Biotechnological Manufacturing Research Articles

Aspects of microbiological monitoring of the production environment in pharmaceutical and biotechnological manufacturing

Aspects of microbiological monitoring of the production environment in pharmaceutical and biotechnological manufacturing

Experimental evaluation of bearingless spinfilter topologies

Rotating filter membranes can increase the long term filtrate flux by efficiently and continuously removing the retained particles on a microfiltration membrane. However, the practical implementation of rotating machinery in high purity applications such as biotechnological manufacturing of proteins raises concerns about the contamination of the process fluids due to submerged bearings and imperfect sealings to the outside environment. Bearingless and hermetically sealed spinfilters provide a solution to these drawbacks, but create challenges due to the contactless operation, and consequentially, the open internal gap between the feed and the filtrate. To address this, five bearingless spinfilter topologies to reduce the remixing across the open gap are proposed, experimentally evaluated, and discussed. Three spinfilter topologies reach a filtrate purity of 100%, while the best performing concept reaches specific filtrate fluxes of over 2300Lh−1m−2.

The Past, Present, and Future of Tubular Melt Electrowritten Constructs to Mimic Small Diameter Blood Vessels - A Stable Process?

Melt Electrowriting (MEW) is a continuously growing manufacturing platform. Its advantage is the consistent production of micro- to nanometer fibers, that stack intricately, forming complex geometrical shapes. MEW allows tuning of the mechanical properties of constructs via the geometry of deposited fibers. Due to this, MEW can create complex mechanicsonly seen in multi-material compounds and serve as guiding structures for cellular alignment. The advantage of MEW is also shown in combination with other biotechnological manufacturing methods to create multilayered constructs that increase mechanical approximation to native tissues, biocompatibility, and cellular response. These features make MEW constructs a perfect candidate for small-diameter vascular graft structures. Recently, studies have presented fascinating results in this regard, but is this truly the direction that tubular MEW will follow or are there also other options on the horizon? This perspective will explore the origins and developments of tubular MEW and present its growing importance in the field of artificial small-diameter vascular grafts with mechanical modulation and improved biomimicry and the impact of it in convergence with other manufacturing methods and how future technologies like AI may influence its progress.

Monitoring of the Enzymatic Reactions Course by Differential Microwave Dielectrometry Method in Real Time

Enzymatic reactions are the basis of many biotechnological manufacturing and biomedical diagnostic procedures that require effective methods of monitoring over the reaction course. In the current paper, we present the results of the development of a new approach within the differential microwave dielectrometry method for the non-invasive monitoring of the course of enzymatic reactions based on the complex permittivity changes of these reactive mixture solutions in real time at a fixed frequency of 31.82 GHz. The dynamic studies of the dielectric parameters of selected enzymatic systems containing a protein substrate (immunoglobulin G, human serum albumin) and enzyme trypsin. The developed differential microwave dielectrometry setup has been performed to verify the proposed approach effectiveness for the enzymatic reaction monitoring in biomedical practice and the food industry. Our microwave dielectrometry results have been validated by the results of the UV-Vis spectrophotometry method for selected enzymatic systems. We propose a new approach to use the differential microwave dielectrometry method with high sensitivity (in average 0.5% and 3÷5% for the real and imaginary parts of the complex permittivity, respectively) to estimate the course of enzymatic reactions in real time.

Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC.

For decades, the ability of detergents to solubilize biological membranes has been utilized in biotechnological manufacturing to disrupt the lipid envelope of potentially contaminating viruses and thus enhance the safety margins of plasma- and cell-derived drugs. This ability has been linked to detergent micelles, which are formed if the concentration of detergent molecules exceeds the critical micelle concentration (CMC). Traditionally, the CMC of detergents is determined in deionized water (ddH2O), i.e., a situation considerably different from the actual situation of biotechnological manufacturing. This study compared, for five distinct detergents, the CMC in ddH2O side-by-side with two biopharmaceutical process intermediates relevant to plasma-derived (Immunoglobulin) and cell-derived (monoclonal antibody) products, respectively. Depending on the matrix, the CMC of detergents changed by a factor of up to ~4-fold. Further, the CMC in biotechnological matrices did not correlate with antiviral potency, as Triton X-100 (TX-100) and similar detergents had comparatively higher CMCs than polysorbate-based detergents, which are known to be less potent in terms of virus inactivation. Finally, it was demonstrated that TX-100 and similar detergents also have virus-inactivating properties if applied below the CMC. Thus, the presence of detergent micelles might not be an absolute prerequisite for the disruption of virus envelopes.

