Infoscience

Thesis

Bioconjugates for bioseparation by affinity precipitation

Recent advances in the fields of biotechnology and medicine have intensified the necessity for pharmaceutical-grade biomacromolecules purification (e.g. enzymes, antibodies, plasmid DNA). One of the key factors for a successful downstream process is the possibility to isolate and concentrate the target biomolecules in one step. Affinity precipitation is a promising technique for the purification of genetically engineered molecules. This method combines the ability of the affinity techniques to specifically capture the target molecule from a diluted feed with the concentration effect typical of a precipitation technique. Affinity precipitation is an interesting alternative to established (chromatographic) technologies. This method uses stimulus-responsive affinity macroligands (AML) to capture and then coprecipitate the target molecule. A typical AML consists of a "smart polymer" to which an affinity ligand is linked. The polymer mediates the response to an external stimulus (e.g. a change in temperature, pH or salt content) and the affinity tag mediates the selective binding of the target molecule. Upon stimulation, the entire affinity complex is precipitated. In this thesis the design and characterization of new AML to be used in affinity precipitation were studied. Moreover, their applications in the purification of protein or plasmid DNA were also described. Free radical chain transfer polymerization of N-isopropylacrylamide (NIPAAm) was used to synthesize the AML precursor, i.e., a semitelechelic thermoresponsive telomer carrying a functional group. This latter has then been reacted, using different bioconjugation techniques, to synthesis the final AML. Telomerization kinetic studies were performed for the determination of the best polymerization conditions. In a first approach, the AML was constructed using the well known avidin/biotin system. A carbodiimid-coupling strategy was used to conjugate an avidin molecule to a poly-N-isopropylacrylamide polymer (PNIPAAm) which carries a carboxylic acid end group. Then, a single stranded poly-CTT biotinylated oligonucleotide was reacted with the avidin/polymer complex to create the final AML (BiotTF-AML). This AML was found to bind tightly and with high specificity to a specific plasmid DNA via a triple helix interaction. Triple helix affinity precipitation (THAP) process was then used for the purification of plasmid DNA from a clear lysate (recovery yield > 70%, no detectable protein contamination). Another AML synthesis strategy (to be used in the THAP process) was proposed. It was based on the chemical ligation between a PNIPAAm carrying a hydrazide moiety and an aldehyde group on the poly-CTT (coupling efficiency 80%). Moreover, it was also shown that the AML employed for the plasmid DNA purification can be used in the transfection of mammalian cells with no loss in protein expression. Finally, a direct telomerization approach using a modified FLAG-peptide as telogen, was shown to be a useful strategy for a one pot synthesis of an AML employed in the affinity precipitation of antibody.

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