Hi, as we know that proteins are highly sensitive molecules and susceptible to degradation at various conditions of temperature, moisture etc.  SInce here monoclonal antibodies are involved, the degradation may be due to critical attributes of its chemical structure or contamination by other microorganisms. You may be required to develop and use a cell bank system. Pls find this article for your kind perusal.

Hemanth,  Without knowing specifics of your system, I can only give you general suggestions.  Aggregation can be dependent upon the concentration, buffer ionic strength, and temperature.  You should aim to keep them at as low concentration as you can and store them at -20.  You should also definitely avoid repeated freeze-thaw cycles.  Try increasing the ionic strength of your storage buffer and also make sure that you add glycerol.

Hi Hemanth, proteins are usually less soluble when they are kept at a pH close to their pI .A rule of thumb is to store your protein in a buffer that is either 2-3 pH units above or below its pI.

As Brian said, freeze-thaw tends to denature proteins, which makes them less soluble.

Have you tried to solubilize your aggregates and see if the Ab is still functional?

Hello Hemanth. When you say you are storing at 20C, do you mean -20C? You mention freeze-thaw further down, so that suggests -20.  When you freeze and then thaw, does this avoid the aggregation? You do specifically say that storage in solution at 2-8C leads to aggregation, so here are two thoughts:

1. It is possible that your protein is cold-labile and it would be worth filter-sterilising a sample to see if it is more stablle in solution at temperatures closer to physiological.

2. You say you have varied buffer conditions but you don't say in what way. If the aggregation is due to hydrophobic interaction, it is worth bearing in mind that phosphate, especially concentrated phosphate, will strongly enhance hydrophobic interaction, so that a Tris-chloride buffer might be better.

Freeze-thaw are generally bad for proteins; however, sometimes it's unavoidable simply because the protein is not stable at 2-8C. Adjusting the pH, adding cryoprotectant and using different buffer species are the only things you can do for -20C storage. Also, be sure you are not shaking or introducing air since the air-water interface can easily cause aggregation. Gently swirl to mix.

Lastly, aggregation is a concentration dependent process by definition. Reduce the concentration as low as possible.

It might be useful to make some more effort to find buffer conditions that allow for 2-8C storage to avoid F/T altogether.

Depending on how refold-able your protein is you could try lyophilizing your protein.  Once lyophilized, protein can be stored at -20 for years at a time without degradation.

Minus 20 isn't enough for storing proteins, you need minus 80 for long-term activity and good structure. Comments on pI important; also, check your buffers and avoid divalent cations-- phosphate usually inferior. Look into the pedigree of your protein and see the comments of those who have parried with it for years-- you need the right buffer, correct ionic strength (some like it salty, others dilute). And what's important at the end, antigenic perfection or enzymic activity?  Is your aggregation irreversible, or actually a good way to keep it stable? You can keep antibodies precipitated in ammonium sulfate for years-- first simple method I'd try!  If you must store a purified concentrated protein ready for use, there's often an excipient or detergent or emulsifier in there to promote suspension.

Thank you very much guys for all the valuable answers and im sry for the late response..im not feeling well these days i was on sick leave..

i have the explanations for some answers..very soon i will get back to you all.... after my recovery.. a big thanks to all once again...

hi harita, we are maintaining a good cell bank system and also we are taking precautions to avoid all kinds of contamination..in fact we have a  validated system for that..we are not facing issue with that.. the article was really good and helpful please send me more articles if you have like this.

Dear Brian, i have some restrictions. i cant explain much about the specifics. hope you will understand. even we tried to keep the concentration low..but there are some practical issues in that. decreasing the protein concentration also does not showed any effect on aggregation formation. increasing the ionic strength of the protein is effecting the biological activity. till now we haven't tried Glycerol.. i will try a buffer with glycerol...

Hi Stefansson, i tried that also... doesnt work...now trying to change the buffer content

Hi Hemanth, do you know if your aggregated Abs are still functional?

You could try to gently disperse your aggregates by putting them in a tube and immerse them into a sonicating water bath.

Sonicating water baths are not as harsh as the sonication techniques used to bust open bacteria. Sonicating water baths have helped me to solubilize protein aggregates without denaturing them.

I never heard about this steffy..these aggregates are almost invisible...I'll try this also...but i need more clarity... I can't experiment much on this

Hi Hemanth, It eventually all boils down to this question: Can you recover enough  Abs from your preps to demonstrate a commercially viable method of Ab production despite aggregation during storage?

If yes, then don't worry about the aggregates. Throw them out.

If no, then you will have to rethink the storage methods and, like other suggestions, include glycerol and freeze your prep at -80oC

Hi all...I have changed the formulation buffer and now its working..thanks for all your suggestions...