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A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand
Bárbara B Sousa1, Marcos F Q Sousa2,3, Marta C Marques1
1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal.
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 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.
Insights
A new scalable production process for human recombinant Bone Marrow Tyrosine kinase in the chromosome X (BMX) was developed. This method efficiently produces highly pure BMX, enabling high-throughput screening for novel cancer therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Production
Background:
- Bone Marrow Tyrosine kinase in the chromosome X (BMX) is implicated in cancer progression.
- Targeting BMX with covalent inhibitors is a promising therapeutic strategy.
- Current methods for producing pure BMX are insufficient for large-scale screening.
Purpose of the Study:
- To develop a scalable and efficient process for producing high-purity human recombinant BMX (hrBMX).
- To facilitate high-throughput screening of covalent BMX inhibitors for cancer therapy.
Main Methods:
- Utilized an insect cell-baculovirus expression vector system for hrBMX production.
- Employed small-scale shake flasks and 5 L stirred-tank bioreactors (STB).
- Implemented a streamlined two-step chromatographic purification process.
Main Results:
- Achieved comparable expression levels in shake flasks and bioreactors.
- Reduced purification time by 75% while maintaining protein stability.
- Obtained a yield of 24 mg/L with >99% purity, generating 100 mg hrBMX in 34 days from 5 L STB.
Conclusions:
- The developed platform enables scalable and rapid production of high-purity hrBMX.
- This process supports in-vitro covalent ligand high-throughput screening for BMX inhibitors.
- Facilitates the development of novel cancer therapeutics targeting BMX.
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