Adsorption of endotoxin on glass in the presence of rhIL-11

Poor recovery of spiked endotoxin in the Limulus amebocyte lysate assay (LAL assay) was observed in the presence of recombinant human interleukin-11 (rhIL-11), a cationic, hydrophobic protein. Detection of endotoxin activity remaining in the empty glass tubes in which endotoxin and rhIL-11 mixtures were incubated indicated adsorption of endotoxin on glass. At low concentrations of rhIL-11, a correlation between endotoxin adsorbed on glass and a decrease of endotoxin in solution was observed. Adsorption of rhIL-11 on glass correlated with adsorption of endotoxin, which indicates that rhIL-11 mediates adsorption of endotoxin on glass. Consequently, adsorption of endotoxin on glass may occur in the presence of other substances which bind to both of endotoxin and glass.     

Direct visualization of protein adsorption to primary containers by gold nanoparticles

Adsorption of proteins to primary containers can result in protein loss, protein denaturation, or aggregation. We report a simple and effective method to directly detect and visualize adsorption of proteins to container surfaces by staining adsorbed proteins with gold nanoparticles, which bind proteins nonspecifically. The gold nanoparticle staining method was applied to study adsorption to siliconized glass prefilled syringes (PFSs) of a therapeutic protein in a liquid formulation. The protein was found to preferentially adsorb to glass surfaces over siliconized surfaces in PFSs. The presence of adsorbed proteins on glass surfaces was confirmed by in situ Raman spectroscopy. Gold nanoparticle staining patterns revealed that adsorption of proteins to hydrophobic cyclic olefin polymer plastic vials was minimized compared with hydrophilic type I glass vials. Bovine serum albumin (BSA) also preferentially adsorbed to glass surfaces compared with siliconized surfaces as revealed by the gold staining patterns in PFS incubated with BSA, supporting the use of albumin to minimize loss of proteins in glass containers. The method is particularly valuable for high-concentration protein formulations in which adsorption of proteins to containers cannot be easily detected by other methods.     

固化组氨酸吸附鲎试验定量检测内毒素

建立一种简便、可靠的内毒素定量检测方法。方法：在含干扰物的样品中,利用固化组氨酸吸附样品中的内毒素,再以基质偶氮显色法鲎试验定量检测被吸附的内毒素。结果：该法能较好地克服氨基酸、抗生素等可致鲎试验假阴性物质的干扰,并具有较高的准确性和精密度。结论：此法是一种简便、特异的内毒素定量检测方法。     

Foam fractionation of binary mixtures of lysozyme and albumin

A nitrogen gas-based foam fractionation method was employed to separate model proteins, bovine serum albumin (BSA) and hen egg white lysozyme, from each other. Fractionation was characterized by the separation ratio and by recovery of proteins in the retentate as a function of the nominal pore size of the gas dispersion frit and solution conditions (pH and ionic strength). For binary mixtures of the proteins at pH 7.4, and ionic strength (mu) of 0.18 M, the recovery of lysozyme and the separation ratio were both dependent on the frit size employed to generate the foam. At low ionic strength (mu = 0.01 M), separation was only somewhat greater with the small pore size frits, although at values significantly lower than those found for high ionic strength. The diminished separations appear to be due to the only slight changes in recoveries observed for BSA and lysozyme.%Separation ratios of lysozyme from BSA in solutions either of high or low ionic strength were maximal at pH values equal to or less than the isoelectric point (pI) of BSA. Separation ratios were lower when foaming was carried out under low compared with high ionic strength. The recovery of lysozyme was enhanced by foaming from solutions of low pH and high ionic strength. Recoveries of BSA were greatest when the molecule was negatively charged. Electrical interactions between the positively charged lysozyme and negatively charged BSA may explain the diminished separation ratios and enhanced recoveries. Enzyme activity studies of lysozyme remaining in the retentate showed no change from prefoam activity.     

