1. Overview of Mixed-Mode Chromatography (MMC)
Ligands on MMC media interact with target proteins via multiple mechanisms, including hydrophobic interaction, electrostatic interaction, hydrogen bonding and other forces. Mixed-mode chromatography refers to a chromatographic technique where two or more interaction modes function independently or synergistically. Unlike ligands with defined specific binding properties, the interactions involved in MMC are mutually dependent.
MMC can be categorized into physical and chemical types. Physical MMC consists of stationary phases blended with two or more packing materials, while chemical MMC adopts a single type of packing medium endowed with dual or multiple functional groups. Commercially available mainstream MMC media are classified as follows:
Ion exchange & hydrophobic composite media: These media integrate hydrophobic, ionic, hydrogen-bonding and thioaffinity interactions, enabling protein capture under high-salt conditions without buffer dilution or buffer exchange procedures. Ion strength, pH value and buffer composition govern ion exchange and elution behaviors, whereas salt type, salt concentration and additives dominate hydrophobic interaction performance.
Sieve-ion exchange-hydrophobic triple-mode media: Featuring a core-shell microsphere structure, such media allow only small biomolecules to penetrate the shell pores and bind to core-coupled ligands, while target proteins flow through the column and are captured in flow-through mode.
Multifunctional composite media: The chromatographic media are functionalized by self-contained diversified interactive ligands. Integration of multiple protein separation modes significantly enhances separation selectivity. Such media can eliminate multiple process-related impurities in a single chromatographic step, greatly shortening downstream purification timelines.
2. Influence Mechanism of Additives on MMC
Different salts exert varying effects on interactive intensity in accordance with the Hofmeister series. Such ionic properties can be utilized to regulate the binding affinity between proteins and chromatographic media, thereby modulating protein retention and elution profiles.
Various additives adjust the retention and elution behaviors of biomolecules during chromatographic processes by altering hydrophobic forces, hydrogen bonding and other intermolecular interactions. Rational selection and application of additives can optimize chromatographic conditions, and further improve separation efficiency and resolution.
3. Application of MMC in Removing Product-Related Impurities
3.1 Protein Aggregates
Therapeutic proteins tend to form aggregates during purification, storage, transportation and administration, which is a prevalent form of protein instability. Aggregation is jointly determined by intrinsic protein properties and extrinsic factors including buffer composition, temperature, protein concentration and pH value.
Protein aggregation can be inhibited via site-directed mutagenesis and solution condition regulation (pH, ionic strength, temperature, pressure, etc.). For soluble protein aggregates, MMC resins such as HA, Capto MMC ImpRes and Capto Adhere exhibit superior removal efficiency compared with conventional single-mode media including Q Sepharose Fast Flow (ion exchange) and Phenyl Sepharose 6 FF (hydrophobic interaction), relying on differential physicochemical properties such as isoelectric point, surface charge density, charge distribution, hydrophobicity and molecular size.
Different salt combinations significantly affect the dynamic binding capacity, resolution and monomer purity performance of TOYOPEARL MX-Trp-650M resin in monoclonal antibody aggregate removal, verifying that salt system optimization is critical for improving downstream bioprocessing efficiency. In addition, MMC resins can serve as ideal alternatives to Protein A resins in capture and intermediate purification stages, effectively upgrading the overall quality and efficiency of downstream purification workflows.
3.2 Misfolded Proteins
Misfolded proteins possess higher surface hydrophobicity than natively folded counterparts, leading to stronger binding affinity to chromatographic media and requiring harsher elution conditions. Comparative studies on the binding and elution characteristics of misfolded proteins on Capto Adhere and Capto MMC have verified their capability to separate misfolded species from Protein A eluates.
At pH 7.5, a tandem downstream purification workflow combining Capto MMC (co-gradient elution via pH and salt modulation) and Capto Adhere (gradient elution with NaCl-arginine mixture) achieves efficient separation of misfolded etanercept. Arginine facilitates effective elution of misfolded proteins; NaCl weakens electrostatic interactions yet enhances hydrophobic interactions, while arginine hydrochloride attenuates both interactive forces simultaneously.
3.3 Charge Variants
Therapeutic proteins are divided into acidic and basic charge variants based on surface charge characteristics. Acidic variants carry excess negative charges generated via deamidation, glycosylation, incomplete disulfide bond formation, tyrosine sulfation, O-fucosylation, oxidation and isomerization. Basic variants are enriched with positive charges derived from C-terminal Lys/Arg retention, residual N-terminal Gln, succinimide intermediate formation, C-terminal amidation and isomerization.
Charge variants adversely affect the safety and efficacy of biotherapeutics, among which acidic variants impose more severe impacts on pharmaceutical potency. MMC resins show prominent application potential in the polishing stage for charge variant separation. Relevant studies confirm that single-step MMC purification platforms can eliminate 30% of acidic charge variants, completely remove monoclonal antibody fragments and deplete host cell proteins (HCP).
Integrated chromatographic workflows combining cation exchange chromatography and MMC enable high-efficiency separation of charge variants, aggregates, HCP and host cell DNA (HCD) in monoclonal antibody production, with qualified impurity clearance rates: aggregate content below 1%, HCP residual level less than 10 ppm and HCD residual level lower than 5 ppb. In summary, MMC-based impurity removal strategies streamline purification procedures and facilitate one-step integrated purification, breaking through the limitations of traditional multi-step processes and providing novel solutions for unresolved downstream purification challenges.
