Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Pharm Res. 2012 Jan;29(1):187-97. doi: 10.1007/s11095-011-0534-2. Epub 2011 Aug 2.
To identify and understand isomerization products and degradation profile of different aspartate residues in an IgG1 monoclonal antibody.
Recombinant IgG1 was incubated for extended periods of time in a formulation buffer at recommended and accelerated storage temperatures. Isomerization reaction products were analyzed using ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), peptide mapping, and LC-MS. Model peptides with sequences containing specific aspartate residues in IgG1 were synthesized and incubated under accelerated conditions. Products of isomerization reactions of peptides were analyzed by reverse phase chromatography (RP-HPLC) and LC-MS. X-ray crystallography data from Fab of IgG1 were used to understand mechanism of isomerization reactions.
A MAb containing labile Asp32-Gly sequence in CDR I region undergoes rapid isomerization reaction and leads to formation of isoaspartate (IsoAsp) and cyclic imide (Asu) forms. Isomerization of aspartate residues was observed in a non-CDR region containing Asp74-Ser sequence. Isomerization reaction at Asp74-Ser led to formation of Asu74 and trace isoAsp74. While isoAsp32 increased linearly with time, isoAsp74 did not increase during storage. Asu32 and Asu74 followed non-linear degradation kinetics and reached steady state over time. Isomerization reaction of two different model peptides containing Asp32-Gly or Asp74-Ser with neighboring amino acid sequences as those found in the MAb result in formation of IsoAsp.
Observed levels of Asu and trace IsoAsp at the Asp74 site are unusual for typical isomerization reactions. In addition to primary sequences, pKa, solvent exposure and high order structure around aspartate residues may have influenced isomerization reaction at Asp74 in MAbI. Different degradation profiles from the two Asp residues can influence shelf life and should be carefully evaluated during product development.
鉴定和了解 IgG1 单克隆抗体中不同天冬氨酸残基的异构化产物和降解情况。
在推荐的加速储存温度下，将重组 IgG1 在制剂缓冲液中孵育较长时间。使用离子交换色谱（IEC）、疏水相互作用色谱（HIC）、肽图和 LC-MS 分析异构化反应产物。合成含有 IgG1 中天冬氨酸残基特定序列的模型肽，并在加速条件下孵育。通过反相色谱（RP-HPLC）和 LC-MS 分析肽的异构化反应产物。使用 IgG1 Fab 的 X 射线晶体学数据来了解异构化反应的机制。
含有 CDR I 区不稳定 Asp32-Gly 序列的 MAb 会发生快速异构化反应，导致形成异天冬氨酸（IsoAsp）和环亚氨酸（Asu）形式。在含有 Asp74-Ser 序列的非 CDR 区观察到天冬氨酸残基的异构化。Asp74-Ser 的异构化反应导致 Asu74 和痕量 isoAsp74 的形成。虽然 isoAsp32 随时间呈线性增加，但在储存过程中 isoAsp74 没有增加。Asu32 和 Asu74 遵循非线性降解动力学，随时间达到稳定状态。含有 Asp32-Gly 或 Asp74-Ser 与 MAb 中发现的相邻氨基酸序列的两种不同模型肽的异构化反应导致 IsoAsp 的形成。
在典型的异构化反应中，观察到 Asp74 位点的 Asu 和痕量 IsoAsp 水平异常。除了一级序列外，pKa、溶剂暴露和天冬氨酸残基周围的高级结构也可能影响 MAbI 中天冬氨酸的异构化反应。两个天冬氨酸残基的不同降解谱可能会影响货架期，在产品开发过程中应仔细评估。
