Extent Of PEGylation Research Articles

Assessing site-specific PEGylation of TEM-1 β-lactamase with cell-free protein synthesis and coarse-grained simulation

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 β-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

Determination of extent of PEGylation using denaturing capillary isoelectric focussing

Conjugated proteins and enzymes are often formed using N-hydroxysuccinimide (NHS) chemistry, which reacts with free primary amines resulting in a loss of charge and a reduction in isoelectric point (pI). Measurement of the extent of reaction of these conjugates is critical for biopharmaceutical developers. Due to this change in protein charge state, denaturing capillary isoelectric focussing (cIEF) offers a potentially straightforward and convenient approach for extent-of-reaction quantification. Here, we demonstrate the potential of this technique with poly(ethylene glycol) (PEG) conjugates of Erwinia chrysanthemil-asparaginase (ErA). Development of an appropriate sample preparation technique is critical to achieving reproducible cIEF electropherograms, particularly for denaturation-resistant proteins such as ErA, and an emphasis was placed on this during development of the PEG-ErA cIEF method. cIEF electropherograms demonstrating a distribution of PEGylation states in a bell-shaped curve were obtained, and assignment of PEGylation states to these peaks was critical to routine use of the method. The method is sensitive enough to resolve non-lysine adducts of PEG (such as those conjugated to histidine residues) and was shown to give reproducible results over a 2 year period. Biopharmaceutical developers should consider cIEF for extent of reaction monitoring and measurement for conjugates of free amine groups.

Optimization of Cationic Nanogel PEGylation to Achieve Mammalian Cytocompatibility with Limited Loss of Gram-Negative Bactericidal Activity.

Tuning the composition of antimicrobial nanogels can significantly alter both nanogel cytotoxicity and antibacterial activity. This project investigated the extent to which PEGylation of cationic, hydrophobic nanogels altered their cytotoxicity and bactericidal activity. These biodegradable, cationic nanogels were synthesized by activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) emulsion polymerization with up to 13.9 wt % PEG (MW = 2000) MA, as verified by 1H NMR. Nanogel bactericidal activity was assessed against Gram-negative E. coli and P. aeruginosa and Gram-positive S. mutans and S. aureus by measuring membrane lysis with a LIVE/DEAD assay. E. coli and S. mutans viability was further validated by measuring metabolic activity with a PrestoBlue assay and imaging bacteria stained with a LIVE/DEAD probe. All tested nanogels decreased the membrane integrity (0.5 mg/mL dose) for Gram-negative E. coli and P. aeruginosa, irrespective of the extent of PEGylation. PEGylation (13.9 wt %) increased the cytocompatibility of cationic nanogels toward RAW 264.7 murine macrophages and L929 murine fibroblasts by over 100-fold, relative to control nanogels. PEGylation (42.8 wt %) reduced nanogel uptake by 43% for macrophages and 63% for fibroblasts. Therefore, PEGylation reduced nanogel toxicity to mammalian cells without significantly compromising their bactericidal activity. These results facilitate future nanogel design for perturbing the growth of Gram-negative bacteria.

Chemical modification of protein A chromatography ligands with polyethylene glycol. I: Effects on IgG adsorption equilibrium, kinetics, and transport

Chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) has been proposed as a strategy to increase the process selectivity and resin robustness by providing the ligand with a steric repulsion barrier against non-specific binding. This article comprises a comprehensive study of IgG adsorption and transport in Repligen CaptivA PriMAB resin with PEGylated ProA ligands that are modified using 5.2 and 21.5 kDa PEG chains. We studied the impact of the molecular weight of the PEG as well as the extent of PEGylation for the 5.2 kDa PEG modification. In all cases, PEGylation of ProA ligands decreases the resin average pore size, particle porosity, and static binding capacity for IgG proportional to the volume of conjugated PEG in the resin. Resin batch uptake experiments conducted in bulk via a stirred-tank system and with individual resin particles under confocal laser scanning microscopy suggests that PEGylation introduces heterogeneity into IgG binding kinetics: a fraction of the IgG binding sites are transformed from typical fast association kinetic behavior to slow kinetic behavior. pH gradient elution experiments of an IgG molecule on the modified resins show an increase in IgG elution pH for all modified resins, implying a decrease in IgG-ProA binding affinity on modification. Despite losses in static binding capacity for all resins with PEGylated ligands, the loss of dynamic binding capacity at 10% breakthrough (DBC10%) ranged more broadly from almost 0–47% depending on the PEG molecular weight and the extent of PEGylation. Minimal losses in DBC10% were observed with a low extent of PEGylation with a smaller molecular weight PEG, while higher losses were observed at higher extents of PEGylation and with higher molecular weight PEG due to decreased static binding capacity and increased mass transfer resistance. This work provides insight into the practical implications for resin performance if PEGylation is considered as a strategy for selectivity enhancement in affinity chromatography with macromolecular ligands.

