View - ResearchGate

94 Roque and Lowe
market, with 14 FDA-approved monoclonal antibodies, 70 in late stage
clinical (Phase II+) trials, and more than 1000 in preclinical development in
2003 (2). Engineering the downstream processing of antibodies has been a
principal task in research and industry, by exploring different types of interactions
and separation techniques. Affinity chromatography is undoubtedly
the most widespread technique in use for the purification of antibodies.
It has seen improvements in classical chromatographic techniques (such as
the expanded bed adsorption mode) and in non-chromatographic techniques,
namely, affinity precipitation and aqueous two-phase systems. Biospecific
affinity ligands, mainly immunoglobulin (Ig)-binding proteins isolated from
the surface of bacteria (proteins A, G, and L), have been the most popular
ligands for antibody purification. The “traditional” pseudobiospecific affinity
matrices include, for example, thiophilic, hydrophobic, and mixed-mode adsorbents,
and are also well liked for antibody purification purposes although
they lack in specificity (3). Combinatorial approaches applied to affinity
chromatography identified a new class of pseudobiospecific ligands, termed
as biomimetics, as an improved version of the natural affinity ligands. Lowmolecular-weight
substances, able to bind Igs in the same fashion as protein
A, have been developed (4). These include the multimeric peptide TG19318
(5) and the artificial protein A (ApA) (ligand 22/8), a triazine-based fully
synthetic ligand (6). The latter belongs to a class of de novo designed nonpeptidic
ligands developed by Lowe and co-workers and represents an appealing
concept for the generation of highly resistant, specifically tailor-made affinity
ligands.
Protein L (PpL) has received special attention since its discovery in 1985,
mainly for being an Ig light chain-binding protein and, as a consequence,
being particularly suitable for the purification of scFv (single-chain variable
fragment), Fab and F(ab´) 2 biomolecules (7). PpL binds with high affinity (K d
of 1 nM) to a large number of Igs with 1, 3, and 4 light chains (but not to
2 and subgroups) and thus recognizes 50% of human and more than 75%
of murine Igs (8). Although displaying high selectivity, PpL adsorbents suffer
from high costs of production and purification, low binding capacities, limited
life cycles, and low scale-up potential, which is attributable to the biological
nature of the ligand. Biomimetic ligands, as the ApA, are fully synthetic in
nature and can circumvent problems associated with biological ligands, while
maintaining the affinity and specificity for the target proteins. In this chapter,
we describe the process followed for the design and development of an Igbinding
ligand, mimicking the interaction of PpL with the light chains (named
as artificial PpL), following the concept of de novo designed biomimetics (9)
(see Fig. 1).