Protein Protocols Protein Protocols

Synthetic Ligands for Ig Purification 1015
Fig. 1. Structure of PAM (TG19318) (GKK 2[YTR] 4).
as recombinant or extractive ligands such as protein A or G. In addition, the low toxicity
of TG19318 and the low molecular weight of the resulting fragments considerably
reduce the problems of contamination by leaked ligand, as is the case for protein A.
Preliminary experiments suggest that the ligand is more stable to proteolytic digestion
when coupled to solid supports, and the enzymatic activity normally found in crude
feedstock derived from cell culture supernatants does not lead to noticeable loss of
capacity. The ligand can be easily immobilized on preactivated solid supports, as the
presence of four peptide chains departing from a central core, of which only a limited
number are involved in the coupling with the solid phase, leaves the others fully available
for the interaction. All the different supports tested so far maintain the ligand
recognition properties for immunoglobulins, even if with different functional capacities.
The affinity columns with immobilized PAM are not affected by the presence of
denaturants, detergents, or other sanitation reagents commonly used for pirogen
removal, and TG19318 columns can withstand a large array of harsh sanitizing agents
with no capacity losses. Immunoglobulin adsorption on TG19318 affinity columns
occurs with neutral buffers at low ionic strength and at room temperature. Elution of
adsorbed immunoglobulins may be achieved under mild conditions not causing immunoglobulin
denaturation, simply by changing the buffer pH to acid or alkaline conditions,
with acetic acid (pH 3) or sodium bicarbonate (pH 9.0). Increasing the ionic strength of
the dissociation buffer favors a more efficient elution of adsorbed antibodies.
Affinity interaction is strong enough to allow purification of antibodies directly from
diluted supernatants in which the immunoglobulin concentration is very low, from 10
to 50 µg/mL. The main contaminant, albumin, is always efficiently removed in the
purification step with any type of support tested for TG19318 immobilization. Column
capacity depends on the type of support used for ligand immobilization, and may range
from 10 to 25 mg of immunoglobulin/mL of support.
Purification of antibodies with clinical and therapeutic applications represents a key
step in the validation process of these molecules, but the complex and the laborintensive
isolation procedures may alter the quality and the safety of the purified product.
In this scenario, the availability of a synthetic ligand able to recognize antibodies
independently from their class is of great importance from an industrial point of view,
as it may simplify the isolation procedures, leading to lower production costs and reducing
considerably the presence of biological contaminants in the purified antibody preparation.