Automation in the pharmaceutical analysis laboratory: a centralized/decentralized approach

It has been over 10 years since robots have appeared in the pharmaceutical analysis laboratory. In the early days, it was common for one selected individual to be responsible for the programming, usage and maintenance of the robots(s). However, the increasing use of robotics has prompted the formation of robotics ‘laboratories’ and/or ‘groups’. This is especially true when multiple robotic systems and applications are involved.Over the past several years at ISLAR, many champions of robotics have given presentations on the setup and usage of robotics within their organizations. These managers have described both the ‘centralized’ and ‘decentralized’ approaches to the implementation of robotics. In the centralized system, a single group is charged with all aspects of the robotic project, including justification, purchase, validation, use and maintenance. Under such an arrangement, samples are usually given to the robotics group for analysis. In contrast, a totally decentralized approach to robotics would have units interspersed throughout the organization, with each individual group responsible for their respective unit(s), in much the same way as liquid chromatographs are considered.At Hoffmann-La Roche, aspects of both the centralized and decentralized approaches to robotics are used which make our combined system the ‘best of both worlds’. This paper describes the Roche philosophy towards robotics and highlights the advantages to the system used.     

Biochemical,Sensory, and Molecular Evaluation of Flavour and Consumer Acceptability in Australian Papaya (Carica papaya L.) Varieties

Inconsistency in flavour is one of the major challenges to the Australian papaya industry. However, objectively measurable standards of the compound profiles that provide preferable taste and aroma, together with consumer acceptability, have not been set. In this study, three red-flesh papayas (i.e., ‘RB1’, ‘RB4’, and ‘Skybury’) and two yellow-flesh papayas (i.e., ‘1B’ and ‘H13’) were presented to a trained sensory panel and a consumer panel to assess sensory profiles and liking. The papaya samples were also examined for sugar components, total soluble solids, and 14 selected volatile compounds. Additionally, the expression patterns of 10 genes related to sweetness and volatile metabolism were assessed. In general, red papaya varieties had higher sugar content and tasted sweeter than yellow varieties, while yellow varieties had higher concentrations of citrus floral aroma volatiles and higher aroma intensity. Higher concentrations of glucose, linalool oxide, and terpinolene were significantly associated with decreased consumer liking. Significant differences were observed in the expression profiles of all the genes assessed among the selected papaya varieties. Of these, cpGPT2 and cpBGLU31 were positively correlated to glucose production and were expressed significantly higher in ‘1B’ than in ‘RB1’ or ‘Skybury’. These findings will assist in the strategic selective breeding for papaya to better match consumer and, hence, market demand.     

Physiological and Molecular Analysis Reveals the Differences of Photosynthesis between Colored and Green Leaf Poplars

Leaf coloration changes evoke different photosynthetic responses among different poplar cultivars. The aim of this study is to investigate the photosynthetic difference between a red leaf cultivar (ZHP) and a green leaf (L2025) cultivar of Populus deltoides. In this study, ‘ZHP’ exhibited wide ranges and huge potential for absorption and utilization of light energy and CO₂ concentration which were similar to those in ‘L2025’ and even showed a stronger absorption for weak light. However, with the increasing light intensity and CO₂ concentration, the photosynthetic capacity in both ‘L2025’ and ‘ZHP’ was gradually restricted, and the net photosynthetic rate (Pn) in ‘ZHP’ was significantly lower than that in ‘L2025’under high light or high CO₂ conditions, which was mainly attributed to stomatal regulation and different photosynthetic efficiency (including the light energy utilization efficiency and photosynthetic CO₂ assimilation efficiency) in these two poplars. Moreover, the higher anthocyanin content in ‘ZHP’ than that in ‘L2025’ was considered to be closely related to the decreased photosynthetic efficiency in ‘ZHP’. According to the results from the JIP-test, the capture efficiency of the reaction center for light energy in ‘L2025’ was significantly higher than that in ‘ZHP’. Interestingly, the higher levels of light quantum caused relatively higher accumulation of Q_A^- in ‘L2025’, which blocked the electron transport and weakened the photosystem II (PSII) performance as compared with ‘ZHP’; however, the decreased capture of light quantum also could not promote the utilization of light energy, which was the key to the low photosynthetic efficiency in ‘ZHP’. The differential expressions of a series of photosynthesis-related genes further promoted these specific photosynthetic processes between ‘L2025’ and ‘ZHP’.     

Jumping into the 20th century before it is too late: is laboratory robotics still in its infancy?

