Paper Title
Optimizing Hybridoma Technology for Efficient Monoclonal Antibody Production: Enhancing Fusion, Cloning, and Expansion Processes

Abstract
Monoclonal antibodies (mAbs) play a crucial role in diagnostics, therapeutics, and biomedical research, and their production through hybridoma technology remains a cornerstone of biotechnology. However, this process requires rigorous optimization to achieve efficiency, particularly in ensuring the successful expansion and stabilization of the producing clone. This study aimed to enhance hybridoma technology by optimizing key stages such as cell fusion, monoclonality establishment, and clone expansion. We compared two different culture media for cell fusion—one containing complete DMEM/HEPES with serum and the other using serum-free DMEM without HEPES or proteins. The results showed that the latter medium achieved a 100% hybridoma formation rate compared to 63% with the serum-containing medium, significantly improving fusion efficiency. Limiting dilution cloning was successfully applied to isolate and expand single hybridoma clones, and these clones were further cultured in conditioned media enriched with growth factors derived from mouse thymus. Finally, a macrophage feeder layer was employed to support the cell expansion of hybridomas. The optimizations implemented here significantly improved hybridoma stability and productivity, contributing to a more reliable and efficient monoclonal antibody production process. Keywords - Hybridoma Technology, Monoclonal Antibody Production, Cell Fusion, Limiting Dilution Cloning.