11 Key Contributing Factors For Maintaining Sterility Assurance

By Yadnyesh Patel and Vaibhav Patel

The first article of this series on sterility assurance covered the fundamentals, including terminologies and key components such as aseptic processing, terminal sterilization, and post-aseptic processing terminal sterilization. In this article, we continue the series by discussing 11 key contributing factors for maintaining sterility assurance.

1. Containers And Closures

The container and closure for a sterile formulation are integral parts of the sterile product. Container and closure systems are critical for maintaining sterility in pharmaceutical products. They ensure protection against microbial contamination throughout the products’ shelf life and are chosen to minimize interaction with, preserve the quality attributes of, and facilitate dispensing of the sterile product.¹

Selection of containers and closures should consider the following factors:

• The container and closures must be integral, durable, nonreactive, and impermeable to contamination.
• They should not react with formulation materials.¹
• Containers and closures should have tight seals that prevent microbial ingress, validated by container closure integrity testing (CCIT).
• Components must withstand and be compatible with sterilization/depyrogenation methods without compromising integrity.¹
• Components must be clean to prevent particulate-induced contamination. They must be free of particulates, endotoxins, and leachables or extractables.¹
• Packaging must withstand environmental stresses while maintaining sterility.
• Containers and closures must adhere to standards that ensure reliability and safety.

2. Decontamination

Decontamination is a term used to describe a variety of processes that systematically remove or destruct microbial contaminants from equipment surfaces and environments to minimize the contamination risk without an expectation of a total kill.¹

Decontamination is integral to sterility assurance, ensuring environments and materials remain free of contaminants. Through validated cleaning methods, effective disinfectants, and continuous monitoring, it supports the production of safe, sterile products.

Key contributions of decontamination are:

• Decontamination lowers the microbial load on surfaces, equipment, and environments, reducing the risk of contaminants entering sterile processes. This supports sterile product manufacture.¹
• Effective decontamination ensures cleanrooms and controlled areas remain free from viable microorganisms, supporting aseptic operations.
• Disinfectants with broad-spectrum activity are used to eliminate bacteria, fungi, and viruses. Non-sporicidal and sporicidal disinfectants are used on rotation basis to implement effective decontamination practices. Rotation of disinfectants prevents microbial resistance. ^{2, 3}
• Autoclaving, gas sterilization, and vaporized hydrogen peroxide decontaminate equipment and tools used in sterile production; this helps to remove contaminants that might interfere with the manufacturing process.
• Decontamination protocols/SOPs for personnel, including best aseptic practices, maintaining personnel hygiene, hand washing/sanitization practices, and sterile gowning, reduce contamination risks from human operators.
• Risk assessments and routine audits refine decontamination strategies, ensuring sustained sterility assurance.

3. Depyrogenation

Depyrogenation is the process of removing or inactivating pyrogens, such as bacterial endotoxins, from materials, equipment, and products to ensure sterility assurance. Depyrogenation is critical because pyrogens, even in sterile products, can cause harmful reactions in patients. Minimizing pyrogen content is a requirement for injectable products. During the production of sterile products, depyrogenation processes are used in a variety of ways to minimize pyrogenic contamination of surfaces, materials, and products.¹

Key contributions of depyrogenation include:

• Endotoxins, primarily from gram negative bacteria, can remain even after sterilization. Depyrogenation ensures these contaminants are destroyed or reduced to acceptable levels.
• Validated depyrogenation methods such as dry heat sterilizers, depyrogenation tunnels, and chemical treatments effectively reduce the minimum 3 log of endotoxins from equipment, materials, glassware, utensils, machine surfaces, and environments. This optimizes the product quality and minimizes the risk to the patients.⁴
• Regulatory agencies like FDA and EMA require endotoxin control in sterile product manufacturing, making depyrogenation essential for compliance.
• Regular endotoxin testing using compendial methods like the limulus amebocyte lysate (LAL) test ensures the effectiveness of depyrogenation processes to meet the product specification requirements and patient safety.

Depyrogenation is vital for sterility assurance as it eliminates endotoxins that sterilization alone cannot address. By integrating validated depyrogenation methods and rigorous testing, manufacturers ensure product safety, regulatory compliance, and patient protection.

4. Equipment

Properly designed, maintained, and validated equipment ensures that production processes remain free from microbial contamination, safeguarding product quality and patient safety.

Equipment used for sterile product manufacturing varies in its impact on the manufacturing process and on product quality and should have several important characteristics.

