Deep integration of low-cost liquid handling robots in an industrial pharmaceutical development environment.

The pharmaceutical industry is increasingly embracing laboratory automation to enhance experimental efficiency and operational resilience, particularly through the integration of automated liquid handlers (ALHs). This paper explores the integration of the low-cost Opentrons OT-2 liquid handling robot with F. Hoffmann-La Roche AG's in-house workflow orchestration software, AutoLab, to overcome barriers to lab automation. By leveraging the OT-2's development-oriented interfaces and AutoLab's modular architecture, we achieved a user-friendly, cost-efficient, and flexible automation solution that aligns with FAIR (findable, accessible, interoperable, reusable) data principles. We demonstrate an advanced workflow development methodology, utilizing the software architecture, that facilitates the creation of two flexible pipetting protocols and medium complexity assays. This deep integration approach diminishes the learning curve for novice users while simultaneously enhancing the overall efficiency and reliability of the experimental workflow. Our findings suggest that such integrations can significantly mitigate the challenges associated with lab automation, including cost, complexity, and adaptability, paving the way for more accessible and robust automated systems in pharmaceutical research.

Construction of a Calibration Curve for Lycopene on a Liquid-Handling Platform─Wider Lessons for the Development of Automated Dilution Protocols.

Liquid-handling is a fundamental operation in synthetic biology─all protocols involve one or more liquid-handling operations. It is, therefore, crucial that this step be carefully automated in order to unlock the benefits of automation (e.g., higher throughput, higher replicability). In the paper, we present a study, conducted at the London Biofoundry at SynbiCITE, that approaches liquid-handling and its reliable automation from the standpoint of the construction of the calibration curve for lycopene in dimethyl sulfoxide (DMSO). The study has important practical industrial applications (e.g., lycopene is a carotenoid of industrial interest, DMSO is a popular extractant). The study was also an effective testbed for the automation of liquid-handling. It necessitated the development of flexible liquid-handling methods, which can be generalizable to other automated applications. In addition, because lycopene/DMSO is a difficult mix, it was capable of revealing issues with automated liquid-handling protocols and stress-testing them. An important component of the study is the constraint that, due to the omnipresence of liquid-handling steps, errors should be controlled to a high standard. It is important to avoid such errors propagating to other parts of the protocol. To achieve this, a practical framework based on regression was developed and utilized throughout the study to identify, assess, and monitor transfer errors. The paper concludes with recommendations regarding automation of liquid-handling, which are applicable to a large set of applications (not just to complex liquids such as lycopene in DMSO or calibration curves).

Improvement of bioanalytical parameters through automation: suitability of a hand-like robotic system.

Commercial automation systems for small- and medium-sized laboratories, including research environments, are often complex to use. For liquid handling systems (LHS), development is required not only for the robot's movements but also for adapting the bioanalytical method to the automated system. This study investigates whether a more human-like automation strategy-using a robotic system (RS)-is more suitable for research laboratories than a professional automation approach utilizing a commercial automated LHS. We conducted a series of measurements for protein determination using a Bradford assay manually, with a fully automated LHS, and with our human-like RS. Although the hand-like RS approach requires more than twice the time of the LHS, it achieved the best standard deviation in this setup (RS = 0.5, manual = 0.71, LHS = 0.86). Due to the low limit of detection (LOD) and limit of quantification (LOQ), most protein samples could be quantified with the RS (samples below LOQ = 9.7%, LOD = 0.23; LOQ = 0.25) compared to manual (samples below LOQ = 28.8%, LOD = 0.24; LOQ = 0.26) and the LHS (samples below LOQ = 36.1%, LOD = 0.27; LOQ = 0.31). In another time-dependent enzymatic assay test, the RS achieved results comparable to the manual method and the LHS, although the required time could be a constraint for short incubation times. Our results demonstrate that a more hand-like automation system closely models the manual process, leading easier to accurate bioanalytical results. We conclude that such a system could be more suitable for typical research environments than a complex LHS.

Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1 ng/µL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Practical deployment of automation to expedite aqueous two-phase extraction.

The feasibility of bioprocess development relies heavily on the successful application of primary recovery and purification techniques. Aqueous two-phase extraction (ATPE) disrupts the definition of "unit operation" by serving as an integrative and intensive technique that combines different objectives such as the removal of biomass and integrated recovery and purification of the product of interest. The relative simplicity of processing large samples renders this technique an attractive alternative for industrial bioprocessing applications. However, process development is hindered by the lack of easily predictable partition behaviours, the elucidation of which necessitates a large number of experiments to be conducted. Liquid handling devices can assist to address this problem; however, they are configured to operate using low viscosity fluids such as water and water-based solutions as opposed to highly viscous polymeric solutions, which are typically required in ATPE. In this work, an automated high throughput ATPE process development framework is presented by constructing phase diagrams and identifying the binodal curves for PEG6000, PEG3000, and PEG2000. Models were built to determine viscosity- and volume-independent transfer parameters. The framework provided an appropriate strategy to develop a very precise and accurate operation by exploiting the relationship between different liquid transfer parameters and process error. Process accuracy, measured by mean absolute error, and device precision, evaluated by the coefficient of variation, were both shown to be affected by the mechanical properties, particularly viscosity, of the fluids employed. For PEG6000, the mean absolute error improved by six-fold (from 4.82% to 0.75%) and the coefficient of variation improved by three-fold (from 0.027 to 0.008) upon optimisation of the liquid transfer parameters accounting for the viscosity effect on the PEG-salt buffer utilising ATPE operations. As demonstrated here, automated liquid handling devices can serve to streamline process development for APTE enabling wide adoption of this technique in large scale bioprocess applications.

Anastrozole and Tamoxifen Impact on IgG Glycome Composition Dynamics in Luminal A and Luminal B Breast Cancers.

This study examines the intricate relationship between protein glycosylation dynamics and therapeutic responses in Luminal A and Luminal B breast cancer subtypes, focusing on anastrozole and tamoxifen impacts. The present methods inadequately monitor and forecast patient reactions to these treatments, leaving individuals vulnerable to the potential adverse effects of these medications. This research investigated glycan structural changes by following patients for up to 9 months. The protocol involved a series of automated steps including IgG isolation, protein denaturation, glycan labelling, purification, and final analysis using capillary gel electrophoresis with laser-induced fluorescence. The results suggested the significant role of glycan modifications in breast cancer progression, revealing distinctive trends in how anastrozole and tamoxifen elicit varied responses. The findings indicate anastrozole's association with reduced sialylation and increased core fucosylation, while tamoxifen correlated with increased sialylation and decreased core fucosylation. These observations suggest potential immunomodulatory effects: anastrozole possibly reducing inflammation and tamoxifen impacting immune-mediated cytotoxicity. This study strongly emphasizes the importance of considering specific glycan traits to comprehend the dynamic mechanisms driving breast cancer progression and the effects of targeted therapies. The nuanced differences observed in glycan modifications between these two treatments underscore the necessity for further comprehensive research aimed at thoroughly evaluating the long-term implications and therapeutic efficacy for breast cancer patients.

Development and evaluation of three automated media pooling and molecular diagnostic systems for the detection of SARS-CoV-2.

