the purification process, as well as the sensibility to cleaning solutions. These wellknown phenomena necessitate the development of analytical methods for the identification and the quantification of the released material. As a corollary implication,
these questions open another file about the toxicity of released ligands. Naturally,
these questions are most exclusively asked when the aptamer affinity chromatography is applied to the preparation of biopharmaceuticals, which is probably not yet a
reality. Nevertheless, studies should move toward conditions for both the sorbent
and the process validation.
Since experimental data have not yet been published, what can be suggested here
are few speculative hypotheses. Two points are to be considered. The first is the
potential hydrolysis of the chemical link between the oligonucleotide and the
sorbent. One of the most stable bonds is alkylamine (generated, for instance,
between an epoxy group and a primary amine), while the weakest are esters or
carbamates – especially under alkaline treatments. Amide bonds are also relatively
fragile, but their hydrolysis depends on the molecular structure of neighboring
chemical residues. Whatever the hydrolysis reason, the release mechanism can be
extremely slow, and therefore only traces of oligonucleotide would be desorbed.
Regular analytical detections are ineffective to evidence the leakage phenomena;
however, it could be possible in certain conditions to amplify the signal by means of
PCR technologies.
Once analytical methods are well validated, it will be also of importance to
determine when the major ligand release phenomenon occurs. Most generally, it
can happen either during the loading phase when possible deleterious enzymes may
be present in the crude sample or during the sanitization phase when harsh cleaning
agents are used (e.g., sodium hydroxide).
To fully validate a chromatographic process, the potential toxicity of possible
released material should also be determined.
6 Comparison Between Aptamer- and Antibody-Based
Affinity Chromatography
Aptamers are frequently compared to antibodies as extraordinary selective molecule
to recognize protein targets. It is thus natural to analyze the properties of these two
affinity ligands and then compare their performance as affinity sorbents.
On a functional standpoint, DNA or RNA aptamers exhibit affinities to target
proteins relatively similar or even higher compared to antibodies, with dissociation
constants from the low nanomolar to the picomolar range, and are described as
equivalent ligands [170]. Antibodies are proteins; they are sensitive to proteases that
are frequently present in the crude extracts. Acidic conditions may induce partial
denaturation that is most of the time reversible, depending on the pH, but also
contribute to antibody aggregation. Moreover, in alkaline conditions, irreversible
glutamine deamidation processes are possible with a reduction of the affinity
126
G. Perret and E. Boschetti
these questions open another file about the toxicity of released ligands. Naturally,
these questions are most exclusively asked when the aptamer affinity chromatography is applied to the preparation of biopharmaceuticals, which is probably not yet a
reality. Nevertheless, studies should move toward conditions for both the sorbent
and the process validation.
Since experimental data have not yet been published, what can be suggested here
are few speculative hypotheses. Two points are to be considered. The first is the
potential hydrolysis of the chemical link between the oligonucleotide and the
sorbent. One of the most stable bonds is alkylamine (generated, for instance,
between an epoxy group and a primary amine), while the weakest are esters or
carbamates – especially under alkaline treatments. Amide bonds are also relatively
fragile, but their hydrolysis depends on the molecular structure of neighboring
chemical residues. Whatever the hydrolysis reason, the release mechanism can be
extremely slow, and therefore only traces of oligonucleotide would be desorbed.
Regular analytical detections are ineffective to evidence the leakage phenomena;
however, it could be possible in certain conditions to amplify the signal by means of
PCR technologies.
Once analytical methods are well validated, it will be also of importance to
determine when the major ligand release phenomenon occurs. Most generally, it
can happen either during the loading phase when possible deleterious enzymes may
be present in the crude sample or during the sanitization phase when harsh cleaning
agents are used (e.g., sodium hydroxide).
To fully validate a chromatographic process, the potential toxicity of possible
released material should also be determined.
6 Comparison Between Aptamer- and Antibody-Based
Affinity Chromatography
Aptamers are frequently compared to antibodies as extraordinary selective molecule
to recognize protein targets. It is thus natural to analyze the properties of these two
affinity ligands and then compare their performance as affinity sorbents.
On a functional standpoint, DNA or RNA aptamers exhibit affinities to target
proteins relatively similar or even higher compared to antibodies, with dissociation
constants from the low nanomolar to the picomolar range, and are described as
equivalent ligands [170]. Antibodies are proteins; they are sensitive to proteases that
are frequently present in the crude extracts. Acidic conditions may induce partial
denaturation that is most of the time reversible, depending on the pH, but also
contribute to antibody aggregation. Moreover, in alkaline conditions, irreversible
glutamine deamidation processes are possible with a reduction of the affinity
126
G. Perret and E. Boschetti