Bacterial Endotoxin Testing—Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide

Abstract

For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in “Guidance for Industry: Pyrogen and Endotoxins Testing.” It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.

Low endotoxin recovery and its impact on endotoxin detection

“…Tsuchiya [27] Charles River The reduction of activity of LPS is due to the replacement of LPS molecules on the surface of aggregates by detergent molecules Orving et al [28] FUJIFILM Diosynth Biotechnologies, Biogen, Microcoat Biotechnologie GmbH, University of Southern Denmark Lauryldimethylamine oxide (LDAO), a component used in the virus inactivation step alone, could lead to a LER effect. Unlike the traditional LER effect that is time-dependent, the LDAO-induced LER is instant and time-independent Bu et al [29] BeiGene Guangzhou Biologics Manufacturing Co., Ltd.…”

“…There are two main reasons for the reduced detectability of endotoxins: test interference and the masking effect, the latter of which is generally thought to be the cause of LER. [20,28] Test interference describes the interference of sample constituents with the LAL-based detection system. Since the LAL system is enzymatic, components in the samples, such as heavy metals and protease inhibitors, may stop activation of the detection system.…”

“…Several sample treatment methods have been shown to overcome test interference, for example, dilution, filtration, and dialysis, although there might be some potential problems associated with them such as dilution folds and membrane binding loss when using filtration and dialysis. [20,28] The masking effect describes the interference of sample constituents with the endotoxin itself. Components within the samples, such as nonionic surfactants and proteins, are likely to interact with endotoxin and lead to activity loss.…”

“…[19,25,39] However, an opposite opinion also exists arguing that industry should still use CSEs as the analyte because they represent the worst-case scenario: if LER can be overcome on purified LPS, it can be applied to unpurified endotoxins too. [28] Interestingly, before the term LER was mentioned, the 2012 U.S. FDA Guidance for Industry Pyrogen and Endotoxin Testing: Questions and Answers suggested that "Protocols should consider the source of the endotoxin used in the study, bearing in mind that purified endotoxins might react differently from native sources of endotoxins. [40] "…”

“…After the concept of LER was introduced, research groups have tried to address the LER phenomenon as shown in Table 1. [19][20][21][22][23][24][25][26][27][28][29] T A B L E 1 Selected studies of the low endotoxin recovery phenomenon…”