As the question. During the formualation development, one IgG2 of CE result shows 3 main peaks. We assume the first 2 are isoforms. The third one is on the right side of the first 2 with a relativly small wide peak. SEC shows one main peak. SDS-PAGE doesnt show any evidence of 3 peaks according to CE.
Can anyone share some experience and knolwedge on that?
Hi Yue Huang,
Can you provide more details about the CE procedure... Is it reduced or non-reduced CE.
The third peak might correspond to structural isomer (disulphide isoform)...
Hi Saracanan:
Thank you for your reply.
It is non-reduced one. It is a good point to challenge the CE precedure. Currently we are now investigating into whether the sample preparation( increase the heating duration) will affect the result.
Any thoughts onto that？
Share two reference here.
Article Human IgG2 Antibodies Display Disulfide-mediated Structural Isoforms
Article Electrophoretic evidence for the presence of structural isof...
Hi Yue Huang,
If its non-reduced sample, then there are chances of disulfide isoforms. Increasing the heating duration might help you to prevent non-covalent dimers and aggregates.
Do you have a mass spec that can be coupled to CE... if Yes, then this would solve the concern of identifying the third peak..
Also, consider running the IgG sample in NATIVE PAGE along with NATIVE PAGE markers... This might give you some clue.
All the Best for your efforts.
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