Heterologous Protein Purification from Egg White via pH-Adjusted Chromatography
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Solution Overview
Problem
Current methods for isolating heterologous proteins from egg whites are inefficient and costly for large-scale production, as they rely on immunoaffinity procedures suitable only for small-scale processes, making them impractical for bulk production due to high costs, labor, and time requirements.
Innovation Solution
A method involving pH adjustments of egg whites to specific ranges followed by hydrophobic interaction chromatography, cation exchange chromatography, and additional filtration steps to efficiently purify heterologous proteins from large volumes of egg whites, including nanofiltration and ultrafiltration/diafiltration, allowing for the processing of volumes greater than 1 liter.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If immunoaffinity procedures are used to isolate heterologous proteins from egg whites, then high protein purity can be achieved, but the process becomes impractical for large-scale production due to high costs, labor, and time requirements
Solution Approach 1:
The patent employs multiple pH adjustments throughout the purification process. The pH is adjusted to 5.8-6.5 for initial filtration, then to 5.0-5.6 for viral inactivation, and finally to 3.0-4.0 for elution from the cation exchange column. These parameter changes enable efficient separation at large scale while maintaining high purity
Solution Approach 2:
The patent introduces a cation exchange chromatography matrix as an intermediary medium to facilitate protein separation. This matrix acts as a mediator between the egg white mixture and the target heterologous protein, enabling scalable purification through binding and selective elution based on charge interactions
2Quantity of substance
If traditional isolation methods are used, then small-scale production can be achieved, but the process becomes inefficient and costly for large-scale production
Solution Approach 1:
The purification process is divided into distinct sequential steps: initial filtration, viral inactivation, cation exchange chromatography, and nanofiltration. This segmentation allows each step to be optimized independently for large-scale processing while maintaining overall process efficiency
Solution Approach 2:
The patent implements a continuous flow process where egg whites are processed through multiple columns in sequence without interruption. The process handles large volumes continuously, with buffers and samples flowing through the system without batch interruptions, significantly improving productivity
3Productivity
If large volumes of egg whites are processed, then bulk production can be achieved, but the complexity of the purification process increases
Solution Approach 1:
The cation exchange chromatography matrix serves multiple functions simultaneously: it captures heterologous proteins based on charge, enables viral inactivation through pH control, and facilitates concentration through selective binding. This multi-functionality reduces the need for separate specialized steps, managing complexity while handling large volumes
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 efficient purification of heterologous proteins from large volumes of egg whites, reducing costs and time while maintaining high protein purity and stability, suitable for industrial-scale production.
Implementation Method 1
subjecting the first filtrate of (b) to a hydrophobic interaction chromatography matrix
Implementation Method 2
subjecting the neutralized solution to a cation exchange chromatography matrix
Data Source
AI summary
The present invention is related a method of purifying a heterologous protein from an egg white. In some embodiments, the disclosure is directed to a method of purifying a heterologous protein from an egg white comprising the heterologous protein, the method comprising, (a) adjusting the pH of the egg white to a pH of 5.8 to 6.5 to form a pH-adjusted egg white; (b) filtering the pH-adjusted egg white of (a) and collecting a first filtrate; (c) subjecting the first filtrate of (b) to a hydrophobic interaction chromatography matrix, and collecting a first eluate comprising the heterologous protein; (d) adjusting the pH of the first eluate of (c) to a pH of 5.0 to 5.6 to form a pH-adjusted eluate; (e) filtering the pH-adjusted eluate to obtain a second filtrate; (f) adjusting the pH of the second filtrate to a pH of 3.0 to 4.0 to form a pH-adjusted second filtrate; (g) neutralizing the pH-adjusted second filtrate of (f) to a pH of 5.0 to 8.0 to form a neutralized solution; (h) subjecting the neutralized solution to a cation exchange chromatography matrix and collecting a second eluate comprising the heterologous protein.