Endotoxin removal from protein solutions

Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration. A general method for the removal of endotoxins from protein solutions is not available. Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements. Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success. Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose. After giving an overview of the properties of endotoxins and the significance of endotoxin contamination, this review intends to provide an overall picture of the various methods employed for their removal. Avenues are pointed out how to optimise a method with regard to the specific properties of endotoxins in aqueous solution.     

Current trends in endotoxin detection and analysis of endotoxin–protein interactions

Endotoxin is a type of pyrogen that can be found in Gram-negative bacteria. Endotoxin can form a stable interaction with other biomolecules thus making its removal difficult especially during the production of biopharmaceutical drugs. The prevention of endotoxins from contaminating biopharmaceutical products is paramount as endotoxin contamination, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even lead to death. Highly sensitive and accurate detection of endotoxins are keys in the development of biopharmaceutical products derived from Gram-negative bacteria. It will facilitate the study of the intermolecular interaction of an endotoxin with other biomolecules, hence the selection of appropriate endotoxin removal strategies. Currently, most researchers rely on the conventional LAL-based endotoxin detection method. However, new methods have been and are being developed to overcome the problems associated with the LAL-based method. This review paper highlights the current research trends in endotoxin detection from conventional methods to newly developed biosensors. Additionally, it also provides an overview of the use of electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and docking programs in the endotoxin–protein analysis.     

Endotoxin removal by anion-exchange polymeric matrix.

The effects of anion-exchange polymeric matrices on endotoxin removal from albumin and gamma-globulin solutions are evaluated. The positively charged cellulose acrylic media carrying DEAE or QAE functional groups remove significant amounts of endotoxin from tap water, but are less effective in protein solutions. With properly controlled pH levels and salt concentrations, the endotoxin level in a protein solution can be reduced; however, low endotoxin concentrations, less than 100 pg/ml, are more difficult to remove. The endotoxin removal capacity depends on the number of functional groups existing in the matrix, expressed as the number of milliequivalents (meq), and on the pH operable range, which is directly related to the pK alpha value of the matrix. The effects of pH and salt on endotoxin removal from albumin and gamma-globulin solutions by an anion-exchange polymeric matrix were evaluated statically in test tubes. In addition, a dynamic flow was performed under statically defined conditions on a 250-ml DEAE cartridge for the removal of endotoxin from albumin at a flow rate of 40 ml/min. A greater than 75% reduction in the endotoxin can be achieved, with protein loss occurring only in the early stage of removal. Such processes are useful for the reduction of endotoxin from biological solutions produced by natural sources or recombinant DNA technology.     

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.     

Evaluation of the applicability of the bacterial endotoxin test to antibiotic products.

Fundamental conditions for applying the bacterial endotoxin test to antibiotic products were investigated so as not to affect the level of regulation by the rabbit pyrogen test. According to accuracy evaluation of test methods, the kinetic-turbidimetric and kinetic-colourimetric assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the gel-clot method. In total, 102 antibiotic products were evaluated on their interfering effect to show that the antibiotics could be categorized into three groups depending on intensity of the interference. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Accordingly, lists of antibiotics are presented to provide limits of concentration for eliminating interference and endotoxin limits for approval to facilitate effective bacterial endotoxin tests.     

A rapid highly-sensitive endotoxin detection system

This paper presents a rapid, highly-sensitive, and low-cost method of endotoxin quantification based on the use of stress-responsive magnetoelastic sensors, that monitor the gel formation (viscosity change) of the Limulus Amoebocyte Lysate (LAL) assay in response to endotoxin. Ribbon-like magnetoelastic sensors, 12.7 mm × 6 mm × 28 μm, were immersed in a LAL assay after mixing with test samples of variable endotoxin concentration, and the decrease in resonance amplitude of the sensor was recorded as a function of time. Experimental results show excellent correlation between endotoxin concentration and the maximum clot rate, determined by taking the minimum point of the first derivative of the amplitude–time curve, as well as the clotting-time, defined as the time that corresponds to the maximum clot rate. Using a LAL gel–clot assay with a sensitivity of 0.06 EU/ml (EU: endotoxin unit), the magnetoelastic sensor based technology can detect the presence of endotoxin at 0.0105 EU/ml in test requiring approximately 20 min. Unlike optical methods used for determining endotoxin concentration, the color of the test solution does not impact the magnetoelastic sensor measurement. Due to the small size of the sensor reader electronics and low cost, the magnetoelastic sensor based endotoxin detection system is ideally suited for wide-spread use in endotoxin screening for sepsis prevention.     

