Size-Exclusion Chromatography of Proteins

Further information about problems that may occur in amino acid analysis by ion-exchange chromatography can be found in the review paper published by Williams [57] in 1986. 

The biopharmaceutical industry has continued its focus on the development of bio-therapeutic monoclonal antibody (mAb) drugs. mAbs produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates 

05% formic acid, SEC enables a baseline-resolved separation of the heavy and light chains. The separation of a papain digest under nondenaturing conditions is shown in Eigure 5.35. 

200pL/min (a) extracted ion chromatogram of mAb, (b) mass spectrum of mAb, and (c) decon-voluted spectrum of mAb (Reproduction with permission from Thermo Fisher Scientific, Sunnyvale, CA, USA, [58]). 

5 pm column dimensions 150 mm x 2.1 mm i.d. eluent 20mmol/L and (e) deconvoiuted spectrum of mAb monomer (Reproduciton with permis- 

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Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

Special Considerations for the Size Exclusion Chromatography of Proteins... 

For the size exclusion chromatography of proteins on silica-hased diol packings, it is generally recommended to use fully aqueous mobile phases with a salt concentration between 0.1 and 0.3 M. In general, a phosphate buffer around pH 7 is used as the mobile phase. Under these circumstances, the tertiary structure of most proteins is preserved without difficulty and the interaction of proteins with each other is minimized. However, other inorganic buffers or combinations of buffers with organic solvents can be used without difficulties for special applications. 

L. Size exclusion chromatography of proteins Na = M = i 03 D e [E] Slow mass transfer with large molecules. Aqueous solutions. Modest increase in NSk with increasing velocity. [79]... 

Ion-exchange and size-exclusion chromatography of proteins M.W. Bushey and J.W. Jorgenson, Anal. Chem.. 62, 161 (1990). 

Kopaciewicz W, Regnier FE. Nonideal size-exclusion chromatography of proteins effects of pH at low ionic strength. Anal Biochem 1982 126(1) 8—16. 

There are two main application areas for size exclusion chromatography of proteins desalting or group separation and fractionation. Both areas are industrially of equal importance but the prerequisites for their ability of being sealed-up are quite different... 

Chemically attached copolymers of iV-vinylpyrrolidone (N-VP) and N-(2-hydroxyethyl)acrylamide (N-HEAA) steeply decrease the inherent glass adsorp-tivity which is observed for proteins in aqueous buffer solutions. Thus, it became possible to apply the prepared materials to the size exclusion chromatography of viruses and ribosomes. 

Nave, R., Weber, K., and Potschka, M., Universal calibration of size-exclusion chromatography for proteins in guanidinium hydrochloride including the high-molecular-mass proteins titin and nebulin,. Chromatogr. A, 654, 229, 1993. 

Dubin, P. L. and Principi, J. M., Failure of universal calibration for size exclusion chromatography of rodlike macromolecules vs. random coils and globular proteins, Macromolecules, 22, 1891, 1989. 

Batas, B. and Chauduri, J.B., Considerations of sample application and elution during size-exclusion chromatography-based protein refolding, /. Chromatogr. A, 864, 229, 1999. 

Figure 9. The effect of storage on protein and peptide composition in cooked ground beef stored in a refrigerator of 4 days (adapted from 7). Upper graph represents the size exclusion chromatography of acidic extracts of fresh, cooked, and cooked-stored beef. Lx)wer graph represents the reverse phase HPLC of peak II from the size exclusion chromatography.

E. Watson and W. C. Kenney, High-performance size exclusion chromatography of recombinant derived proteins and aggregated species, J. Chromatogr., 436 289-298 (1988). 

Figure 14.4. Selectivity curve from SEC of DNA ( ) and protein ( ) molecular weight standards on a 106 x 10 mm Superose 6 gel filtration column with 0.02 M Tris-HCl pH 7.6 containing 0.15 M NaCl as the eluent.4 [Reprinted, with permission, from H. Ellegren and T. Laas, Journal of Chromatography 467, 1989, 217-226. Size - Exclusion Chromatography of DNA Restriction Fragments. Fragment Length Determinations and a Comparison with the Behaviour of Proteins in Size-Exclusion Chromatography . 1989 Elsevier Science Publishers B.V.]...

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