Scaling analytical RP-HPLC to semi-preparative for fractionation and characterization of pegfilgrastim oxidized variants

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2024-12-11 DOI:10.1016/j.jpba.2024.116633

Haley Sutton, Teresa Youngberg, Christian Perez, Anke Hartung, Xuemei Han, Navin Rauniyar

{"title":"Scaling analytical RP-HPLC to semi-preparative for fractionation and characterization of pegfilgrastim oxidized variants","authors":"Haley Sutton, Teresa Youngberg, Christian Perez, Anke Hartung, Xuemei Han, Navin Rauniyar","doi":"10.1016/j.jpba.2024.116633","DOIUrl":null,"url":null,"abstract":"<div><div>Pegfilgrastim, a 40 kDa PEGylated form of recombinant human granulocyte colony-stimulating factor (rhG-CSF), is a biotherapeutic protein used to treat chemotherapy-induced neutropenia. To ensure the product is safe and effective, stringent monitoring of product-related impurities, particularly those arising from oxidative degradation, is necessary. This study focuses on the isolation and characterization of oxidized variants in pegfilgrastim using a multi-step approach that includes method transfer to semi-preparative High-Performance Liquid Chromatography (HPLC), mass spectrometry, and an in vitro cell-based potency assay (CBPA). The analytical reversed-phase (RP)-HPLC method was successfully scaled up and optimized for isolating oxidized variants in H<sub>2</sub>O<sub>2</sub>-treated pegfilgrastim. Mass spectrometry analysis identified the degree and specific sites of oxidation, with Met1 being the most susceptible. CBPA showed that oxidation at Met1 alone had minimal impact on functional activity, while oxidation at both Met127 and Met138 led to significant reductions in activity. The impact of oxidation at all four sites in pegfilgrastim could not be assessed due to significant degradation. These findings highlight the importance of robust analytical strategies in the characterization and control of pegfilgrastim impurities.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116633"},"PeriodicalIF":3.1000,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of pharmaceutical and biomedical analysis","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0731708524006757","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}

引用次数: 0

Abstract

Pegfilgrastim, a 40 kDa PEGylated form of recombinant human granulocyte colony-stimulating factor (rhG-CSF), is a biotherapeutic protein used to treat chemotherapy-induced neutropenia. To ensure the product is safe and effective, stringent monitoring of product-related impurities, particularly those arising from oxidative degradation, is necessary. This study focuses on the isolation and characterization of oxidized variants in pegfilgrastim using a multi-step approach that includes method transfer to semi-preparative High-Performance Liquid Chromatography (HPLC), mass spectrometry, and an in vitro cell-based potency assay (CBPA). The analytical reversed-phase (RP)-HPLC method was successfully scaled up and optimized for isolating oxidized variants in H₂O₂-treated pegfilgrastim. Mass spectrometry analysis identified the degree and specific sites of oxidation, with Met1 being the most susceptible. CBPA showed that oxidation at Met1 alone had minimal impact on functional activity, while oxidation at both Met127 and Met138 led to significant reductions in activity. The impact of oxidation at all four sites in pegfilgrastim could not be assessed due to significant degradation. These findings highlight the importance of robust analytical strategies in the characterization and control of pegfilgrastim impurities.

查看原文本刊更多论文

聚非格拉西汀氧化变异体半制备的定量分析RP-HPLC。

Pegfilgrastim是一种40 kDa聚乙二醇形式的重组人粒细胞集落刺激因子（rhG-CSF），是一种生物治疗蛋白，用于治疗化疗诱导的中性粒细胞减少症。为了确保产品安全有效，必须严格监控产品相关杂质，特别是氧化降解产生的杂质。本研究的重点是利用多步骤方法分离和表征聚乙二醇非格拉斯汀的氧化变异，包括方法转移到半制备型高效液相色谱（HPLC）、质谱分析和体外细胞效价测定（CBPA）。采用反相高效液相色谱法（RP -HPLC）分离h2o2处理的聚非格昔汀的氧化变异体。质谱分析确定了氧化的程度和特定位点，其中Met1是最易受影响的。CBPA表明，Met1的氧化对功能活性的影响最小，而Met127和Met138的氧化导致活性显著降低。pegfilgrastim中所有四个位点的氧化影响由于明显的降解而无法评估。这些发现突出了强大的分析策略的重要性，在表征和控制聚乙二醇格拉西汀杂质。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.