Endotoxin Contamination Research Articles

An Escherichia coli Strain for Plasmid DNA Production with a Low Endotoxin Level

Plasmid DNA (pDNA) is one of the possible types of vectors used in gene therapy. Escherichia coli is the most common bacterium for the production of pDNA. Lipopolysaccharides are the main component of the outer membrane of E. coli. Lipopolysaccharides are strong endotoxins, and their presence in pharmacological substances is highly undesirable. One of the approaches to eliminating endotoxin contamination of target substances is knocking out the genes of the lipopolysaccharide biosynthesis pathway. Using the CRISPR/Cas9 genome editing technology, we obtained a mutant form of the E. coli strain SCS110. We knocked out the genes kdsD, gutQ, lpxM, lpxL, lpxP, pagP, and eptA. As a result, lipopolysaccharide contamination of pDNA samples isolated by different methods from the mutant E. coli SCS 110 was significantly lower (19 to 295 times) than in pDNA samples from the original strain.

Plant-derived materials for biomedical applications.

With exceptional biocompatibility and biodegradability, plant-derived materials have garnered significant interest for a myriad of biomedical applications. This mini-review presents a concise overview of prevalent plant-derived materials, encompassing polysaccharide-based polymers, protein-based polymers, extracellular vesicles, mucilage, decellularized scaffolds, and whole plant-based biomass. Through different processing techniques, these plant-derived materials can be tailored into a variety of forms, such as nanoparticles, nanofibers, and hydrogels, to address the nuanced requirements of biomedical applications. With the emphasis on wound healing, tissue engineering, and drug delivery, this review underscores the unique advantages of plant-derived materials, such as lower risk of endotoxin and virus contamination, reduced ethical concerns, scalability, and eco-friendly attributes. However, challenges such as the need for the development of standardized isolation methods of these materials, and further transition from preclinical to clinical applications still remain to be solved.

Methyl-esterification, degree of polymerization and ∆4,5-unsaturation of galacturonic acid oligosaccharides as determinants of immunomodulation

In recent years, immunomodulation by pectin and pectin-derived galacturonic acid oligosaccharides has been the subject of wide-spread scientific research due to the potential of different pectin structures as bioactive biomolecules. Yet, gaps remain in understanding the structure-dependent immunomodulation of galacturonic acid. This study describes in vitro immunomodulatory effects of well-characterized galacturonic acid oligosaccharides. Both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a saturated non-reducing end (degree of polymerization 1–10) significantly induced cytokine production by THP-1 macrophages and directly activated TLR2 and TLR4 in transfected HEK-293 cells, even when accounting for minor endotoxin contamination. In contrast, both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a Δ4,5-unsaturated non-reducing end (degree of polymerization 1–7) did not activate TLR2 and TLR4 and led to significantly reduced cytokine production (p < 0.05), suggesting Δ4,5-(un)saturation as a pivotal factor for immunomodulation by galacturonic acid oligosaccharides. Exposure to non-methyl-esterified saturated galacturonic acid oligosaccharides resulted in significantly lower TNF-α production, IL-1β production and TLR4 activation (p < 0.05) compared to methyl-esterified saturated galacturonic acid oligosaccharides, while IL-10 production and TLR2 activation remained unchanged. These findings establish galacturonic acid oligosaccharides as versatile immunomodulators with TLR2 and TLR4 binding capacity, fit for different immunomodulatory applications depending on their structural characteristics.

Multifactor Quality and Safety Analysis of Semaglutide Products Sold by Online Sellers Without a Prescription: Market Surveillance, Content Analysis, and Product Purchase Evaluation Study.

Over the past 4 decades, obesity has escalated into a global epidemic, with its worldwide prevalence nearly tripling. Pharmacological treatments have evolved with the recent development of glucagon-like peptide 1 agonists, such as semaglutide. However, off-label use of drugs such as Ozempic for cosmetic weight loss has surged in popularity, raising concerns about potential misuse and the emergence of substandard and falsified products in the unregulated supply chain. This study aims to conduct a multifactor investigation of product quality and patient safety risks associated with the unregulated online sale of semaglutide by examining product availability and vendor characteristics and assessing product quality through test purchases. We used a complex risk and quality assessment methodology combining online market surveillance, search engine results page analysis, website content assessment, domain traffic analytics, conducting targeted product test purchases, visual quality inspection of product packaging, microbiological sterility and endotoxin contamination evaluation, and quantitative sample analysis using liquid chromatography coupled with mass spectrometry. We collected and evaluated 1080 links from search engine results pages and identified 317 (29.35%) links belonging to online pharmacies, of which 183 (57.7%) led to legal pharmacies and 134 (42.3%) directed users to 59 unique illegal online pharmacy websites.Web traffic data for the period between July and September 2023 revealed that the top 30 domains directly or indirectly affiliated with illegal online pharmacies accumulated over 4.7 million visits.Test purchases were completed from6 illegal online pharmacies with the highest number of links offering semaglutide products for sale without prescription at the lowest price range. Three injection vial purchases were delivered; none of the 3 Ozempic prefilled injection pens were received due to nondelivery e-commerce scams.All purchased vials were considered probable substandard and falsified products, as visual inspection indicated noncompliance in more than half (59%-63%) of the evaluated criteria. The semaglutide content of samples substantially exceeded labeled amounts by 28.56%-38.69%, although no peptide-like impurities were identified. The lyophilized peptide samples were devoid of viable microorganisms at the time of testing; however, endotoxin was detected in all samples with levels ranging between 2.1645 EU/mg and 8.9511 EU/mg. Furthermore, the measured semaglutide purity was significantly low, ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels by manufacturers. Glucagon-like peptide 1 agonist drugs promoted for weight loss, similar to erectile dysfunction medications more than 2 decades ago, are becoming the new blockbuster lifestyle medications for the illegal online pharmacy market. Protecting the pharmaceutical supply chain from substandard and falsified weight loss products and raising awareness regarding online medication safety must be a public health priority for regulators and technology platforms alike.

