Antigen-Binding Molecule Production With Protein A Disulfide Control
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Solution Overview
Problem
Existing methods for producing antigen-binding molecules with disulfide bonds between amino acid residues in regions other than the hinge region face challenges of low yield, long reaction times, and heterogeneity, particularly in forming appropriate disulfide bonds during cell culture expression.
Innovation Solution
A method involving the use of a reducing agent followed by affinity chromatography to form and stabilize disulfide bonds between amino acid residues in regions other than the hinge region, including steps for reducing agent treatment and removal, to enhance yield and homogeneity of antigen-binding molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If reducing agent treatment followed by re-oxidation is performed to form disulfide bonds, then appropriate disulfide bonds are formed between heavy chains, but the reaction time is long and productivity is low
Solution Approach 1:
Protein A affinity column serves as an intermediary that immobilizes antibody molecules in a controlled orientation, enabling efficient disulfide bond formation between heavy chains while facilitating rapid reducing agent treatment and quick removal of the reducing agent through column washing, thus resolving the contradiction between bond formation accuracy and production efficiency
Solution Approach 2:
The antibody molecules are pre-immobilized on the Protein A affinity column in a specific orientation before reducing agent treatment, which prepares the molecules for optimal disulfide bond formation and enables subsequent rapid processing by simply washing away the reducing agent, thereby improving both bond formation accuracy and productivity
2Manufacturing precision
If reducing agent treatment is performed to convert capped molecules to LINC format, then appropriate disulfide bonds are formed, but heterogeneity of disulfide bonds remains
Solution Approach 1:
The Protein A affinity column acts as a mediator that holds antibody molecules in a uniform orientation during reducing agent treatment, ensuring consistent disulfide bond formation between heavy chains and eliminating heterogeneity, while the column's washing capability removes residual reducing agent to prevent further unwanted reactions
Solution Approach 2:
By immobilizing all antibody molecules on the Protein A affinity column in the same orientation and treating them uniformly with the reducing agent followed by uniform washing, the method achieves homogeneous disulfide bond formation across all molecules, resolving the heterogeneity issue
3Manufacturing precision
If buffer exchange is performed for re-oxidation to form disulfide bonds, then appropriate disulfide bonds are formed, but the process complexity increases
Solution Approach 1:
The method combines the re-oxidation step with the column washing step by performing both operations in a single buffer flow-through process on the Protein A affinity column, eliminating the need for separate buffer exchange operations and reducing process complexity while maintaining disulfide bond formation
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 approach significantly improves the production efficiency and structural homogeneity of antigen-binding molecules with disulfide bonds, increasing the proportion of molecules with appropriate disulfide bonds and reducing the presence of mis-disulfide bonded forms.
Implementation Method 1
treating a preparation containing molecules having disulfide bonds between the heavy chains and molecules in which appropriate disulfide bonds are not formed with a reducing agent
Implementation Method 2
contacting antigen-binding molecules with a reducing agent and then removing the reducing agent, wherein the antigen-binding molecules are bound to an affinity column
Data Source
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AI summary
In one embodiment, it was discovered that a preparation containing antigen-binding molecules that have at least one disulfide bond formed between amino acid residues in regions other than the hinge region can be produced more efficiently and with high reproducibility by carrying out the following in chromatography: contacting a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in regions other than the hinge region and mis-disulfide bonded forms and/or non-disulfide bonded forms of the antigen-binding molecules with a solution containing a reducing agent; and subsequently, removing the reducing agent.