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Additive manufacturing, particularly Vat photopolymerization, presents a promising technique for producing complex, tailor-made structures, making it an attractive option for generating single-use components used in biopharmaceutical manufacturing equipment or cell culture devices. However, the potential leaching of cytotoxic compounds from Vat photopolymer resins poses a significant concern, especially regarding cell growth and viability in cell culture applications. This study explores the potential of parylene C coating to enhance the inertness of a polyurethane-based photopolymer resin, aiming to prevent cytotoxicity and improve biocompatibility. The study includes an analysis of extractables from the resin and photoinitiator to evaluate the resin's composition and to define selected marker compounds for investigating the coating efficiency. The time-dependent accumulation of relevant extractable compounds over a 70-day period are assessed to address the long-term use of the coated components. The impact of irradiation on the material and the coating was evaluated, along with an accelerated aging study to address the long-term performance of the coating. Biocompatibility in terms of in vitro cell growth studies is evaluated using Chinese hamster ovary cells, a standard cell line in biopharmaceutical manufacturing. Results demonstrate that parylene C coating significantly reduces the release of cytotoxic compounds, such as the photoinitiator diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO). Although accelerated aging indicates a reduction in the barrier properties of the coating over time, the parylene C coating still effectively slows the release of extractables and significantly improves cell compatibility of the 3D printed parts. The findings suggest that parylene C-coated components can be safely integrated into biopharmaceutical manufacturing processes, with recommendations to minimize storage times between coating application and use to ensure optimal performance.

Plastic components are essential in the pharmaceutical industry, encompassing container closure systems, laboratory handling equipment, and single-use systems. As part of their material qualification process, studies on interactions between plastic contact materials and process solutions or drug products are conducted. The assessment of single-use systems includes their potential impact on patient safety, product quality, and process performance. This is particularly crucial in cell and gene therapy applications since interactions with the plastic contact material may result in an adverse effect on the isolated therapeutic human cells. We utilized the cell painting assay (CPA), a non-targeted method, for profiling the morphological characteristics of U2OS human osteosarcoma cells in contact with chemicals related to plastic contact materials. Specifically, we conducted a comprehensive analysis of 45 common plastic extractables, and two extracts from single-use systems. Results of the CPA are compared with a standard cytotoxicity assay, an osteogenesis differentiation assay, and in silico toxicity predictions. The findings of this feasibility study demonstrate that the device extracts and most of the tested compounds do not evoke any measurable biological changes on the cells (induction  ≤ 5%) among the 579 cell features measured at concentrations  ≤ 50 µM. CPA can serve as an important assay to reveal unique information not accessible through quantitative structure-activity relationship analysis and vice versa. The results highlight the need for a combination of in vitro and in silico methods in a comprehensive assessment of single-use equipment utilized in advanced therapy medicinal products manufacturing., (© 2024. The Author(s).)

