Aseptic Manufacturing Research Articles

A Novel Technique of Aseptic Manufacture of Autologous Serum Eye Drops (ASEDs) and Sterility Analysis of the Bottled Ophtioles.

To introduce a novel technique of the aseptic manufacture of autologous serum eye drops (ASEDs) with a prefiltered closed system and to analyze the sterility of the produced ophtioles between 2018 and 2022. This is a prospective single-center study conducted at the Department of Ophthalmology at a Swiss University Hospital between 2018 and 2022. For regulatory reasons, closed systems for manufacturing ASEDs are strongly recommended. We attached an upstream sterile filter (Sterivex PES0.22 µm Burlington, USA) to a commercially available closed system (COL System Modena, Italy) for manufacturing ASEDs. The goal of this novel approach was to reduce the microbiological contamination of the donated autologous blood. Using the presented manufacturing method, we are able to produce, on average, 56 ophtioles per batch, containing either 1.45 mL or 2.5 mL of autologous serum per ophtiole. For each batch of ASEDs, we performed a microbiological analysis by automated blood culture testing (BACTEC). This system examines the presence of bacteria and fungi. We analyzed all manufactured batches between 2018 and 2022. None of the 2297 batches and the resulting 129 060 ophtioles showed bacterial or mycotic contamination. During the analyzed period, two batches were discarded: one due to fibrin-lipid aggregations, further microbiological and histological work-up excluded any contamination; another due to false-positive HIV in serological testing. Overall, the contamination rate was 0%, and the batch discharge rate was 0.09%. The combination of upstream sterile filtration with a commercial closed system for manufacturing ASEDs proved to be effective in ensuring sterility without any contamination over the past 4 years. This is becoming crucial, as the demand for autologous blood products for treating ocular surface disorders, such as refractory dry eyes or nonhealing defects of the corneal epithelium, is on the rise.

Operator-derived particles and falling bacteria in biosafety cabinets

IntroductionTo ensure the sterility of cell products that cannot undergo conventional sterilization processes, it is imperative to establish and maintain a clean room environment, regulated through environmental monitoring, including particle counts. Nevertheless, the impact of particles generated by operators as potential contaminants remains uncertain. Thus, in this study, we conducted an accelerated test to assess the correlation between particles generated by operators and airborne bacteria, utilizing biosafety cabinets within a typical laboratory setting. These biosafety cabinets create a controlled environment with air conditioning and high-efficiency particulate air (HEPA) filters, offering fundamental data relevant to cell production. Materials and methodsWe conducted a simulation followed by real-time experiments involving human operations to explore the quantity of particles, particle sizes, and the percentage of bacteria within these particles. This investigation focused on conditions with heightened particle generation from operators within a biosafety cabinet. The experiment was conducted on operators wearing textile and non-woven dustless clothing within biosafety cabinets. It entailed tapping the upper arms for a duration of 2 min. ResultsObservations under biosafety cabinet-off conditions revealed the presence of various particles and falling bacteria in textile clothing. In contrast, no particles or falling bacteria were detected in operators wearing dustless clothing within biosafety cabinets. Notably, a correlation between 5 μm particles and colony-forming units in textile clothing was identified through this analysis. The ratio of falling bacteria to the total number of particles within the biosafety cabinet was 0.8 ± 0.5 % for textile clothing, while it was significantly lower at 0.04 ± 0.2 % for dustless clothing. ConclusionThis study demonstrated that the number of particles and falling bacteria varied depending on the type of clothing and that quantitative data could be used to identify risks and provide basic data for operator education and evidence-based control methods in aseptic manufacturing areas. Although, this study aims to serve as an accelerated test operating under worst-case conditions, the results need to make sure the study range in general research.

