Chromatography Forum

Hi All,

Has anyone used RP-HPLC for analysis of large proteins such as monoclonal antibodies (MW ~160 kDa)? I was told that RP-HPLC is not suitable for this type of analsis due to the large size. Is that true?

Thanks a lot!

Never have done it myself but a quick Google search revealed (2nd listing!):

Trap for MAbs: characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry;

Le JC, Bondarenko PV.
J Am Soc Mass Spectrom. 2005 May;16(5):797

They were using a POROS column but it might be possible to do it on small, non-porous particles as well.
They used acetonitrile/2-propanol/ethanol/water/formic acid as mobile phase.

There are reversed-phase columns that can handle the large proteins. You need 300-1000A pore size, either silica or polymer, or a monolith. The problem is that RP elution conditions denature the proteins, and that creates other issues. Most people in the business use ion exchange for antibodies. (Dionex ProPac is an excellent choice.) Hydrophobic interaction chromatography is the other good choice.

Never have done it myself but a quick Google search revealed (2nd listing!):

Trap for MAbs: characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry;

Le JC, Bondarenko PV.
J Am Soc Mass Spectrom. 2005 May;16(5):797

They were using a POROS column but it might be possible to do it on small, non-porous particles as well.
They used acetonitrile/2-propanol/ethanol/water/formic acid as mobile phase.

Thank you very much, Rhaefe. I am trying to find out if RP-HPLC itself is good enough to detect any changes to a monoclonal antibody, such as deamidation, degradation or oxidization. With LC/Mass, the LC separation might not be that critical for detection as HPLC alone, am I right? I will look into the study you found.

There are reversed-phase columns that can handle the large proteins. You need 300-1000A pore size, either silica or polymer, or a monolith. The problem is that RP elution conditions denature the proteins, and that creates other issues. Most people in the business use ion exchange for antibodies. (Dionex ProPac is an excellent choice.) Hydrophobic interaction chromatography is the other good choice.

Mark,

Thanks a lot for your reply. I am using Dionex ProPac for the purpose now. The problem I have with this CEX method is that the separation of different form of the antibody is not even close to baseline separation. One possible reason might be the purity of the sample was not good. And also there are a bunch of peaks clattered within 10 minutes in a 60 minute run. There was always a long tail. That's why I am looking for something else to try.

In RP-HPLC, the protein is denatured. But I am not interested in recovering the protein anyway. As long as it detects or separates the change, it works for my purpose.

BTW, I attended an interesting seminar where the presenter said they could use a large column to purify protein with RP-HPLC (protein recovered without denatured). He did not reveal details.

For looking for things like deamidation, you will get very little resolution under reversed-phase conditions for such a large molecule. However, it might still be better than what you can get from other techniques, and the combination of RP with MS can provide answers. I have to look for some older applications, but I remember some examples of that type.

Another approach that I have seen used is to digest the protein first, and then use LC/MS to look for characteristic peptides in the peptide profile.