Endotoxins are heat stable lipopolysaccharide (LPS) complexes that are found in the outer most layer of Gram-negative bacterial cell walls. Patient exposure to this toxin as a result of exposure to a contaminated drug product or device can result in severe adverse reactions including activation of pro-inflammatory cytokines and severe febrile reaction. In the pharmaceutical industry, procedures for endotoxin prevention, detection, and removal are strictly followed. The removal of endotoxin from drug and medical containers is commonly achieved via specialized rinsing and/or dry heat-based destruction methods.

In this study, water for injection (WFI) rinsing, steam sterilization, and dry heat-based methodologies were examined to better understand their effectiveness in reducing endotoxin levels of contaminated industry standard type-1 glass and “alternative” polymer constructed vials commonly used throughout the pharmaceutical industry for drug manufacturing. In addition to the three different glass manufacturers vials that were evaluated here, three “alternative” materials were also evaluated in this study. The alternative vials were all International Organization for Standardization (ISO) standard 10R vials each with a unique composition as follows; 1) polytetrafluoroethylene (PTFE) obtained from REDACTED, 2) cyclic olefin polymer (CoP) marketed as Daikyo Crystal Zenith vials obtained from West Pharmaceutical Services, and 3) proprietary plastic vials with a microscopic glass coating sold by SiO₂ Material Science (SiO₂).

Each empty vial was spiked with endotoxin standard prior to its exposure to the various endotoxin removal treatment(s) discussed throughout this study. Rinsing as a standalone endotoxin removal method achieved an average log reduction of 2.2 for the glass and 5.3 for the alternative vials. Steam sterilization alone delivered an approximate 3 log reduction across all glass types and the PTFE alternative vials. The same steam sterilization process was far less effective on the CoP and SiO₂ alternative vials (1.5 & 1.2 endotoxin log reduction, respectively). A combination of rinsing and steam sterilization achieved approximately 6 logs of endotoxin reduction between all vial types, proving to be an effective endotoxin removal method for pharmaceutical vials across both traditional glass and alternative materials. Dry heat depyrogenation was effective across all glass containers, delivering ≥6 logs of endotoxin reduction. The alternative vials were not exposed to dry heat as they cannot withstand the elevated temperatures required for depyrogenation via heat.

Introduction

The recognition and understanding of endotoxin as a hazardous substance in drug products mark a significant milestone in pharmaceutical research and safety. In the late 20th century, as scientists delved into the intricacies of drug formulation and quality control, they discovered a menacing presence within certain products—endotoxin. This potent human toxin, derived from the cell walls of certain bacteria, possesses the ability to trigger severe inflammatory responses and pose substantial risks to human health.

Today, endotoxins can be more broadly classified as a specific type of pyrogen. While the term endotoxin is specific to the LPS component of Gram-negative bacterial cell walls, the term “pyrogen” or “microbial pyrogen” is a broader and more general characterization referring to any fever-inducing microbial substance.¹ Bacterial endotoxins are the most prevalent and quantifiable pyrogen associated with the contamination of parenteral preparations and implantable devices.² While the normal structure of endotoxin (LPS) within healthy cells is not inherently toxic or problematic, cellular fragments that are released as a result of cell division, damage, and lysis can cause complications.³ These fragments, many of which consist of constituents of the outer membrane, allow for the unchecked exposure of Lipid A, the inner most compound of LPS that is responsible for most of the biologic reactivity associated with endotoxin.⁴ When this type of contamination persists in parenteral or other implantable products, exposed patients may experience a strong pro-inflammatory immune response and a febrile reaction via induction of Interleukin-I and TNF.³ For this reason, it is of critical importance for manufacturers of parenteral drug products and implantable devices to ensure adequate control mechanisms are in place to prevent the accumulation of endotoxin contamination on or within medical devices and drug products. In the pharmaceutical and medical device industries, the term depyrogenation refers to a validated process designed to remove or inactivate pyrogenic material, by a specified quantity, which is monitored by inactivation of endotoxin.⁵

While in-process endotoxin monitoring steps such as the screening of raw materials, critical water systems, and the environment are in place to help reduce contamination risks, it is only after subjecting the final product to a validated depyrogenation process where a 99.9% or 3-logarithmic reduction of endotoxin is achieved that a manufacturer may make a claim of “depyrogenated.”⁶ The two most common methods for depyrogenation of an in-process manufacturing component, finished device, or parenteral product are through dry heat or rinsing. While both methods can be utilized to safely reduce contaminating endotoxin, it is important to understand the pros and cons associated with each method (Table 1).


