Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant.
The isolation and separation of the molecular variants of monoclonal IgA from cell culture supernatants is possible using several filtration and ion-exchange chromatography steps, followed by size-exclusion chromatography for the actual separation of the molecular variants. The latter step is especially time consuming and laborious. This report presents possible improvements of the procedure. Use of the displacement rather than the elution mode may render the ion-exchange step more productive (higher product concentrations and space-time yield). For the final separation of the molecular variants, hydroxyapatite (HA) elution chromatography can serve as an alternative to size-exclusion chromatography. By using an optimized, complex phosphate gradient, the IgA dimers can be separated quantitatively from the monomers and higher oligomers. It may in individual cases be necessary to use a size-exclusion polishing step to reach the required final degree of purity, however, the amount of material to be processed is reduced to such an extend by the HA-step, that the overall process is still more productive. Buffer pH and flow-rate as well as the stationary phase material used were additional factors considered during the optimization of the HA elution chromatography. HA-displacement chromatography resulted only in a concentration of the overall IgA fraction, but not in a separation of the molecular forms.
Luellau
E
E
Institute de Genie Chimique, EPF Lausanne, Ecublens, Switzerland.
von Stockar
U
U
Vogt
S
S
Freitag
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D002614:Chelating Agents / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods*; D003470:Culture Media; D019281:Dimerization; D017886:Durapatite / Q000737:chemistry*; D004533:Egtazic Acid / Q000737:chemistry; D006825:Hybridomas; D006863:Hydrogen-Ion Concentration; D007070:Immunoglobulin A / Q000737:chemistry / Q000302:isolation & purification*; D051379:Mice; D011108:Polymers / Q000737:chemistry / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D003470:Culture Media; D007070:Immunoglobulin A; D011108:Polymers; D004533:Egtazic Acid; D017886:Durapatite