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All Journal Journal of Tropical Life Science : International Journal of Theoretical, Experimental, and Applied Life Sciences
Widekdo, Dwi purno
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Agriculture, Biological Sciences & Forestry Environmental Science

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Effect of Combination of Different Antibiotics and Promoters for Expressing Recombinant Darbepoetin in Stable CHO K-1 Cell Line: Evaluating Antibiotic Combinations to Improve Yield and Quality of Darbepoetin Widekdo, Dwi purno; Widodo, Nashi; Rifa'i, Muhaimin; Jatmiko, Yoga Dwi
Journal of Tropical Life Science Vol. 15 No. 1 (2025)
Publisher : Journal of Tropical Life Science

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.11594/

Abstract

Antibiotics are key for successful molecular cloning techniques. Different antibiotics have different mechanisms of action, which leads to cell heath and a viable number of passages. Moreover, the suitability of the promoter also plays an important role in achieving a higher level of protein titter in the stable cell platform. Therefore, with plenty of options of antibiotics and promoters available, we need to determine the best combination of antibiotics and promoters, particularly for specific proteins of interest. Darbepoetin is a recombinant therapeutics protein with extra glycosylation to increase the half-life in the blood; this drug is used for the administration of CKD and leukemia patients. Blasticidin-S and puromycin were used as antibiotics, and CMV and EF-1 promoters were used in this experiment to evaluate the expression of recombinant darbepoetin for the protein model. CHO K-1 cell line was transfected with a plasmid carrying a combination of promoter and antibiotics genes; after 14 days, the level of specific protein expression was evaluated using the western blot technique. A single clone cell was obtained using the serial dilution method to evaluate the clonality and expression of the protein of interest. This study successfully obtained a single clone from stable pool transfection. This result suggested that a combination of puromycin antibiotics and EF-1 promoter has promising expression compared to Blasticidin-S antibiotics with CMV promoter. For further conclusion, an analytical comparison of both combinations needed to be done.
Development of a Stable CHO Cell Line for High-Level Expression and Purification of Recombinant Darbepoetin Alfa Widekdo, Dwi purno; Widodo, Nashi; Rifa'i, Muhaimin; Jatmiko, Yoga Dwi
Journal of Tropical Life Science Vol. 15 No. 3
Publisher : Journal of Tropical Life Science

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.11594/jtls.15.03.12

Abstract

Recombinant Darbepoetin alfa (DARB), an erythropoiesis-stimulating agent, is widely used in treating anemia linked to chronic kidney disease and chemotherapy. This study presents a systematic approach for developing a high-yield, stable Chinese hamster ovary (CHO) cell line capable of producing recombinant DARB. A codon-optimized gene construct under dual CMV/EF1α promoters and puromycin resistance selection was transfected into CHO cells. To isolate high-producing monoclonal populations, transfected cells were subjected to limiting dilution cloning in 96-well plates, allowing single-cell-derived colonies to expand and be individually assessed. Clone DARV IV Pool 2 Clone 2E6 demonstrated the highest and most consistent DARB expression, validated through Western blot using anti-human EPO antibodies. The culture supernatant underwent two-step clarification via centrifugation and tangential flow filtration (TFF), followed by purification using anion-exchange chromatography on a HiTrap Q HP column. Gradient elution enabled effective separation, with SDS-PAGE and Western blot confirming high purity and molecular integrity of the recombinant protein. Variability in clone expression highlighted the influence of genomic integration sites and potential epigenetic silencing, emphasizing the importance of screening and stability validation. This study demonstrates that rational vector design, antibiotic-based clone selection, and robust purification strategies can produce CHO-derived DARB suitable for large-scale production. The integrated workflow supports scalability, product consistency, and regulatory readiness for biosimilar therapeutic manufacturing
Co-Authors Nashi Widodo Rifa'i, Muhaimin YOGA DWI JATMIKO