Live Biotherapeutic Products (LBPs) are drug products designed to prevent, treat, or cure disease using living microorganisms. These therapeutics present unique challenges for quality control, especially in the development of microbial safety methods. This blog post outlines the challenges of quality control for LBPs and offers alternative testing strategies for optimal safety and efficiency.

Why Traditional QC Methods Fail for Live Biotherapeutic Products

USP <61> and USP <62> are compendial methods that have been designed and qualified to detect presence of contaminants in pharmaceutical products. USP <61> focuses on determining the number of contaminating bacteria or yeast and mold in a product, while <62> looks for very specific organisms that can be dangerous for exposure in certain product types. These are safety methods to ensure that the product will not cause a patient to become sick or develop infections due to a bacterial or fungal contaminant.

To use these methods, quality control laboratories, like those at List Labs, must prove that these methods work with each specific product. The USP chapters define how to perform method suitability testing, including using challenge organisms to ensure that if a contaminant is present, you can accurately and consistently detect that contaminant. The suitability evaluation essentially makes sure that the product will not mask a contaminant while using these methods.

When it comes to Live Biotherapeutic Products (LBPs), complications arise when attempting method suitability testing for USP <61> and <62>. In USP <61>, several method techniques are available. LBPs present problems with each of these:

• In membrane filtration, the filter traps bacteria, including the product organism. The filter can become clogged with the sheer number of cells, preventing filtration.
• The other method techniques of pour plating and spread plating introduce a different problem. The generic growth medium prescribed for the test will typically encourage the growth of not only the challenge organisms, but the product organism itself. A “lawn” of growth or cloudiness from the product can inhibit or prevent visualization and accurate enumeration of challenge organisms.

For USP <62>, the most common complication for suitability is in detection of organisms where the initial incubation, or enrichment period, is in a non-selective broth like TSB. Suitability evaluation requires a small inoculum of the challenge organism of <100 CFU, tested with a minimum required amount of 1g or 1mL of the product itself. During this enrichment period, the product organism is likely subjected to its own ideal growth conditions and nutrients. This results in the LBP outcompeting the small inoculum of challenge organisms for the available nutrients, making it difficult, if not impossible, to recover the challenge organism.

Regulatory Gaps – Differences between USP and EP guidance

The USP chapters include some examples and procedures for method modifications when suitability for a product does not meet the method requirements. However, these modifications often fall short of addressing the problem, since these methods are not designed for products with high concentrations of live organism. Regulators, however, still require contaminant enumeration and detection methods to be established for LBPs to ensure product safety. In 2019, the European Pharmacopoeia published monograph 3053 to provide guidance on LBP manufacturing, including how to set quality requirements. The monograph also included two chapters specific to contamination detection methods.

Chapter 2.6.36 is the EP’s equivalent to USP <61> for LBPs. Instead of the USP’s TAMC, or total aerobic microbial count, this chapter defines the test as AMCC, which stands for aerobic microbial contaminating count, and likewise YMCC, yeast and mold contaminating count. This implies the method looks for contaminating morphologies, with the expectation that the product organism is among the growth on the plate. Chapter 2.6.38 is the USP <62> equivalent, as an enrichment-based method for detection of objectionable organisms.

Both chapters provide a decision tree for method suitability, with suggested next steps and modifications to attempt when recovery of the challenge organisms is unsuccessful. Many of the suggested modifications are like those outlined in the USP, but the EP chapters dive more into suggested media, neutralizers, and specialized techniques that can be used for specific LBP organisms.

QC Roadblocks – Practical issues labs face (strain interference, lack of neutralizers, resource intensity)

Even with the guidelines for modifications given by the USP and the EP, quality labs can run across many roadblocks during method development for contamination detection, as follows:

Differentiation: When the product organism(s) is subjected to growth-promoting testing conditions, there will always be the issue of interference from the product, either preventing, inhibiting, or impacting accuracy in detection of contaminants. This becomes especially difficult when the product organism grows similar in appearance, or grows on the same selective media, as a challenge organism.

