A concise guide to choosing suitable gene expression systems for recombinant protein production

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems

How Plants Can Contribute to the Supply of Anticancer Compounds

Plants were the first sources of medicines used by humankind, with evidence of herbal remedies dating back at least 60,000 years. Many plants have been used medicinally because they produce secondary metabolites with pharmacological properties, including compounds such as paclitaxel (Taxol) that inhibit cell division and can therefore be used as a treatment for cancer. With the advent of recombinant DNA and molecular biotechnology in the 1970s, plants have also been modified genetically to produce more of their native pharmaceutically active substances, or even nonnative compounds. The scope of medicinal plants has also expanded beyond secondary metabolites to include pharmaceutical recombinant proteins, such as human antibodies. This chapter provides an overview of the anticancer compounds naturally produced in plants and how gene technology has been used to facilitate their production. It also considers how plant-based expression systems can help to supply modern healthcare systems with protein-based anticancer compounds such as monoclonal antibodies, lectins, and anticancer vaccines

Plants as sources of natural and recombinant anti-cancer agents

Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines targeting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the introduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active substances, and even the production heterologous recombinant proteins, including human antibodies, lectins and vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side effects compared to small molecule-based drugs

Procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design- of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity

An overview of descriptors to capture protein properties – Tools and perspectives in the context of QSAR modeling

Proteins are important ingredients in food and feed, they are the active components of many pharmaceutical products, and they are necessary, in the form of enzymes, for the success of many technical processes. However, production can be challenging, especially when using heterologous host cells such as bacteria to express and assemble recombinant mammalian proteins. The manufacturability of proteins can be hindered by low solubility, a tendency to aggregate, or inefficient purification. Tools such as in silico protein engineering and models that predict separation criteria can overcome these issues but usually require the complex shape and surface properties of proteins to be represented by a small number of quantitative numeric values known as descriptors, as similarly used to capture the features of small molecules. Here, we review the current status of protein descriptors, especially for application in quantitative structure activity relationship (QSAR) models. First, we describe the complexity of proteins and the properties that descriptors must accommodate. Then we introduce descriptors of shape and surface properties that quantify the global and local features of proteins. Finally, we highlight the current limitations of protein descriptors and propose strategies for the derivation of novel protein descriptors that are more informative

Hitze stabilisiert Malariaimpfstoffkandidaten in komplexen Proteinextrakten

Plants can be an alternative to mammalian cell cultures in terms of vaccine production due to low upstream production costs and a good safety profile. However, product purification can be challenging because of abundant host cell proteins (HCPs) in raw plant extracts. Here we report the optimization of blanching, a heat-based method, that allowed to remove over 90 percent of HCPs and to stabilize a malaria vaccine candidate by carefully adjusting the temperature in the step and combined it with solid-liquid separation and chromatographic purification

Molecular farming - the slope of enlightenment

Molecular farming can be defined as the use of plants to produce recombinant protein products. The technology is now >30 years old. The early promise of molecular farming was based on three perceived advantages: the low costs of growing plants, the immense scalability of agricultural production, and the inherent safety of plants as hosts for the production of pharmaceuticals. This resulted in a glut of research publications in which diverse proteins were expressed in equally diverse plant-based systems, and numerous companies were founded hoping to commercialize the new technology. There was a moderate degree of success for companies producing non-pharmaceutical proteins, but in the pharmaceutical sector the anticipation raised by promising early research was soon met by the cold hard reality of industrial pragmatism. Plants did not have a track record of success in pharmaceutical protein manufacturing, lacked a regulatory framework, and did not perform as well as established industry platforms. Negative attitudes towards genetically modified plants added to the mix. By the early 2000s, major industry players started to lose interest and pharmaceutical molecular farming fell from a peak of expectation into a trough of disillusionment, just as predicted by the Gartner hype cycle. But many of the pioneers of molecular farming have refocused their activities and have worked to address the limitations that hampered the first generation of technologies. The field has now consolidated around a smaller number of better-characterized platforms and has started to develop standardized methods and best practices, mirroring the evolution of more mature industry sectors. Likewise, attention has turned from proof-of-principle studies to realistic techno-economic modeling to capture significant niche markets, replicating the success of the industrial molecular farming sector. Here we argue that these recent developments signify that pharmaceutical molecular farming is now climbing the slope of enlightenment and will soon emerge as a mature technology

