Hello,
I am analyzing purified antibodies using a superdex 200 increase 10/300 GL column. Occasionally, I observe tailing at the peak, particularly with antibodies that have high surface concentrations of Lysine and Arginine.
The figure below shows data from size exclusion chromatography analysis conducted under the same conditions, where tailing was observed in antibodies with high positive charge.
I'm wondering if increased positive charge on the surface could enhance interactions with the column.
Are there any references demonstrating this phenomenon?
Thank you!
Hi,
Yes, such surface charges can influence the on-column behavior of proteins, for example in SEC. This is referred to as secondary column interactions: in addition to the main separation mechanism in SEC, through pores, your protein of interest also interacts with the column material through electrostatic interactions. This usually negatively influences the resolution, as peak shapes worsen, like the tailing you observe.
Secondary interactions can also occur in other types of chromatography, like seeing hydrophobic secondary interactions in an ion exchange separation.
Have you considered adding the amino acid lysine or arginine to your SEC mobile phase to reduce secondary interactions of your antibody, thereby optimizing its SEC analysis?
Hope this helps!
The chromatograms you included have no scales of any kind on them, so do not help us understand what your peak shape represents. Please include chromatograms with labeled scales.
Please share with us some basic information about your method so we can provide constructive suggestions:
(1) What is your mobile phase composition ?
(2) What is the method's Flow Rate ?
(3) What are the columns exact dimensions, particle size ?
(4) What is the temperature of the column ?
(5) What is the sample concentration AND INJECTION VOLUME (*These columns are very easy to overload and if you inject too much sample, you will always see what appears to be "tailing").
(6) What are the analysis wavelength and bandwidth?
(7) You say you are using SEC mode. If so, then please tell us what the measured void volume is for your method using the column you selected? This is a critical value that should be known before running any samples.
I really appreciate your interest. I'll share the information you requested.
1) pH 7.4 PBS
2) 0.75 mL/min
3) Particle size, d50v : ~ 8.6 μm
Bed dimensions (mm) : 10 × 300-310
Approximate bed volume (mL) : 24
4) RT
5) 1mg/mL, 10μg
6) 280nm
7) The void volume was measured by a senior, not by me, but I'm unsure of the exact value.
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