ISVD Purification via Stress-Induced Variant Conversion

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Solution Overview

Problem

The production of structurally homogeneous immunoglobulin single variable domains (ISVDs) is challenging due to the presence of product-related conformational variants, particularly when expressed in lower eukaryotic hosts like yeast, which affect the homogeneity and stability of the final product.

Innovation Solution

A method involving low pH treatment, chaotropic agents, or heat stress is applied during the isolation and purification process to convert and remove conformational variants, utilizing chromatographic techniques to distinguish and separate the desired polypeptide from its variant form, thereby enhancing homogeneity and stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If polypeptides comprising at least three or four ISVDs are produced in lower eukaryotic hosts like yeast, then production yield is improved, but product-related conformational variants are formed that reduce homogeneity

Engineering Contradiction:

Improveproduction yieldVSAvoidproduct homogeneity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies preliminary action by treating the polypeptide with low pH conditions, chaotropic agents, or heat stress before final purification to convert conformational variants into the desired native form. This pre-treatment prevents the accumulation of heterogeneous structures and ensures high homogeneity in the final product while maintaining high production yields from yeast expression systems.

Inventive Principle:

Principle #10Preliminary action

2Ease of manufacture

If conventional purification methods are used without conformational variant conversion, then production cost is reduced, but product homogeneity and stability are compromised

Engineering Contradiction:

Improveproduction costVSAvoidproduct stability

Core Design Contradiction:

Ease of manufactureVSStability of the object's composition

Solution Approach 1:

The patent employs parameter changes by subjecting the polypeptide to specific physical and chemical conditions (low pH, chaotropic agents, heat stress) that induce conformational variant conversion. These controlled parameter changes transform unstable conformational variants into stable native structures, improving product stability without requiring complex additional purification steps, thus maintaining cost-effectiveness.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If complex purification strategies are implemented to remove conformational variants, then product homogeneity is improved, but device complexity and production cost increase

Engineering Contradiction:

Improveproduct homogeneityVSAvoidpurification strategy complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent converts the harmful effect of conformational variants into a benefit by using the same stress conditions (low pH, heat, chaotropic agents) that initially cause variant formation to subsequently convert those variants into the desired native form. This approach eliminates the need for complex separation strategies while achieving high product homogeneity through a unified treatment process.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

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 method significantly reduces the percentage of conformational variants to less than 5%, resulting in more homogeneous and stable polypeptides suitable for therapeutic applications by converting the variants into the desired form using specific conditions and techniques.

Implementation Method 1

applying conditions that convert the conformational variant into the (desired) polypeptide

Methodology Applied

Scientific EffectLow pH treatment:

Implementation Method 2

applying conditions that convert the conformational variant into the (desired) polypeptide

Methodology Applied

Scientific EffectChaotropic agent effect:

Implementation Method 3

applying conditions that convert the conformational variant into the (desired) polypeptide

Methodology Applied

Scientific EffectHeat stress: Heating

Implementation Method 4

utilizing chromatographic techniques to distinguish and separate the desired polypeptide from its variant form

Methodology Applied

Scientific EffectChromatography: Chromatography

Data Source

PatentUS20230136595A1Method for the production and purification of multivalent immunoglobulin single variable domains

Publication Date: 2023.05.04 ABLYNX NV

AI summary

The present disclosure relates to an improved method for the manufacture of polypeptides comprising at least three or at least four immunoglobulin single variable domains (ISVDs). More specifically, an improved method is provided of producing, purifying and isolating polypeptides comprising at least three or at least four ISVDs in which an undesired product-related conformational variant is reduced or absent.