Antibodies and recombinant active substances of the second generation, which also include the
Fc-fusion protein, represent a high percentage of the global market of biologically active
substances as there is a wide range of their use in numerous applications. Because biological
medicines are produced by living organisms, the healing substance in the final biological
medicine can express a certain lower stage of variability (microheterogeneity), which has to be
within a certain acceptable range. One of the more important post-translation modifications,
that has a vital influence on their structure, solubility, stability, function, locality, folding, and
interactions with other proteins, is glycosylation. The production of therapeutic glycoproteins
thus takes place under controlled conditions, which must be constantly monitored to ensure a
consistent, reproducible and predictable pattern of glycosylation. With this aim, we wanted to
develop chromatographic techniques for determining different isoforms o antibodies, which we
could include in a bioprocess analysis. In the first part of the doctorate dissertation, we
characterized lectin ligands for affinity chromatography. For this purpose, we modified the
lectins for a more successful immobilization to carriers and better exposure of the active region
for binding. Biological activity of lectins was checked by biolayer interferometry (BLI). The
modified lectin rPA-ILNME6, which we named rPE6, bonded to a carrier showed better
bonding kinetics in comparison to the first. In the second part of the doctorate dissertation, we
further determined the conditions for partial antibody unfolding, which is needed, for the
carbohydrates to expose themselves and with that become accessible to bond with lectins. We
exposed the antibodies to temperature stress with the presence of a reductant and detected
changes with the help of tryptophane intrinsic fluorescent measurements, DLS measurements,
size-exclusion chromatography, and ionic exchange chromatography. According to the results
of intrinsic fluorescence and BLI measurements, the unfolding of antibodies was achieved, but
the proportion of unfolded molecules, as well as their stability, was low, as they could not be
detected by chromatographic methods. Because of the unsuccessful unfolding of the anty bodies
we used Fc-fusion proteins, as representatives of glycoproteins, with known glycan structures
exposed for bonding, for the development of chromatographic carriers with lectins. This way
we were able to characterize a poliHIPE carrier with the bonded lectin rPE6 in the third part of
the doctorate dissertation. We estimated the carrier capacity to 0.57 mg/ml and the limit of
detection between 0.19 mg/ml and 8 mg/ml. We showed that the binding kinetics of the
immobilized lectin rPE6 is fast enough so that it doesn't affect the Fc-fusion protein binding.
This allows us to use the developed carrier for fast and efficient separation of glycoprotein
isoforms. We optimized the separation time to 10 min, at a mobile phase rate of 8 ml/min, and
the carrier maintained stability for a further four months after immobilization. In the fourth part
of the doctorate dissertation, we showed an example of the use of this carrier for monitoring a
perfusion bioprocess production of an Fc-fusion protein. The concentration pattern obtained
with poliHIPE carrier matched with the results obtained with the protein A column isolation.
The components of the medium, HCPs and DNA molecules did not interfere with the detection
with our carrier, which makes it suitable for separating isoforms of glycoproteins that have
oligosaccharides bound by lectin ligand in the glycan structure. The carrier was also used to
determine layer thickens by measuring pressure drop, which allows us to track the aggregate
formation of the Fc-fusion protein, but their concentration was below the limit of detection.
However, we used this method to show lectin aggregation during immobilization.
|
