Low Endotoxin Recovery (LER) and Biologic Drugs
Introduction
Biologic drugs are transforming the pharmaceutical landscape, constituting 50% of recent FDA approvals. (Figure 1).
Figure 1. Fresh from the biotech pipeline; record-breaking FDA approvals.1
Endotoxin detection is an integral part of the drug quality control process, and all sterile pharmaceutical and medical device products that come into contact with a patient’s blood, or with cerebral fluids must be tested for endotoxins before their release. However, the production of stable biologic formulations pose a unique challenge: the detection of masked endotoxins (including those subject to LER, is essential for ensuring product quality and patient safety. FDA requires a demonstration of LER absence in all submitted Biologic License Applications (BLA’s) containing surfactants underscoring the importance of this issue (See PDA Technical Report 82).
Understanding Masked Endotoxin
Over 20 years ago, researchers demonstrated that proteins in biologics formulations can mask endotoxin, undermining traditional detection methods' reliability (Petsch et al.1998).2 A more recently observed phenomenon, known as low endotoxin recovery (LER), presents another significant roadblock in biologic drug quality control. Studies by Schwarz et al. (2017, 2014) highlighted a surfactant-specific version of endotoxin masking. Using LER samples Schwarz et al. showed that even LER masked endotoxins can activate immune responses.3, 4
- Endotoxin detection methods: Tests like Limulus amebocyte lysate (LAL)and recombinant factor C (rFC) rely on the Factor C protein to detect endotoxins. Limulus-based tests bind with LPS molecules and set off a reaction or cascade of reactions enabling endotoxins to be detected and measured. However, Factor C only works with naturally aggregated endotoxins. Biologic manufacturing processes, involving surfactants and buffers, can disrupt this aggregation.
- Manufacturing impact: Surfactants and chelating buffers maintain biologics molecular stability. However, they can also break down the aggregate structure of LPS and thus lose the ability to activate Factor C, resulting in false negatives.
- Detecting LER: To assess the presence of LER in a specific sample, a Hold Time Study (HTS) protocol is recommended.
A Hold Time Study protocol involves spiking a series of identical sample aliquots with a control standard endotoxin (CSE) or a reference standard endotoxin (RSE) and assessing detection over time, typically 7 days after spiking. This is usually done in “reverse” fashion to test all time points at once (Fig. 2).
Figure 2. Reverse Hold Time Study spike days. The staggered recovery will demonstrate the presence/absence, kinetics and strength of the phenomenon.
A typical case study will be shown to demonstrate the bioMerieux demasking approach in some detail.
bioMérieux Demasking Case Study:
a. Reagents: ENDO-RS and ENDOLISA
The ENDO-RS solution uses a series of sample treatments to coax the disassociated endotoxin back into aggregated form (see Fig.3 for the content of the ENDO-RS kit). There are multiple endotoxin-masking conditions and mechanisms, a specific ENDO-RS sample preparation protocol must be developed for each product.
Figure 3. The Endo-RS LER test kit includes solutions A – E “mixed and matched” in a trial manner to achieve the best result for a specific sample protein content and formulation matrix.
A demasking study follows a series of steps (as shown in Figure 3):
a) Endotoxin spike and hold to create LER sample
b) ENDO-RS component screening
c) Optimization of the demasking system
d) Lowering of the endotoxin concentration (increasing the endotoxin test detection level)
e) Optimization of the demasking system at the specification limit.
The HTS answers the basic question: “Do we have LER?” In addition to using Endo-RS, the kit contains an Endolisa plate that is used to gain initial recovery before optimization in a regular 96-well plate.
