Immobilized ADAMTS13 Purification for Virus Inactivation Without Aggregation
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Solution Overview
Problem
Purification of recombinant ADAMTS13 protein is challenging due to its sensitivity to solvent-detergent chemicals and the difficulty in separating it from non-ADAMTS13 impurities, while also addressing virus contamination issues.
Innovation Solution
A method involving hydroxyapatite chromatography to separate ADAMTS13 from impurities, followed by tandem chromatography with a mixed mode cation exchange/hydrophobic interaction resin, and an optional virus inactivation step using a solvent-detergent mixture while the protein is immobilized on a cation exchange resin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional solvent-detergent treatment is used for virus inactivation, then virus contamination is reduced, but ADAMTS13 protein aggregate formation increases and purification efficiency decreases
Solution Approach 1:
The protein is immobilized on a chromatography resin before virus inactivation treatment. This preliminary immobilization prevents aggregate formation during the subsequent solvent-detergent treatment, allowing effective virus inactivation while maintaining protein quality.
Solution Approach 2:
The chromatography resin acts as an intermediary between the protein and the solvent-detergent mixture. By binding the protein to the resin, the treatment conditions can be applied without direct contact between the protein and aggregating agents, thus inactivating viruses while preventing aggregates.
2Manufacturing precision
If traditional chromatography methods are used for ADAMTS13 purification, then separation from impurities is achieved, but purification yield and efficiency remain insufficient
Solution Approach 1:
The invention employs multiple chromatography steps with varying parameters (resin types, buffer compositions, flow rates, temperature conditions) to optimize both separation precision and overall yield. Each step is tuned to maximize recovery of active ADAMTS13 while removing specific impurity classes.
Solution Approach 2:
The purification process is divided into multiple sequential chromatography steps, each targeting different impurity types. This segmentation allows progressive enrichment of ADAMTS13, achieving high purity while maintaining cumulative yield through optimized recovery at each stage.
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
Achieves high yields of purified ADAMTS13 with reduced aggregate formation and effective virus inactivation, with yields ranging from 60% to 95% depending on the embodiment.
Implementation Method 1
enriching for ADAMTS13 protein by chromatographically contacting the sample with hydroxyapatite under conditions that allow ADAMTS13 protein to appear in an eluate from the hydroxyapatite
Implementation Method 2
chromatographically contacting the eluate with a mixed mode cation exchange/hydrophobic interaction resin that binds ADAMTS13 protein
Implementation Method 3
chromatographically contacting the eluate with a mixed mode cation exchange/hydrophobic interaction resin that binds ADAMTS13 protein
Implementation Method 4
treating a sample to be purified with a solvent-detergent mixture in solution. Incubation of the sample with the solvent-detergent chemicals led to deactivation of lipid-coated viruses
Data Source
AI summary
Provided herein are methods for purifying recombinant A Disintegrin-like and Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13) protein from a sample. The method comprises enriching for ADAMTS13 protein by chromatographically contacting the sample with hydroxyapatite under conditions that allow ADAMTS13 protein to appear in the eluate or supernatant from the hydroxylapatite. The methods may further comprise tandem chromatography with a mixed mode cation exchange/hydrophobic interaction resin that binds ADAMTS13 protein. Additional optional steps involve ultrafiltration/diafiltration, anion exchange chromatography, cation exchange chromatography, and viral inactivation. Also provided herein are methods for inactivating virus contaminants in protein samples, where the protein is immobilized on a support. Also provided herein are compositions of ADAMTS13 prepared according to said methods.