3.1.1 Ion exchange chromatography
Ion Exchange Chromatography (IEC) separates ionizable compounds by immobilizing the ions reversibly to adsorb to oppositely charged molecules. IEC is a broadly employed liquid chromatography(LC) technique due to its relatively simple and adaptable procedures along with notable sample-handling capacity, immense applicability, specifically concerning proteins and enzymes, cost-effectiveness, and remarkable resolving capability, making it more adaptable [14].
IEC depends upon Van der Waals forces among proteins containing charged amino acids and a solid matrix. Matrix holds ion-load opposite to a specific protein that is being extracted and the protein’s affinity for the column is achieved through ionic interactions. By altering the pH, concentrating ion salts, or increasing the amount of ions in the buffer solution, proteins are capable of being removed from the column. Anion exchange matrices (positively charged) readily adsorb to the proteins carrying a negative charge and cation-exchange matrices (positively charged) adsorb to the proteins having a positive charge [15]. Ion Exchange Chromatography (IEC) has two types: anion exchange chromatography (AIEC) and cation exchange chromatography (CIEC). AIEC is commonly used for acidic proteins and for removing negatively charged molecules like DNA and RNA during virus purification and endotoxin removal. IEC is versatile for capturing, purifying, and polishing recombinant proteins. It helps with capturing capacity and yield in the initial stages and prioritizes purity in later purification steps. It can also concentrate low-molecule concentrations before purification. In monoclonal antibody (mAb) purification, IEC assists in at least one of two polishing steps after the protein an affinity step [16].
RNA, DNA, host cell proteins, leached Protein A, and endotoxins being negatively charged are removed by blind and elute mode in CIEC during mAb polishing. Dissociate charged Ab variants are also eluted with CIEC. AIEC can efficiently purify simpler mAbs from impurities like DNA, RNA, acidic HCPs, and leached protein A in flow-through (FT) mode even in case of loose binding to resins. [16].