We are working on monoclonal antibodies using a G1 synapt HR mass. we can take a good charge state envelope for proteins with molecular weight of less than 40 KD such as Gh hormone or CAD enzyme. However for monoclonal antibodies with molecular weight of more than 100 KD such as Ritoximab, charge state envelope has low sensitivity and resolution so that the Maxent software can not calculate molecular weight of MABs. Our instrument is 8K.
any comment is greatly appreciated.
What is the mass range of the instrument?... It should be up to 8kDa to see both high and low charge states to fully characterize the mab. Nowadays vendors are adding biopharma options (software and ESI/Mass filter configuration) to increase the mass range of the mass analyzer to see the lower charge states therefore higher m/z responses...In this case, you may try supercharger agents to be added into the mobile phases to increase the z to get lower m/z ratios. It may be partially helpful in terms of observing a wider charge envelope (higher m/z signals can shift to lower m/z values due to the loaded protons and resulting higher z levels) and the lost signal might get back a bit.
@Dear Ismail
Thanks for your comment. Mass range or our device is 8k and we set the range from 1000-8000.
We used formic acid or TFA in mobile phase however not good charge state envelope is obtained.Would you please introduce other supercharger agents?
Thanks for your comment again
Assuming your mass calibration is OK and the calibration linearity covers the higher masses as well. I suggest confirming this initially...
Theoretically, you are only able to monitor charge variants that embark more than 18-19 protons (Rituximab counted as 145 kDa). Thus efficient ESI ionization and more versatile adduct is needed...
In some cases denatured form of the mab can be ionized more efficiently and adduct abundances improve...Therefore try to denature your mab (w/o reducing) and rather than folded structure try to monitor the denatured intact mab to load more hydrogens onto it...This is an empirical output seen at HDX application of IgG after treatment with guanidin to increase the deuterium uptake.
Here are some experimentally proven superchargers to decrease the m/z ratio of the HMW proteins. There are many supercharger alternatives such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol, sulfolane, ortho-nitroanisole, propane sultone, ethylene carbonate, propylene carbonate...
Additionally, I would not prefer TFA because as you know it suppresses ionization much and causes signal loss.
Which states are not possible to see higher m/z or lower m/z and what was the loading concentration used as on column or as infusion mode)?
Take into account that since the charge profile is getting expanded due to the higher MW proteins (e.g. Mabs) sensitivity decreases in reverse proportionally. Hence more sample amount is needed to see minor charge states and complete the profile. G1 synapt HR mass is a relatively old TOF version and therefore, it may need more proteins to reflect what you have...
It may be more beneficial if you are able to share your Ritoximab raw data (mass spectra image) to elucidate the issue.
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