The Key to Quality and Consistency: A Comprehensive Guide to Vial Washing Machine Performance Qualification in Sterile Manufacturing

Introduction;

In the pharmaceutical industry, ensuring the safety and efficacy of drug products is paramount. A crucial step in achieving this goal is maintaining clean and contaminant-free containers and closures. Regulatory GMP codes like the FDA (21 CFR Part 211.94 9 -c) require drug product containers and closures to be clean, sterilized, and free from pyrogenic properties to ensure their suitability for intended use. While Depyrogenation methods such as heating tunnels or ovens can help address pyrogenic concerns, it's equally important to remove particles, endotoxin, and chemical contaminants that might cause adverse reactions in humans or animals. In this regard vial washer machines, is identified as one of critical equipment based on the output of critical assemblies, and to ensure their effectiveness and adherence to regulatory standards, validation qualification studies should be conducted in accordance with available guidelines such as Annex 15 EU GMP.

Vial washing machines qualification consists of Installation Qualification (IQ) and Operation?Qualification (OQ) which confirms that the equipment is installed as specified and utilities are sufficient to maintain operation as expected respectively. However, Performance Qualification (PQ) of vial washer machines plays a pivotal role in demonstrating the actual effectiveness of washing processes in removing contaminants and meeting acceptance criteria.

Glass Vial Types;

There are two primary types of vials used in pharmaceutical manufacturing; bulk vials and Ready to Use/Ready to Fill (RTU/RTF) vials. Both types are made of Type I borosilicate glass, a type of glass that is extremely resistant to chemical attack and which suffers minimal thermal expansion.

• ?Bulk Vials;

Bulk vials are typically supplied in trays or shrink-wrapped packs and require additional processing before being filled with a drug product. These steps include:

Washing: Thorough cleaning to remove contaminants, particles, and other impurities.

Depyrogenation: A process to eliminate pyrogens, which are fever-inducing substances.

After these processes, bulk vials are suitable for filling with drug products.

• Ready to Use/Ready to Fill (RTU/RTF) Vials;

RTU/RTF vials have already undergone washing and Depyrogenation before being placed in secondary packaging, which is further sterilized. This eliminates the need for additional washing at the pharmaceutical manufacturing facility. These vials are cleaner than as-received bulk vials and are specifically designed for use in aseptic processing environments.

Vial Washing Machine Types in Pharmaceutical Manufacturing;

?By nature, the Type I glass itself is of high quality however after the vial manufacturing process they are often subjected to uncontrolled environments and may become contaminated with particulates and micro-organisms. For this reason, it is necessary to decontaminate the vials before usage. The aim of washing is to reliably remove particulates, chemical contaminants, and bacterial endotoxins and hence such washing process shall be validated and documented.

The most effective way to remove these contaminants from vials is through “scrubbing” with pure water. Water for Injection (WFI) is the most widely used process water which is not only devoid of particulates, but also almost without microorganisms and Endotoxin and is therefore pure enough to clean without leaving contaminants in the vials. This scrubbing process is accomplished by high pressure water jets and then blowing filtrated compress air in order to dry the vials.

There are three primary types of vial washing machines employed in manufacturing facilities;

1. In-Line Washing Machines: These machines clean vials in a linear, continuous motion. Vials move through various cleaning stages, including pre-wash, detergent cleaning, and rinsing.
2. Rotary Washing Machines: The most commonly used type, these machines employ a circular motion for vial cleaning. Vials are loaded onto a rotating platform that moves them through cleaning stages, such as pre-rinse, detergent wash, and final rinse.
3. Exterior Vial Washers: Designed for cleaning the exterior surfaces of vials, these machines ensure the removal of contaminants before further processing.

In this article, we will just focus on Rotary Washing Machines due to their widespread use in pharmaceutical manufacturing.

