Cell Selection for Recombinant Protein Stability
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Solution Overview
Problem
Current methods for producing biopharmaceutical proteins, such as antibodies, face challenges in suppressing the reducing activity of thioredoxin reductase in host cells, leading to protein degradation or denaturation during production, and existing methods for selecting cells with reduced reducing activity are inefficient and not easily scalable.
Innovation Solution
A method involving the measurement and comparison of gene or protein expression levels of specific genes (e.g., Plet1) to identify and select cells with suppressed reducing activity, using a relative quantitative value or relative expression level to evaluate and select cells capable of producing recombinant proteins with reduced degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If thioredoxin reductase inhibitor or oxidizing agent is added to culture broth, then reducing activity is suppressed, but device complexity and manufacturing cost increase
Solution Approach 1:
The patent extracts and eliminates the source of the harmful reducing activity by knocking down or knocking out the thioredoxin reductase gene in the host cell. This removes the problematic enzyme from the system entirely, avoiding the need to add inhibitors or oxidizing agents during culture, thus simplifying the manufacturing process while maintaining protein stability
Solution Approach 2:
The patent performs gene modification (knockdown or knockout of thioredoxin reductase) in advance during host cell preparation, before the culture and protein production stages. This preliminary action permanently suppresses reducing activity throughout the production process, eliminating the need for subsequent additions of chemical agents
2Reliability
If thioredoxin reductase gene is knocked down to suppress reducing activity, then protein degradation is reduced, but cell growth is significantly deteriorated
Solution Approach 1:
The patent applies local quality modification by specifically targeting and knocking down only the thioredoxin reductase gene while leaving the rest of the cell genome intact. This localized genetic modification suppresses reducing activity in the cytoplasm without affecting overall cell growth mechanisms, thereby maintaining productivity while improving protein stability
Solution Approach 2:
The patent changes the expression level parameter of the thioredoxin reductase gene through knockdown or knockout, reducing its activity to a level that no longer causes significant protein degradation. This parameter change is achieved through genetic modification that selectively reduces enzyme activity while maintaining cell viability and growth capacity
3Productivity
If conventional cell selection methods are used, then production cells are obtained, but reducing activity evaluation is inefficient and not easily scalable
Solution Approach 1:
The patent performs gene modification (knockdown or knockout of thioredoxin reductase) in advance during host cell preparation, before the culture and protein production stages. This preliminary action permanently suppresses reducing activity throughout the production process, eliminating the need for subsequent additions of chemical agents
Solution Approach 2:
The patent replaces complex chemical evaluation methods with a simpler genetic marker-based selection system. By using cells with knocked down or knocked out thioredoxin reductase gene, the selection process becomes more efficient and scalable, as it relies on genetic characteristics rather than complex biochemical assays
Data Source
AI summary
A method for selecting a cell strain in which reduction of a recombinant protein is suppressed, and uses of the selected cell are disclosed. The method for selecting a cell, includes a first step: measuring the expression level of a gene in a cell, the gene being at least one gene selected from genes comprising any one of the base sequences represented by SEQ ID NOS: 1 to 16 or orthologous genes thereof; and a second step: comparing the expression level of the gene measured in the first step with a control value of the expression level of the gene in a control cell, and evaluating the expression level capable of suppressing reduction of a recombinant protein based on a difference therebetween.