Purification and characterization of recombinant human anti-HAV monoclonal antibody.

In order to obviate the drawbacks o f plasma immunoglobulins, the whole molecular recombinant human anti_HAV (hepatitis A virus)monoclonal antibody (anti_HAV IgG) produced and secreted by rCHO cells was purified and its physicochemical properties were extensively c haracterized. The rCHO cells were cultured in serum_free medium and the supernat ants were collected. The recombinant human IgG molecules were sequentially purif ied by ultrafiltration, rProtein A Sepharose Fast Flow affinity chromatography, ion exchange chromatography and diafiltration. In affinity chromatography, prior to the target protein elution, an intermediate high salt wash step was inserted , different pH and salt concentrations were evaluated for the capacity of removi ng host cell DNA. The yield of the downstream purification process was approxima tely 40%. The purity of anti_HAV IgG thus generated was assayed with SEC_HPLC me thod, integration result showed that the monomeric IgG content was more than 99% . Western_blot was carried out with AP_antiHuman IgG (Fab specific) and AP_antiH uman IgG (Fc specific) respectively, the blot result demonstrated that the anti_ HAV IgG is human antibody with Fab and Fc structure. The specific anti_HAV activ ity determined by ELISA was 100 IU/mg, with anti_HAV immunoglobulin as the worki ng standard reference. Ligand leakage in the eluate of the affinity column was a pproximately 32 ng/mg IgG, while after further purification steps, it was decrea sed to less than 2 ng/mg IgG. Residual host cell DNA was monitored with solid do t blot assay, DNA can be removed effectively with intermediate high salt wash st ep in the affinity chromatography. Free sulfhydryl content of anti_HAV IgG was a ssayed with fluorescent spectrophotometer, the low molecular weight bands appear ed in non_reducing SDS_PAGE may be caused by the presence of free sulfhydryl. Th e endotoxin content was less than 1EU/mg examined by standard LAL test procedure s. Anti_HAV IgG prepared with this process is able to fulfill the regulatory req uirements of State Food and Drug Administration for recombinant products.