Affinity Chromatography

The choice of purification strategy is largely contingent on its performance and economics, which is related to the effectiveness of the separation strategy employed and its robustness. Conventional purification techniques based on precipitation with salts, temperature, pH and high molecular weight polymers are now being substituted by highly selective and sophisticated strategies based on affinity chromatography. This highly selective technique simulates natural processes such as biorecognition. Molecular recognition encompasses interactions such as those between enzyme and substrates, antigens and antibodies, ligands and receptors, DNA and protein interactions, viral proteins and cell surface glycopro-teins, hormones and transmitters that generate a cascade of events to carry out important biological activities. Exploitation of this affinity for interaction between target proteins and their complementary ligands or binding partners is the essence of all affinity techniques. The concept can be traced back to 1910 when Starkenstein first reported the isolation of a-amylase by adsorption onto insoluble starch, and was subsequently followed by several remarkable examples. Only in the second half of the 20th century did the use of the technique gain momentum and the technique was termed affinity chromatography in 1968 by Cuatrecasas. Table 2 outlines the historical perspectives in the development of affinity chromatography. Ever since, techniques in affinity chromatography

Table 1 Quality concerns of purified recombinant protein products

Purity, efficacy, potency, stability, pharmacokinetics and pharmacodynamics Toxicity and immunogenicity

Presence of contaminants such as nucleic acids, pyrogens, viruses, residual host cell proteins, cell culture media contaminants, leachates from seperation media and unknown impurities

Post-translational modifications; mainly glycosylation and proteolytic processing Protein folding and aggregation

Table 2 Important dates and achievements in the history of affinity techniques

1910 Starkenstein purified a-amylase on insoluble starch

1923 Lipase enrichment on powdered stearic acid

1951 Immunoaffinity chromatography

1967 Cyanogen bromide activation, Staphylococcus nuclease purified

1968 Term affinity chromatography coined by Cuatrecasas, biospecific adsorbents;

a new era of modern affinity chromatography begins

1970 Group-specific adsorbents (coenzymes, lectins, nucleic acids)

1972 Boronates in affinity chromatography

1978 Textile dyes

1979 High performance liquid affinity chromatography (HPLAC)

1984 Biomimetic dyes

1985 Phage display

1986 Purification tags 1990 De novo ligand design have been considerably refined for compliance with present-day strict quality control demands such as end product purity, safety, potency and stability. The choice of a stable and efficient solid support, activation and coupling chemistry and selection of a ligand have been investigated in detail since they contribute to efficient recoveries of the target protein.

The most commonly employed adsorbents in affinity chromatography are based on biomolecules such as monoclonal antibodies that offer selectivity and specificity. However, these adsorbents, besides being expensive, are prone to chemical and biological degradation, and themselves need purification prior to immobilization on a solid support and may have issues such as viruses and nucleic acids associated with them. Although biologicals have high selectivity, they have low capacities, a limited life cycle and a low scale-up potential. To counteract these problems, more durable and controllable peptides, pep-tidomimetics and synthetic ligands were introduced.

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