Modeling Endotoxin Reduction in Depyrogenation Oven and Tunnel Studies

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Abstract

Endotoxin results have been a major area of concern, especially in the biologics area due to issues with low endotoxin recovery. In some recent years, there were regulatory concerns about whether we were properly inoculating endotoxin challenge units in oven and tunnel studies, specifically whether we were directly inoculating the surfaces of these units for validation. When we perform oven and tunnel validation studies for depyrogenation, there is a requirement for a minimum three-log reduction of endotoxin. There have been many occurrences in the last few decades of initial studies meeting these requirements and then with no warning the endotoxin studies failing in subsequent qualifications. Calculations developed by Dr. Michael Akers have been useful in determining the theoretical endotoxin reduction using the heat penetration data from the study. Using these numbers in routine qualifications allows the user to determine how close the parameters fall in predicting the three-log reduction. When the numbers are close to the specified limit one may desire to adjust the cycle to ensure a greater safety factor, reducing the risk of failed studies later in the re-qualification process.

Background

USP (2018) has a general monograph (number 1) that has a section on bacterial endotoxins which states the following:

All articles intended for parenteral administration should be prepared in a manner designed to limit bacterial endotoxins as defined in Bacterial Endotoxins Testor Pyrogen Test.

This requirement is applicable for both human and veterinary drug products. Additionally, the Code of Federal Regulations Title 21 §211.113 (b) requires documentation for the validation of all aseptic and sterilization processes. (CFR, 2017)

The first injectable drugs introduced into the market were considered crude and unsafe. When these drugs were used, there was an increase in infections, adverse drug reactions, fevers of unknown origin, and even deaths from shock. Some were confused by the sporadic fevers resulting from the injection of sterile drugs. (FDA, 1979)

While pharmaceuticals are much safer now, there is still the possibility of receiving toxins known to be fever producing from parenteral products. (FDA, 1979)

Pyrogens

By the latter part of the 19th century, it was known that some parenteral solutions caused a marked rise in body temperature. While at this time, they did not know what caused the fevers, it is now known to cause health issues in humans. As such, it is important to ensure that pyrogens are not present in the pharmaceutical manufacturing process that could result in contaminated product. (FDA, 1979)

Parenterally administered drugs allow a pyrogen, if present, to bypass the normal body defenses, resulting in a febrile response and other biological reactions. There are three basic effects caused by pyrogens: fever production, shock and changes in physiological functions. The term “endotoxin” refers to a pyrogen that can be extracted from Gramnegative bacteria. This term is frequently used interchangeably with pyrogen, even though it may not be true in all cases. (FDA, 1979)

Make Your Product Pyrogen Free

The best way to ensure the production of non-pyrogenic product is to eliminate pyrogens in your process. Typical procedures like heating, filtration, or adsorption techniques are not effective in pyrogen removal. It is important to keep ingredients pyrogen-free from the start. It is important to screen ingredients and ensure proper storage. Testing and monitoring must be conducted to ensure that pyrogen contamination does not occur and if it occurs that it is eliminated before product release. (FDA, 1979)

One of the critical process controls used in pharmaceutical manufacturing is depyrogenation of the primary packaging components. This can be accomplished in a variety of ways, including for example: washing to dilute the endotoxin from surfaces (frequently used on things like stoppers), high temperature depyrogenation (typically temperatures in excess of 170°C). High temperatures are used in ovens and tunnels for glass depyrogenation. When depyrogenation processes are used, they must be validated/qualified for use.

Sandle (2013) evaluated different methods for endotoxin reduction and found dry heat to be most efficient in depyrogenation. As such, this method is used most frequently for items which withstand high temperatures.

Qualification Processes

Depyrogenation ovens and tunnels are typically qualified in the same basic way. The equipment qualification model is routinely used to perform the qualification process. The installation qualification (IQ) typically documents the design and physical portions of the system. Also included are examples of the supporting documentation available, system definitions, and drawings, and so forth.

