Downstream processing of recombinant proteins from transgenic feedstock

The search for inexpensive production systems capable of producing large quantities of recombinant protein has resulted in the development of new technology platforms based on transgenic plants and animals. Over the past decade, these transgenic systems have been used to produce several products and potential therapeutic proteins. Improvements continue to be made, not only in how the proteins are expressed but also in how the end products are obtained. As improvements in expression are realized, cost-saving measures will increasingly focus on downstream processing.     

Production of foreign proteins using plastid transformation

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.     

Manufacturing of recombinant therapeutic proteins in microbial systems

Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.     

Superoxide dismutase: an industrial perspective

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.     

PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.     

Genomics, molecular genetics and the food industry

The production of foods for an increasingly informed and selective consumer requires the coordinated activities of the various branches of the food chain in order to provide convenient, wholesome, tasty, safe and affordable foods. Also, the size and complexity of the food sector ensures that no single player can control a single process from seed production, through farming and processing to a final product marketed in a retail outlet. Furthermore, the scientific advances in genome research and their exploitation via biotechnology is leading to a technology driven revolution that will have advantages for the consumer and food industry alike. The segment of food processing aids, namely industrial enzymes which have been enhanced by the use of biotechnology, has proven invaluable in the production of enzymes with greater purity and flexibility while ensuring a sustainable and cheap supply. Such enzymes produced in safe GRAS microorganisms are available today and are being used in the production of foods. A second rapidly evolving segment that is already having an impact on our foods may be found in the new genetically modified crops. While the most notorious examples today were developed by the seed companies for the agro-industry directed at the farming sector for cost saving production of the main agronomical products like soya and maize, its benefits are also being seen in the reduced use of herbicides and pesticides which will have long term benefits for the environment. Technology-driven advances for the food processing industry and the consumer are being developed and may be divided into two separate sectors that will be presented in greater detail: 1. The application of genome research and biotechnology to the breeding and development of improved plants. This may be as an aid for the cataloging of industrially important plant varieties, the rapid identification of key quality traits for enhanced classical breeding programs, or the genetic modification of important plants for improved processing properties or health characteristics. 2. The development of advanced microorganisms for food fermentations with improved flavor production, health or technological characteristics. Both yeasts and bacteria have been developed that fulfill these requirements, but are as yet not used in the production of foods.     

Industrial production of recombinant therapeutics in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its recent advancements

Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.     

Fluorescent proteins in microbial biotechnology—new proteins and new applications

The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.     

Solid-state fermentation: a promising microbial technology for secondary metabolite production

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.     

Are we prepared for animal cell technology in the 21st century?

Large scale animal cell culture for the production of complex therapeutic proteins has been a major success of the biotechnology industry. Today, approximately half of the $ 5 billion annual turnover of the biotechnology industry is based upon this technology, in many cases with reactors of more than 10 m³. As we look towards the 21 st century, however, we can see novel approaches to the production of therapeutic proteins, by means of gene and cellular therapies. These technologies present new engineering challenges to the animal cell technologist. Are we prepared to meet these challenges? The needs include: small-scale reactors for the preparation of autologous cell lines, methods for the production of viruses to be used as vectors in gene therapy, artificial organ and the processing of xenogenic cell lines and tissues for cellular implants in humans. More attention should be given to three-dimensional cell cultures. Mass transfer considerations need to be extended beyond just oxygen transfer, to include cellular communication in small systems; this is becoming increasingly important for the control and optimise growth and product formation. Apart from improvements of large-scale systems, substantial advantages could be gained by studying new methods for the production and delivery of therapeutic proteins, using small-scale cell culture systems. We should adapt teaching, regulatory, patent and clinical infrastructure to meet this challenge in a harmonious way.     

