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    Home > Biochemistry News > Biotechnology News > The fermentation liquid clarification process in single-resistance production.

    The fermentation liquid clarification process in single-resistance production.

    • Last Update: 2020-09-15
    • Source: Internet
    • Author: User
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    Antibody therapy is considered to be the most popular recombinant protein treatment.
    can be expressed at high levels in soluble and stable forms in cell cultures.
    humanized antibodies that use mammalian cells to express ionization have been widely used, and cell removal and fermentation solution clarification are of great significance for the development of subsequent processes.
    typical fermentation liquid clarification process Mammalian cultured cells are susceptible to shearing forces, easily broken to release proteases and other host cell proteins, thereby affecting the stability and purity of antibodies.
    addition, processing time is important for cell removal and clarification, as cell cultures are nutritious and easy to germ, resulting in contamination of proteins and process facilities.
    centrifugation and cut flow filtration are often used in industry to remove cells, and in recent years deep filtration has become more and more widely used.
    flocculants are also sometimes used to enhance the effects of cell removal and centrifugation.
    sterilization filtration is generally used as the last step in cell removal and clarification before the column.
    the main process structure of the current cell removal and fermentation fluid clarification is shown in Figure 1.
    1 Common clarification process for large-scale animal cell fermentation fluids First-level deep filtration, second-level deep filtration, sterilization filtration Centrifuges Are mostly used in the Disk-stack centrifugation industry to remove cells and their fragments.
    Disk-Stack centrifuges can be continuously centrifuged, increasing processing volume and reducing processing time.
    Figure 2 shows the structure of the Disk-stack centrifuge.
    In the process of centrifugal process development, there are many parameters to be optimized, such as the speed of the filling, the speed of the centrifuge, the removal frequency of the filter cake layer, etc., these parameters will affect the stability of the cells, processing time and antibody yield.
    can be used to reasonably balance claims by optimizing these parameters.
    2 Disk-stack centrifuge structure (courtesyofWestfalia Inc.) (1) feed, (2) drain outlet (light phase), (3) centrifugal pump, (4) disc, (5) slag storage space, (6) slag drain, (7) drain Slag piston valve, (8) centrifugal trap, (9) discharge outlet (heavy phase), (10) nozzle, (11) metering water injection/opening water, (12) timer. Cut-flow filtration Cut-in flow filtration is also widely used in cell clean collection, as is centrifugation.
    the advantage of cutting flow filtration is that particle-free cells can be cleared in one step, without the need for additional filtration steps.
    the concentrated polarization layer that is shown to form in the membrane can seriously affect the treatment of the membrane and become a major problem of filtration of the cut-in flow.
    addition, irreversible membrane clogging caused by particles can reduce membrane life and become another major problem.
    There is a lot of research on weakening the concentrated polarization layer, such as the use of rotating the membrane plate to increase the flow rate near the membrane surface, thereby washing off the concentrated polarization layer, and one way is to use periodic backwashing to wash off the polarization layer.
    for the cut-in flow filtration system, the ratio and yield are two important factors that must be considered.
    the film area required to process cell fermentation fluid, which is of great economic significance.
    too high will speed up the clogging of the membrane and reduce the life of the membrane, so the optimization of the vector is very important.
    and aperture of the film are also very important and decisive for the amount of communication and the ability to clean.
    addition, the load of the membrane, i.e. the fermentation liquid product treated per membrane area, is also an important parameter, which needs to be optimized in depth, and the load optimization of the membrane is very important for the amplification of the process.
    Deep filtration Deep filtration refers to the use of porous media to intercept solid particles from the flow phase instead of surface intercepts, and the surface of the general medium has a positive charge, which can be electrostated to intercept impurity particles.
    principle is shown in Figure 3.
    use deep filtration when there are more particles in the fluid.
    deep filter media generally consists of cellulose or polypropylene fiber column beds, filter media (such as diatom soil) and adhesives (used to create flat interfaces).
    because centrifugation does not completely remove cells and their fragments, and the cut flow is too expensive, deep filtration is often used after centrifugation, can also be used alone for the clarification of cell fermentation fluids.
    because the aperture of the deep filter medium is not equal, a sterilization filtration process is followed immediately after the deep filtration to benefit the purification of the column.
    3 The development of the filtering process of deep filter media needs to select the filter medium first, and the chemical properties, aperture and charge properties of the medium are the factors to be considered.
    in the process of selecting the medium, the turbidity of the filtered liquid and the increase of the pressure difference of the filtered medium will be two important parameters.
    important parameter for selecting a medium is the change in pressure difference after deep filtration and then sterilization.
    3M's deep filtration products, as shown in Figure 4, offer different sizes of products required for small, pilot and mass production from the laboratory.
    Figure 4 is suitable for all-in-one deep filtration products of different sizes For the clarification process of cell fermentation fluid, the 3M recommended solutions are single-stage deep filtration and two-stage deep filtration two modes, can completely replace centrifugal steps, filter out liquid turbidity is less than 10NTU, the current is generally more than 100L/M2.
    specific selection scheme is shown in Figure 5 below.
    5 cell fermentation solution clarification process, 3M also has a strongly charged ZB series of products that can be used in clarification processes with specific requirements for DNA or HCP removal.
    flocculation flocculation effect through a variety of ways is the fermentation liquid in small particles into large particles to enhance the subsequent filtration effect.
    flocculants reduce the small particles that clog the membrane holes by adsorbing small particles, while reducing the compressibleness of the particles, thereby reducing the filter resistance of the filter cake layer.
    commonly used flocculants are diatomite and pearl rock.
    but adding these soil materials to the fermentation fluid may not be a problem for the products expressed in the primary core, but there may be application limitations for clean eun fermentation fluids.
    so fixing these materials with deep filtration is often used to clarify fermentation fluids.
    the appropriate process unit for cell removal and clarification depends on the size of the production and the process facility.
    large-scale process has led to a protein yield of more than 98% in the cell removal and clarification process.
    , centrifugation has become a popular method of cell removal (larger than 2000L), disk-stack centrifugation is easy to clean and sterile than many large cut-in flow devices.
    the use of centrifugal devices will inevitably require a secondary clarification process of fermentation fluids, as well as the need to provide relevant verification information and relatively high maintenance costs.
    deep filtration is increasingly accepted by biopharmaceutical customers because of its easier process amplification, lower cost, short processing cycle, and one-time deep filtration process because of its more flexible and simple operation.
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