Purification Techniques | Ion Exchange Chromatography to Remove Endotoxins

During R&D and production, when endotoxin is mentioned, is the first thought that comes to mind "a difficult opponent"? So, how to give the "opponent" a powerful and effective fatal blow? Don't worry, let's continue chatting.

Endotoxins are complex lipopolysaccharides (LPS) that constitute an intrinsic part of the outer cell wall of all Gram-negative bacteria and are responsible for the organization and stability of the cell wall. They consist of three distinct regions: bacterial cell-specific polysaccharides, non-specific core polysaccharides and lipid A. Among them, lipid A is its main toxic component.

After bacterial cells die and dissolve or the bacterial cell structure is destroyed by artificial means, endotoxins will be released and cause toxic effects on the host. Toxic reactions caused by endotoxins mainly include febrile reactions, multiple organ failure, septic shock, sepsis, meningococcemia, etc.

The molecular weight of endotoxin monomer is about 10KD. In aqueous solutions with different components, macromolecular aggregates can be formed, with the maximum size exceeding 1000 KD. Endotoxins are extremely resistant to heat. Only high temperatures above 160°C for 2 to 4 hours, or heating and boiling with strong acids, alkalis, or strong oxidants for more than 30 minutes can destroy their biological activity.

Due to the harmfulness of endotoxins, the Chinese Pharmacopoeia and the United States Pharmacopoeia have clearly stipulated the inspection and requirements for endotoxins: During the R&D and production processes, in addition to ensuring aseptic operation and all equipment, consumables, and reagents that come into contact with the feed liquid While there is no risk of introducing exogenous endotoxin contamination, necessary and effective technological means or methods should be adopted to reduce or remove endogenous endotoxin contamination, meet process requirements, and improve product safety.

There are many techniques for removing endotoxins, and chromatography is currently one of the more prominent methods in terms of cost and effectiveness. Using the different properties of endotoxin molecules, chromatography methods with different principles can be used. Among them, this article focuses on ion exchange chromatography among the chromatography methods.

Endotoxin contains lipid A, which contains multiple phosphate structures and has a low isoelectric point, usually 3.1. When the solution pH is above its isoelectric point, endotoxins have a partial negative charge and can bind to positively charged anion exchange chromatography media. Therefore, strong anion exchange chromatography media can be used to bind endotoxins and remove endotoxins by allowing target proteins to flow through.

Among them, BioLink can provide different choices of strong anion exchange chromatography media, such as MaXtar Q, MaXtar Q HR, MaXtar Q HR XL, Q Chromstar BB, Q Chromstar FF, Q Chromstar HP, Q Chromstar XL, Q Chromstar BB XL, etc., different chromatography media can be selected according to different process requirements.

Parameter introduction of MaXtar Q HR

• Measurement conditions for dynamic capacity:

Column height: 10 cm, test flow rate 150 cm/h, test buffer: 0.05 M sodium acetate solution, pH 4.75, test sample: 6 mg/ml BSA sample, when the penetration of BSA reaches 10%, amount of BSA loaded (mg) per unit medium volume (ml).

• Working range: Within this pH value range, the function of the medium does not change significantly.

• v/v: volume ratio

Applications:

Endotoxin removal using MaXtar Q HR in plasmid purification

• Chromatography column: Chrom-Screen MaXtar Q HR 4.7ml; 10 cm

• Loading: affinity purified plasmid sample (retention time: 5 min)

• Balance solution: 0.4M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5

• Eluent: 1.0M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5

MaXtar Q HR chromatogram

Summary: After purification with MaXtar Q HR packing, the endotoxin content is <5EU/mg, which meets the quality requirements for endotoxin control.    

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References:

1. Kim, S.E., Su, W., Cho, M., Lee, Y., Choe, W.S., 2012. Anal Biochem. 424, 1, 12-20.

2. Su, W., Lin, M., Lee, H., Cho, M., Choe, W., Lee, Y., 2012. Biosensors and Bioelectronics. 32, 32-36.

3. Ding, S., Chang, B., Wu, C., Chen, C., Chang, H., 2007. Electrochem. Commun. 9, 5, 1206–1211.

4. Gutsmann, T., Howe, J., Zahringer, U., Garidel, P., Schromm, A.B., Koch, M.H., Fujimoto, Y., Fukase, K., Moriyon, I., Martinez-de-Tejada, G., Brandenburg, K., 2010. Innate Immun. 16, 39–47.

5. National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: three parts [M]. Beijing: China Medical Science and Technology Press, 2020: 42-48.