The purity of your recombinant proteins is critical to the success of your research. A number of contaminants can affect both in vitro and in vivo systems. Some of the results are predictable, but others are not. All can affect your experimental data.
This article will focus on one of the most common contaminants: endotoxins or lipopolysaccharides. List Labs provides certificates of analysis showing the very low levels of endotoxins in our products.
What Are Endotoxins?
Lipopolysaccharides, also known as endotoxins, are a class of complex hydrophobic molecules found in the cell membranes of Gram-negative bacteria such as Escherichia coli. They are released in large quantities following cell death and during cell division, so they are a common component of recombinant protein production.
The general structure of an endotoxin is one or more Lipid A molecules bonded to one end of a short polysaccharide oligomer. The oligomer has polysaccharide side chains that carry O-antigen. Endotoxins are generally not inactivated by heat and must be removed during purification.
Endotoxins have a variety of deleterious effects on mammalian systems. These can vary widely even in similar systems. In fact, at least one case is known in which two insensitive T-cell lines were cloned from an endotoxin-sensitive parent line. One factor seems to be the presence of the CD14 receptor protein on the surface of the affected cells. Higher expression of CD14 seems to correlate with greater endotoxin sensitivity. In especially sensitive systems, even picomolar concentrations of endotoxin can lead to anomalous experimental results.
Effects in vitro
Documented effects in vitro include:
- Distortion of cell membranes by interactions with Lipid A, up to and including cell death.
- Lesser membrane distortions still affect gene expression and protein production, with specifics varying by different types of cells.
- Stimulation of leukocyte cultures to produce tissue factors.
- Inducing production of IL-6 in equine macrophages.
- Activating primary human immune cells, especially CD1c+ dendritic cells, to produce inflammatory cytokines. Sensitive cells can be activated by endotoxin concentrations as low as 0.02 ng/ml.
- Enhancing production of prostaglandins, acid phosphatase, fibrinolytic inhibitor, collagenase, nerve growth factor, and adhesion molecule 1, depending on the cell type.
- Inhibiting angiotensin-converting enzyme activity, synthesis of proteoglycan, and synthesis of alpha2 macroglobulin.
Effects in vivo
In live animals, endotoxins produce an inflammatory response in almost all tissues that are exposed to them. The pyrogenic nature of endotoxins produces effects ranging from fever to fatal septic shock.
All of the above effects, both in vivo and in vitro, may be synergistic with other contaminants or (in live animals) endogenous products. This, combined with widely varying cell sensitivity, make the experimental effects of endotoxin contamination difficult to predict.
Purification to Remove Endotoxins
The standard method of purifying recombinant proteins and removing endotoxins is affinity chromatography, using affinity tags on the target proteins, eluting the bound target, and then cleaving the tags in post-processing. Affinity chromatography is the method of choice at List Labs and gives our recombinant products exceptional purity.
Endotoxin contamination is a potentially serious problem in recombinant proteins, with highly variable and difficult to predict experimental effects. Even low levels of contamination may produce anomalous effects, which may vary across different cell lines and test subjects. Only reliable purification can prevent contamination. List Labs provides certified products with known purity and very low levels of endotoxin.
Sigma Aldrich, “Cell Culture FAQs: Bacterial Endotoxin Contamination;”
- Dawson, “The Significance of Endotoxin to Cell Culture and Biotechnology;” LAL Update, March 1998, Vol 16, No. 1, p. 1-4; Associates of Cape Cod Incorporated; https://www.acciusa.com/pdfs/newsletter/updt0398.pdf
- Schwarz, M. Schmittner, A. Duschl, and J. Horejs-Hoeck, “Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells;” PLoS One. 2014; 9(12): e113840; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257590/
By: Md. Elias, Ph.D, Senior Scientist
What are Endotoxins?
Endotoxins (aka lipopolysaccharide, LPS or lipoglycan) are part of the outer membrane of Gram-negative bacteria consisting of a lipid moiety and a polysaccharide moiety, the latter is composed of an inner core, outer core and O-antigen joined by covalent bonds1. In animals the lipid part of endotoxin (known as lipid A) often elicits strong immune responses mediated by Toll-like receptor 4 complex (TLR4/MD2/CD14) on the surface of immune cells2. Uncontrolled activation of such immune responses is often associated with production of inflammatory mediators. This may lead to capillary leak syndrome, which causes damage and dilation of the endothelial layer of blood vessels, a decrease in cardiac function and an increase in body temperature (fever); commonly referred to as fatal septic shock1,2.