Protective Airflow including ‘First Air’ in GMP Aseptic manufacturing

Good Manufacturing Practices (GMP) related to sterile product aseptic manufacturing environments rely on protective airflow mechanisms to ensure the control over airborne contamination. Alongside the protective airflow, pressure differentials that drive airflow cascade ensure GMP compliant contamination control of airborne contamination that may include Microbial carrying particles (MCPs). Airflow visualisation qualification by smoke studies has been clarified as a GMP requirement in EU & PICS Annex 1. The scope of Annex 1 applies to manufacture of sterile medicinal products together with bioburden control processes where bioburden in intermediates, substances, APIs and non-sterile products can also impact patient safety so a wide scope of protective airflow applications is applied. As a GMP requirement (included in 2008 version of Annex 1): It should be demonstrated that air-flow patterns do not present a contamination risk, e.g. care should be taken to ensure that air flows do not distribute particles from a particle generating person, operation or machine to a zone of higher product risk.' Within Annex 1: 2023, the concept of First Air has emerged as a critical element. First Air is defined as “filtered air that has not been interrupted prior to contacting exposed product and product contact surfaces with the potential to add contamination to the air prior to reaching the critical zone.” The requirement for extensive smoke studies and the integration of First Air protection as a mandated aspect in GMP regulations has brought to the fore an imperative need for a deeper comprehension of airflow dynamics and their implications on sterile product Aseptic manufacturing. This shift beckons us to explore the nuances of protective airflow, particularly in the context of Computational Fluid Dynamics (CFD) analyses, to discern the efficacy of airflow patterns in different scenarios. Stakeholders involved in Annex 1 implementation have been impacted by introduction of the First Air protection requirement, considering design and in qualification via smoke study airflow visualisation. Such requirements must follow QRM principles where process understanding and knowledge of contamination hazards are essential to mitigate risks in compromise of product quality and potential harm to patients. Risk mitigations must take a Quality by Design (QbD) approach as monitoring alone does not provide assurance of product sterility or in meeting defined bioburden limits.

Robotics and Aseptic Processing in View of Regulatory Requirements.

Several nanomedicine based medicinal products recently reached the market thanks to the drive of the COVID-19 pandemic. These products are characterized by criticality in scalability and reproducibility of the batches, and the manufacturing processes are now being pushed towards continuous production to face these challenges. Although the pharmaceutical industry, because of its deep regulation, is characterized by slow adoption of new technologies, recently, the European Medicines Agency (EMA) took the lead in pushing for process improvements using technologies already established in other manufacturing sectors. Foremost among these technologies, robotics is a technological driver, and its implementation in the pharma field should cause a big change, probably within the next 5 years. This paper aims at describing the regulation changes mainly in aseptic manufacturing and the use of robotics in the pharmaceutical environment to fulfill GMP (good manufacturing practice). Special attention is therefore paid at first to the regulatory aspect, explaining the reasons behind the current changes, and then to the use of robotics that will characterize the future of manufacturing especially in aseptic environments, moving from a clear overview of robotics to the use of automated systems to design more efficient processes, with reduced risk of contamination. This review should clarify the regulation and technological scenario and provide pharmaceutical technologists with basic knowledge in robotics and automation, as well as engineers with regulatory knowledge to define a common background and language, and enable the cultural shift of the pharmaceutical industry.

Validation of a Sporicidal Bio-Decontamination Process for use in Pharmaceutical Isolators in an NHS Aseptic Manufacturing Unit

The recently published revision of EU GMP Annex 1 Manufacture of Sterile Medicinal Products includes a requirement for pharmaceutical isolators to undergo a bio-decontamination process of the interior which should be automated, validated and controlled within defined cycle parameters and include a sporicidal agent. It is widely accepted that the easiest way to achieve this is by converting Hydrogen Peroxide (H2O2) liquid into a state that will diffuse readily through the closed isolator chamber leading to bio-decontamination of all surfaces which it comes into contact with. The technology to do this has been around for years and can be validated to give a high level of assurance of surface bio-decontamination comparable with sterilization when used on surfaces in the isolator critical work zone. This type of activity is routinely used in large-scale sterile manufacturing that takes place in the pharmaceutical industry where the amount of materials and timescales involved make this viable. In the NHS, aseptic preparation and manufacture of sterile products is carried out in isolators both for immediate use and for stock in anticipation of demand. Automated bio-decontamination systems are available but their use is much less than in the pharmaceutical industry and reliance is still placed on manual spraying and wiping of surfaces with disinfectant agents such as alcohol and hydrogen peroxide. This brings a degree of variability and is more difficult to validate. For sterile manufacturing units holding an MHRA Specials Manufacturing licence the expectation is that proper consideration is given to using an automated bio-decontamination process or there is justification for not doing it. For units preparing products for immediate use there is not the same expectation but as standards are improved, it is reasonable to assume it may become the case in the future. To use this type of process requires it to be integrated into the isolator, meaning either buying a new isolator or retrofitting to an existing one. The relatively high cost of this is a barrier to pharmacy department aseptic units using the technology. There is a range of bio-decontamination systems on the market from Devea (www.devea environnement.com)which are claimed to be suitable for use in hospital pharmacy clean room facilities. One model from this range, the Phileas Genius is a simple, stand-alone battery powered unit that generates vapourized hydrogen peroxide of particle size 5-10 µm from 7.4% solution using novel spinning disc technology. This generator can be transferred into and out of any closed isolator work zone through the transfer hatch (as long as the hatch door is wide enough) and can be used to do pre-programmed cycles (called zones) to routinely decontaminate inner surfaces.

