large-pore rpHPLC 12/15/2000
Last month, there was a discussion about rpHPLC of large proteins (e.g. mAbs). Andy Alpert instead recommended methods in which the tertiary structure of the protein is retained, like ion exchange or HIC. He also recommended that large-pore or no-pore chromatography materials be used for these large proteins (see below).
Which brings me to the point of this letter. Is there any use for large-pore rpHPLC in protein analysis? One person in our lab has ordered a SynChropak RP4-1000 for analysis of a recombinant antibody-like molecule (MW ~125,000), wanting to know if the column would be useful in a stability-indicating assay. Is there any literature supporting (or disparaging) the use of these columns?
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12033
(518)488-6012
(518)488-6030 FAX
vernon.shoup@regpha.com
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From: POLYLC
Dear Wei,
To come right to the point... you are out of your mind to attempt to use reversed-phase chromatography for a protein as large as a MAb! Reversed-phase HPLC is not synonymous with HPLC. As a rule, for proteins larger than 25 KDa, you get better analytical results (and better recoveries) with modes of chromatography designed to conserve the tertiary structure of the protein. This means ion-exchange or hydrophobic interaction chromatography. For something as large as a MAb (150 KDa), you'd better use a chromatography material that either has pores 1000-A or larger or else no pores at all. Otherwise, you will get skewed peak shapes as a result of steric hindrance.
Cation-exchange HPLC works well for MAb's in general. Weak cation-exchange (WCX) materials work best for human MAb's, while strong cation-exchange (SCX) materials work better for mouse (=murine) MAb's for some reason. A MAb typically elutes in 3 major peaks and numerous minor peaks. The last major peak is the native sequence. The second big peak is the form missing the C-terminal Arg- or Lys- from one of the heavy chains, while the first big peak is the form missing the Arg- or Lys- from the C-termini of both heavy chains. The minor peaks are oxidation products, forms where the tertiary structure is degraded, forms with disulfide bridges the wrong way, carbohydrate side chain variants, and the like. It isn't surprising that you saw a number of bands in SDS-PAGE.
If you supply your FAX#, I'll FAX you a typical chromatogram of a MAb on a WCX column along with running conditions.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045 USA
tel: (410) 992-5400 FAX: (410) 730-8340
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<< Subj: RP-HPLC for mAb
Date: 11/07/2000 2:40:40 PM Eastern Standard Time
From: WXZhang@genetics.com (Wei Zhang)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Item Type: Task
Start Date: 11/07/2000 (Tuesday) Due: 11/07/2000 (Tuesday) We are developing RP-HPLC methods for mAbs. We've tried C18, C4, -CN and phenyl columns from different vendors, none of them gave good results in terms of peak shape and resolution. We saw some minor bands in SDS-PAGE and are trying to use RP-HPLC to resolve them. Could you please share with us your experience with RP-HPLC of mAbs or other large proteins? Thanks a lot!
Wei Zhang, Ph.D.
Genetics Institute
wxzhang@genetics.com
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