在提议的生物类似药 GP2013 利妥昔单抗与原研药利妥昔单抗之间的理化性质和功能可比性。

BACKGROUND: Regulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab. OBJECTIVE: To compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods. METHODS: Primary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D (1)H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays. RESULTS: Intact mass analysis of GP2013 and the heavy and light chains using RP HPLC-ESI-MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab's functional activities, GP2013 could not be distinguished from originator rituximab. CONCLUSION: GP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

背景：生物类似药产品的监管批准是基于其与原研产品的可比性。彻底的理化性质和功能可比性研究是证明生物相似性的关键要素。在此，我们报告了拟议的生物类似药利妥昔单抗（GP2013）和原研利妥昔单抗的特征描述。

目的：使用广泛的常规分析和扩展特性鉴定方法比较 GP2013 与原研利妥昔单抗。

方法：使用包括高效液相色谱电喷雾电离质谱（HPLC-ESI-MS）、用 UV 和 MS 检测的肽图、圆二色性（CD）、傅里叶变换红外（FTIR）光谱、氢氘交换（HDX）MS、一维（1）H 核磁共振（NMR）光谱、X 射线晶体学和差示扫描量热法（DSC）在内的多种方法分析一级和高级蛋白质结构。使用阳离子交换色谱（CEX）和反相（RP）HPLC 中的肽图评估电荷和氨基酸修饰。使用硼酸盐亲和色谱法确定相对糖化程度。使用 2-氨基苯甲酰胺（2-AB）标记和正相 HPLC 分离后，分别使用荧光和 MS 检测鉴定和定量聚糖。使用带有十二烷基硫酸钠（SDS）的还原毛细管电泳（CE-SDS）测定糖基化位点占有率。使用还原和非还原 CE-SDS、尺寸排阻色谱（SEC）和不对称流场流分离（AF4）测定尺寸异质性。生物学特性鉴定包括一系列生物测定（体外靶标结合、抗体依赖性细胞介导的细胞毒性[ADCC]、补体依赖性细胞毒性[CDC]和细胞凋亡）和表面等离子体共振（SPR）Fc 受体结合测定。

结果：使用 RP HPLC-ESI-MS 对 GP2013 及其重链和轻链进行完整质量分析显示出利妥昔单抗的预期分子质量。GP2013 与原研利妥昔单抗的氨基酸序列完全相同。使用 RP-HPLC-UV/MS 肽图进行进一步的序列确认显示，Lys-C 消化的 GP2013 和原研利妥昔单抗的色谱图不可区分。使用大量冗余和正交方法表明，GP2013 的高级结构与原研利妥昔单抗无法区分。GP2013 和原研利妥昔单抗在电荷变异体、特定氨基酸修饰和聚糖模式方面具有可比性。与原研产品相比，GP2013 的纯度、聚集体和颗粒水平也相似。在功能方面，通过使用涵盖利妥昔单抗广泛功能活性的综合生物测定和结合测定套件，GP2013 与原研利妥昔单抗无法区分。

结论：GP2013 在一级和高级结构、翻译后修饰和尺寸变异体水平上被证明在理化性质上与原研利妥昔单抗高度相似。广泛的功能特性鉴定套件表明，GP2013 具有与原研利妥昔单抗相同的生物学特性。