Machine Key Components;

A typical Rotary Vial Washing Machine used in pharmaceutical manufacturing consists of several key components as follows to ensure thorough cleaning of vials:

?a) Ultrasonic bath: Some vial washing machines may be equipped with an ultrasonic bath to enhance the cleaning process. Ultrasonic cleaning technology uses high-frequency sound waves to create cavitation bubbles in a liquid medium, which implode and generate powerful shock waves. These shock waves effectively remove contaminants, dirt, and particles from the surfaces of the vials.

The ultrasonic tank in a vial washing machine typically consists of a bath filled with water or a cleaning solution. It should be made of stainless steel 316 L with surface roughness of 0.8 micron. The vials are immersed in the solution, and ultrasonic transducers generate high-frequency sound waves. As the ultrasonic waves propagate through the liquid, they create millions of tiny cavitation bubbles that collapse and release energy, dislodging dirt and debris from the vials' surfaces. The intensity of vibration can be easily adjusted by the operator.

To prevent damage to the ultrasonic bath and its components, particularly the screw, the temperature of the water should be maintained between 50°C and 60°C. The screw is designed to securely hold the vials and facilitate their transportation from inside the bath to the grippers.

To ensure the ultrasonic bath's performance, it should be checked regularly using an aluminum foil test. This involves submerging a piece of aluminum foil in the bath and running the ultrasonic cleaner. The resulting perforations and erosion on the foil indicate good cavitation activity and effective cleaning performance besides this, other methods can use ultrasonic sound intensity measuring instrument. In brief, regular testing helps maintain optimal cleaning efficiency and identifies any potential issues with the ultrasonic bath.

b) Arms and grippers; In a vial washing system, multiple arms or manipulators are typically suspended from the top sheet metal. Each of these arms is normally equipped with grippers which are designed to securely hold and rotate vial glasses during the washing process (Pic. III). ?The number of grippers is usually half the number of needles This configuration enables efficient handling and cleaning of the vials, ensuring a thorough and effective wash.

b) Washing Stations; The washing machine is usually equipped with multiple washing stations. Typically, there are 4 to 7 internal washing stations dedicated to cleaning the interior of vials. Additionally, there are 2 to 4 external washing stations focus on cleaning the exterior surfaces of the vials. These stations work in a sequential manner to ensure comprehensive cleaning.

c) Washing Needles and Nozzles; Each internal washing station features multiple washing needles (typically ranging from 4 to 8 needles (or channels) per station that spray fresh water for injection (WFI) or recycled water (RW) or filtered compressed air (CA) as cleaning media onto the internal surfaces of the vials. This process effectively removes contaminants and residues from the vials' interiors.

Typically, the container-specific immersion depth of the needles is a part of the vial size configuration and can be adjusted either through the Human Machine Interface (HMI) or manually using specific bolts.

Similarly, each outer wall washing station is equipped with multiple “nozzles” that are suspended from the top sheet metal ring of the machine and spray recycled water (RW) or filtered compressed air (CA) onto the external surfaces of the vials. This ensures thorough cleaning and removal of contaminants and residues from the vials' exteriors.

The nozzles can be adjusted in height, angle and lateral distance to the container.?

The washing process;

In a typical vial washing process, bulk vials are commonly placed on an infeed conveyor belt and introduced to an ultrasonic bath for initial cleaning. Following this standard procedure, the vials are then transferred to grippers, where an infeed rotary screw moves them from the housing to the grippers. The grippers securely hold the vials as they undergo washing at the various stations.

The internal and external washing stations follow a specific sequence of steps to clean and dry the vials' interiors and exteriors. This configuration allows for the simultaneous washing of 8 vials ensuring both efficiency and effectiveness in the cleaning process. While the external washing stations operate continuously, ensuring a steady flow of cleaning action, the internal washing and drying and external drying stations take a more methodical approach, working sequentially to ensure each vial is dried perfectly.

With such a comprehensive and efficient cleaning process, we can trust that every vial is thoroughly cleaned and dried inside and out, ready for the next step in its journey.

