Alkyldimethylamine Oxide Lysis for AAV Purification

Solution Overview

Problem

Current methods for releasing adeno-associated virus (AAV) and adenovirus particles from cells are inefficient, often requiring ecologically questionable reagents like Triton X-100, which can introduce impurities and are difficult to scale, especially for serotypes like AAV2 that are tightly associated with cellular components.

Innovation Solution

Using a composition comprising alkyldimethylamine oxides, such as Lauryldimethylamine-N-Oxide (LDAO) and Tetradecyl Dimethylamine-N-Oxide (TDAO), in combination with salts like sodium chloride to promote cell lysis and release of viral particles without affecting their integrity, followed by purification steps like filtration and chromatography.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If Triton X-100 is used for cell lysis, then cell lysis efficiency is improved, but ecotoxicity increases and virus purification becomes difficult

Engineering Contradiction:

Improvecell lysis efficiencyVSAvoidecotoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent substitutes Triton X-100 with alkyldimethylamine oxides (such as LDAO and TDAO), which have different chemical properties including lower ecotoxicity. This reagent substitution maintains effective cell lysis while eliminating the harmful environmental effects of octylphenol degradation products.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs detergents that can be easily removed or degraded without leaving persistent harmful residues. Alkyldimethylamine oxides are selected because they can be effectively eliminated during purification processes, unlike Triton X-100 which leaves persistent ecotoxic degradation products.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Productivity

If Triton X-100 is used for cell lysis, then cell lysis efficiency is improved, but virus purification complexity increases

Engineering Contradiction:

Improvecell lysis efficiencyVSAvoidpurification process complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent employs detergents that can be easily removed or degraded without leaving persistent harmful residues. Alkyldimethylamine oxides are selected because they can be effectively eliminated during purification processes, unlike Triton X-100 which leaves persistent ecotoxic degradation products.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Ease of manufacture

If physical lysis methods are used, then scalability is improved, but time consumption increases

Engineering Contradiction:

ImprovescalabilityVSAvoidtime consumption

Core Design Contradiction:

Ease of manufactureVSLoss of time

Solution Approach 1:

The patent replaces mechanical/physical lysis methods with a chemical lysis approach using alkyldimethylamine oxides. This chemical method acts more rapidly than physical methods while maintaining scalability, as the detergent can be easily mixed throughout the cell suspension in controlled bioreactors.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Productivity

If conventional cell lysis reagents are used, then virus release is limited, but ecotoxicity is reduced

Engineering Contradiction:

Improvevirus release efficiencyVSAvoidecotoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent substitutes Triton X-100 with alkyldimethylamine oxides (such as LDAO and TDAO), which have different chemical properties including lower ecotoxicity. This reagent substitution maintains effective cell lysis while eliminating the harmful environmental effects of octylphenol degradation products.

Inventive Principle:

Principle #35Parameter changes

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 releases and purifies viral particles with high efficiency, exceeding the yields of traditional methods, particularly for serotypes like AAV2 and AAV9, while avoiding the use of environmentally harmful reagents, and allows for the removal of detergent residues through tangential flow filtration.

Implementation Method 1

contacting a suspension of the cells with an effective amount of a composition comprising an alkyldimethylamine oxide to promote cell lysis and release of the AAV particles from the cells

Methodology Applied

Scientific EffectDetergent lysis: Surfactant

Implementation Method 2

allows for the removal of detergent residues through tangential flow filtration

Methodology Applied

Scientific EffectFiltration: Filter (physical)

Data Source

PatentUS20230183656A1Methods and compositions for purifying adeno associated virus particles or adenoviruses

Publication Date: 2023.06.15 MERCK PATENT GMBH

AI summary

The present invention relates to compositions and methods for lysing cells and isolating and/or purifying adeno-associated virus particles or adenovirus particles using a detergent selected from the group of alkyldimethylamine oxides.