Setup of a Contamination Control Strategy Using the Hazard Analysis Critical Control Point (HACCP) Methodology.

A Contamination Control Strategy (CCS) is a document that focuses on how to prevent contaminations with microorganisms, particles, and pyrogens within sterile and/or aseptic and preferably also in nonsterile manufacturing facilities. This document determines to what extent measures and controls in place are efficient in preventing contamination. In order to efficiently evaluate and control all potential hazards associated with sources of contamination within a CCS, the Hazard Analysis Critical Control Point (HACCP) methodology could be a useful tool to monitor all Critical Control Points (CCPs) related to various sources of contamination. This article describes a way to set up the CCS within a pharmaceutical sterile and aseptic manufacturing facility (GE HealthCare Pharmaceutical Diagnostics) by applying the HACCP methodology. In 2021, a global CCS procedure and a general HACCP template became effective for the GE HealthCare Pharmaceutical Diagnostics sites having sterile and/or aseptic manufacturing processes. This procedure guides the sites through the setup of the CCS by applying the HACCP methodology and helps each site to evaluate whether the CCS is still effective taking all (proactive and retrospective) data following the CCS into account. A summary of setting up a CCS using the HACCP methodology, specifically for the pharmaceutical company GE HealthCare Pharmaceutical Diagnostics Eindhoven site, is provided in this article. Use of the HACCP methodology enables a company to include proactive data within the CCS, making use of all identified sources of contamination, associated hazards, and/or control measures and CCPs. The constructed CCS allows the manufacturer to identify whether all included sources of contamination are under control and, if not, which mitigatory actions need to be performed. All current states are reflected by a traffic light color to reflect the level of residual risk, thereby providing a simple and clear visual representation of the current contamination control and microbial state of the manufacturing site.

Comparison of one-step with two-step production of Bacillus atrophaeus spores for use as bioindicators.

The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two-step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one-step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one-step BI production is technically possible but at the expense of spore yield. The two-step manufacturing process can realize almost 10-fold higher spore yields.

Everolimus-Loaded Reconstituted High-Density Lipoprotein Prepared by a Novel Dual Centrifugation Approach for Anti-Atherosclerotic Therapy.

The conventional techniques for the preparation of reconstituted high-density lipoprotein (rHDL) are hampered by long process times, the need for large amounts of starting material, and harsh preparation conditions. Here, we present a novel rHDL preparation method to overcome these challenges. Furthermore, we propose a dual mode of action for rHDL loaded with the immunosuppressant drug everolimus (Eve-rHDL) in the context of atherosclerosis and cardiovascular disease. We use dual centrifugation for rHDL nanoparticle preparation and characterize the physicochemical properties by NS-TEM, N-PAGE, DLS, AF4, and HPLC. In addition, we determine the biological efficacy in human and murine cell culture with regard to cellular uptake, cholesterol efflux, and proliferation. We confirm the characteristic particle size of 10 nm, discoidal morphology, and chemical composition of the rHDL preparations and identify dual centrifugation as an ideal method for cost-effective aseptic rHDL manufacturing. rHDL can be prepared in approx. 1.5 h with batch sizes as little as 89 µL. Moreover, we demonstrate the cholesterol efflux capacity and anti-proliferative activity of Eve-rHDL in vitro. The anti-proliferative effects were comparable to free Eve, thus confirming the suitability of rHDL as a capable drug delivery vehicle. Eve-rHDL shows great efficacy in vitro and may further be employed to target atherosclerotic plaques in vivo. Highly effective anti-atherosclerotic therapy might be feasible by reducing both inflammatory- and lipid burden of the plaques. Dual centrifugation is an ideal technique for the efficient application of the rHDL platform in cardiovascular disease and beyond.