Polysiloxanes are considered one of the most important commercial families of synthetic elastomers. They are frequently employed in biopharmaceutical manufacturing equipment as flexible single-use solutions due to superior material properties and compatibility with diverse sterilization methods. Extractables and leachables (E&L) testing is essential in qualifying such equipment, involving extraction studies to assess the potential release of compounds from plastic components for risk assessment. Silicone releases oligomeric siloxanes and small hydrolysis products, with dimethylsilanediol (DMSD) being the main hydrolysis product found in significant concentrations in aqueous process solutions. DMSD presents challenges for analysis, requiring specifically tailored analytical methods to detect it, which are commonly not applied in standard E&L screening tests. In biopharmaceutical manufacturing, it is relevant to consider the potential of DMSD to repolymerize into silicone oil when specific process parameters are altered. This may lead to interactions with drug ingredients, including proteins, resulting in the formation of aggregates. We synthesized and characterized DMSD using X-ray structure analysis and established an HPLC method with a refractive index detector to investigate the release of DMSD from commercially available silicone tubing used in drug manufacturing following autoclaving and irradiation. Subsequently, we assessed typical biopharmaceutical downstream operations for effectively removing this compound from the process stream., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Roberto Menzel, Tanja Maier, Ina Pahl, and Armin Hauk report a relationship with Sartorius Stedim Biotech GmbH that includes: employment., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Single-use (SU) devices and assemblies used as manufacturing equipment in the biopharmaceutical industry require comprehensive qualifications. These qualifications include the assessment of compounds released from SU devices in contact with the process fluids, and how these leachable compounds potentially influence process performance, drug product quality, and patient safety. SU suppliers need to provide comprehensive qualification data for several parameters, for both new products and product changes, such as changes in the sterilization process applied to the SU device. The introduction of X-ray irradiation as an alternative to the currently used and established gamma irradiation of SU devices represents a situation where robust data is required to demonstrate equivalency between these two radiation technologies. Here, we present the results of a comprehensive extractables study for three SU components, bags, tubing, and sterilizing grade filters, evaluated after X-ray and gamma-ray irradiation. The selected study conditions were set up to allow a direct comparison of the results from the two sterilization methods, and to allow conclusions to be made on the impact of irradiation type on the polymers and their additives. Orthogonal analytical methods are applied to identify and quantify all organic compounds present. The data package provided here supports risk assessments for application of irradiated SU equipment in biopharmaceutical manufacturing. The formation of reaction products and the fundamental chemical pathways are discussed and found to be independent of the irradiation type. The results demonstrate the equivalency of both irradiation methods for extractables from plastic components used in pharmaceutical and biopharmaceutical manufacturing., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Conflict of Interest Declaration The authors declare that they have no conflict of interest; the authors are employees of Sartorius Stedim Biotech]., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of 60 Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables-one concern connected with the use of single-use process equipment-the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization., (© 2021 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Filtration is universally used in biopharmaceutical processing. For example, in upstream processing for sterilizing-grade filtration of cell culture media or in various downstream operations, such as clarification, filtration of intermediates, and in critical final filling applications. It is well known that filtration devices can release a certain level of organic compounds within the first filtrate fractions, which can be measured as total organic carbon (TOC). The compounds are primarily released from the surface of its construction materials. This includes typical polymer constituents that migrate from the material, as well as compounds which are formed during sterilization by irradiation. The level of compounds present on a surface is reduced significantly during rinsing of filters. Therefore, these can be defined as "rinsables". A deeper understanding of filter rinsing characteristics and chemical composition of a rinse solution is relevant for process design and risk mitigation, especially in high-risk applications. This publication provides the analytical and mathematical tools to measure and evaluate rinsing curves obtained from different sterilizing-grade membrane filter capsules. Total organic carbon (TOC) content, high-resolution mass spectrometry, ion chromatography, and headspace GC-MS were used to determine the composition of rinsing fractions and to follow the course of the rinsing curve. The required, filter-specific parameters Bulk Volume per Surface area (BVS) and Rinsing Volume per Surface area (RVS) are introduced. They are used for calculating minimum bulk and rinsing volumes of filters that lead to TOC concentrations below the threshold of 500 µg/L for Water for Injection. Three relevant filtration cases in biopharmaceutical manufacturing are discussed together with best practices for evaluation and use of BVS and RVS parameters. Results of a verification test are presented and discussed., (Copyright © 2021. Published by Elsevier B.V.)

Quantitative information on process equipment-related leachables (PERLs) is required for process qualification and within a safety assessment. Extractables data for single-use equipment are suitable and applicable if the extractables study conditions fit or are bracketing the expected conditions of use. It is necessary to extrapolate extractables data when the expected in-use conditions are not covered by the test conditions. Methods for such quantitative extrapolation of extractables data toward potential PERLs are therefore needed. They are comprehensively described in this publication and include: scaling of extractables data for devices of different sizes adjusted to process-volumes, extrapolation to temperatures different from the extraction temperature, extrapolations to different solvent compositions, extrapolation to various contact times, and the combination of extractables data from individual components to assess assemblies. These extrapolation methods yield extractables data as if an extractables study had been performed. The methods presented are consistently derived from basic physicochemical principles. The relevant, underlying physical parameters are obtained from extractables experiments and are compared with published data. The applicability and justification of the proposed calculation methods are discussed and benchmarked against experimental findings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

