Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein

SDS‐PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein‐containing conjugates, mostly pegylated proteins. PEG‐maleimide (MAL) is frequently used to site‐specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C–S linkage between protein and PEG‐MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein was observed. It is a thiol‐independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG‐MAL modified conjugates although PEG‐vinylsulfone conjugates to thiol‐containing proteins also through a C–S linkage. Cleavage kinetics of PEG40k‐MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide‐thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG‐MAL modified protein/peptide by SDS‐PAGE analysis.     

Quantitative display of the redox status of proteins with maleimide‐polyethylene glycol tagging

Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide‐polyethylene glycol (m‐PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG‐switch. With PEG‐switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG‐switch in quantitation of the extent of protein thiol oxidation in cells in response to H₂O₂ and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m‐PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.     

合成分枝型聚乙二醇的简便新方法

以赖氨酸和mPEG5000为起始物,利用多肽合成中常用的保护、缩合和脱保护等方法合成了在生物医学领域中具有重要应用价值的分枝型聚乙二醇.用该方法形成的分枝型PEG在有机相中以缩合反应的方式一步完成,反应条件温和,且有较高的产率(61%).各步产物的表征都与其结构一致.最终产物分枝型PEG的¹HNMR的表征结果与其结构吻合.     

Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry was investigated as a method for the rapid determination of the extent of polymer coupling in polyethylene glycol- (PEG) conjugated superoxide dismutase (SOD). PEG-conjugated SOD, an antioxidant with an extended in vivo circulation lifetime compared to that of superoxide dismutase, is being evaluated as an effective therapeutic agent for the treatment of injuries and arthritis. The mass spectra of a standard batch of PEG-conjugated bovine SOD showed the presence of identifiable and well resolved peaks that correspond to 0–7 PEG molecules attached to bovine SOD. The area of each of the peaks provides a determination of the amount of PEG-conjugated SOD with a given number of bound PEG groups. SOD is a noncovalent dimer of two identical subunits that dissociates in MALDI. The information obtained in the mass spectra thus corresponds to a monomer of SOD. Each SOD monomer contains 10 lysines, which are the sites of PEG-conjugation. Multiple MALDI determinations of two batches of samples indicated good reproducibility for routine determination of the extent of polymer content. The amount of PEG-conjugated SOD that contained a given number of PEG molecules, determined by MALDI, was compared with the value deduced from the amount of PEG-conjugation at each attachment site measured by a peptide mapping method. Agreement between the data obtained in the two techniques (MALDI and peptide mapping) indicates that MALDI may be used to obtain quantitative information on PEG-conjugated SOD to determine the amounts of PEG-conjugated protein each with a different number of PEG groups attached. Measurement of several batches of samples stored at a higher temperature showed a lower extent of PEG-conjugation in PEG-conjugated SOD. This reduction in the PEG content resulted from the PEG-deconjugation of PEG-conjugated SOD at a higher temperature. Thus, MALDI can be used to examine the stability of PEG-conjugated SOD. The high sensitivity, relatively straightforward data interpretation, speed of analyses, and good reproducibility in measurements make this technique a useful analytical tool for fingerprinting PEG-conjugated SOD as well as potentially other polymer-conjugated proteins.     

Zeta potentials of PDMS surfaces modified with poly(ethylene glycol) by physisorption

Controlling zeta potential of PDMS surface coated with a layer of PEG is important for electroosmosis and electrophoresis in PDMS made microfluidic chips. Here, zeta potentials of PDMS surfaces modified by simple physisorption of PEG of different concentrations in phosphate buffer solutions, pure water, and PEG solution were reported. Coating PEG on PDMS surfaces was achieved by immersing a PDMS layer into the PEG solution for 10 min and then taking it out and placing it in an oven at 80℃ for 10 h. To avoid damaging the PEG layer on the PDMS surface, an induction current method was employed for zeta potential measurement. Zeta potentials of PEG modified PDMS in electrolyte solutions were measured. The results show that 2.5% PEG can effectively modify PDMS surface with positive zeta potential value in phosphate buffer solutions, pure water and 10% PEG solution. Further increase in PEG solution beyond 5% for surface modification has no obvious effect on zeta potential change.     

