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Understanding both the challenges and solutions of aseptic manufacturing.
Medicinal products such as parenteral, ophthalmic, and some inhalation preparations require sterilization (i.e., entirely free from undesired living organisms of all types). However, it has been recognized for decades that the sterility of a product batch cannot be assured by testing. As expressed in chapter 2.6.1 of the European Pharmacopoeia (Ph.Eur.), “a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test” (1).
This challenge is a result of several limitations of sterility testing. The first one was recognized and highlighted by D. Bryce in an article published in 1956; the sample size was so restricted that it provided only a gross estimate of the state of “sterility” of the product lot (2). This limitation could be circumnavigated by testing the entire batch. However, this solution is highly impractical and economically not viable, as there would not be any product left to be released because the sterility test is a destructive assay. Even if it was possible to test the entire batch of a product, negative results would still not fully guarantee that the batch was sterile. The limitation lies in the ability of micro-organisms to grow under the provided circumstances, as extensively discussed in an article from 1969 by Frances Bowman (3).
As per regulations, terminal sterilization of medicinal products in their final container is the preferred option where possible, as it gives the highest level of assurance that a product is free from viable micro-organisms. This can be achieved via steam and dry-heat or ionizing sterilization. When a fully validated terminal sterilization method is used, the manufacturer can avail of parametric release of a product when meeting the criteria as outlined in Annex 17 of the EudraLex (4). The European Medicines Agency guideline, Guideline on the Sterilization of the Medicinal Product, Active Substance, Excipient, and Primary Container, provides a decision tree to assist with the selection of optimal sterilization (5).
Due to the stability and robustness of some small-molecule products, terminal sterilization of the final product is feasible. However, unlike their small-molecule counterparts, large molecules or biologics are not as physically robust. The sterilization process would irreversibly damage the molecular structure and thereby its effectiveness. Sterility assurance of biologics comes with its own challenges, and manufacturers must move to aseptic processing to reduce the risk of microbial contamination or contamination of microbial byproducts like endotoxins to a minimum.
Strictly speaking, aseptic processing is not deemed a sterilization process. Often, products are subject to a final sterile filtration step followed by an aseptic process step, such as filling and lyophilization. It is after this final sterile filtration that contamination can occur and cannot be further eliminated because there is no additional step to remove any microbial contamination once the product is filled in its primary container.
Significant risks from microbiological contamination are presented by various sources. These include materials, container closures, equipment, the facility itself, and personnel. The ability to manufacture a biological product that is free from microbial contamination consistently depends on the design of the aseptic process and the contamination control elements in place.
Each element of contamination control must be able to prevent contamination. However, it is the sum of all elements acting together that dictates the overall success. These contamination control elements should be captured in a contamination control strategy. To identify the most vulnerable or critical parts within the aseptic processing chain, a contamination control risk assessment (CCRA) is a helpful tool. By applying quality risk management (QRM) tools, such as a failure mode and effect analysis, in combination with a process flow, the various risks across the phases of the aseptic process are made visible.
As there are often multiple elements and different utilities involved with aseptic manufacturing, a cross-functional team that collaborates to create the CCRA is recommended. In addition, the use of a multi-disciplinary team consisting of subject matter experts in their field of interest prevents bias and encourages the challenging of perceptions of the participants. Altogether, it enables one to draw an overview of the various interdepending feeders of the process, such as equipment, utilities, and consumables, each with their potential sources of contamination. Based on current controls present in the process, an assessment can be made to determine if the risk is sufficiently under control. Where needed, controls can be implemented to reduce the contamination risk from happening or increase detectability if such contamination occurs. Risk assessments and risk management are to be considered throughout the aseptic manufacturing and require in-depth knowledge of the processes, technologies used, and concepts of contamination control.
When conducting a risk assessment for existing processes, the use of historical data and information gathered throughout the manufacturing life cycle allows for the identification of design flaws or shortcomings. The information exposes faults with equipment vulnerabilities and/or practices that could pose a contamination risk. Based on the identified areas of concern, proposals for control measures and process improvements can be made that enhance the overall control of contamination. The risk assessment also provides an opportunity to assess if the current aseptic process design still meets regulatory expectations and highlights the most critical or urgent issues to be addressed. Being involved in a comprehensive risk review process can often show how perceived controls and reality are not always aligned.
