Purpose

To understand the impact of methionine oxidation in GCSF on efficacy (neutrophil production/activation) and safety (biochemical and histopathological changes).

Methods

Nine GCSF biosimilars were analyzed for the levels of residual iron and copper content. Oxidation in GCSF was induced by H₂O₂ 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.

Results

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.

Conclusion

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.

中文翻译：

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了解GCSF中的氧化倾向并评估其安全性和有效性

目的

了解GCSF中蛋氨酸氧化对功效（中性粒细胞产生/激活）和安全性（生化和组织病理学变化）的影响。

方法

分析了九种GCSF生物仿制药的残留铁和铜含量。通过H ₂ O ₂处理诱导GCSF中的氧化，并制备了四个样品：wtGCSF（无氧化），MetO（1138），MetO（1,138,127）和MetO（1138,127,122）。这些样品用于通过生物层干涉术评估与GCSF受体（GCSFR）的结合亲和力，使用圆二色性评估热稳定性，并使用相关的基于细胞的分析方法评估体外效力。体内药效学检查了GCSF蛋氨酸氧化后嗜中性粒细胞产生的变化，其结果与GCSF介导的嗜中性粒细胞活化/成熟相关基因的差异表达有关。还进行了包括生化和组织病理学改变在内的临床前安全性研究。

结果

Met 122和Met 127对效能的影响最大。与GCSFR的较低结合亲和力被确定为降低功效和效能的根本原因。已经阐明了Asp 110在GCSF中作为蛋氨酸氧化过程中对功效产生不利影响的关键残基的作用。通过中性粒细胞成熟所需下游基因的差异表达，与GCSF的体外结合亲和力受损表现为体内药效学差异。

结论

来自本研究的数据表明，GCSF中的蛋氨酸氧化是至关重要的质量属性，在商业生产和随后的供应链阶段需要仔细监控。