DEPYROGENATION
occur but does not necessarily follow linear regression, it is ‘bi-phasic’ reduction [10].
Endotoxin reduction is assessed using Endotoxin Indicators, which are prepared using a preparation of Control Standard Endotoxin (CSE) derived from Escherichia coli O113:H10. The CSE used should be similar to endotoxin used to perform routine the Bacterial Endotoxin Test (BET) using Limulus Amoebocyte Lysate (LAL) methodology and traceable to a reference standard. There is some debate within the industry regarding how an Endotoxin Indicator is prepared; this is either by adding endotoxin to the surface of the item to be depyrogenated and drying it, or by using a pre-prepared endotoxin indicator. Of the two approaches, the first approach is the preferred by the author. This is because it is considered the greater challenge as the endotoxin is applied directly to the surface of the device to be depyrogenated [11].
Study Design
The aim of the study was to determine if successful depyrogenation could be achieved practicably using methods other than dry heat. Two alternative methods were investigated: moist heat (autoclaving) and a caustic rinse, and the results compared with dry heat depyrogenation.
Acceptance Criteria
There is no compendial endotoxin tolerance limit (K) published for glass final product containers so the limit for medical devices was used in this study, which is 20 EU/device [12].
Methodology
The method selected for the study was the kinetic-turbidimetric LAL (Limulus amebocyte lysate) test (as per the Bacterial Endotoxins Test, European Pharmacopoeia <2.6.14>). The LAL test is based on the lysate enzyme isolated from the horseshoe crab, which clots in the presence of endotoxin [3]. In practice, for the kinetic-turbidimetric test, the LAL reaction rates are expressed in terms of the time taken to reach a predetermined ‘threshold’ optical density, known as the onset time [13]. Higher endotoxin concentrations result in shorter onset times. Endotoxin concentrations are calculated from a standard curve (E. coli endotoxin standard), constructed by linear regression of log onset time versus log endotoxin concentration [14]. Samples, standards and controls are tested at least in duplicate. Positive product control recovery should be in the range of 50 – 200%.
The study used glass vials that had been rinsed with Water-for-Injection. A 50, 000 EU/ml solution of endotoxin was prepared. Vials were inoculated with 0.1ml of the solution to give a theoretical 5, 000 EU spike and were air-dried in a unidirectional airflow cabinet overnight. A 0.1 ml inoculation volume was chosen because the literature reports that smaller volumes of inocula reduce adsorption leading to higher and more consistent endotoxin recoveries from the vials.
Once prepared, Endotoxin Indicators were placed in defined locations in a depyrogenation device (10 Endotoxin Indicators were considered sufficient to assess the depyrogenation capabilities), along with two positive controls.
Treatment and Testing Three treatments were performed:
1. Dry heat: vials were treated in a Carbolite oven at 250°C for 30 minutes. Heating at 250°C for not less than 30 minutes to
16 American Pharmaceutical Review | Endotoxin Supplement 2013
depyrogenate glassware and utensils is stated in USP Pyrogen Test Chapter <151> [3].
2. Moist heat: vials were autoclaved at 121°C for 30 minutes; this autoclave cycle is a standard one used to sterilize equipment.
3. Caustic: each vial was rinsed with 10ml of 0.1N NaOH for 1 minute. This concentration of caustic is reported in the literature as capable of depyrogenation [17]. A 1-minute wash was performed as it was practicable and could be easily implemented and carried out if shown to be successful.
Three runs were performed for each treatment, except for dry heat as it was shown that depyrogenation was 100% successful after the first two runs (as might be expected from an established and mature technology).
After treatment, each vial had 5 ml of buffer added and was sonicated in an ultrasonic bath for 15 minutes, then placed onto an orbital shaker until tested. All bottles were vortex mixed for one minute just before testing. A combination of ultrasonication plus vortexing is suggested in the literature to give optimal endotoxin recovery.
All samples were tested in duplicate against a standard curve ranging from 5.0 EU/ml to 0.005 EU/ml. Positive controls, post moist heat treatment samples and post caustic treatment samples required dilution so that detectable endotoxin was within the range of the standard curve. For each run at least one treated sample and all controls were also tested spiked with 0.5 EU/ml of endotoxin. To test the samples, a 1:2 dilution with LAL reagent water was performed in the lysate reaction tube (for samples and controls requiring dilution to fall within the range of the standard curve, this additional 1:2 dilution was performed as part of testing). For the positive product control the 1:2 dilution was performed with a 1.0 EU/ml endotoxin standard solution resulting in a dilution the same as the test sample but with a 0.5 EU/ml endotoxin spike. Positive product control spike recoveries between 50-200% indicate the absence of interference, and the suitability of dilutions for testing.
Results
Data Analysis Data analysis was performed using Microsoft Excel™ and Systat 11™. A two-way ANOVA was performed to test for signifi cant diff erences between the three treatments. Diff erences were confi rmed using Student’s t-test. As the post treatment endotoxin concentrations measured are dependent on the initial spike for each run, statistical analysis was performed on the log endotoxin reduction, rather than the actual endotoxin measured. This approach reduces the risk of detecting false signifi cances and does not assume that the data is from the same population.
Results of the Three Treatments
Log endotoxin reduction
Dry Heat Treatment
Mean
Maximum Minimum
5.2 5.4 5.0
Table 1.
A summary of the results from the three glassware treatments are listed in the table below:
Moist Heat Treatment
1.7 2.5 1.0
Caustic Treatment
1.7 2.9 1.0
Statistical analysis indicated that dry heat was the most effective treatment for depyrogenation, reducing endotoxin significantly more
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