目的: 建立一种基于荧光法测定重组蛋白药物中微量游离巯基的检测方法。方法: 采用基于荧光法巯基检测试剂盒进行游离巯基检测,首先对蛋白变性条件进行摸索优化,确定了变性剂种类及变性条件,并采用重组蛋白药物进行了方法验证,结果均符合验证标准。但在采用该方法对我公司PEG-GCSF和KGF产品(分子序列中理论均含有1 mol巯基/mol蛋白)进行检测时,发现游离巯基检测值均显著低于理论值,检测值最高仅为理论值的29%,可能与蛋白分子内部包埋的游离巯基未充分暴露有关。因此,本研究在已有分析方法的基础上采用蛋白酶酶解的方式,充分暴露蛋白分子中的游离巯基,并对样品的酶切条件及其他关键参数进行了优化,对优化后的分析方法进行了方法学验证。结果: 通过筛选,确定了最佳变性条件:终浓度为8 mol·L-1尿素37 ℃孵育30 min。针对已知理论游离巯基含量的重组蛋白1(PEG-GCSF)和重组蛋白2(KGF)产品游离巯基检测值明显低于理论值的问题,采用蛋白酶切方式,酶切条件为蛋白酶K酶切(目的蛋白:蛋白酶K(20∶1,w:w),37 ℃孵育1 hr,酶解后检测巯基实测值与理论值一致。经验证该方法实测结果RSD均<15%;各浓度的加标回收率均在80%~120%范围内;在1~15 μmol·L-1游离巯基范围内线性良好-1定量限(LOQ)为1 μmol·L-1,各项验证指标均符合验证标准,满足检测需求。结论: 本研究采用蛋白酶酶切的方式进行样品前处理,充分暴露重组蛋白药物分子内包埋的游离巯基,解决了复杂重组蛋白药物产品中游离巯基无法准确定量的难题,为该类蛋白分子中微量游离巯基的定量测定和表征研究提供了方法和参考。
Objective: To establish a fluorometric method for the determination of trace amount free sulfhydryl groups in recombinant protein drugs. Methods: The Measure-iTTM Thoil Assay Kit based on fluoresence was used to detect free sulfhydryl groups. First, the protein denaturation conditions (urea, guanidine hydrochloride) were explored and optimized to determine the types and conditions of denaturation agents. The optimized method was verified by recombinant protein drugs. The results met the verification standards. However, in the testing of PEG-GCSF and KGF (molecular theory contains 1 mol free sulfhydryl/mol protein) products by the above method, it was found that the detection value of free sulfhydryl group was significantly lower than the theoretical value. The highest detection rate was only 29% of theoretical value, which may be related to the insufficient exposure of free sulfhydryl group inside protein molecule. In this study, free thiol in recombinant protein drug molecules was fully exposed by protease enzyme creative way. Other key parameters were optimized, and the methodology verification of the optimized analysis method was completed. Results: By screening the denaturation conditions of the samples before treatment, the optimal pre-treatment conditions were using 8 mol·L-1 urea to incubate for 30 min at 37 ℃. To solve the problem that sulphur content of recombinant protein 1 and recombinant protein 2 was much lower than the theoretical value, PEG-GCSF and KGF were first treated by proteases cut at the optimal protease conditions. Proteinase K [protein: protease K (20∶1,w∶w)], 37 ℃ incubation for 1 h. The measured values after enzymolysis were consistent with the theoretical values. It has been verified that the precision of the results is less than 15%. The recovery rate of each concentration is in the range of 80%-120%. The linear range of free sulfhydryl group was 1-15 μmol·L-1 (r>0.99). The limit of quantification (LOQ) was 1 μmol·L-1. All the verification indicators are in line with the verification standards and met testing requirements. Conclusion: In this study, protease digestion was used for pre-treatment of samples to fully expose free sulfhydryl groups in protein drug molecules. It provides a method and reference for quantitative determination and characterization of trace free sulfhydryl groups in this kind of protein molecules.
