I'm trying to reduce the loading time of desired protein from clarified harvest on affinity columns, but I'm not getting it. We should select the resin based on the binding capacity and also consider the residence time. Considering all these we must give some time for the process to go on and I'm trying to reduce this time but I'm failing. I want to this with a lesser cost. Please give me your valuable suggestions.
Hi Hemanth, as Jan said, more details about your problem will help in any answers given.
But a rule of thumb for affinity chromatography is that the longer your column is, the better your chances are to bind your protein to the affinity resin at high flow rates.
To help you more details are required. What kind of affinity are you using? What resin are you using now (cat no.)? What are the properities of your protein? At which step exactly you fail?
ready to help - jan;)
Hi Hemanth, as Jan said, more details about your problem will help in any answers given.
But a rule of thumb for affinity chromatography is that the longer your column is, the better your chances are to bind your protein to the affinity resin at high flow rates.
Hi Hemanth, as far as I can tell, the resin you are using is immobilized protein A.
Many antibodies require special buffers to bind effectively to protein A, G or A/G. I would recommend getting prepared buffers for your purification.
http://www.piercenet.com/product/binding-elution-buffers-igg-purification
Secondly, packing your resin into a long and thin column will increase the chance of your mAb binding to the resin.
I'm using bpg 300/500 column...i tried sodium acetate and citrate buffers...they are working fine..total resin volume is 14 litres
Hi Hemanth, you should have included some minor details in your question, such as that you are isolating Abs on an industrial scale.
OK, so everything is working well except that it takes a long time to load the 14 liter column with your clarified harvest, right?
How much volume are you loading?
I have been involved in some medium scale Ab purification processes and the best way to speed up the process is to concentrate the Ab containing media before you apply it to the column.
U got me...exactly I'm processing for industrial scale..up to 2kl..loading volume is 1500-1900 lit...it may vary sometimes...but till now I haven't tried concentrating my desired protein of interest... I'll try that...and suggest me which is the best way
Wow! You are not messing around! Kinda dwarfs my puny 5 liter preps.
Anyway, I only know of 2 techniques that can concentrate this amount of volume:
Hollow fiber concentration
http://www.gelifesciences.com/downloads/Download/29013303AB.pdf
Spray freeze drying
http://www.pharmtech.com/pharmtech/article/articleDetail.jsp?id=648017
There have to be other techniques out there that I don't know of.
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