Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA, 02142, USA.
Amino Acids. 2019 Sep;51(9):1353-1363. doi: 10.1007/s00726-019-02772-9. Epub 2019 Aug 24.
Interferons are signaling proteins that belong to the large class of cytokines and human interferons which are classified based on the type of receptor interactions: type I, II and III. IFNα2b belongs to the type I interferon class with a major therapeutic application for the treatment of hepatitis B and C infections. A recombinant form of IFNα2b expressed in E. coli, known as IntronA, has been approved by US Food and Drug Administration (FDA). IFN γ, also known as type II interferon, plays a significant role in the inhibition of viral replication. Actimmune is a US Food and Drug Administration (FDA) approved version of IFN γ for the indication of reducing infections associated with chronic granulomatous disease and severe malignant osteopetrosis. In this study we have applied advanced analytical methods for the characterization of IFNα2b and IFN γ produced from Pichia pastoris. The multi-enzyme digestion approach has been developed to allow measurement of 100% sequence coverage and detailed analysis of post-translational variants and degradation products. In this manner, we identified the following variants in IFN α2b: N-terminal residual leader sequence, an amino acid substitution, oxidation of methionine residues and two sites of high mannose N-glycosylation. In the Pichia IFN γ produced material, our approach detected variants resulting from glycosylation, C-terminal proteolysis, oxidation of methionine residues and deamidation. In this manner, the analytical program was able to support rapid process development as well as identify product variants and degradation products in the resulting product.
干扰素是一类信号蛋白，属于细胞因子大家族，根据与受体相互作用的类型，人类干扰素可分为 I 型、II 型和 III 型。IFNα2b 属于 I 型干扰素，主要用于治疗乙型和丙型肝炎感染。一种在大肠杆菌中表达的 IFNα2b 重组形式，称为 IntronA，已获得美国食品和药物管理局（FDA）的批准。IFNγ，又称 II 型干扰素，在抑制病毒复制方面发挥重要作用。Actimmune 是一种经美国食品和药物管理局（FDA）批准的 IFNγ，用于减少与慢性肉芽肿病和严重恶性骨质增生症相关的感染。在这项研究中，我们应用了先进的分析方法来表征毕赤酵母表达的 IFNα2b 和 IFNγ。我们开发了多酶消化方法，以实现 100%序列覆盖，并对翻译后变体和降解产物进行详细分析。通过这种方法，我们在 IFNα2b 中鉴定出以下变体：N 端残留的前导序列、一个氨基酸取代、甲硫氨酸残基的氧化和两个高甘露糖 N-糖基化位点。在毕赤酵母 IFNγ 产生的物质中，我们的方法检测到了糖基化、C 端蛋白水解、甲硫氨酸残基氧化和脱酰胺作用导致的变体。通过这种方式，分析方案能够支持快速的工艺开发，并识别出最终产品中的产品变体和降解产物。
