Abstract
Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called “Low Endotoxin Recovery (LER)”. LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration’s (FDA’s) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.
Highlights
Lipopolysaccharides (LPS), the endotoxins of most Gram-negative bacteria, belong to the strongest immune-stimulating compounds known in nature
We have investigated the: (i) (i) gel to liquid crystalline phase transition of the hydrocarbon chains of LPS, and with that, the fluidity of the acyl chains, with Fourier-transform infrared spectroscopy (FTIR); (ii) three-dimensional aggregate structure of LPS by using synchrotron radiation small-angle X-ray scattering (SAXS); (iii) LPS aggregate sizes by dynamic light scattering and have related these data to the biological activities in the macrophage activation test (MAT); (iv) the interaction of LPS with a synthetic anti-LPS peptide Pep19-2.5 was monitored to find out whether differences in head group binding are observed
The current work has laid out a number of analytical approaches to study the LPS system and provide insight into the structural changes that the LPS might be going through, subsequently leading to the Low Endotoxin Recovery (LER) effect
Summary
Lipopolysaccharides (LPS), the endotoxins of most Gram-negative bacteria, belong to the strongest immune-stimulating compounds known in nature This property may be beneficial at low concentrations, but pathophysiological at high concentrations, leading to severe sepsis and septic shock with high lethality [1]. In clinical studies of Opal and co-workers, it was found that sepsis patients belonging to the survivors had a medium LPS serum concentration of 0.3 ng/mL and the non-survivors 0.7 ng/mL [2]. The reason for this is the fact that LPS induces a “cytokine storm” (interleukins, tumor-necrosis-factor-α (TNF-α) and many others), leading to a septic shock. It is of uttermost importance to control LPS-load, in particular, in parenteral pharmaceutical formulations, such as recombinant therapeutic proteins, which are anticipated to be injected
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