Downstream purification and analysis of the recombinant human myelin basic protein produced in the milk of transgenic cows : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University (Palmerston North) New Zealand.

Abstract

Downstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.