Application of Biofluorescent Particle Counters for Real-Time Bioburden Control in Aseptic Cleanroom Manufacturing

This experimental study examines the use of a real time viable particle counter in pharmaceutical cleanroom operations under ISO class 8/Annex 1 Class C ‘in operation’ conditions. The used measuring system, a Biofluorescent Particle Counter (BFPC), continuously detected particles >1.0 µm as well as microbe carrying (viable) particles (MCPs). In addition, classic air samplers for the evaluation of Colony Forming Units (CFU) were installed to enable a comparison of counter-provided values with classic, agar-based methods of microbial air sampling required by regulatory guidelines. A test room comparable to typical cleanrooms in the pharmaceutical industry was operated under three different air change rates (ACR). At these ACRs, the operators wore three different garments. With test repetition, 18 experiments were conducted simulating different pharmaceutical process conditions. The results show correlations of measured particles to MCPs and MCPs to CFUs. Furthermore, albeit an industry-wide used and regulatory accepted minimum ARC value of 20 h−1, the study results indicate that an ACR of 10 h−1 is capable of keeping cleanrooms within regulatory limits for particles and CFUs, and thus provides the opportunity to save energy for cleanroom operations. The implementation of real time viable particle counters can be used to achieve a quality advantage for ISO class 8 cleanrooms for a continuous, documented control of the cleanroom status, and thus enable a reduction of ACRs with the aim of energy saving. Further long-term studies should validate this.

TECHNOLOGICAL CHALLENGES IN STERILE PRODUCTOS MANUFACTURING: IS PHARMACEUTICAL INDUSTRY TRAINED?

Pharmaceutical industry, in addition to assuming the sanitary commitment, as has been demonstrated with the manufacturing of COVID-19 vaccines, must comply with the European Union Good Manufacturing Practices, for that reason it´s subject to a continuous updating demanded by the regulatory requirements. This commitment is even, if possible, greater for injectable drug’s manufacturing companies due to their intrinsic characteristics of safety for the patient, all this together with the maximum productivity objective. A review about the main challenges that the pharmaceutical industry must face for the manufacture of injectable products is carried out, that comprised the incorporation of new biological drugs and advanced therapies into the therapeutic arsenal, the increase in terms of the requirements in regulatory and inspection tasks by health authorities and the leading role that Contract Manufacturing Organisations are playing. On the other hand, issues such as the analytical methods used to evaluate product sterility validity, the impact of possible changes in the different processes and elements, especially in the case of aseptic manufacturing, and the evolution, with the implementation of Sterility and Quality by Design, of sterile products manufacturing science are discussed. Finally, the implementation of disruptive factors such as continuous manufacturing and robotization, which will have, with a high probability, a greater role in the not-so-distant future, is proposed. Key Words: Sterile Drugs, Inspection, Aseptic Manufacturing, Isolators, Good Manufacturing Practices, PAT, Pharmaceutical Industry

Interventions Risk Evaluation and Management in Aseptic Manufacturing.

Interventions performed by personnel during an aseptic process can be a key source of microbiological contamination of sterile biopharmaceutical products, irrespective of the type of manufacturing system used. Understanding the relative risk of this source of contamination provides valuable information to help make decisions for the design, qualification, validation, operation, monitoring, and evaluation of the aseptic process. These decisions can be used to improve the aseptic process and provide assurance of the sterility of the products. To achieve these goals, an assessment of the contamination risk is needed. This risk assessment should be objective, accurate, and useful. This article presents an Intervention Risk Evaluation Model (IREM) philosophy and an objective, accurate, and useful method for intervention risk determination. The IREM uses a key word approach to identify, obtain, measure, and evaluate intervention risk factors. This article presents a general discussion of the method with the help of a case study to illustrate the development of the model, whereas subsequent parts would focus on application of this model with practical examples. This not only attempts to create objectivity of the entire process, but it develops awareness of the associated risks among shop floor operators, which can lead to a reduction of the overall risk level of the process and an improvement in the sterility assurance level.

