Protein concentration effect on protein-lipopolysaccharide (LPS) binding and endotoxin removal

It is known some proteins can disaggregate endotoxins and form complexes with lipopolysaccharide (LPS). Nevertheless, how protein concentration affects protein-LPS binding and endotoxin removal is unknown. In this study, protein samples at various concentrations were incubated with endotoxin samples at a fixed concentration. The mixtures were filtered by ultrafiltration membranes. As protein concentration increased, the amount of endotoxin detected in the filtrates increased too. This result indicates protein concentration has significant effect on protein-LPS binding and the amount of endotoxin disaggregated.     

The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseudomonas aeruginosa as revealed by monoclonal antibodies and Western blotting

Abstract Monoclonal antibodies raised against single serotype components of a Pseudomonas aeruginosa vaccine have been shown to bind to the O antigen region of lipopolysaccharide (LPS). Outer membrane (OM) proteins, prepared by detergent treatment of envelope fractions and by EDTA/sonication treatment of whole cells, were separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred to nitrocellulose membrane and reacted with LPS-specific monoclonal antibodies. The patterns produced revealed that many of the protein bands were in fact protein-LPS complexes.     

Understanding the interaction of proteins to ion exchange chromatographic supports: A surface energetics approach

Ion exchange chromatography is one of the most widely used chromatographic technique for the separation and purification of important biological molecules. Due to its wide applicability in separation processes, a targeted approach is required to suggest the effective binding conditions during ion exchange chromatography. A surface energetics approach was used to study the interaction of proteins to different types of ion exchange chromatographic beads. The basic parameters used in this approach are derived from the contact angle, streaming potential, and zeta potential values. The interaction of few model proteins to different anionic and cationic exchanger, with different backbone chemistry, that is, agarose and methacrylate, was performed. Generally, under binding conditions, it was observed that proteins having negative surface charges showed strong to lose interaction (20 kT for Hannilase to 0.5 kT for IgG) with different anionic exchangers (having different positive surface charges). On the contrary, anionic exchangers showed almost no interaction (0–0.1 kT) with the positively charged proteins. An inverse behavior was observed for the interaction of proteins to cationic exchangers. The outcome from these theoretical calculations can predict the binding behavior of different proteins under real ion exchange chromatographic conditions. This will ultimately propose a better bioprocess design for protein separation.     

基于分子模拟的离子交换层析中静电相互作用研究

将分子模拟方法引入到蛋白质离子交换层析中的静电相互作用研究。选用蛋清溶菌酶和牛胰凝乳蛋白酶为模型蛋白质,阳离子交换吸附剂SP Sepharose FF等为模型层析介质。从蛋白质数据库(PDB)中获得蛋白质三维结构数据,分析了介质孔径和配基分布,以点电荷模拟离子交换层析介质的功能配基,构筑了蛋白质-介质配基模拟表面体系。采用MCCE、Delphi和GRASP等程序包进行了分子模拟计算,考察了作用方向、作用距离、盐浓度、pH等对蛋白质和模拟配基平面间静电相互作用的影响。结果表明,宏观的层析平衡常数与微观分子模拟计算的相互作用能量参数间存在良好的线性关系。     

Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high‐efficiency protein refolding. We have herein found that electrostatic repulsion between like‐charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion‐exchange resins when the protein and ion‐exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like‐charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like‐charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.     

Rapid separation of proteins and their higher-molecular fragments by means of Spheron ion-exchanges.

Ion-exchange derivatives are described. of a hydrophilic rigid macroporous glycolmethacrylate gel called Spheron, suitable for rapid high-performance liquid chromatography (HPLC) of proteins and their fragments. Their flow parameters are compared with those of ion exchange derivatives of cellulose and polydextran. The conditions for work with them are described (regeneration, cycling, equilibration, column packing) as well as the construction of a simple apparatus for medium-pressure ion exchange chromatography of proteins. The efficiency of these ion exchangers for the separation of proteins is illustrated with examples of chromatography of an artificial mixture of serum albumin, chymotrypsinogen and lysozyme. Chromatography of cyanogen bromide fragments of serum albumin and the A and B chains of oxidized insulin showed that the method can be applied in chromatography on higher molecular protein fragments. A review of all proteins, including technical enzymes, which have already been chromatographed on Spheron ion exchangers is also given. The prospects of Spheron ion exchangers for HPLC of proteins and their fragments are briefly discussed.     

Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions

Whole cells of Escherichia coli strains 0111, K12 and B as well as the ampicillin-resistant mutant K12 D21 and several lipopolysaccharide (LPS) mutants derived from this strain were analyzed for their molar LPS content per mg dry weight. An increase of the LPS concentration in some LPS mutants was substantiated by analyzing isolated cell walls and relating the molar LPS content to the murein subunit as measure of cell surface area. The increase of LPS was paralleled by increasing amounts of phospholipid while the overall protein content in the outer membrane decreased.According to the pattern of major outer membrane proteins in the various strains and the respective LPS structures, protein-LPS interactions are discussed as important requirements for outer membrane assembly and stability.Abbreviations LPS lipopolysaccharide - SDS sodium dodecyl-sulfate Dedicated to Dr. Otto Lüderitz on the occasion of his 60th birthday     

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process

Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to 7‐fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins.     

