目的 重组抗埃博拉病毒抗体是由3种抗埃博拉病毒包膜糖蛋白(GP)的单抗组成的复方抗体,本实验分别对3种重组单抗的质控方法进行研究。方法 利用ELISA方法测定重组抗埃博拉病毒单抗的结合活性;利用LC-MS肽图法对其进行鉴别;CE-SDS和SE-HPLC测定纯度;iCIEF法测定等电点及电荷异质性;切糖后对N-糖进行2AB标记,用正向色谱结合质谱对其糖型和含岩藻糖修饰糖型比例分析;利用高效液相色谱法测定其聚山梨酯20含量。结果 3种重组抗埃博拉病毒单抗结合活性的EC₅₀为(12.53±1.62)、(11.10±0.62)、(6.09±0.35) ng·mL^-1;LC-MS肽图法测定一级序列覆盖率均大于95%;非还原CE-SDS主峰纯度分别为(94.41±0.05)%、(95.58±0.17)%、(96.11±0.05)%;还原CE-SDS重链和轻链纯度之和分别为(98.19±0.06)%、(97.97±0.03)%、(98.57±0.03)%;SE-HPLC主峰分别为(99.59±0.01)%、(99.56±0.01)%、(99.74±0.01)%;iCIEF分析主峰等电点为(8.70±0.01)、(8.26±0.01)、(8.85±0.01);3种单抗聚山梨酯20含量分别为(0.34±0.00)、(0.35±0.00)、(0.35±0.01) mg·mL^-1;LC-MS对N糖谱分析相对灵敏,3种单抗含岩藻糖糖型比例均小于0.5%。结论 本实验的质控方法研究运用了现代化质控理念,建立的质控方法具有保证产品安全、有效、质量可控的特点,为突发状况下埃博拉疫情防控提供了用药储备的技术支持,也为其他组合抗体的质量控制积累了经验。

OBJECTIVE To establish quality control methods for recombinant anti-EBOV mAbs which are comprised by three anti-EBOV glycoprotein mAbs. METHODS The binding-activity of recombinant humanized anti-EBOV mAbs to EBOV glycoprotein was evaluated by ELISA.Peptide map by LC-MS was used in the identification tests. Purity was analyzed by CE-SDS and SEC-HPLC. iCIEF was performed to measure the PI value and the charge heterogeneity. 2AB was labeled on the released glycan, and was analyzed by NP-HPLC and mass spectrometry. The concentration of polysorbate 20 was tested by HPLC. RESULTS The EC_50s of recombinant anti-EBOV mAbs were (12.53±1.62), (11.10±0.62) and (6.09±0.35) ng·mL^-1, respectively. The theoretical sequence coverage rates of three mAbs were all above 95%. The main peak area percentages shown by non-reduced CE-SDS were (94.41±0.05)%, (95.58±0.17)% and (96.11±0.05)%. The peak area percentages of both heavy and light chain shown by reduced CE-SDS were (98.19±0.06)%, (97.97±0.03)% and (98.57±0.03)%. The main peak area percentages shown by SE-HPLC were (99.59±0.01)%, (99.56±0.01)% and (99.74±0.01)%. The isoelectric points of the main peak shown by iCIEF were (8.70±0.01),(8.26±0.01) and (8.85±0.01). The concentrations of polysorbate 20 were (0.34±0.00),(0.35±0.00) and (0.35±0.01) μg·mL^-1, respectively. The glycan mapping analysis was relatively sensitive, and the percentage of fucosylated N linked glycan was less than 0.5%. CONCLUSION Up-to-date quality control methods for recombinant anti-EBOV mAbs are established in this study, which may be used to ensure the safety, effectiveness and quality controllability of the product. The methods can provide technical assists to Ebola outbreak and be a reference of the quality control of other domestic cocktail monoclonal antibody products.