Reversed-phase Chromatography (RP-HPLC / RP-UPLC) Technology

Reversed-phase chromatography (RPC) is a liquid chromatography technique widely used for protein separation and purification. Its basic principle is to use the hydrophobicity of proteins for separation. CD Formulation integrates cutting-edge RP-HPLC / RP-UPLC 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, including formulation development, process development, drug release testing, and stability testing.

What is Reversed-phase Chromatography (RPC)?

RPC is a widely used chromatographic technique, especially in analytical and preparative chemistry for the separation and purification of compounds. In RPC, the stationary phase is hydrophobic (non-polar) and the mobile phase is a polar liquid, usually containing water and an organic solvent such as acetonitrile or methanol. The technique is frequently used for peptide and protein separations due to its excellent resolution, good reproducibility, and compatibility of the eluent with MS analysis. In the biopharmaceutical industry, RP-HPLC is frequently used to identify large-molecule drugs before release.

Reversed-phase Chromatography (RPC) Principle

The principle of RPC separation and purification of protein or peptide molecules is based on the different interactions of these molecules with a nonpolar stationary phase in the presence of a polar mobile phase. In RPC, the stationary phase consists of a hydrophobic nonpolar material, such as C4, C8, or C18 alkyl chains (where the number indicates the carbon chain length) chemically bonded to silica particles. This nonpolar surface interacts with the hydrophobic regions of the protein. When a protein sample is injected into the column, the more hydrophobic proteins interact more strongly with the stationary phase and are retained longer, while the less hydrophobic proteins elute faster. By gradually increasing the concentration of organic solvent in the mobile phase, the proteins are eluted according to their hydrophobicity. More hydrophobic proteins elute at higher concentrations of organic solvents than less hydrophobic proteins. Similarly, more water can be added to the mobile phase to increase the retention time of all protein molecules in the sample.

Fig.1 Diagram of Reverse-phase chromatography separation. (Salvato F, et al., 2012)

Our Services Related to Reversed-phase Chromatography (RPC) Technology

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

Our experienced team of experts has completed hundreds of RP-HPLC / RP-UPLC 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 RP-HPLC / RP-UPLC technology, we support the following protein/peptide development and characterization plans, including but not limited to:

• Purify and initially characterize proteins from complex mixtures such as cell lysates or serum.
• Isolate specific protein isoforms or conformations that differ in hydrophobicity.
• Couple to mass spectrometry for detailed structural characterization to identify proteins and their post-translational modifications (PTMs).
• Analyze how different conditions (such as pH, temperature, or the presence of denaturants) affect the hydrophilicity of proteins, providing insight into the folding state of proteins.
• Investigate how the presence of ligands alters the hydrophobic properties of proteins (protein-ligand interactions), which can indicate binding events or conformational changes.
• Screen and characterize a variety of protein mutants or variants, either from natural evolution or directed engineering, such as in therapeutic protein development.
• Assess protein stability under different conditions to guide formulation development.
• Monitor and control product quality during manufacturing to ensure that therapeutic proteins have the correct folding and structural integrity.

Our Equipment

Our analytical laboratory is equipped with two RPC technologies, i.e. high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UPLC), to support the separation, purification, qualitative identification, and quantitative analysis of protein drugs. The following detectors are used to detect various molecular species:

• Ultraviolet (UV) absorption detector.
• Fluorescence detector.
• Refractive index (RI) detector.
• Multi-angle laser light scattering (MALLS) detector.
• Charged aerosol detection (CAD) detector.

Advantages of Reversed-phase Chromatography (RPC) Technology

• Excellent separation efficiency, allowing separation of closely related protein species, such as isoforms and PTMs.
• Scalability, both for analytical and preparative applications.
• RPC can be used for various protein characterizations, including proteins with different hydrophobicities.
• RPC can reduce sample complexity by separating proteins based on hydrophobic interactions, facilitating downstream analysis, such as mass spectrometry.
• RPC is generally compatible with a variety of analytical detectors, such as UV/VIS, fluorescence, and mass spectrometry, allowing sensitive detection and characterization of proteins.
• RPC can be easily integrated with other chromatographic methods, such as size exclusion or ion exchange chromatography, for comprehensive protein characterization.

Custom Reversed-phase Chromatography (RPC) Services

Why Choose Our Reversed-phase Chromatography (RPC) Technology?

• We have a team of experts with rich experience in RP-HPLC / RP-UPLC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using RP-HPLC / RP-UPLC technology to support protein/peptide biopharmaceutical development.
• We utilize state-of-the-art RP-HPLC and RP-UPLC systems that ensure high resolution, sensitivity, and accuracy, enabling us to deliver reliable results that exceed regulatory standards.
• Our validation procedures are rigorous and align with industry guidelines, ensuring that our analytical methods are reproducible and compliant with both GLP and GMP requirements.
• We offer end-to-end support, from initial method development through to regulatory submission, helping clients navigate the complexities of biopharmaceutical testing.
• 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 RP-HPLC / RP-UPLC 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.