Membrane technology for purification of therapeutic proteins

Membrane systems are used throughout the downstream purification in the commercial production of high value therapeutic proteins. Over the last two decades, new membranes, modules, and systems have been developed specifically to meet the requirements of the biotechnology industry. These developments have been facilitated by an improved fundamental understanding of: (1) the effects of electrostatic interactions and concentration polarization on protein transmission during ultrafiltration and (2) the role of membrane morphology on protein fouling during both sterile and virus filtration. This perspective highlights some of the key work in this area and provides insights into possible future improvements in membrane technology for the purification of recombinant therapeutic proteins. Biotechnol. Bioeng. 2009;103: 227–230. © 2009 Wiley Periodicals, Inc.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

重组抗体工程及其在肿瘤靶向及癌症治疗中的应用

在过去的十几年中,重组抗体工程在基础研究、医学和药物生产上已经成为最有希望的领域之一。重组抗体及其片段在正在进行诊断和治疗的临床试验中占所有生物蛋白的30％以上。研究集中在抗体作为理想的癌症靶向试剂方面,最近由于FDA批准使用第一个工程化治疗抗体而使热度达到极点。过去的几年中,在设计、筛选及生产新型工程化抗体方面已经取得了重大进展。改革的筛选方法已经能够分离出高亲和力的癌－靶向及抗病毒的抗体,后能够抑制病毒用于基因治疗。癌症诊断和治疗的另一个策略是将重组抗体片段与放射性同位素、药物、毒素、酶以及生物传感器表面进行融合。双特异性抗体及相关融合蛋白也已经生产出来用于癌症的免疫治疗,在抗癌疫苗以及T细胞补充策略上有效地增强了人免疫应答。     

Foreign protein production in plant tissue cultures

Foreign proteins synthesised by plants are now in the marketplace, and clinical trials for plant-derived therapeutic proteins are underway. Economic analysis of plant production systems has helped identify the types of protein that would be most suitable for manufacture using tissue culture methods. The major advantages associated with in vitro plant systems include the ability to manipulate environmental conditions for better control over protein levels and quality, the rapidity of production compared with agriculture, and the use of simpler and cheaper downstream processing schemes for product recovery from the culture medium.     

Molecular Farming in Plants: A Current Perspective

The low cost of production makes plants an ideal candidate for producing many high value compounds through genetic engineering. Expression of vaccines, therapeutic proteins, nutraceuticals, industrial enzymes, and other bio-polymers has been achieved in different plants. A few products for human health care that have been produced in plant systems are currently undergoing human clinical trials. Some recombinant molecules produced in plants for diagnostic use are currently available in the market and several other compounds are in the pipeline for commercialization. The involvement of several biotechnology companies and the successes achieved provide promise for the growth of this emerging field, “Molecular Farming”.     

Field-assisted extraction of cells, particles and macromolecules

Improved bioseparation techniques are increasingly important for biotechnology because separation is often the limiting factor for the success of biological processes. Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products. Similarly the isolation, purification and concentration of many biomolecules produced in fermentation processes is extremely important. Often such downstream processing contributes a large portion of the product cost and thus efficient and economical alternative approaches to bioseparation processes are needed to eliminate, reduce or facilitate the handling of solids. Field-assisted separations, which hold immense potential for providing a major improvement in bioseparation in the near future, are considered in this review. Special emphasis is given to multistage methods, which are cost-effective compared with competing technologies. Commercial applications of these methods are detailed, we present suggestions for future work and we analyse the scale-up and economic aspects of these processes.     

Industrial biotechnology: Tools and applications

Industrial biotechnology involves the use of enzymes and microorganisms to produce value-added chemicals from renewable sources. Because of its association with reduced energy consumption, greenhouse gas emissions, and waste generation, industrial biotechnology is a rapidly growing field. Here we highlight a variety of important tools for industrial biotechnology, including protein engineering, metabolic engineering, synthetic biology, systems biology, and downstream processing. In addition, we show how these tools have been successfully applied in several case studies, including the production of 1, 3-propanediol, lactic acid, and biofuels. It is expected that industrial biotechnology will be increasingly adopted by chemical, pharmaceutical, food, and agricultural industries.