Why is endotoxin contamination common in labs?
Since bacteria are widely present in nature, coexisting with plants and animals, endotoxins are ubiquitous. Endotoxins are naturally released from dead bacteria or as vesicles/blebs as part of the normal bacterial life cycle3. One of the critical properties of endotoxin is its high heat stability; it is found to be very difficult to deactivate/destroy using normal sterilizing conditions. In fact, steam sterilization, while eliminating live microbes, inadvertently increases the endotoxin level on glassware4. Biochemically, endotoxins are hydrophobic in nature and they have a tendency to stick to other hydrophobic materials such as common plastic lab wares. As a result of these properties, endotoxin contamination is common in laboratory procedures. US and European Pharmacopeia guidelines state that complete destruction of endotoxins requires 30 minutes of dry sterilization at 250℃5.
What are the FDA limits on endotoxin concentration?
Humans are found to be much more sensitive to endotoxins than the other animals. While a dose of 1 µg of endotoxins per Kg body weight induces septic shock in humans, mice can tolerate a thousand times higher dose. Since bacterial endotoxins are the most prevalent pyrogenic contaminants, the US Food and Drug Administration (FDA) has set limits on the concentration of endotoxin for human and veterinary parenteral drugs and medical devices. Endotoxin levels are measured as EU/ml where EU stands for endotoxin units. One EU equals approximately 0.1 to 0.2 ng endotoxin/ml of solution, depending on the reference standard used; this is the amount of endotoxin present in 105 to 1010 bacteria. FDA guidelines state that endotoxins unit, rather than weight should be used for testing comparisons because the potency of an endotoxin for causing pyrogenic effects depends on a variety of factors: polysaccharide chain length, aggregation, solubility in biological fluids, bacterial source, associated substances, etc. Current USP endotoxin limits in drugs for parenteral administration is 5 EU/kg of body weight per hour and for intrathecal it is 0.2 EU/kg. Endotoxin limits for medical devices is 0.5 EU/ml or 20 EU/device and for cerebrospinal fluid contacted devices it is 0.06 EU/ml or 2.15 EU/device6-9.
What is the rabbit pyrogen test?
The rabbit pyrogen test, which was introduced during 1940’s, was very successful in screening water and solutions used to validate parenteral drugs. However, this test is expensive, time consuming and not very quantitative. In the 1970’s an in-vitro assay method was developed based on the observation that horseshoe crab (Limulus polyphemus) amebocyte lysate would clot in the presence of a very low level of endotoxins. This is known as Limulus Amebocyte Lysate or LAL assay. The LAL assay was approved by the FDA during 1970’s to measure the LPS in parenteral drugs, devices and products that come in contact with the blood8. There are at least three forms of the LAL assay, each having different sensitivities: 1) LAL gel clot assay, 2) LAL kinetic turbidimetric assay, and 3) LAL chromogenic assay. The former one can detect endotoxins down to 0.03 EU/ml while the later two can detect endotoxin down to 0.01 EU/ml7,8.
What is Low Endotoxin/Lipopolysaccharide recovery (LER/LLR)?
Although LAL is a powerful assay to detect the presence of endotoxin at very low levels, concerns have grown in the recent past when measurable endotoxin concentration was found in decline over time (such as during storage) in products or in-process materials despite the fact that the samples may maintain pyrogenicity in the USP pyrogen test. This phenomenon is termed as low endotoxin/lipopolysaccharide recovery or LER/LLR. It was revealed that LER/LLR phenomenon can occur from masking of endotoxins by pharmaceutical excipients such as widely used polysorbate and citrate or by added/contaminated proteins8,10,11.
