Creative BioMart Chromatography has a professional team to provide endotoxin removal services in process of protein purification. We have a wealth of experience and advanced instruments. We can design a customized protocol according to the characteristics of the target protein, and perform a variety of detection assay with high accuracy and sensitivity to meet the individual needs of customers. We also provide resin and media for endotoxin removal.
What Is Endotoxin
Endotoxins, also known as lipopolysaccharides (LPS), are found mainly in the outer membrane of Gram-negative bacteria. The general structure of endotoxin is a polar heteropolysaccharide chain, which consists of three parts: The O-antigen region, the core oligosaccharide part, and the Lipid A part. (fig. 1)
Fig. 1 Structure of bacterial LPS (Source: http://en.wikipedia.org/wiki/Lipopolysaccharide, accessed on 31 Jul 2020).
Endotoxin has both positive and negative effects. Endotoxins can stimulate particular immune systems but can also affect the function of proteins. As a major contaminant in commercially available proteins or bioactive substances, endotoxin often complicates the study of the biological effects of major components. The presence of a small amount of endotoxin in the recombinant protein may cause the host endotoxin shock, tissue damage and even deathc. Therefore, it is necessary to remove endotoxin.
Chromatography-Based Endotoxin Removal Strategies
Chromatography is the most reliable and widely used method for endotoxin removal. The chromatographic methods currently under study include affinity, dimensional exclusion, ion-exchange, hydrophobic interaction, reverse-phase, particle-based adsorption, and so on. Important factors to consider are the affinity of endotoxin with proteins and the different types of solvents, the affinity of endotoxin and proteins with media, temperature, pH, and so on.
| Methods | Superiority Characteristics | Inferior Characteristics |
|---|---|---|
| Affinity chromatography | 1. High selectivity saves several purification steps and reduces production costs. 2. Super-coiled pDNA can be specifically identified and purified by arginine affinity chromatography. Since arginine is non-immunogenic, it can avoid interfering with endotoxin assay. |
1. Low yield. 2. Elution requires a high salt concentration. |
| Immobilised metal affinity chromatography (IMAC) | 1. It can be used to remove RNA, pDNA and endotoxin. 2. It can be used in situations involving special affinity interactions, such as pure plasmid DNA that binds specifically only to Fe3+ charged chelating compound. (Tan L., et al, 2007) |
1. Binding depends on the molecular weight and conformation of DNA. 2. Due to steric hindrance, pDNA does not bind in the presence of RNA. |
| Size-exclusion chromatography (SEC) and ultrafiltration | 1. Simple, inexpensive and reproducible 2. When proteins are not present, the ultrafiltration method enables the removal of large quantities of endotoxin aggregates by using alkanediol. |
1. Size-exclusion has limited pDNA capacity and selectivity. 2. A large size difference between the product and contaminant is required to effectively remove endotoxin. |
| Anion-exchange chromatography (AEC) | 1. High separation speed and wide selection of AEC media. 2. As an alternative medium, expanded bed AEC can easily purify highly viscous solutions. |
1. It applies only to the purification of positively charged proteins, as biological product loss may occur when using negatively charged proteins. 2. Poor selectivity to pDNA due to nonspecific binding. |
| Cation-exchange chromatography (CEC) | 1. Some researchers claimed that CEC is more efficient than AEC in terms of endotoxin removal. 2. Poly-L-lysine (PLL) works well for protein recovery and still usable after binding capacity exhaustion. |
1. Some agents used for CEC, such as Zirconia-immobilised PLH, are expensive and unstable under alkaline conditions. |
| Hydrophobic Interaction Chromatography (HIC) and reverse-phase chromatography (RPC) | 1. They effectively remove endotoxins by interacting with non-polar protein surfaces through van der Waals forces. 2. RPIPC is reproducible and effective for the commercial production of therapeutic pDNA. |
1. RPC is toxic and requires organic solvents for operation. |
| Paramagnetic Particles | 1. It was able to achieve a 90% reduction in endotoxin levels when paramagnetic particles were used as an additional step of LPS removal. (White D., et al, 2003) | 1. It needs to operate under low salt conditions. |
What We Offer
- Evaluation of endotoxin removal efficiency and protein loss rate.
- Customize protocol for target protein.
- Endotoxin removal with the customized protocol.
- Endotoxin detection, including Limulus amoebocyte lysate (LAL) gel-clot assay, LAL turbidimetric assay, LAL chromogenic assay.
- Deliver less than 0.01-0.1 EU/μg protein products.
Our Specialty
- Advanced platform, professional technology.
- Competitive prices within the industry.
- Faster response, higher efficiency.
- Keep in touch and respond to all your questions.
Creative BioMart Chromatography has a professional technical team and a cwealth of experience in endotoxin removal. For each project, experts will be arranged to evaluate the removal efficiency and the loss rate of the target protein, and the most suitable solution will be customized. Then, we perform the customized solution and LAL assay to ensure project quality. Our customers have direct access to our experts and give timely feedback to any online inquiries. If you are interested in our services, please contact us.
How to Place an Order
*If your organization requires the signing of a confidentiality agreement, please contact us by email.
References:
- Tan L., et al. (2007). Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions. Journal of Chromatography A. 1141 (2), 226-234.
- White D., et al. (2003). Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System. Journal of the Association for Laboratory Automation, 8 (4), 50–53.
For Research Use Only. Not intended for any clinical use. No products from Creative BioMart may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative BioMart.