CHO Cell PKM Knockout for Reduced Lactate Production
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Solution Overview
Problem
Chinese hamster ovary (CHO) cells used in therapeutic protein production exhibit lactogenic behavior, leading to lactate accumulation that adversely affects cell growth, viability, and productivity, and alters product quality, with existing process condition optimizations being limited in effectiveness and applicability.
Innovation Solution
Modulating pyruvate kinase muscle (PKM) expression, particularly knocking down or knocking out PKM-1 and PKM-2 polypeptide isoforms, using genetic engineering systems like CRISPR/Cas, ZFN, or TALEN, to reduce or eliminate lactogenic activity in mammalian cells, thereby reducing lactate production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If process conditions are optimized to reduce lactate generation, then lactate accumulation is reduced, but the approach cannot apply to all lactogenic CHO cell lines and complicates large-scale manufacturing
Solution Approach 1:
The patent extracts and targets the specific enzymatic mechanism (PKM) responsible for lactate generation in CHO cells. By using CRISPR/Cas9 to knock out the PKM gene, the invention directly removes the harmful lactate-generating pathway at its molecular source, rather than attempting to control lactate levels through complex process conditions. This resolves the contradiction by providing a cell line modification that eliminates lactate accumulation without complicating manufacturing processes.
Solution Approach 2:
The patent fundamentally changes the metabolic parameter of PKM enzyme activity in CHO cells by completely knocking out the gene. This genetic modification permanently alters the cell's metabolic behavior to eliminate lactate production, providing a universal solution that works across different cell lines and scales without requiring complex process optimization.
2Object-affected harmful factors
If PKM expression is knocked down or knocked out, then lactogenic activity is reduced or eliminated, but genetic engineering is required
Solution Approach 1:
The patent employs CRISPR/Cas9 genome editing technology, which allows the cell's own molecular machinery to perform the modification. The system uses guide RNA to direct Cas9 nuclease to the PKM gene, where it creates double-strand breaks that lead to gene knockout. This self-service approach eliminates the need for complex external control systems or continuous process monitoring, as the modified cells inherently lack the ability to produce lactate through the PKM pathway.
Data Source
AI summary
The present disclosure relates to methods, cells and compositions for producing a product of interest, e.g., a recombinant protein. In particular, the present disclosure provides improved mammalian cells expressing the product of interests, where the cells (e.g., Chinese Hamster Ovary (CHO) cells) have modulated lactogenic activity. The present disclosure also relates to methods and compositions for modulating pyruvate kinase muscle (PKM) expression (e.g., PKM-1 expression) in a mammalian cell to thereby reduce or eliminate the lactogenic activity of the cell, as well compositions comprising a cell having reduced or eliminated lactogenic activity and methods of using the same.