Over the last 5-10 years, remarkable advances have been made in endotoxin research. For example, irrefutable data have illuminated the biogenesis and structural differences of lipopolysaccharides concordant with Gram-negative microorganisms’ strain and the conditions they experience. Although the term autochthonous has not been well received in pharmaceutical microbiology, it is important to understand that endotoxins derived from autochthonous microorganisms in manufacturing environments are structurally different from those derived from laboratory-grown Gram-negative microorganisms. Moreover, this term provides an extremely useful microbiological adjective for originating in a particular place. As microbiology advances, we must also advance our language and definitions with fidelity to current art.
Without a doubt or exception, the drug product and drug substance manufacturing processes, which incorporate aqueous environments, possess populations of Gram-negative microorganisms that gear their physiology toward survival. This leads us to many potential consequences and questions regarding the sampling, testing and specifications critical to supplying finished drug products (and devices) that are safe for the recipient patient population.
Of late, the commercial availability from multiple vendors of recombinant alternatives to Limulus amoebocyte lysate, derived from the horseshoe crab, has opened significant opportunities. Equally, accompanying questions about their true equivalence to naturally sourced LAL continue, with some vendor publications reporting a continuing need for optimization, along with recently improved formulations but without an accompanying explanation of why or how (1). Recent research studies by Burgenson combined endotoxins derived from Gram-negative microorganisms autochthonous to a manufacturing process, different LAL-based assays (recombinant and naturally sourced) and a selection of typical and model drug products (2). The outcome of these studies illustrates the need for careful consideration of analytical reagents, test methods and products in establishing a drug product for an endotoxin test method. There remains a way to go in bottoming out some of the remaining kinks in testing and technology for drug- and device-borne endotoxins.
Any endotoxin test is predicated upon an established specification limit for endotoxins founded upon patient safety. In 2002, USP <85> was subsequently harmonized with the Japanese and European pharmacopeias, and the first harmonized chapter appeared in USP 25–NF 20 (3). Although much of the compendial content between the harmonized chapters is identical, there are some region-specific differences. Additionally, the European Medicine Agency’s ICH Guideline Q4B Annex 14 Note for Evaluation and Recommendation of Pharmacopeial Texts on bacterial endotoxins tests, which became effective in May 2013 (4), recommends the use of the harmonized compendial chapters in the European, Japanese and United States pharmacopeias for the bacterial endotoxin test (BET). This is noteworthy because it encourages the possible adoption of a single means of generating endotoxin specifications. At the same time, the U.S. FDA issued complementary guidance confirming the same recognition (5). As a result, the methodology of setting endotoxin specification limits for finished drug products and devices is globally harmonized upon a calculation incorporating consideration of a data-supported threshold pyrogenic dose and patient body mass.
Recently, some health authorities had erroneously suggested the inclusion of an arbitrary safety factor into an endotoxin specification limit. This could be due to a misunderstanding of inherent variability in the BET method. If this supposition is correct, then this is misattributed to the nature of the gel clot method, which is qualitative (either a clot is formed or a clot is not formed), coupled with its use of two-fold dilutions (reflected in 50-200% acceptance criteria of product spike samples). In comparison, the kinetic and endpoint BET methods generally employed are quantitative with an assay variability of approximately 10%. It has also been suggested that the coadministration of multiple therapies and medical devices could result in an aggregate endotoxin load that might breach the threshold for eliciting a pyrogenic reaction. Until now, there has been limited, objective and documented analysis evaluating if reservations in the compendial method of setting endotoxin limits are warranted.
- Stevens, Ingrid, Norihiko Ogura, Madeline Kelley, Robert L. D’Ordine, Hikaru Mizumura, Toshio Oda, Junko Akiyoshi, and Edwin G. Jahngen. “Advanced Recombinant Cascade Reagent Pyrosmart Nextgen® for Bacterial Endotoxins Test as Described in the Pharmacopeias.» BPB Reports 5, no. 5 (2022): 105-14. https://doi.org/10.1248/bpbreports.5.5_105.
- Burgenson, A L. «Comparison of Four Endotoxin Detection Reagents in Measuring Autochthonous Endotoxin Levels in Four Representative Parenteral Products.» Pharmacopeial Forum 49, no. 2 (2023). DocID: GUID-39D7842E-76C1-4CF2-B2F6-A10B2857B74A_10101_en-US.
- U.S. Pharmacopeial Convention. “General Chapter <85> Bacterial Endotoxins Test.” In USP 41-Nf 36, 6011. Rockville, Md.: USP, 2018. DocID: GUID-39D7842E-76C1-4CF2-B2F6-A10B2857B74A_10101_en-US.
- European Medicines Agency. “ICH Guideline Q4b, Annex 14 to Note for Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on Bacterial Endotoxins Tests – General Chapter.” EMA/CHMP/ICH/529785/2010. London: EMA, 2013. https://www.ema.europa.eu/en/documents/regulatory-procedural-guideline/international-conference-harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en.pdf.
- U.S. Food and Drug Administration. “Guidance for Industry: Q4b Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions. Annex 14: Bacterial Endotoxins Test General Chapter.” Silver Spring, Md.: U.S. Department of Health and Human Services, October 2013. https://www.fda.gov/media/79183/download.
About the Author
Edward C. Tidswell, PhD, is Executive Director of Microbiology Quality and Sterility Assurance, for Merck Inc. He is responsible for sterility assurance and microbiology issues across all sterile and nonsterile products and manufacturing. His prior appointments include senior global R&D, and quality leadership roles across diverse drug, device and biologics portfolios for Baxter Healthcare, Eli Lilly and Evans Vaccines. Tidswell, PhD, actively publishes and is a leading authority on pharmaceutical microbiology, risk, aseptic and sterile manufacturing. Currently, a member of the PDA Journal of Pharmaceutical Science and Technology Editorial Board, he also continues to serve on the USP Expert Committee for Microbiology.