Depyrogenation via heat is the most common and effective way to destroy pyrogenic material. This is the preferred method of choice for heat-resistant materials because it results in destruction and inactivation of the endotoxin as opposed to physical separation.⁵ Additional benefits to using dry heat as the depyrogenation method include: 1) this level of processing also sterilizes the material and 2) the high temperature operating ovens and the filtered air that heat them are not common sources of Gram-negative bacteria and/or endotoxin contamination (compare to water). To achieve three logs or greater of endotoxin destruction via heat, temperatures ≥ 180ºC for exposure periods between 30 minutes to 3 hours are required.⁵

When depyrogenation via heat is not an option, rinsing with pharmaceutical quality water may be used as an alternative technique to remove endotoxin from heat-labile products.² Plastic bottles and vials, elastomeric stoppers, heat-sensitive seals/closures, and certain medical devices are examples of products or product contact components that may be good candidates for depyrogenation via rinsing. This process works by physically removing/separating contaminating endotoxin from the product through multiple rinse cycles. Because depyrogenation via rinsing is not a destructive method and relies on the physical removal of endotoxin through dilution, it’s of critical importance to ensure the water source itself is not a contributing factor of endotoxin contamination. Water can support the growth of many different microorganisms and is therefore a potential endotoxin contamination source. As such, rinsing as a standalone depyrogenation method should be considered a last resort option and if chosen the water used should be routinely monitored. For example, water used for the removal of endotoxin should be of high-purity (water for injection (WFI)) and preferably above 60ºC.² Nonsterile water that is below 55ºC and above 8ºC should be considered as a potential risk for microbial contamination and therefore potentially contaminated with endotoxins.² Additionally, the water source(s) should be frequently evaluated for bacterial (bioburden) and endotoxin (pyroburden) contamination, ultimately maintaining an endotoxin level of < 0.25 endotoxin units (EU/mL).

Throughout the pharmaceutical industry, vigilant efforts have been undertaken to identify, measure, and eliminate endotoxin from drug formulations, as even minute quantities could lead to adverse reactions and jeopardize patient safety. The diligent investigation and implementation of rigorous testing methodologies and strict regulatory standards have paved the way for comprehensive endotoxin control strategies in pharmaceutical manufacturing. In this paper we compare the effectiveness of two commonly employed endotoxin removal and inactivation methods for glass and “alternative” polymer vials. Additionally, an industry standard steam sterilization method is also evaluated for endotoxin reduction effectiveness.

Materials & Methods

Results

Discussion

Dry heat is a proven endotoxin destruction method. As expected, this process was highly effective at reducing endotoxin contamination levels from the glass vials in excess of 6 logs regardless of manufacturer type. Since the alternative containers are not compatible with dry heat depyrogenation temperatures, no dry heat data was collected for this group.

Rinsing alone resulted in approximately 2.2 logs of endotoxin reduction for each glass vial type (Figure 1). However, for the alternative vials, rinsing alone achieved a 4.1 endotoxin log reduction for the PTFE vials, a 6.2-log reduction for the CoP Daikyo vials, and a 5.7-log reduction for the SiO₂ vials (Figure 1). The latter two vial types remarkably achieved endotoxin removal to that obtained with glass vials subjected to dry heat. This dramatic difference in the effectiveness of endotoxin removal between the glass and alternative vials is a substantial finding of this paper.

We note that endotoxin is both hydrophilic and hydrophobic. We wonder if the results obtained are explained accordingly. Because glass is hydrophilic, it is reasonable that the endotoxin molecules have an affinity to orient themselves to the glass surface via their hydrophilic terminus. Therefore, the Lipid A terminus is water facing, and as expected, is more difficult to remove when rinsing with water. Plastics, on the other hand, are typically hydrophobic and can be expected to behave in the opposite manner. Thus, the hydrophobic nature of endotoxin can result in its hydrophilic terminus being oriented outwardly from the surface making it more accessible to rinsing. A further evaluation exploring the chemical construct of endotoxin and it’s hydrophobic/hydrophilic affiliation between it and various different substances including glass and plastics will be an interesting follow up to this study and may better explain some of the findings.

