Low Endotoxin Recovery Case Studies

INTRODUCTION

Since CHEN and VINTHER first described a unique drug formulation phenomenon that renders the detection of endotoxin using Limulus-based methods ineffective, the debate has raged as to how the phenomenon should best be mitigated.1 The phenomenon was labelled “Low Endotoxin Recovery” or LER. It should be noted that LER can be complicated by the presence of protein masking owing to the differential of the isoelectric points of endotoxin and many proteins.

Some methods of mitigation used have included:

• the addition of simple surfactants eg, magnesium chloride
• and/or a pH change to mitigate the dissociation of endotoxin aggregates in quality control (QC) samples
• and/or the use of the ENDO-RS® sample preparation kit to essentially reaggregate dissociated endotoxin in QC tests.

The removal of activity from endotoxin in QC samples (via LER) may initially seem like a good thing; however, some have shown that dissociated endotoxin remains biologically active from an immunological (non-pyrogen) perspective. Schwarz et al found that LER dissociated endotoxin-activated key immune cell reactions. Additionally, detoxified LPS is used as an adjuvant in vaccines to stimulate immune reactions. Today, many important drugs, especially biologics, act via immunemodulating properties such as suppressing autoimmune reactions or activating cancer immune response activities.

Pharmaceutical companies and the US Food and Drug Administration (FDA) have published papers on the use of the ENDO-RS kit to overcome LER (see further background paper references below). The ENDO-RS kit (Figure 1) was created as a means of testing samples for endotoxin with difficult-to-test matrices. A case study is presented here.

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