Olivier Pitiot

A potential set up based on histidine hollow fiber membranes for the extracorporeal removal of human antibodies

Abstract Poly ethylene vinyl alcohol (PEVA) based hollow fiber membrane grafted with l -histidine has been found to be particularly useful for the preparative scale separation of immunoglobulin G and potential clinical applications. In this paper, we present a set-up designed for the extracorporeal removal of immunoglobulin G. Biochemical and hydrodynamic conditions were optimized so as to perform in vitro removal of 50% of plasma immunoglobulin G.

Structure-function relationship in glycosylated α-chymotrypsin as probed by IMAC and IMACE

Abstract Chemical glycosylation of bovine α-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of α-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of α-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods.

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: I. Study of the mechanistic aspects of adsorption for the separation of human serum albumin from its non-enzymatic glycated isoforms (advanced glycosylated end products)

The characteristics of albumin adsorption on histidyl-aminohexyl-Sepharose 4B were investigated. In particular, the adsorption capacity of the gel was studied as a function of conductivity and pH of the running buffer. The adsorption was maximum at low salt concentration around neutral pH, involving electrostatic and hydrophobic interactions. Kinetic aspects were also investigated. Dissociation constant (KD) and maximum capacity (Qx) were, respectively, estimated to be 4.5 x 10(-5) M (medium affinity) and 93.3 mg (high capacity) of human serum albumin per ml of adsorbent. According to these preliminary results, separation of HSA and its non-enzymatically glycated isoforms (conventionally named advanced glycated end products: AGEs) was achieved. Chromatographic potential of this separation tool is discussed.