Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India.
Pharm Res. 2020 Sep 29;37(10):207. doi: 10.1007/s11095-020-02928-3.
To understand the impact of methionine oxidation in GCSF on efficacy (neutrophil production/activation) and safety (biochemical and histopathological changes).
Nine GCSF biosimilars were analyzed for the levels of residual iron and copper content. Oxidation in GCSF was induced by HO treatment and four samples were prepared: wtGCSF (no oxidation), MetO (1138), MetO (1,138,127) and MetO (1138,127,122). These samples were used to evaluate binding affinity with the GCSF receptor (GCSFR) using biolayer interferometry, thermal stability using circular dichroism and in vitro potency using a relevant cell-based assay. In vivo pharmacodynamics examined changes in neutrophil production upon GCSF methionine oxidation, with the outcome correlated with the differential expression of genes implicated in the GCSF mediated neutrophil activation/ maturation. Pre-clinical safety studies including biochemical and histopathological changes were also performed.
Met 122 and Met 127 have the most deleterious effect on the potency. Lower binding affinity with GCSFR was identified as the underlying cause for lower efficacy and potency. Role of Asp 110 in GCSF as the critical residue having adverse impact on efficacy in context of methionine oxidation has been elucidated. Impairment of in vitro binding affinity with GCSF manifests as in vivo pharmacodynamic differences via differential expression of downstream genes required for neutrophil maturation.
The data from the present study suggests that methionine oxidation in GCSF is a critical quality attribute that needs careful monitoring and control during commercial manufacturing and subsequent supply chain stages.
了解 GCSF 中甲硫氨酸氧化对疗效（中性粒细胞生成/激活）和安全性（生化和组织病理学变化）的影响。
分析了 9 种 GCSF 生物类似物的残留铁和铜含量水平。通过 HO 处理诱导 GCSF 氧化，并制备了 4 个样品：wtGCSF（未氧化）、MetO（1138）、MetO（1138、127）和 MetO（1138、127、122）。使用生物层干涉法评估这些样品与 GCSF 受体（GCSFR）的结合亲和力，使用圆二色性评估热稳定性，使用相关的基于细胞的测定法评估体外效力。体内药效学研究检查了 GCSF 甲硫氨酸氧化后中性粒细胞生成的变化，结果与 GCSF 介导的中性粒细胞激活/成熟相关基因的差异表达相关联。还进行了包括生化和组织病理学变化在内的临床前安全性研究。
Met 122 和 Met 127 对效力的影响最大。与 GCSFR 的结合亲和力降低被确定为效力和效力降低的潜在原因。已阐明 Asp 110 在 GCSF 中作为关键残基在甲硫氨酸氧化背景下对疗效产生不利影响的作用。体外结合亲和力的损害通过对中性粒细胞成熟所需的下游基因的差异表达在体内药效学上表现为差异。
本研究的数据表明，GCSF 中的甲硫氨酸氧化是一个关键的质量属性，在商业制造和随后的供应链阶段需要仔细监测和控制。