Lysozyme stability in primary emulsion for PLGA microsphere preparation: effect of recovery methods and stabilizing excipients

Purpose. To investigate the conformational stability of a model protein, lysozyme, in the primary emulsion phase of the microsphere preparation process. Methods. The conformational stability of lysozyme during primary emulsification was assessed by differential scanning calorimetry (DSC) and enzymatic activity assay. PEG 400 was used to separate lysozyme from water-in-oil (w/o) emulsion containing poly(lactide-co-glycolide) (PLGA). Results. No significant changes in the recovery of lysozyme were observed due to increasing sonication time from 20 to 60 s at 40 W or increasing intensity from 40 to 60 W for 20 s. By using the method involving PEG 400, lysozyme recovery in the presence of PLGA was increased from 11.8% to 70%. Hydroxypropyl--cyclodextrin (HP--CD) increased lysozyme recovery from 35% to 70% at low lysozyme concentration (20 mg/ml), and from 70% to 77% at high lysozyme concentration (100 mg/ml) in the presence of PLGA. Sugars such as trehalose and mannitol failed to increase lysozyme recovery. DSC results suggested the retention of the conformational structure of the recovered lysozyme, which was supported by an enzymatic activity assay. Conclusions. HP--CD was found to be a promising stabilizer that protected lysozyme during the primary emulsification. Protein recovery method with the help of PEG 400 allowed the study of protein stability in w/o emulsions in the presence of PLGA. DSC provided supplementary information on the conformational changes of lysozyme during emulsification.     

Interference in the Limulus amebocyte lysate assay for endotoxin determination in peritoneal dialysis fluids and concentrates for hemodialysis

The interference of the saline concentration of fluids for peritoneal dialysis and concentrates for hemodialysis on the limulus amebocyte lysate (LAL) assay for endotoxins was investigated. The experiments were carried out individually with each substance that compose fluids for hemodialysis, to determine the possible inhibition or enhancement effects that they could cause on the LAL assay. The compositions were also assayed to investigate the possibility of synergistic effect. They were assayed by the gel-clot method from two different suppliers, and the samples that showed inhibition effect were also assayed by the chromogenic method. The samples were analysed at successive dilutions, with different LAL sensitivities, to satisfy the endotoxin limits of 5 EU/ml for the concentrate and 0.25 EU/ml for the fluid for dialysis peritoneal. The results showed that the major interference on the gel-clot assay occurs in presence of acetic acid and in concentrates containing acid acetic, even the pH being adjusted between 6.5 and 7.5. However, the test, after an adequate dilution, could be validating for all samples. Chromogenic test can be used for peritoneal dialysis fluids considering a limit of 0.25 EU/ml and sample dilution of eight times, but it cannot be used for concentrates for hemodialysis without further dilution. Considering the results and that the chromogenic is a more time-consuming method, endotoxins in fluids for hemodialysis can be satisfactorily assayed by the gel-clot method.     

Competitive interfacial adsorption of blood proteins

The competitive adsorption of blood proteins is of great importance for the treatment of thrombosis using a colloidal drug delivery system. The aim of this study is to investigate competitive adsorption of albumin (BSA) and human immunoglobulin G (HIgG) against fibrinogen (Fb). The competitive adsorption of blood proteins was investigated using interfacial rheology at physiological pH. The influence of bulk concentration, temperature and pH on the interfacial adsorption of protein molecules was determined at the air/aqueous interface. As expected, the results indicated that increase in bulk concentration enhanced the interfacial adsorption. Structure and molecular weight of the protein molecules under investigation had influence on interfacial adsorption leading to a competition at the interface. HIgG is more flexible and surface active molecule than BSA. Thus, HIgG replaced BSA and Fb at the air/aqueous interface. In the presence of Fb, BSA adsorbed rapidly initially and then, was replaced by Fb at the interface. The kinetics of displacement of albumin at the interface was rather slow. In conclusion, the investigation of competitive adsorption of blood proteins may be useful biotechnologically, as it will provide useful information for the production of an antithrombogenic material, which will adsorb albumin rather than Fb.     

Characterization of endotoxin and cationic liposome interaction

The objective of this study was to characterize the interaction of endotoxin with cationic liposomes used in nonviral gene delivery. Endotoxin-cationic liposome interaction was characterized using fluorescent anisotropy, and the Limulus amebocyte lysate (LAL) assay. Cellular toxicity of endotoxin-cationic liposome complex was examined using a dimethylthiazol diphenyltetrazolium bromide (MTT) assay. The effect of endotoxin on the lipid-DNA complex and subsequent transfection into COS-1 cells was also examined. A competitive interaction occurred between fluoroscein isothiocyanate (FITC)-labeled endotoxin and plasmid DNA for binding dioleoyl glycero trimethylammonium propane:dioleoyl glycero phosphoethanolamine (DOTAP:DOPE) liposomes using fluorescent anisotropy techniques. The LAL assay demonstrated no change in endotoxin activity upon interaction with liposomes. No loss of COS cell viability was detected via the MTT assay during a 5-hr exposure to endotoxin. Transient transfection studies indicate that increasing levels of endotoxin lowered activity more than 90% at 50,000 endotoxin units (EU)/ml. Endotoxin and cationic liposomes interact mainly by an electrostatic attraction. Endotoxin contamination can potentially impact transfection efficiency via competition with plasmid DNA for cationic liposome binding by increasing transfection variability at 50 EU/ml, a concentration of endotoxin contamination that can occur with small-scale plasmid preparations used for in vitro cell transfections, but would not be expected with typical GLP or GMP preparations used in clinical studies.     