Pooled testing combined with molecular diagnostics for the detection of SARS-CoV-2 is a promising method that can increase testing capacities and save costs. However, pooled testing is also associated with the risks of decreased test sensitivity and specificity. To perform reliable pooled testing, we developed and validated three automated media pooling and molecular diagnostic systems. These pooling systems (geneLEAD-PS, Panther-PS, and Biomek-PS) comprised existing automated molecular detection platforms, corresponding automated media pooling devices, and laboratory information management systems. Analytical sensitivity analysis and mock sample evaluation were performed, and the obtained data were used to determine the sizes of the pool for the validation study. In the validation study, a total of 2,448, 3,228, and 6,420 upper respiratory samples were used for geneLEAD-PS, Panther-PS, and Biomek-PS, respectively, and the diagnostic performances were compared with the reference RT‒PCR assay. A pool size of 6 for geneLEAD-PS and a pool size of 4 for Panther-PS and Biomek-PS were selected for the validation studies. All three systems showed high positive percent agreement values of ≥90.5% and negative percent agreement values of ≥99.8% for any specimen type. Pooled testing resulted in a 65%-71% reduction in cost per sample. The testing capacities of geneLEAD-PS, Panther-PS, and Biomek-PS were 144 samples in 3 hours, 384 samples in 5.5 hours, and 376 samples in 4 hours, respectively. The developed pooling systems showed robust diagnostic performances and will increase the testing capacities of molecular diagnostic tests while saving costs and may contribute to infection control of COVID-19.IMPORTANCEDuring the COVID-19 pandemic, there have been surges in demand for accurate molecular diagnostic testing and laboratory supply shortages. Pooled testing combined with highly sensitive molecular testing, which entails mixing multiple samples as a single sample, is a promising approach to increase testing capacities while reducing the use of consumables. However, pooled testing is associated with risks that compromise diagnostic performance, such as false negatives due to dilution of positive samples or false positives due to cross-contamination. To perform reliable pooled testing, three different pooling systems (an automated pooling device, an automated molecular detection platform, and a laboratory information management system) were developed to accurately interpret pooled testing results. These three systems were validated using multiple clinical samples and showed high concordance with individual testing. The developed pooling systems will contribute to increasing reliable molecular testing capacities while using fewer consumables and saving costs.

TidyTron: Reducing lab waste using validated wash-and-reuse protocols for common plasticware in Opentrons OT-2 lab robots.

Every year biotechnology labs generate a combined total of ∼5.5 million tons of plastic waste. As the global bioeconomy expands, biofoundries will inevitably increase plastic consumption in-step with synthetic biology scaling. Decontamination and reuse of single-use plastics could increase sustainability and reduce recurring costs of biological research. However, throughput and variable cleaning quality make manual decontamination impractical in most instances. Automating single-use plastic cleaning with liquid handling robots makes decontamination more practical by offering higher throughput and consistent cleaning quality. However, open-source, validated protocols using low-cost lab robotics for effective decontamination of plasticware-facilitating safe reuse-have not yet been developed. Here we introduce and validate TidyTron: a library of protocols for cleaning micropipette tips and microtiter plates that are contaminated with DNA, E. coli, and S. cerevisiae. We tested a variety of cleaning solutions, contact times, and agitation methods with the aim of minimizing time and cost, while maximizing cleaning stringency and sustainability. We tested and validated these cleaning procedures by comparing fresh (first-time usage) versus cleaned tips and plates for contamination with cells, DNA, or cleaning solutions. We assessed contamination by measuring colony forming units by plating, PCR efficiency and DNA concentration by qPCR, and event counts and debris by flow cytometry. Open source cleaning protocols are available at https://github.com/PlantSynBioLab/TidyTron and hosted on a graphical user interface at https://jbryantvt.github.io/TidyTron/.

Can I benefit from laboratory automation? A decision aid for the successful introduction of laboratory automation.

The large volumes of samples to be analysed every day would be impossible to manage without laboratory automation. As laboratory procedures have progressed, so have the tasks of laboratory personnel. With this feature article, we would like to provide (bio)chemical practitioners with little or no knowledge of laboratory automation with a guide to help them decide whether to implement laboratory automation and find a suitable system. Especially in small- and medium-sized laboratories, operating a laboratory system means having bioanalytical knowledge, but also being familiar with the technical aspects. However, time, budget and personnel limitations allow little opportunity for personnel to get into the depths of laboratory automation. This includes not only the operation, but also the decision to purchase an automation system. Hasty investments do not only result in slow or non-existent cost recovery, but also occupy valuable laboratory space. We have structured the article as a decision tree, so readers can selectively read chapters that apply to their individual situation. This flexible approach allows each reader to create a personal reading flow tailored to their specific needs. We tried to address a variety of perspectives on the topic, including people who are either supportive or sceptical of laboratory automation, personnel who want or need to automate specific processes, those who are unsure whether to automate and those who are interested in automation but do not know which areas to prioritize. We also help to make a decision whether to reactivate or discard already existing and unused laboratory equipment.

