Gurel Busra, Berksoz Melike, Capkin Eda, Parlar Ayhan, Pala Meltem Corbacioglu, Ozkan Aylin, Capan Yılmaz, Daglikoca Duygu Emine, Yuce Meral
SUNUM Nanotechnology Research and Application Center, Sabanci University, Istanbul 34956, Turkey.
ILKO ARGEM Biotechnology R&D Center, Istanbul 34906, Turkey.
Pharmaceutics. 2022 Jul 28;14(8):1571. doi: 10.3390/pharmaceutics14081571.
Avastin is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants' in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.
阿瓦斯汀是一种人源化重组单克隆抗体，通过靶向血管内皮生长因子-A（VEGF-A）来抑制血管生成，从而用于治疗癌症。SIMAB054是本研究中开发的一种阿瓦斯汀生物类似药候选物，其电荷变体谱与其原研药不同。因此，它被分离为酸性、主要和碱性异构体，并通过阳离子交换色谱法（CEX）进行物理收集，以进行全面的结构和功能分析。将原研产品分离为相同的种类并采用相同的方法收集，用作比较分析的参考。采用超高效液相色谱（UPLC）电喷雾四级杆飞行时间质谱（ESI-QToF）在完整蛋白质和肽水平上分析导致电荷异质性的修饰。通过完整单克隆抗体分析监测样品的C末端赖氨酸切割和糖基化谱。通过对选定信号肽进行肽图分析来确定包括氧化、脱酰胺和N末端焦谷氨酸形成在内的翻译后修饰。通过表面等离子体共振（SPR）研究揭示了分离的电荷异构体对抗原VEGF-A和新生儿受体FcRn的相对结合亲和力。结果表明，原研产品和SIMAB054的所有CEX级分都具有相同的结构变体，尽管比例不同。常见的糖型和翻译后修饰是相同的，但某些样品的百分比不同。差异主要源于额外的C末端赖氨酸残基的存在，这些残基在体内易于酶解，因此之前被评估为临床无关。另一个关键发现是不同电荷种类中存在不同的糖蛋白形式，例如酸性种类中半乳糖基化增加，碱性种类中岩藻糖基化缺失。对分离的电荷变体进行的SPR表征进一步证实，在生物类似药候选物的CEX分析中发现的碱性种类在原研产品中也存在，尽管含量较低。电荷变体的体外抗原和新生儿受体结合活性在样品之间有所不同，可以用更大的样本集在体内进一步研究，以揭示对候选药物药代动力学的影响。微小的结构差异可能解释了分离的电荷变体中的抗原结合差异，这是可比性研究中的一个关键参数。因此，除非观察到的结合差异得到合理解释并证明没有任何临床影响，否则这种生物类似药候选物可能不符合严格的监管标准。