Detection of endotoxins using nanomaterials

An endotoxin is a lipopolysaccharide complex that is associated with the outer membrane of gram negative bacteria, and even a small amount can trigger strong exothermic activity. Pharmaceutical and food products can be contaminated with endotoxins, so there is a strict need to control them. A limulus amoebocyte lysate (LAL) assay is a standard endotoxin detection method that is highly sensitive and easy to use, but it has disadvantages in that the sample preparation is a complex process, it takes a long time to confirm the presence of endotoxins, and it requires a controlled workplace. Recently, endotoxin detection using nanomaterials has been gaining attention, and this review reports on the latest endotoxin detection methods using gold nanoparticles, magnetic nanoparticles, quantum dots, and carbon materials, among different nanomaterials, and it also introduces the future prospect of endotoxin detection.     

An assay for measurement of protein adsorption to glass vials

Protein adsorption to primary packaging is one of the problems faced by biopharmaceutical drug companies. An assay was developed to quantify loss of proteins to glass vial surfaces. The assay involves the labeling of protein with a fluorescent dye, incubation of the labeled protein with the vial surface, elution of the adsorbed protein using a stripping buffer, and determination of fluorescence of the adsorbed protein using a fluorometer. The assay is simple to set up, accurate, sensitive, and flexible. The assay can be modified for indirect measurement of protein adsorption and offers an attractive alternative for researchers to quantify protein adsorption to glass vials and syringes.     

Role of a novel multifunctional excipient poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)-block-poly(l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42-88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36-60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte-protein complexation may play a role in protein-controlled delivery.     

Toxic cyanobacteria and cyanotoxins in public hot springs in Saudi Arabia.

Toxic cyanobacteria are well reported in rivers, lakes and even marine environments, but the toxin production of cyanobacteria in hot springs is largely unexplored. Therefore, the present study investigated the presence of toxic cyanobacteria and cyanotoxins in public hot springs in Saudi Arabia. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that Saudi spring cyanobacterial mats contained microcystins (MCYSTs) at concentrations ranging from 468 to 512.5 microg g(-1). The Limulus amebocyte lystae (LAL) assay detected lipopolysaccharide (LPS) endotoxins in these mats at concentrations ranging from 433.3 to 506.8 EU g(-1). MCYSTs and endotoxins were also detected in spring waters at levels of 5.7 microg l(-1) and 640 EU ml(-1), respectively, exceeding WHO's provisional guideline value for MCYST-LR in drinking-water. High-performance liquid chromatography (HPLC) analysis revealed that only Oscillatoria limosa and Synechococcus lividus can produce MCYSTs with a profile consisting of MCYST-RR and -LR. Based on the LAL assay, 12 out of 17 cyanobacterial species contained LPS at concentrations ranging from 0.93 to 21.06 EU g(-1). However, not all LPS of these species were toxic to mice. This study suggests that the hot springs in the world including Saudi Arabia should be screened for toxic cyanobacteria to avoid the exposure of people recreating and bathing in spring waters to cyanobacterial toxins.     

Bacterial endotoxin retention by inline intravenous filters

Filters used in i.v. administration sets were tested for their ability to retain bacterial endotoxins for up to 96 hours of continuous infusion. Inline filters composed of cellulose ester, polyacrylate, polypropylene, polyethylene, or Posidyne Nylon 66 were used during continuous infusion of 5% dextrose injection at 83 mL/hr. One milliliter of inoculum containing 10(8) Escherichia coli was injected through a port upstream from the filter. A bacterial filter was used to monitor the sterility of effluent from the inline filters. The effluent was tested with limulus amebocyte lysate (LAL) that could detect endotoxin concentrations greater than 50 pg/mL. A control solution was monitored for viability of the bacteria throughout the course of the study, and positive endotoxin controls were used to confirm the sensitivity of the LAL. Samples of effluent were tested at 0, 4, 19, 24, 48, 72, and 96 hours. Effluent from all filters was sterile throughout the study. LAL assay indicated that only the effluent from filters containing Posidyne Nylon 66 was free of endotoxins for 96 hours. Effluent from the other filters contained endotoxins immediately after injection of the E. coli. Of the inline filters tested, only the one composed of Posidyne Nylon 66 was able to retain E. coli endotoxin for 96 hours. Further study is needed with E. coli and other microorganisms that are likely contaminants of i.v. infusions.     

