Mammalian cell protein expression for biopharmaceutical production

Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. While expression and production have been fully developed and Chinese hamster ovary cells are used for the majority of products both on the market and in clinical development, significant progresses in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting, have been reported in the last several years. With the latest analytical methods development, more attention is being devoted towards product quality including glycol profiling, which leads to better understanding the impact of culture condition during production. Additionally, transient gene expression technology platform plays more important role in biopharmaceutical early development stages. This review focused on the latest advancements in the field, especially in active areas such as expression systems, glycosylation impact factors, and transient gene expression.     

Cell-free technologies for biopharmaceutical research and production

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转基因动物的检测方法

转基因动物的研究目前尚处于实验室研究阶段,获得转基因动物难度大,检测转基因比较困难,从染色体和基因水平、转录、翻译、整体表型等不同角度介绍了转基因动物的不同检测手段和方法,并对各水平存在的问题及应用前景作了阐述。     

Exploring the potential of Saccharomyces cerevisiae for biopharmaceutical protein production

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An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.     

New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris

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Highlights► The application of Pichia pastoris for biopharmaceutical production is described. ► Synthetic biology approaches and perspectives to improve production are reviewed. ► Glycoengineering efforts to produce humanized, uniform glycoproteins are covered. ► The design and application of synthetic promoter variants are highlighted. ► The molecular toolbox available for synthetic biology in P. pastoris is discussed.     

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by-products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.     

High-level expressing YAC vector for transgenic animal bioreactors

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.     

The application of transgenic techniques for the improvement of domestic animal productivity

The application of transgenesis techniques to domestic animals has now been achieved in all the major species of domestic animals. In this review, the progress towards genuine practical applications of the technique is examined. Areas which appear to have made progress during the past several years include the evaluation of animals with elevated growth hormone levels, the introduction of novel metabolic pathways, the expression of transgenes in the mammary glands of pigs and sheep, and the real possibility that functional immunoglobulin genes might be used to confer genetically-inherited disease resistance to commercially-valuable animal breeds.     

Seaweeds for animal production use

Early scientific studies conducted at the turn of the twentieth century failed to support the inclusion of seaweeds into animal rations at high inclusion rates. At that time, based on proximate analysis and energy availability studies, dried seaweeds or kelp meal largely fell out of favor as a recommended animal feed source. Nevertheless, kelp meal was still regarded by some as having properties which improved animal health and productivity which were not conveniently explained by conventional feed analysis. In the 1970s, research leads to the discovery that chelated micromineral sources were more efficient for the delivery of microelements than conventional inorganic sources. This prompted renewed interest in seaweeds as rich sources of over 60+ microelements. However, it was only in the early 2000s, when detailed analysis of the complex structure of the polysaccharides associated with seaweeds was tied to their prebiotic actions, that a clear explanation for the basis of productivity and health enhancement was attained. Further analysis indicated that other constituents in various brown seaweeds such as phlorotannins and antioxidants also contributed to the observed bioactivities. Of all of the brown seaweeds cited in studies, the one most scientifically documented is Ascophyllum nodosum, and of all of these sources, Tasco®, a sundried, high-quality macroalgal product, produced by Acadian Seaplants has been the most studied. The latest studies of Tasco® suggest prebiotic potencies at least five times that of the reference prebiotic inulin with additional performance-enhancing benefits in animal rations that rival antibiotic inclusions.     

Examining the sources of variability in cell culture media used for biopharmaceutical production

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.     

动物转基因新技术研究进展

动物转基因技术是21世纪发展最为迅速的生物高新技术之一, 它是指通过基因工程技术将外源基因整合到受体动物基因组中, 从而使其得以表达和遗传的生物技术。动物转基因的关键限制因素是转基因效率和基因表达的精确调控。目前有多种转基因技术, 每一种技术各有其优缺点, 仍然需要进一步研究。随着研究的深入, 转基因技术必将在探讨基因功能、动物遗传改良、生物反应器、动物疾病模型、器官移植等领域有广阔的应用前景。文章综述了近年发展的提高转基因效率的生殖干细胞法、提高转基因精确性的基因打靶法、RNA干扰(RNAi)介导的基因沉默技术和诱导多能干细胞(iPS)转基因技术。新的转基因技术为转基因动物的研究提供了更好的平台, 可以加快促进人类医药卫生、畜牧生产等领域的发展。     

Model-based design and integration of a two-step biopharmaceutical production process

This paper presents the design of a two-step process in which the first step is PEGylation of a protein, and the second step is chromatographic purification of the target mono-PEGylated protein from the unreacted and the di-PEGylated impurities. The difference between optimizing each process step separately and optimizing them simultaneously is studied. It was found that by optimizing the steps simultaneously up to a 100 % increase in productivity could be obtained without reduction in yield. Optimizing both steps at the same time makes it possible for the optimization method to take into account that the di-PEGylated protein is much easier to separate than the non-PEGylated protein. The easier separation makes it possible to get a higher yield and productivity at the same time. The effect of recycling was also studied and the yield could be increased by 30 % by recycling the unreacted protein. However, if maximum productivity is required, batch mode is preferable.     

Transgenic animal production and animal biotechnology

Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.     

Process analytical technology (PAT) tools for the cultivation step in biopharmaceutical production

The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.     

The signifiance of meteorology in animal production

The production of meat, milk and eggs is highest and occurs at a maximal efficiency if the meteorological elements are within a certain range (zone of indifference). Outside this range the animal has to combat meteorological stress. This requires extra energy, so that less energy is available for productive processes. It is therefore important to find out at which levels the various meteorological elements become stressful to the animal organism. This study has to take into consideration the diversity of domestic animals, both with regard to structural features and functional traits. Responses of various categories of domestic animals to the following potentially stress producing meteorological conditions are briefly reviewed: cold, heat, solar radiation, high altitude and indoor environment. Knowledge so derived can be applied either by adapting the animal to the environment by breeding and selection, or by adapting the environment to the animal by technical and managerial means. Some suggestions are made for future considerations in the field of biometeorology of domestic animals.