Search

The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non‐proteolytic release mechanism for HMCES‐DNA‐protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA‐dsDNA junctions, HMCES‐DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES‐DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re‐crosslinks or dissociates. Our study reveals that the protective role of HMCES‐DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum. Synopsis: The protein HMCES forms covalent crosslinks with abasic sites in ssDNA to prevent the formation of toxic dsDNA breaks. This study reports a non‐proteolytic release mechanism that restricts formation of HMCES‐DNA crosslinks to a necessary minimum, and that is regulated by DNA context. HMCES‐DNA crosslinks are stable in ssDNA and at ssDNA‐dsDNA junctions, thereby protecting abasic sites against incisions.In dsDNA, HMCES‐DNA crosslinks are released, allowing APE1 to incise the abasic site to initiate repair.Release requires a conserved glutamate residue within HMCES' active site, which catalyses reversal of the crosslink.Affinity to the underlying DNA structure determines whether HMCES re‐crosslinks or releases the abasic site. [ABSTRACT FROM AUTHOR]

Background: Knowledge on immunity after SARS-CoV-2 infection in patients with multiple sclerosis (pwMS) and the impact of disease-modifying treatment (DMT) is limited. Objective: To evaluate degree, duration and potential predictors of specific humoral immune response in pwMS with prior COVID-19. Methods: Anti-SARS-CoV-2 antibody testing was performed in pwMS with PCR-confirmed diagnosis of symptomatic COVID-19 from a nation-wide registry. Predictors of seropositivity were identified by multivariate regression models. Results: In 125 pwMS (mean age = 42.4 years (SD = 12.3 years), 70% female), anti-SARS-CoV-2 antibodies were detected in 76.0% after a median of 5.2 months from positive PCR. Seropositivity rate was significantly lower in patients on IS-DMT (61.4%, p = 0.001) than without DMT or immunomodulatory DMT (80.6%; 86.0%, respectively). In multivariate analysis, IS-DMT was associated with reduced probability of seropositivity (odds ratio (OR): 0.51; 95% confidence interval (95% CI): 0.17–0.82; p < 0.001). Predefined subgroup analyses showed marked reduction of seropositivity in pwMS on rituximab/ocrelizumab (OR 0.15; 95% CI: 0.05–0.56; p < 0.001). Rate of seropositivity did not change significantly over 6 months. Conclusions: Humoral immunity is stable after SARS-CoV-2 infection in MS, but is reduced by immunosuppressive DMT, particularly anti-CD20 monoclonal antibodies. This provides important evidence for advising pwMS as well as for planning and prioritizing vaccination. [ABSTRACT FROM AUTHOR]

Abstract: Heterologous proteins are often poorly expressed in Escherichia coli and especially small peptides are prone to degradation. Npro autoprotease fusion proteins, deposited as inclusion bodies in E. coli, are a versatile tool for peptide and protein overexpression and generate an authentic N terminus at the target molecule. Autoproteolytic cleavage and subsequent release of the fusion partner are initiated upon refolding. Fusion proteins with the Npro mutant EDDIE follow a monomolecular reaction. The reaction rate was only dependent on chaotrope concentration, decreasing exponentially by a factor of 1.2–1.5 for urea and by a factor of 2.1–5.3 for GuHCl. The first amino acid of the target peptide had a major impact on the reaction rate studying a set of model peptides. Reaction rates were in the range of 2.2×10−4 to 7.3×10−5 s−1 and could be increased up to fivefold by exchanging the first amino acid of the target peptide. A panel of biophysical methods was used to assess EDDIE secondary and tertiary structure. Immediate formation of secondary structure and slight increase in β-sheet content of approximately 5% over the course of the cleavage reaction was observed and interpreted as aggregation. Aggregation and cleavage occurred simultaneously. EDDIE has a relatively loose structure with the cleavage site exhibiting the lowest solvent exposure. We hypothesize that this is the mechanism for establishing a spatial proximity between cleavage site and the catalytic centre of the autoprotease. Fluorescence measurements revealed that further structural changes did not occur after the initial hydrophobic collapse. Thus, the overall reaction is predominantly controlled by cleavage kinetics and refolding kinetics does not play a major role. [Copyright &y& Elsevier]

