Comparison of affinity membrane adsorbers for the purification of recombinant human erythropoietin (rhEPO)

Abstract

In this work affinity membrane adsorbers were investigated for the chromatographic purification of recombinant human erythropoietin (rhEPO) produced in mammalian cells. Cibacron Blue (CB), IDA-Cu +2 , wheat germ agglutinin (WGA), concanavalin A (ConA) and an anti-EPO monoclonal antibody (MAb) were tested as affinity ligands, attached to microporous Sartobind  membranes. In experiments carried out with cell culture supernatant, the best results were obtained with Sartobind-CB, Sartobind-WGA and Sartobind-MAb membranes. The thermodynamic parameters were determined by adsorption isotherms of rhEPO onto the membranes. Sartobind-ConA presented the lowest affinity for rhEPO, as evidenced by a lower association constant. For Sartobind-CB, Sartobind-IDA-Cu +2 and Sartobind-MAb K A was in the order of 10 5 L mol −1 , whereas for Sartobind-WGA it was 10 6 L mol −1 . Sartobind-CB eluates were also investigated by RP-HPLC. The purity level achieved in this one-step purification strategy was 55%, indicating that the Sartobind-CB membrane is a promising affinity membrane for rhEPO purification.

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

“…31,[35][36][37][38][39] No caso de membranas e géis empregados em leito expandido, as técnicas de ativação e imobilização dos agentes quelantes são similares àquelas utilizadas para os géis cromatográficos tradicionais.…”

“…37,49,[57][58][59][60][61][62][63] A literatura mostra que, além destas aplicações, IMAC foi utilizada para separação de diferentes tipos de células microbianas, uma vez que estas, quando possuíam resíduos de histidina acessíveis, apresentavam a capacidade de serem adsorvidas em uma coluna monolítica de estrutura macroporosa composta por criogéis de poliacrilamida com IDA-Cu 2+ imobilizado. 65 Proteínas que contêm naturalmente resíduos de histidinas acessíveis (recombinantes ou não) e apresentam importância comercial no campo terapêutico e diagnóstico, como proteínas do soro humano, 29 imunoglobulinas, 31,35,38,[66][67][68][69][70][71] interferon, 72 proteína C humana, 73,74 fator IX 73,75 e hormônio de crescimento humano, 76 foram purificadas por IMAC utilizando os metais de transição Cu 2+ , Ni 2+ , Zn 2+ , Fe 3+ e Co 2+ imobilizados.…”

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

“…Due to the limited pressure stability of Cellufine 1 sulfate under operating conditions (<2 bar, recommendation of manufacturer) the flow rate is restricted resulting in suboptimal productivity. Membrane adsorbers, which may overcome such drawbacks, have been already used for affinity separation of different target molecules, for example, recombinant proteins (Cattoli et al, 2006;Mellado et al, 2007), antibodies (Boi et al, 2006b;Castilho et al, 2002;Platonova et al, 1999), lectins (Boi et al, 2006a;Sorci et al, 2006) or influenza virus particles (Opitz et al, 2007a). In addition, membrane-based ion-exchange chromatography was successfully applied for purification of several other virus species, including for example insect baculoviruses (Wu et al, 2007), mosquito-specific parvoviruses (Czermak et al, 2008;Specht et al, 2004), alphaherpesviruses (Karger et al, 1998), adenovirus vectors (Peixoto et al, 2008) or influenza viruses (Kalbfuß et al, 2007b).…”

Purification of monoclonal antibodies in a stirred cell with polyethyleneimine‐modified polyethersulfone ultrafiltration membrane

“…These polishing steps usually comprise two chromatographic steps, normally a cation exchange and an anion exchange chromatography . However, chromatography, particularly in packed bed mode, is typically associated with several major limitations, mostly due to the high pressure drop across the bed and the dependence on intra‐particle diffusion for the transport of the biomolecules to their binding sites, resulting in slow mass transfer . Hence there is significant interest in alternatives that decrease the number of processing steps without the loss of purity of the final product.…”