Mixed Mode Chromatography with Nonionic Polymers for Antibody Purification
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Solution Overview
Problem
Current mixed mode chromatography methods for protein purification, such as hydroxyapatite and fluorapatite, face challenges in binding capacity and selectivity, particularly for antibodies, due to phosphate and salt concentration effects, leading to economic limitations and inefficiencies in separating non-aggregated antibodies from aggregates and contaminants.
Innovation Solution
The use of aqueous-soluble nonionic organic polymers, like polyethylene glycol (PEG), enhances binding capacity and selectivity on mixed mode chromatography supports by delaying elution and preferentially retaining antibodies and large molecules, allowing for improved separation and removal of contaminants, including viruses, across various chromatography methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If mixed mode chromatography is used for protein purification, then selectivity for separating proteins is improved, but binding capacity is reduced due to phosphate and salt concentration effects
Solution Approach 1:
Aqueous-soluble nonionic organic polymers act as intermediaries that modify the chromatographic environment. These polymers preferentially interact with contaminants and aggregated proteins, allowing the mixed mode chromatography support to maintain its selectivity while the polymer handles the capacity limitations by selectively retaining interfering substances in solution.
2Ease of operation
If phosphate salts are used for elution in mixed mode chromatography, then calcium affinity binding is disrupted for elution, but binding capacity is reduced
Solution Approach 1:
The aqueous-soluble nonionic organic polymer serves as a mediator that allows elution to proceed without requiring high phosphate concentrations. The polymer maintains selective retention of contaminants while the chromatography support can be eluted with lower phosphate concentrations or alternative elution strategies, preserving binding capacity for subsequent cycles.
3Manufacturing precision
If mixed mode chromatography supports are used, then unique selectivities are achieved, but method development becomes complicated and requires extensive resources
Solution Approach 1:
The addition of aqueous-soluble nonionic organic polymers creates a new controllable parameter in the chromatographic system. This parameter provides an additional degree of freedom for method development, allowing optimization of selectivity and capacity independently. The polymer concentration and type can be adjusted to fine-tune the separation without requiring complex support modifications.
4Manufacturing precision
If mixed mode chromatography is used for antibody purification, then separation of antibodies from contaminants is improved, but productivity is reduced due to lower binding capacity
Solution Approach 1:
The aqueous-soluble nonionic organic polymer acts as a capacity-enhancing intermediary. By selectively binding to contaminants and aggregated antibodies in the feed solution, the polymer increases the effective binding capacity of the chromatography support for monomeric antibodies. This allows higher antibody loads to be processed while maintaining purification quality.
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 presence of nonionic organic polymers like PEG increases protein binding capacity, delays elution, and enhances selectivity, enabling more effective purification of antibodies and viral clearance, regardless of the specific chromatography method used, thus improving productivity and purity.
Implementation Method 1
Mixed mode chromatography involves the use of solid phase chromatographic supports that employ multiple chemical mechanisms to adsorb proteins or other solutes
Implementation Method 2
chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity
Implementation Method 3
The presence of nonionic organic polymer preferentially enhances the retention of antibody on mixed mode chromatography supports in comparison to most contaminating proteins
Implementation Method 4
aqueous-soluble nonionic organic polymers, like polyethylene glycol (PEG), enhances binding capacity and selectivity
Implementation Method 5
The presence of nonionic organic polymer preferentially enhances retention of aggregated antibody and other very large molecules (e.g., viruses) on mixed mode chromatography supports in comparison to non-aggregated antibody
Data Source
AI summary
This invention relates to the use of mixed mode chromatography for purification of a protein from a mixture containing other materials, including fragmented or aggregated antibodies, host cell proteins, DNA, endotoxin, and/or virus. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies or other proteins suitable for in vivo applications.