PEG Reagents Research Articles

The Development and Application of a HPTLC-Derived Database for the Identification of Phenolics in Honey.

This study reports on the development and validation of a HPTLC-derived database to identify phenolic compounds in honey. Two database sets are developed to contain the profiles of 107 standard compounds. Rich data in the form of Rf values, colour hues (H°) at 254 nm and 366 nm, at 366 nm after derivatising with natural product PEG reagent, and at 366 nm and white light after derivatising with vanillin–sulfuric acid reagent, λ max and λ min values in their fluorescence and λ max values in their UV-Vis spectra as well as λ max values in their fluorescence and UV-Vis spectra after derivatisation are used as filtering parameters to identify potential matches in a honey sample. A spectral overlay system is also developed to confirm these matches. The adopted filtering approach is used to validate the database application using positive and negative controls and also by comparing matches with those identified via HPLC-DAD. Manuka honey is used as the test honey and leptosperine, mandelic acid, kojic acid, lepteridine, gallic acid, epigallocatechin gallate, 2,3,4-trihydroxybenzoic acid, o-anisic acid and methyl syringate are identified in the honey using the HPTLC-derived database.

Extraction and Analyses of Flavonoids and Phenolic Acids from Canadian Goldenrod and Giant Goldenrod

Invasive alien plant species Canadian goldenrod (Solidago canadensis L.) and giant goldenrod (Solidago gigantea Aiton) were investigated as a source of phytochemicals and yellow dyes. Flavonoids and phenolic acids were extracted from the inflorescence of Canadian goldenrod with thirteen extraction solvents ethanol, methanol, acetone, water, and mixtures of organic solvents (70%, 80%, and 90%) with water. High performance thin-layer chromatography (HPTLC) coupled to densitometry and high-performance liquid chromatography with photo-diode array detector (HPLC-PDA) were used for analyses of the obtained sample test solutions (STSs), which showed the best and comparable extraction efficiencies for 70% acetone(aq), 70% methanol(aq), and 70% ethanol(aq). HPTLC combined with image analyses in fluorescent mode resulted in different chromatographic fingerprints for Canadian goldenrod and giant goldenrod STSs (70% acetone(aq)) after development, after post-chromatographic derivatization with NP reagent and after use of PEG reagent. The developed HPLC methods enabled analyses of phenolic acids and flavonoids (aglycones and glycosylated) in STSs and hydrolyzed STSs form inflorescence of Canadian and giant goldenrod. Different contents of chlorogenic acid, rutin, hyperoside, isoquercetin, and quercetin were observed in STSs of both goldenrod species. The analyses of hydrolyzed STSs confirmed that glycosylated flavonoids in Canadian and giant goldenrod inflorescence are mainly glycosides of quercetin, kaempferol, and isorhamnetin. Additional analyses using HPTLC and HPLC coupled to tandem mass spectrometry (MS/MS; HPTLC-MS/MS and LC-MS/MS) enabled tentative identification of phenolic acids and flavonoids (10 with HPTLC-MS/MS and 15 with LC-MS/MS), from which several were identified in Canadian (4 with HPTLC-MS/MS and 8 with LC-MS/MS) and in giant (7 with HPTLC-MS/MS and 9 with LC-MS/MS) goldenrod for the first time.

Evaluation of sporadic bovine alkaline phosphatase interference in the Beckman Access unconjugated estriol (uE3) assay affecting maternal serum screening results

