We are currently working on purification of human recombinant Factor IX (rhFIX) using three independent chromatographic steps. We experience significant loss of activity during purification and would like to try different buffer compositions to maintain the stability of hrFIX throughout the process. What would you suggest? I have read that arginine works well (among other aminoacids), do you agree? At which concentration would you use it? Thanks a lot!
Dear Dr. Luciana Sampieri
The pH and ionic strength of the buffer can significantly impact rhFIX. Buffers with neutral pH values and moderate ionic strength are generally preferred for rhFIX purification. Commonly used buffer is Tris-HCl. It is an effective buffer for protein purification, providing a stable pH environment during various purification steps, including chromatography. The pH of Tris-HCl buffer is typically adjusted to a range suitable for protein stability and separation, often around pH 7.0-8.0.
You should add NaCl to Tris-HCl buffer. The addition of NaCl helps to increase the ionic strength of the buffer. Higher ionic strength can sometimes promote the binding of FIX to the resin, especially in ion exchange chromatography. NaCl can help stabilize FIX, particularly during the purification process, by reducing protein aggregation and maintaining its native conformation. NaCl can be used to elute FIX from the resin by increasing the salt concentration in the buffer. You can do this stepwise, with increasing NaCl concentrations to achieve a desired elution profile. The concentration of NaCl used in purification buffers for rhFIX, range from 50 mM to 500 mM, but this will depend on the specific resin and purification protocol.
There are other buffers which can also be used like sodium citrate or phosphate buffer in conjunction with NaCl during rhFIX purification. But I would prefer Tris-HCl.
I agree with you regarding the use of arginine. Arginine, with its positively charged guanidino group, can interact with negatively charged components in the rhFIX formulation, helping to maintain the protein's structure and prevent aggregation or degradation. It can form hydrogen bonds with hydroxyl groups in other molecules, which can help to stabilize the protein structure by influencing the formation of tertiary structure.
Arginine can be used in buffers to maintain the correct pH and ionic strength, which is important for protein stability. Arginine can be used in buffers at concentrations ranging from 0.1 to 2 M to stabilize human recombinant Factor IX (rhFIX) during purification.
Regards,
Malcolm Nobre
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