Words Matter: A Commentary and Glossary of Definitions for Microbiological Quality.

In the design, control, and regulation of the manufacturing and supply of microbiologically controlled devices (including sterile devices) and drug products (including cleaning, disinfection, and sterilization processing and/or aseptic process manufacturing), different terms and/or definitions are often used for similar processes or applications internationally. With product innovations (including combination products and cell-based therapy) and global regulatory influences, there is a growing need to harmonize these definitions. The objective of the Kilmer Regulatory Innovation microbiological quality and sterility assurance glossary is to clarify and harmonize the practical use of terms employed by the different parts of regulated healthcare product industries internationally and by regulators of the manufacturing and supply of microbiologically controlled healthcare products internationally. The glossary is expected to continue to evolve, and further industry, academic, and regulatory input is encouraged.

Words Matter: A Commentary and Glossary of Definitions for Microbiological Quality

In the design, control, and regulation of the manufacturing and supply of microbiologically controlled devices (including sterile devices) and drug products (including cleaning, disinfection, and sterilization processing and/or aseptic process manufacturing), different terms and/or definitions are often used for similar processes or applications internationally. With product innovations (including combination products and cell-based therapy) and global regulatory influences, there is a growing need to harmonize these definitions. The objective of the Kilmer Regulatory Innovation microbiological quality and sterility assurance glossary is to clarify and harmonize the practical use of terms employed by the different parts of regulated healthcare product industries internationally and by regulators of the manufacturing and supply of microbiologically controlled healthcare products internationally. The glossary is expected to continue to evolve, and further industry, academic, and regulatory input is encouraged.

Effects of different air change rates on cleanroom ‘in operation’ status

The objective of this experimental study is to analyze non-viable and viable particle loads in a pharmaceutical cleanroom under ‘in operation’ conditions using different air change rates (ACRs). Regulatory guidelines give limit values for particles/m3 and colony forming units (CFUs)/m3. A widely used ACR is 20 h−1 as this value is recommended by the Food and Drug Administration (FDA) in its guidance for industry on sterile drug products. However, this value may be too high, resulting in increased costs for energy. A typical pharmaceutical cleanroom was used for this study, and operations were simulated with a process unit and two operators in the room. The experiments were conducted twice with four different ACRs and four different types of operator garments, resulting in 32 trials in total. Particle load and CFUs were measured by calibrated particle counters and microbial air samplers. The results give evidence that an ACR of 20 h−1 is not required. ACR 10 h−1 is sufficient without compromising the demanded air quality. Furthermore, it was found that regulatory agencies should reevaluate the expected limits as these currently give a high buffer between the required and actual values, which potentially cover up problems in aseptic manufacturing.

Monitoring the Sterility of Aseptic Manufacturing Equipment

Monitoring the Sterility of Aseptic Manufacturing Equipment

Buprenorphine loaded PLGA microparticles: Characterization of a sustained-release formulation

Buprenorphine is a short-acting analgesic drug. Its use in veterinary medicine requires several injections per day to alleviate pain in animals. We, therefore, designed a poly-lactic-co-glycolic acid (PLGA) based depot formulation of the opioid to prolong the analgesic effect. We characterized our novel microparticulate depot formulation with emphasis on a potential future product development in the present work.Microparticles showed low residual moisture levels and did meet bacterial endotoxin requirements defined by the European Pharmacopeia. Depot formulation showed physicochemical stability over 6 months at 4 °C, indicating sufficient shelf life. Reconstituted formulation stored at 4 °C showed unchanged release properties for 24 h. Terminal sterilization by x-rays with a dose of 30 kGy revealed the sensitivity of buprenorphine towards radiation, resulting in substantial drug degradation. However, microparticle properties were not affected. Our experiments indicate that sterilization of the final product is not possible, therefore requiring aseptic manufacturing protocols.In conclusion, our novel buprenorphine depot formulation based on PLGA microparticles can be stored for at least 6 month as a lyophilisate and can be used for at least 24 h after reconstitution. Pilot experiments suggest that scale-up and a future aseptic production should be feasible. We conclude that the novel depot formulation shows promising attributes, a prerequisite for future industrial production and commercialization.

