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 sepn. of the mol. variants of monoclonal IgA from cell culture supernatants is possible using several filtration and ion-exchange chromatog. steps, followed by size-exclusion chromatog. for the actual sepn. of the mol. variants. The latter step is esp. 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 concns. and space-time yield). For the final sepn. of the mol. variants, hydroxyapatite (HA) elution chromatog. can serve as an alternative to size-exclusion chromatog. By using an optimized, complex phosphate gradient, the IgA dimers can be sepd. quant. 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 amt. 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 addnl. factors considered during the optimization of the HA elution chromatog. HA-displacement chromatog. resulted only in a concn. of the overall IgA fraction, but not in a sepn. of the mol. forms. [on SciFinder (R)

Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA mols., as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatog., and size exclusion chromatog. for the simultaneous and qual. sepn. of the three mol. forms. Milligram quantities of purified IgA mols. were recovered allowing for direct comparison of the biol. properties of the three forms. LPS binding specificity was tested after purifn.; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding expts. using LPS monolayers indicated that purified reconstituted sIgA and IgA mols. recognize LPS with identical affinity (KA 1.0 * 108 M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 * 105 M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is crit. for recognition by the antibody. [on SciFinder (R)