As shown in the table No.I, there are seven washing stations in each run of the vial washing machine considered as A typical washing cycle. The machine utilizes 8 needles to perform the internal cleaning tasks. Each vial is engaged with a single needle as it progresses through the washing stations.

For example, if a vial is washed by Needle 1 in Station 1, the same Needle 1 will be used to wash the vial in subsequent stations (Stations 2 through 7). This ensures that each needle consistently handles the same vials throughout the entire washing process, maintaining uniformity and consistency in the washing procedure.

It is important to note that, in contrast to the aforementioned process for internal washing and drying, all nozzles in the outer wall washing station are engaged during the washing process. This means that we cannot specify a particular nozzle for each vial, as all nozzles are actively involved in the cleaning procedure.

Essential Installation Qualification (IQ) Aspects for Vial Washing Machines;

During Installation Qualification (IQ) for a vial washing machine, it is crucial to ensure that the equipment, facility, or utility is installed according to the design requirements and User Requirement Specifications (URS). As part of the IQ process, various aspects should be verified and documented, including equipment identification, proper installation, connections, calibration of measurable instrument and gauges (according to the instrument list), filters integrity (according to the component list), safety features, cleaning methods, spare parts, documentation, drawings, cables, connectors, calibration records, and materials of construction (MOC).

An essential component of the IQ process is emphasizing the need for an appropriate cleanroom environment. The vial washing process should be carried out in a Grade C or at least Grade D cleanroom with high-efficiency particulate HEPA filtering to minimize contamination risk.

Moreover, the installation should include a properly designed and controlled exhaust duct-work system connected to the vial washing machine and to ensure safety and prevent unintended access from the technical area, a pneumatic valve is installed within the exhaust duct-work. This system comprises a dehumidifying exhaust fan to manage humidity levels and maintain an optimal environment for the washing process by preventing the back-flow of air or contaminants into the cleanroom area.

?Apart from the above points the design of washing machines should consider:

? Easy draining, cleaning, and drying of the equipment and pipes, with appropriate gradients and drains

? Post-wash drying with air passed through a sterilizing grade filter

? Protection of washed components prior to the depyrogenation phase

? Air breaks at washer drain to prevent back flow

Verifying these factors during the design and Installation Qualification phase are essential to ensure the vial washing machine's proper installation, safety, and performance within the pharmaceutical manufacturing environment

Operational Qualification (OQ) for Vial Washing Machines: Ensuring Performance and Safety in Unloaded Conditions

Operational Qualification (OQ) is a critical step in the validation process for vial washing machines in pharmaceutical manufacturing. It involves documenting evidence that the equipment, facility, or utility operates according to predefined requirements and specifications. OQ primarily focuses on testing the individual components and operations of the equipment without load conditions, ensuring all supplies and connections function correctly. Key aspects of OQ for a vial washing machine include:

• Evaluating low, medium, and high operational ranges for various aspects of the equipment in unloaded conditions.
• Availability of operational Standard Operating Procedures (SOPs) and appropriately trained personnel for equipment operation in unloaded conditions.
• Confirmation of satisfactory calibration, cleaning, and operational parameters for the equipment when it is operating.
• A vital element of OQ for a vial washing machine is the verification of safety alarm system functionality checks. It should be included for alarms for low/high pressure and low/high temperature

During OQ, technicians should rigorously test these alarms to ensure they activate appropriately in response to various abnormal conditions or equipment malfunctions when the machine is running

?Performance Qualification (PQ) and Critical Quality Attributes (CQAs);

The primary goal of the performance qualification (PQ) is to assess the efficiency and consistency of the vial washing machine under various operating conditions. This process involves testing the machine using specific parameters and challenges to ensure it meets predefined quality standards and performs reliably.

Critical quality attributes (CQAs) are key characteristics of the cleaning process that can significantly impact product quality, safety, efficacy, purity, strength, drug release, and stability. Note that 'critical' in this context means that any variations of the parameters under consideration can have an adverse effect (CQAs on patient health, CPPs on adherence to CQA acceptable limits).

