Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers

Gašperšič, Jernej; Podgornik, Aleš; Kramberger, Petra; Jarc, Marko; Jančar, Janez; Žorž, Mirjan; Krajnc, Nika Lendero

doi:10.1016/j.jchromb.2016.07.020

Cited by 6 publications

(6 citation statements)

References 18 publications

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“…They possess highly connected open pores allowing convective mass transfer and high flow rates. The ability of monoliths to separate PEGylated proteins by IEX and HIC modes has been demonstrated in previous reports ( Gašperšič et al, 2016 ; Mayolo-Deloisa et al, 2016 ).…”

Section: Pegylationmentioning

confidence: 59%

“…IEX has been extensively used in downstream processes for capture, intermediate and polishing steps ( Pfister and Morbidelli, 2014 ). This chromatographic technique allows the simultaneous separation of the native protein, unreacted PEG, mono- and multi-PEGylated species, and even positional isomers ( Seely and Richey, 2001 ; Gašperšič et al, 2016 ; Nguyen et al, 2016 ). The PEGylation reaction can alter the superficial charge of the protein.…”

Section: Pegylationmentioning

confidence: 99%

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Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Sánchez‐Trasviña

Flores-Gatica

Enriquez-Ochoa

et al. 2021

Front. Bioeng. Biotechnol.

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Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

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Section: Pegylationmentioning

confidence: 59%

Section: Pegylationmentioning

confidence: 99%

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Sánchez‐Trasviña

Flores-Gatica

Enriquez-Ochoa

et al. 2021

Front. Bioeng. Biotechnol.

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“…Since proteins have several available reactive groups, the mPEG is probably binding to di erent residues on the protein [1]. e isomer separation of PEGylated proteins has been reported earlier using ion exchange chromatography [3,8]. Nevertheless, to our knowledge, its separation by HIC has not been reported.…”

Section: Capacity Of Pegylated Monoliths To Separate Pegylatedmentioning

confidence: 85%

“…In this work, the DBC in HIC mode for each PEGylated monolith was determined using BSA as model protein under di erent operative conditions: ow rate, salt concentration, and feed protein concentration. Since the EDA monolithic disk is a weak anionic support [8], it was not possible to use it as a control in saturation curve experiments under the same conditions. e breakthrough curves, which show the rise of protein concentration in the e uent with time until this value reaches the same concentration of the feed [22], are presented in Figure 1.…”

Section: Adsorption Capacity Of Pegylated Monolithsmentioning

confidence: 99%

“…In this context, chromatographic methods have represented the most attractive and e ective alternative for their separation since it can exploit the di erent properties altered during the PEGylation reaction [4]. e chromatographic methods that have been used to separate mono-PEGylated proteins are hydrophobic interaction (HIC), anion exchange (AEX), size exclusion (SEC), and cation exchange (CEX) chromatographies [4,6,8]. Our research group has been working extensively in the separation of PEGylated proteins.…”

Section: Introductionmentioning

confidence: 99%

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Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

Sánchez‐Trasviña

Rito‐Palomares

González‐Valdez

2019

Advances in Polymer Technology

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PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.

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A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins

De los Santos‐González,

Ibarra‐Herrera,

Valencia‐Gallegos

et al. 2023

Electrophoresis

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Separation of PEGylated protein mixtures into individual species is a challenging procedure, and many efforts have been focused on creating novel chromatographic supports for this purpose. In this study, a new monolithic stationary phase with hyperbranched nanostructures was chemically synthesized. For this, monoliths with a support matrix of poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) and ethylenediamine chemistry were modified with third‐generation dendrons with butyl‐end groups. The new monolith was analyzed by infrared spectroscopy, confirming the dendron with butyl ligands and exhibited low mass transfer resistance as observed by breakthrough frontal analysis. This support was able to separate mono‐PEG ribonuclease A from the PEGylation mixture, indicated by a single band (∼30 kDa) in the electrophoretic analysis. Moreover, the separation of mono‐PEGylated positional isomers was probably observed, as the protein with ∼30 kDa was found in two separate peaks. Interestingly, the dendronized monolith allowed the separation of the reaction mixture into individual PEGylated species when using high ammonium sulfate concentrations (2 M). A correlation between the PEGylation degree and the strength of the hydrophobic interactions on the monolith was observed. This chromatographic approach combines the natural branched architecture of dendrons and the higher capabilities of the monoliths enhancing the hydrophobic surface area, and therefore the interaction between the PEGylated proteins and ligands. Thus, the novel support represents a novel platform for the purification of PEGylated from non‐PEGylated proteins with biotechnological applications.

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Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers

Cited by 6 publications

References 18 publications

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins

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