Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

Therapeutic Rationale for Endotoxin Removal with Polymyxin B Immobilized Fiber Column (PMX) for Septic Shock.

Endotoxin removal therapy with polymyxin B immobilized fiber column (PMX) has been clinically applied for sepsis and septic shock patients since 1994. The effectiveness and usefulness of this therapy have been demonstrated for more than a quarter of a century. However, a documented survival benefit has not yet been demonstrable in a large, multicenter, randomized and controlled trial. Following the findings derived from a large sepsis clinical trial with PMX in North America, a new trial is ongoing to determine if PMX has a long-term survival benefit when administered to septic patients. Another approach to support a survival benefit from intervention with PMX is to utilize a detailed analysis available from a large clinical data base. The endotoxin adsorption capacity of PMX columns in vitro and the effectiveness of PMX columns can be further demonstrable in animal models. The capability of PMX and details of its mechanism of action to intervene in the sepsis cascade and impede organ dysfunction in septic patients is not fully understood. The surface antigen expression in monocytes and neutrophils are improved after PMX therapy. Immunomodulatory effects as a result of endotoxin removal and/or other mechanisms of action have been suggested. These effects and other potential immune effects may explain some of the improved effects upon organ dysfunction of sepsis and septic shock patients. Endotoxemia may be involved in the pathophysiology of other diseases than sepsis. A rapid diagnostic method to detect and target endotoxemia could allow us to practice precision medicine and expand the clinical indications of endotoxin removal therapy.

Rationale of Blood Purification in the Post-Resuscitation Syndrome following Out-of-Hospital Cardiac Arrest: A Narrative Review.

Even today, little is known about the pathophysiology of the post-resuscitation syndrome. Our narrative review is one of the first summarizing all the knowledge about this phenomenon. We have focused our review upon the potential role of blood purification in attenuating the consequences of the post-resuscitation syndrome. Blood purification can decrease the cytokine storm particularly when using a CytoSorb absorber. Acrylonitrile 69-based oXiris membranes can remove endotoxin and high-mobility group box 1 protein. Blood purification techniques can quickly induce hypothermia. Blood purification can be used with veno-arterial extracorporeal membrane oxygenation to remove excess water. Further trials are needed to provide more concrete data about the use of blood purification in the post-resuscitation syndrome.

Selective Polymyxin Hemoperfusion in Complex Therapy of Sepsis in Children after Cardiac Surgery.

BACKGROUND: To date, sepsis remains one of the main challenges of intensive care in pediatrics. Newborns with low birth weight and infants with chronic diseases and congenital disorders are particularly at risk. The incidence of infectious complications in pediatric cardiac surgery is known to be approximately 15-30%. The main etiological factor of sepsis is endotoxin. AIM: To evaluate the efficiency and safety of polymyxin (PMX) B-immobilized column-direct hemoperfusion in complex intensive therapy of sepsis in children after cardiac surgery with cardiopulmonary bypass. DESIGN: Prospective cohort study. METHODS: This study enrolled 15 children, aged 9-96 months, with congenital heart diseases and with body weights of 6.2-22.5 kg. The criteria for admission were body weight >6 kg and clinical and laboratory signs of sepsis (microbiological analysis, procalcitonin [PCT] >2 ng/mL, and endotoxin activity assay [ЕАА] >0.6). Intensive care included inotropic and vasopressor support, mechanical ventilation, broad-spectrum antibiotic therapy, and PMX hemoperfusion procedures. Extracorporeal therapy was initiated within 24 h following the sepsis diagnosis. Every patient underwent 2 hemoperfusion sessions with the use of a PMX B-immobilized column; the session duration was 180 min. RESULTS: We noted improvements in hemodynamic parameters, oxygenation index, and laboratory signs of sepsis, with decreases in the endotoxin concentration according to the EAA, PCT, and presepsin levels. The 28-day survival of the patients in this severely affected group was 80%. Main Conclusion: The inclusion of extracorporeal methods of blood purification, aimed at the selective elimination of circulating endotoxin, in the treatment of sepsis increases the survival rates of children after open heart surgery. Second Conclusion: The obtained results of sepsis therapy with PMX hemoperfusion in children after cardiac surgery enable us to suggest the sufficient safety and efficiency of the procedures in this category of severely affected patients.

