Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Characteristics and applications of adsorbents for pyrogen removal

Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated. Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range. The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent. The apparent dissociation constant was 1.57 X 10(-9) M. The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed. The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length. Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.     

Studies on adsorption isotherms of endotoxin and BSA using an affinity column

In this paper, an adsorbent with dimethylamine ligand for endotoxin removal was prepared and used to study the adsorption isotherms of endotoxin and BSA. The results showed that as the introducing of endotoxin, the maximum adsorption capacity q_m of BSA increased from 7.24 to 7.74 mg/mL and the apparent association constant K_A of BSA decreased from 14.06 to 11.48 mL/mg. The adsorption isotherms of BSA changed from Langmuir model to Multilayer model. All these gave the direct evidences to construct the adsorption process. By comparing adsorption isotherms of endotoxin with BSA, it was found that the apparent association constant K_A of endotoxin was much higher than that of BSA.     

Recovery of Acinetobacter radioresistens lipase by hydrophobic adsorption to n-hexadecane coated on nonwoven fabric

A simple and clean adsorption/desorption process was proposed for recovering Acinetobacter radioresistens lipase from fermentation broth. The adsorbent used was n-hexadecane coated on a hydrophobic nonwoven fabric (NWF). n-Hexadecane has a melting point of 16-18 degrees C, and its affinity for lipase decreases markedly from liquid to solid state. Accordingly, performing the adsorption and desorption above and below, respectively, the melting point would need no extraneous materials for separation. The adsorption isotherms at various temperatures were found to follow the Langmuir model. Simulation of the batch adsorption/desorption process showed that there exists an optimal amount of adsorbent for both concentration factor and enzyme recovery; the process is restrained by equilibrium. The performance of column adsorption/desorption could also be simulated using the adsorption isotherm, and it was shown that the concentration factor was proportional to the amount of adsorbent used. The benefits of this process include easy preparation of adsorbent, low operational cost, no extraneous materials needed, negligible enzyme denaturation, high efficiency, and simple process simulation.     

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

Endotoxin removal using a synthetic adsorbent of crystalline calcium silicate hydrate

A synthetic adsorbent of crystalline calcium silicate hydrate, the product LRA by Advanced Minerals Corp., has been studied for endotoxin removal from aqueous solutions. This adsorbent removes endotoxin effectively, and the removal is greatly enhanced by the presence of an electrolyte such as NaCl, Tris-HCl, or Na2HPO4. It has an endotoxin removal capacity as high as 6 million endotoxin units (EU) per gram. Its endotoxin removal kinetics is fast, and for instance, over 99.9% endotoxin in a 5000 EU/mL solution was removed by mixing for 2 min at an adsorbent usage of 10 g/L. Using the chromatographic column method to treat a 5000 EU/mL solution, an endotoxin log-reduction factor of 6.2 was achieved with a single pass. This adsorbent also demonstrated significantly better performance when compared to many commonly used endotoxin removal agents, such as ActiClean Etox Endotoxin Removal Resin, Affi-Prep Polymyxin Support, Detroxi-Gel Endotoxin Removing Gel, Q Sepharose Fast Flow Media, and Sigma Endotoxin Removal Solution. Furthermore, it demonstrated a high selective removal of endotoxin from a solution of lambda DNA. This adsorbent provides opportunities for developing disposable, scaleable, and cost-effective methods for endotoxin reduction in many biotechnological and pharmaceutical processes.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

Chromatographic removal of endotoxin from protein solutions by polymer particles

Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents.     

Breakthrough behavior of 17.5 mol% water in methanol, ethanol, isopropanol, and t-butanol vapors passed over corn grits

Ground corn is now used in industry as an adsorbent to remove water from ethanol vapors. It is stable and inexpensive at 10 cents/lb (22 cents/kg). For regeneration it requires less than 2000 Btu/gal of 190 proof ethanol processed. If necessary, it could be readily saccharified and fermented into ethanol after use. This renewable resource has further exciting potential as an inexpensive adsorbent for water removal from other alcohols, including methanol, isopropanol, and t-butanol. Water sorption capacity in a fixed bed, nonisothermal adsorption column appears to be a function of the heat capacity of the non-adsorbed alcohol vapor, relative to the heat capacity of the corn adsorbent. Methanol, ethanol, isopropanol, and t-butanol containing 17.5 mol% water gave 105,151, 284, and 358 g anhydrous product/kg adsorbent, respectively, per adsorption cycle. This adsorbent, having operational temperature ranges between 80 and 100 degrees C, is indicated to be of potential utility in solvent recycle processes using these industrially important alcohols. Observed adsorption characteristics are discussed in terms of the alcohol properties of molecular size, heat capacity, and diffusivity. The adsorption mechanism is hypothesized to include transport of water molecules into the structure of adjacent starch molecules present in small spherical bodies (diameter of several microns) immobilized on the surface of the corn grit particles.     

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Efficient removal of both cationic and anionic dyes from aqueous solutions using a novel amphoteric straw-based adsorbent

In the current paper, a novel amphoteric straw-based adsorbent was prepared and applied to adsorb various dyes from aqueous solutions. The amphoteric adsorbent was proven effective in eliminating both cationic and anionic dyes (methylene blue and acid green 25), especially at corresponding favored pH conditions. The fundamental adsorption behavior of the adsorbent on removing various dyes was also investigated at different temperatures. The adsorption isotherms were all best-fitted by the Langmuir equation, whereas the adsorption kinetics was well-described by both the pseudo-second order model and the Elovich model. The experimental result revealed that the adsorption mechanism followed the monolayer chemical adsorption with an ion-exchange process.     

