Confined bioprinting and culture in inflatable bioreactor for the sterile bioproduction of tissues and organs.

The future of organ and tissue biofabrication strongly relies on 3D bioprinting technologies. However, maintaining sterility remains a critical issue regardless of the technology used. This challenge becomes even more pronounced when the volume of bioprinted objects approaches organ dimensions. Here, we introduce a novel device called the Flexible Unique Generator Unit (FUGU), which is a unique combination of flexible silicone membranes and solid components made of stainless steel. Alternatively, the solid components can also be made of 3D printed medical-grade polycarbonate. The FUGU is designed to support micro-extrusion needle insertion and removal, internal volume adjustment, and fluid management. The FUGU was assessed in various environments, ranging from custom-built basic cartesian to sophisticated 6-axis robotic arm bioprinters, demonstrating its compatibility, flexibility, and universality across different bioprinting platforms. Sterility assays conducted under various infection scenarios highlight the FUGU's ability to physically protect the internal volume against contaminations, thereby ensuring the integrity of the bioprinted constructs. The FUGU also enabled bioprinting and cultivation of a 14.5 cm3 human colorectal cancer tissue model within a completely confined and sterile environment, while allowing for the exchange of gases with the external environment. This FUGU system represents a significant advancement in 3D bioprinting and biofabrication, paving the path toward the sterile production of implantable tissues and organs.

Single-Use System Integrity I: Using a Microbial Ingress Test Method to Determine the Maximum Allowable Leakage Limit (MALL).

An aerosol microbial ingress test was specifically designed and used to create a predictive model in order to determine the maximum allowable leakage limit (MALL) of single-use systems (SUSs). The MALL is defined as the greatest leak size that does not pose any risk to the product. The procedure involved taking test samples of film material from single-use bags. As test samples, an ethylene vinyl acetate multilayer film (300 µm thick) and a polyethylene multilayer film (400 µm thick) were cut into 50 mm patches. Artificial defects of 1-100 µm were laser-drilled in the middle of each film patch. The patch was assembled on a holder and properly sealed. The test units were filled aseptically with culture media and placed inside an aerosol chamber. Various pressures were applied to the test unit to simulate the constraints that single-use systems may be subject to under real-world conditions. After an aerosolization cycle with spores of Bacillus atrophaeus, a minimum concentration of 106 CFU/cm2 was reached on the film surface. The test units were incubated for 14 days at 30°C-35°C and visually inspected for bacterial ingress. Thirty samples per defect size were tested. Logistic regression was used to indicate the MALL for a single-use system according to the required risk level. With this method, the probability of the occurrence or absence of ingress for a specific defect size was reported according to the experimental data. In addition to physical parameters, such as the pressure applied and the film material, the effect of the probabilistic nature of the microorganisms in determining the MALL is considered. Although finding an experimental model to predict the MALL for real-life process conditions was the ultimate objective, this paper also presents the microbial ingress test data obtained so far for two extreme conditions. Potential constraints, such as vibration, shock, acceleration, liquid movement, and pressure differentials, observed during normal usage were simulated using two extreme differential pressures, 0 mbar and 300 mbar. The estimated MALL for typical use-case conditions are 10-20 µm for storage applications and 2-10 µm for shipping conditions. The microbial integrity test method used in this article was able to detect bacterial ingress down to 3 µm defect size.LAY ABSTRACT: As use of single-use systems (SUSs) is increasingly expanding into all process steps of commercial manufacturing, integrity failure can significantly impact drug safety, availability, and costs. Consequently, growing industry scrutiny on single-use system integrity (SUSI) is raising the need to develop good science behind reliable determinations of liquid leakage and microbial ingress as well as the appropriate physical integrity testing technologies. In the current study, microbial ingress testing by the aerosol method is used to determine the maximum allowable leakage limit (MALL) for SUSs. To define the MALL, it is generally assumed that a system or product will not show any microbial ingress or leakage at a certain defect size. Statistical analysis of the experimental data in this study indicated the MALL with probability at a certain defect size for each system. As a result, the method studied provides a more accurate way of predicting ingress and increasing safety down the line for drug manufacturers and patients alike.

Reconciliation of pH, conductivity, total organic carbon with carboxylic acids detected by ion chromatography in solution after contact with multilayer films after γ-irradiation.

