DEAMIDATION AND ISOASPARTIC ACID FORMATION IN ANTIBODIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 重组糖蛋白药物的正常降解途径，如单抗(MAbs)，可能会导致各种不必要的产物，从而对蛋白质有效发挥作用的能力产生不利影响。抗体的降解途径之一是天冬酰胺残基脱酰胺为异天冬氨酸和天冬氨酸(通常分别以3：1的比例)。蛋白质和多肽中天冬酰胺的脱酰胺反应是通过琥珀酰亚胺中间体，然后琥珀酰亚胺环的部分水解形成产物。最常见的脱酰胺反应发生在天冬酰胺残基上，在氨基酸序列中紧随其后的是非空间氨基酸，如甘氨酸或丝氨酸。一个典型的IgG1mAb在每个重链上含有15~20个天冬酰胺残基，在每个轻链上含有4~7个天冬酰胺残基。然而，并不是所有的天冬酰胺残基都被去胺化，因为它们并不都与紧凑的氨基酸相邻，而且抗体的三维结构可以帮助防止某些位置的降解。此外，天冬氨酸异构化为异天冬氨酸也可以通过类似的途径发生，这是不可取的，而且更难检测到。脱酰胺和异天冬氨酸的存在对生物制药制造是一个令人担忧的问题，因为这些降解产物可能导致三级结构变化和活性丧失，特别是如果存在于轻链或重链的CDR区域。奥康纳的研究小组最近发表的结果表明，利用电子捕获和电子转移解离可以区分天冬氨酸和异天冬氨酸残基。这提供了一种可能性，即在单抗中对这些修饰进行严格的结构表征是可能的。此外，来自O‘Connor小组的最新结果表明，ASN：ASP：IsoAsp比率的实际位点特异性定量也是可能的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The normal degradation pathway for recombinant glycoprotein pharmaceuticals, such as monoclonal antibodies (mAbs), can lead to a variety of undesired products that can adversely affect the ability of the protein to function efficiently. Among the degradation pathways for antibodies is the deamidation of asparagine residues to isoaspartic acid and aspartic acid (typically in a 3:1 ratio, respectively). The deamidation of asparagine in proteins and peptides occurs through a succinimide intermediate followed by partial hydrolysis of the succinimide ring to form the products. Deamidation occurs most frequently at asparagine residues that are followed in the amino acid sequence by a non-steric amino acid such as glycine or serine. A typical IgG1 mAb contains 15  20 asparagine residues on each heavy chain and 4 - 7 asparagine residues on each light chain. However, not all of the asparagine residues are expected to be deamidated since they are not all adjacent to a compact amino acid and the three-dimensional structure of the antibody can aid in protection against degradation at some locations. In addition, isomerization of aspartic acid to isoaspartic acid can also occur through a similar pathway and is both undesirable and considerably more difficult to detect. The presence of deamidation and isoaspartic acid is a concern for biopharmaceutical manufacturing, as these degradation products may lead to tertiary structural changes and loss of activity, especially if present in the CDR regions of either the light or heavy chain. The recently published results by O'Connor's research group showed that differentiation of aspartic and isoaspartic acid residues is possible using electron capture and electron transfer dissociation. This offers the possibility that rigorous structural characterization of these modifications in monoclonal antibodies may be routinely possible. Furthermore, recent results from the O'Connor group suggest that actual site-specific quantitation of the Asn:Asp:isoAsp ratio may be possible as well.