新型长效重组促卵泡素的纯化和性质及药代动力学研究

Purification,Characterization and Pharmacokinetic Study of a Novel Long-acting Follicle-stimulating Hormone

促卵泡素(Follicle-stimulating hormone,FSH)用于治疗男女不孕不育症,由于FSH的半衰期较短,需要频繁注射给药,因此制备长效FSH制剂一直是该类药物的研发方向。首次将来自绒促性素(human chorionic gonadotropin,HCG)中的羧基端肽(carboxy-terminal peptide,CTP)和人免疫球蛋白G2(immunoglobulin G2,IgG2)Fc片段变体(vFc)与单链FSH有序连接,通过基因工程和哺乳动物细胞培养技术实现了其在中国仓鼠卵巢细胞中的稳定表达,然后利用Protein A柱层析手段进行纯化,制备了新型重组FSH-CTP-vFc融合蛋白,同时分析了不同细胞培养时间表达的重组融合蛋白的理化性质和体内外活性。动物试验结果表明,纯化的FSH-CTP-vFc融合蛋白具有显著的体内生物活性,药代动力学分析显示该重组融合蛋白的半衰期是重组FSH的近10倍。这种新型长效重组FSH-CTP-vFc融合蛋白在临床上可大大减少注射次数和患者痛苦,提高患者用药依从性。

Follicle-stimulating hormone （FSH） has been utilized to treat male and female infertility. However, frequent injection of FSH to patients is required due to its short serum half-life. Developing a long- acting recombinant FSH is clinically desirable. A DNA construct containing FSH-CTP-vFc was made by fusing the coding sequence of carboxyl-terminal peptide （CTP） of the β-subunit of human chorionic gonadotropin and a variant of human Immunoglobulin G2 （ IgG2 ） Fc fragment （ vFc ） into FSH gene. The DNA construct was transfected into Chinese Hamster Ovary （CHO） cells, and cell clones with highly and stably expressed FSH- CTP-vFc fusion protein were selected. The cultured medium of cell was collected for protein purification by protein A based chromatography. Biochemical and physical characterization were performed for the fusion proteins purified from the samples of different culture time. ELISA assay and animal study shows that the purified FSH- CTP-vFc protein has significant activity in vitro and in vivo. Pharmacokinetic research demonstrates that the serum half-life of the fusion protein is almost ten fold of the recombinant FSH＇ s. Obviously, this novel long-acting FSH- CTP-vFc protein could greatly reduce the injection frequency and improve the patient compliance.