[1] 田悦, 杜军保. 二硫键和巯基在蛋白质结构功能中的作用及分析方法 [J]. 实用儿科临床杂志, 2007, 22(19):1499
TIAN Y, DU JB. Effect of disulfide bond and sulfhydryl group on structure and function of protein and analytical method [J]. J Appl Clin Pediats, 2007, 22(19):1499
[2] TAO XIANG, CHRIS CHUMSAE, LIU H. Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis [J]. Anal Chem, 2009, 81(19):8101
[3] GEVONDYAN NM, VOLYNSKAIA AM, GEVONDYAN VS. Four free cysteine residues found in human IgG1 of healthy donors [J]. Biochemistry (Moscow), 2006, 71(3):279
[4] ZHANG W, CZUPRYN MJ. Free sulfhydryl in recombinant monoclonal antibodies [J]. Biotechnol Progr, 2002, 18(3):509
[5] LIU H, GAZA-BULSECO G, CHUMSAE C, et al. Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE [J]. Biotechnol Lett, 2007, 29(11):1611
[6] HARRIS RJ. Heterogeneity of recombinant antibodies: linking structure to function [J].Develop Biol,2005 (2):117
[7] FRANEY H, BRYCH SR, KOLVENBACH CG, et al. Increased aggregation propensity of IgG2 subclass over IgG1: role of conformational changes and covalent character in isolated aggregates [J]. Protein Sci, 2010, 19(9):1601
[8] LACY ER, BAKER M, BRIGHAM-BURKE M. Free sulfhydryl measurement as an indicator of antibody stability [J]. Anal Biochem, 2008, 382(1):66
[9] ZHANG T, ZHANG J, HEWITT D, et al. Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody [J]. Anal Chem, 2012, 84(16):7112
[10] 张力文,尚中博,常志显,祁志冲,李德亮. 巯基检测方法研究进展 [J]. 河南大学学报(自然科学版), 2018, 48(4):430
ZHANG LW, SHANG ZB, CHANG ZX, et al. Research progress on the determination of thiol compounds [J].J Henan Univ (Nat Sci), 2018, 48(4):430
[11] ELLMAN GL. Tissue sulfhydryl groups [J]. Arch Biochem Biophys, 1959, 82(1):70
[12] RIENER CK, KADA G, GRUBER HJ. Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine [J]. Anal Bioanal Chem, 2002, 373(4-5):266
[13] LACY ER, BAKER M, BRIGHAM-BURKE M. Free sulfhydryl measurement as an indicator of antibody stability [J]. Anal Biochem, 2008, 382(1):66
[14] 杨庆珍, 徐琳玲. 血中巯基总量测定及临床意义 [J]. 福建医药杂志, 2000, 22(4):40
YANG QZ, XU LL. Determination of total amount of thiol in blood and its clinical significance [J]. Fujian Med J, 2000, 22(4):40
[15] CHIKU T, PULLELA PK, SEM DS. A dithio-coupled kinase and ATPase assay [J]. SLAS Discov, 2006, 11(7):844
[16] MAFORIMBO E, SKURRAY GR, NGUYEN M. Evaluation of l-ascorbic acid oxidation on SH concentration in soy-wheat composite dough during resting period [J]. LWT-Food Sci Technol, 2007, 40(2):338
[17] OWUSU-APENTEN R. Colorimetric analysis of protein sulfhydryl groups in milk: applications and processing effects [J]. Crit Rev Food Sci Nutr, 2005, 45(1):1
[18] 邓春平, 杨波, 梅雄, 等. 重组碱性成纤维细胞生长因子游离巯基的测定分析 [J]. 中国生物工程杂志, 2016, 36(6):76
DENG CP, YANG B, MEI X, et al. Measurement and analysis of recombinant basic fibroblast growth factor's free sulfhydryl [J]. China Biotechnology, 2016, 36(6):76
[19] WOODWARD J, TATE J, HERRMANN PC, et al. Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei [J]. J Biochem Biophys Methods, 1993, 26(2):121
[20] 刘欣荣, 洪维伟, 邓意辉. 4,4'-二吡啶基二硫法测定巯基化壳聚糖中的巯基含量 [J]. 沈阳药科大学学报, 2013, 30(2):120
LIU XR, HONG WW, DENG YH. Determination of thiol content in thiolater chitosan by 4,4'-dithiodipyridine [J]. J Shenyang Pharml Univ, 2013, 30(2):120
[21] LU J, SUN C, CHEN W, et al. Determination of non-protein cysteine in human serum by a designed BODIPY-based fluorescent probe [J]. Talanta, 2011, 83(3):1050
[22] CHUMSAE C, GAZA-BULSECO G, LIU H. Identification and localization of unpaired cysteine residues in monoclonal antibodies by fluorescence labeling and mass spectrometry [J]. Anal Chem, 2009, 81(15):6449
[23] SHAO J, SUN H, GUO H, et al. A highly selective red-emitting FRET fluorescent molecular probe derived from BODIPY for the detection of cysteine and homocysteine: an experimental and theoretical study[J]. Chem Sci, 2012, 3(4):1049
[24] ROBOTHAM AC,KELLY JF. Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry[J]. mAbs, 2019, 11(4):757
[25] HUTTERER KM, HONG RW, LULL J, et al. Monoclonal antibody disulfide reduction during manufacturing[J]. mAbs, 2013, 5(4):608
[26] ZHANG W, CZUPRYN MJ. Free sulfhydryl in recombinant monoclonal antibodies[J]. Biotechnol Prog, 2002, 18, 509
[27] WEI BC, JIA WT, YANG Y, et al. Development of a rapid reversed-phase liquid chromatographic method for total free thiol quantitation in protein therapeutics[J]. J Pharm 2020, 189: 113434
[28] ZHANG YL, QI P. Determination of free sulfhydryl contents for proteins including monoclonal antibodies by use of Solo VPE[J]. J Pharm Biomed Anal,2021,201:114092