ANDeS: An automated nanoliter droplet selection and collection device.

The Droplet Microarray (DMA) has emerged as a tool for high-throughput biological and chemical applications by enabling miniaturization and parallelization of experimental processes. Due to its ability to hold hundreds of nanoliter droplets, the DMA enables simple screening and analysis of samples such as cells and biomolecules. However, handling of nanoliter volumes poses a challenge, as manual recovery of nanoliter volumes is not feasible, and traditional laboratory equipment is not suited to work with such low volumes, and small array formats. To tackle this challenge, we developed the Automated Nanoliter Droplet Selection device (ANDeS), a robotic system for automated collection and transfer of nanoliter samples from DMA. ANDeS can automatically collect volumes from 50 to 350 nL from the flat surface of DMA with a movement accuracy of ±30 µm using fused silica capillaries. The system can automatically collect and transfer the droplets from DMA chip into other platforms, such as microtiter plates, conical tubes or another DMA. In addition, to ensure high throughput and multiple droplet collection, the uptake of multiple droplets within a single capillary, separated by air gaps to avoid mixing of the samples within the capillary, was optimized and demonstrated. This study shows the potential of ANDeS in laboratory applications by using it for the collection and transfer of biological samples, contained in nanoliter droplets, for subsequent analysis. The experimental results demonstrate the ability of ANDeS to increase the versatility of the DMA platform by allowing for automated retrieval of nanoliter samples from DMA, which was not possible manually on the level of individual droplets. Therefore, it widens the variety of analytical techniques that can be used for the analysis of content of individual droplets and experiments performed using DMA. Thus, ANDeS opens up opportunities to expand the development of miniaturized assays in such fields as cell screening, omics analysis and combinatorial chemistry.

Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing.

IMPORTANCE: Accurate and fast molecular testing is important for the diagnosis and control of COVID-19. During patient surges in the COVID-19 pandemic, laboratories were challenged by a higher demand for molecular testing under skilled staff shortages. We developed an automated multipurpose molecular testing system, named PCRpack, for the rapid, high-throughput testing of infectious pathogens, including SARS-CoV-2. The system is provided in an all-in-one package, including a liquid handling instrument, a laboratory information management system, and other materials needed for testing operation; is highly customizable; and is easily implemented. PCRpack showed robust liquid handling performance, high clinical diagnostic performance, a shorter turn-around time with minimal hands-on time, and a high testing capacity. These features contribute to the rapid implementation of the high-performance and high-throughput molecular testing environment at any phase of the pandemic caused by SARS-CoV-2 or future emerging pathogens.

Automated cell culture system for the production of cell aggregates with growth plate-like structure from induced pluripotent stem cells.

Programmable liquid handling devices for cell culture systems have dramatically enhanced scalability and reproducibility. We previously reported a protocol to produce cell aggregates demonstrating growth plate-like structures containing hypertrophic chondrocytes from human induced pluripotent stem cells (hiPSCs). To apply this protocol to large-scale drug screening for growth plate-related diseases, we adapted it to the automated cell culture system (ACCS) consisting of programmable liquid handling devices connected to CO2 incubators, a refrigerator, and labware feeders, designed for up to 4 batches with several cell culture plates culturing for several months. We developed a new program preparing culture media with growth factors at final concentration immediately before dispensing them to each well and precisely positioning the tip for the medium change without damaging cell aggregates. Using these programs on the ACCS, we successfully cultured cell aggregates for 56 days, only needing to replenish the labware, medium, and growth factors twice a week. The size of cell aggregates in each well increased over time, with low well-to-well variability. Cell aggregates on day 56 showed histochemical, immunohistochemical, and gene expression properties of growth plate-like structures containing hypertrophic chondrocytes, indicating proper quality as materials for basic research and drug discovery of growth plate related diseases. The established program will be a suitable reference for making programs of experiments requiring long term and complex culture procedures using ACCS.