As LER has been observed in a range of different sample matrices, the specific mechanism of LER has not been explained; although a number of hypotheses have been proposed. Chen and Vinther suggested that a chelating agent and polysorbate may mask the endotoxins and form the LER/LLR complex that inhibits endotoxin binding to its receptor, Factor C, needed for LAL reaction12. However, results of other studies do not support any specific mechanism. In one study, at low concentration of surfactant (0.0001% v/v polysorbate 20), LAL activity is enhanced to approximately 180%. At increasing concentration of surfactant, the LAL activity went down and reduced to almost zero at about 0.0025% v/v polysorbate 20. Other studies suggest that there are interplays in between endotoxins and the formulation in terms of aggregation, solubilization and masking. Time and temperature are also reported to have effects on LER. The LER phenomenon was reported to occur more rapidly at room temperature than at 2-8℃, and a seven day incubation is sufficient to determine whether a drug exhibits LER or not. LER is also reported when organic compounds such as citrate, acetate and MES buffers including the benzamidine (protease inhibitor) or EDTA or dimethyl sulfoxide was present as excipient in the product12,13,17.
What are the current FDA recommendations and guidelines for Low Endotoxin/Lipopolysaccharide recovery?
As the LER/LLR phenomenon in pharmaceutical formulations became more evident from a large number of studies, the FDA became concerned about LER/LLR in drugs and medical devices, and came up with new USP guidelines albeit with old guidelines in place. The USP guidelines recommend that drug producers should perform hold time studies to detect LER/LLR for all new drugs. The hold time studies should be done by adding known quantity of endotoxins to undiluted product and then measure the concentration of detectable endotoxin over time under appropriate storage conditions13. A decline in endotoxin concentration is indicative of LER. In addition to the hold time studies, the USP is proposing a new Reference Standard (RS), Naturally Occurring Endotoxin (NOE) from a well characterized as Gram negative bacteria. Reasons to use NOE as an RS are described in details in a recent review written by Dr. Radhakrishna S. Tirumalai, who is a Principal Scientific Liaison in the Science Division, USP14. The reasons to use NOE in future for LPS quantification are very thoughtful: First, natural endotoxins are vesicles or ‘blebs’ of the outer membrane of gram negative bacteria. Second, cell wall fragments that are generated from naturally dead bacteria are real-life contaminants that might be present in pharmaceutical raw materials, water systems, in process samples and final drug products. Third, chemically extracted LPS which is often called ‘endotoxin’ does not exist in nature and it is biochemically dissimilar to the native endotoxin. Fourth, extracted LPS is stripped off from cell walls, will be absorbed to surfaces, and will form micelles and other aggregates in solution. Fifth, different product formulations and factors such as temperature, pH, salt, detergents, chelating agents also have effects on aggregation. Clearly, extracted LPS may be an inappropriate choice as a RS as it is chemically, biologically, and structurally different from natural gram negative bacterial cell wall fragments. The new USP guidelines also included a recommendation for bacterial strains, along with methodology for preparation, storage and documentation of ‘NOE’ that mimics the ‘real world’ endotoxin contamination14.
What are strategies to overcome low endotoxin/low lipopolysaccharide recovery?
A number of strategies for overcoming LER/LLR have been suggested. Sample dilution to 1/1000 showed significant improvement in the recovery of added endotoxin to overcome LER/LLR in endotoxin assay15. Addition of magnesium sulfate in two antibodies formulated with polysorbate 80, citrate or sodium phosphate was shown to mitigate LER/LLR in an endotoxin assay15,16. A freeze thaw regime was reported to mitigate LER/LLR in one study. Other studies have shown that protease treatment to unmask endotoxin worked to mitigate LER in endotoxin assays16,17. Given the different interactions with different products and excipients, it is conceivable that one specific strategy will not work for all and strategies need to be developed for each product16-18.
Since endotoxins are abundant, highly heat stable and difficult to remove, two general strategies are recommended for addressing and mitigating the LER/LLR phenomenon. The first strategy would be to minimize endotoxin contamination at all levels i.e. in the materials that go into a product, in all the process involved in its manufacture, prevention of bio-burden in manufacturing process and ensuring endotoxin removal at relevant process steps. Second, develop strategies to test for LER and develop methods to overcome LER/LLR.