Abstract: Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin–FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays. [Copyright &y& Elsevier]

Abstract: Epithelial cell adhesion molecule (EpCAM) is an attractive target for monoclonal antibody serotherapy because it is over-expressed in ∼70% of epithelial cancers and their metastatic lesions. IGN101, the immunogenic formulation of the murine monoclonal anti-EpCAM antibody Mab17-1A, has been shown to evoke a strong humoral immune response in both monkey studies and early clinical trials. Notably, there was a reduction in the number of circulating EpCAM-positive tumor cells in the peripheral blood of treated cancer patients. In contrast to earlier publications by other groups, we could not detect an anti-EpCAM immune response upon treatment with Mab17-1A using a conventional but optimized anti-EpCAM ELISA. Therefore, in a novel experimental setup, sera of healthy immunized monkeys, normal human donors and cancer patients immunized with IGN101 were tested for reactivity against a series of overlapping synthetic peptides encompassing the entire sequence of EpCAM prepared by SPOT synthesis on cellular supports. Using this method, sera from normal donors reacted with different peptides compared to sera from healthy monkeys. However, the peptides were clustered in the same regions of EpCAM. Cancer patients generally had a lower reactivity to EpCAM peptides and immunization with IGN101 induced reactivity against a different set of peptides. Antibodies cross-reacting with both the IgG2a framework and with the Mab17-1A idiotype were identified. In summary, our data indicate that some EpCAM peptides may be recognized in a species-specific manner. At least seven EpCAM-derived peptides could be of diagnostic interest (QCQCTSVGAQ, ERVRTYWIII, ALQKEITTRY, TYWIIIELKH, IADVAYYFEK, AYYFEKDVKG, GQTLIYYVDE), while four out of these seven peptides may also possess therapeutic relevance (TYWIIIELKH, ALQKEITTRY, IADVAYYFEK, AYYFEKDVKG). [Copyright &y& Elsevier]

Abstract: The growing range of applications for peptide arrays prepared by SPOT synthesis confirms that they are a powerful proteomics technique to study numerous aspects of molecular interaction events. The most frequent application for peptide arrays prepared by SPOT synthesis is the identification of linear epitopes that are recognized by antibodies. In the conventional format using secondary antibodies for detection unspecific binding and high background have been observed. This leads to difficulties in evaluation of developed membranes. Especially for application with combinatorial libraries false positive results are to be avoided. To circumvent this issue, we directly labeled compounds of interest with biotin and detected binding by incubation with streptavidin–horseradish peroxidase via chemiluminescence. Optimization of method conditions led to a very sensitive detection technique with no or low number of unspecific spots, which is superior to conventional detection with secondary antibodies. As one consequence, evaluation of competitive assays got more reliable. [Copyright &y& Elsevier]

• Equivalence tests assess changes in product quality of biopharmaceutical processes. • Test performance is influenced by the groups' sample sizes, standard deviations and distributions. • Data should be investigated for possible outliers before applying an equivalence test. • A sample size of 4 leads to a significantly higher test performance compared to 3. Efficient development for multistep downstream processing of biopharmaceuticals depends on fast and robust transfer from bench to manufacturing-scale. Equivalence tests are used to assess the impact of changes in product quality and quantity during process development, optimization and production. This article presents the results of a simulation study comparing the impact of sample sizes, data distributions, variances and equivalence acceptance criteria (EAC) on the performance of equivalence tests. Our results show that a sample size of at least four in the reference group was the minimum recommended size to obtain an acceptable overall performance. Reference data should be evaluated for outliers prior to the equivalence tests and reliable test decisions are only taken with a low variance in the data of the reference group using the EAC proposed by Limentani. Considering these recommendations we obtained a reliable product yield comparability after chromatographic purification of a biopharmaceutical in bench and manufacturing-scale. [ABSTRACT FROM AUTHOR]

