Purification
of Gonadotropin from
Horse Serum Applying Magnetic Microsorbents (Report on
the ProGonado
Joint Project)
Within the
framework of the ProGonado project, a novel method of magnetic purification of
proteins is demonstrated by the direct extraction of gonadotropin from horse
serum. It is the objective to develop the components required for the
industrial use of the process and to establish the first technical application of
product extraction from animal serum based on magnet technology. For this
purpose, magnetic microsorbents are used in a magnetic separator with a total
capacity of 80 g particles.
By the specific
attachment of the target protein to the surface of the sorbents and subsequent
magnetic separation of the loaded sorbents directly from the serum or from a
process flow treated by simple precipitation, an effective purification scheme
of a few steps only shall be implemented. Magnetic particles with anion
exchange functionality and with gonadotropin-specific antibodies are applied.
Results
After having identified the optimum ligand
concentration of 50 µg antibodies/mg magnetic particles, gonadotropin was
isolated directly from horse serum by coupling monoclonal antibodies to the
particles. At an initial concentration of 20 – 30 IU/ml equine gonadotropin in
serum, an eCG yield of 50 – 60 % was reached using the separation process on
the laboratory scale.
The magnetic particles with anion exchange
functionality were tested for their capacity of separating gonadotropin from
precleaned diafiltrated serum on the laboratory scale. Using a solution of 342
IU/ml in contact with diethyl-aminopropyl (DEAP)-functionalized
magnetic particles, about 80 % of the gonadotropin
were bound and subjected to complete elution.
The purification factors relative to the
initial total protein in the serum are currently reaching 300 in the case of
anion exchange beads and up to 1000 for magnetic beads with coupled antibodies.