Eshmuno^® CMX Resin: A guide to mixed mode chromatography

Section overview

• What is mixed mode chromatography?
• How do mixed mode resins improve purification?
• Mixed mode resins for hydrophobic mAb purification
• Mixed mode resins for fusion protein purification
• ADC purification and glycoprotein purification using mixed-mode resins
• Key takeaways on Eshmuno^® CMX mixed-mode chromatography resin
• Frequently asked questions
• Related products

What is mixed-mode chromatography?

In mixed-mode chromatography, a sorbent is engineered with ligands that interact with proteins through multiple binding mechanisms. This multi-mode interaction increases selectivity and often provides cleaner separations than either interaction mode alone. Interaction mechanisms can include ion exchange, hydrophobic interaction, affinity, and size exclusion. For mixed-mode chromatography, the precise spatial positioning of the ligand’s structural elements is important to achieve binding of the target protein. Although the ligands enable different interactions with the target protein, the interactions are not independent as adjusting conditions to alter one interaction, may affect binding through the other mode.¹

How do mixed-mode resins improve purification?

Mixed-mode resins can enhance selectivity and product binding though interactions based on more than one binding mechanism. Because binding relies on multiple interaction  mechanisms, mixed-mode resins can offer improved binding under a broad range of process conditions. In addition, the dual binding mechanisms offer opportunities for reducing the number of steps needed for protein purification, and in some cases can solve protein purification challenges.²

Eshmuno^® CMX is a mixed mode chromatography resin that combines weak cation exchange (CEX) with moderate hydrophobic interaction properties and provides high selectivity for monoclonal antibodies (mAb), fusion proteins and antibody drug conjugate (ADC) separation, with effective removal of low molecular weight impurities and host cell proteins (HCPs).

This high-performance resin uses Eshmuno^® bead technology which has an innovative surface tentacle structure that effectively binds the target while the rigid hydrophilic polyvinyl ether base matrix enables high flow rates and shorter processing times.

Eshmuno^® CMX resin enables users to:

• Streamline workflows and reduce costs by reducing the number of chromatographic purification steps
• Improve purification performance through higher selectivity, superior dynamic binding capacity (DBC) and increased recovery 

The broad operational window for Eshmuno^® CMX resin means that pH and conductivity conditions can be adapted to maximize product binding, Figure 1. Our High Throughput Screening Protocol provides guidance on how best to quickly identify suitable purification conditions for your molecule.

Eshmuno^® CMX resin is supported by the Emprove^® Program which provides comprehensive documentation and regulatory information and helps streamline resin selection and qualification.

For more information on our resins for purification, visit our ion exchange chromatography resins webpage.

Mixed mode resins for hydrophobic mAb purification

The development of next-generation antibody therapeutics is increasingly focused on complex molecular formats, including bispecific antibodies (BsAbs), multispecific molecules, antibody fragments, fusion proteins and ADCs. While these advanced modalities offer significant therapeutic advantages, they also present new purification challenges compared with mAbs, and many exhibit greater hydrophobicity and a higher propensity to form aggregates, making them difficult to purify using conventional chromatography workflows.²

Eshmuno^® CMX resin combines weak cation exchange with moderate hydrophobic interactions to provide enhanced selectivity for these challenging molecules. By leveraging two complementary binding mechanisms, the resin enables efficient separation of product-related impurities while maintaining high product recovery.

To demonstrate its performance, Eshmuno^® CMX resin was evaluated for the purification of a highly hydrophobic BsAb. Following high-throughput screening to identify optimal operating conditions, the resin achieved a dynamic binding capacity (DBC_10%) of 64 g/L while enabling effective removal of low-molecular-weight (LMW) species, high-molecular-weight (HMW) aggregates, and host cell proteins (HCPs).²

Chromatography columns (bed height 20 cm, diameter 1 cm) packed with Eshmuno^® CMX resin were equilibrated in 5 column volumes (CV) of equilibration buffer (50 mM sodium acetate, 25 mM sodium sulfate, pH 5.0) to promote antibody capture. Clarified antibody harvest material (loading: 10 g/L) was applied to reach a DBC_10% of 64 g/L resin.

After loading, the column was washed with 3.5 CV of washing buffer 1 (100 mM MES, pH 5.5) to saturate resin buffering capacity, followed by 5 CV of wash buffer 2 (15mM Tris/acetate, pH 9.0) to reduce low molecular weight (LMW) and HCP bound to antibody. A third wash (6 CV, 50 mM sodium citrate, 100 mM sodium sulfate, pH 6.1) conditioned the resin for elution with 10 CV of elution buffer (100 mM sodium succinate, 300 mM sodium sulfate at both pH 6.0 or 6.2), performed in two separate runs. All chromatography run conditions are as described in Koehnlein et al (2022).^² Figure 3 shows the purification profile after elution at pH 6.2.

Table 1 summarizes impurity removal and process performance. Eshmuno^® CMX resin reduced LMW impurities by ~10-fold and HMW species by 5-fold, resulting in main peak purity of >97% and product recovery of ~70% at pH 6.2. HCP were reduced by > 3 logs to levels near the assay detection limit. Although residual DNA was substantially reduced, a subsequent anion exchange (AEX) polishing step remained necessary to achieve final DNA clearance.

Overall, Eshmuno^® CMX resin delivered impurity removal comparable to that typically achieved using a conventional two-step purification sequence combining CEX and HIC. By consolidating the CEX and HIC functionalities into a single chromatography step, Eshmuno^® CMX resin has the potential to simplify downstream processing, reduce operating costs, improve process efficiency, and minimize product losses associated to additional purification steps.³

For additional experimental details and results from studies with other challenging antibody formats, see Koehnlein et al. (2022).

Mixed mode resins for fusion protein purification

Fusion proteins comprise the Fc part of a human IgG and a receptor and are often developed to prolong therapeutic efficacy or improve stability or solubility of a therapeutic. Protein aggregates represent a major purification challenge for fusion proteins because they are considered critical quality attributes that must be minimized during downstream processing to avoid unintended immune responses.⁴

A study was performed with a biomanufacturer to evaluate the performance of Eshmuno^® CMX resin for purification of a fusion protein and to compare performance with their existing purification template with competitive products.

The eluate (25 mg/mL) from a Protein A capture column containing Eshmuno^® A resin was loaded on a column (0.5 cm i.d., 10 cm length) containing Eshmuno^® CMX resin. Efficient separation of the fusion protein from high molecular weight aggregates was achieved using a gradient elution (A: 300 mM phosphate buffer pH 4.5 and B: 300 mM phosphate buffer pH 10.0), Figure 4.