• The presented hybrid model predicts the duration of crossflow ultrafiltration. • The hybrid model exhibits superior predictions compared to the film theory. • The hybrid model can predict batch and fed-batch filtrations. • Training the hybrid model is a simple and fast to automate process. Cross-flow ultrafiltration is a powerful tool used in bioprocesses to concentrate and separate biopharmaceuticals. However, there is a major challenge for its effective use: the fouling rate and decrease in the permeate flux vary substantially depending on the concentration and characteristics of the incoming feed, which causes variations in the process durations. We developed a hybrid model for use in predicting the flux evolution and duration of cross-flow ultrafiltration processes for various proteins, membrane types, input parameters, and filtration modes. The trained hybrid model is able to determine the process duration with an average normalized root-mean-square error of less than 6.2. It has superior adaptation to varying filtration characteristics compared to the mechanistic film theory model and can handle both batch- and fed-batch ultrafiltration using the same training set. In addition, the presented hybrid model can be used as a digital twin to simulate virtual processes with varying input parameters and different batch modes, which is a valuable tool for efficient process development or optimization. [ABSTRACT FROM AUTHOR]

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored. [ABSTRACT FROM AUTHOR]

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media. [ABSTRACT FROM AUTHOR]

Abstract: Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse‐derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro‐orifice, ultra‐sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro‐orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation‐mediated bubble growth, not by hydroxyl radical release or shear stress as often reported. [ABSTRACT FROM AUTHOR]

• Model for prediction of volumetric power in put in microtiter plates established. • Geometrical relationship for scale up/down from microtiter plates established. • Model based scale down from continuous bench scale stirred reactor to microscale. • Scale up from microscale to pilot scale for buffer preparation proved. • Direct scale up of mixing from microscale to stirred tank reactors by factor 50.000. Many unit operations in bioprocessing engineering rely on adequate mixing. During process development, scale-up of unit operations is a critical, often laborious multistep task. Volumetric power input is used as one scale-up criteria for operations in stirred systems. While for stirred tank reactors the volumetric power input can be calculated from the Power Number Ne or extracted from literature, currently microtiter plates (MTPs) require experimental determination of power input for each process condition and geometry. We established data-driven models to predict the volumetric power input in shaken 24-, and 96-well MTPs without further experiments. The models were then applied to predict process conditions for the efficient solubilization of buffer components. Dissolution kinetics obtained in a continuous 10 L bench-scale system with baffles and a Rushton turbine were similar to those determined in 96-well MTPs. Combining the developed model with a geometrical relationship enables the prediction of operating conditions for mixing in a stirred tank reactor up to 10 L from shaken MTPs with 200 µL working volume. This granted a scale-up factor of 50.000, leading to a significant reduction of material and time consumption during process development and makes prediction of conditions more robust compared to the current approaches. [Display omitted] [ABSTRACT FROM AUTHOR]

Pre-packed small scale chromatography columns are increasingly used for process development, for determination of design space in bioprocess development, and for post-licence process verifications. The packing quality of 30,000 pre-packed columns delivered to customers over a period 10 years has been analyzed by advanced statistical tools. First, the data were extracted and checked for inconsistencies, and then were tabulated and made ready for statistical processing using the programming language Perl ( https://www.perl.org/ ) and the statistical computing environment R ( https://www.r-project.org/ ). Reduced HETP and asymmetry were plotted over time to obtain a trend of packing quality over 10 years. The obtained data were used as a visualized coefficient of variation analysis (VCVA), a process that has often been applied in other industries such as semiconductor manufacturing. A typical fluctuation of reduced HETP was seen. A Tsunami effect in manufacturing, the effect of propagation of manufacturing deviations leading to out-of-specification products, was not observed with these pre-packed columns. Principal component analysis (PCA) showed that all packing materials cluster. Our data analysis showed that the current commercially available chromatography media used for biopharmaceutical manufacturing can be reproducibly and uniformly packed in polymer-based chromatography columns, which are designed for ready-to-use purposes. Although the number of packed columns has quadrupled over one decade the packing quality has remained stable. [ABSTRACT FROM AUTHOR]