Users of single-use (SU) components need extractables data to demonstrate material safety for regulatory bodies before incorporation in the biopharmaceutical process. In this context, the correct use of such extractables data is key to deriving realistic risk assessments for SU devices. In this paper, a standardized extractables approach was used that provides comprehensive extractables information including identity and quantity. The combination of extractables data obtained from different components of a sterile filter capsule, such as the filter cartridge and housing, and the scaling thereof is presented. A sterile filter type including polyethersulfone membrane was extracted with pure water and pure ethanol at 40°C for 24 h. The organic extractables were identified and their concentration quantified using state-of-the-art analytical methods such as gas chromatography-mass spectrometry for semivolatile compounds together with headspace sampling for volatile compounds, and liquid chromatography coupled with high-resolution mass spectrometry. The extractables detected were assigned to the materials of filter construction. The evaluation showed that the extractables quantities per device depended on the surface areas of the contact materials, such as the filter membrane, and also on the plastic parts. This paper confirms the validity of a so-called component approach and a scaling concept to calculate extractables data for SU filters of different sizes with short-term contact. LAY ABSTRACT: In the biopharmaceutical industry, a large number of SU system combinations with a variety of different sizes are used. Suppliers of such diverse SU systems and assemblies cannot perform extraction studies for all of the different configurations and sizes individually. It is acceptable in this industry to use component approaches and scaling concepts to provide extractables data for SU systems and assemblies derived from a dedicated extraction experiment. This paper shows the applicability of a so-called component approach and of a scaling concept to calculate extractables data for sterile polyethersulfone membrane filters and filter capsules of different sizes. Selected extraction conditions allow scaling calculations according to underlying physical principles. The extractions were performed under short-term contact, for example, 24 h, to ensure that the release of extractables was diffusion-controlled. The results demonstrated that extractables quantities depend on the surface area of the contact material. Membrane-related compounds were scalable with the membrane area, whereas polypropylene (PP)-related compounds were scalable with the PP contact area., (© PDA, Inc. 2019.)

This publication describes the development of an experimental set-up and testing protocol to test the hypothesis that filters used for sterile filtration can act as scavengers of leachables. The filter materials polyethersulfone (PESU) and cellulose acetate (CA) were tested. These membrane materials are used commonly in downstream operations during biopharmaceutical manufacturing. A solution containing a mixture of eight typical leachables was filtered through the respective filter and the eluate was monitored by HPLC-UV in order to quantify these leachables model compounds (LMC). The results show that substances are efficiently scavenged from an aqueous solution depending on their molecular structure and the filter type used. A mass balance was established by recovering the LMCs from the filters by rinsing the membranes with an organic solvent. Breakthrough curves were determined experimentally and substance specific filter capacities for the individual LMCs are presented. The surface specific filter capacity for the different LMCs range from <0.7 to 45 μg/cm 2 . An extrapolation of these filter scavenging capacities to process conditions, where the filtration areas can be many square-meters in size, gives indication that the potential removal of expectable process-related leachables during filtration in downstream processing should not be underestimated. The capacity of a filter for the leachable bDtBPP, which is known to inhibit cell growth, was determined in samples after a buffer sterile-filtration using a standardized cell-culture test with CHO cells. The specific filter capacity of bDtBPP obtained with the cell-culture test was nearly identical to the analytically derived result. As outlook, the scavenger effect of a filter is demonstrated for media solutions containing buffer and model proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sterile filters are ubiquitous in biopharmaceutical manufacturing processes. Because such filters are in direct contact with the process fluid, profiling of the extractables is of utmost importance. The work presented here reveals the extractables profile from filter cartridges for sterilizing-grade filtration, which were obtained from six different vendors. All filters contain a 0.2 μm polyethersulfone membrane for sterile filtration combined with a polyethersulfone pre-filter with retention rates spanning from 0.4 to 0.6 μm. These filter cartridges are designed for use in stainless steel housings which allow for in-line steam sterilization. A combination of different analytical techniques such as (headspace) gas chromatography-mass spectrometry, ultra-performance liquid chromatography-high-resolution mass spectrometry (electrospray ionization), inductively coupled plasma mass spectrometry, total organic carbon, non-volatile residue, conductivity, and pH value were applied to develop a comprehensive extractables profile on a qualitative and semi-quantitative basis. Pure ethanol and purified water were used as extraction media. The extractables profile consisted of various polyolefin-related extractables, additives such as antioxidants and degradation products thereof, hydrocarbons, and processing aids in addition to membrane-related extractables. LAY ABSTRACT: Filter cartridges or other filter products for sterile filtration are currently most commonly made of polymeric materials such as polypropylene, and a filter membrane material such as polyethersulfone. These materials will usually release chemical substances upon extraction in the laboratory ( extractables ), or upon application in biopharmaceutical processing ( leachables ). Potential extractables and leachables are additives used to tailor the physicochemical properties and to protect the polymeric materials, or degradants of these substances, or they arise from substances used during the manufacturing of the filter cartridges. Multiple analytical techniques were applied here to investigate the concentration and chemical nature of extractables obtained upon application of two distinct extraction solvents. Typical extractables found were antioxidants or releasing agents in addition to compounds originating from the polyethersulfone membrane., (© PDA, Inc. 2018.)