蛋白质和肽类分子的聚乙二醇化化学

蛋白质和肽类生物大分子聚乙二醇化后，溶解行为改善，稳定性增强，免疫原 性消除或降低，循环半衰期延长，药物动力学优化，因而在生物技术和生物医学中 具有重要的应用前景，聚乙二醇化也逐渐发展成为一种平台技术．随着聚乙二醇化 蛋白质和肽类进入临床试验阶段，聚乙二醇化化学越来越引起研究者的重视．简要 评述第一代和第二代聚乙二醇化化学，主要包括聚乙二醇化学修饰剂的类型与性能 比较等．     

Synthesis of water-soluble dendrimers based on melamine bearing 16 paclitaxel groups

The design, synthesis, and characterization of triazine dendrimers derivatized with the anticancer agent paclitaxel are described. The precursor generation two dendrimer 1 is prepared in six linear steps in 64% overall yield and presents 16 amines and two groups for radioiodination. This macromolecule is subsequently derivatized with a paclitaxel conjugate to yield a generation three dendrimer, 2, which is then pegylated in two steps. The pegylated final products, 4a and 4b, with molecular weights of 46 and 77 kDa, respectively, solubilize paclitaxel in water. Pegylated dendrimer 4a is 30 wt % paclitaxel, 52 wt % PEG, and 18 wt % dendrimer. Target 4b is 18 wt % paclitaxel, 71 wt % PEG, and 11 wt % dendrimer.     

Covalent binding of polyethylene glycol to the surface of red blood cells as detected and followed up by cell electrophoresis and rheological methods

Cyanuric chloride activated polyethylene glycol (PEG)-5000 was covalently coupled to murine and human red blood cells (pegylated RBC). Our purpose was to camouflage RBC receptors, which is necessary for parasite invasion, a process essential to sustain parasitemia. Cell electrophoretic mobility analysis (CEM) of pegylated RBC distinguished a new population of cells bearing characteristic CEM. Pegylation of RBC also modified their rheological properties, which were documented by evaluation of cell deformability (based on cell transit time through calibrated micropores) and cell aggregation (as measured by ultrasonic interferometry). Homologous transfusion of pegylated RBC into murine malaria-infected mice had no significant effect on the cerebral malaria death rate in Plasmodium berghei-infected mice, but it reduced the peripheral blood parasitemia by a factor 2 while in Plasmodium yoelii infected mice, the parasitemia was dramatically reduced by a factor of 4. These experiments demonstrate that transfusion of pegylated RBC may inhibit peripheral parasitemia. Cell electrophoresis appears to be a useful tool to allow in vivo detection and to investigate the fate of transfused pegylated RBC.     

Pharmaceutical performance of solid dispersions containing poly(ethylene) glycol 6000 and diazepam or temazepamInfluence of grinding

The effect of grinding on the physical properties and pharmaceutical performance of solid dispersions made of poly(ethylene) glycol 6000 (PEG6000) and temazepam or diazepam was studied using differential scanning calorimetry (DSC), X-ray powder diffraction and dissolution experiments. DSC-analysis of flash-cooled dispersions revealed that amorphous PEG present immediately after grinding crystallised upon aging mainly into the twice folded modification and to a small extent into the extended form. DSC-analysis of dispersions kept in the slab form for 1 month and subsequently ground, revealed that in the abscence of the grinding impulse crystallisation of PEG6000 takes place in the same way as in dispersions ground immediately after preparation and then aged for 1 month. Grinding solid dispersions immediately after preparation resulted in superior dissolution properties compared with solid dispersions kept in the monolith-slab form and subsequently ground. This difference in dissolution properties was found to be attributed to the drug and not to the polymer, more precisely, it was suggested that the drug particle size in ground dispersions was smaller than in dispersions kept in the slab form and subsequently ground. These findings suggest that grinding of solid dispersions immediately after preparation is the preparation method of choice instead of liquid filling of hard gelatin capsules resulting in monoliths. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bacterial polyesters grafted with poly(ethylene glycol): Behaviour in aqueous media