The equipment should:

• be made of nonreactive smooth and easily cleanable materials (e.g., stainless steel), sterilizable to prevent microbial growth¹
• have hygienic design features, such as rounded corners and seamless surfaces, to reduce contamination risk
• withstand sterilization methods like steam autoclaving, dry heat, or chemical sterilization without compromising functionality
• require minimum human intervention through physical separation during operation, automated systems equipped with sensors and alarm monitors, robotics, critical parameters (temperature, pressure), and provide real-time alerts for deviations¹
• be qualified (initial and periodic, i.e., IQ, OQ, PQ, and RQ), calibrated, and have a maintenance program in place (i.e., preventive maintenance and breakdown maintenance)
• have a closed system or isolators that reduce exposure to external contaminants, enhancing sterility assurance
• not adversely impact essential product quality attributes.¹

Equipment is a cornerstone of sterility assurance, enabling controlled and contamination-free manufacturing processes. Proper design, validation, maintenance, and operation of equipment ensure consistent sterility, regulatory compliance, and the production of safe, high-quality products.

5. Facilities

Proper design, maintenance, and management of facilities minimize contamination risks and support aseptic operations.

The impact of manufacturing operations on the location and overall design of the sterile manufacturing area must be considered. Emphasis should be given in facility design to the flows of materials, components, personnel, equipment, and waste streams throughout the facility and to the orderly transition of items between environments of different classifications to prevent mix-up and avoid product contamination.¹

The contributions of facilities to sterility assurance include:

• Facilities must be designed to minimize microbial, chemical, and particulate contamination.¹
• The facility design must be supported by practices and procedures such as cleaning and decontamination, gowning, and material transfer. The architectural details of the facility infrastructure must consider the means for cleaning and disinfection.¹
• Facilities must include cleanrooms with controlled environments (classified ISO 5 to ISO 8) to prevent contamination.
• The manufacture of sterile products should be carried out in appropriate cleanrooms, entry to which should be through change rooms that act as airlocks for personnel and airlocks for equipment and materials.⁶
• Cleanrooms and change rooms should be maintained to an appropriate cleanliness standard and facilitated with clean air.⁶
• High efficiency particulate air (HEPA) filters ensure a continuous supply of clean air, removing particulates and microorganisms.
• Unidirectional airflow systems provide a homogeneous air speed in a range of 0.36 to 0.54 m/s at the working position.
• Controls and monitoring should be scientifically justified and should effectively evaluate the state of environmental conditions of cleanrooms, airlocks, and pass-through hatches. Cleanrooms should have proper air pressure differentials (e.g., positive pressure in critical areas) that prevents cross contamination.⁶
• Regular monitoring of environmental conditions (i.e., temperature, humidity, differential pressure, particulate level, and microbial presence) ensures facility compliance with sterile conditions.
• Airflow visualization studies should correlate with the air speed measurement to effectively maintain the critical area.
• Facilities must undergo regular maintenance and validation to ensure systems such as HVAC airlocks and cleanroom integrity remain effective.
• Facilities must adhere to guidelines from regulatory agencies (i.e., FDA, EMA, WHO) to meet sterility standards. Validation and documentation demonstrate ongoing compliance.
• The various operations of component preparation, product preparation, and filling should be carried out with appropriate technical and operational separation measures within the cleanroom or facility to prevent mix-up and contamination.
• The core activities for sterile product manufacture should be carried out in classified environments operating in conformance with the ISO 14644 series of standards.^{1, 5}
• A positive pressure should be maintained from higher to lower from the more critical areas to less critical areas.¹
• The manufacture of sterile products should be performed in cleanrooms/zones with four grade-classified environments.⁶

Conventional Cleanrooms

• Grade A: This is the critical zone for high-risk operations (e.g., aseptic processing line, filling zone, stopper bowl, open primary packaging, or for making aseptic connections under the protection of first air). Normally, such conditions are provided by localized airflow protection, such as unidirectional airflow workstations within RABS or isolators.
• Grade B: For aseptic preparation and filling, this is the background cleanroom for grade A (where it is not an isolator). Air pressure differences should be continuously monitored. Cleanrooms of lower grade than grade B can be considered where isolator technology is used.
• Grades C and D: These are cleanrooms used for carrying out less critical stages in the manufacture of aseptically filled sterile products or as a background for isolators. They also can be used for the preparation/filling of terminally sterilized products.

