Hi everyone,
We are producing chimeric antibodies in the lab using HEK293 cells.
I'm currently struggling with an antibody - which binding activity varies greatly between batches.
I use the same plasmid for expression and have tried different expression systems (including EXPI). I've tried concentrating the antibody using both G and A beads (both worked well).
The yield of the antibody is fairly good in any system, but the target binding (assessed by flow cytometry) changes drastically with different batches.
Can anyone suggest why this should happen to the same antibody? and how can I troubleshoot this?
Much appreciated!
Ayelet
Which solution do you use for the elution during your purification ? Acidic condition can be very detrimental to the quality and functionality of your eluted antibody. If you need stringent conditions try to neutralise immediately your elution with some Tris pH9 buffer hat might help. Also increasing salinity might have a slightly beneficial effect on the protection of antibodies properties
Jean-Pierre Dangy Thanks a lot for the reply!
We indeed elute the antibodies with Glycine pH2, and immediately neutralize them with Tris pH9. What other elution strategy do you recommend?
The thing is I use the same elution/storage conditions for all the batches, and still the "good" ones retain their activity over time, and the "bad" ones are problematic from the get go.
Well we had these type of issues you could try to go with milder conditions for the elution and for example trying Citrate buffer it's usually less detrimental
Elution Buffer: 0.1 M citric acid, pH 3.0
Which isotype is your antibody ?
I think most of my IgG1 were purified using Protein A sepharose but yes it is very sticky
Oups I didn't ask you, are those Mouse IgG1 ? If yes then you should use the High salt binding buffer if you use Protein A
OK then they should bind very strongly to both type of Sepharose. You could have a look to the Gentle elution solutions kit provided by PIERCE. This has helped me for several difficult antibodies
Dera Ayelet Avin
Do you observe this problem often or just for a specific mabs that may have some intrinsic issues?
I'm agree that acid pH could be detrimental and i suggest to you to increase a little your elution pH.
Generally using proteinG, elution with Glicine 100mM pH=2.7 (at pH3 the elution is not so efficient) dropped into a tris 1M pH=9. concentrated with ultrafiltration, immediatelly buffer exchanged (with PD-10) and stored in PBS we have good batch to batch reproducibility in terms of affinity (estimated by FACS and/or BLI) for igG1-chimeric human-mouse mabs produced from Expi293 and ExpiCHO.
Did you try to run the differenct batches in a SEC coloumn or perform DSF analisys to see if you have different aggregation %?
good luck
ciao
Manuele
Dear Ayelet Avin
For optimization the critical parameters contribute to the “ production efficacy yield” and these are:
1. Determination of the specific growth rate.
2. Accumulation of the recombinant protein from high density culture.
3. Specific production rate in overall product process duration.
Check the links below:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803805/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958569/
Some platforms are there in regards with purification and with the use of high throughput screening for further developments. Please use the standard protocol for optimized buffers.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958570/
Dear Manuele Martinelli and Anju Kaushal,
Thank you so much for the detailed reply.
We indeed observe this issue for a specific antibody, potentially suggesting an intrinsic problem. We plan to run a few more validations on small scale, to understand if it's worth the troubleshooting.
If yes, I'll have a go at your suggestions.
Much appreciated.
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