NOE
Selection of Organisms
A variety of Gram-negative organisms were screened for production of NOE in order to determine potential candidates for use in generating an NOE preparation. All NOE solutions were made by preparing a lawn culture on Tryptic Soy Agar (TSA) and incubating at 32°C for 24-48 hours. Colonies were picked from the plate, inoculated into Tryptic Soy Broth (TSB) and incubated at 32°C in a shaking incubator overnight. The resulting culture was centrifuged, fi ltered through a 0.45 μm fi lter and tested for endotoxin using the kinetic chromogenic LAL assay. Table 1-1 indicates the resulting EU/mL for each organism assessed.
Previous NOE spiking studies at the Pfi zer Andover, MA site using Escherichia coli and Serratia marcescens preparations were useful since they produced high EU/mL stock solutions that remained stable over long periods of time. Organisms that produce high concentrations of endotoxin are particularly benefi cial for use in spiking studies as these preparations allow for fl exibility of testing both low and high spiking concentrations.
Because the preparations produce endotoxin concentrations that are signifi cantly higher than levels seen during routine use, the concentration of laboratory prepared NOE stocks must be confi rmed prior to each use in order to prepare an accurate spiking solution.
LAL Validation vs. LAL Spiking Studies
When LAL assays are validated for routine use during product manufacture and release, endotoxin in the form of CSE is spiked into already diluted sample as the PPC in order to evaluate matrix inhibition/enhancement. Validation activities focus on the interfering factors test, which assesses whether the sample matrix may be interfering with endotoxin detection by determining the PPC spike recovery (i.e., the amount of endotoxin spiked into the PPC must be recovered at 50-200%). A sample dilution is selected where the spike recovery falls within the acceptable PPC spike recovery range and also aligns with the parameters set in USP Chapter <85>.
LAL validation studies are typically designed to consider the eff ect of the sample matrix after the sample has been diluted to a level whereby interference is no longer seen in the assay, as determined by the recovery of the PPC spike into the diluted sample. In this case, the endotoxin in the PPC only interacts with the sample after dilution, not at the onset.
To determine the eff ect the sample matrix has on endotoxin, the endotoxin must interact with the undiluted sample matrix prior to performing the routine LAL assay. Using NOE to initially spike undiluted sample better represents a contamination event, since the source of such a contamination would most likely be from bacterial sources and not from a processed endotoxin source such as CSE.
Table 1-1: Organisms Assessed for NOE production Organism
Klebsiella pneumoniae Salmonella typhimurium Moraxella osloensis Proteus mirablis
Stenotrophomonas maltophilia Sphingomonas paucimobilis Escherichia coli
Serratia marcescens Acinetobacter genomospecies
36,789 15,062 12,934 26,779 35,564 <50
28,075 56,830 4,640
22 American Pharmaceutical Review | Endotoxin Supplement 2013 EU/mL Figure 1. Routine LAL Assay vs. NOE Spiking Study
The main diff erence between the role of the PPC spike and NOE spike is that the PPC spike provides information as to whether the sample matrix is interfering with the detection measures of the assay itself, while the NOE spike provides information on the direct interaction of the sample matrix with endotoxin. Figure 1 illustrates the diff erence between routine testing and NOE spiking studies using the kinetic LAL assay.
Design of Hold Time Studies
In many cases, LAL samples are delivered to the testing laboratory and held for some period of time prior to initiating testing. Studies should be performed to evaluate the impact of this hold time on the recovery of endotoxins in a particular sample type. NOE is useful for these studies since high EU/mL stocks can be easily prepared and stored by the laboratory, making it relatively simple to create spiked samples at various endotoxin concentrations
Hold time studies are performed by spiking NOE into undiluted samples prior to performing the LAL assay. The spiked samples are assayed immediately to determine the starting concentration, and aliquots are stored and assayed at set time points. By spiking NOE into undiluted sample, the eff ect of the product and the matrix on any endotoxin that may be present may be determined, both at the initial time point and over the storage time and temperature for the sample. The kinetic LAL assay is used in these studies since the results are quantitative, allowing for a determination of the endotoxin amount at each time point.
Table 1-2 shows the results of a hold time study using NOE spiked into two diff erent buff ers that were held at 2-8°C for 3 weeks, and Table 1-3 shows the results of a hold time study using NOE spiked into the same two buff ers, this time held at -80°C for 3 months These results indicate that endotoxin may be recovered from the buff er matrices over the entire time period of the studies at both temperatures.
Initial Time Point Testing and Use of NOE for Troubleshooting
An important consideration for hold time studies is confi rmation of the amount of endotoxin initially spiked. When the initial time point is assayed
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