Xiao Gang, Bondarenko Pavel V
Amgen, Department of Pharmaceutics, One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
J Pharm Biomed Anal. 2008 May 12;47(1):23-30. doi: 10.1016/j.jpba.2007.11.050. Epub 2007 Dec 14.
Two degradations of aspartate residues located in Asp-Asp motifs in the CDR3 region of a recombinant monoclonal antibody were identified and quantified after the antibody was aged in a mildly acidic buffer at elevated temperatures. The degraded sample aged at 25 degrees C for 1 month generated 1.8% antibody molecules that had isomerization in the aspartate residues, while the degraded sample after aging at 45 degrees C for 1 month contained 7% isomerization. Peptide bond cleavages at the aspartate residues were also detected and characterized. The percentage of clipped antibody molecules after 1 month of storage was 1% at 25 degrees C and 4.4% at 45 degrees C. The generated cleaved polypeptides were noncovalently attached to the intact antibody molecule and were not involved in the aggregation formation. They were not detected by native size-exclusion chromatography because of their strong non-covalent association to the rest of the antibody molecules. On the other hand, the cleaved polypeptides were dissociated and detected as fragments under denaturing conditions of reversed-phase HPLC, denaturing size-exclusion chromatography and MALDI-TOF mass spectrometry. It was demonstrated that the cleavages at the above aspartate residue sites occurred due to the aging of the sample at elevated temperatures and were not method-induced by the reversed-phase HPLC and other methods used in this study.
在重组单克隆抗体的互补决定区（CDR3）中，位于天冬氨酸-天冬氨酸基序的两个天冬氨酸残基在抗体于温和酸性缓冲液中高温老化后，其降解情况被鉴定并定量。在25℃下老化1个月的降解样品产生了1.8%天冬氨酸残基发生异构化的抗体分子，而在45℃下老化1个月后的降解样品含有7%的异构化。天冬氨酸残基处的肽键断裂也被检测并表征。在25℃下储存1个月后，截短抗体分子的百分比为1%，在45℃下为4.4%。产生的裂解多肽通过非共价键与完整抗体分子相连，且不参与聚集物形成。由于它们与抗体分子其余部分的强非共价缔合，在天然尺寸排阻色谱中未被检测到。另一方面，在反相高效液相色谱、变性尺寸排阻色谱和基质辅助激光解吸电离飞行时间质谱的变性条件下，裂解多肽解离并作为片段被检测到。结果表明，上述天冬氨酸残基位点的断裂是由于样品在高温下老化所致，而非本研究中使用的反相高效液相色谱和其他方法所诱导。
