Mixed-mode chromatography (MMC) uses ligands that combine two or more interaction mechanisms - most commonly ion-exchange and hydrophobic interaction - to separate product-related impurities that share enough biophysical similarity with the target antibody to defeat single-mode purification strategies. As mAb pipelines expand to include bispecific antibodies, Fc-fusions, and antibody-drug conjugates (ADCs), and as intensified upstream processes drive higher impurity loads through the downstream train, MMC has moved from a niche polishing option to a strategically important step in modern biopharmaceutical purification platforms.
Quick Take
- Mixed-mode resins exploit simultaneous ionic, hydrophobic, hydrogen-bonding, and in some ligand architectures pi-pi interactions, giving a selectivity window that no single-mode technique can replicate
- MMC operates in either bind-and-elute or flow-through mode depending on the target impurity profile and process position - mode selection is as important as resin selection
- High-molecular-weight (HMW) aggregates, low-molecular-weight (LMW) fragments, host cell proteins (HCPs), and acidic charge variants are all addressable within a single MMC step under the right conditions
- Salt tolerance is a structural advantage of many MMC resins - the ability to process clarified harvests at elevated conductivity without buffer exchange reduces process complexity
- Complex modalities including bispecifics and ADCs are driving increased interest in MMC as a polishing step capable of handling the structural heterogeneity these molecules introduce
What Makes Mixed-Mode Chromatography Different from Conventional Polishing
The selectivity of any chromatography step is determined by the interactions available between the stationary phase ligand and the proteins in the feedstock. Ion-exchange chromatography (IEX) separates on charge; hydrophobic interaction chromatography (HIC) separates on surface hydrophobicity; size-exclusion chromatography (SEC) separates on hydrodynamic radius. Each mechanism is powerful within its domain and limited outside it. Product-related impurities - particularly aggregates and charge variants - often share enough similarity with the target mAb in charge, hydrophobicity, or size that no single mechanism provides adequate resolution.
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MMC ligands are designed to engage multiple mechanisms simultaneously. A typical multimodal cation exchange (MMCEX) ligand presents both a charged group and a hydrophobic aromatic or aliphatic moiety on the same scaffold, creating a binding interaction that depends on the combined surface properties of the protein rather than any single attribute. This cooperative binding mechanism is what enables MMC to resolve species that are nearly indistinguishable by IEX or HIC alone. A comprehensive 2026 review in Biotechnology and Bioengineering covering all commercially available multimodal resins confirmed that the integration of ionic, hydrophobic, hydrogen-bonding, and pi-pi interactions gives MMC a broader selectivity window than conventional single-mode techniques, enabling efficient impurity removal across diverse mAb formats and feed compositions.
Bind-and-Elute vs. Flow-Through: Choosing the Right Operating Mode
MMC resins can be deployed in either bind-and-elute or flow-through mode, and the choice fundamentally changes what the step is designed to accomplish. In bind-and-elute mode, the target protein is captured on the resin and eluted under conditions that release it while retaining impurities. This approach offers the highest resolving power and is appropriate when the target molecule needs to be concentrated or when specific impurity species require targeted separation through differential elution. The trade-off is method development complexity: defining the binding, wash, and elution conditions that maximize yield while achieving the required clearance is a multi-variable optimization problem.
Flow-through mode positions the target protein as the non-binding species. Load conditions - typically controlled by pH and conductivity - are set so that the target antibody passes through the column while impurities including HCPs, aggregates, and charge variants bind to the resin and are retained. This approach is operationally simpler, tolerates higher load challenges, and is more readily integrated into platform downstream processes. The published data for a single-step flow-through polishing workflow following Protein A capture showed mAb yields above 93% at loading densities approaching 1000 mg/ml resin, while reducing aggregate content from 2.3% to below 1.0% - a clearance performance that would require two conventional polishing steps to achieve.
Impurity Classes That Mixed-Mode Chromatography Addresses
MMC's multi-interaction selectivity makes it effective against several distinct impurity classes within a single step, which is its principal economic and operational advantage over traditional two-step polishing sequences.
HMW aggregates are the most clinically significant product-related impurity, carrying immunogenicity risk that makes their removal a regulatory requirement. MMC resins operating in flow-through mode have demonstrated robust aggregate clearance, with a 2025 study in Molecules examining multiple commercial multimodal resins and reporting that cooperative hydrophobic and ionic interactions allow aggregate species to be selectively retained while monomeric antibody passes through - a separation that is difficult to achieve by IEX alone because aggregates and monomer share similar charge density.