Risk assessment is an integral aspect of the Quality-by-Design strategy to identify potential obstacles at every stage of biopharmaceutical production, from process development to tech transfer. We explored flow process chart, root cause analysis, and failure mode and effects analysis, to assess the scale-up of bacterial cell disruption and its influence on centrifugation and filtration steps. The Ishikawa diagram suggests that data on the impact of homogenizer valve design on product release, impurity profile, particle size distribution, viscosity, and dsDNA fragment size are missing which were collected experimentally for this study. Cell lysates from micro-, lab- and pilot scales cell disruption were analyzed for the above-mentioned parameters. Process parameters affecting these output parameters were identified on each individual scale. Cell disruption on the micro scale was performed in a bead mill. High pressure homogenization was used on lab- and pilot scales. Cell disintegration by bead milling delivers homogenates of product and impurity content comparable to those on bench scale but with 3-fold higher viscosity and significantly larger dsDNA fragments, 8.0 instead of 1.0 kbp, respectively. Miniaturized pressure flow curves identified dsDNA fragment sizes as critical for filter performance during clarification. Combining risk assessment, micro scale cell disintegration and bench scale pressure flow curves allows for selective and efficient process development, and scale up for primary recovery steps. • Risk assessment for primary recovery of biopharmaceuticals recombinantly produced in Escherichia coli. • Comparative analysis between microscale bead milling and high-pressure homogenization conducted. • Importance of risk assessment and emphasizing its utility from development to scale-up and tech transfer. • Selected microscale experiments and miniaturized pressure flow curves allow efficient process development and scale up. [ABSTRACT FROM AUTHOR]

Background and purpose: SARS‐CoV2 vaccination is recommended for patients with multiple sclerosis (pwMS), but response may be limited by disease‐modifying‐treatments (DMTs). The aim of this study was to compare the rates of humoral immune response and safety of SARS‐CoV‐2 vaccines in pwMS and healthy controls (HCs). Methods: In this multicenter prospective study on 456 pwMS and 116 HCs, SARS‐CoV‐2‐IgG response was measured 3 months after the first vaccine dose. The primary endpoint was defined as proportion of patients developing antibodies (seroconversion). Secondary endpoints included antibody level, safety and efficacy. Results: Compared to 97.4% in HCs, seroconversion occurred in 96.7% (88/91) untreated pwMS, 97.1% of patients (135/139) on immunomodulatory DMTs and 61.1% (138/226; p < 0.001) on immunosuppressive DMTs. Seroconversion was lowest in patients on antiCD20 monoclonal antibodies (CD20 mAbs; 52.6%) followed by sphingosine‐1‐phosphate‐receptor‐modulators (S1PMs; 63.6%). In the S1PM subgroup, seroconversion increased with lymphocyte count (odds ratio [OR] 1.31 per 0.1 G/L; p = 0.035). In pwMS on CD20 mAbs, B‐cell depletion decreased seroconversion (OR 0.52; p = 0.038), whereas time since last DMT did not. Safety of SARS‐CoV‐2 vaccines in pwMS was excellent. Conclusions: Humoral response to SARS‐CoV2 vaccines in pwMS is generally excellent. While reduced by immunosuppressive DMTs, most importantly by B‐cell‐depleting CD20 mAbs and S1PMs, seroconversion is still expected in the majority of patients. SARS‐CoV2 vaccination should be offered to every MS patient. [ABSTRACT FROM AUTHOR]