Early use of endotoxin absorption by oXiris in abdominal septic shock: A case report.

RATIONALE: Septic shock leads to multiple organ failure and increases mortality rate. We reported a critical patient with abdominal septic shock, which was the first case successfully treated with continuous renal replacement therapy (CRRT) and a newly designed endotoxin removal device oXiris in mainland China. PATIENT CONCERNS: A 51-year-old man developed gastric ulcer perforation after resection of a benign peritoneal tumor and had a second abdominal surgery. His blood pressure decreased to 70/40 mm Hg with oliguria, requiring large doses of noradrenaline and intravenous fluid for resuscitation. The abdominal cavity was not sutured after the second open surgery due to severe abdominal infection and distention. His leukocyte count was over 30109/L, while the blood lactic acid was 12.5 mmol/L and procalcitonin (PCT) was >100 ng/mL. DIAGNOSIS: Since the bacterial culture of peritoneal exudate showed positive with Enterobacter aerogenes and Pseudomonas aeruginosa after the second surgery, and the patient had severe low blood pressure, hyoxemia and oliguria, combined with the laboratory tests results, he was diagnosed with Gram-negative related septic shock, acute kidney injury, and multiple organ dysfunction. INTERVENTIONS: CRRT with oXiris membrane was performed for 80hours and followed by AN69 ST membranes during the subsequent 27 days. Antibiotics together with other medical treatment were applied to the patient in the meantime. OUTCOMES: At the end of 80 hours treatment with oXiris, PCT of the patient had decreased to 14.52 ng/mL and lactic acid decreased to 4.2 mmol/L. The total sequential organ failure assessment (SOFA) score decreased from 15 to 11. Urine output steadily increased to 250 mL/h, and vital signs and blood pressure were stable without noradrenaline. At the end of the 27 days of conventional CRRT, his kidney function had completely recovered with a total sequential organ failure assessment score (SOFA score) of 6. LESSONS: oXiris, with its enhanced endotoxin adsorption, appeared to accelerate improvement in organ dysfunction and ultimate survival in our patient. In critical patients with abdominal septic shock, oXiris is an important adjunctive consideration to supplement definitive source control and antimicrobial therapy.

Anticoagulant chitosan-kappa-carrageenan composite hydrogel sorbent for simultaneous endotoxin and bacteria cleansing in septic blood.

Here, we describe a green approach to fabricate genipin crosslinked chitosan-kappa-carrageenan composite hydrogels (C-K hydrogels) aiming at reducing endotoxin level and bacteria burden in septic blood synchronously. The chemical compositions and morphologies of the developed C-K hydrogels were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The C-K hydrogels significantly inhibited adverse blood-material interactions such as hemolysis, complement activation, platelet activation and contact activation, and exhibited better anticoagulant properties than raw chitosan hydrogels. Most importantly, the optimized C2-K1 hydrogels were competent to eliminate 63.3 % of endotoxin in septic blood with a maximum adsorption capacity of 95.0 EU/g during a 3-h simulative hemoperfusion procedure. Bacteria cleansing experiments further demonstrated that the optimized C2-K1 hydrogels effectively decreased 46.0 % of E. coli and 68.7 % of S. aureus load, respectively. It is believed that the C-K hydrogels are promising hemoperfusion sorbents to treat severe septic patients.

Optimization of ultrasonic-assisted ultrafiltration process for removing bacterial endotoxin from diammonium glycyrrhizinate using response surface methodology.

Ultrasonic-assisted ultrafiltration (UAU) removing bacterial endotoxin from diammonium glycyrrhizinate, was firstly applied to surfactant separation. Separation efficiency was related with four variables, including ultrafiltration molecular weight cut off (MWCO), ultrasonic power, concentration and pH. The SCQ-9200E ultrasonic system was provided for the study with adjustable ultrasonic power 80 W to 800 W, and the ultrasonic frequency was 40 KHz. On the basis of response surface methodology (RSM), the optimal separation conditions were determined to be the ultrafiltration MWCO as 10 kDa, the ultrasonic power as 570 W, diammonium glycyrrhizinate concentration as 150.00 µg/mL and the pH as 4.70. The experimental rejection of bacterial endotoxin was 94.08%, meanwhile the transmittance of diammonium glycyrrhizinate was 93.65%. Based on the ultrasonic power, solution volume, and ultrasonic container size, the experiments with UAU at different power intensities showed that ultrasonic at a power intensity of 57 W/L and the power density of 0.32 W/cm2 could solve the separation contradiction between diammonium glycyrrhizinate and bacterial endotoxin. This study indicated that UAU could be an innovation in ultrasonic separation fields, and had a vast range of prospects for making use in pharmaceutical preparation area.