Pb2+-NaOH介质中蛋白质水解物的极谱吸附波及其应用Pb2+-NaOH介质中蛋白质水解物的极谱吸附波及其应用

目的建立一种快速、简便、灵敏的蛋白质含量测定的新方法。方法含有巯基或二硫基的蛋白质与0.5 mol·L^-1氢氧化钠、1.5×10^-4 mol·L^-1 Pb²⁺和0.02%四丁基碘化铵的混合溶液在沸水浴中反应5 min，采用单扫描极谱法，测定-0.66 V(vs SCE)处一价导数波高。结果牛血清白蛋白（BSA）、人血清白蛋白（HSA）的浓度在7.5×10^-10～3.0×10^-7 mol·L^-1与其波高呈线性关系，BSA和HSA检测限均为3.0×10^-10 mol·L^-1；溶菌酶(Lyso)的浓度在1.4×10^-8～1.3×10^-6 mol·L^-1与其波高呈线性关系，Lyso的检测限为7.0×10^-9 mol·L^-1。结论该方法取样量少、体系简单、灵敏度高，可用于人血清样品中蛋白质含量的测定。     

Determination of Protein Binding of a Highly Lipophilic Drug,Isocarbacyclin Methyl Ester (TEI-9090), Using a Polydimethylsiloxane-coated Glass Beads Assay

Abstract— An adsorption technique with polydimethylsiloxane-coated glass beads (PDMS-GB) was developed to determine the protein binding of a highly lipophilic and hydrophobic drug. The present assay method is based on the quantitative adsorption of unbound drug to the PDMS-GB. This method of batch separation in a glass assay tube has an advantage of simplicity and rapidity. To evaluate the reliability of PDMS-GB assay, we compared the protein binding of diazepam in serum in-vitro measured by ultrafiltration and PDMS-GB assay. There was no significant difference between the extent of binding measured by each method. Using PDMS-GB assay, we determined the protein binding of the prostaglandin I₂ (PGI₂) analogue isocarbacyclin methyl ester (TEI-9090), whose binding cannot be measured by commonly employed techniques (equilibrium dialysis, ultrafiltration, gel filtration or ultracentrifugation) because of a high degree of adsorption to membranes, resins or tubes. The percentage of TEI-9090 bound in human serum, 4% human serum albumin (HSA, fatty acid-free) and dog serum were ～98, ～87 and ～95%, respectively, and these values were independent of TEI-9090 concentration up to 10 ng mL^?1. The binding of isocarbacyclin (TEI-7165) to serum protein in man, dogs, rabbits and rats, determined by ultrafiltration, was also high (>90%). While the displacement of TEI-9090 and TEI-7165 binding to HSA by aspirin, salicylic acid and indomethacin was not observed, clofibric acid and free fatty acids significantly inhibited the protein binding of both compounds. These results indicate that the binding site of TEI-9090 and TEI-7165 on HSA could be identical with the possible binding site of PGI₂.     

Model Protein Adsorption on Polymers Explained by Hansen Solubility Parameters

Hansen solubility parameters (HSP) theory has been successful in explaining the wettability of organic solvents on polymer surfaces and miscibility of different polymers. Here, we demonstrate that the amount of bovine serum albumin (BSA) protein adsorption on different polymer surfaces can also be explained by HSP. Interestingly, the HSP of the adsorbed BSA proteins calculated from the protein adsorption data is different than the HSP of native BSA protein itself. The HSP of the adsorbed BSA proteins are more hydrophobic than the native BSA protein. This observation suggested adsorbed BSA proteins are partially denatured and exposed their hydrophobic core toward the polymer surfaces. These results highlight a new strategic direction to understand interaction of protein with a surface: a theoretical approach that compliments experimental approach. The model in this study could be used to predict the amount of BSA adsorption on a polymer or any other solid surface, if the HSP of that surface is known. Further, the model can serve as a prescreen method to identify surfaces that are problematic at the outset and inform subsequent empirical studies to select packaging that will have the least adsorption for the specific biologic application.     

