TNFR2:Fc Purification via Misfolded Variant Quantification
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Solution Overview
Problem
Current methods for producing TNFR2:Fc fusion proteins struggle to effectively separate and quantify wrongly disulphide bridged variants, leading to impurities that reduce the potency and purity of therapeutic preparations.
Innovation Solution
A method involving non-reducing peptide mapping, specifically trypsin digestion followed by reversed phase chromatography, is developed to quantify the wrongly disulphide bridged TNFR2:Fc variant (T7), allowing for improved purification through hydrophobic interaction chromatography to achieve higher purity and potency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional production methods are used to manufacture TNFR2:Fc, then production cost and time are reduced, but wrongly disulphide bridged variants cannot be effectively separated leading to reduced purity and potency
Solution Approach 1:
The patent segments the purification process into distinct steps: (1) affinity chromatography to capture TNFR2:Fc, (2) hydrophobic interaction chromatography to separate wrongly bridged variants, and (3) ion exchange chromatography for further purification. This multi-stage segmentation enables effective removal of misfolded variants while maintaining production efficiency
Solution Approach 2:
The patent introduces an intermediary detection method using specific antibodies that recognize wrongly disulphide bridged variants. This intermediary detection system enables precise identification and quantification of misfolded proteins, allowing for targeted removal during purification without affecting overall productivity
2Manufacturing precision
If hydrophobic interaction chromatography is used to separate wrongly bridged variants, then purity is improved, but the complexity of the purification process increases
Solution Approach 1:
The patent employs hydrophobic interaction chromatography with a universally applicable resin that can separate wrongly bridged variants from correctly folded TNFR2:Fc in a single step. This multi-functional approach combines separation and purification functions, reducing overall process complexity while achieving high purity
Solution Approach 2:
The patent utilizes parameter changes in hydrophobic interaction chromatography by adjusting salt concentration and pH to optimize separation of misfolded variants. By changing these parameters, the process achieves high purity without requiring multiple complex purification steps
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 enables the relative quantification of the T7 variant, enabling the separation of correctly and incorrectly bridged TNFR2:Fc, resulting in a more potent and pure TNFR2:Fc composition suitable for therapeutic use.
Implementation Method 1
subjecting the sample resulting from step (b) to tryptic digestion
Implementation Method 2
subjecting the sample resulting from step (c) to HPLC, thereby separating fragments indicative of Cys78-Cys88 disulphide bridged TNFR2:Fc
Implementation Method 3
A method for purifying TNFR2:Fc using said method for determining the relative amount of said specific wrongly disulphide bridged TNFR2:Fc
Data Source
AI summary
The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.