Alizadeh Bahareh, Zarghi Afshin, Mahboubi Arash, Aboofazeli Reza
Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2023 Jan 30;21(1):e134282. doi: 10.5812/ijpr-134282. eCollection 2022 Dec.
As a widely used therapeutic protein, recombinant human erythropoietin (rhEPO) is currently one of the most effective biopharmaceuticals on the market for the treatment of anemia in patients with chronic renal disease. Increasing in vivo rhEPO half-life and its bioactivity is a significant challenge. It was hypothesized that the application of self-assembly PEGylation retaining activity, named supramolecular (SPRA) technology, could prolong the protein half-life without a significant loss of bioactivity.
This study aimed to assess the stability of rhEPO during synthetic reactions, including the conjugation with adamantane and the formation of the SPRA complex. To do this, the secondary structure of the protein was also evaluated.
FTIR, ATR-FTIR, Far-UV-CD, and SDS-PAGE methods were employed. Thermal stability studies of SPRA-rhEPO complex and rhEPO were investigated at 37°C for ten days using a nanodrop spectrophotometer.
The secondary structure of lyophilized rhEPO, AD-rhEPO, and rhEPO (pH 8) was compared to rhEPO. Results showed that the secondary structure of the protein was unaffected by lyophilization, pH change, and the formation of covalent bonds in conjugation reaction. SPRA-rhEPO complex was also stable for seven days in phosphate buffer (pH 7.4) at 37°C.
It was concluded that the stability of rhEPO could increase by complexation using SPRA technology.
重组人促红细胞生成素（rhEPO）作为一种广泛应用的治疗性蛋白质，是目前市场上治疗慢性肾病患者贫血最有效的生物药物之一。提高rhEPO在体内的半衰期及其生物活性是一项重大挑战。据推测，应用具有保留活性的自组装聚乙二醇化，即超分子（SPRA）技术，可以延长蛋白质半衰期而不显著丧失生物活性。
本研究旨在评估rhEPO在合成反应过程中的稳定性，包括与金刚烷的缀合以及SPRA复合物的形成。为此，还对蛋白质的二级结构进行了评估。
采用傅里叶变换红外光谱（FTIR）、衰减全反射傅里叶变换红外光谱（ATR-FTIR）、远紫外圆二色光谱（Far-UV-CD）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）方法。使用纳米滴分光光度计在37℃下对SPRA-rhEPO复合物和rhEPO进行了为期十天的热稳定性研究。
将冻干rhEPO、AD-rhEPO和rhEPO（pH 8）的二级结构与rhEPO进行了比较。结果表明，蛋白质的二级结构不受冻干、pH变化以及缀合反应中共价键形成的影响。SPRA-rhEPO复合物在37℃的磷酸盐缓冲液（pH 7.4）中也能稳定七天。
得出结论，使用SPRA技术进行复合可以提高rhEPO的稳定性。
