Membrane adsorber for endotoxin removal

ABSTRACT The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.

RESUMO A superfície de membranas planas de nylon foi modificada utilizando-se bisoxirano como espaçador e poli(álcool vinílico) para recobrimento das membranas. O aminoácido histidina foi utilizado como ligante para endotoxinas, visando à sua aplicação na remoção de endotoxinas a partir de soluções aquosas. São discutidas as etapas de caracterização do adsorvedor com membranas, análise do procedimento de despirogenização e avaliação da eficiência de remoção em modo estático. A densidade de ligantes nas membranas foi em torno de 7 mg/g membrana (massa seca), permitindo uma remoção de endotoxinas de até 65%. O desempenho das membranas preparadas com nylon e recobertas com poli(álcool vinílico) contendo histidina como ligante foi superior ao de outros adsorvedores com membranas descritos na literatura. A ausência de adsorção de endotoxinas em membranas sem histidina confirma que a remoção das endotoxinas deve-se exclusivamente à presença do ligante na superfície da membrana. As membranas modificadas mostraram-se bastante estáveis, exibindo um tempo de vida superior a 30 dias.

Hexa-histidine tag as a novel alternative for one-step direct labelling of a single-chain Fv antibody fragment with 99m Tc.

From genetic material of hybridoma cells, we have generated a recombinant single-chain antibody fragment (scFv antibody) specific to carcinoembryonic antigen (CEA), which can substitute an intact murine monoclonal immunoglobulin G1 (IgG1) antibody, also developed by our group, and used in clinical practice for many years. In this paper, we examine a novel one-step method for direct 99mTc labelling of a recombinant anti-CEA scFv fragment through a C-terminal peptide tag containing a six-histidine sequence. This C-terminal peptide tag does not affect antigen binding, and was employed as a strategy for the one-step method of direct 99mTc labelling of a recombinant antibody fragment, based on the criteria of Zamora and Rhodes (Zamora PO, Rhodes BA. Imidazoles as well as thiolates in proteins bind technetium-99m. Bioconj Chem 1992; 3: 493-498). This is a novel technique for the rapid labelling of molecules, suitable for in vivo trials. The method yields >95% labelling efficiency without major effects on biological or in vitro stability.