The purification of enveloped viruses, such as baculovirus, remains a significant challenge in bioprocessing. This study explores various factors affecting recombinant baculovirus purification using functionalized non-woven fibers in bind-elute mode, including contact time during adsorption, buffer pH and conductivity and ligand density and chemistry. Non-woven fibers with grafted quaternary ammonium groups demonstrated flow ratedependent binding behavior, with mass transfer limitation observed for larger particles. Reducing the residence time decreased the binding for capacity particles due to mass transfer limitation. Adjusting the pH and conductivity of the equilibration, wash and elution buffers changed the elution profile; however infectious virus recovery could not be improved. Alternatively, reduction of the binding strength was attempted by reducing the ligand density. Surprisingly, fibers with reduced quaternary ammonium ligand density showed lower overall recovery of dsDNA, protein, total and infectious particles, suggesting unspecific binding interactions with the fiber backbone. Finally, a cation exchange fiber with carboxylic acid ligands was used and improved the total infectious virus recovery to 30%, with a yield of 26% in the main product fraction, doubling the results achieved with anion exchange fibers. This suggests that weaker binding interactions help maintain baculovirus infectivity. These findings highlight the potential of non-woven fiber materials for the purification of enveloped viruses such as baculovirus and provide strategies to further enhance recovery and preserve viral infectivity.

Techno-economic analysis (TEA) and life-cycle assessment (LCA) are essential tools for evaluating manufacturing processes, but the use of proprietary software creates barriers to accessibility and reproducibility. We present the BioProcessNexus, an open-source platform that democratizes access to process modeling through surrogate models trained on Monte Carlo data from proprietary TEA software. The platform facilitates model generation, analysis, and optimization while promoting standardization and collaboration across the scientific community. We demonstrate BioProcessNexus's capabilities through a comprehensive analysis of enzymatic PET recycling, comparing three surrogate modeling types: partial least squares, random forest, and Gaussian Process regression. Our analysis revealed that enzymatic PET recycling faces economic challenges, with a unit production cost of $1.74 kg-1 TPA and an expected negative gross margin of -49.5 %. Sensitivity analysis identified feedstock cost and purification strategy as key areas for optimization. BioProcessNexus enables accessible, reproducible process modeling even when proprietary software was used for the initial model development. This approach advances the open innovation initiative and promotes transparent scientific collaboration while reducing barriers to advanced process modeling and optimization techniques. In this article, we will (i) introduce the BioProcessNexus platform and (ii) showcase a use case of an enzymatic PET recycling process.

The European Union’s Green Deal emphasizes life science and biotechnology as key drivers for achieving a circular economy. However, the prevalent use of non-sustainable single-use plastics derived from petrochemical sources in life science research and development contradicts this ecological goal. This study presents a viable alternative through on-site sterile 3D printing of single-use plastics using poly(lactic acid) (PLA). The sterile printing of PLA demonstrates a substantial reduction in CO2eq emissions per shake flask, decreasing from 260 g CO2eq to 145 g CO2eq. Suitability for cell culture was demonstrated for sterile printed PLA, autoclaved high-temperature PLA and for suspension cell cultures in shake flasks using CHO and insect cells as well as adherent Vero cell cultures in cell culture wells. Further optimization of sustainable shake flasks is achieved by utilizing recycled PLA, resulting in a remarkable reduction to 73 g CO2eq, constituting a 72% decrease in CO2eq emissions compared to conventional single-use plastics. Moreover, by employing geometric optimization to minimize material usage, emissions can be further reduced to 44 g CO2eq, representing an 83% reduction in CO2eq emissions. Anticipated advancements in PLA production suggest future carbon net negative PLA production, potentially achieving zero carbon emissions for single-use plastics using PLA. The exceptionally cost-effective nature of 3D printed single-use plastics, coupled with negligible capital costs for implementation, positions sterile 3D printing as a practical solution aligned with the 2030 Green Deal goals. Moreover, ongoing improvements in bioplastic production underscore the feasibility of meeting the 2050 targets of carbon neutrality for single-use plastics in life science research and development.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal microscopy, but also as a tracer for determination of residence time distribution in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. The site occupancy of two labeled antibodies, subclass 1 and 2 conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the IgG1 and IgG2 molecules, in positions 188 and 189, respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling despite the secondary region responsible for binding to the protein A was partly labeled. Fluorescent-labeled antibodies are a good compromise as an inert tracer in protein A affinity chromatography because they are much cheaper than isotope-labeled antibodies.

