Endotoxins can already have a damaging influence at research level, leading to altered cellular responses and misinterpretation of results. Rousselot® has developed a range of highly purified pharmaceutical gelatins with low endotoxin levels, called X-Pure®. The use of low-endotoxin gelatin can speed up bench-to-clinic transfer by reducing variability in your data, increasing reproducibility and enabling you to avoid regulatory hurdles in further development. It can make your product safer and hasten the route to the market. Here is the inside story.
A closer look at endotoxins
Endotoxins, or lipopolysaccharides (LPS), are characteristic components of the outer membrane of Gram-negative bacteria, where they provide protection and structural integrity. They are large molecules, consisting of three main units (Figure 1): a lipid head (lipid A); an oligosaccharide (core); and a sequence of repetitive subunits (O-specific antigen). Disruption of this membrane, which can be caused by cell growth, division or death, causes large amounts of LPS to be released in the environment[1]. Consequently, endotoxins are EVERYWHERE, making them contaminants that are hard to avoid.
Figure1 - Impact of endotoxins or lipopolysaccharides (LPS) on research and development phases and clinical applications
IS THE PRESENCE OF ENDOTOXINS A BAD THING FOR BIOMEDICAL RESEARCH OR CLINICAL APPLICATIONS? YES! ENDOTOXINS CAN HARM YOUR BODY, YOUR CELLS AND YOUR RESEARCH
Endotoxins are, as the name says, toxins. When endotoxin molecules are released in the human body, their lipid head is recognized by a variety of receptors. The ensuing interactions can cause moderate to severe immune reactions (2, 3), with symptoms like fever, chills, shaking, and respiratory difficulties (Figure 1). Because of this, endotoxins are a known problem in the world of biomaterials and medical devices. Their presence can potentially induce harmful effects and is therefore highly regulated by authorities around the world, especially the United States Food and Drug Administration, the FDA(4).
On a cellular level, endotoxins can impact cell viability, proliferation, functionality and differentiation(2). Since LPS are toxins, cell types that are part of the immune defence – such as epithelial, endothelial and structural component cells - will react to their presence by initiating and driving inflammation. LPS activates M1 macrophages, which play a crucial role in secreting inflammatory cytokines and generating reactive oxygen species(3).Other cell types that, at first sight, do not play a role in the immune system - like stem cells, adipocytes, astrocytes, oligodendrocytes, fibroblasts, intestinal cells, and pancreatic alpha and beta cells - can also be affected by endotoxins(2).
A consequence of these interactions is that endotoxins can muddle the results of your research in its earliest phase (Figure 1).
Why are endotoxins so hard to get rid of?
As mentioned before, endotoxins are part of bacteria and therefore difficult to avoid. Once present, they are extremely hard to remove due to the fact that endotoxins can form large supramolecular structures (up to 100 nm) that are UV- and heat-stable (2). Successful removal requires temperatures over 180 °C, which raises the risk of altering or damaging the biomaterial itself. This is why most commercially available biomaterials are non-endotoxin purified.
Most commercially available biomaterials are non-endotoxin purified
Benefit from minimized endotoxin levels with highly purified gelatins
With X-Pure, Rousselot has developed a range of highly purified pharmaceutical gelatins and collagens with low endotoxin levels. Thanks to our proprietary process (Patent WO2016085345), we maintain the product’s characteristics, while minimizing the presence of endotoxins (<10 EU/g).
Purified gelatins minimize data variability and improve reproducibility
A number of scientific studies have highlighted the fact that the presence of LPS can interfere with data reproducibility and lead to misinterpretation of results.
- A first example is shown in Figure 2. Stem cells were grown on GelMA, either made from type A X-Pure (blue) or commercial gelatin (grey), to study the impact of endotoxins on their differentiation by measuring glycosaminoglycan (GAG) production(5). Cells grown on GelMA made from X-Pure show consistent results, while results from cells grown on GelMA from commercial gelatin seem to be donor-dependent. The presence of LPS causes a large variation in response of the cells. This confirms that using non-purified gelatins can reduce the reliability of your results. Data variability and misinterpretation can lead to unpublishable results, a problem you can overcome by using ultra-purified pharmaceutical gelatins.
The effect of endotoxins on cell viability and performance can lead to data variability and misinterpretation of results.
Figure 2: The impact of endotoxins on stem cell differentiation. Mesenchymal stem cells were cultured on GelMA, either produced from type A X-Pure gelatin or from commercial gelatin. Chondrogenic differentiation was evaluated by measuring GAG production on day 28 (5).
- Another experiment showed the influence of endotoxins on the production of inflammatory markers (TNF-α and CCL-2). Equine peripheral blood mononuclear cells (PBMC) exposed to commercial, non-purified gelatin show an increased production of inflammatory markers compared to cells cultured on X-Pure gelatin (Figure 3). This demonstrates that endotoxins can lead to undesirable immune responses. LPS presence in biomaterials is a major concern in the context of (immune) cell research and in-body applications. Analogously to Figure 3, cells grown on commercial gelatin (Figure 3A and 3B) show a higher data variability than cells cultured on X-Pure gelatin.
Figure 3: Influence of LPS on TNF-α (A) and CCL-2 (B) production in equine peripheral blood mononuclear cells (PBMC) cultured on gelatin hydrogels for 24 h (5).
- More recently, a study led jointly by the University of Twente and Rousselot has shown for the first time that the accuracy and predictive value of 3D in vitro cancer models is significantly impacted by the presence of endotoxins.
The findings suggest that biomaterials with low endotoxin content could lead to a more accurate representation of the safety and potency of novel therapeutics, helping to improve the validity of 3D in vitro models and reduce the need for animal testing.
Rousselot X-PURE gelatin – from research to patient
Imagine having conducted your preliminary testing with endotoxin-contaminated biomaterials and having to change that material to enter non-clinical safety evaluation or clinical trials. This is where you need a pharmaceutical gelatin that allows your development to meet the legal limits for clinical use (Table 1).
Unfortunately, the now purified biomaterial cannot be considered equivalent to the impurified one used before. This will necessitate extra revalidation work and could raise questions about the results and conclusions from preliminary testing.
Data obtained with high, variable LPS-containing biomaterials is at risk risk of falling short of endpoints of non-clinical safety evaluations, jeopardizing timings to clinical trials.
Table 1: FDA regulations concerning permitted LPS-levels for particular applications (4)
X-Pure gelatin research grade is functionally equivalent to its GMP grade, minimizing the need to revalidate R&D results ahead of non-clinical or clinical trials. It will help you accelerate the process of getting your biomedical innovation to the people who need it.
The presence of LPS should be minimized at all cost when working in research, non-clinical and clinical environments. Using low-endotoxin X-Pure gelatin during R&D can enable a faster and more reliable track to the market.
Stay tuned for the next episode of Endotoxins, an inside story: LPS, the wrench in the works of 3D drug screening models.
(1) Gorbert, M.B. and M.V. Sefton, Endotoxin: The uninvited guest. Biomaterials, 2005. 26(34): p. 6811-6817
(2) Heinrich, M.A., M. Mangia, and J. Prakash, Impact of endotoxins on bioengineered tissues and models. Trends Biotechnol, 2021.
(3) Zhao, J., et al., Releasing Behavior of Lipopolysaccharide from Gelatin Modulates Inflammation, Cellular Senescence, and Bone Formation in Critical-Sized Bone Defects in Rat Calvaria. Materials (Basel), 2019. 13(1).
(4) FDA. Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers 2012; Available from: https://bit.ly/4748QTZ
(5) Groen, W., et al., Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro. Int J Mol Sci, 2020. 21(22).