Hebbi Vishwanath, Kathiresan P, Kumar Devendra, Komives Claire, Rathore Anurag S
Department of Chemical Engineering, Indian Institute of Technology, 110016, Hauz Khas, India.
Department of Biomedical, Chemical and Materials Engineering, San Jose State University, 951920082 San Jose, USA.
J Chem Technol Biotechnol. 2018 Apr;93(4):959-967. doi: 10.1002/jctb.5537. Epub 2017 Dec 5.
Purification of peptides offers unique challenges with respect to obtaining the desired process yield and selectivity. Lethal Toxin Neutralizing Factor (LTNF) is a peptide that is known to neutralize snake venom in mice when the peptide is preincubated with the venom prior to intravenous injection. A process for producing highly purified recombinant LTNF has been developed. The process has been modelled in SuperPro designer using laboratory data for a plant capable of producing 10 Kg of purified rLTNF. Economic analysis has been performed for manufacturing 3 ton of purified rLTNF.
The process developed produces peptide in the form of concatemer that has been specifically designed to accumulate as insoluble inclusion bodies (IB) during expression in . A cation exchange chromatography step has been developed to capture the rLTNF concatemer at 140 g/L dynamic binding capacity. Further, the purified concatemer is cleaved completely into monomeric rLTNF using alpha-chymotrypsin enzyme. Finally, a reversed phase high performance liquid chromatography has been designed to purify rLTNF with a recovery of more than 90% and purity greater than 98%. The overall process recovery is 78±2% resulting in 3.36 g of purified product per batch. Techno-economic evaluation of the process has been performed to demonstrate its economic feasibility against currently marketed antivenom products.
The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.
肽的纯化在获得所需的工艺产率和选择性方面面临独特挑战。致死毒素中和因子（LTNF）是一种肽，已知在静脉注射前将该肽与蛇毒预孵育时，它能在小鼠体内中和蛇毒。已开发出一种生产高度纯化重组LTNF的工艺。该工艺已在SuperPro Designer中使用一家能够生产10千克纯化rLTNF的工厂的实验室数据进行建模。已对生产3吨纯化rLTNF进行了经济分析。
所开发的工艺以串联体形式生产肽，该串联体经过专门设计，以便在大肠杆菌中表达期间作为不溶性包涵体（IB）积累。已开发出一个阳离子交换色谱步骤，以140克/升的动态结合容量捕获rLTNF串联体。此外，使用α-胰凝乳蛋白酶将纯化的串联体完全切割成单体rLTNF。最后，设计了一种反相高效液相色谱法来纯化rLTNF，回收率超过90%，纯度大于98%。整个工艺回收率为78±2%，每批可得到3.36克纯化产物。已对该工艺进行技术经济评估，以证明其相对于目前市场上销售的抗蛇毒血清产品的经济可行性。
所开发的工艺能够以78±2%的回收率生产纯化的rLTNF。该研究表明，重组技术可用于经济高效地生产rLTNF，并显示出作为目前可用的抗蛇咬抗蛇毒血清替代品的潜力。
