超高效液相色谱法同时测定细胞培养液中17种游离氨基酸浓度

Simultaneous determination of 17 kinds of free amino acid concentrations in cell culture medium by UPLC

建立柱前衍生-超高效液相色谱同时测定高表达程序性死亡蛋白-1(programmed death protein-1,PD-1)抗体的细胞株培养液中17种游离氨基酸浓度的方法,根据浓度检测结果,对表达抗PD-1抗体的细胞株进行氨基酸代谢分析。使用6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯(6-aminoquinoline-N-hydroxysuccinimidyl carbamate,AQC)作为衍生剂,将不同浓度的氨基酸标准品或细胞株培养液中的游离氨基酸转化为稳定的紫外衍生物,采用ACQUITY UPLC BEH C18(2.1 mm×100 mm,1.7μm)色谱柱梯度洗脱分离,流速为0.7 mL·min^(-1),柱温55℃,进样量为1μL,在紫外检测器检测波长为260 nm的条件下进行检测,使用外标法计算氨基酸浓度,并对高表达抗PD-1抗体的细胞株培养液中游离氨基酸浓度进行测定,根据细胞培养周期内氨基酸浓度变化进行代谢分析。结果表明,柱前衍生-超高效液相色谱法能在11 min内将17种氨基酸进行完全分离,在0.75~500μmol·L^(-1)浓度范围内线性关系良好(相关系数R2≥0.9993),检测限为0.1~0.3μmol·L^(-1),定量限为0.3~1μmol·L^(-1),建立的方法具有快速、重现性高的优点,氨基酸代谢分析显示,蛋氨酸、异亮氨酸和亮氨酸可能为细胞株生长和表达限制性成分。建立的柱前衍生-超高效液相色谱法快速、准确,可用于同时检测细胞培养液中17种游离氨基酸浓度的变化。

To establish an ultra high performance liquid chromatography coupled with pre-column derivatization method for simultaneous determination of 17 kinds of free amino acid concentrations in CHO cell which expressed high levels of programmed death protein-1(PD-1)antibody,and its amino acid metabolism was analyzed by this method.Using 6-aminoquinoline-N-hydroxysuccinimidyl carbamate(AQC)as a derivatization reagent,the free amino acids in different concentrations of amino acid standards or cell lines were transformed into stable UVabsorbent compounds,which were separated by gradient elution through ACQUITY UPLC BEH C18(2.1 mm×100 mm,1.7μm)column.The flow rate was set at 0.7 mL·min^(-1),the column temperature at 55℃,the loading amount of sample at 1μL,the UV detection wavelength at 260 nm,and then the free amino acid concentration in cell lines which expressed PD-1 antibody was determined by external standard method.According to the changes of amino acids concentration during the process of cell culture,the amino acid metabolism was analyzed.The results showed that pre-column derivatization high performance liquid chromatography could completely separate 17 kinds of amino acids within 11 minutes,has good linear relationship(R2≥0.9993)in the range of 0.75-500μmol·L^(-1),the limits of detection was 0.1-0.3μmol·L^(-1),the limits of quantitative was 0.5-1μmol·L^(-1).The established method is quickly and reproducibility.Amino acid metabolism revealed that methionine,isoleucine and leucine may be the restrictive factors of expression for cell line.This method can be used for detection changes of free amino acid concentration in cell line.