The operational qualification (OQ) includes tests that assess the functionality of the system comparing the performance against a prescribed set of specifications. Some typical inclusions are: temperature distribution empty chamber studies, evaluation of other control parameters and the like. The required number of evaluation studies vary, but frequently at least three studies are performed. There are also specified limits for calibration accuracy and limits for thermocouple temperatures compared to the controls in the study. Other typical requirements may include: minimum and maximum chamber temperature during exposure. Depending upon the company and type of depyrogenation unit, there may be a variety of other parameters that must be met, e.g., belt speed, stabilization times, mean temperatures, agreement of temperatures between probes, how many probes must be functional in the study, and the like.

Performance qualification (PQ) studies are performed to provide documented evidence that the replicate studies performed demonstrate reproducibility, and provide an accurate measure of the variability among successive runs. (FDA, 2011)

Microbiological validation is performed as part of the PQ. This includes challenging the components with Escherichia coliendotoxin at a level that is sufficient to quantitate a three-log reduction of the endotoxin as required by the Food and Drug Administration “Guideline on Sterile Drug Products Produced By Aseptic Processing”. (FDA, 2004) Typically, biological indicator spore strips or ampoules are not employed in the validation for two reasons: 1) if using spore strips, they may be incinerated by the heat generated by the process, and 2) Escherichia coli endotoxin has been reported to provide a challenge equivalent to 10100 bacterial spores.

In addition to the biological challenges, typically heat penetration, temperature distribution studies are also conducted. One of the common depyrogenation temperatures used is 250°C. While not the only temperature utilized, the calculations can be adjusted for the temperatures utilized in your process.

If the system is software controlled, software validation is also performed, either separately or concurrently.

Modeling Endotoxin Recovery for Qualification Studies

Several companies have established a depyrogenation F_250°C value of > 16 minutes. Some companies have chosen to designate this value as Fp. This value was established based upon a publication by Akers, et al. (1982) which indicated that a two-log reduction of Escherichia coli endotoxin occurred with a F_170°C of 500 minutes using an exposure temperature of 250°C.

Assuming a linear degradation, a three-log reduction of endotoxin would be expected following an F_170°C accumulation of 250°C. This F1_70°C value was converted to F_250°C using the z-value of 47.6°C established by Tsuji and Lewis (1978) in the following equation: (Akers, et al., 1982 and Stumbo, 1982) (Note: While there are several different D-values published, if you are using a different D-value, the calculation should be adjusted accordingly.)

F_170°C/F_250°C = 10^(T-Tref)/Z

F_170°C/F_250°C = 10^(250-170)/Z

750/F_250°C = 10^{(250-170)/47.6}

750/F_250°C = 10^1.6807

1/F_250°C = 47.9372 x 1/750 = 0.0639

F_250°C = 15.6

(This value was rounded to the whole minute of 16.0)

F_Tref = value of F at the reference temperature

FT = value of F at the desired temperature

T_ref = reference temperature = 170°C

T = desired temperature = 250°C

z = z value of E. coli endotoxin = 47.6°C

This calculation results in the F_250°C value of 16.0 minutes (after rounding) , which, when delivered, will theoretically result in a three log reduction of the Escherichia coli endotoxin challenge.

The benefit of using the theoretical reduction level is that it provides the user with some information on how likely you are to meet the limits in future studies. It also aids in addressing issues when either the heat penetration or the biological challenge does not meet the specified requirements.

It is easy to implement this type of procedure as when the calculation have been calculated for the cycles used, companies are used to performing F_o evaluations, which also measure heat penetration probe temperatures, entering in D-values and z-values. The same equipment is used for measurement of both types of F-value.

Conclusions

Use of modeling to predict the theoretical endotoxin recovery can be a useful tool in assessing the capability of your depyrogenation process and to ensure that you have properly developed a depyrogenation cycle that will withstand the test of time.

Acknowledgements

Many thanks to the following individuals who were instrumental in the development of these modeling calculations: Dave Kuchta, Larry Mikelionis, Ray Wild, and Kathy Kraft.

Literature Cited

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2. CFR (2017) Code of Federal Regulations Title 21 §211.113 (b). Code of Federal Regulations. GMP Publications, Inc. http://www.gmppublications.com.
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