Endotoxin removal from human-like collagen by allantoin

ABSTRACT

Human-like collagen(HLC) has an important role in biomedical field, while endotoxin contamination limits it use. In this study, the saturated allantoin as an adsorbent was applied in adsorbing endotoxins from human-like collagen in Tris-acetate-EDTA(TAE) buffer.Finally, the endotoxins carried by allantoin and soluble allantoin could be removed by centrifugation and ultrafiltration. Treated by 400mg/ml of allantoin, at pH6.5 and 25mM NaCl the endotoxins residual in HLC was decreased to 2.38+0.11EU/mg and protein recovery achieved 98.7% in TAE buffer. Endotoxins could be adsorbed on the surface of allantoin crystal by the hydrogen-bond. At pH8.0, the main force was hydrophobic and bivalent bridge between HLC and endotoxins. When the pH was 6.5, the negative-electron repulsion became the main force. And then, Transmission Electron Microscopy(TEM) was used to observe the ultrastruction of HLC,illustrating the protein ultrastruction had not been affected in TAE buffer. In addition, the method of endotoxins removed by allantoin adsorptoin in TAE buffer was safety and greatly reduced the costs,time consumption in contrast with affinity chromatography. As a whole, this paper provided a good idea to establish a large-scale industrial process to remove endotoxin in biomedical field for its simple operation and low cost.

KEYWORDS:

• Allantoin
• Endotoxin removal
• hydrophobic interaction
• Tris-Acetate-EDTA buffer

Additional information

Funding

This study was financially supported by the National Natural Science Foundation of China (21676214,21576222);Shaanxi Key Laboratory of Degradable Biomedical Materials Program(2014SZS07-K04,2014SZS07-P05,16JS106,2014SZS07-Z01, 2014SZS07-Z02, 2015SZSj-42,2016SZSj-35, 2014SZS07-K03).