Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, USA.
Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, USA.
Pharm Res. 2023 Jun;40(6):1425-1433. doi: 10.1007/s11095-023-03527-8. Epub 2023 May 1.
An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.
氨基酸序列变异（SV）被定义为蛋白质药物产品中的非预期氨基酸取代。SV 导致产品异质性，并可能影响产品质量、安全性、免疫原性和疗效。在整个产品生命周期中，包括克隆选择、营养饲料策略的开发、商业制造工艺以及批准后监测产品质量的变更，都需要对生物治疗药物进行 SV 分析。SV 分析的建议分析程序包括定性（鉴定 SV）和定量（定量鉴定的 SV）两部分。SV 分析的复杂性和当前程序的多样性突出表明需要采用系统方法来评估这些方法在可靠鉴定和定量生物治疗药物中的 SV 的能力。我们在这里描述了一种用于评估 SV 分析程序的“加标控制”方法。该概念源自分析程序验证中常规使用的质量控制样品。一种 FDA 批准的单克隆抗体（mAb）中加入了准确数量的高度同源 mAb，以创建含有低但准确水平“人工”SV 的 mAb 样品。加标控制样品进行变性、还原、烷基化、消化，然后通过高分辨率轨道阱质谱进行分析。通过计算机分析，在两种 mAb 之间发现了四个单氨基酸差异，这些差异可用于代表加标控制样品中的 SV。目前的工作流程可靠地鉴定了所有四个“人工”SV。还评估了鉴定的 SV 的分析范围（0.01%至 2%）、准确性和精密度。总体而言，加标控制样品有助于证明 SV 分析程序（即样品制备、LC 分离、质谱测定和生物信息学软件）符合目的，适合在预定阈值下鉴定和定量 SV。