ENDOLISA® features a 96-well plate pre-coated with a specific endotoxin-binding phage protein, and is used in conjunction with ENDO-RS to overcome LER. The process allows for the removal of interfering substances through a washing step prior to detection with recombinant Factor C fluorescence method. (Figure 4)
Figure 4. Endotoxin detection using Endolisa after Endo-RS treatment for overcoming LER.
b. Initial screening experiments
Initial screening tests are performed with high endotoxin spike concentrations, (e.g., 50 or 100 EU/mL CSE). A known LER triggering solution (citrate buffer and polysorbate 20) is spiked and held for seven days. The ENDO-RS components are added to the sample according to the guidance in the package insert.
c. Method Details
For each 1mL sample, components A-E are added in specific volumes and incubated. The sample is then diluted and assessed with the rFC assay using the ENDOLISA plate. For successful demasking, the recoveries should be between 50 and 200 percent.
For demasking of 50 EU/mL of the citrate/ polysorbate 20 buffer, the screening was performed as shown in Table 1:
- The table shows the results of the various combinations tested allowing to choose the most performant one.
- Here the best results were obtained with the combination A, B, C, D2 and E (1:10 or 1:100 pre-diluted). With 70- and 120-percent endotoxin recovery of the initial 50 EU/ mL spike.
Results guide optimization efforts, focusing on enhancing endotoxin recovery. Refining the test involves the addition of progressively smaller endotoxin spikes for the HTS samples (in our case study 50, 25, 12.5 and 6.25 EU/mL) to determine which concentration can be demasked. A new “foothold” on a method capable of demasking a smaller amount of endotoxin contamination can be achieved. In decoding Table 2 results, optimization experiments should be done for the endotoxin spike of 12.5 EU/mL (25 percent recovery, Table 2, #9).
The reconfigurator (E) is the most important ENDO-RS component, supported by multiple modulators. During the optimization experiments, where the concentrations of these buffers were changed, it was observed that the higher the pre-dilution of component E, the higher the endotoxin recoveries. In our case, the subsequent assay pre-dilutions of up to 1:200 were tested in combination with different concentrations of D2. When using a higher diluted component D2 for demasking, endotoxin recoveries of 70-90 percent were obtained (Table 3, #8 #11). A demasking protocol was thus developed that could detect 12.5 EU/mL in the citrate/ polysorbate 20 buffer, 90 percent of the 12.5 EU/mL CSE spike was detectable using the ENDOLISA assay.
Through systematic testing and optimization, a reliable and stable sample preparation can be developed with the ENDO-RS kit to detect possible endotoxin contaminations in drug products.
Table 1. Determining a starting point for overcoming LER in citrate/polysorbate 20 buffer using ENDO-RS components A-E.
Table 2. Endotoxin titration results in improved %recovery relative to the initial screening shown in Table 1. The results are improved because recoveries are obtained for spikes as low as 25 to 12.5 EU/mL.
Table 3. Optimization experiments using a lower endotoxin spike concentration of 12.5 EU/mL. The 12.5 EU/mL spike recoveries are noticeably improved relative to Table 2 (12.5 EU/mL recoveries).
Conclusion:
Understanding masked endotoxins is critical to ensuring the safety of biologic drugs. By proactively addressing LER through innovative detection methods including LER Hold Time Studies (HTS), manufacturers not only meet regulatory standards but also enhance product quality and patient safety.
Embracing these state-of-the-art protocols elevates manufacturers brand's reputation as leader in pharmaceutical safety, driving innovation and setting new standards for excellence. Connect with our LER specialists today to enhance product safety and quality standards.
Discover Additional rFC Endotoxin Resources
References
1 Low Endotoxin Recovery case studies, Christian Faderl, and Kevin L. Williams, European Pharmaceutical Review, 2019. (2). Senior, M. Fresh from the biotech pipeline: record-breaking FDA approvals. Nat Biotechnol 42, 355–361 (2024). https://doi.org/10.1038/s41587-024-02166-7.
2 Petsch et al., Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay; Application for endotoxin removal from cationic proteins, Analytical Biochemistry, 1998;259z91):42-7.
3 Schwarz et al., Biological Activity of Masked Endotoxin, Scientific Reports, March 20, 2017.
4 Schwarz et al., Residual endotoxin contaminations in recombinant proteins are sufficient to activate human cd1c+ dendritic cells, PLoS One, 2014;9(12).