Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities.

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