The preparation of endotoxin-free genetically engineered murine B1 antisense RNA.

One of the major limitations in the production of genetically engineered RNA from Escherichia coli (E. coli) is contamination by endotoxin. Here we report the first method that is capable of removing endotoxin from genetically engineered RNA. As a proof of concept, we transformed E. coli with a plasmid containing a tandem short interspersed nuclear elements from the mouse genome (SINE B1 elements). We then evaluated several extraction methods (SDS-NaCl centrifugation, SDS-NaCl filtration, TRIzol and SDS hot-phenol) and refinements thereof, and measured the resulting RNA yield, RNA purity, RNA integrity and endotoxin content. SDS-NaCl filtration with 2 mol/L NaCl, incorporating DEPC as an RNA protective agent, effectively removed endotoxin and resulted in a good RNA yield. Triton X-114 phase separation further reduced the endotoxin content of SDS-NaCl filtration-extracted RNA. RNA extracted by SDS-NaCl filtration with Triton X-114 phase separation did not cause adverse reactions in BALB/c mice and did not induce fever in rabbits when injected into these animals. The RNA met the requirements of nucleic acid reagents for in vivo experiments on animals.

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.

Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach.

Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides.IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Detection and Removal of Small Molecule and Endotoxin Contaminants in ADC Preparations.

Incomplete removal of free (unconjugated) drug or drug-linker species used to prepare ADCs results in contaminated ADC samples which may pose a risk for toxicity. Due to the extreme potency of typical small molecule toxins employed in ADCs, even relatively low levels of free drug contaminants in ADC samples have been hypothesized to result in nonspecific (i.e., off-target) activity in biological systems. It is possible for trace levels of certain free drug species to persist in final ADC samples despite the inclusion of common purification steps during the preparation processes. Therefore, methods for the detection, quantification, and removal of residual free drug present in ADC samples are ultimately required for the preparation of safe and efficacious final ADC drug products. Herein we report general methods for the detection and removal of such contaminants.

Adsorptive filtration: A case study for early impurity reduction in an Escherichia coli production process.

Primary recovery of intracellular products from Escherichia coli requires cell disruption which leads to a massive release of process-related impurities burdening subsequent downstream process (DSP) unit operations. Especially, DNA and endotoxins challenge purification operations due to their size and concentrations. Consequently, an early reduction in impurities will not only simplify the production process but also increase robustness while alleviating the workload afterward. In the present work, we studied the proof of concept whether a nonwoven anion exchange filter material decreases soluble impurities immediately at the clarification step of E. coli DSP. In a first attempt, endotoxin burden was reduced by 4.6-fold and the DNA concentration by 3.6-fold compared to conventional depth filtration. A design of experiment for the adsorptive filtration approach was carried out to analyze the influence of different critical process parameters (CPPs) on impurity reduction. We showed that depending on the CPPs chosen, a DNA lowering of more than 3 log values, an endotoxin decrease of approximately 7 logs, and a minor HCP clearance of at least 0.3 logs could be achieved. Thus, we further revealed a chromatography column protecting effect when using adsorptive filtration beforehand.

Cry1A Proteins are Cytotoxic to HeLa but not to SiHa Cervical Cancer Cells.