Restricted Access Barrier Systems (RABS)

The design of RABS should ensure grade A conditions with unidirectional airflow and first air protection in the critical zone. A positive airflow from the critical zone to the supporting background environment should be maintained.⁶

Isolators

The bio-decontamination process of the interior should be automated, validated, and controlled within defined cycle parameters and should include a sporicidal agent in a suitable form (e.g., gaseous or vaporized form). Isolators are commonly decontaminated using automated systems. ⁶

Isolators provide complete separation between personnel and the enclosed ISO 5 processing environment. A defined pressure differential is maintained between the ISO 5 environment and the surrounding area.¹

Blow-fill-seal (BFS) And Form-fill-seal (FFS)

These technologies form, fill, and seal flexible walled containers in an ISO 5 environment. The critical activities are performed within a unidirectional airflow environment. Decontamination is performed as is common for the background environment.¹

6. Materials​

Sterile products are manufactured with combinations of a wide range of materials, including active pharmaceutical ingredients (small and large molecules), excipients, solvents (usually water), process gases, and processing aids, all of which contribute to the microbiological quality attributes of the product.¹

Selecting, handling, and validating appropriate materials minimizes contamination risks and supports the production of high-quality sterile products.

• Raw materials and packaging materials should be adequately controlled and tested to ensure that levels of bioburden and endotoxin/pyrogen are suitable for use.⁶
• Primary and secondary packaging materials must form a sterile barrier while protecting products during storage and transportation.
• Materials should not interact with or degrade the product. For example, glass containers must resist pH changes and rubber stoppers must minimize extractables and leachables.
• Sterile materials must be stored and handled in controlled environments to prevent contamination. Use of appropriate containers, cleanroom garments, and aseptic handling techniques is critical.
• Gases used in aseptic processes should be filtered through a sterilizing grade filter (with a nominal pore size of a maximum of 0.22 μm) at the point of use.⁶
• Materials must meet regulatory guidelines and testing specifications to use in sterile manufacturing.
• Microbial contamination may be present on/in active pharmaceutical ingredients, excipients, and primary packaging materials. Hence, controlling bioburden in materials and formulated product is a critical aspect of sterility assurance.¹

7. Personnel

Manufacturing/operations personnel are considered the primary source of microbial or particulate contamination, as the many essential activities they perform include cleaning, assembly, equipment operation, material transfer, environmental monitoring, and decontamination^. While personnel are often necessary for the performance of these activities, the contamination derived from them must be prevented from entering the production materials before and after sterilization.¹

The personnel involved in the preparation of sterile products must:

• be well trained in aseptic techniques, hygiene practices, and gowning procedures and understand the principles of microbiology, sterilization/depyrogenation, aseptic processing, and contamination control
• minimize unnecessary movement, talking, and actions that generate particulates or disturb cleanroom airflow
• understand cleanroom dynamics, such as airflow patterns, to avoid disrupting sterile zones
• be authorized, qualified, and adequately trained to enter into aseptic areas to reduce contamination risk
• be instructed to report any specific health conditions or ailments that may cause the shedding of abnormal numbers or types of contaminants and therefore preclude cleanroom access. They should periodically be subject health checks to ensure that personnel with illnesses, open wounds, or infections are restricted from sterile environments.
• be prepared to address contamination events promptly, including understanding decontamination and sterilization procedures
• seek feedback from supervisors/seniors that helps then to refine their techniques and identify areas for improvements.
• undergo training for the basic elements of microbiology and hygiene, with a specific focus on cleanroom practices, contamination control, aseptic techniques, and the protection of sterile products (for those operators entering the grade B cleanrooms and/or intervening into grade A).⁶

Only the minimum number of personnel required should be present in cleanrooms. The maximum number of operators in cleanrooms should be determined, documented, and considered during activities such as initial qualification and aseptic process simulation (APS), so as not to compromise sterility assurance.⁶ All personnel, including those performing cleaning, maintenance, and monitoring and those that access cleanrooms, should receive regular training, gowning qualification, and assessment in disciplines relevant to the correct manufacture of sterile products.