: Quality management systems are, as a rule, tightly defined systems that conserve existing processes and therefore guarantee compliance with quality standards. But maintaining quality also includes introducing new enhanced production methods and making use of the latest findings of bioscience. The advances in biotechnology and single-use manufacturing methods for producing new drugs especially impose new challenges on quality management, as quality standards have not yet been set. New methods to ensure patient safety have to be established, as it is insufficient to rely only on current rules. A concept of qualification, validation, and manufacturing procedures based on risk management needs to be established and realized in pharmaceutical production. The chapter starts with an introduction to the regulatory background of the manufacture of medicinal products. It then continues with key methods of risk management. Hazards associated with the production of medicinal products with single-use equipment are described with a focus on bioreactors, storage containers, and connecting devices. The hazards are subsequently evaluated and criteria for risk evaluation are presented. This chapter concludes with aspects of industrial application of quality risk management.

In biopharmaceutical processes, in the area of food and medical technology a variety of devices is used. These devices consist of various polymers. The detection and identification of potential extractables from these polymers during application are requested by the regulatory bodies. For risk and toxicity assessment, both identification and quantification of extractables are necessary. This article describes the development of a LS-MS methodology transfered from an established HPLC-UV-VIS method for full extractables analysis of sterile-grade filtration cartridges.

Numerous animal studies suggest that acute and chronic exposure to opioids can be associated with the development of hyperalgesia, i.e. an increased sensitivity to noxious stimuli. Hyperalgesia has been documented during withdrawal and on occasion while animals were still exposed to opioids. A pivotal role in the genesis of opioid-associated hyperalgesia has been attributed to a pain facilitating system involving the N-methyl-D-aspartate (NMDA)-receptor. In humans little direct evidence documents opioid-associated hyperalgesia, albeit observational data suggest that such hyperalgesia may be relevant in a clinical context. This study used a double blind, randomized, crossover and placebo-controlled design to test in opioid-naïve, healthy human volunteers whether hyperalgesia would develop within 30 min of stopping a 90-min infusion with the mu-opioid agonist remifentanil, and whether co-administration of the NMDA-receptor antagonist S-ketamine would prevent such hyperalgesia. We found that a skin area with pre-existing mechanical hyperalgesia was significantly enlarged after stopping the remifentanil infusion. However, the pain response to heat assessed in regular skin was not different before and after the infusion of remifentanil. Co-administration of the NMDA-receptor antagonist S-ketamine abolished observed enlargement of the hyperalgesic skin area. This study provides direct evidence in humans that short-term administration of an opioid can enhance hyperalgesia as observed during withdrawal and points to a potential role of the NMDA-receptor system in mediating such a hyperalgesic response. This study also points to a differential susceptibility of different pain modalities for the expression of hyperalgesia associated with opioid administration.

