C225 (also known as Cetuximab) is a chimeric monoclonal antibody (MAb) with specificity for the human epidermal growth factor receptor (EGFr). C225, grown in serum-free production medium, is currently undergoing clinical trials. Maloney ectropic murine leukemia virus (Maloney-MuLV) was chosen as the model virus for the initial virus clearance validation of the C225 purification process. MuLV is an enveloped retrovirus, approximately 80 to 110 nm in size. The purification process, a five-step chromatographic procedure, utilizes Macro-Prep® High Q anion and Macro-Prep High S cation exchange supports. For the validation, conducted at an off-site location, load samples were spiked with known titers of MuLV. The spiked load samples were run on scaled-down versions of process chromatography columns. The spiked anion load had a titer of 1.9 x 106 plaque forming units (PFU)/ml and the cation load a titer of 5.75 x 105 PFU/ml. The anion exchange feed stream (10.6 ml, 107.3 PFU total) was loaded onto the anion exchange support under pH and conductance conditions where C225 does not bind to the matrix and contaminants such as DNA and RNA bind strongly. The anion flow-through process intermediate was loaded onto the cation exchange support under conditions where C225 binds and is then eluted (10.6 ml, 106.8 PFU total. To determine the amount of viral reduction, samples were taken of the load, flow-through (anion exchange step) and eluate (cation exchange step) for each run. The virus level in the samples was quantified by XC plaque-forming activity. The entire purification scheme netted a total of 15 log10 of MuLV clearance. The two ion exchange supports in combination resulted in a total of a nearly 8 log10 reduction of MuLV. The anion exchange step gave a 5.7 log10 reduction of the viral load and the cation exchange step a 2 log10 reduction.
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