Control of Protein Adsorption to Cyclo Olefin Polymer by the Hofmeister Effect

Cyclo olefin polymer (COP) is an attractive plastic because it has low protein adsorption despite its hydrophobic chemical structure. Here, the adsorption of model proteins to the COP was evaluated in comparison with a representative plastic, polystyrene (PSt), using reflectometry interference spectroscopy (RIfS) technology. The effects of different salts on adsorption were then examined. The adsorption of bovine serum albumin onto COP increased in the presence of kosmotropic salts, whereas adsorption of IgG increased in the presence of chaotropic salts. By contrast, the adsorption of these 2 proteins to PSt was unaffected by these Hofmeister salts. Langmuir–Freundlich model of COP adsorption suggested that the COP surface is more homogeneous for protein binding than the PSt surface. Furthermore, RIfS and sum frequency generation analyses indicated that water molecules bind more weakly to COP than to PSt. Our data propose a novel viewpoint of the way protein binds to COP surface that is different from the way it binds to PSt.     

Influence of polymeric effectors on binding of 3H-dihydroalprenolol to beta-adrenergic receptor of rat brain

The significance of anionic and cationic charges of glycocalyx, phospholipid or protein, etc. on the cell surface of the rat brain was examined for beta-adrenoceptors using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of polymeric effectors, DNA, heparin, polymyxin B, histone, gelatin, colominic acid and bovine serum albumin (BSA), on the affinity of beta-adrenoceptors. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a radioligand. Polymyxin B, DNA and heparin significantly caused a reduction in the maximum number of beta-adrenoceptors (Bmax), but only small changes were observed with histone, gelatin, BSA and colominic acid. Only DNA induced a decrease in the value of the dissociation constant (Kd) of beta-adrenoceptors. These results suggest that anionic or cationic charges in the environment of the receptor sites could have a crucial role in drug-receptor interaction.     

Influence of surfactants upon protein/peptide adsorption to glass and polypropylene

This paper explores the use of surfactants as a pharmaceutical excipient to reduce adsorptive losses of protein/peptide drugs. The predominant adsorption mechanism for protein/peptide drugs is shown to change with the surface and conditions of study. In the presence of surfactants, where the surfactant-surface interaction is greater than the surface-protein/peptide interaction, drug adsorption is reduced and/or eliminated. Anionic (sodium dodecyl sulfate), cationic (dodecyltrimethylammonium chloride and benzalkonium chloride) and nonionic surfactants (Polysorbate 20 and Poloxamer 188) are evaluated as possible protein/peptide adsorption controlling excipients. For protein/peptide adsorption onto glass, where an electrostatic interaction predominates, only the most hydrophobic surfactants (Polysorbate 20 and benzalkonium chloride) were significantly effective. Protein/peptide adsorption to polypropylene, where a hydrophobic/dehydration mechanism predominates, allows additional surface-active agents to be effective in reducing drug adsorption.     

Cyanobacteria and their toxins in treated-water storage reservoirs in Abha city, Saudi Arabia.

Occurrence of toxic cyanobacteria in drinking and recreational waters poses human health at risk as they can release potent toxins into the water. In the present study, open and covered treated-water storage reservoirs as well as their relevant tap waters in Abha city, Saudi Arabia, were surveyed for the presence of cyanobacteria and their toxins. The results revealed the contamination of most open reservoir and tap waters by algae and cyanobacteria, with an abundance of toxigenic species of cyanobacteria. Depending on the results of the Limulus amebocyte lysate (LAL) assay and enzyme linked immunosorbent assay (ELISA), endotoxins and microcystins (MCYSTs) were found in most open reservoir and tap waters at concentrations up to 32EUml(-1) and 0.3mugml(-1), respectively. The extracts of axenic cultures of most cyanobacterial species isolated from these reservoirs showed activity to LAL assay, with large endotoxin amounts obtained in Calothrix parietina (490EUg(-1)) and Phormidium tenue (210EUg(-1)). Based on ELISA and HPLC analysis for these extracts, only C. parietina can produce MCYSTs (202mugg(-1)) with a profile consisting of MCYST-RR and -LR. This study suggests that open treated-water storage reservoirs should be covered to prevent the presence of cyanobacteria and their toxins in such drinking and recreational waters.