New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

Endotoxin Detection in Magnetic Resonance Imaging Contrast Agent Using Optimising Chromogenic Limulus Amebocyte Lysate Assay.

Endotoxin contamination in magnetic resonance imaging (MRI) contrast agents can pose a risk to patient safety causing immune reactions. Strict endotoxin limits are enforced for implants and catheters inserted into the body, but there are not clear rules for MRI contrast agents. Here, we investigated the efficacy of chromogenic LAL assay test for screening endotoxin activity in MRI contrast media manufactured in Malaysia. The powdered agent was dissolved in water for injection and endotoxin levels were measured. The coefficient of efficiency value for the standard curve, exhibiting r 2 ≥ 0.98, along with the absence of interfering substances and endotoxin activity below the regulatory threshold of 0.5 EU/mL, support the conclusion that the agent is unlikely to be pyrogenic or induce pyrogenic effect.

The Removal of Endotoxins in the Actual Production Process

Endotoxins, also called lipopolysaccharides (LPS), are mostly found in the outer membrane of Gram-negative bacteria, which are widely used as a common expression system to produce some biological products. The presence of small amounts of endotoxin can cause side effects in host organism such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins in the final products. In the actual production process, endotoxin contamination can occur at any process unit. In this article, we review the structure and characteristics of endotoxins and the effects that it causes in vivo. The endotoxin removal strategies are also elaborated according to the relevant mechanism. Besides, the endotoxin removal also faces enormous challenges in the actual production process.

Human milk oligosaccharides produced by synthetic biology

The benefits of human breast milk (HBM) to newborn growth, development, and overall health have been well investigated. As breastfeeding rate has declined significantly, the use of infant formula has risen and remains a significant component of the infant's diet. HBM contains several essential nutritional components, with human milk oligosaccharides (HMO) being the third most abundant following lactose and lipids. A diverse array of HMOs is known to exert great benefits in intestinal, immune, and cognitive functions. In contrast to HBM, infant formula containing mainly bovine milk lacks the diversity of HMOs. Efforts have been made to replicate this characteristic in infant formula through chemical and chemoenzymatic synthesis as well as microbial production. Utilizing microbial hosts appears to be more favourable due to its accessibility and cost-efficiency. Escherichia coli has been preferably used due to its high incorporation of DNA, high-level expression capability, and rapid growth. However, potential endotoxin contamination raises health concerns and prevents approval as Generally Recognized as Safe (GRAS) by the FDA. This prompts the use of other microbes such as Bacillus subtilis. Future research in this area is needed to optimize effective procedures using microbial hosts, high yield production, and economic feasibility. This may lead to infant formulas closely mimicking HBM and its health benefits.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine.

Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Lessons learned from contamination with endotoxin originated from the supplement in the cell culture medium

IntroductionEndotoxin is a typical pyrogen derived from the outer membrane of Gram-negative bacteria. In fabricating cell-based medicinal products, it is necessary to control endotoxin in the process and the products. In the quality control tests of our clinical study, endotoxin concentration in the culture supernatant of autologous oral mucosal epithelial cell sheets exceeded the criterion value. Therefore, endotoxin measurements were conducted to clarify the cause of the endotoxin contamination. MethodsThe reagents used to prepare the culture medium, the unused culture medium, and the culture supernatants were diluted with pure water. Endotoxin concentrations in the diluted samples were measured. ResultsEndotoxin was detected in both the unused culture medium and the culture supernatant of the epithelial cell sheets at higher concentrations than the criterion value. Therefore, endotoxin concentrations in the reagents used to prepare the culture medium were measured and were found to be below the criterion value, except for cholera toxin. On the other hand, three lots of cholera toxin products were used for the measurement, and the endotoxin concentrations were higher than the criterion value. The results indicate that the endotoxin contamination is caused by the cholera toxin product. ConclusionsTo prevent endotoxin contamination in cell-based medicinal products, endotoxin concentrations in reagents used for the fabrication should be measured in the facility conducting clinical research or confirmed by an adequate certificate of analysis from the manufacturers of the reagents.

Reducing Endotoxin Contamination in Biopharmaceuticals for Happier Biotechnologist?