LOQ: a typical method modification includes diluting the product to reduce the interference of the LBP with the growth of the challenge organism. If this is done in an enumeration method, that dilution must factor into final calculations. The total amount of product that ends up on the plate will determine the limit of quantitation or LOQ for detection of contaminants. If the LOQ surpasses the specification for the product type, the test will not be acceptable.

Neutralizers: Neutralizers can be added into media or diluent to suppress the activity of the product. However, any neutralizer that is specific to the live organism is typically not just selective for that one strain. Antibiotics or selective media components can also inhibit the growth of the challenge organisms, and other bacteria that may be of concern as a contaminant in the product, preventing detection.

Resources: For a USP <62> method, increasing the dilution of the product is an option to mitigate the inhibition caused by the growth of the product organism. But contrary to USP <61>, this must be done without decreasing the required amount of product to be tested, leading to increased volumes of enrichment media. There are limits to this approach due to incubator space and autoclave size for labs making their own media.

All of these roadblocks become exponentially problematic when the LBP contains a consortium of organisms, rather than a single strain.

Future Solutions – Fit-for-purpose controls, alternative methods, and sequencing-based tools

One solution for the obstacles in contamination method development is to utilize alternative challenge organisms. Different challenge organisms can be used in method suitability when the LBP falls into the same objectionable organism category or is the same organism as the challenge organism defined for method suitability. If the LBP is an E. coli, for example, Enterococcus strains can be substituted as challenge organisms to replace E. coli. Depending on the product type, a lab might instead use a pathogenic E. coli, like an STEC or an EHEC for the challenge, along with chromogenic media, to demonstrate the ability to recover and differentiate between pathogenic E. coli and the product organism.

Alternative culture methods should be explored when the USP or EP methods and suggested modifications remain unsuccessful. In order to design an appropriate method, a quality control team needs to understand how the product organism behaves under the intended or potential testing conditions. Product organism traits that are helpful to know include the ideal and non-ideal growth conditions, pH tolerance range, antibiotic resistance profile, carbohydrate fermentation capabilities, and growth appearance on a variety of selective media.

Culture methods which evaluate carbohydrate utilization, nitrate reduction, or unique growth on selective media can be combined to establish a successful contaminant detection panel. Other analytical tools may be used to supplement a culture method, and can serve as confirmatory or supplemental testing to the traditional methods. Molecular methods, including PCR, DNA Microarrays, MALDI-TOF, ELISA, FISH, ESI-MS, and LFIA can all be useful in identifying specific DNA sequences or antigens of challenge organisms, to differentiate a contaminant from the product organism.

The right materials and methods for safe LBPs

Advancing live biotherapeutics will require new tools and collaboration across the industry. Microbiologics supports this effort by providing challenge organisms, LBP strain characterization, and microbiome testing services that help innovators and CDMOs like List Labs develop reliable, regulatory-ready QC methods. In any method qualification, there are important parameters to consider and evaluate, including LOD/LOQs, Repeatability, Specificity, Intermediate Precision, Robustness, Equivalency, etc. Since the panel of challenge organisms establishes specificity, choosing the right organisms is essential. The intended patient population, immunocompromised or pediatric patients for example, may require evaluation of additional high-risk organisms. A challenge organism panel that includes USP and EP-required organisms, environmental contaminants, slow-growing organisms, and organisms that may be difficult to distinguish from the product, will help to support the robustness of any alternative method. CDMOs like List Labs, with years of expertise in biologic products, can assess risks, provide guidance, and develop the right methods for safety and contaminant detection to comply with regulatory requirements.

Guest Author:

Sarah Henning Senior Manager, Quality Control List Labs

Sarah Henning is the Senior Manager of Quality Control at List Biological Labs, where she specializes in QC method development for bacterial products and recombinant proteins. With nearly a decade of experience in public health and pharmaceutical microbiology, she is passionate about advancing the field in Live Biotherapeutics. A key opinion leader in the industry, Sarah recently presented at a national conference for microbiome research, sharing her insights on creating novel processes for contaminant detection and ensuring product safety. When not at the office, you can find her exploring the California coast with her two dogs, playing volleyball, and volunteering for animal sanctuaries. Connect with Sarah on LinkedIn.com/in/sarahchenning