Supramolecular assembly of micellar aggregates is the basis of low endotoxin recovery (LER) in a drug formulation that can be resolved by a whole blood assay.

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.

Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon.

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.

Multiple nonidentical reductive-dehalogenase-homologous genes are common in Dehalococcoides.

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.

Novel mass spectrometry-based tool for genotypic identification of mycobacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.

Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid bacterial identification.

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria.