Comparison of different types of ceramic hydroxyapatite for the chromatographic separation of plasmid DNA and a recombinant anti-rhesus D antibody.
The chromatographic properties of three types of ceramic hydroxyapatite (HAP) are compared. All three materials were prepared by sintering the original precipitate, albeit at different temperatures (400, 700 and 1000 degrees C for type I, II and III HAP, respectively). The three materials differed in pore size and pressure limits (both lowest for type I and highest for type III). Type I and II HAP had an average particle diameter of 20 microm. The particle size of the type III material was 40 microm. HAP-beads were slurry-packed into 4 x 25-mm stainless steel columns and investigated for the chromatographic isolation of plasmid DNA from clarified E. coli lysates and of a recombinant human antibody from CHO cell culture supernatants respectively. The chromatographic performance of the three types of HAP showed significant differences, which were correlated to the binding capacities of the materials for (linearized) plasmids of different size (4.7, 10.3 and 11.4 kb) and proteins of different isoelectric point (lysozyme, pI = 10.5; anti RhD antibody, pI = 8.3; beta-lactoglobulin, pI = 4.9). The accessibility of the adsorptive surface (pore size) but also the types of binding sites on the HAP-surface (P/C-site ratio) are proposed as determining factors for the chromatographic behavior.
Giovannini
R
R
Center of Biotechnology, Department of Chemistry, Swiss Federal Institute of Technology, Lausanne.
Freitag
R
R
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D016466:CHO Cells; D002516:Ceramics; D002847:Chromatography, Agarose / Q000379:methods*; D006224:Cricetinae; D006801:Humans; D006882:Hydroxyapatites; D010957:Plasmids / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012204:Rh-Hr Blood-Group System
D000906:Antibodies; D006882:Hydroxyapatites; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System