Successful management of laboratory robotic automation programmes in the environment of research and drug discovery within the pharmaceutical industry may perhaps be best compared to a chef preparing the perfect hollandaise sauce. All the ingredients must be available at the same time and be of highest quality for the right price. However, if components are not added in the right quantities and in the proper order, no amount of whipping together by the product champion will create the best product. In the past, managerial scepticism surrounding useful implementation of cost-effective, high-throughput robotic systems often placed these ‘modern toys’ at low priorities for research development laboratories. Management now recognizes the unique contributions of robotics in the research environment. Although the scientific director must still play the role of product champion, new questions are being proposed and new commitments are being made to bring the potential of robotic automation to every laboratory where repetitive functions can benefit from new applications. Research laboratory directors have become both the key ingredient, as well as the rate-limiting determinant in the development of new applications. Having fulfilled the promise of robotic automation to release talented personnel, the challenge now is for the ‘end users’, the bench scientists, to be provided with opportunities to invest the time and effort required for future applications and new career functions.     

Jasmonic Acid and Ethylene Participate in the Gibberellin-Induced Ovule Programmed Cell Death Process in Seedless Pear ‘1913’ (Pyrus hybrid)

Seedless fruit is a feature appreciated by consumers. The ovule abortion process is highly orchestrated and controlled by numerous environmental and endogenous signals. However, the mechanisms underlying ovule abortion in pear remain obscure. Here, we found that gibberellins (GAs) have diverse functions during ovules development between seedless pear ‘1913’ and seeded pear, and that GA₄₊₇ activates a potential programmed cell death process in ‘1913’ ovules. After hormone analyses, strong correlations were determined among jasmonic acid (JA), ethylene and salicylic acid (SA) in seedless and seeded cultivars, and GA₄₊₇ treatments altered the hormone accumulation levels in ovules, resulting in significant correlations between GA and both JA and ethylene. Additionally, SA contributed to ovule abortion in ‘1913’. Exogenously supplying JA, SA or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid promoted ‘Bartlett’ seed death. The regulatory mechanism in which ethylene controls ovule death has been demonstrated; therefore, JA’s role in regulating ‘1913’ ovule abortion was investigated. A further study identified that the JA signaling receptor MYC2 bound the SENESCENCE-ASSOCIATED 39 promoter and triggered its expression to regulate ovule abortion. Thus, we established ovule abortion-related relationships between GA and the hormones JA, ethylene and SA, and we determined their synergistic functions in regulating ovule death.     

Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides

Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar ‘ZhongHuai1’ (‘ZH1’, triploid) and ‘ZhongHuai2’ (‘ZH2’, diploid) generated from the same parents. The mature pollen was sterile in triploid poplar ‘ZH1’. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with “S-adenosylmethionine metabolism”, “actin cytoskeleton organization”, or “translational elongation”. The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties (‘ZH1’, ‘ZH2’, and ‘2KEN8‘). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity.     

Non-Invasive Preimplantation Genetic Testing for Aneuploidy and the Mystery of Genetic Material: A Review Article

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated.     

Reality in the 1990s and beyond—more with less

It is no secret that the pharmaceutical industry is undergoing rapid and revolutionary change. The impact of managed care and threatened pricing caps by the US Congress on new products has caused many companies to re-evaluate their short and long term business and research strategies. In too many cases, this has resulted in down-sizing by both lay-offs and attrition. For most analytical laboratories, this has meant doing more with less resources. There are several major ‘realities’ that are having a significant influence on the amount and type of analytical support required to bring a new product to the market place in today''s regulatory climate. They are pre-approval inspections, the Barr decision and the proposed ICH guidelines. Other realities of the 1990s are also influencing the operation of the analytical laboratory. To cope with these realities, wise use of resources is mandatory. The strategies employed by each company differ, but laboratory automation is usually one of the important elements of the equation. Other elements include contract laboratories, consultants and temporaries. Each of these elements provides part of the solution to doing more with less, but each has its own positives and negatives which must be considered. This paper looks at the relationships between these factors and their impact on the analytical laboratory.     

Automation - down to the nuts and bolts

Laboratories that once viewed automation as an expensive luxury are now looking to automation as a solution to increase sample throughput, to help ensure data integrity and to improve laboratory safety. The question is no longer, ‘Should we automate?’, but ‘How should we approach automation?’ A laboratory may choose from three approaches when deciding to automate: (1) contract with a third party vendor to produce a turnkey system, (2) develop and fabricate the system in-house or (3) some combination of approaches (1) and (2). The best approach for a given laboratory depends upon its available resources. The first lesson to be learned in automation is that no matter how straightforward an idea appears in the beginning, the solution will not be realized until many complex problems have been resolved. Issues dealing with sample vessel manipulation, liquid handling and system control must be addressed before a final design can be developed. This requires expertise in engineering, electronics, programming and chemistry. Therefore, the team concept of automation should be employed to help ensure success. This presentation discusses the advantages and disadvantages of the three approaches to automation. The development of an automated sample handling and control system for the STAR™ System focused microwave will be used to illustrate the complexities encountered in a seemingly simple project, and to highlight the importance of the team concept to automation no matter which approach is taken. The STAR™ System focused microwave from CEM Corporation is an open vessel digestion system with six microwave cells. This system is used to prepare samples for trace metal determination. The automated sample handling was developed around a XYZ motorized gantry system. Grippers were specially designed to perform several different functions and to provide feedback to the control software. Software was written in Visual Basic 5.0 to control the movement of the samples and the operation and monitoring of the STAR™ microwave. This software also provides a continuous update of the system''s status to the computer screen. The system provides unattended preparation of up to 59 samples per run.     

