MDM2 D300A and E1B19K Gene Co-expression in CHO Cells

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Solution Overview

Problem

High cell death rates due to apoptosis in mammalian cell cultures, particularly in high-density protein-free fed-batch bioreactors, limit viable cell density and heterologous protein yield, necessitating improved methods to enhance cell viability and protein production.

Innovation Solution

Over-expressing MDM2 D300A and E1B19K genes in mammalian cell lines, such as CHO cells, to inhibit apoptosis and increase cell viability and secreted protein production, with co-expression of these genes demonstrating a significant increase in integrated viable cell count and protein titers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If high-density mammalian cell culture is used to increase protein production, then productivity increases, but cell death due to apoptosis increases

Engineering Contradiction:

Improveprotein productionVSAvoidcell viability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention changes the genetic parameters of the mammalian cells by introducing and over-expressing anti-apoptotic genes (Bcl-2, Bcl-XL, Aven, c-IAP-1, c-IAP-2, XIAP, and MDM2). This genetic modification alters the cellular parameters related to apoptosis resistance, allowing cells to maintain viability at high densities during fed-batch culture, thereby resolving the contradiction between high productivity and cell viability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention uses copies of anti-apoptotic genes from cancer cells (which naturally over-express these genes to resist apoptosis) and introduces them into normal mammalian cell lines. By copying and over-expressing these protective genes, the normal cells gain cancer-cell-like resistance to apoptosis, enabling them to survive at high densities required for high protein production

Inventive Principle:

Principle #26Copying

2Productivity

If cell density is increased to improve protein yield, then productivity increases, but the onset of death phase accelerates

Engineering Contradiction:

Improveprotein yieldVSAvoidculture duration

Core Design Contradiction:

ProductivityVSDuration of action of stationary object

Solution Approach 1:

The invention performs preliminary action by genetically modifying the cells before culture to over-express anti-apoptotic genes. This pre-protection against apoptosis allows the cells to withstand the stress of high-density culture and extended culture periods, delaying the onset of the death phase and extending the productive culture duration

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The anti-apoptotic genes act as a protective cushion against apoptotic stressors that accumulate during extended high-density culture. By having these protective mechanisms in place beforehand, the cells can endure longer culture periods and higher densities without entering the death phase, thus extending the productive window

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

Data Source

PatentEP2300600B1Methods for improving viability and productivity in cell culture

Publication Date: 2015.09.30 JANSSEN BIOTECH INC

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AI summary

Methods for increasing viability and production of secreted proteins in fed batch eukaryotic cell culture are disclosed.