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.  

I'm using mab select sure...

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.

Thanks Stefan., I'll get back to you..' think you know about TFF...I'm trying that now...I'll give you the update..