静脉注射用人免疫球蛋白细菌内毒素检测

探索静脉注射用人免疫球蛋白(IVIG)细菌内毒素含量的鲎试验检测方法。根据《中国药典》(2000版)细菌内毒素含量的检测方法进行,将待检品用NaOH调pH至中性,稀释至2.4倍可用标示灵敏度为0.25EU/m l的特异性鲎试剂进行细菌内毒素检测,结果与家兔法进行比较,并且在样品中加入定量内毒素0.6 EU/m l用两种方法进行对比试验。结果表明用细菌内毒素检测法(鲎试验法)检测静脉注射用人免疫球蛋白是可行的。     

Establishment of a new perchloric acid treatment method to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application.

We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.     

Detection of the endotoxin of Gram-negative bacteria in human blood

Recent new data on the important role played by lipopolysaccharides (endotoxin) of Gram negative bacteria in physiology and pathogenesis of the most important human infectious and noninfectious diseases testify to the necessity of wide clinical trials of different methods for LPS detection in blood and other physiological fluids. Among presently available diagnostic methods for endotoxinemia detection, the highly sensitive LAL (Limulus Amebocyte Lysate) test in various modifications is most widely used. The LAL test is known to be non-specific, however many drawbacks of this test have been successfully overcome. The results of clinical studies on the determination of the LPS activity in the systemic blood stream and antibody titers to its most common determinants, as well as the reserves of endotoxin binding with granulocytes give grounds for optimistic evaluation of the future studies on the role of LPS in human physiology and pathology. In clinical practice both positive sides and drawbacks of the presently known methods for LPS detection, including the LAL test, must be borne in mind for the complex evaluation of endotoxinemia levels.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.     

单克隆抗体纯化过程中内毒素去除方法分析

目的：分析单克隆抗体纯化过程中,去除内毒素的不同方法的应用效果,并探讨它们应用于中试规模的可行性。方法与结果：比较了多聚赖氨酸型的内毒素去除填料、20nm膜过滤、将单抗附着在蛋白A柱上后使用精氨酸和组氨酸溶液冲洗等3种方法的内毒素去除效果,发现3种方法都可以将内毒素水平大幅降低,可分别将内毒素去除70％、88％和97％。因为单抗分子等电点较高,所获得的最低内毒素含量为0．2～0-3EU／mg。结论：3种方法均具有一定的工艺放大潜力,进一步提高内毒素去除效果将需要综合使用不同机理的去除技术。     

Removal of tightly bound endotoxin from biological products

The method for endotoxin removal described in this paper is useful for separation of tightly bound endotoxin from biological products, particularly those produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product. We employed guanidine hydrochloride and ammonium sulphate in combination with hydrophobic interaction chromatography (HIC). These conditions enable binding of the endotoxin to the matrix, giving unbound product in the column flow-through. This makes the method generally applicable to biological products. An endotoxin reduction of about 3.7 logs was achieved; from as much as 1,100,000 EU mg(-1) in the solubilised material to about 200 EU mg(-1) in the product purified by this method. The method was developed for a cervical dysplasia vaccine, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus type 16, because both conventional and commercially available methods of endotoxin removal were ineffective in removing the tightly bound endotoxin from this product.     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

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.     

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.     

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.     

Clinical aspects of gastric cancer and Helicobacter pylori--screening, prevention, and treatment

Gastric cancer still represents a global health care burden, and in the absence of strategies implemented for early detection, the disease continues to have a dismal prognosis. Patients presenting with clinical manifestations of gastric cancer have limited options for cure. Thus, early detection and prevention play a key role in the fight against gastric cancer. Serologic-based test methods have the potential to detect a subset of patients at high risk of gastric cancer that require a close clinical and endoscopic follow-up. More data have been produced to support Helicobacter pylori eradication as an efficient strategy to prevent gastric cancer. Treatment options for patients with an advanced disease are still limited, but the introduction of new agents opens a more optimistic perspective for the future.     

Current technologies to endotoxin detection and removal for biopharmaceutical purification

Endotoxins are the major contributors to the pyrogenic response caused by contaminated pharmaceutical products, formulation ingredients, and medical devices. Recombinant biopharmaceutical products are manufactured using living organisms, including Gram-negative bacteria. Upon the death of a Gram-negative bacterium, endotoxins (also known as lipopolysaccharides) in the outer cell membrane are released into the lysate where they can interact with and form bonds with biomolecules, including target therapeutic compounds. Endotoxin contamination of biologic products may also occur through water, raw materials such as excipients, media, additives, sera, equipment, containers closure systems, and expression systems used in manufacturing. The manufacturing process is, therefore, in critical need of methods to reduce and remove endotoxins by monitoring raw materials and in-process intermediates at critical steps, in addition to final drug product release testing. This review paper highlights a discussion on three major topics about endotoxin detection techniques, upstream processes for the production of therapeutic molecules, and downstream processes to eliminate endotoxins during product purification. Finally, we have evaluated the effectiveness of endotoxin removal processes from a perspective of high purity and low cost.     

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 assay by bioluminescence using mutant firefly luciferase

The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005 EU/ml of endotoxin within 15 min.