Method to Differentiate Asn Deamidation That Occurred Prior to and during Sample Preparation of a Monoclonal Antibody
Method to Differentiate Asn Deamidation That Occurred Prior to and during Sample Preparation of a Monoclonal Antibody
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DOI：
10.1021/ac801617u
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发表时间：
2008-12-15
影响因子：
7.4
通讯作者：
Liu, Hongcheng
中科院分区：
文献类型：
--
作者：
Gaza-Bulseco, Georgeen;Li, Biqin;Liu, Hongcheng
天冬酰胺(ASN)去酰胺化是抗体不稳定和微观异质性的主要来源。因此，在治疗性抗体中准确地确定ASN脱酰胺的水平和位置是至关重要的。ASN的脱酰胺化通常是通过用液相色谱-质谱法分析多肽水平的抗体来定量的。这需要在分析之前对抗体进行变性、还原、烷基化和酶消化。样品制备中的这些步骤可能直接影响检测到的ASN脱酰胺化的总水平。因此，为了获得准确的ASN脱酰胺程度和位置，重要的是确定在样品制备步骤中是否发生了任何脱酰胺化。然而，这可能是具有挑战性的，因为在样品制备之前和过程中发生的脱酰胺反应导致多肽具有相同的保留时间和相同的相对分子质量增加1 Da。在本研究中，样品制备是在O-18-水中进行的，以区分ASN脱酰胺的两个事件。用这种方法，样品制备过程中发生的脱酰胺反应导致相对分子质量增加了3Da，而不是1Da。通过检查含有脱酰胺产物、isAsp和Asp残基的多肽的同位素峰簇，可以很容易地检测到这种分子量差异。它能够区分由于分析人工制品造成的脱酰胺化，从而确定样品中存在的脱酰胺化程度。
Asparagine (Asn) deamidation is a major source of antibody instability and micro heterogeneity. For this reason, it is critical to accurately characterize both the levels and the sites of Asn deamidation in therapeutic antibodies. Asn deamidation is normally quantified by analyzing antibodies at the peptide level by liquid chromatography-mass spectrometry. This requires denaturation, reduction, alkylation, and enzyme digestion of the antibody prior to analysis. These steps in sample preparation may directly contribute to the total levels of Asn deamidation detected. Therefore, to obtain accurate levels and sites of Asn deamidation, it is important to determine if any deamidation occurred during the sample preparation steps. However, this could be challenging because deamidation that occurred prior to and during sample preparation resulted in peptides with the same retention times and the same molecular weight increase of 1 Da. Sample preparation was carried out in O-18-water in the current study to differentiate between the two events of Asn deamidation. Using this method, deamidation that occurred during sample preparation resulted in a molecular weight increase of 3 Da instead of 1 Da. This molecular weight difference was readily detected by inspection of the isotopic peak cluster of the peptides containing the deamidation products, isoAsp and Asp residues. It enabled discrimination of deamidation that was due to analytical artifacts and thus determination of the level of deamidation that was present in the samples.