Size Exclusion Chromatography (SEC) Technology

Size exclusion chromatography (SEC) is the gold standard for separating protein aggregates from their monomers and has been widely used in the development and production of biopharmaceuticals. CD Formulation integrates cutting-edge SEC 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 Size Exclusion Chromatography (SEC) Technology?

SEC, also known as gel filtration chromatography (GFC) or gel permeation chromatography (GPC), is one of the most effective methods for separating and purifying different proteins based on their molecular size (actually the Stokes radius of the particle). This technique has the advantages of simple operation, rapid separation, and no effect on biological activity, and is widely used in the separation and purification of proteins or peptides. Unlike other types of chromatography, such as ion exchange chromatography (IEX) or affinity chromatography (AC), SEC doesn't require a binding reaction between the sample components and the chromatographic medium for separation, so the sample buffer composition does not directly affect the separation. In addition, the buffer, temperature, pH, and other conditions can be changed according to different types of samples or further purification requirements. SEC can be performed directly before or after other chromatography methods.

Size Exclusion Chromatography (SEC) Principle

In SEC analysis, samples are usually injected into a chromatographic column that is continuously flushed with mobile phase. Since the chromatographic column itself contains tiny porous beads, these beads are composed of dextran, agarose, silica or polyacrylamide (stationary phase). During the elution process, proteins with large molecular weights first flow out along the gaps between the gel particles, while proteins with small molecular weights can enter the gel mesh, causing flow blockage and slow outflow. Therefore, larger molecules elute faster than smaller molecules. In other words, the smaller the molecule, the longer the retention time.

Fig. 1 Principle of size exclusion chromatography (SEC). (Du M, et al., 2022)

Our Services Related to Size Exclusion Chromatography (SEC)

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

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

Protein/Peptide Molecular Weight Determination

SEC provides information about the size and shape of a protein and can be used to estimate its molecular weight. We determine the molecular weight of a sample by comparing the elution volume of a protein of unknown size to the elution volume of a standard protein of known molecular weight.

Protein/Peptide Aggregates Characterization

SEC is the method of choice for detecting and quantifying mAb aggregates, and we use SEC to distinguish between monomeric and oligomeric forms of a protein, as well as to identify any possible aggregates.

Protein/Peptide Conformational Changes Analysis

Protein conformational changes can affect elution behavior in SEC. By comparing SEC curves under different conditions (e.g., pH, temperature, ligand binding), we can infer structural changes in the protein.

Protein/Peptide Stability Assessment

SEC can be used to assess protein stability by monitoring the aggregation state of a protein over time or under various stress conditions (e.g., temperature, pH, or the presence of denaturants). Changes in the elution profile may indicate protein degradation or aggregation, helping to assess the stability of the formulation.

Protein/Peptide Purity Assessment

SEC can effectively separate proteins from smaller contaminants such as salts, nucleotides, and small molecules. Our scientists assess the purity of protein samples by analyzing elution profiles. A single sharp peak generally indicates high purity, while multiple peaks may indicate contamination or the presence of aggregates.

Our Equipment

Our analytical laboratory is equipped with two SEC techniques, HPLC-SEC and UPLC-SEC, to support the separation and purification of protein drugs, including size determination, quantitative analysis, and molecular weight determination of fragments, monomers, and aggregates. 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.

HPLC-SEC

A mature, stable, and widely used technique for the size determination, quantitative analysis, and molecular weight determination of fragments, monomers, and aggregates. The size range of HP-SEC is determined by the pore size of the column and the method settings (e.g. mobile phase, flow setting, and column dimensions).

UPLC-SEC

This technique can be used in all stages of biopharmaceutical development, as well as batch release testing. However, HP-SEC is the preferred choice, if increased sample throughput or better resolution is not required.

Advantages of Size Exclusion Chromatography (SEC) Technology

• SEC is performed under mild conditions and is suitable for characterizing sensitive biomolecules such as proteins and polysaccharides.
• High-resolution separations enable the distinction of closely related species in a mixture, such as proteins of different molecular weight forms.
• Compared to other chromatographic techniques, SEC does not rely on the chemical nature or polarity of the sample, but only on differences in molecular size, thereby minimizing the risk of sample degradation or loss associated with other chromatographic methods.
• Relatively fast separations can be achieved, making it suitable for high-throughput applications.
• Sample preparation is generally simple and does not usually require complex chemical steps.
• Compatibility with a variety of buffer systems.
• SEC can be applied to a wide range of proteins and peptides, adapting to various types of samples, from small-scale studies to large-scale protein production.

Custom Size Exclusion Chromatography (SEC) Services

Why Choose Our Size Exclusion Chromatography (SEC) Technology?

• We have a team of experts with rich experience in SEC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using SEC technology to support protein/peptide biopharmaceutical development.
• Our state-of-the-art SEC instrumentation ensures high resolution and reproducibility, enabling accurate sizing and quantification of biomolecules.
• Our customizable SEC solutions are tailored to meet the specific needs of your project, accommodating a wide range of biomolecular sizes and properties.
• We offer extensive support services, from initial consultation to method optimization and troubleshooting, to ensure seamless integration of SEC technology into your workflow.
• We adhere to strict quality control and regulatory standards to ensure the integrity and compliance of your data.

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 SEC 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.