Regulatory authorities in biopharmaceutical industry emphasize process design by process understanding but applicable tools that are easy to implement are still missing. Soft sensors are a promising tool for the implementation of the Quality by Design (QbD) approach and Process Analytical Technology (PAT). In particular, the correlation between viable cell counting and oxygen consumption was investigated, but problems remained: Either the process had to be modified for excluding CO2 in pH control, or complex kLa models had to be set up for specific processes. In this work, a non-invasive soft sensor for simplified on-line cell counting based on dynamic oxygen uptake rate was developed with no need of special equipment. The dynamic oxygen uptake rates were determined by automated and periodic interruptions of gas supply in DASGIP® bioreactor systems, realized by a programmed Visual Basic script in the DASware® control software. With off-line cell counting, the two parameters were correlated based on linear regression and led to a robust model with a correlation coefficient of 0.92. Avoidance of oxygen starvation was achieved by gas flow reactivation at a certain minimum dissolved oxygen concentration. The soft sensor model was established in the exponential growth phase of a Chinese Hamster Ovary fed-batch process. Control studies showed no impact on cell growth by the discontinuous gas supply. This soft sensor is the first to be presented that does not require any specialized additional equipment as the methodology relies solely on the direct measurement of oxygen consumed by the cells in the bioreactor.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Virus-like particle vaccines have emerged as a promising and innovative approach to combat infectious diseases. Despite their immense potential as platform technology, challenges related to their production process need to be addressed to harness their full capabilities and make them widely assessible for human application.

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.

The main objectives of bioprocesses are to reliably deliver drugs in a relatively short time frame with high quality within a tight regulatory framework. Bioprocesses are highly complex, the level of automation is moderate, and there is constant pressure to improve efficiency and costs. In addition, climate change and resource scarcity mandate a reduction in the environmental footprint of bioprocesses and production facilities. In the biopharmaceutical industry, two extreme production scenarios are applied: a fully disposable factory with the characteristics of full flexibility and speed, or a fixed large-scale plant with high capacity. Forward-looking solutions and ideas will be discussed how to combine new processes and environmental friendliness for the benefit of the patient, security of supply and profitability. The concept will be extended to large scale production of proteins for food and non-pharma applications, e.g., in material science and a roadmap towards a future plant will be laid out.

3D printing has become widespread for the manufacture of parts in various industries and enabled radically new designs. This trend has not spread to bioprocess development yet, due to a lack of material suitable for the current workflow, including sterilization by autoclaving. This work demonstrates that commercially available heat temperature stable poly-lactic acid (PLA) can be used to easily manufacture novel bioreactor vessels with included features like harvest tubes and 3D printed spargers. Temperature responsiveness was tested for PLA, temperature stable PLA (PLA-HP) and glass for temperatures relevant for insect and mammalian cell culture, including temperature shifts within the process. Stability at 27 °C and 37 °C as well as temperature shifts to 22 °C and 32 °C showed acceptable performance with slightly higher temperature overshoot for 3D printed vessels. A stable temperature is reached after 2h for PLA, 3h for PLA-HP and 1h for glass reactors. Temperature can be maintained with a fluctuation of 0.1 °C for all materials. A 3D printed sparger design directly integrated into the vessel wall and bottom was tested under three different conditions (0.3 SLPH and 27 °C, 3 SLPH and 37 °C and 13 SLPH and 37 °C). The 3D printed sparger showed a better kLa than the L-Sparger with more pronounced differences for higher flowrates. An insect cell culture run in the novel vessel exhibited the same growth behavior as that in standard glass vessels, reaching the same maximum cell concentration. Being 3D printed from biodegradable materials, these bioreactors offer design flexibility for novel bioreactor formats. Additionally, their autoclavability allows seamless integration into standard workflows.

Water for injection (WFI) for biopharmaceuticals is coming into focus considering the sustainability goals of society. WFI production consumes excessive amounts of water and energy. Strategies to reduce WFI energy cost include cold WFI production or reduction of buffer volumes used in the process. A suggested alternative with proposed saving of up to 90 % was the recycling of buffers, potentially across multiple unit operations. In this work a risk-based assessment of the idea is made to quantify the potential savings. All streams of an antibody purification were classified according to their potential risk. This analysis showed that the process does not produce sufficient recyclable outlet streams to cover all replaceable inlet streams. From a potentially replaceable 42 % of inlet buffers only a total of 23 % can be replaced, even if cross-batch recycling is allowed. If this is not allowed, the savings drop down to 14 %. While this saves some water and energy, it shows that making full use of the theoretical potential is impossible, and 90 % reduction is never achievable when risk-aware design of recycling across unit operations is used. A universal risk-based assessment is important and can build the rationale to convince regulatory authorities in case of implementation of such a strategy.

