Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation.
Ivanovsky Institute of Virology, N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russian Federation.
PLoS One. 2023 Jun 8;18(6):e0286752. doi: 10.1371/journal.pone.0286752. eCollection 2023.
Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 μM while the standard one had 0.0180 μM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.
大肽生物合成是替代传统化学合成的有价值的方法。在我们基于热稳定伴侣的肽生物合成系统中合成了用于 HIV 感染治疗的最大治疗性肽恩夫韦肽，并对其肽质量以及与工艺相关杂质的概况进行了评估。通过 LC-MS 在中间产物中评估宿主细胞蛋白 (HCP) 和 BrCN 切割修饰肽。通过简单的内部算法对齐 LC-MS 图谱后，评估反应过程中的切割修饰，并估计甲酰化/氧化水平。获得的恩夫韦肽的圆二色谱与化学合成的标准品进行了比较。最终产物内毒素和 HCP 的含量分别评估为 1.06 EU/mg 和 5.58 ppm。使用 MT-4 细胞 HIV 感染抑制模型测量了肽的治疗活性。生物合成肽的 IC50 为 0.0453 μM，而标准品的 IC50 为 0.0180 μM。非酰化的 C 末端被认为是导致 IC50 和 CD 光谱差异的原因。否则，该肽在细胞培养和体内实验中均符合原始化学合成恩夫韦肽的所有要求。
