Apo A-I Purification Using GuHCl and Heat Treatment
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Solution Overview
Problem
Current methods for purifying apolipoprotein A-I (Apo A-I) face challenges in efficiently removing small non-enveloped viruses like parvoviruses and picornaviruses due to the hydrophobic nature of Apo A-I, which leads to aggregation and filter fouling, reducing the effectiveness of virus filtration and increasing processing costs.
Innovation Solution
A method involving the use of guanidine hydrochloride (GuHCl) to inhibit aggregation of Apo A-I, allowing for filtration through filters with pore sizes ranging from 15 nm to 35 nm, combined with a heat treatment step for viral inactivation, to achieve high viral clearance while maintaining protein integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If filtration is performed on Apo A-I solutions, then viral removal is achieved, but filter fouling occurs due to protein aggregation
Solution Approach 1:
The patent applies preliminary action by performing a heat treatment step before filtration to inactivate viruses and prevent protein aggregation. The Apo A-I solution is heated to 60-80°C for 10-60 minutes prior to filtration, which pre-treats the protein to prevent hydrophobic face self-association and aggregate formation during the subsequent filtration process, thereby preventing filter fouling and maintaining high throughput
Solution Approach 2:
The patent changes the temperature parameter of the Apo A-I solution from ambient temperature to 60-80°C before filtration. This temperature parameter change stabilizes the protein structure, prevents aggregation, and enables effective filtration without filter fouling, resolving the contradiction between viral removal effectiveness and filtration throughput
2Productivity
If high concentration of Apo A-I is used, then processing efficiency improves, but aggregation increases
Solution Approach 1:
The patent applies preliminary action by performing heat treatment on concentrated Apo A-I solutions before filtration. This pre-treatment step inactivates viruses and prevents protein aggregation even when high concentrations are used, allowing the solution to be filtered without forming aggregates that would reduce processing efficiency
Solution Approach 2:
The patent changes the temperature parameter to 60-80°C for concentrated Apo A-I solutions before filtration. This temperature parameter change stabilizes the protein structure and prevents aggregation, enabling the use of high concentrations without compromising solution stability or increasing aggregation
3Reliability
If small non-enveloped viruses are removed, then viral clearance is achieved, but filter pore size must be very small causing fouling
Solution Approach 1:
The patent applies preliminary action by performing heat treatment before filtration to inactivate small non-enveloped viruses. This pre-treatment step reduces the virus population and prevents protein aggregation, allowing the use of filters with larger pores that are less prone to fouling while still achieving high viral clearance
Solution Approach 2:
The patent changes the temperature parameter to 60-80°C before filtration, which stabilizes the protein structure and prevents aggregation. This enables the use of filters with larger pores that are less susceptible to fouling, reducing device complexity while maintaining high viral clearance effectiveness
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 approach enables efficient removal of small viruses with a high log reduction value (LRV), reducing viral contamination and aggregation, and allows for larger volumes to be filtered quickly, thereby increasing process efficiency and reducing costs.
Implementation Method 1
The invention relates to a method for purifying apolipoprotein, in particular for removing viral pathogens from apolipoprotein A-I (Apo A-I) containing solutions... involving the use of guanidine hydrochloride (GuHCl) to inhibit aggregation of Apo A-I
Implementation Method 2
filtering the solution through a filter having a pore size in a range from 15 nm to 35 nm
Implementation Method 3
combined with a heat treatment step for viral inactivation
Data Source
AI summary
A method for purifying Apo A-I is provided including the steps of providing a solution comprising Apo A-I and guanidine hydrochloride and filtering the solution through a filter having a pore size in a range from 15 nm to 35 nm to thereby reduce viral contamination of the Apo A-I. An Apo A-I preparation is provided having at least a 12 log LRV (log reduction value) for a parvovirus; and/or at least 9 log LRV for a non-enveloped virus; and/or at least 8.5 log LRV for a lipid enveloped virus. Also provided are pharmaceutical compositions and reconstituted high density lipoprotein formulation comprising Apo A-I and methods of treating diseases disorders or conditions.