Capillary Isoelectric Focusing (cIEF) Technology

Capillary isoelectric focusing (cIEF) is a high-resolution analytical technique that can separate protein/peptide mixtures, protein glycoforms, and other charge variants based on isoelectric point (pI). CD Formulation integrates cutting-edge cIEF technology into our protein/peptide characterization technology platform to provide precise insights for your biopharmaceutical development program. With our cIEF technology, you can obtain critical information about protein charge characteristics, glycoform changes, and other structural variants to support formulation development, product stability assessment, and to prove the identity of protein drugs in quality control (QC) and release testing.

What is Capillary Isoelectric Focusing (cIEF) Technology?

cIEF is a powerful analytical technique used primarily to separate and characterize biomolecules, especially proteins and peptides, based on their isoelectric point (pI). The technique is primarily performed in polyacrylamide gels. To separate molecules based on their pI, a pH gradient is established within the gel via a mixture of ampholytes. The target molecules migrate along the electric field until they reach the pH corresponding to their pI, at which point the net charge of the molecule is zero and migration ceases. The UV absorbance is measured across the capillary throughout the separation, allowing real-time observation and eventual quantification.

Capillary Isoelectric Focusing (cIEF) Technology Principle

cIEF is based on the principle of capillary gel electrophoresis (cGE), which combines the principles of isoelectric focusing with those of capillary electrophoresis to achieve high-resolution separation in a very short time. Electrophoresis is an electric field-driven separation technique that uses the property of charged molecules to move in an electric field. When different molecules in a sample, such as proteins, peptides, ions, etc., are subjected to an electric field, they will migrate at different speeds due to their different charges, sizes, and shapes, thereby achieving separation.

Compared with conventional isoelectric focusing (IEF), cIEF has higher resolution, faster sample analysis speed, and a detection limit that is approximately 10 times lower.

Fig. 1 Principle of capillary isoelectric focusing (cIEF). (CD Formulation)

Our Services Related to Capillary Isoelectric Focusing (cIEF)

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

During the development and production of protein biologics, charge variants of the target protein may arise due to post-translational modifications (PTMs) and degradation. Our scientists rely on cIEF to quantify the charge variants present at different stages of protein and peptide drug development and manufacturing, including cell line selection, stability studies, formulation development, and upstream and downstream process monitoring.

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

Protein Isoelectric Point (pI) Determination

cIEF can effectively separate proteins based on their isoelectric point in a pH gradient. Knowing the pI is essential for understanding protein structure, stability, and interactions.

Protein Charge Heterogeneity Characterization

Many therapeutic proteins, including monoclonal antibodies, exhibit charge variants due to post-translational modifications (PTMs) such as glycosylation, phosphorylation, or deamidation. cIEF provides high resolution of these charge variants, enabling detailed characterization.

Protein Purity Assessment

By analyzing the charge distribution of a protein, cIEF can help identify impurities such as aggregates or fragments, thereby enhancing the assessment of protein purity.

Monitoring Post-Translational Modifications (PTMs)

cIEF can be used to study the effects of PTMs on protein behavior and stability. Differences in pI due to modifications can be quantitatively assessed.

Protein/Peptide Stability Studies

The stability of a protein can be linked to its charge properties. By performing cIEF under various conditions (e.g., pH, temperature), researchers can assess how environmental factors affect protein stability and aggregation behavior.

Formulation Development

In biopharmaceutical development, cIEF can help screen formulation conditions to minimize the formation of charge variants, resulting in a more stable protein product.

Quality Control (QC)

cIEF is often part of a routine QC analytical strategy for therapeutic proteins to ensure consistency in product formulation and identify any variations in production batches.

Our Procedure for Capillary Isoelectric Focusing (cIEF) Analysis

In our analytical laboratory, cIEF is typically performed on polyacrylamide gels. A brief working procedure is as follows:

• Sample preparation and loading: A complex mixture of proteins or peptides is carefully loaded into a narrow capillary.
• pH gradient establishment: A pH gradient is carefully created within the capillary using a mixture of ampholytes. This gradient can be customized.
• Isoelectric focusing: An electric field is applied to drive the migration of protein/peptide molecules through the pH gradient established within the capillary. As they migrate, the biomolecules settle at their respective pI values, where their net charge is zero.
• High-resolution detection: At the precise moment when each molecule reaches its pI, the detector records data, providing information about the charge variant and pI value of the protein.
• Data processing and interpretation: The data is analyzed using statistical tools to determine the abundance of individual proteins or peptides in the sample and their isoelectric focusing properties.

Fig. 2 Workflow for cIEF analysis. (CD Formulation)

Advantages of Our Capillary Isoelectric Focusing (cIEF)

• High Resolution: cIEF provides high-resolution separations compared to traditional techniques.
• Minimal Sample Requirements: The technique typically requires only microliter to nanoliter samples.
• Rapid Analysis: cIEF can be completed in a relatively short time.
• Automation and Scalability: cIEF can be easily automated, allowing for high-throughput analysis, which is beneficial for large-scale proteomics studies or quality control processes in biopharmaceutical development.
• Enhanced Reproducibility: The controlled environment of the capillaries can produce more reproducible results compared to traditional slab gels, reducing variability between runs and experiments.
• Compatibility: cIEF allows for the use of a variety of buffer systems, which helps optimize separations for specific sample requirements.
• High Sensitivity: cIEF can achieve detection limits that are often superior to traditional electrophoretic techniques due to the inherent focusing effect, which concentrates the sample in a narrow area.
• Combination with Mass Spectrometry: cIEF can be combined with mass spectrometry for downstream analysis, providing comprehensive information on protein charge and mass, and helping to identify post-translational modifications.

Custom Capillary Isoelectric Focusing (cIEF) Services

Why Choose Our Capillary Isoelectric Focusing (cIEF) Technology?

• We have a team of experts with rich experience in cIEF analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using cIEF technology to support protein/peptide biopharmaceutical development.
• Our cIEF technology is optimized to detect low-abundance proteins, making it easier to analyze samples that may contain trace amounts of biomolecules.
• Our team consists of experts with extensive knowledge and experience in cIEF technology, ensuring that your analysis is performed with the highest level of expertise.
• Our state-of-the-art cIEF equipment is fully automated, ensuring consistent and reproducible results across different runs and experiments.
• In addition to cIEF testing, we provide powerful data analysis services to help you accurately interpret your results, providing insights that can guide your research.
• We adhere to strict biopharmaceutical quality control measures to ensure the accuracy and reliability of our results.
• 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 cIEF 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.