Abstract: Matrix-assisted refolding is an excellent technique for performing refolding of recombinant proteins at high concentration because aggregation during refolding is partially suppressed. The autoprotease Npro and its engineered mutant EDDIE can be efficiently refolded on cation-exchangers. In the current work, denatured fusion proteins were loaded at different column saturations (5 and 50mgmL−1 gel), and refolding and self-cleavage were initiated during elution. The contact time of the protein with the matrix significantly influenced the refolding rate and yield. On POROS 50 HS, the refolding rate was comparable to a batch refolding process, but yield was substantially higher; at a protein concentration of 1.55mgmL−1, an almost complete conversion was observed. With Capto S, the rate of self-cleavage increased by a factor of 20 while yield was slightly reduced. Processing the autoprotease fusion protein on Capto S at a high protein loading of 50mgmL−1 gel and short contact time (0.5h) yielded the highest productivity. [ABSTRACT FROM AUTHOR]

The land area covered by freely available very high-resolution (VHR) imagery has grown dramatically over recent years, which has considerable relevance for forest observation and monitoring. For example, it is possible to recognize and extract a number of features related to forest type, forest management, degradation and disturbance using VHR imagery. Moreover, time series of medium-to-high-resolution imagery such as MODIS, Landsat or Sentinel has allowed for monitoring of parameters related to forest cover change. Although automatic classification is used regularly to monitor forests using medium-resolution imagery, VHR imagery and changes in web-based technology have opened up new possibilities for the role of visual interpretation in forest observation. Visual interpretation of VHR is typically employed to provide training and/or validation data for other remote sensing-based techniques or to derive statistics directly on forest cover/forest cover change over large regions. Hence, this paper reviews the state of the art in tools designed for visual interpretation of VHR, including Geo-Wiki, LACO-Wiki and Collect Earth as well as issues related to interpretation of VHR imagery and approaches to quality assurance. We have also listed a number of success stories where visual interpretation plays a crucial role, including a global forest mask harmonized with FAO FRA country statistics; estimation of dryland forest area; quantification of deforestation; national reporting to the UNFCCC; and drivers of forest change. [ABSTRACT FROM AUTHOR]

Abstract A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/μg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 μg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/μg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV 280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria. Highlights • Impurity ratios in the eluate fractions differ depending on the capture method. • Clearance of host cell proteins and dsDNA under limit of quantification. • Ion exchange chromatographic step fulfills criteria for clinical application. • Two step purification allows direct processing of clarified E. coli homogenate. • Hydrophobic interaction method enables endotoxin clearance without product loss. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fast filter blocking of cell lysates on sterile filter due to genomic dsDNA. • Genomic dsDNA forms complex with positively charged molecules/proteins. • Heterogeneous complex can be dissolved by increasing ionic strength. • Endonuclease treatment enhances filter capacity due to dsDNA fragmentation. • Filter blocking phenomenon observed for different host cell/POI combinations. During biopharmaceutical production, efficient removal of impurities via filtration is essential. It is desirable to achieve high filter capacities along with a high degree of clearance. However, the complexity of bacterial cell lysates makes it difficult to properly estimate the filtration behavior. Here we investigated how host cell-derived impurities impacted the performance of commercially available sterile filters, with a focus on characterizing the dsDNA-induced decrease of filter capacity. Pressure flow curves at a constant flow, combined with particle analytics (e.g., nanoparticle-tracking analysis), indirectly revealed that dsDNA had a major influence on rapid membrane fouling. The observed phenomenon was more pronounced when using filters with small pore sizes (<0.2 µm). Filter capacity could be substantially increased by reducing the size of the present dsDNA fragments, or by decreasing the potential for dsDNA-driven electrostatic interactions with positively charged molecules. In general, our present findings highlight the importance of monitoring dsDNA even during the primary recovery of proteins in cell lysates. [ABSTRACT FROM AUTHOR]

Escherichia colistores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a “design of experiments” study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples. [ABSTRACT FROM PUBLISHER]

