Life Sciences
8 min read

Sterilization validation project at Datwyler



Datwyler Pharma Packaging Belgium teamed up with Agidens to create a steam sterilization process for a sterilizer located in the United States. The addition of steam sterilization to their existing gamma radiation sterilization options provides an alternative method for sterilizing parenteral packaging products. This was developed with the goal of offering the best possible solutions to their customers.

This in-depth article delves into the complex processes, techniques and results of the steam sterilization validation project.

“Datwyler Pharma Packaging has always been driven by a forward-thinking mindset, striving to cater more effectively to the multifaceted needs of the biopharmaceutical industry. We wanted to expand our portfolio with a ready-to-use steam sterilization option that caters to the needs of biopharmaceutical packagers and manufacturers. By offering both steam sterilization and gamma irradiation, we provide our customers the flexibility to select the most suitable sterilization method for their specific application. Together with Agidens, we delivered a beautiful project that was highly anticipated within Datwyler. The first four batches were released at the end of 2022.”

Carina van Eester Global Product Leader at Datwyler

The Agidens approach to validate steam sterilization processes


Before tackling the project’s complexities, Agidens needed to assess the potential risks linked to the steam sterilization process. The team decided to use Failure Mode and Effects Analysis (FMEA) – a systematic, proactive approach for evaluating a process to identify where and how it might fail. To get a first-hand understanding and an on-ground perspective, two of Agidens’ consultants traveled to the Datwyler facility in Middletown, Delaware (US). Their scrutiny spanned from preparing the loading pattern to unloading the autoclave.

2. Cycle development

After completing the initial risk assessment, the next critical step was to assist Datwyler in creating a detailed test protocol for cycle development. This phase had two key objectives: first, to carry out tests verifying the equipment’s effectiveness, both empty and loaded, to ensure it worked efficiently. Second, to carefully monitor and adjust the steam penetration study, ensuring optimal sterilization at every cycle.

3. Proces validation

Agidens went further into the details of sterilization by helping Datwyler conduct D-value studies. Using the BIER (Biological Indicator Evaluation Resistometer) method, a series of components such as stoppers and plungers were examined. This method accurately measures the time needed to achieve a sterility assurance level of 10^-6 (a 99.9999% or log 6 reduction), guaranteeing thorough sterilization.

4. Validated & first batch release

The success of the collaboration between Agidens and Datwyler extended beyond practical tasks and involved substantial documentation efforts. The process was carefully structured: first, establishing the project’s strategy, then creating the protocol and test scripts. After Datwyler reviewed these, they were further refined.

The final stage was an in-depth report summarizing all the findings, along with conclusions and strategic advice. This ensured Datwyler had the necessary information and insights to confidently advance with their sterilization process.

Diving deeper in the project


Failure Modes and Effects Analysis (FMEA), offers a strategy to risks attributed to those failures.

At Datwyler, the FMEA covered a wide range of areas and topics. We explored various aspects including process input, equipment functionality, loading and unloading the sterilizer, the sterilization process itself, as well as storage and packaging. Each of these sections was thoroughly examined to identify specific subjects and potential risks. To assess the impact and causes of these potential issues, we worked closely with a cross-functional team comprising members from different departments.



The FMEA methodology typically follows these crucial steps

Define the system or process for analysis

To conduct a thorough FMEA, we started by clearly defining the system or process to establish a precise scope for analysis.

Spot potential failure modes

List and explain every possible failure mode.

Determine impact of failures

Assess the impact of each failure mode, paying special attention to how they might affect the customer, product or process.

Calculate the probability of failures

Estimate the likelihood of each failure mode occurring and calculate a Risk Priority Number (RPN) for each.

Strategize remedial actions

Based on the RPNs, plan strategies to address the most critical failure modes. These strategies could include preventive measures, redesigning certain aspects or improving procedures.


Action implementation

Implement these strategies to mitigate the most significant failure modes.

Frequent FMEA Revisions

Periodically revisit and refine the FMEA to maintain its relevance and effectiveness over time.