ObjectivesBovine alkaline phosphatase (BALP) mediated interference is a potential issue in the Beckman Access unconjugated estriol (uE3) assay. As the uE3 assay is a component of second trimester maternal serum screening characterizing this interference is essential for delivering accurate trisomy 18 and trisomy 21 risks. Design and methodsResidual serum samples (n = 517) were measured by two different lots of uE3 assay. Scavenger BALP (sBALP) was added to all samples to remove potential BALP dependent interference and assessed using both lots of uE3 reagent. ResultsBALP mediated interference was observed in similar frequency in both lots of reagent (~3%), although the patterns of positive and negative interference differed between the lots. Pretreatment with sBALP improved lot-to-lot comparison. The presence of BALP related interference was not related to the concentration of endogenous human alkaline phosphatase. The use of polyethylene glycol and sBALP treatment appeared to mitigate BALP mediated interference equally well, and resulted in concordance in measured uE3 concentrations between reagent lots. Additionally, heterophile antibody interference was observed in two samples affected with BALP interference, and the heterophile antibody interference was resolved by both PEG and heterophile antibody blocking reagent treatment, but not sBALP treatment. While the maternal screen numeric risk for affected samples changed, the risk classification changed from a negative to positive screen in two samples. ConclusionsInterference in the uE3 assay has the potential to affect maternal serum risk calculations in different reagent lots, and pretreatment of samples with scavenger BALP or PEG should be considered in cases of unexplained uE3 concentrations.

Comparison of chromatographic profiles of flavonoids and hydroxycinnamic acids of some species from Lamiaceae genus from Ukrainian PHARMACEUTICAL MARKET

The plants from the Lamiaceae family are traditional remedies of folk and scientific medicine. They are present at the domestic pharmaceutical market as herbal raw material (HRM); dietary supplements; aromatherapy; as well as being included in the composition of drugs for oral use etc. According to the modern direction of international harmonization of conditions for standardizations of herbal drugs, the topical issue is the development of unified methods of HPTLC identification for HRM, including the Lamiaceae family.
 The aim of the work was the selection of the unified HPTLC chromatographic conditions for identification of chromatographic profiles of flavonoids and hydroxycinnamic acids of HRM of some Lamiaceae species of Ukraine’s flora, presented at domestic pharmaceutical market, such as Sage, Wild Thyme, Thyme, Oregano, Rosemary, Java tea, Peppermint, Lavender, Anise hyssop, Melissa, Hyssop, White horehound, Motherwort; and comparison of their chromatographic fingerprints.
 As objects of investigation, the HRM of Lamiaceae family presented at the Ukrainian pharmaceutical market were used. The investigation was conducted by HPTLC procedure using an automatic herbal system of CAMAG, Switzerland at the base of CAMAG laboratory, Muttenz, Switzerland and laboratory of ESTCCTI ESIPP, NUPh, Kharkiv, Ukraine.
 The harmonized chromatographic conditions for determination of flavonoids and hydroxycinnamic acids for HRM of Lamiaceae species were chosen, these were: stationary phase – HPTLC Si 60 F254; mobile phase – ethyl acetate, formic acid anhydrous, water (8:1:1); sample preparation: solvent – methanol; method of extraction – shaking for 10 min; relative humidity: 33%, temperature 22 ± 5 °C; derivatization with Natural product and PEG reagents; detection – 254 nm, 366 nm, white light before derivatization: 366 nm and white light after derivatization. The reproducibility of results in these chromatographic conditions was ∆RF ≤ 0,05. It was established, that for all investigated species of the family the zone corresponding with the position and colour to rosmarinic acid was detected, thus it can be used as standard substance for «Identification», «Limit-test» and for chromatographic system suitability test. For investigated samples of domestic HRM, the specific zones that can be used for setting acceptance criteria of the «Identification» and «Foreign matters» tests were conducted. After validation investigation involving greater numbers of samples of each species from different growing regions of Ukraine, the obtained HPTLC chromatographic fingerprints could be recommended as reference images for quality control of home-grown HRM of Lamiaceae family.

Modifying an ÄKTApurifier System for the Automated Acquisition of Samples for Kinetic Modeling of Batch Reactions.

