Process Validation and Sterility Assurance: Relations and Requirements

Robert Dream- Managing Director, HDR Company LLC

The purpose of the production and process control subsystem (including sterilization process controls) is to manufacture products that meet specifications. Developing processes that are adequate to produce drug products that meet specifications, validating (or fully verifying the results of) those processes, and monitoring and controlling the processes are all steps that help assure the result will meet specifications. For sterilization processes, the primary product specification is the desired Sterility Assurance Level (SAL). Other specifications may include sterilant residues and endotoxin levels, Figure 1.⁷

EU Point of Consideration

When the parametric release of sterility is proposed, the Guideline on Real Time Release Testing, EMA/CHMP/QWP/811210/2009-Rev1 (human products only,⁴ the Guideline on Parametric Release, EMEA/ CVMP/QWP/339588/2005 (veterinary products only)5 and the text of Ph. Eur. Chapter 5.1.¹⁶ should be taken into account. The bioburden control criteria should be specified prior to all sterilization processes. High bioburden acceptance criteria should not be justified by the capacity of the sterilization process or any bioburden-reducing step before sterilization. The levels of bacterial endotoxins in the finished product can be impacted by the bioburden and bacterial endotoxins in the components (i.e., active substance, excipients, and containers), and by microbiological contaminants introduced during manufacture. To ensure an acceptable level of bacterial endotoxins in the finished product, the level of microbiological contaminants of the components should be minimal. Acceptance criteria for bioburden and, where relevant, bacterial endotoxins in components and bulk solutions should be specified. All filters used in the manufacture of the finished product that come in contact with the finished product, or with any component (substance or intermediate product) incorporated in the finished product should be described and the information stated should be provided in the quality dossier. The information should be in line with the requirements stated in EudraLex GMP Annex 1.² For ATMPs, the Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products should be followed.³ If a secondary container (e.g., secondary pouch for infusion bags or blisters intended to keep the outside of the container sterile) is used to provide specific protection to the medicinal product, the packaging process should be described, including a risk assessment, since it may affect the sterility of the finished product; for example, trapping moisture between the primary and secondary containers. Information should be provided as to when the packaging step is performed (before or after sterilization) and any aseptic techniques employed. The proposed processes should be justified from a microbiological perspective. If the use of a secondary container means additional sterilization of the finished product is performed, this should be justified with regard to sterility assurance and any potential impact on finished product quality.¹

US FDA Point of Consideration

Sterilization Process Controls-Inspectional Objectives

Inspectional Objectives are to confirm that the sterilization process was validated by reviewing the validation study. Review the specific procedure(s) for the sterilization process selected and the methods for controlling and monitoring the process. Verify that the process is controlled and monitored.

The purpose of the production and process control subsystem (including sterilization process controls) is to manufacture drug products that meet specifications (Figure 2), inspectional process controls are to^7,13.

1. Confirm that the sterilization process was validated by reviewing the validation study.

2. Review the specific procedure(s) for the sterilization process selected and the methods for controlling and monitoring the process. Verify that the process is controlled and monitored.

3. If a review of the Drug Product History Records (including process control and monitoring records, acceptance activity records, etc.) reveals that the sterilization process is outside the firm’s tolerance for operating or performance parameters: 

a. Determine whether the nonconformances were handled appropriately; and

b.Review the equipment adjustment, calibration, and maintenance

4. If the sterilization process is software controlled, confirm that the software was validated.

5. Verify that personnel have been appropriately qualified and trained to implement the sterilization process.

Relationship Between the Stages of Process Validation and Sterility Assurance

As part of stage 1 of the lifecycle of process validation is process design, that is the activity of defining the commercial manufacturing process that will be reflected in planned master production and control records. The goal of this stage is to design a process suitable for routine commercial manufacturing that can consistently deliver a product that meets its quality attributes.⁹

During stage one the process design establishes a Strategy for Process Control, US FDA expects controls to include both examination of material quality and equipment monitoring. Special attention to controlling the process through operational limits and in-process monitoring is essential in two possible scenarios:

1. When the product attribute is not readily measurable due to limitations of sampling or detectability (e.g., viral clearance or microbial contamination), for example:

Probability of Detection of Viruses at Low Concentrations: At low virus concentrations (e.g., in the range of 10 to 1,000 infectious particles per liter), it is evident that a sample of a few milliliters may or may not contain infectious particles. The probability, p, that this sample does not contain infectious viruses is:

p = ((V-v)/V) ⁿ

when V (liter) is the overall volume of the material to be tested, v (liter) is the volume of the sample and n is the absolute number of infectious particles statistically distributed in V.

