Bax/Bak Knockout Cell Lines for Apoptosis-Resistant Bioproduction
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Solution Overview
Problem
Existing eukaryotic cell lines, particularly mammalian cell lines like CHO cells, face challenges in producing therapeutic proteins efficiently due to high apoptotic activity, leading to reduced viability and productivity in bioreactors.
Innovation Solution
Development of eukaryotic cell lines with stable integrated loss-of-function or attenuation-of-function mutations in both the Bax and Bak genes, which reduces apoptotic activity and enhances cell viability and productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If eukaryotic cell lines are used to produce therapeutic proteins, then protein folding, assembly, and post translational modifications are achieved, but high apoptotic activity reduces cell viability and productivity
Solution Approach 1:
The patent extracts and removes the harmful apoptotic function by knocking out the Bax and Bak genes, which are responsible for mitochondrial apoptosis. This extraction of the harmful apoptotic pathway allows cells to maintain their protein production capabilities while eliminating the self-destruct mechanism that limited viability and productivity
Solution Approach 2:
The patent changes the functional state of the Bax and Bak genes from active to inactive through genetic modification. By altering the expression parameters of these apoptotic genes, the cells transition from a high-apoptosis state to a low-apoptosis state, thereby improving viability while maintaining therapeutic protein production capabilities
2Quantity of substance
If cell culture is performed in bioreactors to produce significant quantities of therapeutic molecules, then production scale is increased, but apoptotic activity reduces overall yield and efficiency
Solution Approach 1:
The patent converts the harmful effect of apoptotic activity into a benefit by selectively eliminating the apoptotic pathway. The harmful apoptosis mechanism is transformed into a useful tool for improving cell survival and productivity, allowing scaled-up production to achieve higher yields and efficiency without the limiting factor of cell death
3Duration of action of moving object
If wild type eukaryotic cells are cultured for extended periods, then sufficient time is available for protein production, but apoptotic activity accumulates and reduces cell viability
Solution Approach 1:
The patent provides beforehand cushioning against apoptosis by pre-modifying the cells to lack functional Bax and Bak genes. This prior genetic modification creates a buffer or protective effect against apoptotic activation, allowing cells to withstand culture stress and maintain viability over extended production periods without the usual apoptotic decline
Data Source
AI summary
The present disclosure relates to eukaryotic cell lines with a stable integrated loss-of-function or attenuation-of-function mutation in each of the Bax and Bak genes. Also provided are methods of producing such cell lines. This disclosure also relates to compositions and cell cultures comprising such cell lines, as well as methods of producing a product, such as a recombinant polypeptide or viral vector, using said cells, compositions and cell cultures.