I used AKTA purifier 10 for ion exchange chromatography with gradient of 500 mM NaCl 0 to 50 % (or 0 to 70%) for purification of polyethylene glycol modified protein from remaining unmodified protein. If general protocol based on this is helpful, let me know

Hi Byran;

Below is a process I have used for purifying Mouse IgG on a protein A Column (GE Hi Trap Column) using a pH Gradient on a Bio-Rad Douflow system (you Specified above not an Ion Exchange column):

2 Buffer system - 

Buffer 1:   50 mM Sodium Phosphate(monobasic)/50 mM Citric Acid, 150 mM NaCl (Final), adjust to pH 2.5 with Phosphoric Acid.

Buffer 2:   50 mM Sodium Phosphate (Dibasic)/50 mM Sodium Citrate (Tribasic), 150 mM NaCl (Final), Adjust to pH 8.0 with 0.5 M Tri-Sodium Phosphate if necessary.  

 -  I would estimate the Sodium Contribution from both the Phosphate and Citrate then adjust to 150 mM NaCl using 5 M NaCl - Can not remember the conductivity - however - your machine should monitor conductivity and it will remain constant throughout the run.

1.  I have loaded the antibody onto the column using a standard Protein A Loading buffer (Thermo Scientific/Pierce 21007).

2.  Program Run as Flows - (Isocratic Flow 0.5 mL/min for a 5 mL Column):

      - Wash column with 10-15 CVs of Buffer 2 (High pH Wash)

      -  Elution: Select your linear gradient function and program 100% Buffer 2/0% Buffer 1 to 0% Buffer 2/100% Buffer 1 over 20 column volumes.  I had a pH monitor available - the linear gradient is nearly Perfect/Reproducible - I could not draw a straighter line.

      -  Equilibrate Column at the end at pH 7.3 -7.5 (~20 % Blend - Don't Recall exact Percentage) for storage.

      -  Most Ms IgG's I have worked have eluted between pH 6.6 - 5.9  - sharp peaks less than 2 column volumes.

Should be adaptable to an Akta - they are great machines & the best that money can buy - Good Luck

I had Bio-Rad because we could afford more instrumentation at their prices than GE.

Like to hear back on how this works for you;

Best Regard's

Dan Weaver

Thanks for your response Dan!

I was actually looking for more of a text based command method for something in UNICORN 6.4.

What you've shown is quite an interesting method though. I imagine that you're working with IgG2? IgG1 typically elutes at a pH significantly lower than what you've described (~pH3.5) due the higher affinity for ProA. Does your method have good resolution at that pH range for IgG1?

Sodium acetate is normally what I use in this case for the pH gradient. The citrate working pH range is better than acetate though. Do you find better stability in citrate? I'm aware of citrate stabilizing some loops in some protein.

This was work I did between 2009 - 2011 and I don't have access to all the details any longer - especially the script's code - property of a different company.  This code is not difficult to write - the language structure is a form "script" which is the same for the BIAcore/Akta systems.  Depending upon the features you want - the basic code should take only a couple of hours to write and debug.  Additional features are added along the way to improve the process - means improving your life-style around the process.  Slow Flow rates improves your resolution to an extent - yet the value I suggested works quite well - a good comprise. These are standard columns sizes (geometry's) which are not very difficult to scale up - all well designed by the manufacturer.  Time to Validate the working code is dependent upon pushing through a QA system.

All my antibody's looked the same as far as resolution (see below) - I did not have culture supe's or bioreactor media to purify from - to much volume to ship across country - too expensive.

We had a mixture of IgG1 and IgG2 in that library (40 - 45 Ab I prescreened by BIAcore for functional activity) - probably more IgG2's than IgG1's.  The vendor scaled up slightly - purified using the classical method you described - froze them then shipped them to me.  I biotinylated with a Chromogenic Biotin (Solulink).  Once Ab-bio linked to streptavidin latex beads, these latex beads aggregated - bad news for lateral flow assay.  I used the pH linear gradient before biotinylation to solve the aggregation problem at that later - assay development back on track - worked like a champ!  I had about 2-4 mg of antibody to pull this off to demonstrate proof of concept for the assay design. 

Citrate was not a problem in our processes - I went quickly to the biotinylation (raise pH with Borate - Amine chemistry NH ester).  The product was loaded onto a 120 mL Sephacryl column (~0.25 mL sample @ ~10-15 mg/mL, ) and purified by gel-filtration to purify.   Keep in mind that the gel-filtration process was in place and used even with the frozen Ab - so the linear pH gradient solved an issue in our processes.  To this day I do not know what problem I solved - I just could not repeat the method that my vendor used to solve this problem - I needed a different result from my vendor!!!

Sorry I didn't have the code to send off to you - If you want help with the code send it off to me - however - I want to reassure you that it is pretty easy to write and debug.

looking forward to here from you!

Best Regards

Dan

Hi Bryan;

Wanted to follow up with you to see how things were going. 

My Best Regards

dan

Hey Dan, your method worked quite nicely on the Akta. We actually have a couple Biorad instruments, which are slightly easier to work with than the Aktas.

Though to be fair, GE did help me write the code. We have a service plan with them and they actually sent someone out to help me write a program for capture by Mab SelectSuRe and clean up by CHT using a gradient. Pretty standard, all I do is hit start and the following morning I have fractions of the eluted peak. 

Awesome - I am happy everything worked out nicely