A new scaleable method for the purification of botulinum neurotoxin type E

Botulinum neurotoxins belong to the most toxic substances in nature. Well-known as a food poisoning agent for almost two centuries, the beneficial aspects of this bacterial metabolite were rediscovered about 30 years ago. These toxins, which are produced by the anaerobic bacterium Clostridium botulinum are nowadays used to treat a variety of neuro-muscular disorders. The increased demand requires techniques for the production and purification of these toxins on an industrial scale. The method described herein is based on filtration and chromatography procedures only. Precipitation, centrifugation and dialysis steps were consequently excluded to develop a protocol, which can easily be scaled up from the laboratory purification to industrial needs. About 4 mg of BoNT/E were purified from a 10-L batch culture corresponding to an overall recovery of approximately 14%.     

Actin-based bacterial motility: towards a definition of the minimal requirements

At the border line between microbiology and cell biology, the spectacular capacity o f some intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and several Rickettsias, to use actin polymerization as a driving force for intracellular movement, cell-to-cell spreading and dissemination within the infected tissue is being increasingly studied. Now that it is possible to manipulate the bacterial surface proteins involved in this process - ActA o f L. monocytogenes and IcsA of S. flexneri - these bacterial systems are providing experimental models in which to investigate the role o f actin filament dynamics in cell motility.     

Cell death by necrosis: towards a molecular definition

Necrosis has been defined as a type of cell death that lacks the features of apoptosis and autophagy, and is usually considered to be uncontrolled. Recent research suggests, however, that its occurrence and course might be tightly regulated. After signaling- or damage-induced lesions, necrosis can include signs of controlled processes such as mitochondrial dysfunction, enhanced generation of reactive oxygen species, ATP depletion, proteolysis by calpains and cathepsins, and early plasma membrane rupture. In addition, the inhibition of specific proteins involved in regulating apoptosis or autophagy can change the type of cell death to necrosis. Because necrosis is prominent in ischemia, trauma and possibly some forms of neurodegeneration, further biochemical comprehension and molecular definition of this process could have important clinical implications.     

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N'-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.Key words: monoclonal antibody, recovery, purification, chromatography, membrane, filtration, platform process     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.     

Moving from the bench towards a large scale,industrial platform process for adeno-associated viral vector purification

In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno-associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench-scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.     

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10¹¹ vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10¹¹ vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.     

重组腺相关病毒规模化生物包装技术

重组腺相关病毒(Recombinant adeno-associated virus,rAAV)的诸多优点使其成为具有巨大潜力的人类基因治疗载体。人类基因治疗临床前和临床研究以及基因治疗产品的市场化要求rAAV载体生产的规模化。自1989年野生型的腺相关病毒序列被Samulski报道以来,rAAV的生产工艺已经从传统的质粒共转染发展到应用包装细胞系和生产细胞系,包装细胞也从"人体细胞"衍化到"昆虫细胞"。目前rAAV的生产规模和病毒载体质量都完全可以符合临床应用要求,有效地促进rAAV在基因治疗临床上的广泛运用。以下将着重介绍rAAV规模化生物包装技术的发展趋势,尤其各种规模化生产系统的要点。     

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.     

Improving physical activity in COPD: towards a new paradigm

Chronic obstructive pulmonary disease (COPD) is a debilitating disease affecting patients in daily life, both physically and emotionally. Symptoms such as dyspnea and muscle fatigue, lead to exercise intolerance, which, together with behavioral issues, trigger physical inactivity, a key feature of COPD. Physical inactivity is associated with adverse clinical outcomes, including hospitalization and all-cause mortality. Increasing activity levels is crucial for effective management strategies and could lead to improved long-term outcomes. In this review we summarize objective and subjective instruments for evaluating physical activity and focus on interventions such as pulmonary rehabilitation or bronchodilators aimed at increasing activity levels. To date, only limited evidence exists to support the effectiveness of these interventions. We suggest that a multimodal approach comprising pulmonary rehabilitation, pharmacotherapy, and counselling programs aimed at addressing emotional and behavioural aspects of COPD may be an effective way to increase physical activity and improve health status in the long term.     

基于植物重组蛋白产量提高的研究进展

重组蛋白为疾病治疗提供了新手段,同时创造了可观的经济效益。利用经济作物(主要是烟草)、谷类作物、豆科作物和蔬菜作物生产具有药用价值的重组蛋白是“分子农业”最热门的研究内容。尽管许多重组蛋白已在植物中表达,但只有一小部分已成功投入使用。为了极大地克服限制植物生产重组蛋白发展的问题,研究人员改进表达系统以增加重组蛋白的产量。本文从分析植物产生重组蛋白产量低和/或生物活性低等问题入手,综述了近些年来解决这些问题的优化策略,同时提出了提高植物生产重组蛋白产量的研究方向。     

A chemical and biological approach towards the definition of calcareous soils

Summary Several agricultural problems are associated with the presence of certain levels of CaCO₃ in soils. The level of CaCO₃ at which the phosphate fixation becomes an apparent agricultural problem, is thought to be an appropriate margine at which the soil can be considered calcareous. Thus, a set of soil mixtures, ranging in CaCO₃ content from 1 to 96% was prepared and used in a column study to determine the level at which the CaCO₃ fraction becomes a dominant factor controlling. P³² movement and distribution.Increasing the percentage of oolitic sand, in the soil mixture, from 1 to 10% caused a sharp drop in P³² movement with soil solution and any increase in CaCO₃ content above 10% did not show any further drop in P³² movement. The amount of P³² removed with the soil solution was generally low compared to that retained in soil columns. Studying the distribution of P³² in soil columns, after five displacements, has indicated that the migration of P³² from the top soil increased by increasing CaCO₃ from 1 and 2 to 6%. The amount of P³² removed was however retained in lower sections. A very sharp decrease in P³² migration from the top soil was observed when CaCO₃ content was raised from 8 to 10%.A similar picture was shown when the CaCO₃ material used was in clay size fraction. However the sharp increase in phosphate retention in top soil sections took place at CaCO₃ content of 8% rather than at 10%. A limit of 8 to 10% CaCO₃ was proposed as an appropriate margine for defining calcareous soils.     

Black Catholicism and Black Lives Matter: the process towards joining a movement

This ethnographic study examines how Black Catholics identify with and respond to the Black Lives Matter movement. The study follows several national Black Catholic gatherings since the death of Mike Brown. Using an adaptation of Scott Hunt, Robert D. Benford, and David Snow's social movement frame analysis, I explore how Black Catholics define and construct the ongoing political issues within the Black Lives Matter movement. I discuss the conditions which contribute to Black Catholic’s participation, or lack thereof, in this social movement through the processes of diagnostic framing, prognostic framing, and motivational framing. I position the larger Black Catholic belief system within frame analysis, examine the relevance of the frames with the Black Catholic community, and analyse the frames’ timing with the Black Lives Matter cycle of protest. This research has implications for intragroup meaning making as Black Catholics start the process towards identifying with the Black Lives Matter social movement.     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

Protein production and purification

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.