IgG purification: Multimerization... - (Dec/13/2007 )
Hello everybody!!
I have some troubles in my IgG purification...
well I purify rabbit IgG "classically" with protein A matrix (Prosep A, Millipore) and elute them @ pH 2.0 in citric acid. SE HPLC analysis shows a beautiful monomer form.
As I have to adjust pH to 7.5, SE HPLC shows lots of multimerization - dimerization after pH adjustment.
Questions:
How can I lower the multimerization ??
How can I suppress/reduce these mulitmeric forms to recover a maximum of monomeric form IgG??
Thanks!
Goldo.
instead of adjusting the pH after collection, which will allow the antibody to sit at low pH for an extended period of time, you can collect the fractions into a tube containing a cushion of 1M buffer (phosphate or tris), about 1/20th of the fraction volume. this will adjust the pH immediately after collection and may prevent the multimerization that you are seeing.
Thank you for your answer...
In fact, multimerization appears as soon as I raise the pH...
I have tried to raise the pH immediately (on a 20 mls aliquot) to 7.5 with 1 M Tris unadjusted pH and multimers appears anyway.
the Tris adding speed does not influence the mltimerization kinetic.
the concentration of the sample does not influence significantly the multimers ratio
column size: during elution (pH 2.0) , multimers appears using 35 cm bed height columns... and not for 3.5 cm columns. But, while adjusting pH to 7.5, we have similar ratio of monomeric/dimeric/multimeric forms for both column sizes...
I thought about a salt influence...
And I need to settle the pH at about 2.8 for about 1 hour...
Any other idea???
HEEEEEELP!!!
instead of a low pH elution, try a high pH (10) elution. then neutralize with a low pH buffer (i find that high pH elutes better than low, anyway).
Potential Options:
1. Pierce sells a Gentle Elution Buffer - it's salts instead of pH. I used to use it to strip SPR chips, with good results.
2. Try pH 2.8 instead of 2.0... or a pH gradient to see where your minimal elution strength is...
3. Make sure your buffers aren't reducing your IgG apart in any way. Refolding of hemi-reduced IgG's could cause multimerization.
4. Some use 0.2M glycine, pH 2.8 instead of citric acid, pH 2.0 for elution.
If the multimers are only appearing in the longer columns, it sounds like the Ab is falling apart during the time it takes to transit the column while bathing in pH 2.0. So, time at pH 2.0 is killing your Ab. Options are mildly unpleasant, but either move to a smaller column (and pool batches), or use a short, fat column with a higher flow rate (terrible resolution).
During purification, it's standard to collect fractions into tubes already filled with neutralizing buffer (Tris pH 7.5, 1M or more), so the Abs aren't in high pH for a second longer than necessary.
Let us know if anything works out...
~TheRak