Cell disruption is crucial during recovery of biopharmaceuticals overexpressed in E. coli , which tend to be produced intracellularly as insoluble inclusion bodies. Miniaturized high-throughput systems can accelerate the laborious downstream protocol for such biopharmaceuticals and enable integrated process-development. A fast and robust cell disruption method reflecting the protein and impurity profile of homogenates obtained by large-scale methods is required for such an approach. We established a miniaturized bead mill for parallel mechanical cell disruption at the microscale. Its total protein and impurity release, protein pattern, and particle size distribution were compared to results from microscale enzymatic digestion and referred to laboratory-scale high-pressure homogenization. Bead mill disruption led to equivalent protein and impurity release as well as to the same particle size profile as the large-scale reference. In contrast, lysates obtained by enzymatic digestion contained only 30–47% of overall protein, 17% of dsDNA, and 7–10% of endotoxin compared to those obtained by high-pressure homogenization; also larger debris was present in lysates after enzymatic digestion. The established method is fast, efficient, robust and comparable to current large-scale standards, allowing for parallelization of experiments. Thus, it is the method of choice for rapid integrated process development at the microscale. [ABSTRACT FROM AUTHOR]

Citizens are increasingly becoming involved in data collection, whether for scientific purposes, to carry out micro-tasks, or as part of a gamified, competitive application. In some cases, volunteered data collection overlaps with that of mapping agencies, e.g., the citizen-based mapping of features in OpenStreetMap. LUCAS (Land Use Cover Area frame Sample) is one source of authoritative in-situ data that are collected every three years across EU member countries by trained personnel at a considerable cost to taxpayers. This paper presents a mobile application called FotoQuest Austria, which involves citizens in the crowdsourcing of in-situ land cover and land use data, including at locations of LUCAS sample points in Austria. The results from a campaign run during the summer of 2015 suggest that land cover and land use can be crowdsourced using a simple protocol based on LUCAS. This has implications for remote sensing as this data stream represents a new source of potentially valuable information for the training and validation of land cover maps as well as for area estimation purposes. Although the most detailed and challenging classes were more difficult for untrained citizens to recognize, the agreement between the crowdsourced data and the LUCAS data for basic high level land cover and land use classes in homogeneous areas (ca. 80%) shows clear potential. Recommendations for how to further improve the quality of the crowdsourced data in the context of LUCAS are provided so that this source of data might one day be accurate enough for land cover mapping purposes. [ABSTRACT FROM AUTHOR]

N pro fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli , was further developed by splitting the autoprotease N pro into two fragments to generate a functional complementation system. The size of the expression tag is thus reduced from 168 to 58 amino acids, so by 66%. Upon complementation of the fragments auto-proteolytic activity is restored. This process has been shown for three model proteins of different size, a short 16 aa-peptide, MCP-1, and lysozyme. Moreover, the complementation was still functional after immobilization of the N-terminal fragment to a solid support which enables recycling of the immobilized fragment. This strategy enhances overall productivity of N pro Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the N pro complementation system has, depending on the size of the target molecule, potential to increase the productivity up to 4 fold for batch refolding and even more for on-column refolding strategies by the proven possibility of regeneration of the immobilized fragment. [ABSTRACT FROM AUTHOR]

This study analyzed the efficiency of protein refolding with crossflow ultrafiltration for two distinct types of model proteins: denatured bovine α-lactalbumin and a fusion protein that comprised green fluorescent protein coupled with an engineered N pro autoprotease tag. A mechanistic model of the process dynamics that accounted for the refolding kinetics was developed and verified by comparing with experimental data. The model was used to quantify refolding performance under various operating conditions, including different denaturant removal rates, refolding durations, and protein concentrations. The performance of ultrafiltration with the fed-batch and stepwise operating modes for denaturant removal was analyzed and compared to the performance of the batch dilution method. Performance was evaluated in terms of productivity, yield, and buffer consumption. The superiority of one refolding method over another depended on the protein system. When a slow reduction in denaturant concentration suppressed protein aggregation, the best performance was achieved with the ultrafiltration system. For example, α-lactalbumin refolding with ultrafiltration achieved several-fold higher productivity and lower buffer consumption compared to refolding with the batch dilution method. A further reduction in buffer consumption was achieved with permeate recycling. Conversely, when rapid dilution of the denaturant was most efficient, a combination of batch dilution and ultrafiltration was recommended. The latter reduced the buffer consumption with permeate recycling; e.g., over 80% of the refolding buffer could be recycled during fusion protein refolding. [ABSTRACT FROM AUTHOR]

