發(fā)布時間：2018-07-29 19:58

【摘要】： 蛋白藥物的PEG修飾是第二代生物制藥的重要內(nèi)容,PEG修飾蛋白藥物克服了天然蛋白藥物的諸多不足,而被賦予了低免疫原性、高酶穩(wěn)定性、長半衰期等新特性,已逐漸成為臨床上廣泛應(yīng)用的新一代高效、低毒的治療藥物。本項目完成了PEG修飾hCH-2項目臨床前的大部分基礎(chǔ)研究工作,選擇了合適的PEG修飾劑,優(yōu)化并放大驗(yàn)證了修飾工藝和純化工藝,獲得了單鏈PEG-hCH-2目標(biāo)產(chǎn)物,建立了單鏈PEG-hCH-2原液生產(chǎn)的質(zhì)控方法和質(zhì)量標(biāo)準(zhǔn),探索了單鏈PEG-hCH-2的初步特性,為開發(fā)hCH-2的長效PEG修飾藥物奠定了基礎(chǔ)。具體內(nèi)容主要包括以下幾個方面: (1)利用高效液相色譜技術(shù),建立了一種快速、方便、準(zhǔn)確和穩(wěn)定的定量檢測PEG修飾反應(yīng)液中微量PEG-hCH-2和hCH-2的反相高效液相色譜方法。該方法的分離基礎(chǔ)為PEG化hCH-2疏水性的改變,色譜柱為C8反相柱(250mm×4.6mm,300?,5μm),流動相A為水-TFA(999∶1),流動相B為100%乙氰,流速為1.0ml/min,線性梯度洗脫和等度洗脫相結(jié)合,柱溫為30℃,檢測波長為280nm。在0.049-0.456mg/ml和0.112-1.796 mg/ml濃度范圍內(nèi),PEG-hCH-2和hCH-2的濃度與色譜峰峰面積均呈現(xiàn)良好線性關(guān)系,回歸方程分別為y=2.11×10~6x-3.30×10~4和y=1.08×10~6x-8.45×10~3,線性相關(guān)系數(shù)r分別為0.9997和1.0000,最低檢測濃度分別為0.044 mg/ml和0.056 mg/ml,日內(nèi)和日間RSD(n=6)分別為0.50-2.18%和0.40-2.21%。平均回收率分別為98.01% (n=3,RSD為0.72-2.62%)和97.80% (n=3,RSD為0.56-2.32%)。本方法克服了常用的SDS-PAGE、SPF、TNBS等方法的重現(xiàn)性、精度差以及檢測時間長等不足,完全可用于工業(yè)化大生產(chǎn)質(zhì)量控制過程中PEG-hCH-2的測定。 (2)根據(jù)hCH-2本身的結(jié)構(gòu)特性,選擇分子量為20kD的鏈?zhǔn)界晁徵牾啺孵ヮ恗PEG對hCH-2分子中賴氨酸的ε氨基和/或N末端氨基進(jìn)行非定點(diǎn)PEG修飾。通過PEG修飾hCH-2的單因素影響研究,獲得較適宜的起始pH值為8.0-8.5、摩爾比為1:5、緩沖液濃度為25-50mmol/l、hCH-2濃度為0.5mg/ml和反應(yīng)時間為4h;利用L_9(3~4)正交試驗(yàn)對起始pH值、hCH-2與PEG的摩爾比、hCH-2濃度和緩沖液濃度進(jìn)行了優(yōu)化,獲得優(yōu)化的起始pH值為8.5、hCH-2與PEG的摩爾比為1:7、hCH-2濃度為0.75mg/ml和緩沖液濃度為50mmol/l;保持溫度25℃、反應(yīng)時間4h、反應(yīng)總體積0.5ml和轉(zhuǎn)速200r/min,進(jìn)行優(yōu)化修飾條件的驗(yàn)證試驗(yàn)研究和放大試驗(yàn)研究,結(jié)果表明:優(yōu)化的PEG修飾工藝條件具有良好的穩(wěn)定性和可操作性,可使反應(yīng)液中單鏈PEG-hCH-2含量可達(dá)0.25mg/ml,單鏈PEG-hCH-2修飾
[Abstract]:PEG modification of protein drugs is an important part of the second generation biopharmaceuticals. It overcomes many shortcomings of natural protein drugs and is endowed with new characteristics such as low immunogenicity, high enzyme stability, long half-life and so on. It has gradually become a new generation of highly effective and low-toxic drugs that are widely used in clinic. This project has completed most of the basic research work before clinical application of PEG modified hCH-2 project, selected suitable PEG modifier, optimized and verified the modification process and purification process, and obtained the target product of single chain PEG-hCH-2. The quality control method and quality standard for the production of single strand PEG-hCH-2 solution were established, and the preliminary characteristics of single strand PEG-hCH-2 were explored, which laid a foundation for the development of long-acting PEG modified drugs for hCH-2. The specific contents include the following aspects: (1) using the high performance liquid chromatography (HPLC) technology to establish a rapid and convenient, Accurate and stable RP-HPLC method for quantitative determination of trace PEG-hCH-2 and hCH-2 in PEG modified reaction solution. The separation was based on the change of hydrophobicity of PEG hCH-2. The column consisted of C _ 8 reversed phase column (250mm 脳 4.6mm ~ (300) m), mobile phase A was water (999: 1), mobile phase B was 100% acetic acid, flow rate was 1.0 ml / min, linear gradient elution was combined with isometric elution, column temperature was 30 鈩，

本文編號：2153829