Another weird observation with SEC

[Rocnex1]

Method:
2 TSKgel superSW3000 columns in series + guard column
Isocratic elution, 0.35 ml/min, PBS, pH 7.4
30 min run for each sample
10 mg/ml mAb, 10 ul inj

stability samples, so some are clean mAb with little aggregation, some are stressed, with 20% aggregration. all samples were filtered through 0.45um before inj.

reference standards were injected every 10 samples in any sequence, from same vial. a typical sequence runs for 20 hours. samples were kept at 4 degrees C in autosampler.

Observation:

In a sequence of 20 to 40 samples, back pressure started normal, after 6 or 10 or 15 sampels, back pressure started going downing, sometimes it could drop by 20% in a few samples. Then back pressure went back to normal level after being lower for several samples. I believe that when the pressure was low, flow rate was low because the PA of those samples were abnormally higher than usual.

This has been reproducible, observed in many sequences. The HPLC is an Agilent 1100, technician came in a couple times and didn't see anything wrong with the instrument. Tosoh didn;t know what happened.

Once I tried injected 20 injections of water, similar thing happened.

[HW Mueller]

Measure the flow rate , most likely you will see a mobile phase delivery problem.
You never told us what the concentration of your PBS is.

[Rocnex1]

The PBS is GIBCO 1x PBS, 10 mM sodium phosphate and around 155 mM NaCl, pH 7.4. My mAb solvent is PBS + 0.01% Tw80, pH 7.

Yes I think it might be a flow rate problem. However, it is difficult to catch the moment and check the FR, since it happens during a sequence at varying time. I didn't have the luck to find out what caused the flow rate problem. Like I said, the HPLC tech service came in and check the system twice, they didn't see anything wrong.

By the way, you said your sample solvent also contains high salt, I assume you dilute your sample before inj. I don't think your mAb is formulated in such a high salt buffer, right? So did you check if this high salt buffer introduces or reduces aggregation?

Thanks!

[Rande]

I also have problems with pressure decrease, this causes longer retention time. I believe most of this is due to out-gassing and air bubbles in the pump.
How do you degas your mobile phase? Do you have in-line degassing?

I prepare my buffer fresh (from 10X) , 0.2 um filter, then vacuum degas in an ultrasonic bath for 5 to 10 minutes @ ~ 12 to 15 inch Hg vacuum .
I try to limit my run time to < 30 hours. After that I will pause the run and degas the buffer again – I will always get more bubbles out of solution.

I also have noticed that if the room temperature changes – everything goes screwy !!!!

I have a Beckman HPLC system (I think it’s the same design as the old Altex pumps I used in the early 80’s ….. I know, now I’m dating myself).
It does not have in-line degassing or He sparge, I’ve considered an aftermarket in-line degasser but have not tried that yet.

Anyone have experience with them for aqueous buffers?

Another comment, earlier you mentioned you were changing your guard column frequently. If your problem with peak area is similar to mine, this will cause you to have more adsorption of the HMW aggregate and need to condition the columns all over again.

[HW Mueller]

Rocnex1, if you don´t have a flow problem licked, it seems running a series is a bit premature? (Could it be that your tech service didn´t use your MP, or modified it, like taking the air out of it?).
Injecting something like 5 µL of the mab in its original (unknown to us) solution produced peak spreading, so I replaced it with my mobile phase (150mM both phosphate and chloride, so chloride was isotonic, phosphate at pH ~7 is probably chaotropic) via ultrafiltration. No aggregation!
Intuitively, I am suspicious of coinjecting TWEEN80. But actually, it must not be that bad as a 6% variation is easy to get (the trend is a bit disconcerting, though).

On the side: The last mab I ran on the column cost 10 000 Euro for a few mg. If it aggregated in any of the solutions to which it was subjected here, I would have had an extensive and intensive talk with the manufacturer.