Three membrane materials were tested under similar conditions to determine the effects of membrane material on the performance and cleanability of the filters. The membrane materials investigated were stabilized cellulose (Hydrosart, Sartorius Corporation), cellulose triacetate, and polyethersulfone; all having a 10 kilodalton molecular weight cutoff. Stabilized cellulose is a cellulose-based membrane material, modified for low non-specific protein adsorption combined with high pH-resistance. When analyzing the data, three phenomena were considered: adsorption of protein to the membrane, gel layer formation, and osmotic pressure due to concentration polarization. Throughout these studies, the effects of a gel layer and the osmotic pressure were approximately equivalent in all cassettes. However, the stabilized cellulose was resistant to protein adsorption while the other two membranes exhibited significant decreases in permeate flux due to adsorption. Using a 0.2% BSA (MW = 67,000 kD) solution with a crossflow rate of 5 L/min and transmembrane pressure of 35 psi, the permeate flux through the stabilized cellulose membrane was 3% lower than the baseline saline flux, whereas the cellulose triacetate and polyethersulfone membranes lost 33% and 60% of baseline flux, respectively. The decrease in flux occurring in the latter two membranes is due to adsorption. Another study evaluated adsorption by alternating between crossflow and static operation. After three cycles, the stabilized cellulose maintained the original crossflow flux level. The polyethersulfone lost cumulatively 17% of its crossflow flux after three cycles and the cellulose triacetate lost 13%. The stabilized cellulose and polyethersulfone membranes were also evaluated using a milk solution. The results indicate that the stabilized cellulose is not susceptible to adsorption of any of the milk components while the polyethersulfone permeate flux was limited by adsorption. The saline flux of the stabilized cellulose immediately after testing with the milk solution was 3% lower than the baseline flux, while the polyethersulfone membrane saline flux was 81% lower. The results consistently indicated that, unlike the cellulose triacetate and polyethersulfone membranes, the stabilized cellulose membrane was not subject to adsorption.

Chinese Hamster Ovary (CHO) cells are used for fermentation of high value proteins in the pharmaceutical- and biotechnology industry. During the fermentation process the cells are grown under specific conditions in a defined media. The CHO cells produce extra cellular proteins. Separation and purification of these proteins result in the final product of high value. The production steps utilized in any bio-pharmaceutical process must deliver the target protein with high purity and yield. The first step in the production process is the separation of cells from the rest of the fermentation broth. Currently, stacked depth filters are used in this separation. The disadvantages with this method are:- 1) low product yield (high hold-up/wetting volume) and 2) the messy "clean-up" that is inherent with stacked filters. One of the main process requirements is that the CHO cell removal be accomplished with low cell mortality. This eases the subsequent process steps to purify the target protein with high yields free of DNA and other intracellular proteins. Additionally, the CHO cells can be reused.

The expression of c-myb mRNA and protein was analyzed in fresh leukemic cells by Northern-blot analyses and by immunofluorescent staining using monoclonal c-myb specific antibodies. Staining of the cells was evaluated by flow cytometry. The results demonstrate c-myb mRNA expression predominantly in acute lymphocytic leukemia (ALL, 4/4 cases), acute myeloic leukemia (AML, 17/17) and chronic myeloic leukemia (CML, 12/12) but rarely in chronic lymphocytic leukemia (CLL, 1/17). Immunofluorescent analyses revealed expression of c-myb protein in the nucleus of ALL (5/7) and AML (9/9) with a good correlation of c-myb-positive cells and with the number of proliferating (Ki67-positive) blast cells.

The midtrimester abortion program at a large community hospital was evaluated. During the 3-year study, 1839 patients aborted in the midtrimester by intraamniotic injection of hypertonic saline, prostaglandin F2alpha or a combination of saline and prostaglandin F2alpha. The method, using a combination of saline and prostaglandin F2alpha together with intracervical laminaria, showed significant reduction in the number of failures (4.3 to 1.0%), reduction in the injection-abortion interval from 33.9 to 14.6 hours, shortening of the hospital stay from 2 1/2 to 1 1/3 days, minimum incidence of live abortions (0.9%), infrequent need for oxytocin to effect delivery (7.7%); and low rates of hemorrhage (1.5%) and fever (2.8%). The main disadvantage was an increased rate of incomplete abortions (32.3%), which could be reduced to 27% by patient selection.