If V >> v, this equation can be approximated by the Poisson distribution:

p = e^-cv

when c is the concentration of infectious particles per liter. Or,

c = ln (p/-v)

As an example, if a sample volume of 1 ml is tested, the probabilities p at virus concentrations ranging from 10 to 1,000 infectious particles per liter are:

This indicates that for a concentration of 1,000 viruses per liter, in 37% of sampling, 1 ml will not contain a virus particle.

If only a portion of a sample is tested for viruses and the test is negative, the amount of virus which would have to be present in the total sample to achieve a positive result should be calculated and this value taken into account when calculating a reduction factor. Confidence limits at 95% are desirable. However, in some instances, this may not be practical due to material limitations.¹⁴

2. When intermediates and products cannot be highly characterized and well-defined quality attributes cannot be identified.

As part stage 2 of the process validation lifecycle, A written protocol that specifies the manufacturing conditions, controls, testing, and expected outcomes is essential for this stage of process validation. It is recommended that the protocol discuss the following elements:

The manufacturing conditions, including operating parameters, processing limits, and component (raw material) inputs.

• The data to be collected and when and how it will be evaluated.
• Tests to be performed (in-process, release, characterization) and acceptance criteria for each significant processing step.
• The sampling plan, including sampling points, number of samples, and the frequency of sampling for each unit operation and attribute. The number of samples should be adequate to provide sufficient statistical confidence of quality both within a batch and between batches. The confidence level selected can be based on risk analysis as it relates to the particular attribute under examination. Sampling during this stage should be more extensive than is typical during routine production.
• Criteria and process performance indicators that allow for a science- and risk-based decision about the ability of the process to consistently produce quality products. The criteria should include:

» A description of the statistical methods to be used in analyzing all collected data (e.g., statistical metrics defining both intra-batch and inter-batch variability).
» Provision for addressing deviations from expected conditions and handling of nonconforming data. Data should not be excluded from further consideration in terms of PPQ without a documented, science-based justification.¹⁵

Design of facilities and the qualification of utilities and equipment, personnel training and qualification, and verification of material sources (components and container/closures), if not previously accomplished.

• Status of the validation of analytical methods used in measuring the process, in-process materials, and the product.
• Review and approval of the protocol by appropriate departments and the quality unit.

These controls are established in the master production and control records (see § 211.186(a) and (b)(9)).⁸

The sterility assurance is incorporated into the design during Stage 1 and built into the design and is demonstrated through PPQ protocol execution, Stage 2. Sterility assurance a perquisite to support the execution of PQ and PV to establish product continued process verification lifecycle, stage 3, Figure 3.

Sterility assurance cannot be seen as an entity independent from the process/product qualification and validation. Sterility assurance is generally a prerequisite to starting the PPQ process, however, the process should be designed not only taking into consideration the drug substance/drug product, but also the combination of sterility assurance and the drug product quality.

Process Validation is Well Defined by Regulations

Generally, early process design experiments do not need to be performed under the cGMP conditions required for drugs intended for commercial distribution that are manufactured during Stage 2 (process qualification) and Stage 3 (continued process verification). They should, however, be conducted in accordance with sound scientific methods and principles, including good documentation practices. This recommendation is consistent with ICH Q10 Pharmaceutical Quality System. Decisions and justification of the controls should be sufficiently documented and internally reviewed to verify and preserve their value for use or adaptation later in the lifecycle of the process and product.⁹

For parenteral drug product the essential element of aseptic processing as defined in Figure 6.

1. It means ensuring a product is sterile, with

a. Very low endotoxins
b. Very low foreign particles
c. The entire manufacturing process shall be designed to fit the characteristic of the product:

1. 1. Excipients
   2. Drug Substance
   3. Container
   4. Platform
   5. Administration route

2. The process can be divided in steps (Figure 4 and 5), and for each step shall have as a minimum control of bioburden, endotoxins and particles

3. At some point in time the main product components.

a. Formulation
b. Equipment in contact with product
c. Container closure
d. Environment where product is exposed

Aseptic processes are designed to minimize exposure of sterile articles to the potential contamination hazards of the manufacturing operation. Limiting the duration of exposure of sterile product elements, providing the highest possible environmental control, optimizing process flow, and designing equipment to prevent entrainment of lower quality air into the Class 100 (ISO 5) (and or grade A) clean area are essential to achieving high assurance of sterility.

