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.
Grellet
S
S
Instituto Butantan, Centro de Biotecnologia, Av. Vital Brasil 1500, CEP 05503-900, São Paulo, SP, Brazil. sgrellet@usp.br
Martins
E A
EA
Maimoni Gonçalves
V
V
Yague Lopes
A P
AP
Raw
I
I
Cabrera-Crespo
J
J
D001769:Blood; D001774:Blood Chemical Analysis; D002374:Catalase / Q000097:blood / Q000302:isolation & purification*; D002847:Chromatography, Agarose / Q000379:methods; D006461:Hemolysis; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D010920:Placenta / Q000737:chemistry / Q000201:enzymology; D012709:Serum Albumin / Q000302:isolation & purification*; D013482:Superoxide Dismutase / Q000097:blood / Q000302:isolation & purification*
D007074:Immunoglobulin G; D012709:Serum Albumin; D002374:Catalase; D013482:Superoxide Dismutase