Microstructure control during twin roll casting of an AZ31 magnesium alloy

The existing twin roll casting technique for magnesium alloys suffers heterogeneity in both microstructure and chemistry and downstream processing is required to improve the strip quality, resulting in cost rise. In the present work, twin roll casting was carried out using an AZ31 magnesium alloy, with the application of intensive shearing melt conditioning prior to casting. The effect of process parameters such as pouring temperature and casting speed on microstructure control during casting and subsequent downstream processing was studied. Experimental results showed that the melt conditioning treatment allowed the production of AZ31 strips with uniform and refined microstructure free of centreline segregations. It was also shown that an optimized combination of pouring temperature and casting speed, in conjunction with a strip thickness control operation, resulted in uniformly distributed stored energies due to enhanced plastic deformation, which promoted recrystallization during casting and subsequent heat treatment. Strips prepared by twin roll casting and homogenization developed similar microstructural features to those prepared by twin roll casting followed by lengthy downstream processing by homogenization, hot rolling and annealing and displayed a weaker basal texture, exhibiting a potentially better formability.The EPSRC (UK

Downstream processing of co-amorphous olanzapine

Abstract of poster presented at the 12th PBP World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Wien, Austria, 11-14 May 2021 (virtual meeting)N/

Downstream processing effects on microbial viability

An increasing number of biotechnology production processes require a whole cell recovery step during downstream processing. Viability loss at this stage is generally undesirable as it can lead to reduced yields and separation difficulties further downstream. Comparisons between the effects of different types of equipment on a single strain of cells is rare. Such information can be invaluable when designing new processes or equipment. This work was carried out in the following stages: • Production of identical batches of cells for testing; • Use cells to develop a reproducible assay test system; • Use the assay system to assess the damage of various concentration techniques on the viable cells. A reproducible batch fermentation for the production of Pseudomonas putida ML2 cells was developed. Both the inoculum and fermentation stages were carried out in fully defined media. The fermentation used a minimum salts media and fructose as a carbon source. The bioluminescent ATP assay was tested as a novel method for measuring cell damage. The action of separation was simulated at laboratory scale by the use of a high pressure homogeniser. The ATP assay was tested on growth and stationary phase cells and results were compared to standard assay methods such as plate counts and protein release. Cells in stationary phase appeared to undergo a metabolic activation and so ATP analysis proved to be unreliable. Protein assay and plate count techniques were developed to increase reproducibility and accuracy by the use of multiple repeats and statistical analysis. Batch centrifugation was examined, multichamber bowl centrifugation proved to be less damaging to cells than tubular bowl centrifugation. Continuous centrifugation was carried out using a disk stack centrifuge with intermittent solids discharge. Damage was observed between 0–20% loss of viability. Cells were sampled directly from the separation bowl as well as from the discharged solids. Little difference was observed, suggesting that damage occurs in either the separation or feed zones prior to discharge. Microfiltration was carried out using a specially devised laboratory scale rig, designed at UCL. Runs were carried out under a number of different operating conditions. Increased cell damage was observed at higher cell concentrations and transmembrane pressures. Unlike centrifugation, results the protein release levels are less than the measured loss of viability suggesting a different mode of inactivation. Hydraulic shear stresses were estimated across the range of operations tested, as well as in the fermenter. Examination of the microfiltration results suggested that the action of the system was similar to that of a high speed fermenter