Surface Modification of Cisplatin-Complexed Gold Nanoparticles and Its Influence on Colloidal Stability, Drug Loading, and Drug Release.

Cisplatin-complexed gold nanoparticles (PtII-AuNP) provide a promising strategy for chemo-radiation-based anticancer drugs. Effective design of such platforms necessitates reliable assessment of surface engineering on a quantitative basis and its influence on drug payload, stability, and release. In this paper, poly(ethylene glycol) (PEG)-stabilized PtII-AuNP was synthesized as a model antitumor drug platform, where PtII is attached via a carboxyl-terminated dendron ligand. Surface modification by PEG and its influence on drug loading, colloidal stability, and drug release were assessed. Complexation with PtII significantly degrades colloidal stability of the conjugate; however, PEGylation provides substantial improvement of stability in conjunction with an insignificant trade-off in drug loading capacity compared with the non-PEGylated control (<20% decrease in loading capacity). In this context, the effect of varying PEG concentration and molar mass was investigated. On a quantitative basis, the extent of PEGylation was characterized and its influence on dispersion stability and drug load was examined using electrospray differential mobility analysis (ES-DMA) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) and compared with attenuated total reflectance-FTIR. Using ES-DMA-ICP-MS, AuNP conjugates were size-classified based on their electrical mobility, while PtII loading was simultaneously quantified by determination of Pt mass. Colloidal stability was quantitatively evaluated in biologically relevant media. Finally, the pH-dependent PtII release performance was evaluated. We observed 9% and 16% PtII release at drug loadings of 0.5 and 1.9 PtII/nm2, respectively. The relative molar mass of PEG had no significant influence on PtII uptake or release performance, while PEGylation substantially improved the colloidal stability of the conjugate. Notably, the PtII release over 10 days (examined at 0.5 PtII/nm2 drug loading) remained constant for non-PEGylated, 1K-PEGylated, and 5K-PEGylated conjugates.

Shedding light on surface exposition of poly(ethylene glycol) and folate targeting units on nanoparticles of poly(ε-caprolactone) diblock copolymers: Beyond a paradigm

Polymeric nanoparticles (NPs) of poly(ε-caprolactone) (PCL) covered with a hydrophilic poly(ethylene glycol) (PEG) shell are usually prepared from diblock PEG-PCL copolymers through different techniques. Furthermore PEG, NPs can be decorated with targeting ligands to accumulate in specific cell lines. However, the density and conformation of PEG on the surface and its impact on the exposition of small targeting ligands has been poorly considered so far although this has a huge impact on biological behaviour. Here, we focus on PEG-PCL NPs and their folate-targeted version to encourage accumulation in cancer cells overexpressing folate receptor α. NPs were prepared with mixtures of PEG-PCL with different PEG length (short 1.0kDa, long 2.0kDa,) and a folate-functionalized PEG-PCL (PEG 1.5kDa) by the widely employed solvent displacement method. In depth characterization of NPs surface by 1H NMR, fluorescence and photon correlation spectroscopy evidenced a PEGylation extent below 7% with PEG in a mushroom conformation and the presence of folate more exposed to water pool in the case of copolymer with short PEG. NPs with short PEG adsorbed HSA forming a soft corona without aggregating. Although limited, PEGylation overall reduced NPs uptake in human macrophages. Uptake of NPs exposing folate prepared with short PEG was higher in KB cells (FR+) than in A549 (FR−), occurred via FR-receptor and involved lipid rafts-dependent endocytosis. In conclusion, the present results demonstrate that PEG length critically affects protein interaction and folate exposition with a logical impact on receptor-mediated cell uptake. Our study highlights that the too simplistic view suggesting that PEG-PCL gives PEG-coated NPs needs to be re-examined in the light of actual surface properties, which should always be considered case-by-case.

Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

In Vitro Cellular Gene Delivery Employing a Novel Composite Material of Single-Walled Carbon Nanotubes Associated With Designed Peptides With Pegylation

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical fields including application for drug delivery system. In this study, we developed a novel gene delivery system employing SWCNTs associated with polycationic and amphiphilic H-(-Lys-Trp-Lys-Gly-)7-OH [(KWKG)7] peptides having pegylation. SWCNTs wrapped with (KWKG)7 formed a complex with plasmid DNA (pDNA) in aqueous solution based on polyionic interaction but later underwent aggregation. On the other hand, a complex of pDNA and SWCNT-(KWKG)7 modified with polyethylene glycol (PEG) chains of 12 units [SWCNT-(KWKG)7-(PEG)12] afforded good dispersion stability for 24 h even in a cell culture medium. The in vitro cellular uptake of SWCNT-(KWKG)7-(PEG)12/pDNA complex prepared with fluorescence-labeled pDNA was evaluated with fluorescent microscopic observation and flow cytometry. The uptake by A549 human lung adenocarcinoma epithelial cells increased along with the extent of pegylation, suggesting the importance of dispersion stability in addition to the cationic charge which facilitates ionic cellular interaction. The expression of pDNA encoding the monomeric Kusabira-Orange 2 fluorescent protein in the form of the SWCNT-(KWKG)7-(PEG)12/pDNA complex demonstrated remarkable enhancement of transfection depending also on the extent of pegylation and the N/P ratio. The potential of the SWCNT composite wrapped with polycationic and amphiphilic (KWKG)7 with pegylation as a carrier for gene delivery was demonstrated.

Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

Two synthetic approaches that allow one to control PEG content within spherical nucleic acids (SNAs) have been developed. One approach begins with RNA-modified gold nanoparticles followed by a backfill of PEG 2K alkanethiols, and the other involves co-adsorption of the two entities on a gold nanoparticle template. These two methods have been used to explore the role of PEG density on the chemical and biological properties of RNA–SNAs. Such studies show that while increasing the extent of PEGylation within RNA–SNAs extends their blood circulation half-life in mice, it also results in decreased cellular uptake. Modified ELISA assays show that constructs, depending upon RNA and PEG content, have markedly different affinities for class A scavenger receptors, the entities responsible, in part, for cellular internalization of SNAs. In designing SNAs for therapeutic purposes, these competing factors must be considered and appropriately adjusted depending upon the desired use.

Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins

Abstract PEGylation has successfully improved the pharmacological properties of therapeutic proteins. However, polyethylene glycol (PEG) has been burdened by immunogenicity that renders a negative clinical effect on therapeutic proteins. The anti-PEG immune response to PEGylated proteins possibly depends on the nature of proteins and the conjugated methoxy PEG (mPEG). Thus, it is necessary to investigate the effects of protein immunogenicity, the extent of PEGylation, the molecular weight (Mw), and the branching of mPEG on the anti-PEG immune response. Ovalbumin, tetanus toxoid (TT), TT–TT conjugate, and TT–bovine serum albumin conjugate were used as target proteins. PEGylated proteins with different extents of PEGylation were obtained by fractionation of the PEGylated TT with size exclusion chromatography. The PEGylated proteins with different Mw and branching of mPEG were obtained by modification of TT with linear mPEG (5 kDa and 20 kDa) and branched mPEG (20 kDa). The PEGylated proteins elicited high levels of anti-PEG antibodies (predominantly IgM and IgG1). The anti-PEG immune response depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of mPEG. In contrast, branching of mPEG had an insignificant effect on the anti-PEG immune response to the PEGylated proteins.

Abstract 2956: Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties

Abstract As antibody-drug conjugates (ADCs) continue to emerge as an important therapeutic modality for the treatment of cancer, there is an increased effort to elucidate critical design parameters and devise improved linker technologies. The impact of drug-to-antibody ratio (DAR) on conjugate plasma pharmacokinetics (PK) is known to be an important attribute, and accelerated clearance induced by high levels of drug loading has served as a barrier to translating increased in vitro potency to in vivo xenografts. We have recently demonstrated that the incorporation of a discrete PEG24 unit into an auristatin drug-linker can greatly diminish the impact of drug loading on ADC PK. In an effort to optimize the antibody-mediated delivery of monomethylauristatin E (MMAE) as a homogeneous DAR 8 conjugate, we prepared a series of MMAE linkers using PEG units of varying lengths to identify constructs that preserve antibody PK properties and provide enhanced in vivo activity. The extent of PEGylation and linker chemistry was found to impact conjugate PK properties, biodistribution, antitumor activity, and tolerability. From that effort, a cleavable MMAE linker incorporating the glucuronide-based trigger, a self-stabilizing maleimide, and 12 PEG units emerged as the optimal design. ADCs prepared with this linker have now undergone further preclinical characterization in activity and toxicology models in which they have demonstrated an increase in therapeutic index relative to other MMAE-based ADCs. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Julia H. Cochran, Nagendra Chemuturi, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2956.

