Characterization of a continuous supermacroporous monolithic matrix for chromatographic separation of large bioparticles

A continuous supermacroporous monolithic chromatographic matrix has been characterized using a capillary model, experimental breakthrough curves, and pressure drop experiments. The model describes the convective flow and its dispersive mixing effects, mass transfer resistance, pore size distribution, and the adsorption behavior of the monolithic matrix. It is possible to determine an effective pore size distribution by fitting the capillary model to experimental breakthrough curves and pressure drop experiments. The model is able to describe the flow rate dependence of the experimental breakthrough curves. Mass transport resistance was due to: (i) dispersive mixing effects in the convective flow in the pores; and (ii) slow diffusion in the stagnant film covering the surface within each pore, under adsorption conditions. The monolithic matrix can be described by a very narrow pore size distribution, illustrating one of the advantages of the gel. A broader pore size distribution results in increased band broadening. This can be studied easily using the model developed in this investigation.     

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.     

The functional association of polymyxin B with bacterial lipopolysaccharide is stereospecific: studies on polymyxin B nonapeptide

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.     

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of EDTA or polymyxin with lysozyme for the recovery of intracellular products fromEscherichia. coli

Summary Escherichia coli cells taken from exponential and late stationary (or decline) phases of culture were very susceptible to lysis by EDTA/lysozyme. Log phase cells were most susceptible to lysis by polymyxin/lysozyme. Treatment ofE. coli with EDTA and lysozyme compared favourably with sonication as a method for release of intracellular protein. Concentration ranges for optimal lysis were 100–800 μg/ml for EDTA and 25–50 μg/ml for lysozyme.     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Preparation of a monolithic capillary column with immobilized α-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for α-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N´-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, α-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the α-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Study of the structure and function of endotoxins with the aid of enzymes. 2. Modification of Salmonella paratyphi B endotoxin with lysozyme and lipase]

Endotoxin preparations from the S. paratyphi B cultures, isolated by various methods, were treated with lysozyme (splitting of beta-1,4-glycoside links of lipid A) and lipase of the pancreas (splitting of complex ester links of glycerophosphatides). Lysozyme and, to a lesser extent, lipase, were capable of partial depression of the toxic endotoxin function. The process of enzymatic detoxication coursed selectively, without influencing the serological and immunological activity of the preparations. Suppositions are put forward on the complicated nature of the toxic endotoxin function manifestation and possibility of provision of detoxication effect by specific actions differing by the point of application.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

Towards a rational development of anti-endotoxin agents: novel approaches to sequestration of bacterial endotoxins with small molecules.

Endotoxins, or lipopolysaccharides (LPS), present on the surface of Gram-negative bacteria, play a key role in the pathogenesis of septic shock, a common clinical problem and a leading cause of mortality in critically ill patients, for which no specific therapeutic modalities are available at the present time. The toxic moiety of LPS is a glycolipid called 'lipid A', which is composed of a bisphosphorylated diglucosamine backbone bearing up to seven acyl chains in ester and amide linkages. Lipid A is structurally highly conserved in Gram-negative bacteria, and is therefore an attractive target for developing anti-endotoxin molecules designed to sequester, and thereby neutralize, the deleterious effects of endotoxins. The anionic and amphipathic nature of lipid A enables the interaction of a wide variety of cationic amphiphiles with the toxin. This review describes the systematic evaluation of several structural classes of cationic amphiphiles, both peptides and non-peptidic small molecules, in the broader context of recent efforts aimed at developing novel anti-endotoxin strategies. The derivation of a pharmacophore for LPS recognition has led to the identification of novel, nontoxic, structurally simple small molecules, the lipopolyamines. The lipopolyamines bind and neutralize LPS in in vitro experiments as well as in animal models of endotoxicity, and thus present novel and exciting leads for rational, structure-based development of LPS-sequestering agents of potential clinical value.     

Direct determination of polymyxin B sulfate using resonance Rayleigh scattering and resonance non‐linear scattering methods with hexatungstate

At pH 1.3–1.6, tungstate WO₄^2–, can be converted to hexatungstate W₆O₁₉^2–, which can react with positively charged polymyxin B sulfate (PMB) to result in enhancement of resonance Rayleigh scattering (RRS) and resonance non‐linear scattering, including second order scattering and frequency doubling scattering. Linear relationships can be established between enhanced scattering intensity and PMB concentration. The detection limits (3σ) were 5.5 ng/mL (RRS), 10.1 ng/mL (second order scattering) and 34.6 ng/mL (frequency doubling scattering). The optimum reaction conditions, influencing factors and related analytical properties were tested. The interaction mechanism was investigated via absorption spectrum, circular dichroism spectra and atomic force microscopy imaging. The basis of scattering enhancement is discussed. PMB in eardrops, human serum and urine, were quantified satisfactorily by RRS. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization of a packed bed bioreactor with immobilized cells using experimental design

A central composite design was employed for the optimization of heterogeneous enzymatic hydrolysis of sucrose. The reaction was catalyzed by whole yeast cells of Saccharomyces cerevisiae immobilized in Ca-pectate gel. Bioreactor volumetric productivity was chosen as an optimization criterion, while temperature and gel biomass concentration were optimization parameters. Sucrose inlet concentration of 700 kg m^–3 and outlet conversion of 65% were constant in all experiments. In the temperature range 51–73 °C and biomass concentration range 11–39 kg m^–3 (dry mass of cells), the dependence of bioreactor productivity on the two factors was described by a second order polynom regression equation. No simple optimum was revealed by the experimental design. The bioreactor productivity increased within the whole experimental range of biomass concentration, whereas a temperature optimum was found to be between 60 and 65 °C.List of Symbols b _j jth regression coefficient - c _Si kg m^–3 inlet sucrose concentration - F m³ min^–1 flow rate - F F distribution - f _LF degrees of freedom of lack of fit variance - f _P degrees of freedom of pure error variance - N total number of runs - n ₀ number of runs in the centre of design - P kg m^–3 min^–1 productivity - s _LF ² lack of fit variance - SS _LF lack of fit sum of squares - S _p ² pure error variance - SS _P pure error sum of squares - SS _R total residual sum of squares - V _b m³ bioreactor bed volume - X _O outlet conversion - x ₁ 1st factor - coded temperature - x ₂ 2nd factor - coded biomass concentration - y kgm^–3min^–1 measured response (productivity) - kg m^–3 min^–1 estimated response (productivity) - y _Oi kg m^–3 min^–1 measured response in the centre of design - ¯y ₀ kg m^–3 min^–1 average of response in the centre of design     

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.     

Kinetics of the interaction of endotoxin with polymyxin B and its analogs: a surface plasmon resonance analysis

Lipopolysaccharide, the invariant structural component of Gram-negative bacteria, when present in minute amounts in the circulation in humans elicits 'endotoxic shock' syndrome, which is fatal in 60% of the cases. Polymyxin B (PMB), a cyclic cationic peptide, neutralizes the endotoxin, but also induces many harmful side effects. Many peptide-based drugs mimicking the activity of PMB have been synthesized in an attempt to reduce toxicity while still retaining the anti-endotoxic activity. The study attempts to use the recent technique of surface plasmon resonance (SPR), in determining the kinetics of association and dissociation involved in the interaction of endotoxin with a few selected peptides that have structural features resembling PMB. The results, in conjunction with the thermodynamic data derived using isothermal titration calorimetry (ITC), stress the vital role played by amphiphilicity of the peptides and hydrophobic forces in this biologically important interaction.