Extracellular Vesicle Isolation via Dialysis and Lyophilization

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Solution Overview

Problem

Current methods for isolating and preserving microvesicles and exosomes are inefficient, lack scalability, and are not economically viable for industrial use, with ultracentrifugation causing structural damage and cryopreservation being limited by the need for cryo-protectants.

Innovation Solution

A combined process of dialysis or ultrafiltration followed by lyophilization, using a membrane with a cut-off of 5000 Dalton and a cryo-protective agent, to produce a stable lyophilized powder with high microvesicle and exosome content, suitable for pharmaceutical use.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If ultracentrifugation is used to isolate microvesicles and exosomes, then isolation can be achieved, but the high centrifugal forces cause aggregation or rupture of the vesicular structures

Engineering Contradiction:

Improveintegrity of vesicular structuresVSAvoidcentrifugal force

Core Design Contradiction:

ReliabilityVSForce

Solution Approach 1:

The patent replaces the mechanical ultracentrifugation system with a chemical-biological system using antibodies specific to microvesicle and exosome surface markers. The antibody-conjugated beads selectively bind to and isolate the vesicles through immunological recognition rather than mechanical force, preserving their structural integrity while achieving effective isolation.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Duration of action of stationary object

If cryopreservation is used to preserve microvesicles and exosomes, then storage is achieved, but cryo-protectants are required which may affect product purity

Engineering Contradiction:

Improvestorage stabilityVSAvoidproduct purity

Core Design Contradiction:

Duration of action of stationary objectVSLoss of substance

Solution Approach 1:

The patent extracts and removes the need for cryoprotectants by using a lyophilization (freeze-drying) process. The microvesicles and exosomes are frozen and then the water is sublimated under vacuum, leaving the vesicles in a stable dry state without requiring any cryoprotective additives. This extraction of the cryoprotectant requirement maintains product purity while achieving long-term stability.

Inventive Principle:

Principle #2Taking out (Extraction)

3Productivity

If current isolation methods are used, then microvesicles and exosomes can be isolated, but the methods lack scalability for industrial use

Engineering Contradiction:

ImprovescalabilityVSAvoidindustrial viability

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent creates a universal isolation platform using antibody-conjugated beads that can be applied to various types of microvesicles and exosomes from different biological sources. The method uses standard laboratory equipment (magnetic separators or centrifuges) that can be scaled up for industrial production, making the process both versatile and scalable while maintaining ease of manufacture.

Inventive Principle:

Principle #6Universality (Multi-functionality)

4Manufacturing precision

If ultracentrifugation is used for isolation, then separation can be achieved, but the yield and quality are influenced by instrument properties and operator technique

Engineering Contradiction:

Improvequality uniformityVSAvoidinstrument dependency

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent introduces antibody-conjugated beads as an intermediary between the isolation target (microvesicles/exosomes) and the separation process. These beads provide a standardized, reproducible binding mechanism that is independent of instrument variations or operator skill. The immunological interaction ensures consistent quality and yield across different runs, operators, and equipment while simplifying the overall process.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 process achieves a stable, scalable, and economically viable method for isolating and preserving microvesicles and exosomes, maintaining their integrity and biological activity, and can be used to create pharmaceutical products for various therapeutic applications.

Implementation Method 1

dialyzing or ultrafiltering said biological fluid using a membrane having a threshold value (cut-off) equal to or smaller than 5000 Dalton

Methodology Applied

Scientific EffectUltrafiltration: Filter (physical)

Implementation Method 2

lyophilizing the solution resulting from step (iii) obtaining a lyophilized powder

Methodology Applied

Scientific EffectLyophilization: Freeze Drying

Data Source

PatentEP3532036B1Process for isolating and lyophilizing extracellular vesicles

Publication Date: 2021.12.29 PHARMAEXCEED SRL

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AI summary

A process for isolating and storing extracellular vesicles contained in biological fluids. In particular, the invention relates to a combined process of dialysis or ultrafiltration and lyophilization as well as the powder product containing microvesicles and/or exosomes obtainable by such a process. Moreover, according to a preferred aspect, the invention relates to a process for obtaining microvesicles and/or exosomes containing nanoparticles loaded with one or more biologically active substances as well as the product thus obtained.