Affinity Chromotography - from Textile Dyes to Synthetic Ligands by Design, Part 2

Sythetic ligand chromatography is set to assume a significant position in the bioseparations armory
Aug 01, 2004
Volume 17, Issue 8

John Curling
The first part of this review (BioPharm International July 2004) discussed the development of synthetic ligands with a focus on integrated rational design, combinatorial synthesis, and solid phase screening for ligand discovery and design. This article deals with the properties of synthetic ligands used in affinity chromatography of biopharmaceuticals with emphasis on the safety and the use of triazinyl ligands.

Table 1. Comparison of Synthetic Biomimetic Ligands with Earlier Dye Ligands.
PROPERTIES Initially, dye ligands were pursued not only for their biomimetic function but also because they were readily available from major chemical companies; they were cheap and they were stable when attached to a support matrix. Very significant progress has been made since the mid-1970s when dye ligands were introduced, and the major differences and improvements are given in Table 1.

The advantages of synthetic ligands compared to their biological counterparts have been summarized by Lowe et al.1 and Boyer and Tsu.2 Table 2 is an adaptation of their view, modified to encompass specifically designed ligands.

Table 2. Comparison of Biomimetic Ligands with Conventional (Biological) Ligands.
Because they are based on triazine chemistry, the synthetic ligands under discussion have some similarities with their dye predecessors. These ligands, therefore, display significant stability and resistance to both chemical and biological degradation. This property also confers long-term stability and re-usability, and synthetic ligand adsorbents can be used for at least 100 cycles.3 In this context it may be remembered that the colorfast, reactive (triazine) dyes developed during the 1950s were designed for covalent bonding to cellulosic fabrics (in contrast to earlier animal and synthetic fibers) under mild conditions with minimum leaching.

Figure 1. Triazine Structure with Two Substitution Positions and a Spacer Arm to the Matrix
These affinity adsorbents display dynamic protein-binding capacities in the range 5 up to 40 mg/mL. Most absorbents used in bioprocessing have a capacity of 15 to 20 mg/mL at a linear flow rate of 100 cm/hour. They are comparable with Protein A adsorbents and most ion exchangers. They may be regarded as a particular compound family similar to the families of ion exchangers, and they certainly show similar characteristics in bioprocessing.

LEACHABLES As has been previously mentioned, ligand leakage was noted when Blue dextran was coupled to agarose and significantly reduced when the ligand was coupled directly. Eketorp4 discussed the leakage of ligand from cyanogen bromide-coupled products, which have now generally been discarded in favor of oxirane coupling through a far more stable epoxy linkage. However, as with all adsorbents used in chromatography, leaching with or without degradation of the matrix, is an important consideration.

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