Recording the data necessary to assess the kinetics of a reaction can be labor-intensive. In this technology brief, we show a method to automate this task by utilizing parts of an ÄKTApurifier chromatography system to automatically take samples from a reaction vessel at predefined time intervals and place them in 96-well plates and also enable correlating the samples with in-line spectral data of the reaction solution. Automatic sampling can reduce experimental bottlenecks by enabling overnight reactions or a higher degree of parallelization. To demonstrate the feasibility of the method, we performed batch-PEGylation of lysozyme with varying conditions by changing the molar excess of the PEG reagent. We used analytical cation-exchange chromatography to analyze the samples taken during the batch reaction, determining the concentrations of the individual species present at each time step. Subsequently, we fitted a kinetic model on these data. Fitting the model to four different reaction conditions simultaneously yielded a regression coefficient of R2 = 0.871.

Reaction-Mediated Desorption of Macromolecules: Novel Phenomenon Enabling Simultaneous Reaction and Separation.

Combining chemical reaction with separation offers several advantages. In this work possibility to induce spontaneous desorption of adsorbed macromolecules, once being PEGylated, through adjustment of the reagent composition is investigated. Bovine serum albumin (BSA) and activated oligonucleotide, 9T, are used as the test molecules and 20 kDa linear activated PEG is used for their PEGylation. BSA solid-phase PEGylation is performed on Q Sepharose HP. Distribution coefficient of BSA and PEG-BSA as a function of NaCl is determined using linear gradient elution (LGE) experiments and Yamamoto model. According to the distribution coefficient the selectivity between BSA and PEG - BSA of around 15 is adjusted by using NaCl. Spontaneous desorption of PEG - BSA is detected with no presence of BSA. However, due to a rather low selectivity, also desorption of BSA occurred at high elution volume. A similar procedure is applied for activated 9T oligonucleotide, this time using monolithic CIM QA disk monolithic column for adsorption. Selectivity of over 2000 is obtained by proper adjustment of PEG reagent composition. High selectivity enables spontaneous desorption of PEG-9T without any desorption of activated 9T. Both experiments demonstrates that reaction-mediated desorption of macromolecules is possible when the reaction conditions are properly tuned.

Effects of linear and branched polyethylene glycol on PEGylation of recombinant hirudin: Reaction kinetics and in vitro and in vivo bioactivities

Abstract To improve the therapy efficacy of recombinant hirudin variant-2 (HV2), its PEGylation was investigated using linear mPEG-succinimidyl carbonate (mPEG-SC) and branched mPEG2-N-hydroxysuccinimide (mPEG2-NHS). The reaction mixtures of PEGylation were analyzed by RP-HPLC and the mono-PEG-HV2 products were purified by anion exchange chromatography (IEC). Effects of linear and branched PEG on the hydrolysis kinetics of the PEG reagent, the PEGylation kinetics of HV2 and the in vitro and in vivo bioactivity of mono-PEG-HV2 were investigated. The RP-HPLC and IEC analyses showed that linear and branched PEG-HV2 with identical molecular weight had different chromatographic behaviors. The reaction kinetics showed that branched mPEG2-NHS displayed higher hydrolysis rate but lower PEGylation rates than linear mPEG-SC. Consequently, HV2 conjugated with mPEG2-NHS required a greater molar ratio of PEG to HV2 than that of mPEG-SC to achieve the identically desired yield of mono-PEG-HV2. The in vitro and in vivo bioactivities of mono-PEG-HV2 showed that branched PEG-HV2 had higher therapeutic efficacy than linear PEG-HV2 with identical molecular weight. The in vivo bioactivity of mono-B-PEG40k-HV2 (mono-PEG-HV2 derived from 40 kDa branched mPEG2-NHS) had a markedly longer duration in rabbits than did unmodified HV2, which showed its potential to be developed as a candidate antithrombotic drug.

