PSPH-Knockout Mammalian Cell Lines for Multi-Vector Selection

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Solution Overview

Problem

Existing mammalian cell lines for biomanufacturing lack multiple selection methods, limiting the introduction of multiple vectors for producing molecules like bispecific antibodies and proteins requiring effector proteins, necessitating a need for improved selection systems.

Innovation Solution

Engineering mammalian cell lines with reduced or eliminated expression of the endogenous Phosphoserine Phosphatase (PSPH) and Glutamine Synthetase (GS) genes, using targeted endonuclease-mediated genome editing techniques like CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases to inactivate chromosomal sequences, allowing for multiple selection systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If traditional single-selection methods (GS, DHFR, antibiotic) are used, then cell line engineering is simple, but multiple vectors cannot be introduced for producing bispecific antibodies and multispecific proteins

Engineering Contradiction:

Improveselection system versatilityVSAvoidcell line engineering complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The invention changes the metabolic parameter of the cell line by knocking out the PSPH gene, which blocks the serine biosynthesis pathway. This creates a new selectable phenotype where cells require exogenous serine, establishing a completely new selection system parameter that is independent of traditional GS, DHFR, or antibiotic-based methods

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The PSPH knockout cell line serves multiple functions: it enables serine-based selection for introducing new vectors, can be combined with existing selection systems for multiple vector integration, and provides a metabolic selection mechanism that complements traditional methods. This multi-functional cell line platform supports production of bispecific antibodies, multispecific antibodies, and other complex proteins

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If PSPH gene is knocked out to create serine dependency, then multiple selection systems become available, but cell culture medium must be modified with exogenous serine

Engineering Contradiction:

Improveselection method diversityVSAvoidculture medium preparation

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The invention performs preliminary action by pre-engineering the cell line with PSPH knockout before production. This upfront genetic modification establishes the serine dependency phenotype, allowing standard culture media to be supplemented with serine in advance. The cell line is prepared and validated with this metabolic requirement before actual protein production begins

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If multiple vectors are introduced for bispecific antibody production, then product complexity increases, but selection and screening processes become more difficult

Engineering Contradiction:

Improvevector integration capabilityVSAvoidselection and screening difficulty

Core Design Contradiction:

Adaptability or versatilityVSDifficulty of detecting and measuring

Solution Approach 1:

The invention uses serine supplementation as an intermediary mechanism for selection. By controlling the availability of serine in the culture medium, the system mediates the selection of cells that have successfully integrated the desired vectors. The serine dependency acts as a controllable intermediary that links vector integration status to cell survival, simplifying the detection and measurement of successful transformations

Inventive Principle:

Principle #24Intermediary (Mediator)

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

Enables the production of bispecific antibodies and biotherapeutic proteins by providing cell lines that require exogenous serine, enhancing productivity and facilitating multiple selection systems for efficient expression of recombinant proteins.

Implementation Method 1

The chromosomal PSPH sequence can be inactivated using targeting endonuclease-mediated genome modification, e.g., CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases

Methodology Applied

Scientific EffectCRISPR ribonucleoprotein-mediated genome modification:

Implementation Method 2

using targeting endonuclease-mediated genome editing techniques like CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases to inactivate chromosomal sequences

Methodology Applied

Scientific EffectTargeted endonuclease-mediated genome editing:

Implementation Method 3

Phosphoserine phosphatase (PSPH) catalyzes the final step in the de novo serine synthesis pathway

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS20260085327A1Metabolic selection via the serine biosynthesis pathway

Publication Date: 2026.03.26 EMD MILLIPORE CORP

AI summary

The present disclosure provides an isolated mammalian cell comprising a reduced or eliminated expression of Phosphoserine Phosphatase (PSPH). Further provided are methods for preparing such cells and methods for using such cells for the production of recombinant proteins.