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Note: Adsorbent name: XL stands for extreme load and consists adsorbents with very high loading capacities. Ligand: DEAE, diethyl aminoethyl; Q,quatenary amine; SP, sulphopropyl; IDA, iminodiacetic acid; rProtein A, recombinant protein A. Functionality Functionality of STREAMLINE Heparin was evaluated by its ability to bind antithrombin from bovine plasma. Ligand density Ligand densities for STREAMLINE DEAE, Q XL, SP, and SP XL were determined as millimole charged groups per milliliter of adsorbent. For STREAMLINE Chelating, ligand density is expressed as Cu2+ binding capacity in imole per milliliter adsorbent. Ligand density for STREAMLINE Heparin is milligram of heparin bound per milliliter adsorbent. For STREAMLINE rProtein A, ligand density is expressed as milligram protein A per milliliter adsorbent. Breakthrough capacity For STREAMLINE DEAE, SP, and rProtein A, breakthrough capacity was determined at 300 cm/h linear flow velocity in expanded-bed mode (15 cm settled bed height) at 1% breakthrough of 2 mg/mL bovine serum albumin (DEAE) or lysozyme (SP) or human immunoglobulin G (IgG) (rProtein A). For STREAMLINE Q XL AND SP XL, the breakthrough capacity was determined at 300 cm/h in packed bed mode (10 cm bed height) at 10% breakthrough of 2 mg/mL bovine serum albumin (Q XL) or lysozyme (SP XL). Breakthrough capacity for STREAMLINE Chelating was determined in packed bed mode at 300 cm/h flow velocity (5 cm sedimented bed height) at 1% breakthrough of 2 mg/mL bovine serum albumin. Total binding capacity Total binding capacity was determined in packed bed mode at 50 cm/h flow velocity (15 cm bed height) at 100% breakthrough of 2 mg/mL bovine serum albumin (DEAE, Chelating), lysozyme (SP), or human IgG (rProtein A).

Note: Adsorbent name: XL stands for extreme load and consists adsorbents with very high loading capacities. Ligand: DEAE, diethyl aminoethyl; Q,quatenary amine; SP, sulphopropyl; IDA, iminodiacetic acid; rProtein A, recombinant protein A. Functionality Functionality of STREAMLINE Heparin was evaluated by its ability to bind antithrombin from bovine plasma. Ligand density Ligand densities for STREAMLINE DEAE, Q XL, SP, and SP XL were determined as millimole charged groups per milliliter of adsorbent. For STREAMLINE Chelating, ligand density is expressed as Cu2+ binding capacity in imole per milliliter adsorbent. Ligand density for STREAMLINE Heparin is milligram of heparin bound per milliliter adsorbent. For STREAMLINE rProtein A, ligand density is expressed as milligram protein A per milliliter adsorbent. Breakthrough capacity For STREAMLINE DEAE, SP, and rProtein A, breakthrough capacity was determined at 300 cm/h linear flow velocity in expanded-bed mode (15 cm settled bed height) at 1% breakthrough of 2 mg/mL bovine serum albumin (DEAE) or lysozyme (SP) or human immunoglobulin G (IgG) (rProtein A). For STREAMLINE Q XL AND SP XL, the breakthrough capacity was determined at 300 cm/h in packed bed mode (10 cm bed height) at 10% breakthrough of 2 mg/mL bovine serum albumin (Q XL) or lysozyme (SP XL). Breakthrough capacity for STREAMLINE Chelating was determined in packed bed mode at 300 cm/h flow velocity (5 cm sedimented bed height) at 1% breakthrough of 2 mg/mL bovine serum albumin. Total binding capacity Total binding capacity was determined in packed bed mode at 50 cm/h flow velocity (15 cm bed height) at 100% breakthrough of 2 mg/mL bovine serum albumin (DEAE, Chelating), lysozyme (SP), or human IgG (rProtein A).

where u0 is the superficial velocity, H is the expanded bed height, and e is the bed voidage in expanded mode. e is calculated from H/H0 = (1 — e0)/(1 — e). In a settled bed, the bed voidage (e0) is approximately 0.4. The higher the value for N, the lower the value for Da, indicating a low degree of back-mixing (a flow profile approaching plug flow). The relation between Nand Da is derived from the coupling between the tanks in series and the axial dispersion models by comparing the variances between the models' step-response curves. It is not completely accurate to make this comparison because the responses of the two different models are never identical (56). But for a system with a low degree of back-mixing, this way of calculating the axial dispersion is accurate.

Feedstock Application. The feedstock is applied in expanded bed mode, usually at a constant flow velocity (e.g., 300 cm/h). The adaptor is kept at its uppermost position, or at a position 5-10 cm above the expanded-bed surface. The uppermost position is usually the choice when a new (unknown properties) feedstock is applied or when there are feedstock batch variations, making it difficult to predict how the feedstock will behave in the expanded bed from run to run. If feedstock properties, such as viscosity or biomass or others that affect expansion, are known to be consistent and do not vary, then the adaptor can be left at a position closer to the bed surface. The feedstock is always stirred during application to prevent cells and debris from settling. If there is a build-up of cells and debris on the adaptor, the column is intermittently back-flushed. The duration of a back-flush is about 5-10 seconds, and the interval between back-flushes is usually 10 to 30 minutes, depending on the properties of the feedstock. Another method of applying the feedstock is to change the flow velocity (normally lowering it) during the run, so that the expansion height is kept constant. It has been reported that this approach, under certain conditions, may give a higher productivity than the constant flow velocity method does (58).

Wash. The wash is performed in expanded-bed mode at the highest possible flow velocity. This means that the flow velocity at the beginning of the wash is often the same as during feedstock application, but as the bulk of the cells and debris are washed out, the flow velocity is increased. At the beginning of the wash, the column is usually back-flushed as described in the previous section. During the wash, when the bulk of the particulates has been washed out, the adaptor is lowered closer to the bed surface. Lowering the adaptor significantly reduces the volume of wash buffer and shortens the time required for the wash step. If, instead, the wash is performed with a wide gap between the bed surface and the adaptor, mixing of the liquids is usually severe (a low density wash buffer being introduced after the high density/viscosity feedstock), resulting in a large wash volume. After the wash, when the UV curve has levelled out, the column is back-flushed to remove any trapped cells or debris from the distribution system. The bed is allowed to settle and the adaptor is lowered to the bed surface. The wash is continued in settled-bed mode, in

Product Product Waste

Feedstock

Inlets

(e.g., buffers and cleaning/ saniti-zation fluids)

Product Product Waste

Hydraulic fluid

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