(15a) Chemical Grafting of Chromatographic Resins for the Selective Adsorption of Pegylated Proteins

Chemical
grafting of chromatographic resins for the selective adsorption of PEGylated
proteins.

Hernandez-Martínez
Agustín; Aguilar Oscar*

Centro
de Biotecnología-FEMSA. Tecnológico de Monterrey, Campus Monterrey.

Av.
Eugenio Garza Sada 2501 Sur, Col. Tecnológico Monterrey, México C.P. 64849

*corresponding
author: alex.aguilar@itesm.mx

PEGylation reaction, or covalent attachment of a
polyethylene glycol (PEG) molecule to a therapeutic protein is a recent
strategy still in development aiming to increase the pharmacologic effect and
decrease the degradation rate of the protein during its circulation in the
body. One of the main challenges in the production of PEGylated proteins is
related with the downstream processing, due to the complex mixture of reaction
products, resulting in a population comprising mono- and di-PEGylated species
as well as several positional isomers. The resolution and characterization of
these positional isomers gains higher relevance due to the fact that in most of
the cases only one of the isomers exhibits the desired therapeutic effect.

Recent reports of our group suggest that PEGyilated
proteing exhibit a marked prefference for the PEG-rich phase of a PEG/salt
aqueous two-phase system (ATPS)¹  suggesting a posible affinity
partitioning that could be applied to a PEGylated surface such as the
chromatografic resin.

Sepharose 6B was subjected to chemical modification
according to the protocol reported by Sundberg and Porath² for the
covalent attachment of PEG 550, PEG 2000 and PEG 5000. The amount of polymer
covalently linked to the resin was determined to be from 138 to 180 mg PEG/g
resin. An increase in the static binding capacity in HIC conditions was
observed for the three modified resins (3.0, 3.6 and 2.6 mg BSA/mL resin
respectively), as compared to the unmodified Sepharose 6B (1.2 mg BSA/mL),
suggesting changes in hydrophobicity of the resins due to the PEG molecule.

Dynamic binding capacities were determined for the
three modified supports in 1 mL Tricorn columns using a Äkta Prime system (GE
Healthcare) under the conditions reported by Cisneros-Ruiz et al. (2009)³
(Figure 1), showing a decrease in DBC fro BSA between the unmodified Sepharose
compared to modified resins. Different capacities were observed between the
molecular weight of the polymer used for grafting the chromatographic curface,
mainly due to the difference in the chain length, having a direct impact on the
hydrophobicity of the resin. Additionally a change in the conformation of the
polymer attached to the resin creates a different environment for protein
adsorption.

The potential application of these modified resins for
the isolation and purification of PEGylated products is under study. Preliminaty
results indicate the potential use of these resins for the isolation of
PEGylated products from the reaction mixture, without the need for additional
SEC or IEX previous steps (Figure 2). An optimization of the separation
conditions is needed, but the potential for peak resolution would open the way
for an integrated process for primary extraction and purification of PEGylated
proteins from reaction mixtures.

1- González-Valdez J, Cueto L, Benavides J, Rito-Palomares M. Potential
application of aqueous two-phase systems for the fractionation of RNase A and
a-Lactalbumin from their PEGylated conjugates. J Chem Technol Biotechnol. 86
(2011) 26?33.

2- Sundberg L,
Porath J. Preparation of adsorbents for
biospecific affinity chromatography: I. Attachment of group-containing ligands
to insoluble polymers by means of bifunctional oxiranes. J. Chromatogr. 90
(1974) 87-96

3- Cisneros-Ruiz M, Mayolo-Deloisa
K, Przybycien TM, Rito-Palomares, M.
Separation of PEGylated from unmodified ribonuclease
A using sepharose media. Sep Pur Technol 65 (2009) 105?109