Mixed-Mode Chromatography

Mixed-mode chromatography combines two or more interaction mechanisms, typically ionic and hydrophobic, within a single stationary phase to help achieve enhanced selectivity. These interactions occur simultaneously and are dependent on buffer conditions, such as pH, conductivity, salt species, and additives, allowing scientists to fine-tune binding strength and impurity clearance. This approach enables efficient reduction of aggregates, host cell proteins, and other contaminants while maintaining product recovery. Mixed-mode resins are commonly used in both analytical and preparative chromatography columns, supporting consistent purification from early development through manufacturing-scale processes.

Mixed-mode chromatography integrates multiple interaction modes, such as ionic and hydrophobic interactions, into a single functional group. Ion exchange or hydrophobic interaction chromatography on the other hand utilize a single interaction mode. By combining these interactions, mixed-mode resins can reduce impurities that might otherwise require multiple purification steps. This approach simplifies downstream workflows, reduces processing time and buffer consumption, and enhances overall purification efficiency.

Mixed-mode chromatography can be applied when traditional ion exchange or hydrophobic interaction methods cannot achieve the desired selectivity or impurity reduction, or where standard processes fail to meet productivity goals. It can also be well-suited for moderate-to-high conductivity feedstreams, which can be challenging for traditional ion exchange chromatography. Its multiple interaction mechanisms make it particularly effective for reducing aggregates, host cell proteins, and other impurities in complex feedstocks. By improving selectivity and scalability, as well as offering different operating conditions, mixed-mode chromatography supports efficient, high-quality purification across development and manufacturing stages.

Mixed-mode chromatography can enhance mAb purification by combining multiple interaction modes, such as hydrophobic and ionic interactions, to selectively minimize aggregates, host cell proteins, and other impurities. This approach can improve product quality while maintaining high monomer recovery, even with challenging feedstreams. Because it combines multiple interaction modes into a single chromatography resin, MMC can offer highly efficient impurity reduction and reduce or replace multiple polishing steps.

Performance in mixed-mode chromatography depends on several key process variables, including pH, conductivity, salt species, additives, and load density. These factors can influence the balance between various interaction modes, which determine binding strength and impurity clearance. Optimizing these conditions is crucial for achieving consistent recovery and selectivity across various scales. Researchers can reference application data, design-of-experiments (DoE) studies, or process development tools to fine-tune parameters for optimal performance. Analytical methods, such as UHPLC or mass spectrometry, can further verify aggregate and impurity reduction during development.