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.
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. Comparison of Synthetic Biomimetic Ligands with Earlier Dye Ligands.
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.
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
Table 2. Comparison of Biomimetic Ligands with Conventional (Biological) Ligands.
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.
Figure 1. Triazine Structure with Two Substitution Positions and a Spacer Arm to the Matrix
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.