BACKGROUND: Bacillus thuringiensis toxins are effective against multiple biological targets such as insects, nematodes, mites, protozoa, and importantly, human cancer cells. One of the main mechanisms by which Cry toxins to trigger cell death is the specific recognition of cadherin-like membrane cell receptors. OBJECTIVE: This work aimed to assess the cytotoxicity of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis in HeLa, cervical cancer cell line, as well as their antitumor activity in mouse models. METHODS: We analyzed several biological targets of Cry1Ab and Cry1Ac including erythrocytes, insect larvae, as well as cancer and non-cancer cell lines. The viability of HeLa, SiHa, MCF7 and HaCat cells was assessed by MTT 24 h after the administration of Cry toxins. We also studied apoptosis as a possible cytotoxicity mechanism in HeLa. The capacity of Cry toxins to eliminate tumors in xenograft mouse models was also analyzed. RESULTS: Both toxins, Cry1Ab and Cry1Ac, showed specific cytotoxic activity in HeLa (HPV18+) cervical cancer cell line, with a Cry1Ab LC50 of 2.5 µg/ml, and of 0.5 µg/ml for Cry1Ac. Apoptosis was differentially induced in HeLa cells using the same concentration of Cry1Ab and Cry1Ac toxins. Cry1Ac eliminated 50% of the tumors at 10 µg/ml, and eliminate 100% of the tumors at 30 and 50 µg/ml. CONCLUSION: Bacillus thuringiensis Cry1A toxins show dual cytotoxic activity, in insects as well as in HeLa cancer cell line.

Anti-endotoxin Properties of Polymyxin B-immobilized Fibers.

Polymyxin B is an antibiotic that shows strong bactericidal activity against Gram-negative bacteria, by binding to and inactivating endotoxin. Systemic administration of polymyxin B in humans is restricted because of its nephrotoxicity and neurotoxicity, and this compound was therefore considered a strong candidate ligand for the extracorporeal selective adsorption of circulating endotoxin in the blood. Toraymyxin® is a direct hemoperfusion column that uses polymyxin B attached to an insoluble carrier to bind endotoxin in the blood. In 1994, the Japanese National Health Insurance system approved the use of Toraymyxin for the treatment of endotoxemia and septic shock.In this chapter, we will review the development, clinical use, and efficacy of Toraymyxin, examine the structure of the Toraymyxin column, and comment on the current position of Toraymyxin in the treatment of severe sepsis and septic shock. We will also highlight some potential new applications of Toraymyxin for pulmonary diseases.

Development of boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres with large pores for endotoxin removal.

Endotoxins are found almost everywhere and possess high toxicity in vivo and in vitro. Here we design a novel boronate affinity material, called boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres (Fe3O4@nSiO2@mSiO2-BA) with large pores (pore size > 20 nm) based on the chemical structure and physical properties of endotoxins, for facile and highly efficient removal of endotoxins. Dual modes for endotoxin removal were proposed and confirmed in this work: the endotoxin aggregates with size < 20 nm were bound with boronic acid ligands chemically modified on the inner and outer surface of the large pores of Fe3O4@nSiO2@mSiO2-BA microspheres; while the larger endotoxin micelles (size >20 nm) were absorbed on the outer surface of the prepared material based on boronate affinity. Transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption isotherms and Fourier transform infrared (FT-IR) spectroscopy confirm that Fe3O4@nSiO2@mSiO2-BA microspheres possess core/shell structure, uniform diameter (520 nm), high surface area (205.57 m2/g), large mesopores (21.8 nm) and boronic acid ligands. The purification procedures of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin were optimized, and 50 mM NH4HCO3 (pH 8.0) and 0.05 M fructose were selected as loading/washing, elution buffers, respectively. The binding capacity of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin was calculated to be 60.84 EU/g under the optimized conditions. Finally, the established analytical method was applied to remove endotoxins from plasmid DNA. After endotoxin removal, the endotoxin content in plasmid DNA was reduced from 0.0026 to 0.0006 EU/mL for two-fold concentration, and from 0.0088 to 0.0022 EU/mL for five-fold concentration after binding, respectively. Additional advantages of the prepared boronate affinity material include excellent stability, reusability/repeatability, and low cost. Boronate affinity materials with large pores could thus prove to be powerful adsorbents for endotoxin removal and the potential applications in the aspects of biological research, pharmaceutical industry, and life health.

Adverse immunostimulation caused by impurities: The dark side of biopharmaceuticals.

Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic inflammatory reaction. Biopharmaceuticals that unexpectedly induce an inflammatory response still enter the clinic, even while meeting all regulatory requirements. Impurities (of microbial origin) in biopharmaceuticals are an often-overlooked cause of AI. This demonstrates that the current guidelines for quality control and safety pharmacology testing are not flawless. Here, based on two case examples, several shortcomings of the guidelines are discussed. The most important of these are the lack of sensitivity for impurities, lack of testing for pyrogens other than endotoxin, and the use of insensitive animal species and biomarkers in preclinical investigations. Moreover, testing for the immunotoxicity of biopharmaceuticals is explicitly not recommended by the international guidelines. Publication of cases of AI is pivotal, both to increase awareness and to facilitate scientific discussions on how to prevent AI in the future.

Detection of endotoxin in plasma of hospitalized diarrheic calves.

OBJECTIVES: To investigate whether lipopolysaccharide (LPS) is present in plasma of calves with naturally occurring diarrhea. The second objective was to determine whether plasma [LPS] correlates with clinical, hematological, biochemical, and acid-base variables, and whether [LPS] differs between surviving and nonsurviving diarrheic calves. DESIGN: Prospective observational study (January 2012-May 2014). SETTING: Veterinary teaching hospital. ANIMALS: Thirty-four calves <28 days old admitted for diagnosis and treatment of diarrhea and 30 healthy control calves. MEASUREMENTS AND MAIN RESULTS: Admission demographics, physical examination, blood gas, biochemistry analysis, and outcome data were recorded. Plasma concentration of LPS was determined using a bovine LPS ELISA assay. Plasma [LPS] was detected in both healthy and diarrheic calves. Plasma [LPS] was significantly higher in diarrheic than healthy calves (median: 0.99 ng/mL; Interquartile range (IQR): 0.068, vs 0.88 ng/mL; 0.065 ng/mL, respectively; P < 0.001). Plasma [LPS] was higher in nonsurviving (1.04 ng/mL; 0.07 ng/mL) than in surviving calves (0.98 ng/mL; 0.022 ng/mL; P < 0.001). Plasma [LPS] was higher in beef (1.07 ng/mL; 0.182 ng/mL) than in dairy diarrheic calves (0.99 ng/mL; 0.022 ng/mL; P < 0.001). In diarrheic calves, plasma [LPS] correlated with [l-lactate] (r2 = 0.496; P = 0.002); hypoglycemia (r2 = -0.453; P = 0.007); increased unmeasured strong ions (r2 = 0.332; P = 0.050), [Mg2+ ] (r2 = 0.475; P = 0.004), and [phosphate] (r2 = 0.468; P = 0.005), and increased aspartate aminotransferase activity (r2 = 0.348; P = 0.003). CONCLUSIONS: This study highlights a potential role of LPS in the pathogenesis of metabolic derangements such as hyperlactatemia, hypoglycemia, and increased concentration of unmeasured strong anions in diarrheic calves. Further investigation evaluating the effect of LPS on l-lactate and glucose metabolism in diarrheic calves is warranted.

A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance.

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

Endotoxin reduction in protein solutions using octyl ß-D-1-thioglucopyranoside wash on chromatography media.

Endotoxins are complex molecules and one of the most challenging impurities requiring separation in biopharmaceutical protein purification processes. Usually these contaminants are cleared during the downstream process, but if endotoxin interacts with the target protein it becomes difficult to remove. In the present study we identified a detergent, octyl-ß-D-1-thioglucopyranoside (OTG), that disrupted endotoxin-protein interactions. The integration of this detergent into washes on several chromatography media was demonstrated to provide a separation tool for decreasing endotoxin from target proteins. This study also examined the mechanism of OTG endotoxin-protein disruption through phase modification incubation and chromatographic studies. The non-ionic OTG wash was shown to break both hydrophobic and electrostatic interactions between the endotoxin and protein. This mechanism contrasts with the breaking of hydrophobic interactions by washing with known endotoxin decreasing Triton X-100 detergent. The difference in mechanisms likely results in the ability of OTG to decrease endotoxin to levels less than those resulting from a detergent wash such as Triton X-100. Finally, we show the impact of the OTG endotoxin removal tool on the biopharmaceutical industry. While maintaining monomer purity and activity levels, endotoxin removal from a fusion protein allowed for decreased background levels in a T cell functional assay. The lowered baseline of T cell responses allowed for more effective detection of molecular interaction with the cells. The detergent wash can be used to both decrease the overall level of endotoxin in a purified protein solution and to enable more effective screening of lead candidate molecules.