Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Simultaneous analysis of filgrastim and pegfilgrastim aggregates by size-exclusion chromatography.

A new size-exclusion high-performance liquid chromatographic (SE-HPLC) method for the simultaneous analysis of filgrastim and pegfilgrastim aggregates was developed and validated. A cross-linked agarose and dextran column was used at ambient temperature and an alkaline sodium phosphate buffer as mobile phase eliminated non-ideal interactions with the stationary phase. The robustness of the method was assessed by varying injection volumes, flow rates and sample vehicles. Other reliability assessments include calibration curve, intra and inter-day precision and accuracy, repeatability of retention times, application to real in-process production samples and column lifetime. The method exhibited linearity over the concentration of 0.02-4 mg/ml range for filgrastim and pegfilgrastim monomer with a correlation coefficient of greater than 0.999. The lower limit of quantification was 0.02 mg/ml and the limit of detection was 0.005 mg/ml. This SE-HPLC technique has been successfully used for several years and more than 10,000 samples.

Paper-Based Acoustofluidics for Separating Particles and Cells.

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 µm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis.

Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 µM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.

Unusual effect of flow rate on retention in analytical supercritical fluid chromatography exemplified by polyethylene glycol separation.

We report a case of a peculiar effect of flow rate on retention in a separation of polyethylene glycol oligomers via supercritical fluid chromatography. During method development, we tested flow rate gradients and notices that for some PEG oligomers retention times at flow rate gradient were lower than at constant flow with the largest flow rate value used in a gradient. For instance, at BEH stationary phase and CO2-MeOH gradient from 10 to 35% at 20 min a PEG oligomer having mass of 1225 Da has a retention time 14 min at 1 mL/min flow rate, 10.3 at 2 mL/min and 9.5 min at 1-2 mL/min flow rate gradient. The effect is not unified for all PEG oligomers, it occurs only starting from a particular PEG molecular weight which depends on the stationary phase type and/or mobile phase conditions. We believe that such an unusual flow rate effects can happen in SFC on various occasions, not exclusively for flow rate gradients, and thus should be taken into account during method development or method transfer.

SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins.

PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by decreased receptor-mediated endocytosis and/or delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEPO) used in the treatment of anemia due to chronic kidney disease, has also been abused by athletes as performance-enhancing drug. While it can be detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-EPO antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein and thus leads to a sharper electrophoretic band and enhanced antibody binding. While the method was originally developed for anti-doping purposes, it may also be useful for the electrophoretic separation and immunological detection of other PEGylated proteins. Protocols for urine and serum are presented. They are also applicable for the general detection of EPO-based erythropoiesis-stimulating agents (ESA) in these matrices.

An electrochemical immunosensor for cardiac Troponin I using electrospun carboxylated multi-walled carbon nanotube-whiskered nanofibres.

A sandwich-type nanostructured immunosensor based on carboxylated multi-walled carbon nanotube (CMWCNT)-embedded whiskered nanofibres (WNFs) was developed for detection of cardiac Troponin I (cTnI). WNFs were directly fabricated on glassy carbon electrodes (GCE) by removing the sacrificial component (polyethylene glycol, PEG) after electrospinning of polystyrene/CMWCNT/PEG nanocomposite nanofibres, and utilised as a transducer layer for enzyme-labeled amperometric immunoassay of cTnI. The whiskered segments of CMWCNTs were activated and utilised to immobilise anti-cTnT antibodies. It was observed that the anchored CMWCNTs within the nanofibres were suitably stabilised with excellent electrochemical repeatability. A sandwich-type immuno-complex was formed between cTnI and horseradish peroxidase-conjugated anti-cTnI (HRP-anti-cTnI). The amperometric responses of the immunosensor were studied using cyclic voltammetry (CV) through an enzymatic reaction between hydrogen peroxide and HRP conjugated to the secondary antibody. The nanostructured immunosensor delivered a wide detection range for cTnI from the clinical borderline for a normal person (0.5-2ngmL-1) to the concentration present in myocardial infarction patients (> 20ngmL-1), with a detection limit of ~ 0.04ngmL-1. It also showed good reproducibility and repeatability for three different cTnI concentration (1, 10 and 25ngmL-1) with satisfactory relative standard deviations (RSD). Hence, the proposed nanostructured immunosensor shows potential for point-of-care testing.

The purified vepoloxamer prevents haemolysis in 42-day stored, DEHP/PVC-free red blood cell units.