Primary Progenitor Tenocytes: Cytotherapeutics and Cell-Free Derivatives

Primary progenitor tenocytes are diploid cells that may be cultured in vitro and therapeutically used for allogeneic musculoskeletal regenerative medicine. Firstly, technical aspects of cell banking, biotechnological manufacturing, and extensive preclinical characterization data have confirmed that FE002-Ten primary progenitor tenocytes may be safely considered for human cytotherapeutic use (e.g., in tissue engineering products, standardized transplants). Parallelly, lyophilized progenitor tenocyte extracts (e.g., stabilized cells or cell-free derivatives) were shown to optimally act as potent hyaluronan-based hydrogel functionalizing agents, useful for stability enhancement against oxidative product degradation. Therefore, primary progenitor tenocytes (e.g., FE002-Ten cell source) may potentially be used in diverse clinical presentations of tendon-related pathologies, ranging from volumetric tissue replacement (i.e., for the promotion of enhanced graft bio-integration) to local management of tissular inflammation and pain (i.e., ancillary action of the cellular extracts for the functional enhancement of injectable hyaluronan-based preparations). Overall, the primary progenitor tenocytes investigated under the Swiss progenitor cell transplantation program were shown to represent highly standardized biotechnological materials with a versatility of potential therapeutic uses after formulation into an array of cytotherapeutic preparations or cell-free devices.

Lyophilized Progenitor Tenocyte Extracts: Sterilizable Cytotherapeutic Derivatives with Antioxidant Properties and Hyaluronan Hydrogel Functionalization Effects.

Cultured primary progenitor tenocytes in lyophilized form were previously shown to possess intrinsic antioxidant properties and hyaluronan-based hydrogel viscosity-modulating effects in vitro. The aim of this study was to prepare and functionally characterize several stabilized (lyophilized) cell-free progenitor tenocyte extracts for inclusion in cytotherapy-inspired complex injectable preparations. Fractionation and sterilization methods were included in specific biotechnological manufacturing workflows of such extracts. Comparative and functional-oriented characterizations of the various extracts were performed using several orthogonal descriptive, colorimetric, rheological, mechanical, and proteomic readouts. Specifically, an optimal sugar-based (saccharose/dextran) excipient formula was retained to produce sterilizable cytotherapeutic derivatives with appropriate functions. It was shown that extracts containing soluble cell-derived fractions possessed conserved and significant antioxidant properties (TEAC) compared to the freshly harvested cellular starting materials. Progenitor tenocyte extracts submitted to sub-micron filtration (0.22 µm) and 60Co gamma irradiation terminal sterilization (5-50 kGy) were shown to retain significant antioxidant properties and hyaluronan-based hydrogel viscosity modulating effects. Hydrogel combination products displayed important efficacy-related characteristics (friction modulation, tendon bioadhesivity) with significant (p < 0.05) protective effects of the cellular extracts in oxidative environments. Overall, the present study sets forth robust control methodologies (antioxidant assays, H2O2-challenged rheological setups) for stabilized cell-free progenitor tenocyte extracts. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development (purification, terminal sterilization) allowed for the conservation of critical biological extract attributes.

Need for Expansion of Pharmacy Education Globally for the Growing Field of Nanomedicine.

The emerging landscape of nanomedicine includes a wide variety of active pharmaceutical ingredients and drug formulations. Their design provides nanomedicines with unique features leading to improved pharmacokinetics and pharmacodynamics. They are manufactured using conventional or biotechnological manufacturing processes. Their physical characteristics are vastly different from traditional small-molecule drugs. Pharmacists are important members of the multi-disciplinary team of scientists involved in their development and clinical application. Consequently, their training should lead to an understanding of the complexities associated with the production and evaluation of nanomedicines. Therefore, student pharmacists, post-doctoral researchers, and trainees should be given more exposure to this rapidly evolving class of therapeutics. This commentary will provide an overview of nanomedicine education within the selection of pharmacy programs globally, discuss the current regulatory challenges, and describe different approaches to incorporate nanomedicine science in pharmacy programs around the world.

Evaluation of the Stability of Bacteriophages in Different Solutions Suitable for the Production of Magistral Preparations in Belgium.

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6–9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days.

Recombinant Spidroins as the Basis for New Materials.

