Assessing biologic/toxicologic effects of extractables from plastic contact materials for advanced therapy manufacturing using cell painting assay and cytotoxicity screening.

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.

Dimethylsilanediol from silicone elastomers: Analysis, release from biopharmaceutical process equipment, and clearance studies.

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.

Equivalence study of extractables from single-use biopharmaceutical manufacturing equipment after X-ray or gamma irradiation.

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.

X-ray sterilization of biopharmaceutical manufacturing equipment-Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation.

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.

Rinsing Recommendations for Membrane Filters and Identification of Rinsables.

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.

Using extractables data from single-use components for extrapolation to process equipment-related leachables: The toolbox and justifications.

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.

Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials.

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.

Quantitative characterization of leachables sinks in biopharmaceutical downstream processing.

This article demonstrates, on a quantitative level, that leachables - potentially accumulated during a biopharmaceutical manufacturing process - will be significantly reduced/removed during four key downstream process steps: cell removal using centrifugation or depth filtration, sterile filtration and virus filtration. Eight common leachables model compounds (LMCs) were spiked into typical feed solutions containing buffer and proteins and were analyzed post-processing in the supernatant or filtrates by HPLC-UV. The clearance rates were calculated as the quotient between the scavenged and initially spiked amount of each leachable. High clearance rates were found for hydrophobic LMCs for all investigated downstream operation steps. It is shown that the removal of cells and cell debris from a culture broth reduces the amount of LMCs almost completely after centrifugation or depth filtration. Also, sterilizing-grade and virus filtration provided a high scavenger effect to most of the LMCs. In contrast, only one hydrophilic acid was not significantly scavenged by the described operations. The possibility to include leachables sinks to a process qualification and risk mitigation concept is explained.

Using Extractables Data of Sterile Filter Components for Scaling Calculations.

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.

Filtration membranes - Scavengers for leachables?

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/cm2. 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.

Comparative Extractables Study of Autoclavable Polyethersulfone Filter Cartridges for Sterile Filtration.

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.

Lack of evidence for microplastic contamination in honey.

Honey samples from Switzerland were investigated with regard to their microplastic particle burden. Five representative honey samples of different origin were processed following a standardized protocol to separate plastic-based microparticles from particles of natural origin, such as pollen, propolis, wax, and bee-related debris. The procedure was optimized to minimize post-sampling microplastic cross-contamination in the laboratory. The isolated microplastic particles were characterized and grouped by means of light microscopy as well as chemically characterized by microscopically coupled Raman and Fourier transform infrared spectroscopy. Five particle classes with an abundance significantly above blank levels were identified: black particles (particle count between 1760/kg and 8680/kg), white transparent fibres (particle count between 132/kg and 728/kg), white transparent particles (particle count between 60/kg and 172/kg), coloured fibres (particle count between 32/kg and 108/kg), and coloured particles (particle count between 8/kg and 64/kg). The black particles, which represented the majority of particles, were identified as char or soot and most probably originated from the use of smokers, a widespread practice in beekeeping. The majority of fibres were identified as cellulose or polyethylene terephthalate and were most likely of textile origin. In addition to these particle and fibre groups lower numbers of fragments were detected that were related to glass, polysaccharides or chitin, and few bluish particles contained copper phthalocyanine pigment. We found no indications that the honey samples were significantly contaminated with microplastic particles.

Microplastics profile along the Rhine River.

Microplastics result from fragmentation of plastic debris or are released to the environment as pre-production pellets or components of consumer and industrial products. In the oceans, they contribute to the 'great garbage patches'. They are ingested by many organisms, from protozoa to baleen whales, and pose a threat to the aquatic fauna. Although as much as 80% of marine debris originates from land, little attention was given to the role of rivers as debris pathways to the sea. Worldwide, not a single great river has yet been studied for the surface microplastics load over its length. We report the abundance and composition of microplastics at the surface of the Rhine, one of the largest European rivers. Measurements were made at 11 locations over a stretch of 820 km. Microplastics were found in all samples, with 892,777 particles km (-2) on average. In the Rhine-Ruhr metropolitan area, a peak concentration of 3.9 million particles km (-2) was measured. Microplastics concentrations were diverse along and across the river, reflecting various sources and sinks such as waste water treatment plants, tributaries and weirs. Measures should be implemented to avoid and reduce the pollution with anthropogenic litter in aquatic ecosystems.

A field study of triclosan loss rates in river water (Cibolo Creek, TX).

