Antibody Fragment Expression With Periplasmic Chaperones

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Accumulation of intact, properly assembled, active antibody fragments in Escherichia coli (E. coli) periplasm is challenging due to multiple steps including transcription, translation, non-natural amino acid incorporation, translocation, folding, and disulfide bond formation, with existing methods struggling to achieve high titer expression.

Innovation Solution

Engineering vectors and plasmids that incorporate helper proteins and factors such as chaperones (Skp, FkpA) and genetic elements (parB) to optimize protein expression, specifically for non-naturally encoded amino acids, enhancing folding and assembly of antibody fragments.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional expression systems are used for antibody fragments with non-natural amino acids, then the complexity of multiple processing steps (transcription, translation, folding, assembly) remains high, but the expression titer remains low

Engineering Contradiction:

Improveexpression titerVSAvoidprocessing steps complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent introduces periplasmic chaperones (Skp, FkpA, SurA) as intermediary proteins that mediate the folding and assembly processes of antibody fragments containing non-natural amino acids. These chaperones act as molecular assistants that facilitate proper folding, disulfide bond formation, and chain assembly in the periplasm, thereby resolving the contradiction between maintaining complex processing requirements and achieving high expression titers.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If helper proteins such as chaperones are overexpressed, then the folding and assembly of antibody fragments is improved, but the system complexity increases

Engineering Contradiction:

Improveproper folding and assemblyVSAvoidnumber of helper proteins

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies local quality by selectively overexpressing specific chaperones (Skp, FkpA, SurA) in the periplasm based on their specific functions. Rather than uniformly overexpressing all helper proteins, the invention targets particular chaperones that are most critical for handling antibody fragments with non-natural amino acids, thereby improving folding reliability while minimizing unnecessary system complexity.

Inventive Principle:

Principle #3Local quality

3Productivity

If multiple chaperones and helper factors are introduced to improve antibody fragment yield, then the expression optimization is enhanced, but the vector and strain engineering complexity increases

Engineering Contradiction:

Improveantibody fragment yieldVSAvoidvector and strain engineering

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent merges multiple chaperone genes (skp, fkpA, surA) and helper factors into integrated expression vectors that can be co-introduced into E. coli strains. This combining approach allows simultaneous overexpression of multiple beneficial proteins through coordinated genetic constructs, thereby enhancing antibody fragment yield while streamlining the manufacturing process compared to introducing each component separately.

Inventive Principle:

Principle #5Merging (Combining)

Data Source

PatentUS12404512B2Method for optimizing antibody expression

Publication Date: 2025.09.02 AMBRX INC

AI summary

Disclosed herein are methods, compositions and components for optimizing or increasing expression of a protein, polypeptide or fragment therefrom.