Recombinant Antibody Monomerisation via Denaturation and Reduction

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Solution Overview

Problem

Current methods for purifying recombinantly expressed antibody molecules often result in lower yields due to the formation of unwanted multimers, which are difficult to separate from monomers using conventional chromatography, especially for bispecific antibody formats.

Innovation Solution

A method involving the use of urea and/or Guanidine hydrochloride as denaturants in combination with a reducing agent, such as β-mercaptoethylamine, to convert multimeric species into monomers by reducing disulfide bonds, allowing for the reformation of correct disulfide bonds and increasing the percentage of monomeric antibody molecules.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional chromatography is used to separate monomers from multimers, then purification is achieved, but the yield is reduced due to loss of multimeric species

Engineering Contradiction:

Improvepurification qualityVSAvoidantibody yield

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent applies parameter changes by using denaturants (urea, guanidine hydrochloride) and reducing agents to alter the chemical state of disulfide bonds in multimeric antibody species. This transforms the multimers into monomers through reduction of intermolecular disulfide bonds, allowing all antibody material to be recovered as monomeric product without chromatographic separation losses.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent extracts the problematic multimeric species and converts them into the desired monomeric form. By removing the multimeric state through chemical treatment (denaturation and reduction) and converting it to monomers, the process eliminates the need to discard multimers during purification, thereby increasing overall yield.

Inventive Principle:

Principle #2Taking out (Extraction)

2Stability of the object's composition

If disulfide bonds are used to stabilize variable domain pairing in novel antibody formats, then structural stability is improved, but unwanted multimer formation is stabilized

Engineering Contradiction:

Improvevariable domain pairing stabilityVSAvoidunwanted multimer formation

Core Design Contradiction:

Stability of the object's compositionVSObject-generated harmful factors

Solution Approach 1:

The patent converts the harmful effect of disulfide-stabilized multimers into a beneficial process. By using reducing agents, the intermolecular disulfide bonds that cause multimer formation are reduced, converting the stable multimeric species into monomers. The same disulfide bonding capability then reformes correct intramolecular bonds during purification, stabilizing the desired monomeric structure.

Inventive Principle:

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

Solution Approach 2:

The patent inverts the usual approach by not preventing disulfide bond formation but instead promoting it in a controlled manner. Rather than trying to block multimer formation, the process uses reduction followed by controlled reformation of disulfide bonds under purified conditions, allowing correct pairing to occur while preventing unwanted multimerization.

Inventive Principle:

Principle #13The other way round (Inversion)

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 significantly increases the percentage of monomeric antibody molecules, achieving yields of up to 80% or more, compared to the initial 15% or less, without fully denaturing the antibodies, thus improving the overall yield and quality of recombinant antibody compositions.

Implementation Method 1

a) a conversion step of treating the composition with a denaturant selected from urea and/or Guanidine hydrochloride

Methodology Applied

Scientific EffectDenaturation:

Implementation Method 2

b) wherein step a) is performed in the presence of a reducing agent or after treatment with a reducing agent

Methodology Applied

Scientific EffectReduction: Reduction

Data Source

PatentUS11786593B2Method of monomerisation of recombinant antibody molecules

Publication Date: 2023.10.17 UCB BIOPHARMA SPRL

AI summary

The present invention provides method of increasing the percentage of monomer in a composition of recombinantly expressed antibody molecules characterised in that the antibody molecule comprises at least one Fv with specificity for an antigen of interest comprising one VH and one VL wherein said VH and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprises: a) a conversion step of treating the composition with a denaturant selected from urea and/or Guanidine hydrochloride; b) wherein step a) is performed in the presence of a reducing agent or after treatment with a reducing agent.