Assessing product hold times for endotoxin recovery

In terms of finished product, samples for endotoxin testing cannot always be tested within a short time frame and this means holding samples until testing can be performed.?To verify acceptability, hold times should be established.?The necessity for this is contained within the U.S. FDA “Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers” (2012).?

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Here the FDA states: “The ability to detect endotoxins can be affected by storage and handling. Firms should establish procedures for storing and handling (which includes product mixing) samples for bacterial endotoxins analysis using laboratory data that demonstrate the stability of assayable endotoxins content.?Protocols should consider the source of endotoxins used in the study, bearing in mind that purified bacterial endotoxins might react differently from native sources of endotoxins.”

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Endotoxin hold times should be established by determining the maximum time between filling and testing and assessed through an experiment. The design of the experiment should be based around different product groups and it should contain a sufficient number of replicates in order to demonstrate reproducibility (typically three or more). The hold time assessment relates to industry discussions about ‘low endotoxin recovery’, a phenomenon relating to the difficulty in recovering endotoxin over time. This is typically a failure to recover at a level of below 50% (despite the fact that positive product controls show no evidence of inhibition).?This effect regarded as something distinctly different than the interference commonly seen with the LAL assay that is normally overcome by dilution.?

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Low endotoxin recovery can arise due to:

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1. “Masking” (where endotoxin is present and biologically active but non-recoverable).
2. Binding, where the endotoxin binds to the surface of the container or to product protein.
3. ?Or due to loss of biological activity / inactivation (where the endotoxin has been permanently inactivated and hence it is undetectable using LAL.)?

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The challenge level must also be determined. This can either be achieved by directly challenging a finished product container or a sample aliquot (such as a 10 mL into suitable depyrogenated glassware). ?It is important that the volume of endotoxin solution transferred is minimal, to avoid any excessive dilution effect (such as 0.1mL). In terms of the endotoxin challenge, this will need to fall within the range of the standard curve. Too low a value may lead to recovery complexities due to LAL assay variability. An endotoxin challenge of 0.5 EU/mL using a standard curve covering the range 5.0 to 0.05 EU/mL could be a suitable example.

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To assess the hold time, values should be assessed at day zero, within 30 minutes after the challenge inoculum has been made along with uninoculated samples of the product. The challenged product samples should be subjected to the standard storage conditions applied to product awaiting testing. At the mid-point of the hold time, it is prudent to perform another analysis (if the hold time is a maximum of eight days, testing at day 4 would be suitable as an example). At the final day of the hold time, the sample should be tested.

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The method of preparing samples for testing should be standardised and the same as per normal product testing (such as the time period for removal from an incubator or from cold storage, time required to equilibrate to room temperature, vortex mixing time and so on).

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Each result should meet the requirements for the LAL assay and the endotoxin challenge should be recoverable within 50-200%.?

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If the recovery is not obtained, then different factors require examination. These will include the usual assessments for laboratory error, from endotoxin preparation and inoculation methods through to the technique of the analyst when undertaking the LAL assay. A second area is with the product storage conditions and a third is with sample preparation.

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Another area to consider is with the type of endotoxin used. The ‘pure endotoxin’ (lipopolysaccharide) is not the same as naturally occurring endotoxin (where lipopolysaccharide is bound with other cellular material and is in the form of vesicles). ?Natural endotoxin exists in aggregates (rather than monomers) and this endotoxin is generally more biologically active than endotoxin controls. Pure endotoxin, as provided in the form of Control Standard Endotoxin or Reference Standard Endotoxin, can sometimes be difficult to recover either due to binding to surfaces or due to masking, as might occur through chemical reactions to certain product excipients like polysorbate or citrate. Since lipopolysaccharide in naturally occurring endotoxin is shielded in cell-wall vesicles and generally protected from chelators and detergents; whereas purified lipopolysaccharide, as with CSE, forms micelles and other aggregate types that are unprotected from disaggregation factors, then the path towards the development of a naturally occurring endotoxin standard may be the optimal may forwards.

#endotoxin #microbiology #pharmaceutical