I am currently working on the His SUMO hGH purification process. After the purification of fusion protein, target protein undergoes a digestion stage and is separated from hGH using a SUMO protease enzyme. A NiNTA resin is used to separate His SUMO from hGH. When sample is loaded on the resin His SUMO and enzyme are absorbed to nickel and target protein hGH enters the flow through . According to pharma coupé laws the final product must be transparent and colorless. But the color of protein solution is yellowish. What is the reason for this and is there anybody who can help me remove the yellowish color?
this is the specification of final product
native protein 97%, monomer 99%, HCP< 50ng/mg, HCDNA < 10pg/dose, final concentration is 6.8 mg/ml, buffer is 5mM Histidine in pH 6.2
Have you considered performing a secondary purification step such as ion exchange or gel filtration / buffer exchange? In the former instance I am thinking about capturing the protein and allowing the contaminant to wash away. However, if the colour is due to a small molecule, perhaps the GF column (assuming the size exclusion characteristics are suitable) could suffice to pseudo-adsorb the colour while allowing the GH to flow through.
Secondly, have you considered the possibility that the protein is intrinsically not transparent / colourless?
Best of luck, Robin
You could run 5-10 uL of your final sample on SDS-PAGE to see if it is pure. I see you have good stats, but I don't know how these were determined.
Old imidazole will turn a yellow color.
Do you mean "pharmaceutical" laws?
Usually if proteins samples are yellow, they either have a variety of other proteins in it, have yellow ligands, or an old buffer.
The histidine used in the elution buffer may be stripping reduced niquel from the column turning your solution yellow. Do you need such a low pH? Maybe you could use imidazol instead at a higher pH and then get rid of it by a simple desalting step.
Dear all
This is the whole procedure for purification of hGH
First eluting carried out by use of imidazole 250mM in Ni-NTA, followed by desalting by applied G25 resin, So the final concentration of imidazole decrease to 5mM. Digestion process was carried out by sumo protease enzyme. Final solution load again on Ni-NTA and target protein collected in flow through. base of buffer in this step is 20 mM tis, 150 mM NaCL, 10 mM imidazole. After that buffer of the solution was changed to 20 mM tris, by use of TFF system. Final solution was loaded through Q sepharose column and elution of target protein was carried out by 20mM tris, 250 mM NaCL, pH 6 the buffer of final solution was change to histidine in pH 6.2 to be stored for long time.Properties of final protein was mentioned in question.
I don't know why the final solution is a little bit yellow!!!!!!
According to pharmacopoeia standard tests, the comparability of production scaled of DS with the standard required providing the same protein concentration and same buffer. Personally speaking, as per CQAs you already mentioned (RP, SE, HCD, HCP, ...) that have met the acceptance ranges don't be worried about the color. maybe related to the buffer components. Try to use the fresh raw materials.
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