Chromatographic Separation of Conjugates PolymerProtein
Cisneros Ruíz, Mayra
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The attachment of polyethylene glycol (PEG) molecules, called PEGylation, can improve the therapeutic properties of proteins. The PEGylation product depends on the conditions under which the conjugation reaction takes place. PEGylation reactions often result in a population of conjugate species in terms of the number of attached PEG chains and their locations. As some portion of this population may be biologically inactive the resolution of these protein mixtures represents a challenge to the separation step. Currently, the methods to purify PEGylated proteins have been dominated by size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Research works describing the use of the conjugate hydrophobicity for separation are not very common. It is clear that hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC) have not been fully investigated in the past as separation methods for the resolution of PEGylated proteins. This thesis is focused on the analysis of the chromatographic behavior of PEGylated proteins in RPC and the potential use of a mild hydrophobic support combining HIC and aqueous two phase extraction (ATPE) principles. Two proteins were selected as experimental models: ribonuclease A (RNase A) and apo-?lactalbumin (apo-?Lac). Both proteins were reacted with an activated PEG with a nominal molecular weight of 20 kDa and the reaction mixtures were analyzed by SEC and mass spectrometry. The structure of the PEGylated proteins was analyzed, showing that the attachment of PEG molecules did not modify the structure of the proteins. Reverse phase chromatography (RPC) under neutral pH conditions was used to resolve the populations of PEGylated conjugates. PEG-conjugates were separated with better resolution and in less time using RPC at neutral pH rather than using SEC. RPC also allowed the identification of a tri-PEGylated species produced during the reaction with RNase A; and not identify when SEC was used. The results showed that it is possible to separate PEGylated species by RPC at neutral pH. Changes of pH (at 2.0, 7.0 and unbuffered) of the mobile phase, showed that the pH does not play a significant role in the chromatographic behavior of PEG-conjugates, when the unmodified protein is not retained. However, when the unmodified protein is retained, the effect of the pH on the PEGylated proteins is similar to that observed for the unmodified species. It was demonstrated that temperature affects the chromatographic separation of PEGconjugates in a similar manner in which it affects the separation of the neat polymer. A novel approach to potentially separate PEGylated proteins from the unmodified form using a mild hydrophobic support in which PEG is immobilized in sepharose was addressed. Different behavior retention of the native protein from the PEGylated species was achieved using a gradient elution between 3 M ammonium sulfate in 25 mM potassium phosphate, pH 7.0 and 25 mM potassium phosphate, pH 7.0. Parameters such as pH, salt type and salt concentration had no significant influence on chromatographic behavior of native, monoPEGylated and di-PEGylated RNase A using this separation system. The proposed approach described here provides a simple and practical chromatographic method to separate unmodified proteins from their PEG conjugates.