Words Matter: A Commentary and Glossary of Definitions for Microbiological Quality.

In the design, control, and regulation of the manufacturing and supply of microbiologically controlled devices (including sterile devices) and drug products (including cleaning, disinfection, and sterilization processing and/or aseptic process manufacturing), different terms and/or definitions are often used internationally for similar processes or applications. With product innovations (including combination products and cell-based therapy) and global regulatory influences, there is a growing need to harmonize these definitions. The objective of the Kilmer Regulatory Innovation microbiological quality and sterility assurance glossary is to clarify and harmonize the practical use of terms employed by the different parts of regulated health care product industries internationally and by regulators of the manufacturing and supply of microbiologically controlled health care products internationally. The glossary is expected to continue to evolve; and further industry, academic, and regulatory input is encouraged.

Key points for the aseptic manufacturing of cellular and tissue-based products to meet the good gene, cellular, and tissue-based products manufacturing practice （GCTP）

Key points for the aseptic manufacturing of cellular and tissue-based products to meet the good gene, cellular, and tissue-based products manufacturing practice （GCTP）

Validation of the nant 001 bioreactor system for automated adipose-derived mesenchymal stem cell expansion

Background & Aim Adipose-derived mesenchymal stem cells (ASCs) are an attractive choice for regenerative therapies due to their minimally invasive method of isolation. As with any source, the need for cell expansion to produce therapeutically useful quantities of cells bring challenges. Manual production of stem cells is labor-intensive, expensive and poses a high risk for contamination. The NANT 001 is an innovative bioreactor that has evolved from manual cell culture principles to introduce a high level of automation. This work reports on the validation of this system for the production of ASCs compared with conventional manual expansion processes. Methods, Results & Conclusion ASCs at passage 1 from a healthy donor were suspended in α-MEM supplemented with 10% fetal bovine serum, seeded into either the bioreactor system or Corning 636 cm2 CellSTACK culture chambers at a density of 2000 cells/cm2 and incubated at 37°C, 5% CO2. After 24 hours, and when the cells reached 50% confluency, the monolayer was washed with PBS and the growth media was replaced. Cells were automatically harvested 24 hours after reaching 90% confluence. Three validation runs were performed. Growth kinetics, cell viability, sterility, trilineage differentiation capacity and surface immunophenotype were assessed. Cell growth kinetics and yield from the bioreactor were comparable to those obtained from the manual process. On average, an inoculation of 1.3 × 106 cells yielded 52 × 106 cells from the NANT 001 and 55 × 106 from the manual process in a 6-day expansion. Viability was not affected by the bioreactor process with >95% cell viability recorded from all 3 runs. Supernatants from all cell cultures tested negative for microbial contamination. There were no significant differences in the adipogenic, osteogenic and chondrogenic differentiation propensity of the cells regardless of expansion process. Expression of surface markers in all cases was consistent with IFATS standards for ASC phenotype. Collectively, these data illustrate the potential of using the NANT 001 bioreactor for automated expansion of ASCs. The risk of contamination is decreased by reducing the use of open procedures. Ongoing work consists of a GMP validation of the system for the expansion of ASCs from the stromal vascular fraction of adipose tissue. Based on these observations, the NANT 001 bioreactor represents an effective option for closed, automated and aseptic manufacturing of autologous cell products for applications in cell therapy and regenerative medicine.

Pharmaceutical Cleanroom Classification using ISO 14644-1 and the EU GGMP Annex 1 Part 1: Testing rationale

Cleanroom classification is an essential part of the qualification activities in pharmaceutical cleanrooms that confirm the effectiveness of the cleanroom’s airborne contamination control system. A review of the classification requirements and principles associated with ISO 14644-1:2015 and the 2008 version of Annex 1 of the EU GGMP is contained in this first article, and a suitable classification test method derived for aseptic manufacturing. A second article will consider the application of the method by means of practical examples. ​ Key words: Cleanroom classification, ISO 14644-1, EU GGMP Annex 1