August 5, 2025

By: List Labs

As the field of live biotherapeutic products (LBPs) rapidly advances, so too does the need for rigorous, nuanced microbiological quality control (QC). LBPs—live microorganisms used to prevent, treat, or cure diseases—present unique challenges when meeting regulatory safety standards. Traditional compendial methods were not designed for products teeming with live bacteria. In this blog, we explore the scientific and methodological reasons why standard tests often fail LBPs and what alternative strategies are needed to ensure reliable results.

Standard Methods vs. Microbial Density for LBS

USP <61> and <62> were designed for low-bioburden products. LBPs, by contrast, contain dense populations of live organisms that can interfere with the test system. High CFU counts will clog filters or lead to lawn-like overgrowth on agar, making it nearly impossible to detect contaminants.

The therapeutic organism itself may also outcompete objectionable organisms in enrichment broths, rendering tests like USP <62> ineffective. Even selective agars may be insufficient, failing to suppress the LBP strain.

Real-World Breakdown of USP <61> and <62>

• USP <61> Challenges: Membrane filtration becomes unusable due to the live organism product becoming trapped in the filter. Spread plate and pour plate methods introduce complications due to dense growth of the product on non-selective media. Diluting the product can help, but excessive dilution can prevent detection of contaminants at the necessary levels. Neutralizers that selectively suppress the product organism aren’t typically available.
• USP <62> Challenges: Enrichment broths and selective agars often allow LBP overgrowth. Recovery of challenge organisms at the required low starting inoculum becomes inconsistent, particularly when the LBP shares similar nutrient and environmental preferences. Product dilution requires large volumes of media to maintain the required product sample size.

Performance Metrics: Proving the Case for Alternative LBP Microbial Tests

Validating an alternative method requires strong data and clear performance benchmarks. These include:

• LOD/LOQ: Must meet sensitivity thresholds for product type
• Specificity: Must recover target contaminants amidst the product strain
• Repeatability & Robustness: Must perform across replicates and batches within the potency range
• Equivalency: Must meet or exceed USP performance standards

These metrics are particularly difficult to meet during early-phase clinical trials, where sample volumes are small and timelines are short.

Alternative Methods That Work

When standard tests fail, here are some proven alternatives:

• Metabolic profiling via carbohydrate fermentation or nitrate reduction
• Chromogenic substrates that visually differentiate strains
• Selective media or antibiotics tuned to inhibit specific product strains
• Custom incubation conditions like pH or temperature variation

These techniques require deep knowledge of both the LBP organism and the contaminants being targeted. Developers must also account for growth kinetics, metabolic outputs, and competitive dynamics.

The Need for Product-Specific Method Design

Organism-specific modifications are crucial. An LBP made with Bacillus spores requires different testing protocols than one composed of lactic acid bacteria or yeast. For culture-based methods, adjustments to media, incubation parameters, and detection techniques must be tailored.

The failure to do so results in unreliable testing, regulatory setbacks, or worse—the release of a contaminated product.

Rethinking QC for Living Drugs

Standard methods are a starting point, not a solution. For LBPs, method development must be customized, validated, and supported by microbiological expertise. The risks of relying solely on compendial tests are too great.

At List Labs, we work hand-in-hand with innovators to build QC strategies from the ground up—backed by science, tailored to your organism, and ready for regulatory review.

Contact us today to start your project!