ProteoCool Pills#22: Tips for cleaning the OMNISEC RALS/LALS flow cell if get dirty after the passage of biological samples

 

                                                                           OMNISEC 

is an advanced GPC/SEC system combining a pump, degasser, autosampler for mobile phase delivery and sample injection module with an integrated multi detector incorporating refractive index, UV/Vis, light scattering and viscosity detectors.

I have used the OMNISEC system to analyze the aggregation state of several biological samples (eg. recombinant antibody/protein preparations) in standard Phospate, MES or Tris buffers (pH range 5.5-8).

I found amazing the performances of the OMNISEC RESOLVE module, that thanks to the presence of a temperature controlled Autosampler (4^oC – 60^oC), allow to load in reproducible way an high number of samples and guarantee a very good throughput.

One of the main drawbacks (which i'm not sure if is it is common or not to other light scattering systems) that i found is the high tendency of the LALS detector to get dirty after the passage of some biological samples.

In this post i would like to share with you my experience about the cleaning procedure to use when you  see a strong increase of the LALS baseline signal after the passage of biological samples.

For example in the following video yoo can see the baseline LALS signal that was detected some months ago after the run of about 20 mab samples in a SEC coloumn (all the mabs were expressed from ExpiCHO cells, purified with MAbselect resin and buffer exchanged in PBS by desalting) :

If the RALS signal is not much higher than the optimal (aobut 80mV), the LALS signal was very high (optimal range is 200-300mV) and a cleaning procedure was required.

Since we do not observe any improvement (data not shown) from the passage of any routine cleaning solutions (methanol 10%, acetonitrile 10%, sodium azide 0,02%) suggested on pag106 OMNISEC SYSYEM Basic Guide manual provided with the instrument, we then decided to proceed with Deep cleaning (pag.105) using a 5% HNO3 solution:

        BUT UNFORTUNATELLY WE OBSERVE ONLY A WEAK REDUCTION IN THE LALS BASELINES SIGNAL

we than tested SDS (1% solution) which may be able to resuspend and remove protein aggregates/precipitate:

BUT UNFORTUNATELLY  ALSO IN THIS CASE WE DO NOT OBSERVE A REDUCTION IN THE LALS BASELINES SIGNAL

Finally we tested NAOH 0,1M solution, which is routinelly used for the cleaning of  several chromatographic resins  used for biologic purification  (e,g proteinA, proteinG, sepharose)

NAOH 0,1M works very well 

and restore the LALS BASELINES SIGNAL in the optimal range

 Thanks a lot to 

 Mirco Toccafondi