Downstream processing of recombinant retroviral vectors

Les vecteurs rétroviraux dérivés du virus Moloney de la leucémie murine (MoMLV) ont été utilisés pour livrer des gènes depuis plus de 20 ans et ils continuent d’être le meilleur outil disponible pour transférer de façon stable et efficace des gènes thérapeutiques dans différents types de cellules. Bien que la plupart des études précliniques de thérapie génique utilisent des surnageants bruts ou concentrés de vecteurs rétroviraux, l’étape de purification, pour éliminer le sérum et les impuretés dérivées des cellules hôtes contenus dans ces préparations, est incontournable pour les applications cliniques. Cette thèse décrit le développement de stratégies de purification des vecteurs rétroviraux. Au cours de ce projet, deux procédés complets de purification (à partir d’un surnageant brut de rétrovirus jusqu’au virus de grade clinique) ont été établis, vérifiés, et leurs performances ont été analysées en détail. La filtration sur membrane a contribuée à la clarification, la concentration, à l’échange de tampon et à la purification partielle des particules retrovirales à partir de surnageants à l’état brut sans aucune perte significative d’infectivité virale. Deux nouvelles méthodes de purification, spécifiquement adaptées aux caractéristiques biochimiques et physiques des particules rétrovirales, ont été développées. La première méthode de purification des particules rétrovirales, utilise la chromatographie d’affinité sur colonne d'héparine suivie d’un tamis moléculaire. L’avantage principal d’utiliser les techniques de chromatographie pour la purification des virus, est d’offrir la possibilité de purifier à grande échelle les rétrovirus de façon sélective et efficace. De plus, la chromatographie d’affinité sur colonne d'héparine a donné lieu à des taux de récupération exceptionnels de particules infectieuses et s’est avérée utile pour la purification des vecteurs rétroviraux produits par différentes lignées cellulaires indépendamment de l’enveloppe protéique utilisée pour le pseudo-typage. La deuxième méthode de purification est basée sur la technique de centrifugation zonale transitoire utilisant l’iodixanol comme milieu pour former un gradient. La force de cette technique repose sur les hauts niveaux de pureté obtenus en une seule étape de purification et la capacité à séparer les particules virales des espèces proches telles que les vecteurs défectueux et / ou les vésicules membranaires, qui posent un sérieux défi dans les procédés de purification. Les récupérations finales en particules infectieuses (~38%) et le degré de pureté atteint (plus de 95%) étaient comparables avec l’une ou l’autre des stratégies de purification utilisées. Les méthodes décrites dans cette thèse représentent une amélioration significative sur la méthodologie conventionnelle utilisant un gradient de densité de sucrose pour la purification des rétrovirus et contribuera certainement à l’avancement technologique dans le domaine de la thérapie génique.Retroviral vectors derived from the Moloney murine leukemia virus (MoMLV) have been used as gene delivery vehicles for more than two decades and continue to be the best available tool for stable and efficient transfer of therapeutic genes into various cell types. Although most gene therapy preclinical studies use crude or concentrated retroviral vector supernatants, purification to eliminate serum and host-derived impurities contained in these stocks is a must for clinical applications. This thesis describes the development of downstream processing strategies for retroviral vectors. During the course of this project, two complete multi-step purification schemes (from crude retrovirus supernatant to clinical-grade virus) were designed, tested and their performance analyzed in detail. Membrane filtration contributed to the clarification, concentration, buffer exchange and partial purification of retroviral particles from crude supernatants with essentially no loss in vector infectivity. Two novel purification methods specifically tailored to the biochemical and physical features of retroviral particles were developed. The first method consists of the chromatographic purification of retroviral particles by heparin affinity chromatography followed by size exclusion chromatography. The main advantage of employing chromatography technology for virus purification is that it offers the possibility to selectively and efficiently purify retroviruses on a large-scale. Moreover, heparin affinity chromatography resulted in exceptional recoveries of infective particles and proved to be useful for the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping. The second purification method is based on a rate zonal centrifugation technique using iodixanol as gradient medium. The power of this technique was revealed by the high levels of purity achieved in a single purification step and its potential to separate viral particles from closely-related species such as defective vector forms and/or cell membrane vesicles, all of which pose a serious challenge in downstream processing. The overall yield of infective particles (~38%) and level of purity achieved (over 95%) using either purification strategy was comparable. The methods described in this thesis represent a significant improvement over the conventional sucrose density gradient methodology used for retrovirus purification and will hopefully contribute to the technological progress in the field of gene therapy

Regulatory aspects of continuous downstream processing

Over the past few years, various technologies have been developed to enable a partially or fully integrated continuous bioprocessing platform. Many companies have explored the potentials of these innovative production systems and a lot of data has been published that support the technical viability of integrated continuous bioprocessing. The regulatory agencies, such as the FDA, have repeatedly expressed their support for these developments as they recognize the potentials for enhanced process and product quality control. In addition to this, continuous bioprocessing may allow the industry to move towards a more agile state that would allow biopharmaceutical products to become available to a wider audience at lower costs. Even though the regulators are supportive, no guidance was provided in terms of the regulators’ for submissions and how the guidelines as summarized in ICH Q8 and Q9 can be applied in continuous bioprocessing. Many different companies working towards integrated continuous bioprocessing platforms, all of which are relying on a combination of innovative and existing technologies. In order to facilitate and harmonize the interactions with the regulatory bodies, we have developed strategies and approaches that would cover the typical regulatory aspects of integrated continuous manufacturing platforms, covering both upstream and downstream processing. In this presentation, we will present and discuss regulatory strategies for integrated continuous bioprocessing. This will cover critical quality attributes, bioburden control and virus safety strategies for continuous bioprocessing platforms. In addition to this, some aspects of traceability, lot definitions and deviation management will be addressed

Case Study: Recombinant Bromelain Downstream Processing

This chapter presents details on the purification, formulation and drying of recombinant bromelain. The wide range of applications of recombinant bromelain has increased interest in finding viable purification techniques for large-scale production. An affinity chromatography technique was developed by Amid and co-workers (Expression, purification, and characterization of a recombinant stem bromelain from Ananas comosus, Process Biochem 46:2232–2239, 2011) to purify recombinant bromelain. However, this technique presented low recovery and small sample loading capacity and thus is not suitable as a purification tool in the largescale production of recombinant bromelain. An aqueous two-phase system is one alternative method that we use to purify recombinant bromelain, as it reliable and easy to scale up and has a low cost. As part of avoiding cysteine degradation, spray drying the purified recombinant protein with maltodextrin as an excipient provides the possibility of preserving its activity and creating fine particles that are suitable for end-product application. The processes of the purification, formulation and spray drying of recombinant bromelain are explained briefly

Microbial production of scleroglucan and downstream processing

Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-Biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong globalmarket, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a β-1,3-β-1,6- glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weightdepending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery,cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical(immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selectedstrains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.Fil: Castillo, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina. Universidad Nacional de Tucumán; ArgentinaFil: Valdez, Alejandra Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina. Universidad Nacional de Tucumán; ArgentinaFil: Fariña, Julia Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina. Universidad Nacional de Catamarca; Argentin