PEGylated human serum albumin (HSA) nanoparticles: preparation, characterization and quantification of the PEGylation extent

Modification with poly(ethylene glycol) (PEG) is a widely used method for the prolongation of plasma half-life of colloidal carrier systems such as nanoparticles prepared from human serum albumin (HSA). However, the quantification of the PEGylation extent is still challenging. Moreover, the influence of different PEG derivatives, which are commonly used for nanoparticle conjugation, has not been investigated so far. The objective of the present study is to develop a method for the quantification of PEG and to monitor the influence of diverse PEG reagents on the amount of PEG linked to the surface of HSA nanoparticles. A size exclusion chromatography method with refractive index detection was established which enabled the quantification of unreacted PEG in the supernatant. The achieved results were confirmed using a fluorescent PEG derivative, which was detected by photometry and fluorimetry. Additionally, PEGylated HSA nanoparticles were enzymatically digested and the linked amount of fluorescently active PEG was directly determined. All the analytical methods confirmed that under optimized PEGylation conditions a PEGylation efficiency of up to 0.5 mg PEG per mg nanoparticle could be achieved. Model calculations made a ‘brush’ conformation of the PEG chains on the particle surface very likely. By incubating the nanoparticles with fetal bovine serum the reduced adsorption of serum proteins on PEGylated HSA nanoparticles compared to non-PEGylated HSA nanoparticles was demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Finally, the positive effect of PEGylation on plasma half-life was demonstrated in an in vivo study in mice. Compared to unmodified nanoparticles the PEGylation led to a four times larger plasma half-life.

Dual-Functionalized PAMAM Dendrimers with Improved P-Glycoprotein Inhibition and Tight Junction Modulating Effect

This study aims to surface modify poly(amido amine) or PAMAM dendrimers by sequentially grafting poly(ethylene glycol) or PEG and 4-thiobutylamidine (TBA) so as to reduce PAMAM cytotoxicity while improving the ability of PAMAM to modulate P-glycoprotein (P-gp) efflux and tight junction integrity. Conjugation of functional groups was determined by NMR spectroscopy, FT-IR, thiol group quantification and molecular weight estimation. The yield of the dual-functionalized dendrimers was >80%. The dual-functionalized dendrimer could significantly reduce PAMAM cytotoxicity to <15% as reflected by LDH release in Caco-2 and MDCK/MDR1 cells after 72 h of exposure. Thiolated dendrimers could increase cellular accumulation and permeation of the P-gp substrate R-123, and such effect could be affected by the extent of PEGylation of the dendrimer. Surface-modified PAMAM dendrimers, either by single or dual functionalization, could better modulate tight junction integrity in comparison with unmodified PAMAM, as demonstrated through immunostaining of the tight junction marker ZO-1, permeation of the model compound Lucifer Yellow (LY) and transepithelial electrical resistance (TEER). Of importance, reversible tight junction modulating effect was only observed in the dual-functionalized dendrimers. Collectively, dual functionalization with PEG and TBA represented a promising approach in altering PAMAM dendrimer surface for potential application in oral drug delivery.

Preparation of pegylated lumbrokinase and an evaluation of its thrombolytic activity both in vitro and in vivo

Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities. In clinical practice, LK can only be administered orally because of its antigenicity, immunogenicity and potential to produce anaphylactic reactions after injection. However, many useful drugs such as interferon, insulin, erythropoietin and interleukin have been modified with polyethylene glycol (PEG) to prepare injectable formulations. In this study, LK was modified with methoxy PEG succinimidyl carbonate (mPEG-SC) with molecular weights of 5000, 10,000 and 20,000 and the pegylated products were isolated and purified using the Akta protein purification system. The extent of pegylation was determined by HPLC. Fibrinolytic activities of pegylated and unmodified LK were measured both in vitro against urokinase on fibrin plates and in vivo using a mouse carageenan black tail model. Optimal pegylation was obtained using mPEG-SC5000 in a buffer pH 8.0 with a reaction time of 5h, reaction temperature of 0°C and LK:mPEG-SC molar ratio of 1:25. The results show that mPEG modified LK has strong fibrinolytic and thrombolytic activities both in vitro and in vivo. It is suggested that the pegylated LK is a promising injectable thrombolytic agent for the treatment of thrombotic diseases in clinical practice.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0–400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.

