Process analytics 4.0: A paradigm shift in rapid analytics for biologics development

Analytical testing of product quality attributes and process parameters during the biologics development (Process analytics) has been challenging due to the rapid growth of biomolecules with complex modalities to support unmet therapeutic needs. Thus, the expansion of the process analytics tool box for rapid analytics with the deployment of cutting-edge technologies and cyber-physical systems is a necessity. We introduce the term, Process Analytics 4.0; which entails not only technology aspects such as process analytical technology (PAT), assay automation, and high-throughput analytics, but also cyber-physical systems that enable data management, visualization, augmented reality, and internet of things (IoT) infrastructure for real time analytics in process development environment. This review is exclusively focused on dissecting high-level features of PAT, automation, and data management with some insights into the business aspects of implementing during process analytical testing in biologics process development. Significant technological and business advantages can be gained with the implementation of digitalization, automation, and real time testing. A systematic development and employment of PAT in process development workflows enable real time analytics for better process understanding, agility, and sustainability. Robotics and liquid handling workstations allow rapid assay and sample preparation automation to facilitate high-throughput testing of attributes and molecular properties which are otherwise challenging to monitor with PAT tools due to technological and business constraints. Cyber-physical systems for data management, visualization, and repository must be established as part of Process Analytics 4.0 framework. Furthermore, we review some of the challenges in implementing these technologies based on our expertise in process analytics for biopharmaceutical drug substance development.     

Lipid bilayer arrays: Cyclically formed and measured

Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32‐element array with ～80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn‐key, high‐yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low‐cost, parallelizable, and automatable, allowing high‐throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.     

Temporal decision making in complex environments

The basic hypothesis of the author is that under the influence of technological development and market pressure, situations take on temporal characteristics that are more and more difficult for the operator to control. The temporal strategies traditionally installed by the operator disappear, are transferred or transformed. Far from counterbalancing these phenomena, the displays, as they are designed in the workplace, obliterate the temporal dimension. The errors that are seen to appear are the product of a mismatch between the characteristics of the situation and the operator's resources. Four mechanisms of time estimation are discussed. Field study results on temporal strategies, such as anticipation, assessment of a process evolution and planning adjustment are developed.     

Classifying human operator functional state based on electrophysiological and performance measures and fuzzy clustering method

The human operator’s ability to perform their tasks can fluctuate over time. Because the cognitive demands of the task can also vary it is possible that the capabilities of the operator are not sufficient to satisfy the job demands. This can lead to serious errors when the operator is overwhelmed by the task demands. Psychophysiological measures, such as heart rate and brain activity, can be used to monitor operator cognitive workload. In this paper, the most influential psychophysiological measures are extracted to characterize Operator Functional State (OFS) in automated tasks under a complex form of human–automation interaction. The fuzzy c-mean (FCM) algorithm is used and tested for its OFS classification performance. The results obtained have shown the feasibility and effectiveness of the FCM algorithm as well as the utility of the selected input features for OFS classification. Besides being able to cope with nonlinearity and fuzzy uncertainty in the psychophysiological data it can provide information about the relative importance of the input features as well as the confidence estimate of the classification results. The OFS pattern classification method developed can be incorporated into an adaptive aiding system in order to enhance the overall performance of a large class of safety–critical human–machine cooperative systems.     

The automated synthesis of oligodeoxyribonucleotides

Progress in science is a history of the synergistic relationship between technology and theory: growth in theoretical understanding requires and initiates technological change; technological advance opens up new areas of knowledge. The history of molecular genetics is but a single illustration of this process. Four major achievements in understanding DNA have occurred in recent years: they are the use of X-ray diffraction to posit a model for DNA, the discovery of DNA cutting enzymes, the sequencing of DNA, and the chemical synthesis of DNA. This last achievement, the chemical synthesis of DNA, and, more particularly, its automation, will be the focus of this article.     

Automated applications of sandwich-cultured hepatocytes in the evaluation of hepatic drug transport

Predictions of the absorption, distribution, metabolism, excretion, and toxicity of compounds in pharmaceutical development are essential aspects of the drug discovery process. B-CLEAR is an in vitro system that uses sandwich-cultured hepatocytes to evaluate and predict in vivo hepatobiliary disposition (hepatic uptake, biliary excretion, and biliary clearance), transporter-based hepatic drug-drug interactions, and potential drug-induced hepatotoxicity. Automation of predictive technologies is an advantageous and preferred format in drug discovery. In this study, manual and automated studies are investigated and equivalence is demonstrated. In addition, automated applications using model probe substrates and inhibitors to assess the cholestatic potential of drugs and evaluate hepatic drug transport are examined. The successful automation of this technology provides a more reproducible and less labor-intensive approach, reducing potential operator error in complex studies and facilitating technology transfer.     

