Basal Medium Supplementation to Reduce Trisulfide Bonds

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Solution Overview

Problem

Trisulfide bonds in recombinantly produced therapeutic polypeptides introduce heterogeneity, making it challenging to ensure product quality and consistency due to their formation during manufacturing.

Innovation Solution

A method involving a basal medium with specific concentrations of iron, riboflavin, pyridoxine, folic acid, cyanocobalamin, hypotaurine, and methionine is used to culture host cells, which reduces trisulfide bond levels in polypeptides by supplementing with chelating and reducing agents before or after harvesting.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If standard cell culture media are used for recombinant polypeptide production, then polypeptides are produced efficiently, but trisulfide bonds form causing product heterogeneity

Engineering Contradiction:

Improvepolypeptide production efficiencyVSAvoidproduct homogeneity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies parameter changes by modifying the chemical composition of cell culture media through controlled supplementation with chelating agents (EDTA, DTPA, citrate) and reducing agents (cysteine, glutathione, TCEP) at specific concentrations. These parameter changes in media composition directly reduce trisulfide bond formation while maintaining polypeptide production efficiency, resolving the contradiction between productivity and manufacturing precision

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses chelating agents and reducing agents as intermediary substances that mediate between the cell culture system and the polypeptide product. These intermediaries bind metal ions and maintain reducing conditions in the media, preventing trisulfide bond formation without directly interfering with polypeptide expression, thus maintaining productivity while improving product homogeneity

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If trisulfide bond formation is not controlled, then manufacturing process is simple, but product quality and consistency deteriorate

Engineering Contradiction:

Improvemanufacturing process simplicityVSAvoidproduct quality consistency

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent implements preliminary action by pre-supplementing cell culture media with chelating and reducing agents before initiating polypeptide production. This proactive measure establishes protective conditions that prevent trisulfide bond formation during the entire manufacturing process, ensuring product quality consistency without complicating the manufacturing workflow

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent enables self-service by designing a media supplementation system that automatically maintains reducing conditions throughout the culture process. The chelating and reducing agents continuously scavenge metal ions and maintain low redox potential without requiring external intervention, allowing the system to self-regulate and prevent trisulfide bond formation while maintaining simple manufacturing operations

Inventive Principle:

Principle #25Self-service

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 effectively decreases trisulfide bond levels in polypeptides, enhancing their quality and consistency by reducing heterogeneity and improving manufacturing processes.

Implementation Method 1

supplementing with chelating and reducing agents

Methodology Applied

Scientific EffectChelation:

Implementation Method 2

supplementing with chelating and reducing agents before or after harvesting

Methodology Applied

Scientific EffectReduction: Reduction

Data Source

PatentUS20190169667A1Methods of decreasing trisulfide bonds during recombinant production of polypeptides

Publication Date: 2019.06.06 GENENTECH INC

AI summary

Provided herein are cell culture media and methods culturing host cells expressing polypeptides to reduce the level of trisulfide bonds in polypeptides produced by the host cells.