长效生长激素相关蛋白含量检测反相高效液相色谱法的建立及验证

Development and verification of reverse phase high performance liquid chromatography for determination of content of rhGH-Fc associated protein

目的建立长效生长激素(recombinant human growth hormone-Fc,rhGH-Fc)相关蛋白含量检测的反相高效液相色谱(RP-HPLC)法,并进行验证。方法通过质谱肽图法进行rhGH-Fc相关蛋白结构解析后建立相关蛋白含量检测的方法,优化梯度洗脱方法和流动相方案,并对该方法进行重复性、专属性、耐用性验证,确定检测限和定量限。用建立的方法检测rhGH-Fc氧化加速试验样品、脱酰胺加速试验样品及不同批次纯化液样品。结果建立的RP-HPLC法色谱柱为ZORBAX 300SB-C18(4.6 mm×250 mm,5-Micron),柱温45℃,上样量40μg,流速0.7 mL/min,检测波长280 nm;流动相A为30%乙腈+0.1%三氟乙酸,流动相B为80%乙腈+0.1%三氟乙酸;梯度洗脱方法为0~10 min,10%B→50%B;10~50 min,50%B→90%B。rhGH-Fc的相关蛋白主要产生在4个氧化位点和2个脱酰胺位点处。建立的RP-HPLC检测方法重复性验证RSD为1.8%;蛋白缓冲液对检测结果无干扰;耐用性验证RSD为0.05%;在相同色谱柱条件下,不同设备、不同时间配制的流动相、不同柱温对检测结果无明显影响;方法检测限为0.01 mg/mL,定量限为0.025 mg/m L。结论成功建立了rhGH-Fc相关蛋白含量的RP-HPLC检测方法,该方法重复性、专属性、耐用性良好,可用于rhGH-Fc相关蛋白含量的检测。

Objective To develop a reverse phase high-performance liquid chromatography(RP-HPLC)method for determination of long-acting recombinant human growth hormone-Fc(rhGH-Fc)associated protein and to verify its methodology.Methods The structure of rhGH-Fc associated protein was elucidated by mass spectrometry peptide mapping,and condition for gradient elution and design of mobile phase were optimized,and the developed method was verified for reproducibility,specificity and durability,and determined for limit of detection(LOD)and limit of quantitation(LOQ).The samples for accelerated oxidation test,accelerated deamidation test and the purified samples of various batches were determined by the developed method. Results A RP-HPLC method was developed by using a ZORBAX 300 SB-C18(4. 6 mm × 250 mm 5-Micron)chromatographic column at a temperature of 45 ℃,a sample load of 40 μg,a flow rate of 0. 7 mL/min,and a detection wavelength of 280 nm. The mobile phase A was 30% acetonitrile + 0. 1% trifluoroacetic acid,while the mobile phase B was 80% acetonitrile + 0. 1% trifluoroacetic acid. The optimal condition for gradient elution was as follows:0 ~ 10 min,10% B → 50% B;10 ~ 50 min,50% B → 90% B. The rhGH-Fc associated proteins were mainly produced at four oxidation sites and two deamidation sites. The RSD in verification for reproducibility of the developed method was 1. 8%,while that for durability was 0. 05%. Protein buffer showed no interference to test result. Under the same chromatographic condition,the equipment,mobile phases prepared at different times and column temperature showed no significant effect the determination results. The LOD and LOQ of the developed method were 0. 01 and 0. 025 mg/mL respectively. Conclusion A RP-HPLC method was successfully developed,which showed high reproducibility,specificity and durability,and might be used for the determination of rhGH-Fc associated protein content.