乙型肝炎疫苗中游离抗原对免疫效果的影响

将按《生物制品规程》制备除氢氧化铝吸附方式不同外,其余所有过程均一致的疫苗各１０批,在ＨＢｓＡｇ含量相同前提下,于５００公升大罐内吸附制备的疫苗中游离抗原含量比在１０公升瓶内吸附制备的疫苗高４倍,两者均数分别为３０４．１ｎｇ／ｍｌ和７３．１ｎｇ／ｍｌ,吸附率分别为９７．９５％和９９．５１％;用ＲＰＨＡ测定游离抗原滴度前者也比后者高２倍;动物免疫效价（ＥＤ５０）高４７％。在上述吸附率范围内,游离抗原含量与ＥＤ５０呈正相关（ｒ＝０．７１７９Ｐ＜０．００１）。     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.     

Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 10⁴ EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.     

Protein adsorption and hydrodynamic stability of a dense, pellicular adsorbent in high-biomass expanded bed chromatography

A dense, pellicular UpFront adsorbent (ϱ=1.5 g/cm³, UpFront Chromatography, Cophenhagen, Denmark) was characterized in terms of hydrodynamic properties and protein adsorption performance in expanded bed chromatography. Cibacron Blue 3GA was immobilised into the adsorbent and protein adsorption of bovine serum albumin (BSA) was selected to test the setup. The Bodenstein number and axial dispersion coefficient estimated for this dense pellicular adsorbent was 54 and 1.63×10⁻⁵ m²/s, respectively, indicating a stable expanded bed. It could be shown that the BSA protein was captured by the adsorbent in the presence of 30% (w/v) of whole-yeast cells with an estimated dynamic binding capacity (C/C ₀=0.01) of approximately 6.5 mg/mL adsorbent.     

In situ fabrication of a microfluidic device for immobilised metal affinity sensing

In this work a novel microfluidic device was constructed in situ containing the smallest microscopic co-polymeric immobilised metal affinity (IMA) adsorbent yet documented. This device has for the first time allowed the microlitre scale chromatographic assay of histidine-tagged proteins in a biological sample. To enable this approach, rather than using a high capacity commercial packed bed column which requires large sample volumes and would be susceptible to occlusion by cell debris, a microgram capacity co-polymeric chromatographic substrate suitable for analytical applications was fabricated within a microfluidic channel. This porous co-polymeric IMA micro-chromatographic element, only 27μl in volume, was assessed for the analytical capture of two different histidine-tagged recombinant fusion proteins. The micro-chromatographic adsorber was fabricated in situ by photo-polymerising an iminodiacetic acid (IDA) functionalised polymer matrix around a template of fused 100μm diameter NH(4)Cl particles entirely within the microfluidic channel and then etching away the salt with water to form a network of interconnected voids. The surface of the micro-chromatographic adsorber was chemically functionalised with a chelating agent and loaded with Cu(2+) ions. FTIR and NMR analysis verified the presence of the chelating agent on the adsorbent surface and its Cu(2+) ion binding capacity was determined to be 2.4μmol Cu(2+) (ml of adsorbent)(-1). Micro-scale equilibrium adsorption studies using the two different histidine-tagged proteins, LacI-His(6)-GFP and α-Synuclein-His(8)-YFP, were carried out and the protein binding capacity of the adsorbent was determined to be 0.370 and 0.802mg(g of adsorbent)(-1), respectively. The dynamic binding capacity was determined at four different flow rates and found to be comparable to the equilibrium binding capacity at low flow rates. The sensing platform was also used to adsorb LacI-His(6)-GFP protein from crude cell lysate. During adsorption, laser scanning confocal microscopy identified locations within the adsorbent where protein adsorption and desorption occurred. The findings indicate that minimal channelling, selective product capture and near quantitative elution of the captured (adsorbed) product could be achieved, supporting the application of this new device as a high-throughput process analytical tool (PAT) for the in-process monitoring of histidine-tagged proteins in manufacturing.     

Studies on the Performance of Ethylamine-Modified Chitosan Carbonized Rice Husk Composite Beads for Adsorption of Metal Ion

Heavy metals in the soil and ground water have endangered our environment and human bodies by direct or indirect pathways. Currently, bioremediation is a developing process that offers the possibility to destroy various contaminants using natural biological activity. Biopolymers are industrially attractive because of their capability of lowering transition metal ion concentrations to parts per billion, they are widely available, and they are environmentally safe. This paper deals with the preparation of an ethylamine-modified biopolymer (chitosan) and carbon from biowaste (rice husk) composite beads (EAM-CCRCB) for metal ion removal. The prepared adsorbent was used for the adsorption of hexavalent chromium ions from aqueous solutions. The activation and surface properties of the adsorbent were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and Brunauer-Emmett-Teller (BET) analyses. The effect of process variables such as initial metal ion concentration, adsorbent dosage, and pH of the solution on the performance of percentage removal and adsorption capacity were studied. Various isotherm and kinetic models were fitted with experimental data to describe the solute interaction and nature of adsorption with the adsorbent through batch studies. Mass thermodynamic parameters were determined. Regeneration studies were attempted to check the stability and activity of the adsorbent.