Journal of Chromatography & Separation Techniques
Open Access

Applications of High Performance Affinity Chromatography in the Biopharmaceutical Industry

Christiana Desouza^{* *}Correspondence: Christiana Desouza, Department of Analytics, University of Porto, Praça de Gomes Teixeira, Porto, Portugal, Email:

Author info »

About the Study

In order to describe and research the processes that occur in living systems, it is necessary to analyse biological interactions. Non-covalent processes involving medicines, hormones, proteins, peptides, metal ions, nucleic acids, and lipids are responsible for many biological interactions. Non-covalent interactions allow transport proteins like Acid glycoprotein (AGP) and Human Serum Albumin (HSA) to bind to and transport various medications within the circulatory system, altering drug absorption, distribution, metabolism, and excretion in humans. The purpose of biological interactions, as well as the methods by which they occur, may be determined using information on their kinetics.

To examine the kinetics of a biological system and estimate the rate constants for this sort of process, a number of approaches may be utilised. These approaches are now chosen depending on the kind of system being studied, the complexity of the reaction, the rates of the relevant reactions, and the needed and accessible quantities and concentrations of reactants and products. Surface Plasmon Resonance spectroscopy (SPR), Capillary Electrophoresis (CE), and stopped-flow analysis have all been employed in the past to examine reaction rates in biological systems. However, each of these approaches has its own set of drawbacks. In stopped-flow analysis, for example, a significant concentration of the reactant or product and a quantifiable signal are required; in SPR, a certain type of surface is required; and in CE, a measurable difference in mobilities between the products and reactants is required. Adsorption of biomolecules to the capillary surface in CE must also be considered, which can be reduced by treating the capillary with an appropriate polymer.

HPAC (High-Performance Affinity Chromatography) is another method for determining rate constants in biological systems. HPAC is a type of affinity chromatography in which the stationary phase contains a physiologically relevant binding agent, which is subsequently put in a column appropriate for High-Performance Liquid Chromatography (HPLC). An immobilised protein, enzyme, or antibody; enzyme substrate or inhibitor; antigen; biomimetic colour; and DNA or RNA sequence, among other things, can be used as an affinity ligand. Small and stiff supports, such as silica or monoliths, are utilised in HPAC to produce quick and efficient separations with the immobilised agent. The target analyte experiences selective and reversible interactions with the binding agent in both HPAC and conventional affinity chromatography, allowing the target to be trapped and maintained as it passes through the column. HPAC and affinity chromatography are often employed as separation procedures in application samples to purify or evaluate a specific chemical or group of related solutes. These approaches have been used for sample pretreatment, flow-based immunoassays, chiral separations, and multi-dimensional procedures, among other things. HPAC and other affinity approaches, on the other hand, have been used to describe the strength, binding sites, and rates of biological interactions. Drug interactions with serum proteins, antibody-antigen interactions, enzyme binding with substrates or inhibitors, immunoglobulin-binding protein interactions with antibodies, and lectin binding of glycoproteins are all examples of systems that have been examined using this method.

Several factors will influence the procedure for determining the rates of analyte-ligand binding by HPAC and affinity chromatography. The overall binding strength and pace of the interaction to be monitored, as well as the type of information required, are all major considerations. The amount of analyte and binding agent available for analysis are also factors to consider. For example, some affinity-based procedures need continual application of a target analyte (i.e., frontal affinity chromatography or frontal analysis), whereas others just require injection of a tiny amount of analyte (i.e., an approach known as zonal elution). Non-linear elution circumstances, in which the chromatographic response is dependent on the amount of applied analyte, are frequently used in methods in which the analyte approaches the amount of binding agent. Linear elution circumstances, in which the observed chromatographic behaviour is independent of the amount of analyte, may be used or required in techniques that need only small quantities of analyte.