Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reac tion with either 2,5-dioxopyrrolidinyl 3-{N-[3-(1,3,2-dioxaboran-2-yl)pheny l]carbamoyl}propanoate (PBA-XX-NIIS) or 2,5-dioxopyrrolidinyl 6-{[3,5-di-(1 ,3,2-dioxaboran-2-yl)phenyl]carbonylamino}hexanoate (PDBA-X-NHS) were compa red with respect to the efficiency with which they were immobilized on sali cylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid com plex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 mu mol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown t o be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chro matography. Boronic acid complex formation was compared to covalent immobil ization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP . S HA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, re tention of anti-alkaline phosphatase and recovery of anti-alkaline phosphat ase under alkaline conditions. High capacity SHA-X-Sepharose (greater than or equal to 7 mu mol of SHA/mL of gel) was shown to afford superior perform ance to moderate capacity SHA-X-Sepharose (4.5 mu mol of SHA/mL of gel) wit h respect to stability at pH 11.0 and pH 2.5 when a PDBA-alpha Human IgG co njugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA -X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalen t interaction involving boronic acid complex formation.