MedImmune , Analytical Biotechnology, One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Anal Chem. 2014 Jul 15;86(14):6850-7. doi: 10.1021/ac404218t. Epub 2014 Jul 1.
We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.
我们描述了在近紫外可见光辐照和热应激的单克隆抗体（mAb）药物制剂中导致颜色变化的降解产物的特征。使用反相 HPLC 和尺寸排阻 HPLC 结合吸收光谱法对处理后的样品进行了表征。两种方法都表明，颜色变化是由于 mAb 上形成的生色团引起的，但与光辐照和热应激 mAb 样品中的制剂赋形剂无关。这些生色团通过一种新的肽图方法进一步定位，该方法结合了质谱和吸收光谱法。质谱法鉴定出主要色氨酸氧化产物为犬尿氨酸（Kyn）、N-甲酰犬尿氨酸（NFK）和羟色氨酸（OH-Trp）。吸收光谱表明，每种色氨酸氧化产物在 280nm 以上都表现出一个独特的吸收带，其波长向长波方向移动的顺序为 OH-Trp < NFK < Kyn。在 420nm 处检测到含有 Kyn 的肽。未观察到甲硫氨酸或组氨酸氧化产物的新吸收带。这证实了色氨酸氧化产物（而非甲硫氨酸和组氨酸氧化产物）是导致颜色变化的原因。值得注意的是，在热应激 mAb 样品中鉴定出重链上色氨酸 107 的氢（2 Da 质量减少）丢失的新氧化产物。这种氧化的色氨酸残基在最大吸收波长 335nm 处表现出一个独特的吸收带，这是导致 mAb 药物制剂颜色变黄的原因。本研究表明，结合质谱和吸收光谱的新肽图方法可用于鉴定色氨酸氧化产物作为应激 mAb 药物制剂中导致颜色变化的生色团。