Triclosan (TCS) is an anti-microbial agent used in down-the-drain consumer products. Following sewage treatment some of the triclosan will enter receiving waters. This study was designed to determine the die-away rate of triclosan released into a river as part of the sewage treatment plant effluent matrix. The study was conducted in Cibolo Creek, a moderate sized stream (discharge approximately 0.1 m(3)s(-1)) located in South Central Texas. Triclosan was analyzed from samples collected upstream of the sewage treatment plant, the sewage treatment plant effluent, and the river downstream from the effluent discharge. The first-order loss rate of parent triclosan from the water column was calculated from measured data (0.06 h(-1)) and this rate corresponded to a 76% reduction in triclosan over an 8 km river reach below the discharge. Mathematical modeling indicated that sorption and settling accounted for approximately 19% of total triclosan loss over 8 km. When removing sorption and settling, the remaining amount of triclosan had an estimated first-order loss rate of 0.25 h(-1). This loss rate was presumably due to other processes such as biodegradation and photolysis. These data show that loss of parent triclosan from the water column is rapid. Additional data are needed to fully document loss mechanisms.

Environmental fate of Triclosan in the River Aire Basin, UK.

The concentrations and removal rate of Triclosan, an antibacterial ingredient in consumer products, were measured at advanced trickling filter (TF) and activated sludge (AS) wastewater treatment plants (WWTPs) in the River Aire basin in the UK in September 2000. Additionally, the in-stream removal of Triclosan was measured directly in Mag Brook, the stream receiving the treated effluent from the TF plant, using a fluorescent dye tracer to determine the water plug travel times. The in-stream removal of the dissolved and un-ionized (i.e. bioavailable) fraction of the compound was measured using semipermeable membrane devices (SPMDs) deployed at various distances downstream from the WWTP discharge point. The estimated removal rates were used in the GREAT-ER (Geography-Referenced Regional Exposure Assessment Tool for European Rivers) model to predict the site-specific distribution of Triclosan concentrations in the Aire basin as well as to calculate regional concentrations. High WWTP (approximately 95%) and in-stream (0.21-0.33 h-1) removal rates of Triclosan in Mag Brook confirm that this chemical is rapidly eliminated from the aquatic environment.

Measurement of triclosan in wastewater treatment systems.

The objective of this study was to investigate the fate and removal of triclosan (TCS; 5-chloro-2-[2,4-dichloro-phenoxy]-phenol), an antimicrobial agent used in a variety of household and personal-care products, in wastewater treatment systems. This objective was accomplished by monitoring the environmental concentrations of TCS, higher chlorinated derivatives of TCS (4,5-dichloro-2-[2,4-dichloro-phenoxy]-phenol [tetra II]; 5,6-dichloro-2-[2,4-dichloro-phenoxy]-phenol [tetra III]; and 4,5,6-trichloro-2-(2,4-dichloro-phenoxy)-phenol [penta]), and a potential biotransformation by-product of TCS (5-chloro-2-[2,4-dicholoro-phenoxy]-anisole [TCS-OMe]) during wastewater treatment. These analytes were isolated from wastewater by using a C18 solid-phase extraction column and from sludge with supercritical fluid CO2. Once the analytes were isolated, they were derivatized to form trimethylsilylethers before quantitation by gas chromatography-mass spectrometry. Recovery of TCS from laboratory-spiked wastewater samples ranged from 79 to 88% for influent, 36 to 87% for final effluent, and 70 to 109% for primary sludge. Field concentrations of TCS in influent wastewater ranged from 3.8 to 16.6 microg/L and concentrations for final effluent ranged from 0.2 to 2.7 microg/L. Removal of TCS by activated-sludge treatment was approximately 96%, whereas removal by trickling-filter treatment ranged from 58 to 86%. The higher chlorinated tetra-II, tetra-III, and penta closans were below quantitation in all of the final effluent samples, except for one sampling event. Digested sludge concentrations of TCS ranged from 0.5 to 15.6 microg/g (dry wt), where the lowest value was from an aerobic digestion process and the highest value was from an anaerobic digestion process. Analysis of these results suggests that TCS is readily biodegradable under aerobic conditions, but not under anaerobic conditions. The higher chlorinated closans were near or below the limit of quantitation in all of the digested sludge samples. Based on results from this study, the chlorinated analogues and biotransformation by-product of TCS are expected to be very low in receiving waters and sludge-amended soils.