8. Procedures

Well-defined and rigorously implemented procedures provide a standardized framework that minimizes contamination risks and ensures compliance with regulatory requirements. Written procedures define the operations that have been determined through validation studies and experience to be effective in controlling and facilitating the manufacture and quality of pharmaceuticals, biopharmaceuticals, and medical devices.¹

Contributions of procedures to sterility assurance include:

• SOPs define consistent practices for aseptic techniques, equipment sterilization, and cleaning and sanitization of cleanrooms, cleaning and sanitization of equipment/instruments, entry-exit practices, material handling practices, sampling and analysis practices, maintenance practices, environment monitoring practices, area qualification, equipment qualification/requalification practices, materials, samples, product testing and release practices, etc.
• Regular review, gap assessment, and updates of SOPs ensure alignment with current standards and technologies.
• Procedures for aseptic handling, filling, and sealing minimize microbial contamination during critical operations.
• Documented procedures/protocols for routine cleaning and disinfection of facilities, equipment and surfaces reduce bioburden levels.
• Controlled procedures for transferring materials and components into cleanrooms/sterile areas prevent contamination.
• Established procedures for investigating deviations and implementing corrective and preventive actions (CAPA) ensure continuous improvement in sterility assurance.
• Procedures for personnel training, qualification, and periodic requalification ensure personnel are proficient in sterility-related tasks and protocols.
• Clear documentation of all procedures, operations, and outcomes ensures traceability and compliance with regulatory standards.
• Procedures for handling of interventions (inherent or corrective intervention) should be covered in SOPs to enable personnel to perform the interventions the same way each time.

Well-defined and updated procedures form the backbone of sterility assurance by standardizing critical operations and ensuring consistency, compliance, and effectiveness. Properly developed, implemented, and monitored procedures reduce contamination risks and enhance the reliability of sterile manufacturing.

9. Utilities

Utilities play a critical role in maintaining sterility assurance by supporting sterile processes and controlled environments. Proper design, validation, and maintenance of utilities ensure contamination-free operations and adherence to regulatory standards.

The manufacture of sterile products requires utilities that can have a substantial impact on the final product. Some of the utilities in the facility can become an integral part of the formulated product (e.g., water for injection, nitrogen), and appropriate design of the production and distribution systems for these is essential.¹

Utilities should be designed, installed, qualified, operated, maintained, and monitored in a manner to ensure that the utility system functions as expected. ⁶ Utility system installation records should be maintained throughout the system’s life cycle. Such records should include current drawings and schematic diagrams, construction material lists, and system specifications.⁶

Let’s understand the contributions of specific utilities to sterility assurance:

• HVAC systems: Heating, ventilation, and air conditioning (HVAC) systems control airflow, temperature, and humidity in cleanrooms. HEPA filters remove particulates and microorganisms, maintaining ISO classified environments.⁵
• Water (PW and WFI): WFI is used as a raw material for manufacturing of sterile pharmaceutical products, whereas purified water is used for component /machine parts washing; hence, PW and WFI must meet stringent microbial, chemical, and endotoxin limits. Validated water generation, storage, and distribution systems ensure purity and sterility. To minimize the risk of biofilm formation, sterilization, disinfection, or regeneration of water systems should be carried out according to a predetermined schedule and as a remedial action following out-of-limit or out-of-specification results. ⁶
• Compressed air and gas system: Sterile air and gases (i.e., compressed air and nitrogen gas) used in production must be free from particulates, moisture, and microbial contamination.
• Steam system: Clean steam used for sterilization and steaming-in-place (SIP) must be free of contaminants. Regular validation and monitoring of steam quality ensure compliance with sterility requirements.
• Electrical systems: Uninterrupted power supplies (UPS) ensure consistent operation of critical equipment such as autoclaves, isolators, and HVAC systems, thus maintaining sterility.
• Cleanroom lighting: Properly designed and maintained lighting minimizes particle generation and facilitates thorough visual inspections.

Utilities must meet standards such as cGMP and ISO requirements. Documentation and audits ensure alignment with global regulations.

10. Aseptic Process Simulation

Aseptic process simulation (APS), also known as media fill testing, is a critical tool for maintaining sterility assurance in aseptic manufacturing. By simulating the actual production with sterile growth medium, APS evaluates the ability of the system, equipment, and personnel to maintain sterility under normal operating conditions.