BACKGROUND: Use of the plasticiser di(2-ethylhexyl) phthalate (DEHP) in polyvinyl chloride (PVC) blood bags poses a potential dilemma. The presence of DEHP in blood bags has been shown to be beneficial to red blood cells during storage by diminishing haemolysis. However, DEHP use in PVC may be carcinogenic or estrogenising. Vepoloxamer is a poloxamer with rheological and cytoprotective rheological properties and a favourable toxicity profile in clinical trials. We hypothesised that vepoloxamer may be sufficient to replace the plasticiser DEHP to prevent elevated haemolysis while conserving the biochemical and redox potential++ in RBCs stored for up to 42 days. MATERIALS AND METHODS: Paired analyses of aliquots from pooled RBC suspensions of ABO identical donors were aseptically split into test storage containers (DEHP/PVC or DEHP-free/ethylene vinyl acetate [EVA]) supplemented with or without vepoloxamer (at concentrations of 0.1, 1, 5 or 7.89 mg/mL) and cold stored for up to 42 days. RESULTS: Vepoloxamer significantly prevented the increased haemolysis induced by the absence of DEHP in EVA bags in a dose-dependent manner by days 28 and 42 of storage (approx. 50% reduction of the maximum concentration of vepoloxamer; p<0.001). There was an inverse correlation between the concentration of vepoloxamer used and the haemolysis rate (r2=0.27, p<0.001) and a direct correlation between haemolysis and phosphatidylserine (PS) exposure (r2=0.42; p<0.01). Increased osmotic fragility and shear induced deformability of 42-day stored RBC in EVA bags was significantly corrected by the addition of vepoloxamer. DISCUSSION: Vepoloxamer, in a concentration-dependent fashion, is able to partly rescue the increased haemolysis and PS exposure induced by the absence of the commonly used plasticiser DEHP. These results provide initial but strong evidence to support vepoloxamer use to replace DEHP in long-term storage of RBC.

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.

SCOPE: Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for ß-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. METHODS AND RESULTS: Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. CONCLUSION: The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

Isoform separation and structural identification of mono-PEGylated recombinant human growth hormone (PEG-rhGH) with pH gradient chromatography.

Human growth hormone plays an essential role in the treatment of dwarfism diseases, but it is limited in its short circulating half-life. Nowadays, some manufacturers are trying to take advantage of polyethylene glycol (PEG) conjugated with recombinant human growth hormone (rhGH) to improve its half-life and efficacy. However, the modified products are heterogeneous mixtures composed of reaction products with different modification sites. It is generally known as a challenging task to separate and characterize a PEGylated product, especially for its positional isoforms. In this study, cation exchange high performance liquid chromatograph (IEC-HPLC) based on a pH gradient separation method was presented to separate five position isomers of rhGH conjugated with a 40-kDa branched PEG N-hydroxysuccinimidyl (NHS) functional group. Then Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that each of five materials collected by IEC-HPLC was conjugated with only one branched PEG chain. Furthermore, rhGH and PEG-rhGH were digested by trypsin and peptides were collected by reversed phase high performance liquid chromatography (RP-HPLC). Following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between PEG-rhGH and rhGH. Finally, biological activities of those positional isomers were performed in vivo and very small variations were observed. This method was shown to be suitable for heterogeneity analysis of PEGylated biopharmaceutical products.

Molar mass fractionation in aqueous two-phase polymer solutions of dextran and poly(ethylene glycol).

Dextran and poly(ethylene glycol) (PEG) in phase separated aqueous two-phase systems (ATPSs) of these two polymers, with a broad molar mass distribution for dextran and a narrow molar mass distribution for PEG, were separated and quantified by gel permeation chromatography (GPC). Tie lines constructed by GPC method are in excellent agreement with those established by the previously reported approach based on density measurements of the phases. The fractionation of dextran during phase separation of ATPS leads to the redistribution of dextran of different chain lengths between the two phases. The degree of fractionation for dextran decays exponentially as a function of chain length. The average separation parameters, for both dextran and PEG, show a crossover from mean field behavior to Ising model behavior, as the critical point is approached.

Separation of polyethylene glycols and amino-terminated polyethylene glycols by high-performance liquid chromatography under near critical conditions.

The separation and characterization of polyethylene glycols (PEGs) and amino-substituted derivatives on common silica-based reversed-phase packing columns using isocratic elution is described. This separation is achieved by liquid chromatography under the near critical conditions (LCCC), based on the number of amino functional end groups without obvious effect of molar mass for PEGs. The mobile phase is acetonitrile in water with an optimal ammonium acetate buffer. The separation mechanism of PEG and amino-substituted PEG under the near LCCC on silica-based packing columns is confirmed to be ion-exchange interaction. Under the LCCC of PEG backbone, with fine tune of buffer concentration, the retention factor ratios for benzylamine and phenol in buffered mobile phases, α(benzylamine/phenol)-values, were used to assess the ion-exchange capacity on silica-based reversed-phase packing columns. To the best of our knowledge, this is the first report on separation of amino-functional PEGs independent of the molar mass by isocratic elution using common C18 or phenyl reversed-phase packing columns.

Asymmetrical flow field-flow fractionation for the analysis of PEG-asparaginase.

Monomethoxypolyethylene glycol L-asparaginase (PEG-ASNASE) is the PEGylated version of the enzyme L-asparaginase (ASNASE). Both are used for remission induction in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). The treatment control is generally carried out by performing activity assays, though methods to determine the actual enzyme rather than its activity are rare. Using asymmetrical flow field-flow fractionation (AF4) offered the chance to develop a method capable of simultaneously measuring PEG-ASNASE and PEG. A method validation was performed in accordance with FDA guidelines for PEG-ASNASE from non-biological solutions. The method unfolded a linearity of 15-750 U/mL with coefficients of correlation of r(2)>0.99. The coefficients of variation (CV) for within-run and between-run variability were 1.18-10.15% and 2.43-8.73%, respectively. Furthermore, the method was used to perform stability tests of the product Oncaspar® (PEG-ASNASE) and estimation of the molecular weight by multi-angle light scattering (MALS) of stressed samples to correlate them with the corresponding activity. The findings indicate that Oncaspar® stock solution should not be stored any longer than 24 h at room temperature and cannot be frozen in pure aqueous media. The validated method might be useful for the pharmaceutical industry and its quality control of PEG-ASNASE production.

