Biologics Process Research and Development, Novartis Pharma, Postfach, 4002 Basel, Switzerland.
Biologics Process Research and Development, Novartis Pharma, Postfach, 4002 Basel, Switzerland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Apr 1;955-956:26-33. doi: 10.1016/j.jchromb.2014.02.017. Epub 2014 Feb 18.
Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.
天冬氨酸残基的异构化是生物制药货架期化学降解的主要原因之一。单克隆抗体生物制药通常在轻微酸性 pH 条件下储存，这可能导致异构化反应。这种非酶促化学反应的机制已经被详细研究过。然而，在给定的蛋白质中鉴定和量化异构化位点仍然是一个挑战。我们开发了一种离子对反相高效液相色谱法，用于分离含有单个异构天冬氨酸残基的完整单克隆抗体变体与其天然对应物。我们使用离子对反相高效液相色谱-质谱法对还原和烷基化抗体以及酶切抗体进行了鉴定和表征，从而确定并表征了异构化位点。抗体的 Lys-C 随后是 Asp-N 消化用于鉴定异构化位点。电子转移解离（ETD）质谱用于确认抗体 CDR-H3 区域中天冬氨酸残基上的 DY 基序处的异构化位点。天冬氨酸残基的 C 末端的酪氨酸通常不被认为是异构化的热点。我们的研究结果表明，不能有信心地预测蛋白质中的异构化位点，并且由于这些区域的溶剂暴露或分子的灵活性，抗体的 CDR 区域中所有天冬氨酸残基都必须被视为潜在的异构化位点。此外，还使用离子对反相高效液相色谱法评估了 pH 对异构化速率的影响，结果表明在较低的 pH 值下，异构化速率更快。在 25°C 下储存 6 个月会导致 pH 值为 5.4 时异构天冬氨酸的含量增加到 6.6%，pH 值为 5.8 时增加到 6.0%，pH 值为 6.2 时增加到 5.6%。