2. Cycle development

To create a successful steam cycle, it was crucial to thoroughly understand user needs and the limitations of the materials involved. We established the following key principles:

  • The process’s maximum temperature is capped at 124°C to accommodate the sealing bags.
  • It’s vital to ensure the load is completely dry, particularly for stoppers used in lyophilized products.
  • The versatility of combined loading patterns is crucial. This enables the sterilization of varied products in a single cycle, ensuring that the cart’s loading location does not influence outcomes.

Guided by these essential requirements and focusing on rubber plungers and stoppers as the representative load, Agidens’ senior validation expert designed an effective cycle. Drawing from past experience, a basic cycle was initially created. This initial cycle was thoughtfully designed, requiring only minor adjustments later on.


3. Process validation

In our process validation effort, the concept of the ‘Minimum Acceptable Cycle’ (MAC) was a central focus.

We used the Finn-Aqua GMP BPS (ID 122121-N-DP-BX-BPS-S7) sterilizer at Datwyler Pharma Packaging in Middletown. This autoclave operates on time-based cycles, relying solely on the duration of the process and not factoring in the cumulative lethality as a key process parameter.

For a robust process validation, addressing the most demanding production scenario in terms of sterilization efficacy is crucial. This is covered by the Determination of D-value and Minimal Acceptable Cycle (MAC).

The D-value, which is the time or dose needed to inactivate 90% of the test microorganism (Geobacillus stearothermophilus) at 121°C, was ascertained using a BIER-vessel test (Biological Indicator Evaluation Resistometer). The highest D-value observed for the products under validation was 3.5 minutes. Based on this, the minimal acceptable cycle was defined. For the highest measured D-value, a kill time of 35 minutes was calculated as the minimal acceptable cycle needed to achieve the required kill off of the microbial load. This value is rounded to a kill time of 35 minutes and results in a production sterilization cycle duration of 50 minutes.

The D-value, or decimal reduction time, indicates the time needed to reduce viable microorganisms in a sample by 90% (a one-log reduction). In steam sterilization, it helps set the required time and temperature. A higher D-value means more sterilization time is needed. This value varies based on the microorganism type and sterilization conditions like temperature and pressure.

The BIER (Biological Indicator Evaluation Resistometer) is a sterilization validation tool measuring a bacterial population’s resistance to sterilizing agents, often via electrical resistance, to gauge microorganism viability. Commonly paired with physical and chemical indicators, BIER tests validate sterilization in healthcare, are completely free of any viable microorganisms.

Bracketing approach

After successfully completing the sterilization validation report, we adopted a systematic approach to product categorization using a bracketing method. This entailed grouping products based on specific parameters that might influence sterilization efficacy.

We identified three critical parameters:

  • D-value: a higher D-value results in a higher resistance of the micro-organisms.
  • Total net weight: it is more difficult to reach the required temperature when a higher mass within the autoclave and bag configuration is used.
  • Geometry: a deeper or narrower cavity may pose challenges for steam to effectively reach all surfaces. Additionally, such shapes are more prone to trapping condensate, which can adversely affect the dryness of the load after the sterilization process.

We aimed to define the boundaries — what were the minimum and maximum acceptable limits based on the validated rubber products?

This bracketing approach facilitated the determination of which types of plungers and stoppers (or products in general) can be seamlessly integrated into the existing ‘family’ without compromising on sterilization integrity.


The extensive validation study provided positive and conclusive results, confirming that the validated sterilization cycle effectively and consistently sterilizes all loads within the defined bracket.

  • Each monitored steam penetration location consistently reached F0-values of at least 15 minutes.
  • After the sterilization cycle, we observed complete inactivation in all Biological Indicators used in inoculated stoppers.
  • Measurements of residual moisture post-sterilization consistently stayed at or below the 0.5% target.
  • The equilibration times were efficient, never exceeding 30 seconds.
  • Temperature variations within the room, including the theoretical steam temperatures, were minimal, with differences not surpassing 2°C during holding times.
  • During the holding period, the temperatures recorded by the distribution probes consistently met the established limits.
Pieter Tilkens - Agidens
Pieter Tilkens General Manager
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“Agidens plays it part in increasing patient safety. Our job is more than just technical know-how, it’s about letting manufacturers concentrate on their strengths. In short, we aim to make complicated things simple, allowing our partners and clients to excel in their core.”

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