Column Packing Instructions

Column packing should be done according to standard operating procedures. It is important to ensure that each material is at its working temperature. Seplife® Oligo dT(20) resin is supplied in a slurry in 20% ethanol. In order to determine the necessary volume of resin to pack a defined column, it is necessary to determine or consider the slurry concentration and the compression factor. Seplife® Oligo dT(20) resin has a compression factor of 1.12; factor is determined as the ratio of gravity settled volume to packed bed volume.  

Two column packing procedures may be applied: Constant pressure packing and Constant flow packing.

Column Equilibration

Equilibrate the column with an appropriate buffer applying approx. 5 column volumes (CV). Ensure the  conductivity and pH of the effluent are exactly the same as the equilibration buffer. The equilibration solution  can be a neutral pH buffer such as Tris or phosphate buffer, for example 10mM Tris-HCl, 0.5M NaCl, 1mM  EDTA (pH 7.4). The affinity interaction between the poly DT on the resin and the poly A of the mRNA molecule  is promoted at elevated conductivity, therefore a NaCl concentration of at least 0.2 -0.5M is typically used.  The equilibration buffer should present a suitable conductivity to promote mRNA binding.

Sample preparation and loading

The mRNA sample should be prepared in a buffer solution similar to the equilibration buffer and at the  advantageous conductivity. If the RNA sample requires denaturation, heat the sample at 65-70ºC for 10- 15min and immediately place it in an ice bath. This treatment helps disrupting secondary structures and may  improve purification performance.

mRNA’s can differ greatly in size and structure which influence the binding capacity to the Seplife® Oligo  dT(20). Sample solubility, concentration, impurity profile as well as the buffer composition affect the binding  capacity  to  Seplife®  Oligo  dT(20)  and should  be  optimized.  Typically  the  sample  application  can  be  performed at a retention time of minimum 3 minutes.

Ensure the sample is filtered (0.22µm or 0.45µm filter) and if possible degassed before application to the  column.

After completing the sample application, rinse the column with the equilibration buffer (2-3CV) and with  optimized reduced conductivity buffer to improve impurity removal. For example a solution of 10mM Tris- HCl, 0.2M NaCl, 1mM EDTA (pH 7.4) can be applied for 3-5 CV to encourage impurities removal before the  sample elution.

Elution

At reduced conductivity electrostatic repulsion occurs between the polyA and polyT resulting in separation  of the pairs. This principle is followed for the mRNA elution from the Seplife® Oligo dT(20) chromatographic  resin. Reducing the salt concentration in the buffer or even using water results in the mRNA elution. For  example a solution of 10mM Tris-HCl, 1mM EDTA (pH 7.4) 3-5CV or apply 3-5CV water for sample elution.  The elution conditions should be optimized to ensure the optimum conditions are applied for a high purity and recovery of the mRNA.    

Elution may be enhanced by heating the elution buffer or the column at 65ºC.

Regeneration

The Seplife® Oligo dT(20) chromatographic resin should be rinsed with 3-5CV of water or 20% - 30% ethanol  solution depending on the severity of the contamination. The regeneration process should be followed by  CIP and column wash with the equilibration buffer or equilibrate in storage solution.

Cleaning-in-place (CIP)

The Seplife® Oligo dT(20) chromatographic resin can be sanitized 3-5CV of 0.1M NaOH solution. If a more  severe CIP is require, a 0.5M NaOH solution (3-5CV) can be applied.

After CIP, rinse the column with the equilibration buffer 3-5CVs.

Storage

Ordering information

Production date: See label

Expiry date: 5 years, under proper storage conditions