The economic benefits of continuous PEG precipitation of antibodies compared to continuous capture by protein A affinity chromatography have been always lowered by the excess amount of material needed for the process. PEG is added as a concentrated stock solution and therefore liquid handling is increased during this step. To fully exploit the benefits of PEG precipitation, the precipitant must be added in solid form. We used an in-line feeding device with a screw conveyor delivery system for the continuous addition of PEG6000 in a powder form. The powder feeding device was connected to a tubular reactor where the precipitation occurs. Protein precipitation was continuously performed for 4 h. A yield of 76 % and 79 % with a purity of 98 % was achieved. The total cost of goods and the environmental footprint were compared with typical chromatography-based purification methods; batch and continuous periodic countercurrent protein A affinity chromatography with four columns. Solid PEG precipitation showed a remarkable reduction in water consumption and equipment size, reducing production costs by 45 % compared to liquid PEG and 53 % cheaper than Protein A periodic counter-current chromatography. Process mass intensity was reduced by 55 % and carbon emissions by 60 %. The reduction of water by the direct addition of PEG also impacted the environmental footprint and process costs. This is an attractive approach for a continuous capture step yielding an uninterrupted mass flow of the product and will pave the way for PEG precipitation as a capture step.

The preparation of buffer solutions used in the biopharmaceutical industry is typically performed manually by the addition of one or multiple buffering reagents to water. Recently, the adaptation of powder feeders for continuous solid feeding was demonstrated for continuous buffer preparation. However, the intrinsic charac-teristics of powders can change the stability of the process, due to the hygroscopic nature of some substances and humidity-induced caking and compaction behavior, but there is no simple and easy methodology available for predicting this behavior for buffer species. To predict which buffering reagents are suitable without special precautions and investigate their behavior, force displacement measurements were conducted with a customized rheometer over 18 h. While most of the eight investigated buffering reagents indicated uniform compaction, especially sodium acetate and dipotassium hydrogen phosphate (K2HPO4) showed a significant increase in yield stress after 2 h. Experiments conducted with a 3D printed miniaturized screw conveyor confirmed the increased yield stress measurements by visible compaction and failure of the feeding. By taking additional precautions and adjusting the design of the hopper, we demonstrated a highly linear profile of all buffering reagents over a duration of 12 and 24 h. We showed that force displacement measurements accurately predict the behavior of buffer components in continuous feeding devices for continuous buffer preparation and are a valuable tool to identify buffer components that need special precautions. Stable, precise feeding of all tested buffer components was demonstrated, highlighting the importance of identifying buffers that need a specialized setup with a rapid methodology.

3D printing represents a democratization of manufacturing processes, and inexpensive 3D printed parts for cell culture have been tested as replacements for single-use plastics currently unavailable due to worldwide supply chain issues. In addition, such distributed manufacturing of cell culture laboratory materials helps remote areas and developing countries with limited resources. HEK293 cells were used to test printed shake flasks for cell culture applications and their ease of manufacture. Recorded growth curves showed that renewable biodegradable poly(lactic acid) (PLA) thermoplastic is an excellent and economical replacement for single-use plastic shake flasks, which have shipment lead times during pandemic situations or other supply chain disruptions of over 6 months. With a price of 0.60 € in materials, and printing machines with prices lower than one box of single-use pre-sterilized plastic shake flasks (<350€), the use of PLA is very affordable. Low-cost photopolymerization resins were also tested, but the inherent cytotoxicity of these materials prevented cell growth. This was also true for plant-based resins marketed as having low volatile organic compounds (VOC). Treatment of parts to reduce VOC content was partially successful, but not sufficient to sustain prolonged cell growth. A high-cost medical device IIa-class material showed no improved cell growth. Nevertheless, with PLA a low-cost printing material was identified and the use as cell culture compatible material was demonstrated, providing low-cost supply chain independence. In the future, the printing of pilot-scale bioreactors with PLA as a green sustainable material at the point of its use will be possible.