A number of global and regional maps of forest extent are available, but when compared spatially, there are large areas of disagreement. Moreover, there is currently no global forest map that is consistent with forest statistics from FAO (Food and Agriculture Organization of the United Nations). By combining these diverse data sources into a single forest cover product, it is possible to produce a global forest map that is more accurate than the individual input layers and to produce a map that is consistent with FAO statistics. In this paper we applied geographically weighted regression (GWR) to integrate eight different forest products into three global hybrid forest cover maps at a 1 km resolution for the reference year 2000. Input products included global land cover and forest maps at varying resolutions from 30 m to 1 km, mosaics of regional land use/land cover products where available, and the MODIS Vegetation Continuous Fields product. The GWR was trained using crowdsourced data collected via the Geo-Wiki platform and the hybrid maps were then validated using an independent dataset collected via the same system. Three different hybrid maps were produced: two consistent with FAO statistics, one at the country and one at the regional level, and a “best guess” forest cover map that is independent of FAO. Independent validation showed that the “best guess” hybrid product had the best overall accuracy of 93% when compared with the individual input datasets. The global hybrid forest cover maps are available at http://biomass.geo-wiki.org . [ABSTRACT FROM AUTHOR]

• Adsorption of hFGF2 on Capto S is driven by significant contribution of solid diffusion. • 40-fold reduction of mass transfer for hFGF2 in clarified homogenate compared to pure protein solution. • Increased viscosity and reduction of q max are responsible for reduced mass transfer. • Prediction of BTCs can be obtained with pore diffusion as well as solid diffusion model. • Comparison of adsorption kinetics of hFGF2 on grafted and macro-porous media. Elucidation of intraparticle mass transfer mechanisms in protein chromatography is essential for process design. This study investigates the differences of adsorption and diffusion parameters of basic human fibroblast factor 2 (hFGF2) in a simple (purified) and a complex (clarified homogenate) feed solution on the grafted agarose-based strong cation exchanger Capto S. Microscopic investigations using confocal laser scanning microscopy revealed slower intraparticle diffusion of hFGF2 in the clarified homogenate compared to purified hFGF2. Diffusive adsorption fronts indicated a strong contribution of solid diffusion to the overall mass transfer flux. Protein adsorption methods such as batch uptake and shallow bed as well as breakthrough curve experiments confirmed a 40-fold reduction of the mass transfer flux for hFGF2 in the homogenate compared to pure hFGF2. The slower mass transfer was induced by components of the clarified homogenate. Essentially, the increased dynamic viscosity caused by a higher concentration of dsDNA and membrane lipids in the clarified homogenate contributed to this decrease in mass transfer. Moreover, binding capacity for hFGF2 was much lower in the clarified homogenate and substantially decreased the adsorbed phase driving force for mass transfer. [ABSTRACT FROM AUTHOR]

Highlights: [•] Refolding kinetics of fusion proteins is predictable from μ- to laboratory scale. [•] Surface properties/areas of microtiter plates are relevant for refolding yields. [•] ATR-FTIR enables inline monitoring of protein refolding processes. [•] Scale Up and inline monitoring of protein refolding – important steps toward PAT. [Copyright &y& Elsevier]

Abstract: Inclusion bodies are a source of large quantities of protein with high purity over-expressed in Escherichia coli. Efficient solubilization is the initial step in recovering the protein. We visualized solubilization using high-resolution light and transmission electron microscopy over time and the most common solubilization agents, urea and guanidine hydrochloride, as well as sodium hydroxide. Upon solubilization, the inclusion bodies were penetrated by the solubilization agent, shrinking the densely packed cores as the protein diffused out. Despite the complex process, solubilization could be described by a homogeneous layer model with a high order of reaction. The solubilization of protein aggregates, such as inclusion bodies, does not follow the rules of ordinary solubilization processes, but is controlled by pore diffusion. Consequently, power input and mixing frequency is not relevant to the process. [Copyright &y& Elsevier]

