More and more biomolecules are being produced by the biotechnology industry for applications ranging from medicine and food to engineering materials. Liquid chromatography plays a center-stage role ...in a typical downstream process producing biomolecules such as recombinant proteins. Rigid gigaporous media are porous particles possessing large transecting through-pores with a pore-to-particle diameter ratio of
d
pore/
d
particle> 0.01. They allow convective flow in the large through-pores, while the smaller diffusion-pores (typically several hundred angstroms in size) supply the needed surface areas. Because of the transecting gigapores, a portion of the mobile phase flows through the pores in addition to fluid flow in the interstitial spaces between the particles in a packed-bed column. This considerably lowers the operating column pressure drop. This lower pressure drop makes axial-direction scale-up of chromatographic columns possible to avoid pancake columns that invariably degrade separation resolution. The large gigapores also make the binding sites on the diffusion pore surfaces more accessible, thus increasing the loading capacity of large protein molecules that can be hindered sterically if only diffusion pores are present. This work discusses the development of rigid gigaporous media and their potential impact on the design of multi-stage downstream process from the angle of multi-scale analysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Protein antigens are key components of prophylactic vaccines against infectious diseases. The purification of protein antigens to achieve consistent product purity and quality is an integral part of ...the protein subunit vaccine product development process. Analogous to the biotech and biopharmaceutical companies, the vaccine industry has benefited from the recent progresses made in protein purification technology development or improvement, including disposable technology, membrane technology, high throughput process development, process automation, and process data tracking and analysis. This chapter reviews the methods, technologies, and processes used in protein subunit purification to support vaccine production and manufacturing. Some of the key technical issues encountered during the protein subunit vaccine purification are discussed, as well as the current status and future application of process analytical technology. In addition, an outlook and trends to support future needs in protein subunit vaccine purification are explored.
Models derived from a computational fluid dynamics (CFD) package (CF4X) were used to predict the fate of micro-organisms lost in aerosols as an incidental feature of the normal operation of ...bioprocess equipment. The model predicts the tracks of particles from their assumed source to their point of capture in an Aerojet cyclone impinger. In a set of controlled experiments micro-organisms were sprayed into a small cabinet (volume=0.36m3). The efficiency which CFD predicted for their capture in the cyclone was compared with the actual efficiency measured with a quantitative polymerase chain reaction (QPCR) which was specific for the released organism. The capture efficiency was constant at about 40% over six orders of microbial concentration in the aerosol. This is consistent with the CFD predictions provided the coefficient of restitution of 50% of the particles is about 0.2. This coefficient determines the momentum with which the particles will rebound from the surface and continue their flight. Experiments in a bioprocess pilot plant, where QPCR was used to measure the quantity of process organisms and CFD was used to predict the capture efficiency of the released aerosols, confirm that the release from wellmaintained equipment is very low. Only when the primary containment is broken as part of the normal operation of the process is there a significant release. Even this is small, amounting to about 10 μl of fermentation broth, or 0.2μl of the concentrated process fluid. These uantitativemethods for measuring levels of containment should aid the design of equipment and processes in which a proper balance is struck between the releases which are incidental to the normal operation of a process and the much larger losses which would follow an accidental failure in the equipment. In the long term this balance defines the safety of the containment.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We have modified a standard column chromatography method for the preparation of albumin from human plasma. The proposed method utilizes Sephadex G-25, euglobulin precipitation, DEAE-Sepharose, ...ethanol heat treatment and Sephacryl S-200 HR. The procedure differs from that normally used by the introduction of Schneider's ethanol/heat treatment step and the elimination of a CM-Sepharose step, after the DEAE-Sepharose step. The proposed method was used to produce 15 batches of albumin, 100 liters of plasma per batch. Purity of more than 99% was obtained at an average yield of 26.5 g/l plasma for the albumin produced. All batches presented 99.2 to 99.9% monomer and 0.1 to 0.8% dimer, with no larger polymers detected. The utilization of the final gel filtration step eliminated highly polymerized albumin usually obtained during the process. The advantages of the proposed method are reduction in the overall process time from 6 to 5 days, elimination of the CM-Sepharose step and the reduction of 6 to 4 columns in series for the Sephacryl S-200 HR step.
Lightweight 3D data is useful throughout design and manufacturing. Of course it also benefits local applications. However, it is always important consider overall optimization when promoting 3D data. ...SONY is a company that has practiced this. They started using XVL in 2003 and learned how to use 3D data in their trial production processes. They learned that it was not easy to achieve best practice; people in downstream processes found it difficult to obtain 3D CAD data during the design process as well as identify which is the correct 3D data. To resolve these problems, SONY built a 3D data information distribution system to exchange XVL data with all their employees. Because they had such an information infrastructure, any department could use 3D data freely when they wanted. The company also launched projects to promote use of 3D data at the same time, which helped increase awareness of the usefulness of 3D data amongst their employees. Such endeavors at SONY were pioneered by SONY GLOBAL SOLUTIONS (SGS), which provides the IT infrastructure for the whole of the SONY group. Leading these endeavors were Mr. Watanabe, head of the Engineering Solution Division, and Mr. Sekiya, head of the Engineering Information Solutions Division. The person responsible for building the actual infrastructure was Mr. Yoshii. Mr. Tsukamoto supervised the introduction and promotion of XVL. This chapter discusses how the leading Japanese company SONY started to use 3D data. Their experience in building a 3D data distribution infrastructure should serve as an important reference to companies planning company-wide use of 3D data.
In the manufacturing industry, it is now common practice to implement 3D design using 3D CAD, review the 3D information by virtual simulation on computer (CAE), and transform this into a real object ...on a machine tool (CAM). But such uses of 3D data make up only a small part of the manufacturing process. Leading companies are starting to use 3D data not only in design, but also throughout manufacturing and even in downstream processes such as service and marketing. This chapter discusses the best practices in Japan, how to successfully use 3D data, and the key points for building a 3D data system.
The purification of a (recombinant) protein produced by animal cell culture, the so-called downstream process (DSP), tends to be one of the most costly aspects of bioproduction. Chromatography is ...still the major tool on all levels of the DSP, from the first capture to the final polishing step. In this chapter we will first outline the commonly used methods and their setup, in particular ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography (AC), and gel filtration (gel permeation chromatography GPC, size exclusion chromatography SEC), but also some lesser known alternatives. Then the rational design of a downstream process, which usually comprises three orthogonal Chromatographic steps, is discussed. Finally, process variants deviating from the usual batch-column/gradient elution approach will be presented, including expanded bed, displacement, and continuous annular chromatography, as well as affinity precipitation.