Ultraviolet-B radiation effects on phenolic profile and flavonoid content of Kalanchoe pinnata

Ultraviolet-B radiation is an important abiotic factor that can stimulate the production of secondary metabolites, including polyphenolic compounds. Kalanchoe pinnata (Crassulaceae) is a medicinal plant popularly used in Brazil for treating wounds and inflammation. This species is rich in phenolic compounds, which could account for some of its biological activities, including antileishmanial, antihypertensive and antibacterial properties. We investigated the effects of supplemental UV-B radiation on the phenolic profile, antioxidant activity and total flavonoid content of leaves of K. pinnata. Plants were grown under white light (W – control) and supplemental UV-B radiation (W+UVB). Supplemental UV-B radiation enhanced the total flavonoid content of the leaf extracts, without affecting the antioxidant activity or yield of extracts. Analysis by TLC and HPLC of W and W+UVB leaf extracts revealed quantitative and qualitative differences in their phenolic profiles. W+UVB extracts contained a higher diversity of phenolic compounds and a larger amount of quercitrin, an important bioactive flavonoid of this species. This is the first report of the use of ImageJ® program to analyze a TLC visualized by spraying with NP–PEG reagent. UV-B radiation is proposed as a supplemental light source in K. pinnata cultivation in order to improve its flavonoid composition.

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.

Nonionic fluorosurfactant as an ideal candidate for one-step modification of gold nanorods

In this work, a novel protocol was developed for size tuning and cetyltrimethylammonium bromide (CTAB) removal of gold nanorods using commercially available nonionic fluorosurfactants (FSN), an excellent candidate for PEG and other modification reagents. The tunable gold nanorods can easily be obtained by stopping the ligand replacement reaction at different time intervals. The FSN-coated gold nanorods are stable in the presence of high salt concentrations and over a wide range of pH values. Additionally, the cellular uptake experiments demonstrate that the FSN-coated gold nanorods have superior features in comparison to the widely used PEG-coated gold nanorods, such as high uptake amount, tunable uptake and excellent stability. Our findings suggest that FSN ligands are an ideal candidate for modifying gold nanorods with tunable aspect ratios, excellent biocompatibility, nontoxicity, and high stability, enabling conjugation to biomolecules for specific targeting, uptake, and delivery.

PEGylation of G-CSF in organic solvent markedly increase the efficacy and reactivity through protein unfolding, hydrolysis inhibition and solvent effect

Previous studies demonstrated that hydrophobic proteins could be PEGylated in organic phase rather than water phase. It is still not known what the difference is for a hydrophilic protein's PEGylation in these two different phases. In this study, granulocyte colony stimulating factor (G-CSF) was dissolved in neat dimethyl sulfoxide (DMSO) and was PEGylated. In comparison with the PEGylation in water solution, the PEGylation degree in the organic solvent increased by 33% and 42% for PEG-maleimide (MAL-PEG) and PEG-succinimidyl carbonate (SC-PEG) respectively. Structure analysis revealed that the protein was unfolded in DMSO, which could make the PEGylated sites of G-CSF easily accessible. The hydrolysis half-life in water solution was 40min and 9h for SC-PEG and MAL-PEG respectively. However, in DMSO solvent, PEGs were very stable and no hydrolysis could be detected. Stopped-flow demonstrated that the conjugation speed of G-CSF by MAL-PEG and SC-PEG in DMSO were 1.6×104 and 2×102 times faster than those in aqueous solution. The remarkable acceleration could mainly be attributed to an increase of protein nucleophilicity in DMSO. The results of this study could be referential to industrial application where the cost of PEG reagents and the speed of reaction on large scale are very important.

Thiocarbmoylation Chemistry Based Pegylation Of αα Fumaryl-Hb Is Unique As It Does Not Induce An Increase In Oxygen Affinity: High Oxygen Affinity Pegylated Crosslinked Hbs Are Better As Oxygen Therapeutics