Enhancing protein capacity of rigid macroporous polymeric adsorbent.

A macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin was synthesized and modified with diethylamine to yield an anion-exchange resin suitable for protein adsorption. Efforts were made to enhance protein ion exchange capacity of the resin by investigating the copolymer composition. Different synthesis recipes were attempted, and the resultant resins were characterized by measuring the specific surface area and the adsorption ability using bovine serum albumin (BSA) as a model protein. The intraparticle pore size distribution measured by mercury porosimetry showed that the pores in the range of 40-120 nm took 75% of the total pore volume, indicating that the ion exchanger was favorable for protein adsorption. BSA capacity obtained with an appropriate recipe was as high as 78.6 mg/g wet resin or 50 mg/mL packed volume, which was higher than the capacities of some commercially available ion exchangers. Moreover, by using a pore diffusion model, the effective pore diffusivity of BSA was found to be 5.5 x 10(-12) m(2)/s, similar to those in the commercial ion exchangers.     

Maximizing productivity of chromatography steps for purification of monoclonal antibodies

The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

An exclusion mechanism in ion exchange chromatography

Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes.     

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.     

Protein-lipopolysaccharide interactions. 1. The reaction of lysozyme with Pseudomonas aeruginosa LPS.

Lysozyme (EC 3.2.1.17) complexes with extracted Pseudomonas aeruginosa LPS in two distinct stages. The initial stage does not produce turbidity detectable by nephelometry (measured as nephelos units (N) per time) but does permit low-speed sedimentation of the lysozyme-lipopolysaccharide (LPS) complex. This association is 100% disrupted by the action of 0.1 M Mg2+. Monovalent cations at equal ionic strength to the Mg2+ concentration used for these studies failed to alter significantly the lysozyme-LPS complex, indicating that the role of Mg2+ was not strictly an ionic one. The study of lysozyme-LPS complexes may provide a model system for investigating in vivo protein-LPS interactions.     

High-performance liquid chromatographic separation of platinum complexes containing the cis-1,2-diaminocyclohexane carrier ligand

Platinum drugs with the 1,2-diaminocyclohexane (dach) carrier ligand have shown great promise in cancer chemotherapy, but little is known about their metabolism in the body. Since it is possible to radiolabel the dach ligand, it should be possible to quantitate the biotransformation products of these drugs, provided a method were available to separate the biotransformation products. In this paper we describe a two-column high-performance liquid chromatography system which can be used to separate many likely dach-platinum biotransformation products from the parent compounds, and allow their identification. An initial separation on a reverse-phase Partisil ODS-3 column allowed resolution of the uncharged species. The peak fractions from this column were concentrated 10-fold and reinjected onto a cation exchange Partisil 10 SCX column to allow resolution of the positively charged species. This system allowed resolution of two prototype dach-platinum drugs, (cis-1,2-diaminocyclohexane)dichloroplatinum(II) and (cis-1,2-diaminocyclohexane)malonatoplatinum(II), the aquated species likely to form from these drugs, and the complexes formed when these compounds react with glutathione, metallothionein, and amino acids. By using cation exchange chromatography at pH 2.3 as well as pH 4 and by using 14C-labeled amino acids to determine stoichiometry, it was also possible to determine the most likely structures for some of the amino acid complexes. Most importantly, this system allowed clear separation of many of the likely biotransformation products tested from the biologically important aquated species. This system should prove useful for separating and identifying the biotransformation products of dach-platinum drugs in blood and urine, in tissue culture media, and inside the cell.     

A method for the determination of glucose synthesis in isolated bovine hepatocytes

A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods. Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with (14)C-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin. Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting. Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.     

Protein separations using colloidal magnetic nanoparticles

Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 8 nm are shown to be effective ion exchange media for the recovery and separation of proteins from protein mixtures. These particles have high adsorptive capacities (up to 1200 mg protein/mL adsorbent, an order of magnitude larger than the best commercially available adsorbents) and exhibit none of the diffusional resistances offered by conventional porous ion exchange media. Protein-laden particles are readily recovered from the feed solution using high-gradient magnetic filtration.     

Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion-exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell-shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the pI, and on both sides of the pH scale, the bell-shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI above the pH of the buffer (the 'unadsorbed' fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre-fractionation techniques currently employed in proteomics.     

The impact of protein exclusion on the purity performance of ion exchange resins

Dynamic binding capacity (DBC) decreases with increasing conductivity in the equilibrium regime for ion exchange chromatography. An exclusion regime has been demonstrated in ion exchange resins where DBC increases with increasing conductivity and decreasing protein charge. The purpose of this work was to examine the impact of the exclusion regime on impurity removal. Resin performance was evaluated based on dynamic binding capacities and purity within the exclusion and equilibrium regimes. The results revealed that Chinese hamster ovary proteins (CHOP), a major impurity, exhibit similar exclusion trends as the MAb proteins. The results further the understanding of the exclusion regime and its impact on product purity, a critical area for IEX development and optimization.     

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS‐PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin (∼67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94‐bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94‐bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE‐ion exchange column chromatography. In vitro dissociation tests of the purified PSP94‐bound complexes showed that the binding of serum PSP94‐complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94‐bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker. J. Cell. Biochem. 76:71–83, 1999. © 1999 Wiley‐Liss, Inc.