A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening

Abstract

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is not a simple task with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cells-baculovirus expression system.Comparable expression levels were obtained in small-scale shake flaks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A 2-step chromatographic-based process was implemented, reducing purification times by 75 % when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of >99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno‐associated virus

“…[12] Increasing interest in IC-BEVS has led to numerous efforts to improve production yields, including for AAVs. Strategies such as the design of improved viral expression vectors, [13][14][15][16] development of new bioprocess intensification strategies, [17,18] gene integration, [19] and laboratory evolution of cell host [2] have been successfully employed in IC-BEVS. Despite this, limited progress has been made in understanding the underlying biological mechanisms to enhance recombinant protein expression in IC-BEVS.…”