SEC/GPC Standard Issues

[KAR10]

Hi everyone!

I am developing an SEC method on a Phenomenex BioSep 2000 column 300 x 7.8mm using 1xPBS as the mobile phase and 10% DMSO to remove any residual proteins. The flow rate is 0.3 mL/min. This method is run on a Waters Alliance 2695 system with UV detector at 280 nm. This method will be qualified, transferred to a contract manufacturing facility and validated eventually. From my previous life, I was under the impression that a linear curve and system suitability most be run for each assay. I purchased SEC calibration standards from BioRad containing

Thyroglobulin 670,000 Dalton
Bovine gamma-globulin 158,000 Dalton
Chicken ovalbumin 44,000 Dalton
Equine myoglobin 17,000 Dalton
Vitamin B12 1,350 Dalton

When I set up the calibration curve using GPC software from Empower I got a MW of my protein as 270 kD when it is really 64 kD. My protein elutes at 21 minutes and which came up between the first two standards. My protein is a fusion protein, so I was wondering if it is too different from the standards and that is why I am getting a higher MW? The column I use has a molecular weight cutoff of 300 kD, would this be the problem? It is a requirement that a linear curve be run from a regulatory standpoint?

I appreciate the help! Have a nice day!

[Noser222]

Is the purpose of your method to check the MW or just to look for impurities, excipients, etc.? Differences in charge and shape can affect the elution volume enough to make your MW estimate inaccurate. As long as you don't have to check the MW I would say you could validate the method with a primary standard of your analyte.

[HW Mueller]

Since you are using a phenomenex column my suggestion would be to look at their catalog and see their recommendations, for instance, those comparing the useful ranges for the same protein coming off a SDS elctrophoresis or in its natural state, etc.
Anyway, before starting a new method it is wise to inform oneself of the background.
Where did you get this thing about the removal of "residual proteins" via DMSO?
Why do you expect residual proteins on the column? You don´t think your mobile phase is appropriate?

[Rande]

Greetings and let me see if I can help you.

I have been using SEC-HPLC for proteins (and MAb’s) as my primary HPLC assay for many years over my past 3 or 4 jobs. I use the TOSOH TSK-G3000SWxl , that is what I am used to, but I know many people like the Phenomonex columns.

The BioSep 2000 is OK for a 64 KDa protein, but you have very little resolution for any HMW aggregates. Most of the concerns with my samples have been "dimers - trimers - and larger aggrigates" , yes I often see breakdown products, but they are still easily resolved on the TSK3000.

Also - regulatory concerns are more interested SEC to detect aggrigation, because HMW aggrigates are immunogenic. Degradation products are usually detected by other assays, such as SDS-PAGE, but SEC-HPLC is often the primary assay to see aggrigation.

A few comments and/or questions:

1) Is your product peak a single peak @270 KDa ? This could be a aggrigate of 4 monomers, but I would expect to see Monomer - and increasing aggrigates. Remember that the MW estimate for 270 KDa is a very "soft number" with this column.

Are you sure you are using the GPC software correctly? How does your Std curve look? With your column – do not expect the Thyroglobulin or Vit B12 to be in a linear range. When I need to estimate MW weight, I use Excel with a semi-log plot and use “exponentialâ€