Steam sterilization alone delivered approximately 3-logs of endotoxin reduction across all glass vial types and the PTFE vial while the Daikyo CoP and SiO2 vials yielded 1.5 and 1.2 logs of endotoxin reduction, respectively (Figure 1). While lacking with respect to the CoP and SiO₂ vials, the average 3-log reduction between the three standard glass vials demonstrates effective and adequate removal. Since the vials are open, it was unable to be determined as to whether the endotoxin reduction achieved during steam sterilization is due to destruction/inactivation, further dilution as a result of the steam condensing and draining out of the opened container, or a combination of both. To address this, a follow up study was performed where filled vials were spiked with endotoxin and sealed prior to steam sterilization. The sealing of the vial served to contain the endotoxin within the vial, preventing it from being diluted/drained out of the vial. Following this methodology, the average endotoxin log reduction was 1.5 (data not shown). Only glass vials were used for this analysis. Compared to the ~3-log reduction of endotoxin observed in open vials subjected to steam sterilization, this data suggests that steam sterilization alone works to reduce endotoxin via inactivation and dilution for opened containers (~3-logs) and inactivation alone for closed containers (~1.5-logs).

The combination of rinsing and steam sterilization resulted in highly effective and robust endotoxin reduction similar to that of dry heat depyrogenation. All glass and alternative vial types studied here yielded approximately 6 logs of endotoxin reduction following the combination of these two processes (Figure 1). This data indicates that a combination of rinsing and steam sterilization can be used as an effective method for the depryogenating of medical devices and drug products/components that are otherwise unable to tolerate high levels of heat.

The data collected in this study supports that dry heat and rinsing in combination with steam sterilization were the most effective methods to reduce endotoxin levels of contaminated pharmaceutical glass and plastic vials. The type 1 pharmaceutical glass vials studied here generated similar results, regardless of manufacture type. Both methodologies yielded approximately 6 logs of endotoxin reduction for each container tested (note that the alternative vials could not be dry heat depyrogenated). While the data supports that rinsing alone can be an effective endotoxin removal method, we note that because of its limitations (Table 1) it should only be used when heat-based methods are not an option. We demonstrate here that terminal sterilization via steam at 121ºC for 30 minutes can serve as an effective endotoxin removal process, even in the absence of rinsing and dry heat. The combination of rinsing and terminal sterilization via steam proved to be consistently effective at providing approximately 6 logs of endotoxin removal across all material types and vial sizes studied here. While glass alternatives are available and may serve a niche purpose, there use should be limited as not being able to depyrogenate them via heat poses an unnecessary endotoxin contamination risk. In applications where alternative materials are required as a substitute to glass, the findings in this study stress the importance of supplementing any rinsing methods designed to achieve depyrogenation with steam sterilization.

References
1. El-Radhi AS. Pathogenesis of Fever. Clinical Manual of Fever in Children. 2019 Jan 2:53–68. doi: 10.1007/978-3-319-92336-9_3. PMCID: PMC7122269.
2. USP 43, NF 38, DEPYROGENATION BY RINSING (accessed 2023).
3. Dobbins JJ. Prescott’s Microbiology, Eighth Edition. J Microbiol Biol Educ. 2010 May 20;11(1):64–5. doi: 10.1128/jmbe.v11.i1.154. PMCID: PMC3577227.
4. BLOCK, McDonnel & Hansen, 2020, 6th ed., Disinfection, sterilization, and preservation, Lippincott, Williams and Wilkins.
5. ISO 20857:2013. Sterilization of health care products — Dry heat —Requirements for the development, validation and routine control of a sterilization process for medical devices.
6. PDA Technical Report 3, (TR3) Validation of Dry Heat Processes Used for Depyrogenation and Sterilization, Parenteral Drug Association, Bethesda, MD, 2013.
7. USP 43, NF 38, DEPYROGENATION (accessed 2023).

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Disclaimer: Prince Sterilization Services, LLC (Prince) is a contract sterilization services provider and supplier of ready to use (RTU) pharmaceutical components including vials, stoppers, and seals. Prince is not affiliated with any of the vial or container manufacturers cited within this study. Unless otherwise redacted, all materials used for this study are available to the public. The material and information contained in this study is for general information purposes only. You should not rely upon the material or information in the study as a basis for making any business, legal, or any other decisions.