Concentration-dependent Tailing HPLC-SEC of mAbs

[gwenael.nys]

We observed differences in performance when performing SEC-HPLC for an antibody using Tosoh TSKgel G3000SWXL (7.8 x 300 mm) column.

Mobile phase: 10 mM phosphate, 700 mM NaCl, pH 6.1
Flow rate: 0.25 mL/min.

Problem description: the main peak experiences more tailing than before with specific column lots, in a concentration-dependent manner. We suspect that changes in resin lot might caused column lot-to-lot inconsistencies.
The interesting phenomenon is that when the antibody is diluted to 100 mg/mL (0.5 µL injection volume), the peak is more distorted than when the same is diluted to 25 mg/mL, from which 2 µL is injected (same mass load onto column).
When the sample is injected neat (~130 mg/mL), the peak tailing get even worse.
Dilution is performed with mobile phase.
Tailing causes overestimation of the LMW peak, eluting right after main peak.


Questions:
- How can one explain the concentration-dependent peak tailing, knowing the same mass load is injected?
- How can this be related to specific column lots?
- Can it be related to the extent of mobile phase with which the antibody is mixed, rather than the actual concentration of the antibody?

Instrument, methods, reagents, etc. were verified and are not in cause of this problem.

Thanks for your kind help!

[vmu]

Try to wash the problem column with one of the cleaning solutions (e.g. acidified Na2SO4 solution, as far as I remember) described in the column manual.

[HughW]

The interesting phenomenon and undesirable peak tailing may have been caused by the antibodies clumping together at higher concentrations. This would reduce their effective surface contact area with the stationary phase. The clumps would slowly disperse into individual units as they become diluted by the mobile phase as the chromatographic separation proceeds. The recently dispersed units would have a longer retention time than the clumps.

[LuccasLN]

Hello!

i will agree with the post above me, maybe you could share your sample preparation procedure, such as how you are diluting it (example: do you ad the antibody to the solution or the opposite, how much time it takes, how you mix it,...) and what is the diluent (it is not clear wethere you are using MP or something else). Also, have you taken a look at the pI value of the antibody? Maybe there is something wrong happening because of it, if the pH is too close to pI, i take there could be some poor solubility or clumping going on.

Also, you could take a look at the columns manual and see what are the organic solvent maximum quantities you can use on the column, sometimes a little bit of organic solvent helps on elimination of random secondary interactions. On a similar manner, you could look into the use of sodium perchlorate, Tosoh has some good papers on that.

i hope this helps!