The impact of γ-irradiation on polymers in multilayer films was studied by means of the study of the diffusion and release (spontaneous migration of the molecules from the container into the product) of chemical species in aqueous solution. A series of different measurements have been performed: pH, conductivity, total organic carbon (TOC) and ion chromatography (IC). Their evolution according to γ-irradiation dose was studied. More several rinsings made over several months allowed to quantify well the impact of the irradiation on these polymers. The samples are irradiated at several γ-doses, up to 270 kGy, and compared with a non-irradiated sample used as reference. It shows that quantity of generated carboxylic acids depends on the film material (PE/EVOH/PE and EVA/EVOH/EVA) and increases with γ-dose.

Impact of γ-irradiation, ageing and their interactions on multilayer films followed by AComDim.

To highlight the main factors involved in the degradation of polymers in multilayer films under γ-irradiation, the ANOVA Common Dimensions (AComDim, Analysis of Variance in Common Dimensions) method is applied on spectra recorded with ATR-FTIR (Attenuated Total Reflection-Fourier Transform Infrared). The present study focuses on the stability of γ-irradiated polymers used in single-use plastic bags made of multilayer films for the biopharmaceutical and biotechnological industries. The samples are irradiated at several γ-doses, up to 270 kGy, and compared with a non-irradiated sample used as reference. It shows that the γ-dose, the natural ageing up to six months and the γ-dose × ageing interaction are the most influential factors.

Analysis and evaluation of single-use bag extractables for validation in biopharmaceutical applications.

This paper describes an approach of extractables determination and gives information on extractables profiles for gamma-sterilized single-use bags with polyethylene inner contact surfaces from five different suppliers. Four extraction solvents were chosen to capture a broad spectrum of extractables. An 80% ethanol extraction was used to extract compounds that represent the bag resin and the organic additives used to stabilize or process the polymer films which would not normally be water-soluble. Extractions with1 M HCl extract, 1 M NaOH extract, and 1% polysorbate 80 were used to bracket potential leachables in biopharmaceutical process fluids. The objective of this study was to obtain extractables data from different bags under identical test conditions. All the bags had a nominal capacity of 5 L, were gamma-irradiated prior to testing, and were tested without modification except that connectors, if any, were removed prior to filling. They were extracted at 40 °C for 30 days. Extractables from all bag extracts were identified and the concentration estimated using headspace gas chromatography-mass spectrometry and flame ionization detection for volatile compounds and for semi-volatile compounds, and liquid chromatography-mass spectrometry for targeted compounds. Metals and other elements were detected and quantified by inductively coupled plasma mass spectrometry analysis. The results showed a variety of extractables, some of which are not related to the inner polyethylene contact layer. Detected organic compounds included oligomers from polyolefins, additives and their degradation products, and oligomers from the fill tubing. The concentrations of extractables were in the range of parts-per-billion to parts-per-million per bag under the applied extraction conditions. Toxicological effects of the extractables are not addressed in this paper. LAY ABSTRACT: Extractables and leachables characterization supports the validation and the use of single-use bags in the biopharmaceutical manufacturing process. This paper describes an approach for the identification and quantification of extractable substances for five commercially available single-use bags from different suppliers under identical analytical conditions. Four test formulations were used for the extraction, and extractables were analyzed with appropriately qualified analytical techniques, allowing for the detection of a broad range of released chemical compounds. Polymer additives such as antioxidants and processing aids and their degradation products were found to be the source of most of the extracted compounds. The concentration of extractables ranged from parts-per-billion to parts-per-million under the applied extraction conditions.

Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications.

Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency.

An approach to quality and security of supply for single-use bioreactors.

Single-use systems (also referred to as disposables) have become a huge part of the bioprocessing industry, which raised concern in the industry regarding quality and security of supply. Processes must be in place to assure the supply and control of outsourced activities and quality of purchased materials along the product life cycle. Quality and security of supply for single-use bioreactors (SUBs) are based on a multidisciplinary approach. Developing a state-of-the-art SUB-system based on quality by design (QbD) principles requires broad expertise and know-how including the cell culture application, polymer chemistry, regulatory requirements, and a deep understanding of the biopharmaceutical industry. Using standardized products reduces the complexity and strengthens the robustness of the supply chain. Well-established supplier relations including risk mitigation strategies are the basis for achieving long-term security of supply. Well-developed quality systems including change control approaches aligned with the requirements of the biopharmaceutical industry are a key factor in supporting long-term product availability. This chapter outlines the approach to security of supply for key materials used in single-use production processes for biopharmaceuticals from a supplier perspective.