properties. Antibodies are constructs comprising heavy and light chains that are
associated by disulfur bonds, and as such, they are sensitive to oxidizing agents. All
these weak points are not present with oligonucleotides.
Another comparison is the mode of production of antibodies and aptamers. The
selection of an antibody ligand is performed by hybridoma cell screening, able to
generate the most convenient antibody. This selection process can be directly
compared to the SELEX process. The molecular recognition process is performed
under physiological conditions with antibody screening, while aptamers selection
can occur under various physicochemical conditions, including in the presence of
organic solvents [171]. The SELEX process is very flexible and allows also the
determination of the condition of protein-aptamer dissociation, an advantage that
is not possible with antibody-based ligands. On the contrary the elution in
immunoaffinity chromatography is most of the time obtained using acidic aqueous
buffers. Once identified, the selected antibody is to be produced by cell culture,
whereas the production of aptamers is performed by automatic chemical synthesis
which integrates the purification of the polynucleotide sequence. In comparison, the
production of an antibody by cell culture is a more complex process since, once
expressed in living cells, it needs to be extracted and purified by a sequence of
chromatographic operations. Globally, cell screening, antibody production, and
purification prior to coupling onto a solid support are very laborious steps for a
given preparation of an immunosorbent [172, 173]. These operations impact the
overall final ligand costs with an advantage for aptamer ligands.
Other advantages related to aptamers compared to antibodies are discussed by
Song et al. [174]. An important feature for aptamers highlighted by the authors is
their stability, since they maintain their structure after repeated denaturationrenaturation cycles, against the fact that antibody denaturation is irreversible. Moreover, their stability against degradation enzymes (e.g., nucleases) can be enhanced
by chemical modifications by various chemical reactions [175]. Contrary to antibodies, aptamers have low immunogenic effects and lower toxicity because they are
generally not recognized by the human immune system as foreign agents.
Because of all the above described advantages over antibodies, aptamers appear
as quite well-adapted ligands for preparative affinity chromatography applications.
Even if smaller than antibodies, oligonucleotides are relatively large and are not
favorable to large binding capacities for proteins. It is thus advised to select small
aptamers or to eliminate parts of them that are not vital to the recognition of the target
protein. Antibodies are much larger than aptamers (150–160 kDa and 10–40 kDa,
respectively) and cannot favor large binding capacities for the capture of proteins.
For this reason, they are only marginally used at large scales for protein purification.
Their exploitation is limited to high-value therapeutic proteins and when no other
solutions are available [176–178]. This is the reason why regular antibodies can be
replaced by single-chain antibodies (including camelids) with good probability of
success [47, 179], even though not all issues related to protease sensitivity are fully
resolved.
An important issue, related to the preparation of affinity chromatography sorbents, is the right orientation of the ligand for an optimized docking with the target
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
127
associated by disulfur bonds, and as such, they are sensitive to oxidizing agents. All
these weak points are not present with oligonucleotides.
Another comparison is the mode of production of antibodies and aptamers. The
selection of an antibody ligand is performed by hybridoma cell screening, able to
generate the most convenient antibody. This selection process can be directly
compared to the SELEX process. The molecular recognition process is performed
under physiological conditions with antibody screening, while aptamers selection
can occur under various physicochemical conditions, including in the presence of
organic solvents [171]. The SELEX process is very flexible and allows also the
determination of the condition of protein-aptamer dissociation, an advantage that
is not possible with antibody-based ligands. On the contrary the elution in
immunoaffinity chromatography is most of the time obtained using acidic aqueous
buffers. Once identified, the selected antibody is to be produced by cell culture,
whereas the production of aptamers is performed by automatic chemical synthesis
which integrates the purification of the polynucleotide sequence. In comparison, the
production of an antibody by cell culture is a more complex process since, once
expressed in living cells, it needs to be extracted and purified by a sequence of
chromatographic operations. Globally, cell screening, antibody production, and
purification prior to coupling onto a solid support are very laborious steps for a
given preparation of an immunosorbent [172, 173]. These operations impact the
overall final ligand costs with an advantage for aptamer ligands.
Other advantages related to aptamers compared to antibodies are discussed by
Song et al. [174]. An important feature for aptamers highlighted by the authors is
their stability, since they maintain their structure after repeated denaturationrenaturation cycles, against the fact that antibody denaturation is irreversible. Moreover, their stability against degradation enzymes (e.g., nucleases) can be enhanced
by chemical modifications by various chemical reactions [175]. Contrary to antibodies, aptamers have low immunogenic effects and lower toxicity because they are
generally not recognized by the human immune system as foreign agents.
Because of all the above described advantages over antibodies, aptamers appear
as quite well-adapted ligands for preparative affinity chromatography applications.
Even if smaller than antibodies, oligonucleotides are relatively large and are not
favorable to large binding capacities for proteins. It is thus advised to select small
aptamers or to eliminate parts of them that are not vital to the recognition of the target
protein. Antibodies are much larger than aptamers (150–160 kDa and 10–40 kDa,
respectively) and cannot favor large binding capacities for the capture of proteins.
For this reason, they are only marginally used at large scales for protein purification.
Their exploitation is limited to high-value therapeutic proteins and when no other
solutions are available [176–178]. This is the reason why regular antibodies can be
replaced by single-chain antibodies (including camelids) with good probability of
success [47, 179], even though not all issues related to protease sensitivity are fully
resolved.
An important issue, related to the preparation of affinity chromatography sorbents, is the right orientation of the ligand for an optimized docking with the target
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
127