
Ravil A. Khamitov
601125, Vladimir Region, Petushinskiy District, Vol'ginskiy Village, Vladimirskaya Str., 14
References
1. Timonova S.S., Pavelko V.I., Kirik I.A., Bade V.N., Malygina T.O., Khamitov R.A., Piskunov A.A. Princip operativnogo vybora lidernyh klonov-producentov monoklonal`nyh antitel pri sozdanii stabil`nyh kle tochnyh linij na osnove CHO [Principle of express se lection of leading producing clones of monoclonal an tibodies in the development of stable CHO-based cell lines]. Biotechnology. 2019;35(4):65–72 (In Russian). DOI: 10.21519/0234-2758-2019-35-4-65-72.
2. Adams B.S., Sorhaitz W., Stringham J. Aflibercept. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing LLC, 2025.
3. Cheng G., Ning J., Ahmed S., Huang J., Ullah R., An B., Hao H., Dai M., Huang L., Wang X., Yuan Z. Selection and dissemination of antimicrobial resis tance in Agri-food production. Antimicrob Resist Infect Control. 2019;8:158.
4. Dong W.R., Xiang L.X., Shao J.Z. Novel antibiotic-free plasmid selection system based on complementation of host auxotrophy in the NAD de novo synthesis path way. Appl Environ Microbiol. 2010;76(7):2295–2303.
5. Fann C.H., Guirgis F., Chen G., Lao M.S., Piret J.M. Limitations to the amplification and stability of hu man tissue-type plasminogen activator expression by Chinese hamster ovary cells. Biotechnol Bioeng. 2000;69(2):204–212.
6. Foley J.E., Maeder M.L., Pearlberg J., Joung J.K., Peterson R.T., Yeh J.R. Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat. Protoc. 2009;4(12):1855–1867.
7. Klümper U., Recker M., Zhang L., Yin X., Zhang T., Buckling A., Gaze W.H. Selection for antimicrobial resistance is reduced when embedded in a natural mi crobial community. ISME J. 2019;13(12):2927–2937.
8. Lanzetta P., Korobelnik J.F., Heier J.S., Leal S., Holz F.G., Clark W.L., Eichenbaum D., Iida T., Xiaodong S., Berliner A.J., Schulze A., Schmelter T., Schmidt-Ott U., Zhang X., Vitti R., Chu K.W., Reed K., Rao R., Bhore R., Cheng Y., Sun W., Hirshberg B., Yancopoulos G.D., Wong T.Y., PULSAR Investigators. Intravitreal aflibercept 8 mg in neovascular age-relat ed macular degeneration (PULSAR): 48-week results from a randomised, double-masked, non-inferiority, phase 3 trial. Lancet. 2024;403(10432):1141–1152.
9. Liberski S., Wichrowska M., Kocięcki J. Aflibercept versus Faricimab in the Treatment of Neovascular Age-Related Macular Degeneration and Diabetic Macular Edema: A Review. Int. J. Mol. Sci. 2022;23 (16):9424.
10. Lin P.C., Chan K.F., Kiess I.A., Tan J., Shahreel W., Wong S.Y., Song Z. Attenuated glutamine syn thetase as a selection marker in CHO cells to effi ciently isolate highly productive stable cells for the production of antibodies and other biologics. MAbs. 2019;11(5):965–976.
11. Pallavicini M.G., DeTeresa P.S., Rosette C., Gray J.W., Wurm F.M. Effects of methotrexate on transfected DNA stability in mammalian cells. Mol. Cell Biol. 1990;10(1):401–404. 12. Pana M., ed. Antibiotic resistant bacteria: A continu ous challenge in the new millennium. Rijeka, Croatia: InTech, 2012:531–548.
12. Peubez I., Chaudet N., Mignon C., Hild G., Husson S., Courtois V., De Luca K., Speck D., Sodoyer R. Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microb Cell Fact. 2010;9:65.
13. Ryu A.H., Eckalbar W.L., Kreimer A., Yosef N., Ahituv N. Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene ex pression and regulation. Sci Rep. 2017;7(1):7533.
14. Santiago Y., Chan E., Liu P.Q., Orlando S., Zhang L., Urnov F.D., Holmes M.C., Guschin D., Waite A., Miller J.C., Rebar E.J., Gregory P.D., Klug A., Collingwood T.N. Targeted gene knockout in mam malian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A. 2008;105(15):5809–5814.
15. Yildirim M.E., Karadurmuş N., Ökten İ.N., Türk H.M., Urakçı Z., Arslan Ç., Çelik S., Dane F., Şendur M.A.N., Bilir C., Karabulut B., Cicin İ., Çubukçu E., Karaca M., Ozcelik M., Artaç M., Tanrikulu E., Alacacioglu A., Açıkgöz Ö., Öven B., Geredeli Ç., Çil T., Harputluoğlu H., Kefeli U., Bozkurt O., Tural D., Sakin A., Yalçın Ş., Gumus M. Real-world treatment outcomes from nationwide Onco-colon Turkey registry in RAS wild-type patients treated with biologics second-line mCRC. J. Oncol. Pharm. Pract. 2024:10781552241241004.
Review
For citations:
Timonova S.S., Kirik I.A., Zaripova D.T., Farsieva R.I., Kargopolov I.A., Anisimov R.L., Piskunov A.A., Khamitov R.A. Comparison of Aflibercept Producers Derived from СНО and СНО-GS Cell Lines. Journal Biomed. 2025;21(1):46-57. (In Russ.) https://doi.org/10.33647/2074-5982-21-1-46-57
Views:
132
Email this article 