PyLabRobot: An Open-Source, Hardware Agnostic Interface for Liquid-Handling Robots and Accessories.

Liquid handling robots are often limited by proprietary programming interfaces that are only compatible with a single type of robot and operating system, restricting method sharing and slowing development. Here we present PyLabRobot, an open-source, cross-platform Python interface capable of programming diverse liquid-handling robots, including Hamilton STARs, Tecan EVOs, and Opentron OT-2s. PyLabRobot provides a universal set of commands and representations for deck layout and labware, enabling the control of diverse accessory devices. The interface is extensible and can work with any robot that manipulates liquids within a Cartesian coordinate system. We validated the system through unit tests and several application demonstrations, including a browser-based simulator, a position calibration tool, and a path-teaching tool for complex movements. PyLabRobot provides a flexible, open, and collaborative programming environment for laboratory automation.

In-line product quality monitoring during biopharmaceutical manufacturing using computational Raman spectroscopy.

The implementation of process analytical technologies is positioned to play a critical role in advancing biopharmaceutical manufacturing by simultaneously resolving clinical, regulatory, and cost challenges. Raman spectroscopy is emerging as a key technology enabling in-line product quality monitoring, but laborious calibration and computational modeling efforts limit the widespread application of this promising technology. In this study, we demonstrate new capabilities for measuring product aggregation and fragmentation in real-time during a bioprocess intended for clinical manufacturing by applying hardware automation and machine learning data analysis methods. We reduced the effort needed to calibrate and validate multiple critical quality attribute models by integrating existing workflows into one robotic system. The increased data throughput resulting from this system allowed us to train calibration models that demonstrate accurate product quality measurements every 38 s. In-process analytics enable advanced process understanding in the short-term and will lead ultimately to controlled bioprocesses that can both safeguard and take necessary actions that guarantee consistent product quality.

A handheld plug-and-play microfluidic liquid handling automation platform for immunoassays.

Lab-on-a-chip technologies and microfluidics have pushed miniaturized liquid handling to unprecedented precision, integration, and automation, which improved the reaction efficiency of immunoassays. However, most microfluidic immunoassay systems still require bulky infrastructures, such as external pressure sources, pneumatic systems, and complex manual tubing and interface connections. Such requirements prevent plug-and-play operation at the point-of-care (POC) settings. Here we present a fully automated handheld general microfluidic liquid handling automation platform with a plug-and-play 'clamshell-style' cartridge socket, a miniature electro-pneumatic controller, and injection-moldable plastic cartridges. The system achieved multi-reagent switching, metering, and timing control on the valveless cartridge using electro-pneumatic pressure control. As a demonstration, a SARS-CoV-2 spike antibody sandwich fluorescent immunoassay (FIA) liquid handling was performed on an acrylic cartridge without human intervention after sample introduction. A fluorescence microscope was used to analyze the result. The assay showed a limit of detection at 31.1 ng/mL, comparable to some previously reported enzyme-linked immunosorbent assays (ELISA). In addition to automated liquid handling on the cartridge, the system can operate as a 6-port pressure source for external microfluidic chips. A rechargeable battery with a 12 V 3000 mAh capacity can power the system for 42 h. The footprint of the system is 16.5 × 10.5 × 7 cm, and the weight is 801 g, including the battery. The system can find many other POC and research applications requiring complex liquid manipulation, such as molecular diagnostics, cell analysis, and on-demand biomanufacturing.

End-to-End Data Automation for Pooled Sample SARS-CoV-2 Using R and Other Open-Source Tools.