Science and Risk Based Approaches

These approaches should be used to obtain information needed/ required to make decisions related to the evaluation, design, qualification, validation, operation, and monitoring sterile product manufacturing processes. Risk and science-based approaches should be used to develop and implement control strategies and acceptance criteria designed to assure the establishment and maintenance of manufacturing conditions which affect the sterility of products. Sterile drug product manufacturing processes and testing requirements should have a basis in and relevance to risk to product quality and patient safety. Risk management and assessment methods should be developed to not only identify risk but allow for the improvement of processes and control strategies.

The effectiveness of certain traditional testing and monitoring methods as control strategies should be reevaluated. As technology has been introduced and knowledge acquired, the usefulness and value of testing procedures have changed. Testing and monitoring should be designed, performed, and its results evaluated based on scientific value, risk to product quality and patient safety, and usefulness to the determination of process control. Where testing and monitoring approaches and methods no longer meet the needs or are not optimal, their replacement or modification should be considered. The use of outdated testing and monitoring methods have the potential to add risk, provide false sense of control, be ineffective, and deploy resources in a manner which may not be efficient or optimal. Thus, detracting from the development and use of more effective testing and monitoring approaches.

Precautions Against Microbial Contamination

The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.¹¹

What Should Sterility Assurance Level Demonstrate?

A sterility assurance level of 10^-6 or better (Figure 1) should be demonstrated for a sterilization process. Refer to the FDA guidance entitled “Guideline for the Submission of Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products”.¹² Various industry governing bodies, such as the Association for the Advancement of Medical Instrumentation (AAMI), provide a range of guidance documents, recommendations, and standards for sterility assurance monitoring. The American National Standards Institute (ANSI) is the United States national standard body and all US standards and recommended practices with national recognition must be accepted by ANSI. Professional Associations like the International Association for Healthcare Central Service Material Management (IAHCSSM) provide education on best practices utilizing ANSI/AAMI standards and guidelines.

• ANSI/AAMI ST79: This standard is the comprehensive guide for healthcare steam sterilization. This guide covers a range of 15 sections including Personnel, Material Management, Sterilization, and Process Monitoring and Improvement. Sterility assurance monitoring for steam sterilization is covered within Section 13, the Process Monitoring and Improvement section.¹⁶

• ANSI/AAMI ST58: This standard is the most recognized guideline for vaporized hydrogen peroxide sterilization in healthcare facilities. This standard includes many aspects of the VHP process including packaging and record keeping. It also provides recommendations for an assurance monitoring program under Section 9, Quality Control.¹⁷

Absolute Sensitivity Threshold

Absolute sensitivity threshold is a key metric that represents the theoretical minimum amount of light required to observe any meaningful signal. It is the number of photons required in order to acquire a signal that is equivalent to the noise observed by the sensor. The term “absolute threshold” is also used in experimental research to refer to the smallest level of stimulus that can be detected, usually defined as at least half the time.

Also, absolute sensitivity threshold is the number of photons needed to get a signal equivalent to the noise observed by the sensor. This is an important metric because it represents the theoretical minimum amount of light needed to observe any meaningful signal at all.

EMVA1288¹⁸ is a standard that defines what aspects of camera performance to measure, how to measure them and how to present the results in a unified method. This illustration will help understand the various aspects of imaging performance of an imaging sensor. It will outline the basic concepts that are important to understand when considering how an image sensor converts light into a digital image and ultimately defines the performance of the sensor. Figure 7 presents a single pixel and highlights these concepts.

The graph in Figure 8, shows the relationship between the signal and the noise of a camera. When the noise reaches the noise floor, we have reached absolute sensitivity threshold. IMX249 will reach absolute sensitivity threshold at a lower light density and thus perform better in lower light applications. Mako is an attractively designed GigE (Gigabit Ethernet) Vision-compliant camera in a compact rugged industrial housing. Many models include advanced functionalities such as Precision Time Protocol (PTP), Trigger over Ethernet (ToE) Action Commands, and Power over Ethernet (PoE).

Aseptic Process Simulation (APS)

Aseptic process simulation (APS) is a study that simulates the aseptic filling process by using growth media instead of the actual product. It is required by regulators¹³ to demonstrate the sterility confidence and the aseptic capability of the process. It involves qualified or validated elements such as HVAC systems, cleanroom environment, material transfer, equipment, sterilization processes, and sterilizing filtration. It also requires operator training, skills, supervision, quality assurance, and microbiological monitoring. It is performed as closely to the actual production procedure as possible.

It consists of a minimum of three initial media simulations and repeat media simulations at six-monthly intervals.

