Mixed-Mode Chromatography

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Enhance selectivity with mixed-mode chromatography

Mixed-mode chromatography (MMC), also referred to as multimodal chromatography, combines two or more modes of interaction, such as ionic and hydrophobic, within a single functional group. This multiple-interaction approach can enable orthogonal or enhanced selectivity as compared to single-mode separation techniques. In some cases, it can also help streamline purification by reducing the number of polishing steps needed to achieve product quality targets. Mixed mode chromatography can be a valuable tool for purification of challenging feedstreams, offering enhanced selectivity and enabling scientists to develop robust and scalable downstream processes.

Benefits of mixed-mode chromatography

MMC supports the purification of complex feedstreams and can enhance overall downstream efficiency. By combining multiple interaction mechanisms, MMC can be utilized to promote unique selectivity, product quality, and increased process efficiency.

This approach can:

  • Improve product quality by efficiently reducing aggregates, host cell proteins (HCPs), and other product and process-related impurities
  • Reduce process rework and variability through greater selectivity and robustness
  • Optimize resource utilization with lower buffer consumption and fewer polishing steps
  • Support sustainable scalability by maintaining efficiency from development to cGMP manufacturing

Achieve reliable purification with mixed-mode resins

The Thermo Scientific POROS Caprylate Mixed-Mode Cation Exchange Chromatography Resin combines hydrophobic and weak cation exchange functionality to enable selective impurity reduction in flow-through mode. Designed for efficient reduction of aggregates, HCPs, and other impurities, POROS Caprylate resin supports high monomer recovery and reproducible performance over a large operating space. Its rigid polymer backbone and large throughpores result in low backpressure and high throughput, maintaining scalability from process development through cGMP manufacturing. These properties make POROS Caprylate resin an excellent choice for achieving reliable, high-quality purification outcomes in complex workflows.

Supporting diverse modalities and applications

ADCs

Antibody-Drug Conjugates (ADCs) workflows rely on chromatography steps to separate complex conjugates and reduce impurities. These approaches support consistent production of ADCs for therapeutic research and biomanufacturing.
 

Cell Therapy

Cell therapies utilize living cells as treatments, necessitating strict control over inputs and processing steps. Chromatography is applied to prepare ancillary materials, such as cytokines or viral vectors, that are used during cell manufacturing.
 

Gene Therapy

Gene therapy often relies on viral vectors, such as adeno-associated virus (AAV) or lentivirus, to facilitate the transfer of genetic material. Chromatography separates drug product from product- and process-related impurities in both capture and polishing steps.

mAbs

Monoclonal antibody purification typically starts with Protein A chromatography to capture the antibody from cell culture fluid. Additional steps, such as ion exchange, are used to help reduce aggregates, variants, or process-related impurities.
 

mRNA

Chromatography in mRNA production separates full-length transcripts from double-stranded RNA and truncated products. The purified RNA can then be used in formulation or downstream steps, such as lipid nanoparticle assembly.
 

Vaccines

Chromatography is often used in vaccine manufacturing for both the capture and polishing of the vaccine drug product. The choice of method depends on the platform, for example, protein subunits, viral vectors, or nucleic acid vaccines.
 

Explore related chromatography techniques

Affinity chromatography

Affinity chromatography aims to simplify target capture through specific, reversible interactions between a ligand and its target biomolecule. This selectivity enables efficient isolation of the desired molecule while minimizing co-purified impurities, supporting cleaner recovery and high product integrity during capture.
 

Continuous chromatography

Continuous downstream processing links multiple chromatography steps in sequence, operating with little to no interruption for improved throughput and reduced cycle times. By minimizing downtime and optimizing resin use, this approach supports higher productivity and more sustainable, cost-effective manufacturing.

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step to improve purity and resolution by exploiting differences in hydrophobicity between the target molecule and impurities. HIC can be utilized in bind/elute or flow-through modes.
 

Multi-column chromatography

Multi-column chromatography (MCC) uses several columns running in parallel to perform different purification steps simultaneously. By alternating loading, washing, and elution cycles, MCC can increase resin utilization, shorten process time, and enable continuous, high-efficiency purification.

Explore more chromatography resins

Thermo Fisher Scientific offers POROS ion-exchange, hydrophobic interaction, and mixed-mode chromatography resins for large-scale bioseparations, along with MabCaptureC and CaptureSelect affinity resins designed for efficient purification of mAbs, advanced antibody variants, recombinant proteins, mRNA, viruses, and other biologics.
 

Find the right chromatographic method for your process

Selecting the optimal chromatography approach is important for achieving consistent yield, purity, and scalability in downstream purification. Whether optimizing a polishing step or implementing continuous operation, the right combination of resins and formats can support process intensification, reducing cycle times, improving throughput, and minimizing buffer use. Thermo Fisher Scientific offers chromatography solutions that align with your productivity goals and accelerate progress from development through cGMP manufacturing.

Explore other chromatography resins

Affinity chromatography resins

Affinity resins allow capture with high purity and yield in a single step. While widely used for antibody purification, CaptureSelect technology also supports bispecific antibodies, fragments, Fc-fusion proteins, recombinant proteins, and viral vectors using mild elution conditions.

 

Hydrophobic interaction chromatography resins

HIC separates biomolecules based on surface hydrophobicity. Commonly used during polishing, HIC resins can enhance overall process efficiency




 

Ion exchange chromatography resins

Ion exchange (IEX) chromatography separates biomolecules through charge-based interactions with functional groups. These resins are available in strong and weak base or acid chemistries, suitable for bind/elute or flow-through purification of antibodies, vaccines, viral vectors, and other biologics.

Frequently asked questions

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.

Chromatography resources


Access resources that cover chromatography performance, purification strategies, and method development. These resources empower scientists to select and apply chromatography resins from research to cGMP manufacturing.

Enhance your mixed-mode chromatography process

For Research Use or Further Manufacturing. Not for use in diagnostic procedures.