An easy method for grafting of poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) was developed. Oxidation of the pendant double bonds of PHOU into carboxyl groups to yield poly(3-hydroxyoctanoate-co-3-hydroxy-9-carboxydecanoate) (PHOD) and the esterification of the carboxyl side groups with poly(ethylene glycol) (PEG) were carried out in a single reaction solution. The grafting yield is dependent on the molar mass of the PEG graft. The maximum carboxyl group conversion (52%) was obtained with PEG M_n = 350 and decreased with increasing molar mass of PEG (19% for PEG M_n = 2000). Yields were determined by ¹H and ¹³C NMR. Short PEG grafts lowered the glass transition temperature (PHOD-g-PEG 350 −57 °C) compared to PHOD (−19 °C) and PHOU (−39 °C). This effect depends on the COOH conversion and PEG chain length. Grafting enhanced the hydrophilic character of the modified polymers making them soluble in polar solvents, such as alcohols and water/acetone mixtures. PHOD-g-PEG films were more stable towards hydrolytic degradation as PHOD films. No obvious modification of films was observed after more than 200 days at pH 7.2 and 37 °C. The molar mass of the grafted polymers decreased only slightly during this period, while PHOD films were hydrolyzed into soluble fragments.     

Evaluation of non-specific binding suppression schemes for neutravidin and alkaline phosphatase at the surface of reticulated vitreous carbon electrodes

Non-specific binding (NSB) of high-molecular-weight proteins onto electrode surfaces can complicate the application of electroanalytical techniques to clinical and environmental research, particularly in biosensor applications. We present herein various strategies to modify the surface of reticulated vitreous carbon (RVC) electrodes to suppress non-specific binding of biomolecules onto its surface. Non-specific binding and specific binding (SB) of two enzyme conjugates, neutravidin-alkaline phosphatase (NA-ALP) and biotinylated alkaline phosphatase (B-ALP), and also neutravidin itself, were studied using hydroquinone diphosphate (HQDP) as an enzyme substrate for ALP inside the pores of RVC electrodes that had been subjected to various modification schemes. The extent of NSB and SB of these biomolecules inside RVC pores was assessed by measuring the initial rate of generation of an electroactive product, hydroquinone (HQ), of the enzyme-catalyzed reaction, using linear scan voltammetry (LSV) for HQ detection. Electrodes functionalized with phenylacetic acid and poly(ethylene glycol) (PEG) showed low NSB and high SB (when biotin capture ligands were included in the modification scheme) in comparison with unmodified electrodes and RVC electrodes modified in other ways. A simple sandwich bioassay for neutravidin was performed on the RVC electrode with the lowest NSB. A concentration detection limit of 52 ± 2 ng mL⁻¹ and an absolute detection limit of 5.2 ± 0.2 ng were achieved for neutravidin when this assay was performed using a 100 μL sample size.     

Microporous structure and drug release kinetics of polymeric nanoparticles

The aim of the present study was to characterize pegylated nanoparticles (NPs) for their microporosity and study the effect of microporosity on drug release kinetics. Blank and drug-loaded NPs were prepared from three different pegylated polymers, namely, poly(ethylene glycol)1%-graft-poly(D,L)-lactide, poly(ethylene glycol)5%-graft-poly(D,L)-lactide, and the multiblock copolymer (poly(D,L)-lactide-block-poly(ethylene glycol)-block-poly(D,L)-lactide)n. These NPs were characterized for their microporosity using nitrogen adsorption isotherms. NPs of the multiblock copolymer showed the least microporosity and Brunauer-Emmett-Teller (BET) surface area, and that of PEG1%-g-PLA showed the maximum. Based on these results, the structural organization of poly(D,L)-lactide (PLA) and poly(ethylene glycol) (PEG) chains inside the NPs was proposed and was validated with differential scanning calorimetry (DSC) and X-ray photoelectron spectroscopy (XPS) surface analysis. An in vitro drug release study revealed that PEG1%-g-PLA NPs exhibited slower release despite their higher surface area and microporosity. This was attributed to the presence of increased microporosity forming tortuous internal structures, thereby hindering drug diffusion from the matrix. Thus, it was concluded that the microporous structure of NPs, which is affected by the molecular architecture of polymers, determines the release rate of the encapsulated drug.     