To ensure the machine can clean even heavily contaminated vials, validation officers must establish a suitable initial contamination level for testing. This level should not be less than two times the average test results from three different production lots, following the ANSI/ASQ Z1 standard. For Particulate Matter and Chemical Contamination, the initial contamination level is set by doubling the data derived from the average of test results.? In the case of Endotoxin, a range of 1000-10,000 EU should be used as the initial level. The acceptance criterion should be a three-log reduction of this initial level (1000-10,000 EU).

The following CQAs and associated tests are essential in evaluating the vial washing machine's performance:

-??????? Soluble Matter (Sodium Chloride testing): Determine the presence of soluble residues by measuring sodium chloride levels on cleaned vials, which should fall within acceptable limits.?

Procedure:

A 30% solution of sodium chloride is prepared in water.? Sample vials should be contaminated with 0.1 ml of this sodium chloride solution. Containers are swirled to coat the inner surface and evaporate the solution to dryness at room temperature. Spiked test containers are marked in series with permanent marker on the outer surface. Sample washed vials shall be tested.

Testing method; As per specified method for Sodium Chloride testing in USP

Acceptance Criteria: Precipitation & chloride content should be absent and not detected in the contaminated samples after washing.

-??????? Chemical Contamination (Riboflavin testing): Assess the efficiency of removing chemical contaminants from outside of the vials by using Riboflavin testing.? This test ensures that any residual riboflavin levels are within acceptable limits.

Procedure:

Vials were prepared by the addition of 10ml of Riboflavin 0.02% w/v solution, agitated to ensure coverage both internal and external surface of the vials and then left to dry in an oven at 60??C for 24hours. Washed vials were observed under UV light at 360nm for any signs of riboflavin contamination by appearance of fluorescence.

Acceptance Criteria: The presence of any fluorescent residues indicates that contaminated samples after washing were not properly cleaned.

-??? Particulate Matter Test: When using packaging components for parenteral drug products, it is essential to minimize particulate matter through the washing cycle, ensuring it remains within acceptable levels. Two critical aspects to test are:

• Visible Matter (Glass Particles); Assess the cleanliness of washed vials by inspecting them for visible particles, such as glass fragments or other contaminants. This test typically involves a visual inspection against a light source or a dark background, which can help identify any particulate matter present.
• Sub-visible Matter (Polystyrene Particles); Evaluate the presence of sub-visible particles, such as polystyrene particles, using analytical techniques like light obscuration particle count testing (LO-PC) or microscopy. Samples of rinsing solutions or vials are analyzed for particle counts and size distribution.

Procedure;

a)???? Using Duck particle solution; The duck particle solution is a simulated protein-based soil commonly used in cleaning validation studies to mimic the presence of protein residues in pharmaceutical manufacturing equipment. The solution typically contains duck blood, egg white, and milk. Contaminating vials with duck particle solution allows us to evaluate the vial washer's effectiveness in removing protein-based contaminants, ensuring product quality and safety.

?0.25ml of Duke Particle standard (50μm) added to each vial and agitated to ensure even coverage, covered and dried at 60 °C until no visual water present. Spiked vials were washed and spiked vials and un -spiked to be test as per internal procedure.

b) Skim milk or whey protein solutions; These protein-rich materials can be used as substitutes for duck particle solution, providing a suitable challenge for washing studies.

c) Charcoal slurry; In addition to using duck particle solution for testing sub-visible matter, a 1% charcoal slurry can serve as an alternative testing medium.

10 g of charcoal is dissolved filtrated water to make 1% charcoal slurry. Approximately 0.1 ml of charcoal slurry is used to spike each of vial samples by swirling the container to coat the inner surface and evaporate the solution to dryness at room temperature.