Reducing Endotoxin Contamination in Biopharmaceuticals for Happier Biotechnologist?

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R’s (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

High-grain feeding contributes to endotoxin contamination in dairy milk

To support milk production and milk quality, ruminant animals like dairy cows are particularly fed using concentrate containing high grain and starch. Nonetheless, this type of regimen feeding could induce subacute rumen acidosis condition. Then, these circumstances cause the lysis of gram-negative bacteria accompanied by endotoxin release in gut. More importantly, gut endotoxin could be translocated to mammary gland, whereby this condition negatively affects to milk safety. The aim of the review is to update and summarize the current knowledge regarding high-grain diet and the occurrence of endotoxin in milk of dairy cows. The data suggest that there is interplay between high-grain feeding for dairy cows to endotoxin contamination in milk.

Current Considerations and Practical Solutions for Overcoming Nanoparticle Interference with LAL Assays and Minimizing Endotoxin Contamination.

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

Interlaboratory comparison of endotoxin contamination assessment of nanomaterials.

Endotoxin contamination is a significant hurdle to the translation of nanomaterials for biomedical applications. Multiple reports now describe that more than one-third of nanomaterials fail early pre-clinical assessment due to levels of endotoxin above regulatory requirements. Additionally, most immunological studies or in vivo studies testing nanomaterials in the literature lack inclusion of this assessment, which may lead to false-positive or false-negative results if high levels of the contaminant are present. The currently approved methods for endotoxin contamination assessment rely on enzymatic activity and wavelength absorbance as their endpoint, and many nanomaterials can interfere with such assays. For this reason, we devised an interlaboratory comparison of endotoxin contamination assessment for a range of nanomaterials to challenge the current international organization for standardization and pharmacopeia standards. Herein, we show that detected endotoxin levels could vary considerably between groups, and, in some instances, nanomaterials could both pass and fail regulatory endotoxin limits for medical devices depending on the group undertaking the assessment, all while passing all quality criteria standards. This work emphasises the requirement for multiple assays to fully assess the endotoxin levels in a nanomaterial and highlights the need for additional assays to be developed in this space.

Production and characterization of homologous protoporphyrinogen IX oxidase (PPO) proteins: Evidence that small N-terminal amino acid changes do not impact protein function.

Transgenic soybean, cotton, and maize tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides have been developed by introduction of a bacterial-derived PPO targeted into the chloroplast. PPO is a membrane-associated protein with an intrinsic tendency for aggregation, making expression, purification, and formulation at high concentrations difficult. In this study, transgenic PPO expressed in three crops was demonstrated to exhibit up to a 13 amino acid sequence difference in the N-terminus due to differential processing of the chloroplast transit peptide (CTP). Five PPO protein variants were produced in and purified from E. coli, each displaying equivalent immunoreactivity and functional activity, with values ranging from 193 to 266 nmol min-1 mg-1. Inclusion of an N-terminal 6xHis-tag or differential processing of the CTP peptide does not impact PPO functional activity. Additionally, structural modeling by Alphafold, ESMfold, and Openfold indicates that these short N-terminal extensions are disordered and predicted to not interfere with the mature PPO structure. These results support the view that safety studies on PPO from various crops can be performed from a single representative variant. Herein, we report a novel and robust method for large-scale production of PPO, enabling rapid production of more than 200 g of highly active PPO protein at 99% purity and low endotoxin contamination. We also present a formulation that allows for concentration of active PPO to > 75 mg/mL in a buffer suitable for mammalian toxicity studies.

Upregulation of YciM expression reduces endotoxin contamination of recombinant proteins produced in &lt;i&gt;Escherichia coli&lt;/i&gt; cells

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells.

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

The effect of edible bird's nests on the expression of MHC-II and costimulatory molecules of C57BL/6 mouse splenocytes

The glutinous nest that builds by the saliva secretion of swiftlet is recognizable as an edible bird's nest (EBN). It enriched a medicinal value and was regarded as supplementary food that exerts various beneficial health effects, especially immune boosters. This study's objective was to determine the impact of EBN on the expression of MHC-II and costimulatory molecules (CD86 and CD80) related to the initiation of T-cell activation. Both rEBN and pEBN samples were prepared with simulated gastrointestinal digestion for enhancing the bioaccessibility of bioactive compounds. Our result showed that digested EBN samples slightly influence the upregulation of MHC-II, CD86, and CD80 in gene expression of LPS-stimulated Raw 264.7 cells. The concern of endotoxin contamination in EBN samples, which may cause a false-positive result, was measured by quantitative PCR. We found that the inflammatory genes (IL-1β and TNF-α) were not induced by EBN treatments. Moreover, cell surface protein expression in splenocytes treated with EBN was assessed using flow cytometric analysis. Digested EBN samples demonstrated their capacity to promote the elevation of MHC-II, CD86, and CD80 cell surface protein expression. Finally, the digested-EBN-treated splenocytes only exhibited a specific response in the T-cells population. Thus, EBN is a source of the bioactive compound that has been proposed to exert a role in the stimulation of both MHC-II and costimulatory molecules for TCR/pMHC-II interaction leading to T-cell activation.