Division of Bacterial, Parasitic, and Allergenic Products; Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA.
Appl Microbiol Biotechnol. 2023 Jun;107(12):4069-4077. doi: 10.1007/s00253-023-12558-5. Epub 2023 May 6.
Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland-Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. KEY POINTS: • Enumeration of live biotherapeutics is essential for product quality and safety. • Conventional CFU counting may not differentiate between strains in microbial products. • This approach was developed for direct enumeration of mixed bacterial strains simultaneously.
活生物治疗产品（LBP）批次的特征通常包括对活力的测量，例如菌落形成单位（CFU）。然而，在单一产品中存在具有相似生长要求的多种生物体时，菌株特异性 CFU 计数测定可能会变得复杂。为了克服从多菌株混合物中获得菌株特异性 CFU 值的特定挑战，我们开发了一种将基于质谱的菌落鉴定与传统 CFU 测定相结合的方法。该方法使用由多达 8 种细菌菌株组成的定义混合物进行了评估。在 8 种菌株混合物的四个重复批次中，所有测量的菌株的观察值与预期值的差异小于 0.4 log CFU（差异范围为-0.318 至+0.267）。观察值与预期值之间的平均差异为+0.0308 log CFU，95%一致性区间为-0.347 至 0.408（Bland-Altman 分析）。为了估计精密度，通过三位不同用户对 8 种菌株混合物的单个批次进行了三重复测定，总共进行了 9 次测量。所测量的 8 种菌株的 pooled 标准偏差值范围为 0.067 至 0.195 log CFU，用户平均值没有显著差异。利用新兴的基于质谱的菌落鉴定工具，开发并测试了一种用于同时对混合菌株混合物中的活菌进行计数和鉴定的新方法。这项研究表明，该方法有可能同时对多达 8 种细菌菌株进行准确且一致的测量，并可能为未来的改进和修改提供灵活的平台。关键点：• 活生物治疗产品的计数对于产品质量和安全性至关重要。• 常规 CFU 计数可能无法区分微生物产品中的菌株。• 这种方法是为直接同时计数混合细菌菌株而开发的。
