DEVELOPMENT AND CHARACTERIZATION OF FORMULATION OF RECOMBINANT HUMAN GM-CSF EXPRESSED IN E. COLI.

Abstract

Background: Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is an essential cytokine involved in the regulation of hematopoiesis, particularly in the stimulation of white blood cell production. While GM-CSF has significant therapeutic potential, its clinical application is limited due to its short half-life, instability, and rapid clearance from the body. PEGylation, a process of attaching polyethylene glycol (PEG) chains to a protein, is known to enhance the protein's pharmacokinetic properties by improving stability, solubility, and half-life. This study aimed to optimize the PEGylation process of GM-CSF to enhance its therapeutic potential, stability, and bioactivity. Methods: A Design of Experiments (DoE) approach was utilized to optimize the PEGylation conditions of GM-CSF, evaluating variables such as the PEG-to-protein ratio, pH, reaction temperature, and reaction time. Ion Exchange Chromatography (IEX) was used to purify the PEGylated GM-CSF, isolating it from unreacted protein and by-products. The purified product was analyzed for yield, purity, and bioactivity. Bioactivity was measured using a cell proliferation assay to assess the ability of GM-CSF to stimulate hematopoietic progenitor cell growth. Results: Optimization of the PEGylation conditions resulted in a yield of over 69% mono-pegylated GM-CSF under optimal conditions: a PEG-to-protein ratio of 11.5, pH 5.3, and temperature of 25°C. The purification process via IEX achieved purities of 92-96% for PEGylated GM-CSF. Conclusion: The PEGylation of GM-CSF significantly improves its stability and  purity making it a promising candidate for clinical applications in the treatment of blood disorders, such as chemotherapy-induced neutropenia and other hematopoietic deficiencies. The optimized process offers a scalable and efficient method for producing PEGylated GM-CSF with enhanced therapeutic potential. Further studies are warranted to evaluate its clinical efficacy and safety in human trials.