Hello all,

My institution is having an issue in our analyses of a monoclonal antibody using CE-SDS using Maurice CE-SDS by ProteinSimple:

One of our antibodies mAb X, is showing multiple peaks when analyzed in reduced and non-reduced forms. In the images attached: I am showing that a Trastuzumab standard is showing a single Light Chain and single Heavy chain in reduced form and 1 single peak for intact antibody in non-reduced form.

However, our antibody is showing 3 peaks for LC and 2 peaks for HC for reduced analysis and 3 peaks for intact antibody for non-reduced analysis.

Upon analysis of old lots of mAb X it shows that these are also showing similar peak split patterns.

I was wondering if any of you upon your analyses of monoclonal antibodies have seen a similar phenomenon? - Also if any of you had insight or possible solutions for these analyses.

I am particularly curious as to why the peaks are fragmenting into different species when we don't see similar results with other antibodies (trastuzumab standard)
Sample conditions:
.5 mg/mL in 20 mM Tris Buffer and 1x Sample Buffer by ProteinSimple
2 uL Internal Standard by ProteinSimple

Reduced conditions:
5 % 10 mM TCEP HCl
70 C for 10 minutes
Ice Water 0 C for 5 min
Injection: 4600 V for 20 Seconds
Separation: 5750 V for 25 minutes

Non-Reduced conditions:
5 % 250 mM Iodoacetamide
70 C for 10 minutes
Ice Water 0 C for 5 min
Injection: 4600 V for 20 seconds
Separation: 5750 V for 35 minutes

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