Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry
Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry
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DOI：
10.1021/ac702115b
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发表时间：
2008-02-15
影响因子：
7.4
通讯作者：
Bitsch, Francis
中科院分区：
文献类型：
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作者：
Hagman, Charlotte;Ricke, Darrell;Bitsch, Francis
建立了高效液相色谱-多反应监测质谱法测定血清中单克隆抗体的定量方法。使用人单克隆抗体（HmAb）作为模型蛋白进行方法开发和验证。从其重链的cdr3区域选择一个肽，用于定量整个单抗。该特征肽作为内标模板。在质谱分析之前，大约50%的血清总蛋白含量通过白蛋白消耗去除。方法在食蟹猴血清中的准确度在99 ~ 112%之间。在4 μ g/mL和200 μ g/mL HmAb条件下，试验内变异系数（CV）均小于4% （n = 3）。400 μ g/mL时CV为9% （n = 3）。此外，还对雄性食蟹血清池和雌性食蟹血清池进行了分析。在4 μ g/mL HmAb条件下，雄性食蟹池的CV为7% （n = 3）。从雌性池中获得的CV为8% (n = 3)，为4 μ g/mL。该方法的动态范围为3个数量级。在白蛋白消耗25 μ L血清后，人类和食蟹猴样本中HmAb的最低定量下限均达到2 μ g/mL。
The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intraassay coefficient of variation (CV) was lower than 4% at 4 mu g/mL and 200 mu g/mL HmAb (n = 3). The CV at 400 mu g/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 mu g/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 mu g/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 mu L of serum, a lowest limit of quantification of 2 mu g/mL HmAb was reached in both human and cynomolgus monkey samples.