Testing method; As per USP <788>

Acceptance Criteria: ?It is summarized in the following table as per USP <788>;

-??????? Bio-burden Contamination (Endotoxins): also known as pyrogens, are fever-inducing substances that can be harmful if present in injectable medicines. It is difficult to remove Endotoxins from products once present. Glass, being a more porous and hydrophilic material, tends to retain Endotoxins more strongly than other surfaces like steel or rubber. This makes Endotoxin removal from glass surfaces more difficult. It is far better to keep finished products and components relatively Endotoxin-free rather than have to remove it once present. The most common Depyrogenation procedures for physical components include incineration and removal by washing, also termed dilution Depyrogenation tunnels expose the washed vials to high temperatures (usually above 250°C), which effectively destroys bacterial Endotoxins and reduces their levels to a safe, acceptable range.

Procedure: Spike each testing vial with endotoxin suspension (Endotoxin challenge vials) to be prepared using a final concentration of approximately 1000 to 10000 EU/mL of Endotoxin challenge vials Dry in a vacuum oven at 45°C to 60°C and 30 mmHg about one hour until no visual water present.

Test method; As per USP <85>

Acceptance Criteria:?Not less than three log (99.9%) reduction of endotoxin ranging (1 to 10 EU) or not more than 0.25 EU/ml?

Performance Qualification (PQ) and Critical Process Parameters (CPPs);

In the context of vial washing, Critical Process Parameters (CPPs) play a vital role in ensuring the desired product quality. CPPs are the parameters whose variability can impact the critical quality attributes (CQAs), making it essential to monitor and control them. Key CPPs for the vial washing process may include:

-??????? Cleaning Media temperature (RW, WFI)

-??????? Cleaning Media pressure (RW, WFI)

-??????? Spray pressure (RW, WFI, Compressed Air)

-??????? Intensity of the ultrasonic bath

-??????? Machine speed

To perform Performance Qualification (PQ) effectively, the vial washing machine should be tested under worst-case scenarios, with CPPs set at their lower acceptable range except for machine speed, which should be maximized. This approach enables the identification of potential risks and ensures the equipment's ability to achieve the desired level of cleanliness and performance even under challenging conditions.

Statistically Valid Sample size determination;

In the context of evaluating a vial washing machine's performance in sterile manufacturing, it is crucial to determine an appropriate sample size for reliable results. The binomial distribution is suitable for modeling the number of successes in independent Bernoulli trials, such as the pass/fail outcomes of vial cleaning. This section details the process of determining the sample size for a one-sample proportion test.

Step I: Define Parameters and Hypotheses;

• Estimated proportion of success (p): The expected proportion of vials passing the test. For high-performance equipment, we aim for p = 1, indicating a 100% success rate. However, considering limitations, let's assume p = 0.99.
• Acceptable proportion of success (p?): The desired proportion of vials passing the test, set close to 1 due to the stringent requirements of sterile manufacturing. Let's assume p? = 0.995.
• Unacceptable proportion of success (p?): The performance level below which the machine is deemed ineffective. Let's set p? = 0.98.
• Statistical power (β): The probability of correctly rejecting the null hypothesis when false. For β = 0.80, Z(β) = 0.842.
• Significance level (α): The probability of incorrectly rejecting the null hypothesis. For α = 0.05, Z(α) = 1.645.

Null hypothesis (H?): The proportion of vials passing the test is 0.98 or less.

Alternative hypothesis (H?): The proportion of vials passing the test is greater than 0.98.

Step II: Calculate Sample Size;

The formula for calculating the sample size for a one-sample proportion test is:

n = (Z(α) + Z(β))^2 p (1 - p) / (p? - p?)^2

Plugging in the values:

n = (1.645 + 0.842) ^2 0.99 (1 - 0.99) / (0.995 - 0.98) ^2

n ≈ 272.14

Rounding up, the sample size is approximately 273 vials. However, to ensure thorough testing across the entire washing process, the total sample size to 320 vials. This adjustment improves the reliability of the study and enables a more comprehensive assessment of the machine's performance.

