Current advances in the non‐chromatographic fractionation and characterization of PEGylated proteins

Mayolo‐Deloisa, Karla; González‐Valdez, José; Guajardo‐Flores, Daniel; Aguilar, Oscar; Benavides, Jorge; Rito‐Palomares, Marco

doi:10.1002/jctb.2498

Cited by 27 publications

(27 citation statements)

References 45 publications

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“…The technique has been extremely useful in the separation of conjugates for subsequent mass and structure analysis. However, there are no correlations with quantitative molecular weights because standard protein ladders cannot be used for this purpose [26]. CE has been used to optimize PEGylation reactions, to assess purity and stability [30], to analyze the PEGylation site, and to identify positional isomers [31].…”

Section: Separation and Purification Of Pegylated Conjugatesmentioning

confidence: 99%

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Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

2012

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In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG-protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a "PEGylaided" process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.

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Section: Separation and Purification Of Pegylated Conjugatesmentioning

confidence: 99%

“…Both techniques are widely used to separate PEGylated proteins on the laboratory scale, but are mainly used for analytical purposes [26]. PAGE has been used to analyze and characterize PEGylation reactions using mixtures of stains to visualize reaction products and unreacted PEG [27][28][29].…”

Section: Separation and Purification Of Pegylated Conjugatesmentioning

confidence: 99%

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

2012

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“…As with almost any protein therapeutic, there are issues with the manufacture, characterization, and use of PEGylated proteins [70][71][72][73][74][75], including the potential induction of anti-PEG antibodies [76], especially in the case of hyperPEGylated products that contain large numbers of conjugated PEG molecules [77][78]. Many proteins used as therapeutics display some propensity for immunogenicity [79][80][81][82][83][84].…”

Section: Protein Pegylationmentioning

confidence: 99%

Chemical and Genetic Modification

Farys¹,

Ginn²,

Badescu³

et al. 2013

Pharmaceutical Sciences Encyclopedia

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There are many natural post‐translational modifications of proteins (e.g., glycosylation, ubiquitination, acylation,and amino acid derivatization).Glycosylation is common for therapeutic proteins. Classes of proteins modified by glycosylation include monoclonal antibodies, blood factors, hematopoietic proteins, and cytokines. Excluding monoclonal antibodies, many proteins were, at least when they were first introduced to the clinic, designed to be used as a replacement protein for the corresponding endogenous protein. Maintaining control of the protein structure to be as close as possible to the structure of the endogeneous protein is therefore a key goal. In this review, we focus on (i) optimization of protein pharmacokinetics, (ii) improvement of protein properties to be used as medicines (this primarily includes reduction of immunogenic sequences and improvement of protein stability), (iii) addition of a therapeutic effect, and (iv) use of proteins as diagnostics.

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“…Note that liquid-liquid separation strategies have also been explored. 7,8 Electrokinetics (EKs) is a branch of physics that studies the induced motion of solids and fluids while in the presence of an electric field. EK phenomena have been successfully employed within microfluidic systems to manipulate the movement of a range of bioparticles, among which we can list yeast cells, virus, bacteria, microalgae, and DNA.…”

Section: Introductionmentioning

confidence: 99%

Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures

et al. 2016

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Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step. Published by AIP Publishing. [http://dx

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Current advances in the non‐chromatographic fractionation and characterization of PEGylated proteins

Cited by 27 publications

References 45 publications

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

Chemical and Genetic Modification

Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures

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