Development of a liquid chromatography/mass spectrometry methodology to separate, detect, characterize and quantify PEG-resveratrol prodrugs and the conjugation reaction precursors and intermediates

A simple and reliable liquid chromatography/mass spectrometry (LC/MS) method to monitor pegylation of resveratrol is described. The developed LC/MS method can separate and quantify unmodified MeO-PEG-OH, carboxylic acid terminated PEG, resveratrol and PEG-resveratrol prodrugs. This methodology was able to monitor and determine the extent of conversion of MeO-PEG-OH into respective acidic functional derivatives such as MeO-PEG succinylester acid (MeO-PEGO-SuccOH), which was found to be complete. The developed method was also utilised to determine the extent of conjugation of resveratrol to carboxylic acid terminated PEG. The conversion of carboxylic acid terminated PEG into a PEG-resveratrol conjugate was found to be 100% and 73%, respectively, for MeO-PEG succinylamide resveratrol (MeO-PEGN-Succ-RSV) and MeO-PEG succinylester resveratrol (MeO-PEGO-Succ-RSV). The 100% conjugation of MeO-PEGN-Succ-RSV is consistent with the result obtained from a nuclear magnetic resonance (NMR) study. The average molecular weights determined by LC/MS for MeO-PEG-OH, MeO-PEGO-SuccOH and MeO-PEGO-Succ-RSV were found to be 2108, 2321 and 2423 Da, respectively. These data correlate well with the theoretical values. This methodology proved to be simple and effective in determining the extent of functionalisation of PEG and its conjugation to resveratrol. Overall our LC/MS method coupled with NMR permitted complete characterisation of the polymeric prodrug pegylated-resveratrol and the reaction precursors.     

Adsorption of poly(ethylene glycol)-modified lysozyme to silica

Covalent grafting of poly(ethylene glycol) (PEG) to pharmaceutical proteins, "PEGylation", is becoming more commonplace due to improved therapeutic efficacy. As these conjugates encounter interfaces in manufacture, purification, and end use and adsorption to these interfaces may alter achievable production yields and in vivo efficacies, it is important to understand how PEGylation affects protein adsorption mechanisms. To this end, we have studied the adsorption of unmodified and PEGylated chicken egg lysozyme to silica, using optical reflectometry, total internal reflection fluorescence (TIRF) spectroscopy, and atomic force microscopy (AFM) under varying conditions of ionic strength and extent of PEG modification. PEGylation of lysozyme changes the shape of the adsorption isotherm and alters the preferred orientation of lysozyme on the surface. There is an abrupt transition in the isotherm from low to high surface excess concentrations that correlates with a change in orientation of mono-PEGylated conjugates lying with the long axis parallel to the silica surface to an orientation with the long axis oriented perpendicular to the surface. No sharp transition is observed in the adsorption isotherm for di-PEGylated lysozyme within the range of concentrations examined. The net effect of PEGylation is to decrease the number of protein molecules per unit area relative to the adsorption of unmodified lysozyme, even under conditions where the surface is densely packed with conjugates. This is due to the area sterically excluded by the PEG grafts. The other major effect of PEGylation is to make conjugate adsorption significantly less irreversible than unmodified lysozyme adsorption.     

Physicochemical analysis of ion beam-induced surface modifications on polyethylene glycol films for liquid crystal alignment

ABSTRACT

We investigated the surface modification induced by the ion-beam (IB) irradiation of a polyethylene glycol (PEG) film and its liquid crystal (LC) alignment characteristics. The X-ray photoelectron spectroscopy analysis revealed the chemical modification; as the IB incidence angle increased, the number of surface C–O bonds decreased, inducing an anisotropic dipole moment on the PEG film surface. In addition, the physical modification was demonstrated via atomic force microscopy analysis using three-dimensional images as a function of the IB incidence angle. The surface roughness was analyzed; the modification with the smoothest surface was observed for an IB incidence angle of 45°. This modification affected the LC alignment state of the PEG film, as demonstrated by the polarized optical microscopy analysis with pre-tilt angle measurements. Furthermore, for the same IB incidence angle, the residual DC measured using the capacitance–voltage curves was extremely low. Hence, a PEG film irradiated with an IB incidence angle of 45° could be a suitable LC alignment layer.     

Tailoring of block copolymers based on the stoichiometric control of the end‐functionality of telechelic oligomers and the utilization of large‐scale fractionation by phase fluctuation chromatography: A synthetic strategy for the preparation of end‐functionalized poly(L‐lactide)‐block‐poly(oxyethylene)