Spider web proteins are unique materials created by nature that, considering the combination of their properties, do not have analogues among natural or human-created materials. Obtaining significant amounts of these proteins from natural sources is not feasible. Biotechnological manufacturing in heterological systems is complicated by the very high molecular weight of spidroins and their specific amino acid composition. Obtaining recombinant analogues of spidroins in heterological systems, mainly in bacteria and yeast, has become a compromise solution. Because they can self-assemble, these proteins can form various materials, such as fibers, films, 3D-foams, hydrogels, tubes, and microcapsules. The effectiveness of spidroin hydrogels in deep wound healing, as 3D scaffolds for bone tissue regeneration and as oriented fibers for axon growth and nerve tissue regeneration, was demonstrated in animal models. The possibility to use spidroin micro- and nanoparticles for drug delivery was demonstrated, including the use of modified spidroins for virus-free DNA delivery into animal cell nuclei. In the past few years, significant interest has arisen concerning the use of these materials as biocompatible and biodegradable soft optics to construct photonic crystal super lenses and fiber optics and as soft electronics to use in triboelectric nanogenerators. This review summarizes the latest achievements in the field of spidroin production, the creation of materials based on them, the study of these materials as a scaffold for the growth, proliferation, and differentiation of various types of cells, and the prospects for using these materials for medical applications (e.g., tissue engineering, drug delivery, coating medical devices), soft optics, and electronics. Accumulated data suggest the use of recombinant spidroins in medical practice in the near future.

Skimmed milk as an alternative for IPTG in induction of recombinant protein expression

Cost-effectiveness is an important issue in biotechnological manufacturing industry and using alternative cheap materials with the same benefits has been noticed in most literatures. Isopropyl β-d-1-thiogalactopyranoside (IPTG), a well-known chemical element for induction of protein expression, has several disadvantages such as high expense and toxicity. In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombinant protein expression, which has advantages such as more availability and affordability, in comparison to IPTG supplementation.

Identifying necessary and sufficient conditions for the observability of models of biochemical processes

The notions of observability and controllability of non-linear systems are a cornerstone of mathematical control theory and cover a wide scope of applications including process design, characterization, monitoring and control. Synthetic biology - which aims to (re)-program living functionalities - and bio-based process engineering - which aims to develop biotechnological manufacturing processes based on industrial and natural living agents - remarkably benefit of methodological improvements inspired to control theory for countless reasons including the huge variety of control mechanisms in living organisms, experimental limitations in terms of measurement feasibility, design of controllers - at single cell or population level - of synthetic production processes and process optimization purposes. Many fundamental problems of control theory such as stabilisability of unstable systems and optimal control may be solved under the assumption that the system is observable/controllable. Observability and controllability are mathematical duals, that means that the observability property can be determined analysing the controllability of the dual system and vice versa. Given this duality, we focus on observability. In this work, we revisit a generalization of the Fujisawa and Kuh theorem as a tool to explore the possibility that a system is observable. We show that the theorem, when applicable, is a sufficient but not necessary condition for observability. We revisit the theorem to propose a necessary and sufficient condition for observability for non-linear systems. Finally, we show how it is possible to identify regions of the phase space of the model in which the model is observable.

Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris.

Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH5.9 (64mg/L at 0.02 1/h) compared to pH5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers

BackgroundThermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process.ResultsPhysiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L− 1 and 47.1 U·L− 1·h− 1 for SacPox and to 8700 U·L− 1 and 180.6 U·L− 1·h− 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications.ConclusionsIn this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.

Calcite/aragonite-biocoated artificial coral reefs for marine parks

Natural formation of the coral reefs is complicated by slow biomediated precipitation of calcium carbonate from seawater. Therefore, manufactured artificial coral reefs can be used for the formation of “underwater gardens” in marine parks for the recreational fishing and diving that will protect natural coral reefs from negative anthropogenic effects. Additionally, the coating of the concrete, plastic or wooden surfaces of artificial coral reef with calcium carbonate layer could promote attachment and growth of coral larvae and photosynthetic epibiota on these surfaces. Three methods of biotechnological coating of the artificial coral reefs have been tested: (1) microbially induced calcium carbonate precipitation from concentrated calcium chloride solution using live bacterial culture of Bacillus sp. VS1 or dead but urease-active cells of Yaniella sp. VS8; (2) precipitation from calcium bicarbonate solution; (3) precipitation using aerobic oxidation of calcium acetate by bacteria Bacillus ginsengi strain VSA1. The thickness of biotechnologically produced calcium carbonate coating layer was from 0.3 to 3 mm. Biocoating using calcium salt and urea produced calcite in fresh water and aragonite in seawater. The calcium carbonate-coated surfaces were colonized in aquarium with seawater and hard corals as inoculum or in aquarium with fresh water using cyanobacteria Chlorella sorokiana as inoculum. The biofilm on the light-exposed side of calcium carbonate-coated surfaces was formed after six weeks of incubation and developed up to the average thickness of 250 µm in seawater and about 150 µm in fresh water after six weeks of incubation. The biotechnological manufacturing of calcium carbonate-coated concrete, plastic, or wooden surfaces of the structures imitating natural coral reef is technologically feasible. It could be commercially attractive solution for the introduction of aesthetically pleasant artificial coral reefs in marine parks and resorts.

