ProteoCool Pills #23: Selection of the right post-coloumn filter is essential to detect protein/antibody aggregates in static light scattering

Static light scattering (SLS) is a technique to measure absolute molecular weight using the relationship between the intensity of light scattered by a molecule and its molecular weight and size. 

Some SLS technologies exist: multiangle light scattering (MALS), right-angle light scattering (RALS), low-angle light scattering (LALS) and RALS/LALS hybrid systems 

MALS or RALS/LALS when coupled with other detectors (eg UV-vis, RI, densitometry, fluorimeter) in an advanced GPC-SEC system can be applied to investigate solution properties, stability testing and process development for different kind of  conjugated and unconjugateed biologics

Light scattering detectors (low-angle LALS, right-angle RALS, or multi-angle MALS) are very sensitive to the presence of particulates, when those are used for molecular weight detection in chromatography. Unfortuantelly even the best  columns can shed some particulates from their packing material. Although undetected by most conventional detectors, such as UV and RI, these particles scatter significant amounts of light, produce noise that affect the light scattering signals and baselines. To mitigate this, the MALS, LALS/RALS sistems include an in-line coloumn filter that can significantly improve the quality and thus the accuracy of both the data and results.

In this post i would like to share with you some data acquired loading different mabs in a Cytiva Supedex200 increase 10/30 coloumn in the OMNISEC instrument equiped wih the LALS/RALS detector in the presence/absence of post coloumn filter. 

First of all we compared the baseline signals that can be obtained 

- Without filter;

- With a 0,2um nylon filter;

- With 0,2um cellulose filter; 

The baseline signals suggest that nylon filter is the best in terms of signal/noise ratio 

Is it really the best choiche for analisys of protein/antibody preparations? 

To assess it, a 2% BSA standard and 2 different purified monoclonal antibodies (igG1 human) were loaded in the superdex200 10/30 increase coloumn and analized with the OMNISEC instrumentation  

In all the 3 cases, the Nylon filter guarantee the best signal/noise ratio at LALS but it seems to mask the presence of a significant fraction of high molecular weight proteins.

This was most evident for the mabs, since the dimeric-trimeric and MW aggregates were not detected, even if at UV and RI, using the nylon filter.

ThIs differences may lead to overtimating the quality of a certain mab or protein preparation and it can lead to false positive results in case of stability studies that  would like investigate the mab/protein aggregation propensity under stress (as acid pH, 37°C, freeze/thaw)

For this kind of studies cellulose filter seem to be the best compromise between quality of LALS signal and recovery of the protein polimers. 

As a general comment: Often the diffence is in the details! It is important be able to critically review the Positive results to distinguish the real positives than false positives! 

Suggested links: 

https://www.materials-talks.com/how-to-change-the-light-scattering-post-column-filter-membrane/

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