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Data File 28-9207-54 AA Custom Designed Media

AVB Sepharose High Performance

AVB Sepharose™ High Performance is an affinity medium OH O
H
designed for the purification of adeno associated O N Ligand
virus (AAV). Key characteristics of AVB Sepharose High Matrix N
H
Performance include:
• Fast, one-step purification of adeno associated viruses Fig 1. Partial structure of AVB Sepharose High Performance.
of several subclasses
• Prepacked HiTrap™ columns for simple operation with a Table 1. Main characteristics of AVB Sepharose High Performance
syringe, pump, or chromatographic system
Matrix Cross-linked 6% agarose
• Excellent scalability
Average particle size 34 µm
Adeno associated viruses are of increasing interest as Ligand 14 kD recombinant protein
potential vectors for gene therapy. To enable the use of produced in S. cerevisiae. Binds
AAV in clinical applications, an efficient and high quality AAV of subclasses 1, 2, 3, and 5
production process is needed, including the downstream Capacity Typically > 1012 genome copies/mL gel
purification process. The process needs to be robust, with Recommended flow rate Up to 150 cm/h at 30-cm bed height
high yields, high purity, and low leakage of ligand. In current Maximum back pressure 0.3 MPa, 3 bar
purification protocols density gradient centrifugation is pH stability
typically used, followed by several chromatography steps, Long term 3–10
giving a process with low yield and poor scalability. Short term 2–12

Medium characteristics
AVB Sepharose High Performance is based on a highly chromatographic parameters (e.g., sample load, flow velocity,
cross-linked 6% agarose matrix, which enables rapid bed height, regeneration, cleaning-in-place) to establish the
processing of large sample volumes. The ligand, a 14 kD conditions that will bind the largest amount of target molecule,
recombinant protein, is attached to the base matrix via a in the shortest time and with the highest product recovery.
long, hydrophilic spacer arm to make it easily available for When using AVB Sepharose High Performance the AAV
binding of the virus (Fig 1). can be applied directly from clarified AAV vector cell
Table 1 summarizes the main characteristics of AVB lysate. Typically loading, washing, and elution of the virus
Sepharose High Performance. of the column is applicable with conventional buffers
(e.g., PBS, tris, citrate). Binding of the virus to the column
Functional Principles occurs around neutral pH and elution of the virus is
Affinity chromatography exploits an immobilized ligand done by lowering pH, for example to pH 2 to 5. Since AAV
that adsorbs a specific molecule or group of molecules is sensitive to very acidic conditions, it is important to
under suitable binding conditions and desorbs them under minimize the exposure to low pH during elution. Therefore
suitable elution conditions. These conditions depend on the it is recommended that collected elution fractions are
target molecule, feed composition, and chromatography neutralized immediately.
medium, and must be studied together with other

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Regeneration should restore the original function of Cleaning-in-place and sanitization
the medium. Depending on the nature of the sample,
A cleaning or sanitization protocol has to be designed for
regeneration is normally performed after each cycle,
each application. A suggested cleaning protocol is to use a
followed by re-equilibration in start buffer. To prevent build
solution of low pH, for example 0.1 M phosphoric acid, alone
up of contaminants over time, more rigorous protocols may
or in combination with sodium chloride or ethanol. However,
have to be applied (see Cleaning-in-place and sanitization).
prolonged exposure (i.e., several days) to pH < 2 should be
avoided due to a slow decomposition of the agarose matrix
Stability at low pH. Sodium hydroxide should be used with care due
The ligand is linked to the Sepharose High Performance to the low stability under alkaline conditions.
base matrix via a stable amide bond. In a study where
AVB Sepharose High Performance was stored at room Storage
temperature at different pH values for one week it was
The recommended storage conditions are 20% ethanol at
shown that the leakage is low between pH 2 and 12 (Fig 2).
4°C to 8°C. AVB Sepharose High Performance is supplied
At higher pH there is a leakage of both carbon and nitrogen,
pre-swollen in a 20% ethanol solution.
indicating hydrolysis of the ligand.

300 Ordering information

Carbon
Nitrogen
Product Quantity Code no.
250
AVB Sepharose High Performance* 75 mL 28-4112-01
200 AVB Sepharose High Performance* 1L 28-4112-02
Leakage (ppm)

Prepacked HiTrap column* 5 x 1 mL 28-4112-11

150
Prepacked HiTrap column* 1 x 5 mL 28-4112-12
100
* This product is part of our Custom Designed Media program. If you are interested in large-scale
quantities, please contact your local GE Healthcare representative.
50

Literature
0
0 2 4 6 8 10 12 14 Affinity Chromatography Handbook 18-1022-29
pH
Affinity Columns and Media, Selection Guide 18-1121-86
Fig 2. Stability of AVB Sepharose High Performance at different pH.

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Sepharose is a trademarks of GE Healthcare Companies.
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GE Healthcare Bio-Sciences AB © 2007 General Electric Company – All rights reserved.
First published March 2007.
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28-9207-54 AA 03/2007