Key Words:

• Aseptic process simulation
• Media fills
• Aseptic process qualification
• Aseptic process validation
• Risk assessment

Sterility Assurance in Aseptic Processing

Sterility assurance in aseptic processing requires contributing elements—such as;

• Heating, Ventilation, and Air Conditioning (HVAC) system
• Clean-room environment
• Material transfer
• Equipment
• Manufacturing process steps
• Sterilization processes
• Sterilizing filtration

to be qualified and validated as applicable and for personnel to be trained and qualified.

References

1. Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container; 6 March 2019 EMA/CHMP/CVMP/QWP/850374/2015. Sterilisation guideline - adopted by CxMP - 10.12.2018 (europa.eu)
2. The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Annex 1, Manufacture of Sterile Medicinal Products; 20220825_gmp-an1_en_0.pdf (europa.eu).
3. EudraLex The Rules Governing Medicinal Products in the European Union Volume 4 Good Manufacturing Practice; Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products; EUROPEAN COMMISSION, 22 November 2017. 2017_11_22_ guidelines_gmp_for_atmps_0.pdf (europa.eu)
4. Guideline on Real Time Release Testing (formerly Guideline on Parametric Release), Final; 29 March 2012 EMA/CHMP/QWP/811210/2009-Rev1, 1 October 2012. Real Time Release Testing guideline (europa.eu).
5. COMMITTEE FOR MEDICINAL PRODUCTS FOR VETERINARY USE (CVMP), London, 16 October 2006 EMEA/CVMP/QWP/339588/2005. Microsoft Word - Guideline on Parametric Release (europa.eu).
6. EUROPEAN PHARMACOPOEIA 5.0 5.1.1. Methods of preparation of sterile products; 01/2005:50101. 5.1.1. Methods of preparation of sterile products.pdf (uspbpep.com).
7. Sterilization Process Controls, Production and process controls, subsystem sterilization process controls; US FDA, Content current as of 03/28/2023. sterilization process controls-inspectional objectives - Search (bing.com).
8. § 211.186 Master production and control records. eCFR :: 21 CFR 211.186 -- Master production and control records.
9. Guidance for Industry, Process Validation: General Principles and Practices, January 2011, Current Good Manufacturing Practices (CGMP), Revision 1. Process Validation: General Principles and Practices (fda.gov).
10. Guidance for Industry Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, September 2004 Pharmaceutical CGMPs. U.S. Department of Health and Human Services Food and Drug Administration; Guidance for Industry (fda.gov).
11. ICH Topic Q4B Annex 8 Sterility Test General Chapter, Step 3, ANNEX 6 TO NOTE FOR EVALUATION AND RECOMMENDATION OF PHARMACOPOEIAL TEXTS FOR USE IN THE ICH REGIONS ON STERILITY TEST GENERAL CHAPTER (EMEA/CHMP/ICH/645592/2008). December 2008 (europa.eu)
12. Guidance for Industry for the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products, November 1994. US FDA, For the Submission of Documentation for Sterilization Process (fda.gov).
13. Guidance for Industry Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, September 2004, Pharmaceutical CGMPs, U.S. Department of Health and Human Services Food and Drug Administration; Guidance for Industry (fda.gov).
14. INTERNATIONAL COUNCIL FOR HARMONISATION OF TECHNICAL REQUIREMENTS FOR PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED GUIDELINE VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN, Q5A(R2) Draft version Endorsed on 29 September 2022. https://database.ich.org/sites/default/files/ICH_Q5A%28R2%29_Step2_draft_ Guideline_2022_0826.pdf
15. FDA’s Guidance for Industry, Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production, available at http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm070287.pdf
16. ANSI/AAMI ST79 – Comprehensive Guide to Steam Sterilization and Sterility Assurance in Health Care Facilities.
17. ANSI/AAMI ST58:2013/(R)2018, guideline for vaporized hydrogen peroxide sterilization in healthcare facilities.
18. EMVA Standard 1288 Standard for Characterization of Image Sensors and Cameras Release 3.1 December 30, 2016, Issued by European Machine Vision Association www.emva.org
19. Mako G-234 | 2.35 MP Sony IMX249 CMOS sensor – Allied Vision; https://www.alliedvision. com/en/camera-selector/detail/mako/G-234/
20. Absolute Sensitivity threshold, IMX249 graph, absolute sensitivity - Bing images.
21. SATURATION CAPACITY (also known as Full Well Depth), Teledyne FLIR, MVA 1288 Overview: Imaging Performance | Teledyne FLIR.
22. 21 CFR Part 820 (up to date as of 7/06/2023) Quality System Regulation, 21 CFR Part 820 (up to date as of 7-06-2023).pdf.

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