Dwi Purno Widekdo, Nashi Widodo, Muhaimin Rifa’i, Yoga Dwi Jatmiko
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 indi-vidually 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 epige-netic silencing, emphasizing the importance of screening and stability validation. This study demonstrates that rational vector design, antibiotic-based clone selec-tion, 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. © 2025, Brawijaya University. All rights reserved.
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, 65145, Indonesia
