Linearity in SE-HPLC question

[anandaseneviratne]

We have been eveloping a SE-HPLC method for an antibody aggregate . We have 0.995% R2 linearty for both mAb main peak as well as for the aggregation peak but %aggregate and % main is linearily decreasingin and increasing, respectively. Integration is valey to forcebaseline integration because no basline separation. Still we are trying to optimize the method and trying to understand why the % aggreate increasing while the main peak % is decreasing. So is this method is unacceptable?

Any help, suggestion, comments are highly appreciated.

Ananda

[Meerkat]

sounds like 'antibody degradtion' is occuring; is the protein labile?

[anandaseneviratne]

The protein is in a stable formulaion. This increase of aggregate % is shown by the same peak areas that showed linear in the calibration curve. I mean in the same Chromaleo data, I can se my calibraion curve is linear and at the same time, same peak area shows % aggregates are increasing. The % aggregation shold be the same in all my injections.


Thanks

Ananda

[ScottHorn]

What column are you using? I've read numerous sources that describe similar behavior. The assertion was that the antibody aggregates had a propensity to stick to the column. After a certain number of injections all the active sites on the column were occupied and the aggregate % remained stable. I've dealt with the same problem myself, and the solution I found was to add arginine to the mobile phase. Apparently it interacts with the aggregates in some way that prevents them from sticking to the column. You could also try "conditioning" the column before use with some readily available protein in order to saturate any binding sites present.
Try this link: http://www.researchgate.net/publication ... matography

Scott Horn

[anandaseneviratne]

Hi Scott

Thanks for your reply. We tried several columns viz. Sepax Cenith and Waters Aquity-BEH200. With both columns with various salts and buffers mobile phases, we are seeing the same issue. We tried some NaKCLO4 in the mobile phase and we had some luck but it is not reproducible and we are still seeing %aggregate increases in the standard curve samples.

I was thinking to try arginine too. It is an excellent suggestion. Will do.

Thanks

Ananda

[Andy Alpert]

Putting a chaotrope like NaClO4 in the mobile phase will cause the fractionation range to shift to lower molecular weights. See my book chapter on the subject, cf. the following link: http://www.polylc.com/downloads/SEC_boo ... r_ver1.pdf

Andy Alpert
PolyLC Inc.
aalpert@polylc.com