Microchip capillary gel electrophoresis of multiply PEGylated high‐molecular‐mass glycoproteins

PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.

In this issue: Biotechnology Journal 4/2011

AbstractMutation detection in biopharmaceutical plasmidsOliveira et al., Biotechnol. J. 2011, 6, 378–391The number of applications involving therapeutic plasmid DNA (pDNA) for DNA vaccinations and gene therapy is increasing worldwide. This creates a demand for production of large amounts of stable and purified molecules that conform to Good Manufacturing Practice guidelines and comply with the regulatory policies of the FDA or EMA. Efficient, cost‐effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid‐based biopharmaceutical. In this issue, authors from Lisbon, Portugal, and the MIT (Cambridge, MA, USA) review bioinformatic tools for ab initio detection of potentially unstable DNA regions and techniques used for mutation detection in nucleic acids as well as practical examples from studies using pDNA.Semi‐continuous magnetic PEGylationOttow et al., Biotechnol. J. 2011, 6, 396–409The applications of polypeptide‐based biocatalysts, enzymes, and pharmaceuticals are of growing interest. The most successful technique to enhance their activity and stability is via the addition of polyethylene glycol polymer chains, i.e., PEGylation. In this issue Ottow et al. from the Technical University of Denmark in Lyngby describe a semi‐continuous magnetic particlebased process for the controlled attachment of PEG to proteins for the first time. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing the target protein trypsin and magnetic adsorbents were mixed with activated PEG and the reaction stopped with free amine groups. In a high‐gradient magnetic separator magnetic supports with bound PEGylated trypsin are captured and subsequently eluted. The extent of PEGylation could be controlled by varying the reaction time and PEG concentration.Immuno‐assay on chipHerwig et al., Biotechnol. J. 2011, 6, 420–427Immunoaffinity‐based methods, such as western blottin or ELISA, are crucial analytical tools to detect and quantify proteins. They are however, time‐consuming and expensive. In this issue, the group of Günter Allmaier (Vienna University of Technology, Austria) in collaboration with Agilent Technologies (Waldbronn, Germany) report the development of an immunoprecipitation (IP) method combined with automated microchip capillary gel electrophoresis (IP‐MCGE) with laser‐induced fluorescence (LIF) detection for analysis of endogenous β‐galactosidase in crude E. coli cell lysates. Total cell lysates were derivatized with a fluorescence dye, incubated with anti‐β‐galactosidase antibodies, and the antigen/antibody complex was precipitated with protein G‐coated magnetic beads. The complex was eluted from the beads and loaded directly onto a multisample microchip for analysis. The developed IP‐MCGE method is more sensitive and faster than western blotting, and also requires less reagents.

A magnetic adsorbent‐based process for semi‐continuous PEGylation of proteins

A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.

Kinetic and stoichiometric analysis of the modification process for N-terminal PEGylation of staphylokinase

Staphylokinase (SAK) is a therapeutic protein with promise for thrombolytic therapy of acute myocardial infarction. In this study, polyethylene glycol (PEG) aldehyde was used for N-terminal PEGylation of SAK to improve the pharmacological profiles of SAK. Due to the presence of the competitive PEGylation between the N terminus and the Lys residues, kinetic and stoichiometric analysis was carried out to investigate the process for the N-terminal PEGylation of SAK. To achieve this objective, size exclusion chromatography and tryptic peptide mapping were used to measure the PEGylation extent of SAK molecule and its specific amino acid residues, respectively.

Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel genedelivery vectors

Dendrimers have attracted great interest in the field of gene delivery due to their syntheticcontrollability and excellent gene transfection efficiency. In this work, dendrigraftpoly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivativesof DGLs (generation 2 and 3) with different extents of PEGylation were successfullysynthesized and used to compact pDNA as complexes. The result of gel retardation assayshowed that pDNA could be effectively packed by all the vectors at a DGLs to pDNAweight ratio greater than 2. An increase in the PEGylation extent of vectors resulted ina decrease in the incorporation efficiency and cytotoxicity of complexes in 293cells, which also decreased the zeta potential a little but did not affect the meandiameter of complexes. Higher generation of DGLs could mediate higher genetransfection in vitro. Confocal microscopy and cellular uptake inhibition studiesdemonstrated that caveolae-mediated process and macropinocytosis were involvedin the cellular uptake of DGLs-based complexes. Also the results indicate thatproper PEGylated DGLs could mediate efficient gene transfection, showing theirpotential as an alternate biodegradable vector in the field of nonviral gene delivery.