• Model training was performed on a single membrane in a single experiment. • The presented hybrid models predict the performance of serial and parallel SPTFF. • The methodology enables SPTFF process design based on minimal product wastage. • A Digital Twin enables the comparison of SPTFF with batch and fed-batch TFF performance. Single-pass tangential flow filtration (SPTFF) is a promising and increasingly important tool for continuous biomanufacturing and intensified bioprocessing. SPTFF operates at high product concentrations often limiting the number of process development experiments and therefore process understanding. We developed hybrid model structures to predict the concentration performance of SPTFF in serial and parallel mode with up to three membranes at various pressures, feed flows and protein concentrations. A single experiment to gather the training data for the model was performed in batch mode, significantly reducing the required amount of protein to set up the ideal process design. The hybrid model's excellent interpolation properties further reduce the duration of the training experiments. A set of mass balances controls the model variation of the two SPTFF modes as part of the hybrid model requiring only one training set for both modes. The hybrid model can be used as a Digital Twin to perform in-silico process simulations to gain in-depth process knowledge and enable model-based process control. The Digital Twin facilitates faster process transfer from batch to continuous and gives predictions on how different SPTFF modes perform under changing process conditions. This renders it a valuable tool for efficient process development and process transfer. [ABSTRACT FROM AUTHOR]

• Basic fibroblast growth factor (FGF-2) overexpressed in E. coli degrades in capture eluates during storage at 4 °C. • Hydrophobic interaction chromatography separates such truncated variants of high similarity from full-length FGF-2. • Variants are quantified by an ion exchange – HPLC method using a highly linear pH gradient. • HCP and dsDNA are depleted below the limits of quantification of 1 and 2 ng/mL, respectively. • Endotoxin content is reduced to 0.5 EU/µg FGF-2 which is below the recommended endotoxin level for biopharmaceuticals. Basic fibroblast growth factor (FGF-2) is a labile protein which degrades over time or forms aggregates. A purification process combining a cation ion exchange chromatography (CIEX) capture step with a polishing step by hydrophobic interaction chromatography (HIC) efficiently depleted process-related impurities such as host cell proteins (HCPs), dsDNA and endotoxins from the clarified E. coli homogenate containing the overexpressed FGF-2. Fresh homogenates did not contain verifiable amounts of process-related impurities such as degraded product variants. However, the storage of the capture eluates for longer than two weeks at 4 °C led to product degradation up to 49%. Mass spectrometry showed that the truncated FGF-2 was devoid of the nine N-terminal amino acids. Removal of such product-related impurities is included in state of the art purifications because these byproducts bear the risk of having different biological activities. Therefore, the established purification process has been evaluated for its potential to deplete such truncated FGF-2 variants. The HIC polishing step using Toyopearl® Hexyl-650C with a linear salt gradient over 4 column volumes from 1.65 M trisodium citrate to 0 M separated this variant from the full-length FGF-2. The truncated form eluted before the full length product due to the loss of 9 hydrophobic amino acids. The developed ion exchange HPLC method enabled us to quantify the truncated variant and full-length FGF-2. A purity of > 98% full-length FGF-2 with a step yield of up 86% is achieved. As long as the degree of truncation does not exceed 25% of the stored eluates, the purification process delivers the product in the desired quality of > 98% full-length FGF-2, HCP content below 10 ppm/dose, dsDNA content below 10 ng/mL/dose and endotoxin level below 400 EU/dose. This purification sequence allowed the production of FGF-2 which meets distinctive quality standards defined by regulatory bodies with respect to the depletion of both - process- and product-related impurities. [ABSTRACT FROM AUTHOR]

• Model-based approach for scaling up chromatographic capture step was developed. • Model system for the study was fibroblast growth factor protein from an E. coli homogenate. • Dynamic model was formulated which was used to scale up the operation. • Effects of feed viscosity and competitive adsorption of impurities were accounted for. A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume. [ABSTRACT FROM AUTHOR]