Late Stage Pharmaceutical and Processing Development, Early Stage Pharmaceutical Development, and Protein and Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Bioconjug Chem. 2010 Sep 15;21(9):1588-95. doi: 10.1021/bc900434c.
In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.
在抗体药物偶联物曲妥珠单抗-DM1（T-DM1）的制造过程中，抗体曲妥珠单抗（Tmab）上的赖氨酸残基被修饰以形成中间产物 Tmab-MCC（T-MCC），然后与药物 DM1 缀合。我们的目标是了解修饰和缀合步骤对抗体理化稳定性的影响。采用热诱导应力条件评估 Tmab 与其修饰和缀合形式的结构稳定性，评估含有 Tmab、T-MCC 和 T-DM1 的制剂。采用 DSC、SEC、CE-SDS 和 LC-MS 研究 Tmab、T-MCC 和 T-DM1 对热应激的稳定性。DSC 热图谱显示 CH2 转变的熔点降低，顺序为 Tmab>T-MCC>T-DM1。根据 SEC 分析，在 40°C 下放置 14 天后，T-MCC（约 32%）和 T-DM1（约 5%）的聚集水平显著增加。Tmab 没有明显的聚集形成。CE-SDS 和 LC-MS 数据表明，在 T-MCC 的情况下，聚集主要是共价的，涉及除形成分子间二硫键交联之外的机制。当添加具有亲核侧链的氨基酸（含巯基（Cys）和羟基部分（Ser、Tyr）时，观察到的 T-MCC 聚集被显著抑制。对于 T-MCC 观察到的共价聚集以及亲核氨基酸（特别是 Cys）抑制它的能力表明，MCC 接头中的马来酰亚胺部分可能反应形成 T-MCC 分子之间的分子间共价交联，可能通过迈克尔加成机制。此外，DSC 结果表明，药物部分 DM1 与 Tmab 的缀合导致抗体 CH2 结构域的不稳定。
