HaloTag Ligase Fusion for Scalable Low-Residue Bioconjugation
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Solution Overview
Problem
Conventional bioconjugate production processes are time-consuming, costly, and lack scalability due to the complex nature of biomolecules, and immobilized ligases face challenges with operational stability and reusability, along with residual enzyme contaminants being difficult to remove.
Innovation Solution
A ligase fusion protein comprising a ligase and a Halo tag is developed, with an altered isoelectric point, which is immobilized to a support, enabling efficient and controlled conjugation reactions, and a process involving chromatography steps to remove impurities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional chemistry-based conjugation processes are used, then conjugation can be achieved, but the process becomes time-consuming, costly, and lacks scalability due to multiple upstream and downstream purification steps
Solution Approach 1:
The patent extracts and removes the enzyme from the reaction system using a HaloTag-based affinity purification system. The HaloTag on the enzyme supports specific binding to a HaloTag ligand, enabling selective extraction of the enzyme from the conjugation reaction mixture, thereby eliminating the need for complex downstream purification steps while maintaining high conjugate production efficiency
Solution Approach 2:
The HaloTag serves as an intermediary element that facilitates the removal of the enzyme from the reaction system. By introducing a HaloTag ligand that specifically binds to the HaloTag on the enzyme, the patent creates a controlled interaction that enables selective enzyme extraction without affecting the conjugation reaction or requiring multiple purification steps
2Reliability
If enzyme immobilization is used to improve operational stability and reusability, then enzyme reusability increases, but residual enzyme contaminants remain difficult to remove from the conjugate product
Solution Approach 1:
The patent employs a HaloTag-based extraction system that selectively removes the enzyme from the conjugation reaction mixture. The HaloTag on the enzyme binds specifically to the HaloTag ligand, enabling complete extraction of the enzyme while leaving the conjugate product free of enzyme contaminants, thus resolving the contradiction between enzyme reusability and product purity
Solution Approach 2:
The patent uses a synthetic HaloTag ligand that mimics the natural substrate but with added affinity purification functionality. This copied ligand structure enables both the enzyme to function in the conjugation reaction and be subsequently extracted, eliminating residual enzyme contaminants while maintaining operational stability
3Manufacturing precision
If multiple downstream purification steps are implemented to remove aggregates and impurities, then product purity increases, but cost and time increase simultaneously while yield decreases
Solution Approach 1:
The patent extracts the enzyme from the reaction system using HaloTag affinity binding, which simplifies the downstream purification process. By removing the enzyme in a single step rather than requiring multiple chromatography steps, the patent maintains high conjugate product purity while significantly reducing process complexity, time, and cost
Solution Approach 2:
The patent employs a disposable HaloTag ligand that can be easily removed from the system. This short-lived, easily disposable ligand enables simple enzyme extraction without requiring complex, expensive purification equipment or multiple processing steps, thus maintaining product purity while reducing overall process complexity and cost
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 ligase fusion protein system enhances conjugate production efficiency, reduces residual enzyme contaminants, and improves scalability by minimizing purification steps, thus lowering costs and time.
Implementation Method 1
Ligases, such as Sortase enzymes are applied to catalyze conjugation in a highly substrate-specific and efficient manner under mild conditions
Implementation Method 2
a Halo tag, which binds to a Haloalkyl substrate and forms a covalent linkage thereto
Data Source
AI summary
The present disclosure relates to the field of biotechnology. In particular, provided are a ligase fusion protein and an immobilized ligase comprising the same. Also provided is use of the ligase fusion protein or the immobilized ligase in the preparation of conjugates. Further provided is a process for the preparation of conjugates using a ligase or a ligase unit.