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 challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-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 the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

X 染色体中的骨髓酪氨酸激酶 (BMX) 是一种 TEC 家族激酶，与癌细胞中的许多病理途径相关。BMX 活性的共价抑制有望作为一种治疗癌症的方法。为了筛选有效和选择性的共价 BMX 抑制剂，通常需要大量高纯度的 BMX，这对当前可用的生产和纯化过程来说是一个挑战。在这里，我们使用昆虫细胞-杆状病毒表达载体系统开发了人类重组 BMX (hrBMX) 的可扩展生产工艺。在小型摇瓶 (13 mL) 和搅拌罐生物反应器 (STB, 5 L) 中获得了相当的表达水平。实施了基于两步色谱的工艺，与传统工艺相比，纯化时间减少了 75%，同时保持hrBMX的稳定性。最终产量为每升细胞培养物 24 mg 纯化的 hrBMX，纯度 > 99%。产品质量通过一系列生化和生物物理分析进行评估和确认，包括圆二色性和动态光散射。总体而言，本文开发的平台能够在短短 34 天内从 5 L STB 中产生 100 mg 纯化的 hrBMX，因此具有协助体外共价配体高通量筛选 BMX 活性抑制的潜力。