Protease Inactivation via Two-Step pH Adjustment
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Solution Overview
Problem
Biopharmaceutical products, such as monoclonal antibodies and recombinant proteins, are susceptible to proteolytic degradation due to the presence of proteases in cell-free culture supernatants, leading to reduced yield and quality, especially during purification processes, and existing methods using protease inhibitors are costly and difficult to remove.
Innovation Solution
A method involving a two-step pH adjustment of the cell culture supernatant, first to acidic pH values (3-5) and then to neutral pH values (7-9), to irreversibly inactivate proteases, thereby reducing protein degradation and extending the shelf life of chromatography materials.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If protease inhibitors are added to inactivate proteases, then proteolytic degradation is reduced, but production cost increases and product purification becomes more difficult
Solution Approach 1:
The patent changes the pH parameter of the culture supernatant to inactivate proteases. By adjusting pH to acidic conditions (pH 3-5) and maintaining it for a specific time, proteases are inactivated without adding any chemical inhibitors, thus avoiding the problems of cost and removal difficulty associated with inhibitor-based methods
Solution Approach 2:
The method uses the culture supernatant itself to inactivate proteases through pH adjustment, rather than relying on external additives. The system's own properties are modified to achieve protection, eliminating the need for external inhibitor substances that would require removal
2Productivity
If cell-free culture supernatant is adjusted to acidic pH for purification, then binding conditions for capture step are created, but extensive protein degradation occurs
Solution Approach 1:
The patent applies preliminary pH adjustment to inactivate proteases before the purification process begins. By inactivating proteases in advance, the harmful effect is prevented before it can occur during subsequent acidic pH treatment for capture, allowing efficient purification without degradation
Solution Approach 2:
The method applies a preliminary anti-action by inactivating proteases through pH adjustment before the harmful proteolytic degradation can occur during purification. This preemptive measure counteracts the potential harm before the purification step creates binding conditions
3Stability of the object's composition
If proteases remain active in harvested fluid, then natural state is maintained, but product quality and yield are reduced
Solution Approach 1:
The patent changes the pH parameter to inactivate proteases while minimizing impact on the overall composition stability. By using mild pH adjustment and controlled time exposure, the method achieves protease inactivation with minimal disturbance to other components, balancing natural state preservation with product protection
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 proteolytic activity by up to 75% for acidic proteases and 90% for neutral proteases, improving product quality and yield, and allowing for longer shelf life of chromatography materials without the need for expensive inhibitors.
Implementation Method 1
Proteases that are active at neutral pH values can also be irreversibly inactivated in their activity at neutral conditions by switching to acidic pH values
Data Source
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AI summary
The invention relates to a method for inactivating proteases by multiple change of pH in the cell culture supernatant at the beginning of the operation of purifying biopharmaceuticals. First of all a pH of 3-5, and subsequently a pH of 7-9, is set.