Mixed Mode Chromatography (MMC) Technology

Mixed-mode chromatography (MMC), an alternative or complementary tool to traditional reversed-phase chromatography (RP), ion-exchange chromatography (IEX), and normal-phase chromatography (NP), has been successfully used in research, process development, and bioprocessing to purify proteins and peptide molecules that are difficult to separate by other chromatographic methods. CD Formulation integrates cutting-edge MMC technology into our protein characterization technology platform to provide reliable analytical support for all stages of the development and production of your protein/peptide drugs.

What is Mixed Mode Chromatography Technology?

MMC, also known as multimodal chromatography, involves the combined use of two (or more) retention mechanisms in a single chromatographic system. This technique has great applications in separating compounds that are not retained or not well separated by typical reversed-phase liquid chromatography methods. Since a single MMC column can provide multiple retention modes, higher selectivity and purity can be achieved in a single step than traditional single-mode chromatography by adjusting the mobile phase conditions. In addition, mixed-mode media are an effective way to clean up complex sample matrices for purification or sensitive detection of trace analytes.

Mixed Mode Chromatography (MMC) Principle

In MMC, the ligands fixed on the resin interact with the target protein molecules through multiple types of interactions, among which size exclusion, ionic and hydrophobic interactions are the most important. According to the type of interaction combined, mixed-mode stationary phases can be divided into the following four categories.

• Reversed-phase hydrophilic interaction chromatography (RP-HILIC): It combines the hydrophobicity of RP chromatography and the hydrophilicity of HIC, and is suitable for separating polar and non-polar molecules.
• Reversed-phase ion exchange chromatography (RP-IEX): It uses the characteristics of RP and IEX at the same time, and can selectively separate charged biomacromolecules by adjusting pH and salt concentration.
• Hydrophilic interaction-ion exchange chromatography (HILIC-IEX): It combines the characteristics of HIC and IEX, and is suitable for separating polar molecules, especially at higher salt concentrations.
• Tri-mode mixed-mode high-performance liquid chromatography (MM-HPLC): It can combine the characteristics of RP, HIC, and IEX at the same time, providing higher separation flexibility and selectivity.

Its multi-mode combination not only expands the separation mechanism but also enhances the selectivity and separation efficiency of various proteins. Different types of interactions can effectively separate the target protein when dealing with samples containing multiple similar proteins, thereby improving the degree of purification.

Fig. 1 Types of RP-IEX bimodal mixed-mode columns. (Zhang K, et al., 2016)

Our Services Related to Mixed Mode Chromatography (MMC) Technology

Thanks to decades of experience supporting protein/peptide biopharmaceutical development and manufacturing using MMC technology, our team of highly qualified experts offers a range of MMC-related services to accelerate the implementation and success of your project.

Our experienced team of experts has completed hundreds of MMC separation and purification projects for protein and peptide, supporting all stages of your protein/peptide drug development and manufacturing - from early studies to downstream process monitoring and GMP batch release testing.

Utilizing cutting-edge MMC technology, we support the following protein/peptide development and characterization plans, including but not limited to:

Protein/Peptide Purification

We leverage the unique properties of MMC to efficiently capture target proteins from complex mixtures, thereby increasing yield and purity. In the final polishing step, we use MMC to remove residual impurities, aggregates, and misfolded species.

Aggregate Characterization

By integrating size exclusion technology into MMC, our scientists use MMC to separate variants with different properties to help identify and quantify post-translational modifications (PTMs) such as phosphorylation, glycosylation, and oxidation, as well as analyze protein aggregation states and ensure sample homogeneity prior to functional assays.

Isoform Separation

If a protein exists as multiple isoforms, MMC can facilitate their separation based on subtle differences in charge, hydrophobicity, or other interactions.

Quality Control

Our scientists use MMC technology to monitor the distribution of isoforms and the presence of aggregates to ensure consistent product quality and safety.

High Throughput Screening

In drug discovery and development, MMC provides a powerful platform for high-throughput screening of protein-protein interactions or ligand binding studies, allowing the identification of promising candidates early in the process. We utilize MMC for high-throughput screening to accelerate the development of your therapeutic protein candidates.

When Would You Use Mixed Mode Chromatography (MMC)?

MMC is very beneficial for intermediate purification and polishing in purification schemes/processes.

When the selectivity of traditional resins is not enough to provide the required purity of the target protein, when the sample load conductivity is too high for traditional ion exchange resins, and when the purification steps need to be reduced, CD Formulation will use MMC to separate and purify your target protein/peptide.

In MMC analysis, our scientists will screen pH and conductivity to obtain the best operating conditions and use the design of experiments (DoE) to optimize the separation process.

Advantages of Mixed Mode Chromatography (MMC) Technology

• Alternative selectivity compared to RP, HIC, and IEX.
• Fully compatible with all detection techniques, including mass spectrometry.
• Ability to inject samples with high salt concentrations.
• Ability to separate isomers with high resolution and selectivity.
• Control retention time, resolution, and separation by varying the amount of organic components, buffer properties, buffer concentration, and buffer pH.
• Ability to independently adjust retention time and elution order for various compounds.
• Compatible with organic and aqueous mobile phases.

Custom Mixed Mode Chromatography (MMC) Services

Why Choose Our Mixed Mode Chromatography (MMC) Technology?

• We have a team of experts with rich experience in MMC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using MMC technology to support protein/peptide biopharmaceutical development.
• Our advanced MMC technology allows for enhanced separation efficiency, enabling the purification of complex biomolecules with greater precision and yield.
• We utilize state-of-the-art MMC equipment and innovative methodologies, ensuring that our processes are not only robust but also scalable for large-scale production.
• Our comprehensive support includes not only method development but also troubleshooting, training, and consultation, providing clients with a full spectrum of services throughout their biopharmaceutical development journey.
• We adhere to strict quality control and regulatory standards to ensure the integrity and compliance of your data.
• We provide flexible experimental design and customized solutions.

Publication

CD Formulation aims to provide a powerful analytical tool for the separation, purification, and characterization of proteins and peptides. Please feel free to contact us if you are interested in our services. Learn how our MMC technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.