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Biotechnology and Applied Biochemistry (2001) 34, (135–142) (Printed in Great Britain)

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

Sheyla Grellet¹, Elizabeth A. L. Martins, Viviane Maimoni Gonçalves, Alexandre Paulo Yague Lopes, Isaías Raw and Joaquin Cabrera-Crespo

*Instituto Butantan, Centro de Biotecnologia, Av. Vital Brasil 1500, CEP 05503-900, São Paulo, SP, Brazil

Key words: biopharmaceutical products, blood derivatives, downstream processing, haemoderivatives, protein purification.

Abbreviations used: Cat, catalase; Sod, superoxide dismutase; Alb, albumin; NMML, nominal molecular-mass limit; CV, column vol.

¹To whom correspondence should be addressed (e-mail sgrellet@usp.br).

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 1.8×10⁶ 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 2.1×10⁵ 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.

Received 17 April 2001/19 July 2001; accepted 3 August 2001

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