At its widest definition, affinity chromatography encompasses techniques from immobilized metal chelate affinity to molecular
imprinting and technologies from affinity capillary electrophoresis to affinity precipitation, affinity partitioning, and
affinity membranes.1 A very narrow definition would focus on specific or selective, reversible interactions between the immobilized ligand and
target, dependent on a unique topological relationship involving orientation and molecular reactions. These include biological
as well as biological mimic ligands. This is an area that has seen very significant growth and research development in recent
years. The advent of affinity chromatography is usually attributed to Cuatrecasas, Wilchek, and Anfinsen.2 In 1968 — a year after the introduction of cyanogen bromide activation — they described the purification of certain enzymes
on "inhibitor gels" synthesized by coupling the inhibitor to CNBr-activated agarose. They introduced the notion of protein
purification based on biologically functional pairs, the molecular recognition between a target protein and an immobilized
partner. The technique was rapidly assimilated into the protein purification armory. Three years later, Cuatrecasas' review
cited 100 papers and applications, ranging from immunoaffinity to the isolation of nucleic acids and the separation of complex
cellular structures and cells.3 By 1984, the body of literature had swollen to 1,800 papers, and a 2004 Medline search revealed 35,000 affinity chromatography
citations.4
Figure 1. Triazine Structure with Two Substitution Positions and a Spacer Arm to the Matrix.
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Perhaps affinity chromatography's greatest success at scale has been monoclonal antibody purification. The demand for Protein
A resins is more than 10,000 liters annually, increasing at 50% per year and representing a market in excess of $50 million
in 2002.5 The use of immunoaffinity chromatography enables the production of both plasma-derived6 and recombinant7 coagulation factors VIII and IX, as well as other plasma proteins and biopharmaceuticals from natural and recombinant sources.
Process development is constantly seeking high efficiency processing with the minimum number of steps and maximum output at
the required purity. Larsson discussed customizing ligands to the separation task-at-hand instead of relying on "off-the-shelf,"
group-specific methodologies.8 Indeed the concept of "designer" adsorbents was introduced ten years earlier at the peak of expectations in biopharmaceutical
product development, but at a time (1992) when FDA had approved only 12 recombinant products.9
SERENDIPITOUS ORIGINS
Subramanian described the serendipitous origins of dye affinity chromatography.10 Blue dextran, a 2,000 kDa soluble polymer with the covalently bound dye Cibacron Blue F3GA, was developed to measure the
void volume in gel filtration columns introduced in the early 1960s. It had been noted that some proteins, when co-chromatographed
with Blue dextran, interacted with the marker. It was established in 1968 that the dye chromophore was responsible for binding.11,12
Thus, the evolution of dye affinity is concurrent with the general development of modern separation techniques. The introduction
of the terms mimetic or biomimetic derives from the proposed (and now discarded) mechanism of dye affinity in which the structures
of the chromophores mimic the naturally occurring heterocyclic nucleotides on which many proteins and enzymes depend.13 The synthetic nature of the ligand also led to the introduction of the term pseudo-affinity, referring to the ligand's lack
of biological function.
Undoubtedly the earliest (1973) and most studied ligand is Cibacron Blue F3GA (Ciba-Geigy), which is also available as Procion
Blue H-B (Imperial Chemical Industries) and C.I. Reactive Blue 2 (the Color Index name). These textile dyes from different
manufacturers are no longer available, but single synthetic analogue adsorbents are manufactured by at least two suppliers
of chromatographic media. The first reported separations predate plasma proteome studies by 30 years and concern the depletion
of serum albumin from human plasma to enable identification and purification of low concentration proteins.14 This was carried out using a Procion Blue dextran-Sepharose conjugate. At the time, Travis and Pannell were interested in
a1-antitrypsin and described the difficult separation of this protein from albumin at high ionic strength where any non-specific
ion exchange binding is at a minimum.15
This paper identified an initial problem with dye affinity chromatography, namely the leakage of the dye into the eluate
(properties of synthetic adsorbents will be discussed next month in Part II of this article). In later studies, the authors
used the dye directly conjugated with agarose, thus reducing the leakage problem, improving the binding capacity to 40 mg/mL
gel, and enabling elution of the albumin under non-denaturing conditions.16