BackgroundPEGylated Hbs have emerged as a novel class of nohypertensive, oxygen carrying low viscosity nitric oxide producing resuscitation fluids. The limitations of PEGylation of Hb are that it induces a weakening of its interdimeric interactions and increases oxygen affinity. Besides, the influence of oxygen affinity of PEG-Hb on the tissue oxygen is still a unknown zone in the blood substitute field. In this study, we prepared two PEGylated intramolecularly crosslinked Hb and compared their performance in the extreme hemodilution model. MethodsHb-PEG adducts were prepared by incubation of PEG reagents with Hb to conjugate PEG chains to the amino groups on the surface of Hb molecule. The unreacted PEG was removed by tangential flow filtration against PBS using Minim (PAL Co.) at 4 °C. The oxygen equilibrium curves of Hb-PEG solutions were obtained using a Hemox analyzer (TCS Scientific, New Hope, PA). Extreme hemodilution: By progressive hemodilution, the hamster's systemic Hct is reduced to 18% with a non- O2 carrying colloid plasma expander (dextran 70). It is at the critical transition of Hb where O2 carrying capacity becomes limited that the study materials are introduced. This final hemodilution step is performed with the study material until an 11% hematocrit is reached, a control being generated using dextran 70. O2 delivery to the microcirculation and O2 extraction to the tissue were determined by measured by the Phosphorescence Decay (PD) Technique. ResultsThe extension arm facilitate (EAF) PEGylation as well direct PEGylation using either reductive alkylation chemistry or acylation chemistry based PEGylation of αα fumaryl-Hb generates PEG Hbs with an higher oxygen affinity (∼20 mm Hg), as compared to the oxygen affinity of 30 mm Hg for αα fumaryl-Hb. The thiocarbmoylation chemistry based PEGylation of αα-fumaryl Hb using isothiocyanate phenyl PEG 5K (ITP-PEG5K) has been investigated here. TetraPEGylated αα fumaryl-Hb [(TCP-PEG5K)4 αα-Hb] generated by thiocarbmoylation chemistry generates thiourethane linkage and exhibits an oxygen affinity (34 mm Hg), which is slightly lower than that of parent molecule (30 mm Hg). The tetraPEGylation by ITP PEG exhibits very high site selectivity to the four α-amino groups, which is the same as the reductive PEGylation. The reductive PEGylation results in a significant increase in the oxygen affinity of αα-fumaryl Hb (15 mm Hg). The oxygen affinity of (TCP-PEG 5K)4 ββ-Hb is around 8 mm Hg, which is comparable to that of ββ-Hb. Thus PEG-chains on the amino termini of crosslinked Hb with thiourethane linkage is distinct as it does not increase the oxygen affinity of crosslinjed Hb. Despite the low P50 of (TCP-PEG5K)4 αα-Hb, it neutralized the in vivo hypertensive activity of αα-fumaryl Hb. Besides, the tissue oxygenation by (TCP-PEG5K)4 αα-Hb is 3.5 mmHg, and is lower than that for affinity reductive PEGylated Hb, 5.4 mmHg. Conclusions(i) Conjugation chemistry dictates the influence on the oxygen affinity of PEGylated crosslinked Hbs and not the PEG shell, (ii) The number and amount of PEG-chains in the PEG-shell dictates the plasma expander like properties of PEG Hb; (iii) PEGylation mediated attenuation of the hypertensive activity of Hb is not a correlate of its oxygen affinity; and (iv) The tissue oxygenation PEGylated αα Hb is not correlated with oxygen affinity. Accordingly, the choice of the oxygen affinity for PEG-Hb and chemistry of PEGylation for its application as an oxygen therapeutic will be dictated by the overall efficacy of tissue oxygenation expected for a PEG Hb solution in a given clinical setting. We speculate that (TCP-PEG5KK)4 ββ-Hb will achieve a tissue oxygenation better or comparable to PEG5K2 Hb, the molecule that has given the best tissue oxygenation as an oxygen therapeutic in extreme hemodilution model so far. Disclosures:No relevant conflicts of interest to declare.