Editorial: The necessity of adult learning methods in programs of intensive study

This article presents the educational methodologies that prove effective in adult educational programmes of intensive study. The many facets of a quality educational programme are discussed and I will focus on four topics that any adult educational programme must have: an adult learner, an instructor of adults, a curriculum, and a response to outside forces.These topics become increasingly critical when one examines the components of technical education and, especially, an intensive training programme in laboratory automation systems. The adult will be discussed as a learner and the associated myths and principles. Next, I will focus on the instructor and his/her necessary personal and professional qualities, including essential skills and psychological elements required. Aspects of curriculum will then be studied. The conventional and the innovative approaches to curriculum design, development, and delivery differ markedly. Development and delivery are so closely linked to the curriculum that both will be discussed under the one title of ‘curriculum’. The final discussion will focus on the outside forces that directly and indirectly affect adult education; since these are many, they are limited to a few salient ones.     

Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293^TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.     

The Discovery and Function of Filaggrin

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the ‘histidine-rich protein’. Steinert and Dale named the protein ‘filaggrin’ in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, ‘profilaggrin,’ was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into ‘building blocks’ that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean’s team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.     

Comparison of two data interpretation programs in automated blood cell counters

Two data interpretation programs incorporated into two different automated blood cell counters were evaluated. The programs analysed size distribution histograms for screening for abnormal specimens. The messages given by the programs were ‘normal’, ‘abnormal’ and ‘suspect’. ‘Abnormal’ messages meant abnormalities in the values of the measured parameters. ‘Suspect’ messages were given when the histograms showed either a possibility of the existence of abnormal blood cells, or an abnormal level of such cells; re-examination by a manual method was necessary for such specimens. For reference, the manual differential was done. The false-negative rate for each kind of message was less than 10% and efficiency was about 90%. These programs were helpful in avoiding unnecessary manual observations in reducing the workload in a routine clinical laboratory.     

Designing optimized industrial process analysers for closed loop control

Manufacturers are now looking closely at ways of optimizing ‘quality’ and increasing process efficiency while reducing manufacturing costs. Near infra-red (NIR) technology is a popular solution to this challenge: it provides manufacturers with rapid and reliable in-process analysis and thousands of systems have already been installed in the food, chemical, pharmaceutical and agricultural markets.For over 10 years, NIR has been successfully applied to at-line process analysis. Rugged and easy-to-operate filter analysers are traditionally located in the control room–process operators can then ‘grab samples’ and obtain results in less than a minute. There are many practical advantages to using at-line filter systems. Products from many lines can be run on one system, and, since there is no direct process interface, installation, operation and maintenance are quite simple.Many manufacturers, however, are now striving to achieve on-line closed loop control, in these cases the benefit of obtaining continuous measurement is well worth the effort required to automate the analysis.     

The trials and tribulations of a robotic screening core

It is well recognized within the pharmaceutical industry that high throughput screening is a valuable and rapid tool to identify novel chemical compounds that may lead to tomorrow''s drugs. High throughput screening involves testing as many chemical compounds as quickly as possible against a defined molecular or cellular ‘target’ (for example an enzyme) in the hope that interacting compounds may provide significant therapeutic benefits.At Wyeth-Ayerst Research, a Robotics and Automation Research Core Group has been established which serves as the in-house resource for high throughput screening. The robotics group has three missions: (1) develop and perform high throughput screens for customers in all therapeutic departments in the company; (2) educate customers in issues related to screen design; and (3) help customers to bring automated workstations into their laboratories. The mission, therefore, requires the effective use of automation, as well as building a strong collaboration with customers.The challenges that have been faced fall into two categories: technology limiting and customer relations. Technological challenges arise because it is necessary to develop and implement assays with very different formats and biochemical endpoints within extremely shortened time frames. The primary means to meet these challenges is with flexible robotics and flexible people. Challenges in the area of customer relations include setting realistic expectations, maintaining a sense of collaboration (and not merely service), educating investigators as to how to deal with the huge amount of data generated and seeking feedback. Effective and frequent communication, and an awareness of each individual''s perspective, are essential to provide the most appropriate service.