FACS Producer Cell Sorting Without Drug Selection Pressure
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Solution Overview
Problem
Existing methods for selecting producer cell populations for biologic manufacturing, such as those using methotrexate (MTX) or methionine sulphoximine (MSX), are time-consuming, affect cell viability, and have a negative impact on clonal stability, necessitating a need for rapid and reliable methods to generate high-titer cell populations and clones with minimal adverse effects.
Innovation Solution
The use of fluorescence activated cell sorting (FACS) for batch and clonal selection of producer cells without MTX, involving the sorting of heterogeneous populations based on FACS selectable polypeptides, followed by expansion in drug-free medium, to enhance production of target polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If methotrexate (MTX) or methionine sulphoximine (MSX) is used for selection, then producer cell populations can be selected and amplified, but cell viability is affected and clonal stability is compromised
Solution Approach 1:
The patent removes the harmful selection agent (MTX/MSX) from the system by using FACS-based selection instead. The selectable marker system allows cells to be sorted based on fluorescence intensity without requiring drug pressure, thereby extracting the selection function while eliminating the toxic effects on cell viability and clonal stability
Solution Approach 2:
The patent introduces a fluorescent reporter protein as an intermediary that correlates with target polypeptide expression. This mediator allows indirect selection of high-producing cells through FACS based on fluorescence intensity, rather than directly selecting under drug pressure, thus avoiding the harmful effects while maintaining selection efficacy
2Reliability
If multiple rounds of drug-based selection are performed, then suitable producer clones can be obtained, but the process becomes time-consuming
Solution Approach 1:
The patent performs preliminary sorting of cells based on fluorescence intensity before expansion and cloning. By pre-enriching the population of high-expressing cells through FACS, the subsequent cloning process requires fewer rounds of selection and can be completed more quickly while still obtaining suitable producer clones
Solution Approach 2:
The patent replaces the mechanical/drug-based selection system with a fluorescence-based optical sorting system. FACS allows for rapid, automated separation of cells based on fluorescence intensity, eliminating the need for multiple sequential drug selection rounds and significantly reducing the time required to obtain suitable clones
3Speed
If FACS is used for bulk sorting without MTX, then selection speed increases and cell viability improves, but multiple sorting rounds are needed to achieve high enrichment
Solution Approach 1:
The patent employs periodic or sequential FACS sorting rounds to progressively enrich the population. Each sorting round targets a specific fluorescence intensity threshold, and by performing multiple rounds with increasing stringency, the method achieves high enrichment of target polypeptide producers while maintaining cell viability throughout the process
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 method significantly improves polypeptide production, achieving 1.2- to 30-fold increases in titer compared to traditional methods, with enhanced viability and reduced time, making it suitable for both small and large-scale manufacturing.
Implementation Method 1
fluorescence activated cell sorting (FACS) to batch select producer cells expressing a target polypeptide
Data Source
AI summary
Provided herein are methods and compositions for batch production of producer cells using fluorescence activated cell sorting (FACS). In some aspects, the disclosure provides a drug-selection-free method for batch production of producer cells using FACS. Such batch production methods and compositions can be further utilized to generate clonal populations of producer cells, e.g., for large-scale manufacturing of a polypeptide of interest.