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].