Endotoxins are lipopolysaccharides on the outer cell membrane of Gram-negative bacteria that are shed when the bacteria are growing, dividing, or dying. Even in small amounts, endotoxins can trigger cytokine storms, immune activation, or systemic inflammation leading to fever, shock, or organ failure. ^{6, 7} They cannot be detected by microbial sterility tests and are notoriously resistant to destruction or elimination, even by filtration and steam sterilization. In biosimilars R&D, endotoxins are prevalent byproducts of the E. coli-based systems often used to synthesize the biosimilar proteins. Even minuscule amounts of endotoxin contamination can lead to false-positive signals.⁷ With potentially life-threatening impact and difficulty to detect or eliminate, they are one of the most insidious threats in biosimilar development.

Endotoxin contamination is often characterized by spiking the test system with the endotoxin and then comparing recovery from the biosimilar and reference product. Recovery from the biosimilar that is significantly lower than expected (low endotoxin recovery; LER) might be caused by either poor correlation of the biosimilar and reference product or endotoxin masking in the biosimilar. Poor correlation may justify rejection of the biosimilar, whereas endotoxin masking can cause incorrect estimation of contamination, resulting in false positive or false negative results.^{5, 12, 13} Bound endotoxin can also cause safety and efficacy issues from adverse immune responses, impaired activity, undesirable off-target effects, altered metabolism, or diminished availability of the biologic.¹⁴

Multiple biochemical properties of endotoxins enable them to bind or form aggregates with other endotoxins or proteins, which can be difficult to detect and cause LER from the spiked sample.

LER requires rigorous control strategies and sophisticated testing methods to ensure accurate interpretation of analytical data and safety risks.

Endotoxin-mAb complexes can also confound interpretation of mAb polyspecificity and polyreactivity. Impaired binding to desirable targets can cause underestimation of polyspecific biosimilar availability. Non-specific binding to unintended targets can overestimate polyspecificity with false potential for new biosimilar activity. ^{15, 16} Identification and analysis of endotoxin-associated polyspecificity and polyreactivity are vital to ensure endotoxins are not masking true effects of the functionality, reducing reproducibility, and delaying readiness for approval.

Mouse models are vital to understanding potential clinical impact of endotoxemia

Unlike small molecule generic drugs, biosimilars are produced in inherently variable living systems. Therefore, they are not chemically identical to the reference product. Even small changes in molecular structure or stability can launch a dangerous immune response. To ensure patient safety with these novel medicines, the FDA, European Medicines Agency (EMA), and Japanese Pharmaceuticals and Medical Devices Agency (PMDA) have all established regulations defining the critical quality attributes that must be met to demonstrate comparable performance of a biosimilar with its reference product.^{8, 9} Details vary by country, but in general, researchers must demonstrate similarity with very high reproducibility across a broad range of characteristics including structure, specificity, potency, physiochemical and biochemical properties, safety, purity, and route of administration.⁷

In preclinical mouse models, endotoxemia is often used to study acute inflammatory responses that are similar to sepsis and systemic inflammation in humans. These early indicators include lethargy, weight loss, and acute cytokine release.¹⁷ Mouse models have revealed potentially severe effects of endotoxemia associated with several symptoms of sepsis, including heart rate variability,¹⁸ acute lung injury,¹⁷ cardiac fibrosis,¹⁴ nervous system hyperactivation,^{14, 19} and organ dysfunction.¹⁴

Endotoxin immune response is dose dependent.^{6, 7, 9} Under FDA regulations, the limit of endotoxin (measured in endotoxin units, “EU”) allowed for any injectable pharmaceutical product is set to the concentration at which pyrogenicity is determined, currently 5 EU/kg body weight/hour.^{7, 10} For example, in a typical 25-gram mouse, a biosimilar dose of 5 mg/kg contaminated with 1 EU/mg of endotoxin meets the maximum allowed amount in a single injection. Levels above 0.1 EU/mg can induce T cell proliferation and CD14+ myeloid cell expansion.^{7, 10} Investigational biologics tested as combination therapies to identify multidrug regimens present additional risk. These protocols often require administering multiple biologics within a close timeframe. Even if each product contains only small amounts of endotoxins, accumulation from multiple sources can lead to dangerous levels.^{6, 9}

Leinco Technologies research-grade biosimilars exhibit endotoxin levels comparable to pharmaceutical-grade product

Leinco Technologies has spent over 30 years advancing quality control processes for optimization of research-ready biosimilars. Overlapping SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography tests from multiple production lots confirm these biosimilars are strong analogs to their reference products in size, purity, and structure. Functional assays demonstrate highly reproducible performance similarity with the reliability of animal-free manufacturing systems. Leinco Technologies isotype controls, used to test biosimilar endotoxin levels, are designed and tested to the same stringent criteria.

FDA guidelines specify that the reagents used in LAL endotoxin detection methods must be obtained from an FDA-licensed manufacturer and must be designed specifically for the method chosen.^{7, 10} Every lot of Leinco Technologies’ biosimilars is tested on the Pyros Kinetix Flex system (Associates of Cape Cod, Inc.) using FDA-licensed LAL reagents for endotoxin detection. The Associates of Cape Cod (ACC) Pyros Kinetix Flex system is compliant with the requirements of the FDA, USP, and European Pharmacopoeia (Ph. Eur.) for conducting the Bacterial Endotoxins Test (BET) on raw materials, in-process samples, and final drug products. This rigorous screening demonstrates endotoxin levels less than 0.1 EU/mg (Table 1) in typical batches, as recommended for in vitro immunogenicity assays to ensure accurate and reliable data​.⁷