Recombinant Collagenase Stability via Linker Modification
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Solution Overview
Problem
Current enzyme agents for cell and tissue dissociation, such as collagenase H and G derived from Clostridium histolyticum, have unstable activities due to varying domain structures and mix ratios, leading to low success rates in pancreatic islet separation and potential cell damage, while Grimontia-hollisae-derived collagenase exhibits varying specific activity and stability issues.
Innovation Solution
A recombinant collagenase is developed with a stable specific activity by modifying the Grimontia-hollisae-derived collagenase to lack the prepeptidase C-terminal domain and include a glycine as the third amino acid residue from the C-terminal in the linker region, ensuring stable collagenase activity and rapid dissociation from tissues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a mixture of collagenase H and G derived from Clostridium histolyticum is used for pancreatic islet separation, then the enzyme agent is available and can be used in clinical settings, but the activity is unstable due to varying domain structures and mix ratios, leading to low success rates
Solution Approach 1:
The patent segments the enzyme agent into two distinct components: a stable collagenase component (ColH or ColG with defined domain structure) and a variable supplement component (other collagenases or proteases). This segmentation allows the core stable enzyme to provide consistent baseline activity while the variable component can be adjusted to optimize performance for different pancreatic tissue conditions, thereby resolving the contradiction between availability and activity stability.
Solution Approach 2:
The patent changes the key parameter of enzyme composition by defining specific domain structures (CD-PKD-PKD-CBD for ColH or CD-PKD-CBD-CBD for ColG) and controlling the mix ratio of collagenases. By precisely controlling these parameters, the patent achieves stable baseline activity while allowing flexibility in formulation to adapt to different clinical needs, thus resolving the contradiction between stability and adaptability.
2Reliability
If the mix ratio of collagenase H and G is adjusted to match varying pancreatic tissue composition, then the success rate of islet separation improves, but the complexity of enzyme preparation increases
Solution Approach 1:
The patent simplifies the complex mixture by segmenting it into a defined core component (stable collagenase with specific domain structure) and an optional variable supplement. This segmentation reduces preparation complexity by establishing a standardized base formulation while allowing optional additions, thereby resolving the contradiction between success rate optimization and preparation simplicity.
Solution Approach 2:
The patent controls the complexity by defining specific domain structures and limited mix ratios rather than allowing arbitrary variations. This parameter control standardizes the enzyme preparation process while maintaining the ability to achieve high success rates, thus resolving the contradiction between reliability and complexity.
3Productivity
If Grimontia-hollisae-derived collagenase is used to achieve high specific activity, then the enzyme efficiency improves, but the specific activity varies between production lots, reducing stability
Solution Approach 1:
The patent segments the enzyme source into a primary stable component (ColH or ColG with defined domain structure from Clostridium histolyticum) and an optional secondary component (Grimontia-hollisae-derived collagenase). This segmentation ensures that the core stable enzyme provides consistent baseline activity across all production lots, while the optional secondary component can be added to enhance specific activity when needed, thereby resolving the contradiction between productivity and reliability.
Solution Approach 2:
The patent controls the variability by defining specific domain structures and allowing flexible supplementation. This parameter control ensures that the core enzyme maintains consistent activity across production lots while allowing optimization of specific activity through controlled additions, thus resolving the contradiction between reliability and productivity.
4Productivity
If conventional collagenases are used for cell separation, then the dissociation process can be performed, but cell damage occurs and engraftment rates are reduced
Solution Approach 1:
The patent changes the critical parameters of enzyme structure (domain composition: CD-PKD-PKD-CBD or CD-PKD-CBD-CBD) and controls the enzyme-to-tissue ratio to optimize dissociation efficiency. These parameter changes enable effective cell separation while minimizing cell damage, thus resolving the contradiction between productivity and cell integrity.
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
The recombinant collagenase provides efficient cell separation with stable activity, reducing cell damage and improving engraftment rates, and maintains activity over time, suitable for various cell types including pancreatic islets, liver, and other tissues.
Implementation Method 1
a Grimontia-hollisae-derived recombinant collagenase... enzyme agent for cell and tissue dissociation
Data Source
AI summary
Recombinant collagenases with a stable specific activity and enzyme agents for cell and tissue dissociation such a recombinant are provided. The recombinant collagenase is derived from Grimontia hollisae-derived collagenase is characterized by having, from the N terminus to the C terminus, a collagenase catalytic domain, a linker region sequence, and a prepeptidase C terminal domain, which Grimontia hollisae-derived recombinant collagenase does not comprise at least the prepeptidase C terminal domain. The obtained recombinant collagenase has a high and stable specific activity.