Flow through purification processes for large biomolecules
A technology for biomolecules and pollutants, applied in the field of large biomolecules circulation and purification, can solve problems such as poor total yield and achieve the effect of recovery and improvement
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Embodiment 1
[0100] Example 1: Determination of the outer surface area per unit volume of beads
[0101] The external surface area per unit volume of beads of different sizes can be determined as described herein. In an exemplary experiment, the external surface area per unit volume of the following beads was estimated using the mathematical method described above. The average diameter of Q SepharoseHP was 34 μm, the average diameter of Q Sepharose FF was 90 μm, and the average diameter of Q Sepharose BB was 200 μm (GE Healthcare).
[0102] For example, using Equation I described herein, the external surface area per unit volume of a bead with an average diameter of 34 μm can be calculated as follows:
[0103] A es / V = 6 η d = 6 * 0.74 34 X 10 - 4 = 1305 cm - 1
[0104] The external surface area of all other AEC beads was calculated similarly and summarized in Table 1.
[0105] Table I
[0106] Type of beads
Bead size ...
Embodiment 2
[0116] Example 2: Estimate the internal surface area per unit volume of the beads using static binding capacity
[0117] As described in Example 1, the internal surface area per unit volume of QS4FF (90 μm) and QSBB (200 μm) beads with larger and minimized external surface areas, respectively, was estimated by detecting their static binding capacity to BSA. BSA has been widely used as a model protein in chromatography. It has an average molecular weight of approximately 66KD and a pi of ~5. BSA is representative of the size and charge of most host cell proteins.
[0118] In an exemplary experiment, the static binding capacity of QS4FF and QSBB beads to BSA (Sigma Aldrich Corporation) was measured in two different types of buffers at two different pHs. The test was performed by filling a 1 ml column containing QS4FF or QSBB medium, slurring the medium in 4 ml buffer, and adding 0.5 ml of the slurry to 14.5 ml of a BSA solution having a concentration of 2 mg / ml. After 4 hours, th...
Embodiment 3
[0124] Example 3: Recover virus-like particles (i.e. 100%) from two anion exchange media of different bead sizes. nm BSA coated styrene particles) comparison
[0125] In an exemplary experiment, two separate anion exchange media with different bead sizes were tested for the recovery of virus-like particles. In particular, an anion exchange medium was tested, Q Sepharose 4FF beads (QS4FF, GE Healthcare) with an average diameter of 90 μm and Q Sepharose large beads (QSBB, GE Healthcare) with an average diameter of 200 μm were useful for recovering 100 nm bovine serum albumin ( BSA) Static binding capacity of coated styrene particles (Postnova Analytics), which are exemplary virus-like particles having a size and charge similar to influenza virus or adenovirus. QS4FF and QSBB media contain positively charged quaternary ammonium groups that can bind to BSA-coated latex particles. It is generally assumed that the static binding capacity of these beads for BSA-coated particles shoul...
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