Bispecific Antibody Refolding via Redox Buffer
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Solution Overview
Problem
Current downstream manufacturing processes for bispecific antibody constructs, such as BiTE® antibody constructs, face challenges in reducing aggregation, which leads to reduced yield and increased production costs, particularly in industrial mammalian CHO cell cultures, where about 25% or more of produced constructs are obtained in aggregated form, necessitating inefficient removal steps like cation exchange chromatography.
Innovation Solution
A downstream method involving redox refolding with a buffer containing a chaotropic agent like guanidine and an oxidizing agent like copper (II) is applied to refold bispecific antibody constructs, increasing the monomeric content from 25% to 85% or more, thereby reducing aggregation and enhancing production yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional downstream manufacturing processes are used for bispecific antibody constructs, then production can proceed with standard purification steps, but about 25% or more of constructs are obtained in aggregated form, reducing yield and increasing costs
Solution Approach 1:
The patent applies redox refolding treatment before the final purification step to prevent aggregation from occurring in the first place. By treating the bispecific antibody constructs with reducing agents (DTT or β-mercaptoethanol) followed by oxidizing agents and chaotropic agents (guanidine hydrochloride or guanidine sulfate) at controlled pH and temperature, the constructs are refolded into their native monomeric form before purification, thereby maximizing yield and minimizing aggregation-related losses
Solution Approach 2:
The patent systematically optimizes multiple parameters including pH (maintained between 6.0-8.0 during refolding), temperature (4°C or room temperature), chaotropic agent concentration (1-4 M guanidine), and redox agent concentrations to achieve optimal refolding conditions. These parameter changes enable the conversion of aggregated constructs back to monomeric form, directly improving both productivity and manufacturing precision
2Manufacturing precision
If aggregation removal steps like cation exchange chromatography are added to eliminate aggregated constructs, then product purity is improved, but production costs increase and process complexity increases
Solution Approach 1:
By performing redox refolding treatment before purification, the patent prevents aggregation from persisting into the purification step. This preliminary action ensures that the majority of constructs are already in monomeric form, reducing the burden on subsequent purification steps and minimizing the need for complex aggregation removal protocols
Solution Approach 2:
The patent extracts the aggregation problem from the purification process by addressing it in a separate refolding step. Instead of relying solely on chromatography to remove aggregates, the aggregation issue is resolved through biochemical refolding, allowing simpler and more cost-effective purification steps to be used
3Manufacturing precision
If aggregation removal steps like cation exchange chromatography are added to eliminate aggregated constructs, then product purity is improved, but production costs increase
Solution Approach 1:
The redox refolding treatment is performed as a preliminary step before purification, preventing aggregation-related losses and reducing the need for expensive aggregation removal chromatography steps. This approach maintains high product purity while avoiding the high costs associated with additional purification steps
Solution Approach 2:
The patent converts the harmful aggregated form into the beneficial monomeric form through redox refolding. By using reducing agents to break disulfide bonds followed by controlled oxidation in the presence of chaotropic agents, aggregated constructs are transformed back into functional monomers, turning a quality problem into an opportunity to enhance product yield and purity
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 increases the percentage of monomeric bispecific antibody constructs, improving product quality and reducing production costs by integrating a resource-efficient refolding step into existing downstream processing, specifically by using redox refolding conditions that include chaotropic and oxidizing agents to restore native disulfide bonds.
Implementation Method 1
contacting the construct with a refolding buffer to refold the construct, the buffer comprising a chaotropic agent
Implementation Method 2
buffer comprising an oxidizing agent to form disulfide bonds
Implementation Method 3
redox refolding conditions that include chaotropic and oxidizing agents to restore native disulfide bonds
Data Source
AI summary
The present invention provides an improved manufacturing method for the production of bispecific antibody products, which comprise at least two binding domains. The process comprises at least the method comprising a step of refolding the construct, wherein one or more of the domains comprise a disulfide bond, and wherein the construct is contacted with a refolding buffer to refold the construct to its native form, the buffer comprising (i) a redox and/or oxidizing agent having a concentration of at least 0.1 mM and (ii) a chaotropic agent having a concentration >1 M, and wherein the pH of the refolding buffer corresponds to +/−4.5 of the pI value of the first domain or +/−4.0 of the pI value of the entire construct.