Downstream process development in biotechnological itaconic acid manufacturing.

Itaconic acid is a promising chemical that has a wide range of applications and can be obtained in large scale using fermentation processes. One of the most important uses of this biomonomer is the environmentally sustainable production of biopolymers. Separation of itaconic acid from the fermented broth has a considerable impact in the total production cost. Therefore, optimization and high efficiency downstream processes are technological challenges to make biorefineries sustainable and economically viable. This review describes the current state of the art in recovery and purification for itaconic acid production via bioprocesses. Previous studies on the separation of itaconic acid relying on operations such as crystallization, precipitation, extraction, electrodialysis, diafiltration, pertraction, and adsorption. Although crystallization is a typical method of itaconic acid separation from fermented broth, other methods such as membrane separation and reactive extraction are promising as a recovery steps coupled to the fermentation, potentially enhancing the overall process yield. Another approach is adsorption in fixed bed columns, which efficiently separates itaconic acid. Despite recent advances in separation and recovery methods, there is still space for improvement in IA recovery and purification.

Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

Conformational stability as a design target to control protein aggregation.

Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

Purification of biomolecules combining ATPS and membrane chromatography

Upstream processes for production of therapeutic proteins have been innovated and fermentation processes have been adopted for the use of recombinant microorganisms with high expression, but the downstream process is still the bottleneck in the biotechnological manufacturing process. A combined process consisting of aqueous two phase extraction (ATPE) and membrane chromatography is suggested to debottleneck downstream processing. ATPE has a large capacity, but the yield of the target product is from 74% to 97%. For this reason the product of ATPE waste stream is captured by membrane chromatography. In this work the binding capacity for the protein on protein A, ion exchange and hydrophobic exchange membrane chromatography was investigated experimentally with different concentration of polyethylene glycol (PEG), salt and protein. Protein A membrane was loaded with solutions resembling waste streams of ATPE for purifying IgG. For ion exchange and hydrophobic interaction membrane chromatography, the membrane was loaded with bovine serum albumin (BSA). PEG shows no significant effect on stability and capacity of membrane process. Even for small amount of BSA/IgG and high salt concentrations membrane adsorption is applicable. In this work it is demonstrated experimentally that a total product recovery of 99.9% for the purification of monoclonal antibody is possible.

Environmental risk assessment for ancillary substances in biotechnological production of pharmaceuticals

An increasing number of pharmaceutical active substances are produced through biotechnological processes. For sustained and safe growth of the host organisms as well as optimal expression, purification, and formulation of the product, biotechnological manufacturing processes need optimal and robust environmental conditions, which are attained through the use of buffers, chelators, and antibiotics, beside nutrients. These ancillary substances are drained with the wastewater to a wastewater treatment plant (WWTP) and are released after treatment with the effluent to receiving waters. The potential risks of such substances to WWTPs and surface waters were investigated. Three common buffers (morpholinoethane sulfonic acid [MES], morpholinopropanesulfonic acid [MOPS], 1,4-piperazine (diethanesulfonic acid) [PIPES]), one chelator (ethylenediaminetetraacetic acid [EDTA]), and one antibiotic (gentamycin) were searched in the literature for environmental data or tested for biodegradability and inhibition of activated sludge as well as acute toxicity to algae, daphnids, and fish. Amounts of the ancillary substances used in the European biotechnological production plants of F. Hoffmann-La Roche Ltd in Basle (Switzerland) and Penzberg (Germany), and actual wastewater fluxes through the respective WWTP, as well as realistic dilution factors for the local receiving water, were documented. Based on this information, site-specific predicted environmental concentrations (PECs) for the WWTPs and surface waters in Basle and Penzberg were extrapolated. These PECs were compared with predicted no effect concentrations (PNECs) for the WWTP and surface waters, derived from sludge inhibition and ecotoxicity results, respectively. For all five ancillary substances investigated, all PEC/PNEC risk characterization ratios are <1, indicating no significant risk to the WWTPs or the receiving waters at both sites.