Was ist gesichert bei der diabetischen Retinopathie? Mythen und Fakten

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems

Asynchronous electric drive with digital system of discrete-phase control

Дисертація присвячена вирішенню актуальної наукової задачі створення нового принципу управління швидкістю обертання ротора асинхронного двигуна, який зберігає переваги векторного методу управління, але в той же час принципово відрізняється від чисто скалярного і чисто векторного методів і передбачає розробку системи управління, яка не використовує громіздкий алгоритм координатних перетворень і забезпечує максимальну відповідність режимів роботи асинхронного двигуна заданому оператором алгоритму. Розроблено дискретно-фазовий спосіб управління асинхронним електроприводом і доведено, що така система дозволяє формувати вихідні напруги перетворювача частоти по заданому оператором алгоритму, забезпечує безперервність набігу фази при будь-яких змінах режимів роботи двигуна зі збереженням умови відносини ефективного значення вихідної напруги до частоти обертання ротора. Розроблено нову систему рекуперації енергії з дискретно-фазовим управлінням, яка не містить окремий випрямляч і автономний інвертор напруги з блоком формування широтно-імпульсно модульованого сигналу і автоматично відстежує поточні фази мережі живлення і одночасно є стабілізатором напруги в колі постійного струму. Вперше запропонована потрійна система захисту перетворювача частоти, заснована на вимірі вхідної ємності IGBT з формуванням відповідного захисного інтервалу, що знижує ризик протікання струму короткого замикання, реалізує жорстке завдання максимальних режимів роботи інвертора, а також оперативно формує сигнал його відключення.Диссертация посвящена решению актуальной научной задачи создания нового принципа управления скоростью вращения ротора асинхронного двигателя, который сохраняет преимущества векторного метода управления, но в то же время принципиально отличается от чисто скалярного и чисто векторного методов и предусматривает разработку системы управления, которая не использует громоздкий алгоритм координатных преобразований и обеспечивает максимальное соответствие режимов работы асинхронного двигателя заданному оператором алгоритму с относительным минимальным отклонением по скорости вращения ротора и сохранением устойчивости работы асинхронного электропривода в динамических режимах. Разработан дискретно-фазовый способ управления асинхронным электроприводом и доказано, что такая система позволяет формировать выходное напряжения преобразователя частоты по заданному оператором алгоритму, обеспечивает непрерывность набега фазы при любых изменениях режимов работы двигателя с сохранением условия отношения эффективного значения выходного напряжения к частоте вращения ротора. Кроме того, данная система обеспечивает защиту IGBT модуля от сквозных токов и поддерживает заданное выходное напряжение в звене постоянного тока. Разработана новая система рекуперации энергии с дискретно-фазовым управлением, которая не содержит отдельный выпрямитель и автономный инвертор с блоком формирования широтно-модулированного сигнала, и автоматически отслеживает текущие фазы сети питания и одновременно является стабилизатором напряжения в цепи постоянного тока. Впервые предложена тройная система защиты преобразователя частоты, основанная на измерении входной ёмкости IGBT с формированием соответствующего защитного интервала, что снижает риск протекания тока короткого замыкания, реализует жесткое задание максимальных режимов работы инвертора, а также оперативно формирует сигнал его отключения.The dissertation is devoted to solving an urgent scientific problem of creating a new principle of control of the rotor speed of an induction motor, which retains the advantages of the vector control method, but at the same time is fundamentally different from purely scalar and purely vector methods and provides for the development of a control system that does not use a cumbersome algorithm of coordinate transformations and ensures maximum correspondence of the operating modes of the asynchronous motor to the algorithm specified by the operator. A discrete-phase method for controlling an asynchronous electric drive has been developed and it has been proven that such a system makes it possible to form the output voltage of the frequency converter according to the algorithm specified by the operator, ensures the continuity of the phase incursion for any changes in the engine operating modes while maintaining the condition of the ratio of the effective value of the output voltage to the rotor speed. A new system of energy recovery with discrete-phase control has been developed, which does not contain a separate rectifier with a pulse-width-modulated signal generating unit, and automatically monitors the current phases of the power supply. For the first time, a triple frequency converter protection system was proposed, based on measuring the input capacity of the IGBT with the protective interval

Bioreactor-based production of glycoproteins in plant cell suspension cultures

Recombinant glycoproteins such as monoclonal antibodies have a major impact on modern healthcare systems, e.g., as the active pharmaceutical ingredients in anticancer drugs. A specific glycan profile is often necessary to achieve certain desirable activities, such as the effector functions of an antibody, receptor binding or a sufficient serum half-life. However, many expression systems produce glycan profiles that differ substantially from the preferred form (usually the form found in humans) or produce a diverse array of glycans with a range of in vivo activities, thus necessitating laborious and costly separation and purification processes. In contrast, protein glycosylation in plant cells is much more homogeneous than other systems, with only one or two dominant forms. Additionally, these glycan profiles tend to remain stable when the process and cultivation conditions are changed, making plant cells an ideal expression system to produce recombinant glycoproteins with uniform glycan profiles in a consistent manner. This chapter describes a protocol that uses fermentations using plant cell cultures to produce glycosylated proteins using two different types of bioreactors, a classical autoclavable STR 3-L and a wave reactor