Stracke Jan, Emrich Thomas, Rueger Petra, Schlothauer Tilman, Kling Lothar, Knaupp Alexander, Hertenberger Hubert, Wolfert Andreas, Spick Christian, Lau Wilma, Drabner Georg, Reiff Ulrike, Koll Hans, Papadimitriou Apollon
a Biochemical and Analytical Research; Large Molecule Research ; Roche Pharma Research and Early Development (pRED); Roche Innovation Center ; Penzberg , Germany.
MAbs. 2014;6(5):1229-42. doi: 10.4161/mabs.29601. Epub 2014 Oct 30.
Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.
在药物开发过程中，在制造和储存期间保持治疗性抗体的化学和结构完整性是一项重大挑战。经常观察到Fc甲硫氨酸Met252和Met428的氧化，如果其含量高，会导致与FcRn的亲和力降低和更快的血浆清除率。由于两个位置同时发生氧化，它们对药代动力学性质伴随变化的各自贡献尚未明确确定。一种新型的pH梯度FcRn亲和色谱方法被应用于从氧化的IgG1制剂中分离出三种抗体氧化变体，基于它们的FcRn结合特性。物理化学表征显示，这三种氧化变体主要在每个IgG中氧化型Met252的数量（0、1或2）上有所不同，但在氧化型Met428的含量上没有显著差异。与氧化型Met252含量的增加相对应，在体外观察到FcRn亲和力逐步降低，以及在人FcRn转基因小鼠中清除更快和终末半衰期更短。与未修饰的IgG相比，每个抗体单个Met252氧化对药代动力学（PK）没有显著影响。重要的是，只有在Met252处两条重链都被氧化的分子才表现出明显更快的清除率。相比之下，Met428氧化对PK没有明显的负面影响，甚至导致FcRn结合有所改善和清除减慢。然而，这种微小的影响似乎被Met252氧化的主导作用所消除。通过对IgG氧化变体进行功能色谱分离，随后进行物理化学和生物学表征的新方法，首次为含有相对较高Met252和Met428氧化水平的抗体制剂的药代动力学性质未改变提供了实验支持的解释。
