rProtein A Seplife® Suno Affinity Chromatography Agarose Resin

Affinity Chromatography - Agarose

Appearance: White spherical beads
Type: rProtein A affinity chromatographic resin
Matrix: Highly cross-linked 4% agarose
Ligand: Alkaline tolerant recombinant protein A (E.coli)
10% dynamic binding capacity (mg/ml)**: ≥70
Particle size range (μm): 40-100
pH stability: 3-13 (operational), 2-14 (CIP)
Chemical Stability: Stable in all common aqueous buffers
Flow rate* (cm/h): max 750cm/h
Shipped as: 20% ethanol slurry

*Testing conditions: Chromatography column 16mm×200mm; column bed height 20cm; temperature 25°C; mobile phase water.
** Testing conditions: hIgG, RT=5min, Binding buffer: 20mm PB+0.15M NaCl, pH 7.4; elution buffer: 50mM Tris-HAc pH 3.5, Column 8mm*100mm, room temperature.

Column packing
Column loading should be performed in accordance with standard operating procedures. It is important to ensure that each material is at its working temperature. r-Protein A Seplife® Suno can be loaded the same way conventional Seplife agarose media are loaded.

Equilibration
Equilibrate the column with 3-5 CV of equilibration solution until the pH and conductivity remain unchanged.

Sample feeding
Sample pretreatment: The sample needs to be filtered with a 0.45μm filter membrane before loading to avoid clogging the chromatographic column. The amount of sample loaded is determined according to the nature of the sample and the amount of chromatographic media. Generally, the DBC of the sample on the chromatographic column can be used as a reference for sample volume calculation.

Elution
Generally, a low pH buffer is used for elution, and a buffer with a pH of 3.0-3.5 is selected: such as 0.1mol/L glycine-HCl, pH 3.0. The actual elution conditions can be optimized. After elution, it is recommended to adjust the pH of the mAb solution close to neutral. The neutralization solution could be 1mol/L Tris-HCl, pH 9- 10. Also, the chromatographic media should be returned to a higher pH.
The protein A leaching in the elution fraction can be quantified using Cygnus Protein A ELISA kit F610.
Cleaning
CIP method:
r-Protein A Seplife® Suno is an alkaline-tolerant affinity resin, which can withstand cleaning with 0.1M-0.5M NaOH solution. Generally use more than 3CV of 0.1–0.5 M NaOH, and a contact time around 15 minutes; then rinse with 5CV of equilibration solution or until outlet pH and conductivity are the same as inlet.
Note: The purification process of the fermentation broth may lead to impurities being adsorbed on the chromatographic media, resulting in an increase in pressure during CIP, an “up flow” CIP can be performed every cycle or after a certain number of cycles.

Storage
Sealed and stored at 2-8°C (preservation solution is 20% ethanol) in a ventilated, dry and clean place, do not freeze.