Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia

Abstract

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 log₁₀ CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log₁₀ 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 log₁₀ 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.

Keywords: Advanced manufacturing techniques; Biologics; Live Biotherapeutic Products; Microbial Enumeration.

Fig. 1

Results from preliminary enumeration of four-strain model consortia. Paired data points (connected) represent expected (open circles) and observed (closed circles) log₁₀ CFU values obtained from a single batch enumeration measurement. Consortia consisted of either (A) four different Gram-negative strains from the genus Bacteroides, or (B) four different Gram-positive strains from three different bacterial genera. The genus Bifidobacterium was abbreviated to “Bif.” to avoid confusion with strains from the genus Bacteroides

Fig. 2

Results and agreement analysis from eight-strain consortium enumeration experiments. (A) Expected and observed log₁₀ CFU/mL values with connecting lines indicating paired measurements from the same experimental batch. (B) Correlation analysis of paired expected and observed values with line of identity shown (dashed, red). (C) Bland–Altman plot for analysis of quantitative method agreements with estimates of the estimated bias (solid black line) and 95% limits of agreements (dashed black lines) displayed

Fig. 3

Component strain CFU results from within-laboratory precision study conducted by three different lab users evaluating a single batch of frozen, eight-strain mixture in replicate. Pooled mean and standard deviations shown, with different user values indicated by shape/color of symbols. Overall, differences between users did not contribute significantly to variation in the data (p = 0.775, 2-way ANOVA)