An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

Albumin purification from human placenta.

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin-purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross-flow ultrafiltration (CFUF) membrane; (iv) thermo-coagulation at 70 degrees C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion-exchange chromatography Q-Sepharose; (viii) hydrophobic-interaction chromatography with phenyl-Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A(403) of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.