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Liquid chromatography is a process that separates a sample into its individual components, allowing you to isolate and purify molecules used in downstream bioprocessing steps. The process involves two substances – a stationary phase and a mobile phase. In bioprocessing, a sample is applied to a stationary phase and moves through it by applying the mobile phase.
Chromatography resins are media used to capture and polish mAbs, antibody fragments, vaccines, and other biomolecules using a stationary phase. There are several resin types for both analytical and purification purposes.
You can take advantage of the diverse properties of nucleic acids, proteins, and small and large molecules to exclude or capture targets of interest from sample mixtures using their physical and chemical properties to develop a purification scheme. Different categories of chromatography resins provide many options.
Hydrophobic interaction chromatography (HIC) – separates or purifies proteins, or other biomolecules, based on differences in their surface hydrophobicity. It uses a reversible interaction between the biomolecule and the hydrophobic ligand of a HIC resin.
Multimodal or mixed-mode chromatography (MM) – purifies proteins or other biomolecules in a capture or polishing step. It uses resins functionalized with ligands capable of multiple interactions, which is useful when purifying target molecules without a known specificity.
Size-exclusion chromatography (SEC) – also known as gel filtration. It uses a gel medium to partition proteins based on size. With SEC molecules don’t bind to the chromatography resin.
Affinity chromatography (AC) – separates proteins and other molecules using a strong but reversible interaction between the protein and a specific ligand. Affinity chromatography resins rely on binding interactions between a ligand immobilized to a resin and its binding partner. Because the interaction is so specific, high purity can be achieved in a single step.
Ion exchange chromatography (IEX) – separates molecules based on their overall surface charge. Separation is based on the reversible interaction between a charged protein and a chromatography resin that has an opposite charge.
Chromatography columns are essential to the process of separating the molecule of interest from other components. Chromatography columns contain the resins that the mobile phase passes through. You can choose to pack your own columns or purchase prepacked columns that are ready for use.
In protein purification, DBC is the maximum target protein that can be loaded onto your column before unbound protein breaks through into the elution buffer. In bioprocessing, it’s important to calculate the DBC to determine the best resin and running conditions. DBC measurements are usually done with a chromatography column packed using the flow packing method, and a chromatography system such as an ÄKTA™ system. You will create a method and load increasing amounts of your protein sample in binding buffer. To calculate DBC on an ÄKTA™ system, you can use the DBC calculation extension in UNICORN™ software.
Learn more about calculating DBC using an ÄKTA™ system.