BACKGROUND A major improvement in biomanufacturing will arise with the transition from batch processing to continuous processing. Two important challenges to address in this change are batch definition and the ability to trace raw material through the process. RESULTS We used an established simulation of a process train to compare the conventional batch definition based on a fixed time to a new batch definition method based on the greatest common divisor (GCD) of the time period of the unit operations. We successfully demonstrated that, by using the new concept based on GCD, we will have a constant periodic concentration of product. With this basis, we can define batches in a continuous process, which will lead to higher control over the process, and we will be able to trace the material through the process. CONCLUSION We achieved better control over the process using the batch definition based on the GCD method. In comparison to collecting the outlet products over arbitrary hours or days, collecting the product based on a section using the GCD method meets the criteria for knowing the residence-time distribution of the process, as advised by regulatory authorities. This method can be used in a continuous process or a hybrid process in which there are only a few continuous unit operations along with batch process operations. (c) 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

BACKGROUND: The failure rates and the scheduling of bioprocesses have a substantial impact on process performance and economics but are often overlooked and neglected. Integrated continuous biomanufacturing is more flexible in respect to scheduling and the impact of failures can be reduced by appropriate scheduling and lot definitions. RESULTS: In this work, we used a Monte Carlo approach on an integrated continuous biomanufacturing process with varying daily failure rates in the upstream and scheduling scenarios for seed fermentation (N-1 stage) to quantify the impact on the actual productive uptime of the integrated process. The optimum targeted production time in the continuous upstream ranges between 45 and 90 days depending on the daily failure rate and the lot definition used for the process. We showed that a minimal flexibility for planning of the seed fermentation is necessary to harvest the full potential of integrated continuous biomanufacturing. A comparison with batch manufacturing in the upstream processing showed a higher productive uptime for continuous biomanufacturing regardless of daily failure rates. Computation of productive uptime for different lot definitions showed that a daily lot definition only shows a loss of 3% to a maximum of 5% productivity, depending on the daily failure rate, compared to a real-time release approach. CONCLUSIONS: With this study, we provide a decision-making tool for the scheduling of upstream processes and implementation of integrated continuous biomanufacturing taking failure into account, showing the extent to which planning of flexibility and batch definitions influence the productivity of continuous integrated bioprocesses. (c) 2020 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A fast, automated and accurate millidevice for determination of the apparent solubility of proteins and impurities and different industrially relevant precipitating agents.

Precipitation has gained interest as alternative to the costly protein A chromatography for monoclonal antibody purification. Traditional precipitation processes are based on direct addition of precipitant in a single dose, with limited control on co-precipitation of impurities and not considering batch-to-batch variations. We propose a gradual dosage of polyethylene glycol to prevent co-precipitation and control resulting floc size. We used focused beam reflectance measurement to demonstrate that the PEG6000 dosage time and the final concentration significantly changes the particle size distribution (PSD). We demonstrated that gradual and stepwise precipitant addition was superior to conventional batch PEG precipitation, improving product yield and purity by a factor of 4 for HCP removal, for samples pre-treated with CaCl2 and caprylic acid. We studied the 3D structure of the precipitates by fractal dimension and showed that precipitates exhibited different compactness and density depending on the dosage time, resulting in different filterability in tangential flow filtration and depth filtration. To switch from batch to continuous PEG addition, the 3D structure of precipitates needs to be considered due to its high impact on the resulting process performance. Focused beam reflectance measurement (FBRM) and fractal dimension can be used to adjust the precipitation methodology to improve the product quality attributes and inform about the design of further purification steps.

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an "understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system." Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.

BACKGROUND The development of integrated continuous biomanufacturing processes faces a significant challenge when the parameters for the design of the process cannot be accurately estimated from those of batch experiments. Process design is even more challenging if the outcome of one unit operation highly influences the performance of the subsequent one, such as in harvesting of a precipitate by filtration. In the case of protein precipitation, results from the batch and continuous experiements deviate and their scale-down is limited. Microfluidics suffer from poor mixing characteristics. Thus, milliscale devices were developed to maintain mixing performance but at the expense of a slightly larger scale. RESULTS Milliscale devices were developed for the precipitation of antibodies in continuous tubular reactors to compare different dosage times in small scale. The reactors have multiple addition points for precipitant for the continuous, controlled and precise addition of the precipitating agent without valves. The designed devices have a narrow residence time distribution to achieve fast mixing and a small volume that reduces the time and cost for experiments tenfold. Milliscale devices were used to evaluate the most appropriate dosage time for protein precipitation, thus improving purity by a factor of 3 compared to single addition. The resulting filterability of precipitates in tangential flow filtration and depth filtration was improved. CONCLUSION Results demonstrated that multiple additions were more beneficial than single addition because they help to reduce pressure and increase filter capacity. Such devices can be used to determine and adjust the precipitation methodology to optimize floc formation and improve solid-liquid separation while reducing development time and cost. (c) 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).