Membrane reactor for continuous and selective protein mono-PEGylation

PEGylated proteins are value-added biopharmaceutical products obtained by conjugating poly (ethylene glycol) or PEG with proteins. It is difficult to simultaneously obtain both high selectivity and extent of synthesis of mono-PEGylated protein using a conventional batch reaction. This paper discusses a hollow-fiber membrane reactor (HMR) system suitable for addressing this issue. The HMR system was operated in a continuous mode with the protein being pumped directly into the lumen of the fiber and the PEG reagent being introduced in the hollow-fiber in a distributed manner along its length. The PEG concentration at any location within the reactor thereby was lower than the protein concentration, and thus the synthesis of higher PEGylated by-products was suppressed, leading to an increase in selectivity of mono-PEGylation. An additional factor that contributed towards the enhancement in selectivity was the radial concentration gradient of reactants and product within the hollow-fiber, which resulted in the diffusion of mono-PEGylated protein away from the reaction zone, thus further suppressing the synthesis of the higher PEGylated forms. The extent and selectivity of mono-PEGylation obtained with the HMR system were significantly higher than that obtained in equivalent batch reaction. Furthermore, a continuous process is much more desirable than a batch process from a manufacturing point of view.

Evolution of Reactive mPEG Polymers for the Conjugation of Peptides and Proteins

The covalent attachment of methoxy-poly(ethylene glycol) (mPEG) is a well established strategy used to improve the pharmaceutical properties of several biomolecules. Since the pioneering work of Abuchovsky, PEGylation has emerged as a powerful technology of significant relevance, not only for the development of new and better drugs, but also for application in material science. Peptides and proteins are the most traditional targets for PEGylation due to their intense and diverse biotechnological applications. The terminal amino group, as well as the ε-amino group of lysine and the thiol group of cysteine, are all well known nucleophilic sites that have traditionally been used to couple peptides and proteins to mPEG derivatives. Advances in the methods for preparation of the mPEG starting materials, together with a careful selection of new mPEG functional end-groups have allowed new reactive mPEGs to emerge, which show narrow polydispersity and controlled reactivity, providing more homogeneous conjugates. In the last few years the trend has moved towards site-selective, reversible and enzymatic PEGylation using a new generation of tailor-made reagents and strategies. The main goal of this article is to present some of the most relevant achievements obtained in the PEGylation of peptides and proteins. The chemistry underlying the current methods used for the preparation of mPEG reagents, as well as the chemistry involved in the PEGylation reactions are presented in detail, in order of stimulating the synthetic and polymer chemist to turn their attention in this fascinating multi and interdisciplinary field of research. Keywords: PEGylation, Poly(ethylene glycol), PEG reagents, Conjugation, Protein PEGylation, Controlled drug release, Activated PEG.