LMW fragments, including Fab, Fc, and clipped species in the 25-120 kDa range, present a different challenge. Fragment binding to MMC resins is hindered by the absence or incomplete presentation of the hinge region, which alters the accessible surface area for ligand interaction relative to intact IgG. This structural difference is what MMC exploits to separate fragments from intact mAb at differential ionic strengths - a mechanism that conventional IEX does not reliably replicate because fragments and intact antibody may elute at similar conductivities. Multimodal platforms have demonstrated complete removal of fragment species across multiple IgG1 and IgG4 programs while maintaining step yields above 95% and monomer purity above 99%.
HCP clearance is a third application where mixed-mode chromatography adds value beyond what standard polishing delivers. Persistent HCPs that survive Protein A capture and IEX polishing tend to be those with hydrophobic character or strong electrostatic affinity for the target antibody. MMC resins engaging hydrophobic and ionic interactions simultaneously are better positioned to capture these species than anion exchange (AEX) alone, which relies purely on charge to differentiate HCPs from the target antibody.
| Impurity Class | Primary MMC Mechanism | Preferred Mode | Key Advantage Over Single-Mode |
|---|---|---|---|
| HMW Aggregates | Combined hydrophobic and ionic | Flow-through | Aggregates retained; monomer passes |
| LMW Fragments | Differential surface interaction | Bind-and-elute or flow-through | Hinge-region absence exploited |
| Host Cell Proteins | Hydrophobic and ionic | Flow-through | Persistent HCPs not cleared by AEX |
| Acidic Charge Variants | Ionic, pH-dependent | Bind-and-elute | Resolves species IEX cannot separate |
| Leached Protein A | Hydrophobic and ionic | Flow-through | Removes Protein A ligand carryover |
Method Development: pH, Conductivity, and Load Optimization
The larger design space that MMC ligands offer - a consequence of having multiple controllable interaction parameters - is both an advantage and a development challenge. pH and conductivity interact in MMC in ways they do not in single-mode IEX: reducing conductivity strengthens ionic interactions while simultaneously exposing hydrophobic binding sites, and pH changes alter the ionization state of both the ligand and the protein surface in ways that affect hydrophobic as well as electrostatic contacts. This interdependence means that screening pH and conductivity independently underestimates the design space and that design-of-experiment (DoE) approaches are particularly well-suited to MMC method development.
Elution strategy in bind-and-elute mode is equally important to mixed-mode chromatography method development. For multimodal cation exchange resins, elution is typically achieved at elevated pH - commonly between pH 7 and 9 - where deprotonation of the carboxyl group on the ligand creates charge repulsion that releases the target protein. The pH at which this transition occurs is ligand-specific and must be characterized for each resin-molecule combination. For flow-through applications, load pH and conductivity define the selectivity entirely, and the operating window must be wide enough to accommodate feed variability across batches without compromising impurity clearance.
MMC in Downstream Sequences for Complex Modalities
Bispecific antibodies and ADCs present downstream processing challenges that have accelerated interest in MMC beyond its established role in standard mAb polishing. Bispecifics produced by knob-into-hole or other asymmetric Fc engineering strategies generate homodimer impurities that co-elute with the target in Protein A capture and are difficult to resolve by IEX alone because of their similar charge density. MMC resins with combined ionic and hydrophobic selectivity can differentiate between the target bispecific and homodimer species based on differential surface hydrophobicity - particularly relevant for bispecifics that carry hydrophobic domains in their variable regions.
For ADCs, the drug-antibody ratio (DAR) distribution introduced by conjugation creates a mixture of species that differ in hydrophobicity proportional to the number of attached drug molecules. HIC is the preferred polishing step for DAR-based separation, but unconjugated antibody and low-DAR species that co-elute under standard HIC conditions can sometimes be resolved using MMC resins that add ionic selectivity to the hydrophobic separation. MMC's role in ADC polishing is still being defined in the literature, but as a complementary or alternative polishing step it sits within the same downstream purification sequence as HIC, AEX, and CEX, and the choice between them depends on the specific impurity profile of the molecule in development.
Mixed-Mode Chromatography in mAb Polishing: Key Considerations for Method Developers
Mixed-mode chromatography resolves impurity challenges that ion-exchange and hydrophobic interaction chromatography cannot address independently, making it a strategically important option for purification scientists working with complex mAb formats and high-titer feeds. Its effectiveness depends on selecting the right resin chemistry for the target impurity class, choosing between bind-and-elute and flow-through modes based on the process position and clearance requirements, and using DoE to map the pH-conductivity design space rather than optimizing parameters sequentially. The technique fits naturally into a broader advanced chromatography strategy that also includes optimizing the Protein A capture step upstream, since the load challenge presented to any MMC polishing step is directly shaped by how well the capture step performs.
This article was produced under Separation Science's AI Editorial Guidelines.