To ensure comprehensive testing and account for variability in machine performance throughout the washing cycle, we propose dividing the total sample size into four parts of 80 vials. These parts will be distributed as follows:

• One part at the beginning of the normal batch washing
• Two parts in the middle of the normal batch washing
• One parts at the end of the normal batch washing

Furthermore, to ensure the reliability and reproducibility of the machine’s performance, it is recommended to repeat each validation run three times.

?Consequently, the total number of samples required for each study will be:

320?Vials/run×3?runs=960?Vials total

This comprehensive approach will help validate the machine's performance accurately across multiple runs and different stages of the washing process.

Vial Assignment Table;

Following vial assignment table for 320 vials is created with consideration for 8 needles per station of a vial washing machine, this comprehensive guide is particularly useful in ensuring efficient and accurate processing the PQ study.

Note: For each study, the quantity of vials to be washed is the same as normal production size to demonstrate the performance of the normal process

Re-qualification;

Re-qualification is a critical aspect of maintaining the validated state of vial washing machines in pharmaceutical manufacturing. It ensures that the equipment continues to perform as intended and produces consistent, high-quality results over time. There are two primary types of re-qualification: Scheduled (Planned) and Unplanned.

• Scheduled (Planned) re-qualification:

One qualification run shall be performed as part of routine re-qualification program in the vial washing machine, once in six months (with maximum tolerance of one month) with any of the container, at minimum. All the containers being processed in the vial washing machine shall be routine re-qualified once in 2 years, at minimum, with one run.

• Unplanned re-qualification:

Any modification in the washing process parameters or any major maintenance of equipment, which has potential impact on washing process, shall trigger re-qualification of equipment with appropriate containers and/ or number of runs.

For introduction of any new container (specifications and / or source), the vial washing machine shall be qualified with appropriate studies and or number of runs, based on the impact assessment.

Conclusion;

Performance Qualification (PQ) is a critical aspect of validating the efficiency and consistency of vial washing machines in sterile manufacturing environments. By establishing an appropriate initial contamination level, defining clear acceptance criteria, and thoroughly testing machines under various operating conditions, pharmaceutical manufacturers can ensure their equipment performs reliably and consistently meets predefined quality standards.

To achieve a comprehensive PQ study, collaboration with departments such as Microbiology, Quality Control, and Engineering is essential. This multidisciplinary approach allows for accurate testing, data documentation, and effective monitoring of Critical Quality Attributes (CQAs) and Critical Process Parameters (CPPs).

Statistical justification and an appropriate sample size are crucial components of a robust PQ study. A well-designed sampling plan based on the binomial distribution ensures a reliable performance assessment.

By adhering to industry guidelines and regulations, employing statistically sound methodologies, and focusing on a collaborative approach, pharmaceutical companies can guarantee patient safety and product quality while optimizing their manufacturing processes.

Reference;

1-???? Code of Federal Regulations, Title 21, Food and Drugs, part 211.94, April 2005, Office of Federal Register, Washington, DC, USA,

2-???? United?States?Food?and?Drug?Administration, Process Validation: General Principles and practices.?November 2011, Silver Spring, MD 20993, USA

3-???? Qualification and Validation; Annex 15 of the EU GMP Guidelines, 2015, Brussels, Belgium

4-???? Validation of a Rotary Vial washer for terminally sterilized product manufacture, Andrew Sully Catherine Talbot, Cardiff and Vale University Health Board, SMPU, Cardiff, Wales, UK

5- U.S. Pharmacopeia (USP) Rockvill,12601 Twinbrook Parkway Rockville, MD , USA

6- WHO Technical Report Series 970, 2012, Annex 2. Geneva, Switzerland

7- ISPE Baseline? Guide: Sterile Product Manufacturing Facilities, Volume 3, Third Edition, 2018, 600 N. Westshore Blvd., Suite 900, Tampa, Florida 33609 USA

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