Comparative Binding of Disulfide-Bridged PEG-Fabs

Protein PEGylation is the most clinically validated method to improve the efficacy of protein-based medicines. Antibody fragments such as Fabs display rapid clearance from blood circulation and therefore are good candidates for PEGylation. We have developed PEG-bis-sulfone reagents 1 that can selectively alkylate both sulfurs derived from a native disulfide. Using PEG-bis-sulfone reagents 1, conjugation of PEG specifically targets the disulfide distal to the binding region of the Fab (Scheme 2 ). PEG-bis-sulfone reagents 1 (10-40 kDa) were used to generate the corresponding PEG-mono-sulfones 2 that underwent essentially quantitative conjugation to give the PEG-Fab product 4. Four Fabs were PEGylated: Fab(beva), Fab'(beva), Fab(rani), and Fab(trast). Proteolytic digestion of bevacizumab with papain gave Fab(beva), while digestion of bevacizumab with IdeS gave F(ab')(2-beva), which after reaction with DTT and PEG-mono-sulfone 2 gave PEG(2)-Fab'(beva). Ranibizumab, which is a clinically used Fab, was directly PEGylated to give PEG-Fab(rani). Trastuzumab was proteolytically digested with papain, and its corresponding Fab was PEGylated to give PEG-Fab(trast). Purification of the PEGylated Fabs was accomplished by a single ion exchange chromatography step to give pure PEG-Fab products as determined by silver-stained SDS-PAGE. No loss of PEG was detected post conjugation. A comparative binding study by SPR using Biacore with low ligand immobilization density was conducted using (i) VEGF(165) for the bevacizumab and ranibizumab derived products or (ii) HER2 for the trastuzumab derived products. VEGF(165) is a dimeric ligand with two binding sites for bevacizumab. HER2 has one domain for the binding of trastuzumab. Binding studies with PEG-Fab(beva) indicated that the apparent affinity was 2-fold less compared to the unPEGylated Fab(beva). Binding properties of the PEG-Fab(beva) products appeared to be independent of PEG molecular weight. Site-specific conjugation of two PEG molecules gave PEG(2×20)-Fab'(beva), whose apparent binding affinity was similar to that observed for PEG-Fab(beva) derivatives. The k(d) values were similar to those of the unPEGylated Fab(beva); hence, once bound, PEG-Fab(beva) remained bound to the same degree as Fab(beva). Biacore analysis indicated that both Fab(rani) and PEG(20)-Fab(rani) did not dissociate from the immobilized VEGF at 25 °C, but ELISA using immobilized VEGF showed 2-fold less apparent binding affinity for PEG(20)-Fab(rani) compared to the unPEGylated Fab(rani). Additionally, the apparent binding affinities for trastuzumab and Fab(trast) were comparable by both Biacore and ELISA. Biacore results suggested that trastuzumab had a slower association rate compared to Fab(trast); however, both molecules displayed the same apparent binding affinity. This could have been due to enhanced rebinding effects of trastuzumab, as it is a bivalent molecule. Analogous to PEG-Fab(beva) products, PEG(20)-Fab(trast) displayed 2-fold lower binding compared to Fab(trast) when evaluated by ELISA. The variations in the apparent affinity for the PEGylated Fab variants were all related to the differences in the association rates (k(a)) rather than the dissociation rates (k(d)). We have shown that (i) Fabs are well-matched for site-specific PEGylation with our bis-alkylation PEG reagents, (ii) PEGylated Fabs display only a 2-fold reduction in apparent affinity without any change in the dissociation rate, and (iii) the apparent binding rates and affinities remain constant as the PEG molecular weight is varied.

Impact of Large Aggregated Uricases and PEG Diol on Accelerated Blood Clearance of PEGylated Canine Uricase

BackgroundUricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated.Methods and FindingsRecombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC.ConclusionsThe size of conjugates is important for triggering such phenomena and we speculate that 40–60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

BAX 855, a PEGylated rFVIII product with prolonged half-life

A longer acting recombinant FVIII is expected to serve patients' demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter's rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

PEGylation of Octreotide Using an α,β-unsaturated-β'-mono-sulfone Functionalized PEG Reagent.

PEGylation is a well-established technique utilized to overcome the problems related to the therapeutic applications of peptides and proteins. Reasons for the PEGylation of these biological macromolecules include reducing immunogenicity, proteolytic degradation and rapid clearance from blood circulation. Octreotide is an octapeptide analogue of naturally-occurred somatostatin. This peptide has elimination half-life of less than 2 h that requires frequent daily subcutaneous or intravenous administration. To address this issue, octreotide modification was investigated using bis-thiol alkylating PEG reagent. The required bis-thiol alkylating reagent (V) was prepared from commercially available 4-acetyl benzoic acid in five steps. Octreotide disulfide bond was mildly reduced to liberate the two cysteine sulfur atoms followed by bis-alkylation to form PEGylated peptide. The PEG modification process was monitored through the reverse phase HPLC and (1)H-NMR analysis. According to the HPLC chromatograms of PEGylation reaction, the peak with 30 min retention time was identified to be PEG-octreotide. In addition, (1)H-NMR analysis showed a 7.44% degree of PEG substitution.

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

BackgroundDrug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA.ResultsMonodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature.ConclusionsThis work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

PEGylation of therapeutic proteins

Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.