List Labs is one of the leading manufacturers of high quality endotoxins. Our lipopolysaccharides (LPS) and their derivatives (Products#400, #401, #421, #423, #433, #434) are purified from various bacterial sources with proprietary technology. GMP grade endotoxins are available by custom order. These endotoxins are widely used in the field of immunobiology as immune stimulators/modulators in various cell culture work and as adjuvants. In cell culture studies, endotoxin free media and reagents are considered a routine practice to use because endotoxins have been shown to affect/interfere with cell growth and function, and are known to be the source of significant variability. Each of our toxin products is carefully tested by our QC department using FDA licensed LAL assay kit and FDA approved LAL assay methods to measure the level of endotoxins. Details of endotoxin content are mentioned in the certificate of analysis of the product.
- Rietschel, E.T.,Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Zähringer, U., Seydel, U., Di Padova, F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994, Feb;8(2):217-25. PMID: 8119492.
- Ohto, U.,Fukase, K., Miyake, K., Shimizu, T. Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2. Proc Natl. Acad. Sci. U S A. 2012, May 8;109(19):7421-6. PMID: 22532668.
- Kulp, A., Kuehn, M.J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 2010, 64:163-84. PMID: 20825345.
- Hecker, W.,Witthauer, D., Staerk, A. Validation of dry heat inactivation of bacterial endotoxins. PDA J Pharm. Sci. Technol. 1994, Jul-Aug;48(4):197-204. PMID: 7804819.
- Nakata, T. Destruction of typical endotoxins by dry heat as determined using LAL assay and pyrogen assay. J Parenter. Sci. Technol. 1993, Sep-Oct;47(5):258-64. PMID: 8263663.
- Gorbet, M.B.,Sefton, M.V. Endotoxin: the uninvited guest. 2005, Dec;26(34):6811-7. PMID: 16019062.
- Iwanaga, S. Biochemicalprinciple of Limulus test for detecting bacterial endotoxins. Proc Jpn. Acad. Ser B Phys Biol. Sci. 2007, May;83(4):110-9. PMID: 24019589.
- Chen J, Anders VI. Low Endotoxin Recovery (LER) in Common Biologics Products. Parenteral Drug Association Annual Meeting, Orlando, FL, April 2013.
- US Food and Drug Administration. Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers. June 2012.
- Bolden, J.S.,Warburton, R.E., Phelan, R., Murphy, M., Smith, K.R., De Felippis, M.R., Chen, D. Endotoxin Recovery Using Limulus Amebocyte Lysate (LAL) Assay. 2016, Sep;44(5):434-40. PMID: 27470947.
- Karen Z.M. Current USP Perspectives on Low Endotoxin Recovery (LER). Endotoxin detection part IV. A supplement to American Pharmaceutical Review. 2016. Recovery-LER/.
- Chen, J., and Vinther, A. Low Endotoxin Recovery (“LER”) in Common Biologics Products. Orlando: Parenteral Drug Association Annual Meeting; 2013.
- Karen, Z., Radhakrishna, T., David, H., James, A., Dennis, G., Robert, M., and Donald, S. Endotoxins Standards and Their Role in Recovery Studies: The Path Forward. BioPharma Asia. November/December 2016.
- Radhakrishna, S. T. Naturally Occurring Endotoxin: A new reference material proposed by the US Pharmacopeia. American Pharmaceutical Review. Endotoxin supplement 2016.
- Burgenson, A.L. Endotoxins from different sources: Variability in reactivity and recoverability. Presented at the Pharmaceutical Microbiology Forum. Bacterial Endotoxins Summit Meeting, Philadelphia, PA. 2014.
- Platco, C. Lab Experiences: Low Endotoxin Recovery. Presented at the Pharmaceutical Microbiology Forum. Bacterial Endotoxins Summit Meeting, Philadelphia, PA. 2014.
- Williams, K.L. Endotoxin Aggregation & Binding Properties. Recovering Endotoxin Spikes from Products & Container Clousers. Presentation at the Parenteral Drug Association Conference, Berlin, Germany. 2014.
- Tim, S. Removal of Endotoxin from Protein in Pharmaceutical Processes. Endotoxin detection part IV. A Supplement to American Pharmaceutical Review. 2016.