Liquid Chromatography (LC) Technologies

Liquid chromatography (LC) is the use of chromatographic techniques to separate molecules based on their different physicochemical properties. It is a key part of any protein or peptide structural characterization study and is a requirement of ICH Topic Q6B. CD Formulation integrates a series of cutting-edge liquid chromatographic technologies, including ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), affinity chromatography, reversed-phase chromatography (RP-HPLC/RP-UPLC) and mixed-mode chromatography, 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 Liquid Chromatography (LC) Technology?

LC is a well-established and widely used analytical technique for analyzing compounds in complex mixtures. This separation occurs based on chemical or physical interactions between the sample and the mobile and stationary phases. The ICH Q6B regulatory guideline details the requirements for protein characterization, which is the data that can be obtained about the identity, homogeneity, and purity of a protein product through LC procedures. Common types of LC include:

• High-performance liquid chromatography (HPLC): Use of high pressure to increase separation efficiency, resulting in faster analysis times and better resolution.
• Ultra high-performance liquid chromatography (UPLC): Operates at higher pressure and provides better resolution and speed than HPLC.

Fig. 1 Scheme of a HPLC system. (Salvato F, et al., 2012)

Liquid Chromatography (LC) Principle

The working principle of liquid chromatography is based on the differential partitioning of analytes between the stationary phase and the mobile phase. When the analyte, such as a protein mixture, is introduced into the chromatography system. The mobile phase carries the mixture through a chromatographic column filled with a special material (the stationary phase). The components in the mixture interact with the stationary phase depending on their physicochemical properties, including adsorption, distribution, and ion exchange. The different interactions between the components and the stationary phase cause the components to pass through the chromatographic column at different rates. Components with stronger interactions with the stationary phase are retained in the chromatographic column for a longer time, while components with weaker interactions move faster. The separated components are eluted from the chromatographic column, then detected by the detector and processed by the data system to produce a chromatogram representing the separated components. Ultimately, researchers quantify the concentration of the components in the mixture based on the data obtained from the chromatogram and identify them based on their unique retention time and detector response.

Liquid Chromatography (LC) Technologies at CD Formulation

CD Formulation has extensive experience in separating and characterizing protein or peptide molecules using various liquid chromatographic (LC) techniques. Based on this technology, we provide customized protein or peptide biopharmaceutical solutions so that your product can meet the structural characterization requirements of ICH Q6B.

Our experienced chromatographic team has completed hundreds of protein and peptide LC analysis projects and can support all stages of your protein/peptide drug development and manufacturing - from early research to downstream process monitoring and GMP batch release testing.

To fully support the development of your protein/peptide biopharmaceutical project and impurity analysis of sample purity, we use different LC separation procedures, so any component that cannot be separated by one procedure may be separated by another chromatographic technique.

Our liquid chromatographic techniques include:

Fig. 2 Liquid chromatography (LC) types. (CD Formulation)

Ion Exchange Chromatography (IEX)

IEX separates molecules based on their total charge. Separation of charged proteins or peptides is achieved by using salts or pH gradients in the liquid phase. Usually, different stationary phases (cationic or anionic) are used to separate positively or negatively charged species, respectively.

Hydrophobic Interaction Chromatography (HIC)

In the HIC process, protein molecules are separated based on their hydrophobicity/hydrophilicity. Typically, we use a decreasing salt gradient to elute the components, with more hydrophobic species eluting at lower salt concentrations.

Size Exclusion Chromatography (SEC)

In the SEC process, protein molecules are separated based on size as they pass through a gel column made of beads with specific pore sizes. Larger molecules are preferentially eluted due to exclusion or restricted access to the pore space, while smaller molecules can pass through the pores and bypass the beads and are subsequently eluted.

We also couple SEC with a multi-angle laser light scattering detector (SEC-MALS) to characterize aggregation within protein/peptide samples.

Affinity Chromatography (AC)

In the AC process, protein or peptide molecules are separated by reversible interactions with specific ligands attached to a chromatographic matrix. The technique is well suited for capture or as an intermediate purification step and can be used as long as a ligand suitable for the target protein is available.

Reversed-phase Chromatography (RPC)

RPC uses the interaction of proteins or peptides with a hydrocarbon-coated stationary phase to separate molecules. The technique relies on using a liquid gradient with increasing organic content to promote the elution of each protein component in the sample.

Mixed Mode Chromatography (MMC)

MMC involves combining two (or more) retention mechanisms in a single chromatography system, such as RP-HILIC, RP-IEX, HILIC-IEX, and tri-mode mixed-mode HPLC (MM-HPLC).

MMC is used to separate proteins or peptides that are not retained or not well separated by typical reversed-phase liquid chromatography methods. 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.

Fast Protein Liquid Chromatography (FPLC)

FPLC is an LC method specifically used for protein analysis, which can adopt a variety of separation and purification strategies for proteins of different sizes and properties. This technology can analyze complex protein mixtures at high resolution and can separate them under milder conditions, protecting the activity and function of proteins, which helps to achieve efficient protein separation.

How Can We Help You Design The Optimal Protein Separation Strategy Using Liquid Chromatography (LC)?

The key to protein separation and purification is whether it meets the needs of downstream applications because sample purity, integrity, and yield usually show an inverse relationship. During the development of the entire separation and purification workflow, our scientists will focus on the following four factors based on the characteristics of your product:

Resolution

Resolution refers to the ability of the LC technology to effectively distinguish the target protein from other components. High-resolution separation can ensure a clear division between the target protein and impurities, thereby improving purification efficiency.

Sample Integrity

Sample integrity refers to the degree to which the structure and function of the target protein are maintained during the separation and purification process. If the activity of the protein is to be evaluated in vitro, the purified protein must retain its enzymatic activity. We will maintain the integrity of the sample by screening buffer selection, adding appropriate protease inhibitors, and changing the speed.

Purity

Purity refers to the proportion of the target protein in the final product. For co-eluting compounds, detecting a single peak in the chromatogram does not ensure that the sample is pure. We will also evaluate the sample purity through other analytical techniques such as gel electrophoresis (SDS-PAGE).

Yield

Yield refers to the amount of target protein obtained from the raw material. If the purpose of chromatographic separation is to purify the target protein for downstream applications (i.e. preparative chromatography), then yield (defined as the amount of the desired protein fraction recovered) is an important consideration.

Custom Liquid Chromatography (LC) Services

Why Choose Our Liquid Chromatography (LC) Technology?

• We have a team of experts with rich experience in LC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using LC technology to support protein/peptide biopharmaceutical development.
• Our LC systems feature cutting-edge detectors such as UV/VIS, fluorescence, and mass spectrometry to provide enhanced sensitivity and specificity.
• Our LC technology is optimized for speed, providing fast turnaround time for results without compromising resolution or quality.
• We provide end-to-end support, from sample preparation to data interpretation, to help you make informed decisions based on our results.
• 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 LC technologies 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.