Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran.
Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran.
J Pharm Biomed Anal. 2019 Jul 15;171:1-7. doi: 10.1016/j.jpba.2019.03.011. Epub 2019 Mar 22.
Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.
由于γ-羧化或细胞裂解失败而导致重组激活凝血因子 VII（rFVIIa）在细胞培养介质中缺失 γ-羧基谷氨酸（Gla）结构域的分泌型无 Gla 结构域杂质与天然 rFVIIa 相比，负电荷较少。基于风险评估，这种类型的杂质被认为是 rFVIIa 的关键药物产品质量属性，其在产品批次中的定量分析是质量控制实验室的关键问题。使用三羟甲基氨基甲烷和双三羟甲基丙烷盐缓冲液作为平衡缓冲液和高浓度乙酸铵作为洗脱液，通过强阴离子交换色谱（SAX）对无 Gla 结构域杂质进行分析。在无 Gla 结构域杂质的洗脱时间出现幽灵峰，其强度显著，导致相关峰变形，并对该杂质的稳健和准确定量造成干扰。随后，通过 LC-ESI-MS 对幽灵峰进行分析，以确定其结构，结果显示 m/z 值为 905.27、623.53、341.60 和 563.73。为了找到这些幽灵峰的来源，考虑了水的质量、缓冲盐和 Chelex-100 以及流动相 A 的离子强度（添加 25 mM NaCl）作为影响参数，并使用 DOE 软件设计了几个实验，以优化方法的最佳条件，即具有最低幽灵峰噪声的最高质量。通过 DOE 结果的解释，可以得出结论，高质量的水和缓冲盐以及高质量的 Chelex-100 树脂是实现最低幽灵峰方法的重要因素。然而，向流动相 A 中添加 25 mM NaCl，无论是使用质量较低的缓冲盐还是等级较低的水，都可以得到具有可接受幽灵峰的高质量色谱峰。LC/MS 分析表明，双三羟甲基丙烷的宏观结构可能是由于氢键与彼此或 Tris 分子相互作用而形成的，这可能是幽灵峰的来源。
