protein. The immobilization of antibodies is generally random, involving any
exposed area where primary amine groups are available. This operation generally
reduces the capability of the antibody to recognize the antigen depending on the
accessibility of the active site. In this respect to preserve the highest antibody
recognition capability, an oriented immobilization of the antibody has been proposed. The approach involved a complex chemical process [180] with the necessity
of a first mild oxidation of the glycan of the Fc fragment, generating transient
aldehyde groups, followed by a reaction on a primary amine-derived solid support
[114]. Other attempts have been made to preserve the right orientation of the
antibody. Representative examples are given by the use of protein A to assist for
site-specific immobilization of antibodies on solid surfaces [181–183]. On the
contrary, the aptamer, comprising a spacer placed on one of the two terminal ends,
is by definition oriented, conferring a competitive advantage in affinity chromatography for proteins.
When making a detailed comparative analysis of the chromatographic separation
cycle, critical points of relevance are associated to the loading, elution, and washing/
regeneration phases.
In the loaded crude protein extract, the possible presence of degradation enzymes
(nucleases or proteases) may progressively hydrolyze the aptamer or the antibody
ligands, respectively. Exonuclease action can be counteracted by the presence of a
modified nucleic base at the available extremity of the oligonucleotide. Endonuclease action is however more difficult to stop. If present, the binding capacity of the
aptamer affinity column will be reduced progressively. In the case of immunosorbents, the potential presence of proteases in crude extracts is detrimental to the
integrity of the antibody with consequent degradation of the performance.
The dissociation of the protein-ligand complex (elution phase) implies the use of
more or less mild physicochemical conditions. Acidic pHs are mostly used for
immunosorbents; this elution mode induces little reversible denaturation of the
antibody ligand. In the case of aptamers, the most common elution method is to
wash with buffers comprising chelating agents subtracting thus calcium that stabilizes the aptamer-protein complex. This is a very mild method that is not deleterious
for both the target protein and the affinity ligand. Since the mode of dissociation of
the complex is designed at the level of the SELEX process, alternatives to calcium
ions are possible, such as the replacement with other ions or even the use of pH
change (see Figs. 6, 11, and 12).
Another critical phase is the cleaning – regeneration of an affinity sorbent – that is
necessary at the end of a separation cycle for sanitization purposes and to eliminate
all non-specifically adsorbed material. The most popular chemical agent for this
operation in liquid chromatography is sodium hydroxide at a concentration ranging
from 0.1 to 1 M. Although very effective to remove a large number of impurities,
caustic solutions are also very aggressive to the affinity ligands. Antibodies do not
withstand these treatments, and their cleaning is problematic. In the case of a DNA
aptamer, the sodium hydroxide linearizes the oligonucleotide sequence in a reversible manner. Nevertheless RNA aptamers are sensitive to alkaline conditions
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G. Perret and E. Boschetti
exposed area where primary amine groups are available. This operation generally
reduces the capability of the antibody to recognize the antigen depending on the
accessibility of the active site. In this respect to preserve the highest antibody
recognition capability, an oriented immobilization of the antibody has been proposed. The approach involved a complex chemical process [180] with the necessity
of a first mild oxidation of the glycan of the Fc fragment, generating transient
aldehyde groups, followed by a reaction on a primary amine-derived solid support
[114]. Other attempts have been made to preserve the right orientation of the
antibody. Representative examples are given by the use of protein A to assist for
site-specific immobilization of antibodies on solid surfaces [181–183]. On the
contrary, the aptamer, comprising a spacer placed on one of the two terminal ends,
is by definition oriented, conferring a competitive advantage in affinity chromatography for proteins.
When making a detailed comparative analysis of the chromatographic separation
cycle, critical points of relevance are associated to the loading, elution, and washing/
regeneration phases.
In the loaded crude protein extract, the possible presence of degradation enzymes
(nucleases or proteases) may progressively hydrolyze the aptamer or the antibody
ligands, respectively. Exonuclease action can be counteracted by the presence of a
modified nucleic base at the available extremity of the oligonucleotide. Endonuclease action is however more difficult to stop. If present, the binding capacity of the
aptamer affinity column will be reduced progressively. In the case of immunosorbents, the potential presence of proteases in crude extracts is detrimental to the
integrity of the antibody with consequent degradation of the performance.
The dissociation of the protein-ligand complex (elution phase) implies the use of
more or less mild physicochemical conditions. Acidic pHs are mostly used for
immunosorbents; this elution mode induces little reversible denaturation of the
antibody ligand. In the case of aptamers, the most common elution method is to
wash with buffers comprising chelating agents subtracting thus calcium that stabilizes the aptamer-protein complex. This is a very mild method that is not deleterious
for both the target protein and the affinity ligand. Since the mode of dissociation of
the complex is designed at the level of the SELEX process, alternatives to calcium
ions are possible, such as the replacement with other ions or even the use of pH
change (see Figs. 6, 11, and 12).
Another critical phase is the cleaning – regeneration of an affinity sorbent – that is
necessary at the end of a separation cycle for sanitization purposes and to eliminate
all non-specifically adsorbed material. The most popular chemical agent for this
operation in liquid chromatography is sodium hydroxide at a concentration ranging
from 0.1 to 1 M. Although very effective to remove a large number of impurities,
caustic solutions are also very aggressive to the affinity ligands. Antibodies do not
withstand these treatments, and their cleaning is problematic. In the case of a DNA
aptamer, the sodium hydroxide linearizes the oligonucleotide sequence in a reversible manner. Nevertheless RNA aptamers are sensitive to alkaline conditions
128
G. Perret and E. Boschetti