3.4 Protein Fragments
Protein fragmentation is triggered by enzymatic and chemical cleavage of peptide bonds. Proteases are released during host cell lysis in bioproduction processes, while peptide bond chemical degradation is induced by high temperature, metal ions (copper, iron), extreme pH values and ultraviolet irradiation. Distinct structural domains show varying susceptibility to cleavage; the hinge region is the primary cleavage site of antibodies, and inter-domain interfaces act as secondary vulnerable regions. Protein fragmentation leads to declined biological activity, shortened in vivo half-life and elevated immunogenicity caused by neoepitope exposure.
MMC media including Capto Adhere, HEA Hypercel and PPA Hypercel are validated for removing large hinge fragments (LHF) from intact IgG1 monomers. Experimental results demonstrate that over 75% of LHF can be eliminated via MMC under pH 8.0 and low conductivity conditions, whereas conventional single-mode ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC) fail to achieve effective LHF separation.
4. Application of MMC in Removing Host-Derived Impurities
4.1 Host Cell Proteins (HCP)
The residual limit of HCP in final drug substances is generally controlled below 100 ng/mL, as residual HCP may trigger immunogenic responses, adjuvant effects, abnormal biological activities and enzymatic interference. HCPs are prone to co-purify with target products due to similar physicochemical properties. Clarification of HCP intrinsic properties such as isoelectric point distribution contributes to targeted separation strategy design.
For instance, most E. coli-derived proteins have isoelectric points ranging from 5.0 to 7.0; accordingly, anion exchange media or dialysis are adopted for bulk HCP removal, followed by cation exchange chromatography for residual alkaline protein elimination. EDTA is commonly supplemented in buffers to chelate heavy metal ions and inhibit endogenous protease activity. Based on differential surface charge and hydrophobicity distribution between HCP and target proteins, MMC resins represented by Capto Adhere, Capto MMC and HA resins achieve remarkable HCP clearance effects in bind-elute and flow-through purification modes. Novel multifunctional MMC media are continuously developed to address the complexity of HCP derived from diverse expression hosts.
4.2 Host Cell DNA (HCD)
Genomic DNA and plasmid DNA constitute major DNA-derived impurities in biological samples, with the total residual DNA limit strictly regulated below 10 ng per therapeutic dose. DNA molecules bind to chromatographic media via electrostatic and hydrophobic interactions. Cationic mixed-mode ligands outperform anion exchange media in monoclonal antibody polishing, owing to their inert binding characteristics towards phospholipids and DNA molecules.
Capto Adhere has been successfully applied in plasmid DNA purification from E. coli cell lysates, featuring excellent process robustness and high dynamic binding capacity. Single-step NaCl gradient elution realizes efficient separation of plasmid DNA from host proteins and endotoxins. Although systematic research on MMC-mediated HCD removal in polishing processes remains insufficient, existing application cases confirm the great potential of MMC resins for HCD-targeted separation.
5. Application of MMC in Removing Process-Related Impurities
5.1 Viral Impurities
Viral clearance is an indispensable unit operation in biopharmaceutical manufacturing, which requires combined application of viral inactivation and viral removal strategies. Viral inactivation is achieved via physical means (heat treatment, irradiation) or chemical methods (low-pH incubation, detergent treatment). Viral removal strategies are formulated based on viral structural characteristics: non-enveloped viruses are resistant to solvent-detergent inactivation, and tiny viruses inaccessible to nanofiltration membranes can be rapidly eliminated via chromatographic media.
Given the diversified surface properties of various viruses, systematic characterization of viral physicochemical features lays a foundation for rational chromatographic process design. MMC resins exhibit superior robustness in viral clearance. Capto Adhere eliminates multiple types of viruses through synergistic multi-interaction mechanisms, and Capto MMC can be integrated with detergent-mediated viral inactivation procedures to optimize overall purification workflows. Collectively, MMC resins possess more prominent advantages in viral removal compared with traditional HIC and other single-mode chromatographic media.
5.2 Endotoxins
Endotoxin removal is essential to guarantee the safety of biotherapeutics, and adsorptive chromatography is recognized as one of the most efficient endotoxin removal technologies, whose process optimization relies on clarifying the interaction mechanism between endotoxins and chromatographic media. Although there is a lack of dedicated research on MMC-based endotoxin removal, the unique interactive properties between MMC ligands, target proteins and endotoxins endow MMC with promising application prospects, especially cationic mixed-mode resins with prominent research and application potential in this field.
Conclusion
MMC resins capture and separate target proteins through synergistic electrostatic, hydrophobic, hydrogen-bonding, thioaffinity and other intermolecular interactions. Different types of additives exert differentiated regulatory effects on protein binding affinity and separation selectivity of MMC media, which can be leveraged for systematic purification process optimization.
MMC achieves efficient clearance of protein aggregates, misfolded species and charge variants, and delivers outstanding performance in HCP and viral impurity removal, meanwhile possessing latent application value for HCD and endotoxin elimination. MMC boasts broad application prospects in the biopharmaceutical industry. Further in-depth researches focusing on complex impurity removal mechanisms and systematic purification condition optimization are still required to meet increasingly stringent pharmaceutical quality control standards.