Mapping knowledge translation and innovation processes in Cancer Drug Development: the case of liposomal doxorubicin.

We explored how the knowledge translation and innovation processes are structured when theyresult in innovations, as in the case of liposomal doxorubicin research. In order to map the processes, a literature network analysis was made through Cytoscape and semantic analysis was performed by GOPubmed which is based in the controlled vocabularies MeSH (Medical Subject Headings) and GO (Gene Ontology). We found clusters related to different stages of the technological development (invention, innovation and imitation) and the knowledge translation process (preclinical, translational and clinical research), and we were able to map the historic emergence of Doxil as a paradigmatic nanodrug. This research could be a powerful methodological tool for decision-making and innovation management in drug delivery research.

Temperature dependence of adsorption of PEGylated lysozyme and pure polyethylene glycol on a hydrophobic resin: comparison of isothermal titration calorimetry and van't Hoff data.

The influence of temperature on the adsorption of PEGylated lysozyme and pure PEG on Toyopearl PPG-600M, a hydrophobic resin, is studied by batch equilibrium measurements and pulse response experiments. Differently PEGylated lysozymes are used for the studies, enabling a systematic variation of the solute properties. Either ammonium sulfate or sodium chloride are added. The enthalpy of adsorption is calculated from a van't Hoff analysis based on these data. It is also directly measured by Isothermal Titration Calorimetry. In the investigated temperature range from 5 °C to 35 °C adsorption is favored by higher temperatures and hence endothermic. The results of the van't Hoff analysis of the equilibrium and the pulse response data agree well. Discrepancies between enthalpies of adsorption obtained by calorimetry and van't Hoff analysis are found and discussed. We conclude that the most likely explanation is that thermodynamic equilibrium is not reached in the experiments even though they were carried out carefully and in the generally accepted way.

A new concept in the separation of polyethyleneglycol-modified PAMAM dendrons by liquid chromatography.

In this work, a unique high-performance liquid chromatographic method was developed and applied for monitoring the synthesis of polyethyleneglycol surface modified poly(amidoamine) cystamine core dendrimers (PEG-PAMAMs) and PEG-PAMAM-alkynes with a single alkyne moiety attached to the core of a dendron through a unique sulfhydryl group. The separation of the products was performed on a column with a pentafluorphenylpropyl stationary phase, allowing multiple mechanisms of selectivity. More than 50 peaks were separated in one run, reflecting the degree of dendrimer PEGylation (PEG average molecular mass: 3,000). Moreover, modification of PAMAM with a single alkyne group could be distinguished. The developed method can be used for the general characterization and separation of PAMAM derivatives, in which the degree of modification is critical for final applications.

Cysteine-specific PEGylation of rhG-CSF via selenylsulfide bond.

A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.

Polymer analysis by supercritical fluid chromatography.

Synthetic polymers always have distributions of the degrees of polymerization. Supercritical fluid chromatography (SFC) is almost only one technique to be able to separate pure polymer that has exactly unique degree of polymerization. For typical examples of SFC separation of polymers, four kinds of polymers: polystyrene, poly(ethylene glycol), some nonionic surfactants, and polyprenol are described about the conditions of SFC. Separated pure polymers which have no molecular weight distribution, are called uniform polymer. Uniform polymers are really useful to calibrate or validate various detectors. Here, the applications of calibrating ultraviolet detector, evaporative light scattering detector, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are summarized. These detectors have strong molecular weight dependence of their sensitivity coefficient, in contrast to the good linearity of sample concentration.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Solid-phase extraction combined with dispersive liquid-liquid microextraction, fast derivatisation and high performance liquid chromatography-tandem mass spectrometry analysis for trace determination of short-chained dodecyl alcohol ethoxylates and dodecyl alcohol in environmental water samples.

A new method was developed for preconcentration, derivatisation and analysis of short-chained dodecyl alcohol ethoxylates and dodecyl alcohol. Solid-phase extraction combined with dispersive liquid-liquid microextraction was used for preconcentration of target compounds. Several parameters were optimised including different solid phase extraction sorbents, type and volume of both extracting and dispersive solvents. As a result fast and relatively simple preconcentration method was developed. The analytes were preconcentrated 700 times with the use of small sample volume. The target compounds were derivatised before analysis with the use of newly developed procedure. The derivatisation procedure was made in vial insert and was performed at room temperature with the use of 1-naphthoyl chloride as the derivatisation agent. The developed method was used for the analysis of short-chained dodecyl alcohol ethoxylates and dodecyl alcohol in both sewage effluent from sewage treatment plants and river water samples.