pH Precipitation for DsbC Removal in Protein Purification
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Solution Overview
Problem
Current methods for purifying recombinant proteins from gram-negative host cells, such as E. coli, face challenges in reducing host cell protein contaminants like disulphide isomerase, which are difficult to remove and can interfere with downstream processing, leading to increased costs and reduced yields in therapeutic antibody production.
Innovation Solution
Adjusting the pH of the host cell sample to 5 or less to precipitate recombinant disulphide isomerase, allowing for its separation from the recombinant protein, thereby facilitating downstream processing and improving the yield of the protein of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification methods are used to remove host cell proteins, then purification is achieved, but significant loss of recombinant protein occurs and purification costs increase
Solution Approach 1:
The patent applies preliminary action by precipitating and removing disulphide isomerase (DsbC) before the main purification chromatography steps. By performing this removal step in advance, the patent prevents DsbC from interfering with subsequent purification processes and reduces protein loss that would otherwise occur during chromatography, thereby improving both purification quality and reducing substance loss.
Solution Approach 2:
The patent extracts and removes the harmful contaminant (disulphide isomerase) from the host cell extract using pH-based precipitation. This extraction step separates the contaminant from the recombinant protein before the main purification process, allowing the recombinant protein to pass through chromatography columns without interference, thus reducing loss and improving purification efficiency.
2Productivity
If disulphide isomerase is expressed at high levels to improve antibody expression, then protein yield increases, but contaminant removal becomes more difficult
Solution Approach 1:
The patent utilizes parameter changes by adjusting the pH of the host cell extract to a specific range (pH 3-5) to exploit the differential solubility of disulphide isomerase. At this pH range, DsbC precipitates while the recombinant protein remains soluble, enabling selective removal of the contaminant without affecting the target protein, thus simplifying purification despite high contaminant levels.
Solution Approach 2:
The patent converts the harmful presence of high levels of disulphide isomerase into a benefit by using its unique precipitation behavior at low pH. The contaminant's tendency to precipitate at pH 3-5 is exploited to its advantage, allowing selective removal of DsbC while maintaining the recombinant protein in solution, thereby turning the contamination problem into an opportunity for simplified purification.
3Manufacturing precision
If traditional chromatography is used to remove all contaminants, then purification is achieved, but column fouling occurs and processing time increases
Solution Approach 1:
The patent performs preliminary removal of disulphide isomerase through pH-based precipitation before the chromatography steps. This preliminary action reduces the total contaminant load entering the chromatography system, preventing column fouling and reducing the time required for purification while maintaining completeness of purification.
Solution Approach 2:
The patent extracts and removes the majority of disulphide isomerase contaminant before chromatography using pH precipitation. This extraction step significantly reduces the contaminant burden on subsequent chromatography columns, preventing fouling and reducing processing time while maintaining purification completeness.
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 effectively reduces the quantity of host cell contaminants, minimizes column fouling, and enhances the efficiency of chromatography steps, leading to cost savings and improved protein recovery in therapeutic antibody production.
Implementation Method 1
adjusting the pH of the host cell sample or extract thereof to a pH of 5 or less to precipitate the recombinant disulphide isomerase
Data Source
AI summary
The present invention provides method for purifying a recombinant protein from a gram-negative bacterial host cell sample or extract thereof wherein said host cell expresses a recombinant protein and a recombinant disulphide isomerase DsbC; comprising: a. adjusting the pH of the host cell sample or extract thereof to a pH of 5 or less to precipitate the recombinant disulphide isomerase; and b. separating precipitated recombinant disulphide isomerase DsbC from the recombinant protein to produce a recombinant protein sample.