一种基于半胱氨酸偶联的抗体偶联药物的药物抗体偶联比测定

Determination of drug-antibody ratio in a cysteine conjugation based antibody-drug conjugate

目的:建立了利用反相色谱(RP-HPLC)串联质谱对一种基于半胱氨酸偶联的抗体偶联药物(ADC)的药物抗体偶联比(DAR)进行测定的方法。方法:采用RP-HPLC方法,色谱柱为Varian公司的PLRP-S(50 mm×2.1 mm,5μm,1 000);流动相A为50 mmol·L-1磷酸钠-1.5 mol·L-1硫酸铵(p H 7.0);流动相B为75%50 mmol·L-1硫酸钠(p H 7.0)-25%异丙醇;梯度洗脱;柱温25℃;流速为0.8 m L·min-1;检测波长为280 nm。串联质谱对液相分离各组分进行定性分析。结果:所建立的RP-HPLC串联质谱分析方法可对各个峰组分进行定性定量,所测得的DAR为(4.0±0.1),标准差为2.5%。结论:基于PLRP-S色谱柱的RP-HPLC,无高浓度盐,不会对常规液相色谱系统造成污染,使用的是质谱兼容的流动相,能够兼顾质谱定性与液相定量,可用于基于半胱氨酸偶联的ADC的DAR测定。

Objective： To establish a reverse phase chromatography（ RP-HPLC） method coupled with mass spectrometric analysis for determining drug-antibody ratio（ DAR） of a kind of antibody drug conjugate（ ADC） based on cysteine conjugation. Methods： A RP-HPLC method was established to determine the DAR. The separation was performed on a Varian PLRP-S column（ 50 mm × 2. 1 mm,5 μm,1 000 ） with a gradient elution composed of mobile phase A of 50 mmol·L- 1sodium phosphate and 1. 5 mol·L- 1ammonium sulfate,and mobile phase B of 75% 50 mmol·L- 1sodium sulfate,p H 7. 0,25% isopropanol. The column temperature was 25 ℃,the flow velocity was 0. 8 m L·min- 1,and the detective wavelength was set at 280 nm. Tandem mass spectrometric analysis was performed to qualitatively identify the peaks separated by HPLC. Results： The established RP-HPLC method coupled with tandem mass spectrometry analysis was able to qualitatively and quantitatively analyze each peak. The DAR was measured to be 4. 1 ± 0. 1 with the standard deviation of 2. 4%. Conclusion： The RP-HPLC based on PLRP-S column does not cause pollution to the conventional liquid chromatography system because of no high concentration of salt and the mobile phase used is compatible with mass spectrometry mobile,thus,it can be applied to the qualitative and quantitative analysis of DAR. The method could be successfully applied to determination of the DAR of ADC based on cysteine conjugation.