DEAMIDATION AND ISOASPARTIC ACID FORMATION IN ANTIBODIES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 重组糖蛋白药物（例如单克隆抗体（mAb））的正常降解途径可导致多种不期望的产物，其可不利地影响蛋白质有效发挥功能的能力。抗体的降解途径之一是天冬酰胺残基脱酰胺化为异天冬氨酸和天冬氨酸（通常分别以3：1的比例）。 蛋白质和肽中天冬酰胺的脱酰胺作用通过琥珀酰亚胺中间体发生，随后琥珀酰亚胺环部分水解形成产物。脱酰胺最常发生在氨基酸序列中后面是非空间氨基酸如甘氨酸或丝氨酸的天冬酰胺残基处。 典型的IgG 1 mAb含有15  每条重链上20个天冬酰胺残基，每条轻链上4 - 7个天冬酰胺残基。 然而，预期并非所有天冬酰胺残基都被脱酰胺化，因为它们并不都与紧密氨基酸相邻，并且抗体的三维结构可以帮助保护某些位置免受降解。 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，并且是不期望的并且相当难以检测。 脱酰胺和异天冬氨酸的存在是生物制药生产的一个问题，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。 奥康纳的研究小组最近发表的结果表明，天冬氨酸和异天冬氨酸残基的区分是可能的，使用电子捕获和电子转移解离。 这提供了单克隆抗体中这些修饰的严格结构表征常规上可能的可能性。 此外，奥康纳小组最近的结果表明，对天冬氨酸：天冬氨酸：异天冬氨酸比率进行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.