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.     

A decade of differential display

It has been 10 years since the invention of differential display (DD), a conceptually simple methodology that allows the detection and identification of differentially expressed genes. In the past decade, the number of publications describing successful applications of DD has outnumbered those using any other competing methodologies, including subtractive hybridization, representational difference analysis, serial analysis of gene expression, and DNA microarrays. This review will provide a glimpse of the current progress made in DD technological development, refinement, and automation. Excellent examples of DD applications in studying a variety of biological problems, in such diverse biological systems as bacteria, yeast, flies, plants, and higher mammals, are presented to provide a roadmap for those who would like to pursue a fruitful gene "fishing" expedition. Some of the fundamental differences between DD and DNA microarrays are also discussed.     

Towards an integrative structural biology approach: combining Cryo-TEM,X-ray crystallography,and NMR

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.     

The future of laboratory automation.

Among the many factors that will define the laboratory of the future are the development of advanced computer communications systems, artificial intelligence, robotic systems, and material storage and retrieval systems. This article examines some of these factors and challenges current automation justification procedures in light of the greater competitive environment of today.     

A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples

Materials such as soils, waters, sewage sludges and foods can contain low numbers of salmonellas. A most-probable-number (MPN) method that utilized a bioluminescent-bacteriophage is described that allowed the specific determination of as few as one Salmonella typhimurium cell/100 ml of material within 24 h. The method was developed with soil, lake water and sewage sludge inoculated with Salm. typhimurium and had an efficiency of 100% when tested against a traditional MPN method. The protocol is rapid, sensitive, inexpensive, has a low operator time compared to the traditional MPN method, allows for the repair of injured cells and is amenable to automation.     

High-throughput and automation advances for accelerating single-cell cloning,monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single-cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.     

经济开发区生态规划与建设研究

生态规划和建设是协调经济发展和生态环境关系,创造和谐的人工化环境的调控对策。木文研究了沈阳开发区生态规划和建设的理论、方法,着重探讨了开发区经济、环境协调发展的途径:1)选择理想的协调发展优化方案,2)控制发展热电生产,发展集中供热和低能耗高技术产业,3)节约水资源,提高水循环利用率,4)在生态学原理指导下进行生态管理。     

The discriminatory power of ribotyping as automatable technique for differentiation of bacteria

Since the introduction of ribonucleic acid gene restriction patterns as taxonomic tools in 1986, ribotyping has become an established method for systematics, epidemiological, ecological and population studies of microorganisms. In the last 25 years, several modifications have improved the convenience, reproducibility and turn-around time of this technique. The technological development culminated in the automation of ribotyping which allowed for high-throughput applications e.g. in the quality control of food production, pharmaceutical industry and culture collections. The capability of the fully automated RiboPrinter^® System for the differentiation of bacteria below the species level is compared with the discriminatory power of traditional ribotyping, of molecular fingerprint techniques like PFGE, MLST and MLVA as well as of MALDI-TOF mass spectrometry. While automated RiboPrinting is advantageous with respect to standardization, ease and speed, PCR ribotyping has proved being a highly discriminatory, flexible, robust and cost-efficient routine technique which makes inter-laboratory comparison and build of ribotype databases possible, too.     

中国水青冈分布,生长和更新特点

中国水青冈分布、生长和更新特点吴刚（中国科学院生态环境研究中心,北京１０００８５）Ｄｉｓｔｒｉｂｕｔｉｏｎ,ＧｒｏｗｔｈａｎｄＲｅｖｅｇｅｔａｔｉｏｎａｌＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆＦａｇｕｓｉｎＣｈｉｎａ．ＷｕＧａｎｇ（ＲｅｓｅａｒｃｈＣｅｎ...     

Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images

The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation. An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed. The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method. OTS is based on the statistical properties of local gray-values and gradients in the image. By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition. The automated OTS method was shown to reproduce values obtained manually by an experienced operator.     

Solid phase synthesis of complex natural products and libraries thereof.

Natural products have served as an important source of medicinal compounds and pharmaceutical leads over the last century. Within the last 10 years, significant interest has developed in applying combinatorial chemistry techniques to the study of natural products and their biological activities. In this review, we examine several representative efforts wherein natural product skeletons have been constructed or immobilized on solid support and subsequently derivatized, giving rise to analog libraries useful in understanding the structure-activity relationships of the parent natural product. Issues such as target selection, library design, linker development, automation, and library characterization are addressed.