Choice of an Appropriate Affinity Ligand and Immobilization to a Support

There are a number of factors that have to be considered when choosing an appropriate affinity ligand for use in affinity separations. One critical consideration is the selection of a ligand with appropriate affinity and selectivity towards the target molecule. Although the selection of ligands that form complexes with very low dissociation constants improves capture of adsorbate from feedstocks at low concentrations and permits extensive washing of the adsorbed complex to remove less tightly adsorbed impurities, it may prove difficult to achieve dissociation of the complex during the subsequent elu-tion phase. Elution may need to be achieved as a result of major changes to the physical conditions of the irrigating buffer (e.g. pH, ionic strength) with the possibility of subsequent denaturation of the adsorbate and/or the ligand. In addition, there is the possibility that dissociation of the adsorbed complex will be slow as a result of low values of the rate constant of this step. When being used on a large scale, the cost of the affinity ligand and the number of cycles in which it can be used are also important considerations. Problems and costs associated with the manufacture of ligand in the quantities required to prepare large amounts of affinity adsorbents may become significant, together with any implications that the nature and the source of the ligand may have on the validation of the process. It may prove impossible to find ligand immobilization chemistries that totally prevent low levels of ligand leakage from the affinity adsorbent during all phases of the purification cycle. The use of affinity separations in the production of therapeutic products dictates the need for these processes to be operated in a manner that can be fully validated to comply with good manufacturing practice (GMP). The need for thorough sanitation as part of the inherent clean-in-place (CIP) procedures may necessitate exposure of the affinity ligand to harsh reagents or sterilization protocols not previously encountered during use of the technique in the laboratory, with a consequent increase in the likelihood of deterioration of the affinity adsorbent.

Once a suitable affinity ligand has been chosen, it has to be immobilized on to a suitable support in order to generate an affinity adsorbent. Considerations in the choice of a suitable support are common to those that would be used in other adsorption and chromatographic procedures. In general the use of porous particles has been the most popular in large-scale separations, although the benefits of using membrane materials in achieving fast mass transfer have been demonstrated in some small-scale systems. Important properties of a suitable support include:

• High surface area accessible by the target molecule per unit volume of matrix, to minimize the volume of the adsorbent needed for the separation.

• Spherical particle shape and narrow particle size distribution to facilitate packing the bed to obtain optimal flow characteristics.

• Good mechanical properties of the matrix to resist compression and compaction in tall beds operated at high flow rates at high pressure drops and to resist attrition should removal of the adsorbent from the bed be necessary periodically for thorough sanitation procedures.

A wide variety of matrices, designed specifically for use in process-scale separations, are commercially available. In some cases, they may be purchased with popular, widely used, affinity ligands already covalently immobilized on their surface. Alternatively, base matrices may be available in a chemical form which facilitates customized covalent immobilization of more specialized ligands.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

Get My Free Ebook


Post a comment