Expression Vector PolyA Linkage for High Producer Clone Selection

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for selecting high producer clones for protein expression are labor-intensive, time-consuming, and costly due to the random integration of genes and the need to screen large numbers of cell populations, especially when using conventional dhfr/MTX gene amplification systems in CHO cell lines.

Innovation Solution

The use of an expression vector with a selectable marker gene to which a polyadenylation signal (polyA) has been inoperably linked, allowing only cells with a large number of vector copies to survive under selective conditions, thereby increasing the copy number of the recombinant protein of interest and reducing the number of clones that need to be screened.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional dhfr/MTX gene amplification system is used, then productivity of recombinant protein is improved, but the number of clones to be screened increases significantly, making the process labor-intensive and time-consuming

Engineering Contradiction:

Improveproductivity of recombinant proteinVSAvoidtime for selecting high producer clones

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The invention changes the selection parameter by using a weakened selectable marker (neomycin phosphotransferase gene with reduced expression) instead of the conventional strong marker. This parameter change allows only cells with high copy numbers of the expression vector to survive under selective conditions, directly selecting for high producers without requiring screening of numerous clones

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

Instead of using a strong selectable marker that allows most cells to survive and then screening for high producers, the invention inverts the approach by using a weakened marker that kills most cells, allowing only high copy number cells to survive. This inverts the selection pressure to directly enrich for high producers

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If conventional selectable marker gene is used, then cell survival under selective conditions is improved, but the number of clones requiring screening increases, increasing labor and cost

Engineering Contradiction:

Improvecell survival under selective conditionsVSAvoidease of selecting high producer clones

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The selectable marker gene's expression level is changed from high to reduced/weak expression. This parameter change makes the selection process easier because cells with high copy numbers of the expression vector automatically survive under selective conditions without requiring manual screening of large numbers of clones

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If random integration into host cell genome occurs, then gene expression variability is increased, but the number of clones to be tested increases, making the process expensive and time-consuming

Engineering Contradiction:

Improvegene expression variabilityVSAvoidnumber of clones to be tested

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The invention changes the selection threshold parameter by using a weakened selectable marker that requires high copy numbers for survival. This directly selects for high expression clones from the population, reducing the number of clones that need to be tested while still utilizing random integration mechanisms

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach significantly reduces the time and cost of selecting high producer clones by identifying cells with higher productivity, allowing for more efficient production of recombinant proteins, with productivity levels being 31 times higher in some cases compared to traditional methods.

Implementation Method 1

a polyadenylation signal (polyA) has been inoperably linked

Methodology Applied

Scientific EffectPolyadenylation signal:

Data Source

PatentEP2418282B1Method for selecting a high expression recombinant cell line

Publication Date: 2017.05.10 CELLTRION INC

Figure 1
Figure 2
Figure 3

AI summary

The present invention relates to a method of selecting high producer clones by using an expression vector, the expression vector comprising: (i) a gene expression cassette comprising a selectable marker gene to which polyA has been inoperably linked; and (ii) a gene expression cassette which encodes a recombinant protein of interest and to which polyA has been operably linked. According to the invention, high producer clones can be selected from cell populations at least 10 times fewer than in the existing methods of selecting cell lines. Particularly, high producer clones can be selected using a low concentration of MTX compared to a conventional stepwise gene amplification strategy which comprises carrying out multiple amplification steps while increasing the concentration of MTX. Accordingly, the development period of cell lines can be shortened and the labor and cost required for selection of high-productivity cell clones can be reduced, whereby more efficient production of proteins is possible even when general selectable marker genes other than MTX are used.