BACKGROUND: Due to supply chain shortages of reagents for real-time (RT)-PCR for SARS-CoV-2 and increasing demand on technical staff, an end-to-end data automation strategy for SARS-CoV-2 sample pooling and singleton analysis became necessary in the summer of 2020. METHODS: Using entirely open source software tools-Linux, bash, R, RShiny, ShinyProxy, and Docker-we developed a modular software application stack to manage the preanalytical, analytical, and postanalytical processes for singleton and pooled testing in a 5-week time frame. RESULTS: Pooling was operationalized for 81 days, during which time 64 pooled runs were performed for a total of 5320 sample pools and approximately 21 280 patient samples in 4:1 format. A total of 17 580 negative pooled results were released in bulk. After pooling was discontinued, the application stack was used for singleton analysis and modified to release all viral RT-PCR results from our laboratory. To date, 236 109 samples have been processed avoiding over 610 000 transcriptions. CONCLUSIONS: We present an end-to-end data automation strategy connecting 11 devices, one network attached storage, 2 Linux servers, and the laboratory information system.

AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots.

As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn (DBTL) approach to simultaneously learn how a function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize and algorithmically perform each step of the DBTL cycle. However, there are many fewer options for automating the build step. Build typically involves deoxyribonucleic acid (DNA) assembly, which remains manual, low throughput and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron, an open-source Python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies, beginning from fragment amplification. We show that AssemblyTron can perform polymerase chain reactions across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent in vivo assemblies (IVAs) with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent IVA also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs and wastes associated with synthetic biology, which, along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering.

High-Throughput Expression of Inclusion Bodies on an Automated Platform.

In bioprocesses, which target the production of recombinant proteins as inclusion bodies, the upstream process has a decisive influence on the downstream operations, especially regarding cell disruption, inclusion body purity and composition, and refolding yield. Therefore, optimization of the processes in fed-batch mode is a major issue, and screening for strains and process conditions are performed in highly labor, time and cost intensive shake flasks or multiwell plates. Thus, high-throughput experiments performed similar to the industrial operating conditions offer a possibility to develop efficient and robust upstream processes. We present here an automated platform for Escherichia coli fed-batch cultivations in parallelized minibioreactors. The platform allows execution of experiments under multiple conditions while allowing for real-time monitoring of critical process parameters and a controlled fermentation environment. By this, the main factors that affect yields and quality of inclusion bodies can be investigated, speeding up the development process significantly.

Robotic APTamer-Enabled Electrochemical Reader (RAPTER) System for Automated Aptamer-Mediated Electrochemical Analysis.

Electrochemical aptamer-based (E-AB) sensors using conformational change-induced electron transfer kinetics are sensitive, reagent-less, and cost-effective tools for molecular sensing. Current advances in this technology can allow continuous drug pharmacokinetic monitoring in living animals (Dauphin-Ducharme et al., ACS Sens 4(10):2832-2837, 2019; Idili et al., Chem Sci 10(35):8164-8170, 2019), as well as automated analysis of hormone pulsatility (Liang et al., Nat Commun 10(1):852, 2019). In this chapter, we provide the methodology for an automated E-AB conformational change-based robotic sensing platform. By using an open-source programmable robotic system, this method can be adapted to a wide range of experimental scenarios.

3D Hydrogel Cultures for High-Throughput Drug Discovery.

Our increased understanding of how a cell's microenvironment influences its behavior has fueled an interest in three-dimensional (3D) cell cultures for drug discovery. Particularly, scaffold-based 3D cultures are expected to recapitulate in vivo tissue stiffness and extracellular matrix composition more accurately than standard two-dimensional (2D) monolayer cultures. Here we present a 3D hydrogel cell culture setup suitable for automated screening with standard high-throughput screening (HTS) liquid handling equipment commonly found in a drug discovery laboratory. Further, we describe the steps required to validate the assay system for compound screening.