Simulation of mono‐PEGylated lysozyme separation in heparin affinity chromatography using a general rate model

Abstract

BACKGROUND: The bioavailability of therapeutic proteins is improved through PEGylation. This chemical modification involves the production of isomers with different numbers and sites of attached PEG chains, which are difficult to separate efficiently. Their purification with chromatography requires an understanding of the operation and the evaluation of different operational conditions. The General Rate Model (GRM) was applied for modelling the linear salt gradient elution of mono-PEGylated and native lysozyme in Heparin Affinity Chromatography (HAC) considering mass balance equations for proteins in the bulk-fluid phase, in the particle phase and the kinetic adsorption. length (5, 13, 25 column volumes) and flow (0.8 and 1.2 mL min -1 ) with a relative error in retention times of less than 6% and correlation coefficients greater than 0.78. CONCLUSION: Simulation of the elution curves of PEGylated lysozyme in HAC was performed in this work and the diverse information generated by the model is explained through the physicochemical protein properties. This simulation represents a tool for optimization, prediction and future scale-up of PEGylated proteins purification, which would reduce the investment in time and resources to test several operating conditions. RESULTS: The model was able to simulate the individual proteins and the separation of these in a PEGylation reaction using as proof-of-concept a mono-PEGylated and native lysozyme mixture under changes of operational parameters such as the gradient

Model‐Based Investigation on the Mass Transfer and Adsorption Mechanisms of Mono‐Pegylated Lysozyme in Ion‐Exchange Chromatography

“…The adsorption and desorption rate coefficients and were calculated and are displayed in Figure c and d. With increasing PEG MW, both and showed an exponential increase. A similar trend has been observed by Meja‐Manzano et al for affinity chromatography. The increase of exceeded the increase of by more than two orders of magnitude.…”

“…An approximately linear decrease of the film diffusion coefficient with increasing was determined as displayed in Figure a. A comparable dependency was reported by Meja‐Manzano et al for affinity chromatography. As shown in Figure b, the pore diffusion coefficient decreased reciprocally with increasing according to the Stokes–Einstein equation qualitatively.…”

Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

“…ere are only two works dealing to some extent with affinity chromatography of PEG-modified proteins. In one of these works, the purification of 20 kDa mono-PEGylated lysozyme with heparin affinity chromatography was optimized through an experimental design [32], and the elution curves for the operation were later simulated [33]. e other studies deal with the PEGylation of ligands in AC supports or resins, with the aim of improving their stability using as model concanavalin and protein A [34,35].…”

Is Bioseparation still the limiting step in bioprocess development?

“…Finally, the research papers of this Special Issue present selected advances in the field of primary recovery, purification and bioprocess development . Examples from the primary recovery of products include: a new method to extract recombinant intracellular GFP from E. coli by using aqueous solutions of surface‐active compounds, a novel integrated bioseparation process for the in situ recovery of L‐asparaginase from fermentation broth, a stable and efficient flocculation method associated with depth filtration for continuous cell separation and an approach using ultrafiltration for whey protein separation .…”

“…The pDNA capture using grafted adsorbents is critically discussed by Marcelo Fernandez‐Lahore and co‐workers . A collaboration between the group of Marco Rito‐Palomares and Juan Asenjo resulted in the report of the simulation of a PEGylated protein separation using affinity chromatography. Interestingly, a novel process for the purification of influenza virus‐like particles using cellulose membrane adsorbers is reported by the research group of Manuel Carrondo .…”