One important principle of contamination control is the removal of micro-organisms at the start of the process so as not to solely rely on the final sterile filtration step. This means primary containers and closures in which the product is filled must be sterile. Parenteral products also require these components to be free of pyrogen. This can be achieved in-house by using validated sterilization and depyrogenation processes, or by purchasing pre-sterilized materials that are brought into the critical manufacturing environment.
Besides primary packaging materials, equipment, transfer lines, and other direct product contact materials must be clean and sterile. This can be achieved by using validated clean-in-place methods followed by sterilization-in-place processes with demonstrated hold times. Post sterilization, integrity testing or maintaining a positive pressure of the system can assist in assuring equipment remains sterile. Where possible, equipment is pre-assembled prior to sterilization. This approach reduces the number of manipulations post-sterilization and the risk of introducing contamination once it is sterilized. Minimizing contact by operators has been incentivized by regulators, as has been noticed in the draft revision of Annex 1 (6).
An important aspect of a robust design is to build in redundancies as it prevents reliance on a single control element. The failure of an element does not necessarily lead to contamination. For example, if a clamp connection fails and the system is kept under pressure, it still could prevent ingress. For that reason, vacuum systems or those kept at lower pressure are at increased risk. The preferred option is the use of closed processing systems and, where possible, to minimize connections. Good hygienic design of the system and a well-defined maintenance program as part of the microbial control program are imperative.
Deployment of good engineering practices (GEP) is paramount. Using a combination of GEP, application of good manufacturing practices (GMP), GMP mandates and regulatory guidance, technical standards, and industry monographs are critical. Different sources of information are then augmented with training and education. Collaboration of the cross-functional team defines how to translate these into pragmatic and workable solutions for the design, qualification, and maintenance of the manufacturing system and its utilities. It is an overall important principle that quality cannot be tested in products. No amount of qualification can turn bad engineering into good engineering, and validation must never be used to check that basic engineering is effective. Following GEP with QRM embedded will most likely deliver a system that meets user and regulatory requirements while being cost-efficient, compliant, and well documented.
An alternative to stainless-steel and hard-piped configurations making its way into aseptic processing and sterile manufacturing is single-use technology (SUT). Pre-assembled process systems are delivered gamma irradiated. Applications can be in both upstream and downstream processing as well as in aseptic filling. The big advantage of SUT is the avoidance of investment in time-consuming and costly cleaning, sterilization validation studies, and their periodic revalidation. It also eliminates the need for cleaning and sterilizing systems between batches.
Inappropriate cleaning processes and procedures are still one of the most common findings during GMP inspections. One must be aware of the limitations of SUT, and an assessment must be made to determine if the SUT can handle the pressure of the process, as it has its limitations compared to stainless steel. SUTs are typically made of flexible materials that can handle less pressure compared to stainless steel. Accounting for closure integrity and detection of leaks can be a challenge and—while the need for cleaning and sterilization validation is avoided with SUT—leachables and extractables studies are still required.
The design of a good functioning SUT set-up will involve time and a good relationship with the SUT vendor. Oversight is paramount to assure consistency in quality and supply.
Even with the utilization of closed systems and the use of barrier technology becoming more common, the human factor will continue to play its role. The number one source of contamination for products that cannot be terminally sterilized remains personnel. Hence, a robust training program outlining what to perform, how to perform it, and why to perform activities in a certain way is indispensable. Operator understanding is a key aspect when it comes to adherence to GMP and consistently meeting quality standards.
1. EDQM, Ph.Eur., General Text 2.6.1 pages 191–193 (EDQM, Strasbourg, France, 2021).
2. D. Bryce, J. Pharm. Pharmacol., 8 (1) 561–572 (1956).
3. F. Bowman, J. Pharm. Sci., 58 (11) 1301–1308 (1969).
4. EC, EudraLex, Volume 4—Annex 17: Real Time Release Testing and Parametric Release (2018).
5. EMA, Guideline on the Sterilization of the Medicinal Product, Active Substance, Excipient and Primary Container (March 2019).
6. EC, EudraLex, Draft Guideline, Volume 4—Annex 1: Manufacture of Sterile Products (2020).
Patrick Nieuwenhuizen is a director senior consultant for PharmaLex.
Vol. 35, No. 5
When referring to this article, please cite it as P. Nieuwenhuizen, “Addressing Limitations of Sterility Testing,” BioPharm International 35 (5) 2022.