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

Inactive Publication Date: 2011-08-17
MILLIPORE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, one or more of these techniques are sometimes used in a discontinuous mode, however the overall yield is generally poor

Method used

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  • Flow through purification processes for large biomolecules
  • Flow through purification processes for large biomolecules
  • Flow through purification processes for large biomolecules

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention relates, at least in part, to novel and improved flow-through purification processes for separating large biomolecules, such as, for example, encapsulated viruses, virus-like particles and conjugate vaccines from one or more contaminants in a sample, where the process employs the use of at least one population of a solid porous particle which comprises a minimized external surface area per unit volume of the particles and an internal surface area per unit volume which is not decreased by more than 25% relative to a population of a similar particle which does not have a minimized external surface area.

Description

[0001] Related application [0002] This application claims priority for US Provisional Patent Application No. 61 / 284,368 filed on December 16, 2009, which is incorporated herein by reference in its entirety. Invention field [0003] The present invention relates at least in part to new and improved flow-through methods for separating large biomolecules in a sample, such as, for example, encapsulated viruses, virus-like particles, and conjugate vaccines from one or more contaminants. Purification method. Background of the invention [0004] After large biomolecules such as, for example, encapsulated viruses, infected host cells or virus-like particles in eggs or conjugate vaccines are produced, it is desirable to combine the biomolecules with the infected host cells or other components of the eggs such as DNA, RNA and host Cell proteins are separated to obtain a substantially pure population of the biomolecules. In addition, the produced biomolecule needs to be separated from some...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02
CPCC12N2760/16123C12N2760/16151C12N2795/12023C12N2710/10051C12N2760/16251C12N2710/10023C12N7/00C12N2795/12051C12N2760/16223
Inventor G·耶尔S·拉马斯瓦米K-S·郑
Owner MILLIPORE CORP
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