APS contributes to sterility assurance as follows:⁷

• APS ensures that the aseptic manufacturing process consistently maintains sterility, validating the robustness of equipment, materials, and personnel practices.
• By mimicking real production scenarios, APS identifies potential contamination sources, including human errors, equipment failures, and environmental risks.
• APS evaluates personnel performance during aseptic operations, ensuring compliance with gowning, material handling, and aseptic techniques.
• APS confirms that cleanroom conditions such as airflow patterns, environmental controls, and pressure differentials support sterile operations.
• The process tests the sterility of equipment setup, material transfer, and operational steps, ensuring that all components of the production line contribute to sterility assurance.
• APS challenges the system by incorporating worst-case conditions, such as extended operational times or frequent interventions, to validate its ability to maintain sterility.
• Regulatory agencies, including the FDA and EMA, require APS to demonstrate control over aseptic processes. Successful media fill tests are critical for regulatory approval and ongoing compliance.
• Comprehensive records from APS provide evidence of sterility assurance, supporting audits, and inspections.

11. Cleaning, Sanitization, And Disinfection Program

A robust cleaning, sanitization, and disinfection program is essential for maintaining sterility assurance in pharmaceutical manufacturing and other controlled environments. These processes ensure the removal of contaminants, control microbial load, and maintain aseptic conditions critical for sterile product manufacturing. ³

Key contributions to sterility assurance include:

• Effective cleaning, sanitization, and disinfection practices eliminate residues, particulates, and organic matter that can harbor microorganisms and compromise sterility.
• Sanitization and disinfection reduce microbial bioburden to acceptable levels, minimizing the risk of contamination in sterile environment.
• Disinfection qualification is the most important step for the implementation of a robust sanitization and disinfection program. The validated procedure ensures effectiveness.
• Using validated cleaning agents and disinfectants (e.g., alcohols, quaternary ammonium compounds, hydrogen peroxide) ensures reproducibility and effectiveness against a broad spectrum of microorganisms.³
• Validation studies should demonstrate the suitability and effectiveness of disinfectants in the specific manner in which they are used and on the type of surface material, or representative material if justified, and should support the in-use expiry periods of prepared solutions.⁶
• Use of disinfectant agents on rotation basis prevents development of microbial resistance.
• More than one type of disinfecting agent (i.e., bactericidal and sporicidal) should be employed to ensure they have different modes of action and their combined usage is effective against bacteria, fungi, and spore formers.⁶
• Ready-to-use sterile disinfectants or filtered disinfectants through 0.22 micron should be used for disinfection of grade A and grade B areas.

A well-executed cleaning, sanitization, and disinfection program is integral to maintaining sterility assurance. By effectively controlling contamination risks, these programs uphold the integrity of sterile environments, ensuring the production of safe, high-quality pharmaceutical products.

In our last article of this series on sterility assurance, we will discuss sterility assurance testing methods.

References

1. USP chapter <1211> Sterility Assurance.
2. USP chapter <1072> Disinfectants and antiseptics
3. PDA Technical Report No. 70, Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities.
4. USP chapter <1085>
5. International Organization for Standardization14644:1:2015: Cleanrooms and Associated Controlled Environments
6. EU GMP Annex 1, Sterile Medicinal Products.
7. Guidance for Industry –Sterile Drug products Produced by Aseptic processing –Current Good manufacturing practices- September 2004.

About The Authors:

Yadnyesh Patel earned his master’s degree in microbiology from KBCNM University, Maharashtra, India, in 2009. With over 13 years of extensive experience in quality functions, he has held roles at pharmaceutical organizations such as Claris Otsuka, Zydus, and Sun Pharma. During his tenure, he led microbiological quality functions, quality management systems (QMS), document management, audits, and compliance. His expertise spans QMS, SOPs, documentation management, microbiological test method validations, sterility assurance, aseptic process simulation, and computer system validation. His leadership drives operational excellence.

Vaibhav Patel is a quality assurance professional with over 13 years of experience in drug development. As the director of quality assurance and regulatory affairs at the University of Minnesota, he manages quality systems for clinical productions and ensures FDA compliance. His career covers various biopharmaceutical domains, including radiopharmaceuticals, nanomedicine, cell and gene therapy products, and monoclonal antibodies. Vaibhav's expertise includes developing phase-appropriate quality management systems, leading QA teams, and managing IND product releases as well managing CDMOs in the U.S., China, and India. Previously, he held roles at Elucida Oncology and Memorial Sloan-Kettering Cancer Center. Vaibhav is a certified Quality Auditor and holds a Regulatory Affairs Certification. He is an active member of the American Association of Pharmaceutical Scientists. He holds a master’s degree in pharmaceutical manufacturing from Stevens Institute of Technology and a Bachelor of Pharmacy from Rajiv Gandhi University of Health Sciences.