An AB diblock copolymer of poly(L ‐lactide) (PLLA) and poly(oxyethylene) (PEG) with a cinnamate terminal in the PEG block was prepared by the copolymerization of L ‐lactide and partially end‐modified PEG followed by fractionation. The first step was the terminal modification of PEG with cinnamoyl chloride (CC), in which the degree of cinnamoylation of the hydroxyl terminals of PEG was roughly controlled by the feed ratio of both reactants. The resultant PEG cinnamate was subjected to copolymerization with L ‐lactide to produce a mixture of unreacted PEG dicinnamate (C‐PEG‐C), the diblock copolymer (PLLA‐PEG‐C), and the triblock copolymer (PLLA‐PEG‐PLLA) corresponding to the three components of the PEG cinnamate. This mixture was separated by phase fluctuation chromatography (PFC) to obtain PLLA‐PEG‐C in sufficient purity. This process, involving the stoichiometric control of the terminal reaction of telechelic oligomers and the utilization of PFC for fractionation, can be an efficient method for synthesizing end‐functionalized diblock copolymers from readily available telechelic oligomers. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2405–2414, 2000     

应用光亲和分子进行PEG的光照修饰及蛋白质的光照偶联

合成了一种光活性标记分子-对叠氮苯甲酸,将其偶联到具有双羟基的碳酸酯与乳酸的共聚物P (LA-co-DHP)上,获得了具有光反应活性的可生物降解共聚物P(LA-co-DAP),在光照条件下,可以将蛋白质方便快捷地共价偶联到P(LA-co-DHP)聚合物纤维上.在溶液中进行PEG与对叠氮苯甲酸的光照反应,通过核磁共振光谱...     

Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core–shell reversed phase column and condensation nucleation light scattering detection

A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core–shell column (100 mm × 3 mm, 2.6 μm) and methanol–water–trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10–60 μg/ml and unbound PEG in the range of 2–40 μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.     

Controlled levels of protein modification through a chromatography-mediated bioconjugation

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.     

Size-selective protein adsorption to polystyrene surfaces by self-assembled grafted poly(ethylene glycols) with varied chain lengths

We report about the surface modification of polystyrene (PSt) with photoreactive alpha-4-azidobenzoyl-omega-methoxy poly(ethylene glycol)s (ABMPEG) of three different molecular weights (MWs of approximately 2, approximately 5, and approximately 10 kg/mol) and with two poly(ethylene glycol)/poly(propylene glycol) triblock copolymers (PEG-PPG-PEG) of about identical PEG/PPG ratio (80/20, w/w) and MW(PEG) of approximately 3 and approximately 6 kg/mol, all via adsorption from aqueous solutions. For ABMPEGs, an additional UV irradiation was used for photografting to the PSt. Contact angle (CA) and atomic force microscopy data revealed pronounced differences of the hydrophilicity/hydrophobicity and topography of the surfaces as a function of PEG type and concentration used for the modification. In all cases, an incomplete coverage of the PSt was observed even after modification at the highest solution concentrations (10 g/L). However, clear differences were seen between PEG-PPG-PEGs and ABMPEGs; only for the latter was a nanoscale-ordered interphase structure with an influence of MW(PEG) on the PEG density observed; after modification at the same solution concentrations, the density was significantly higher for lower MW(PEG). The adsorption of three proteins, myoglobin (Mgb), bovine serum albumin (BSA), and fibrinogen to the various surfaces was analyzed by surface plasmon resonance. Pronounced differences between the two PEG types with respect to the reduction of protein adsorption were found. At high, but still incomplete, surface coverage and similar CA, the shielding of ABMPEG layers toward the adsorption of Mgb and BSA was much more efficient; e.g., the adsorbed Mgb mass relative to that of unmodified PSt was reduced to 10% for ABMPEG 2 kg/mol while for both PEG-PPG-PEGs the Mgb mass was still around 100%. In addition, for the ABMPEG layers an effect of MW(PEG) on adsorbed protein mass-decrease with decreasing MW-could be confirmed; and the highest Mgb/BSA selectivities were also observed. A "two-dimensional molecular sieving", based on PEG molecules having a nanoscale order at the hydrophobic substrate polymer surface has been proposed, and the main prerequisites were the use of PEG conjugates which are suitable for an "end-on" grafting (e.g., ABMPEGs), the use of suitable (not too high) concentrations for the surface modification via adsorption/self-assembly, optionally the photografting on the substrate (possible only for ABMPEG), and presumably, a washing step to remove the excess of unbound PEGs. The results of this study also strongly support the hypothesis that the biocompatibility of hydrophobic materials can be very much improved by PEG modifications at surface coverages that are incomplete but have an ordered layer structure controlled by the size and steric interactions of surface-bound PEGs.