Inactivation of viruses using novel protein A wash buffers.

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.

Mitigation of chromatography adsorbent lot performance variability through control of buffer solution design space.

The separation of undesired product-related impurities often poses a challenge in the purification of protein therapeutic species. Product-related impurity species, which may consist of undesirable isoforms, aggregated, or misfolded variants of the desired monomeric form of the product, can be challenging to remove using preparatory scale chromatographic techniques. When using anion exchange chromatography to remove undesirable product-related impurities, the separation can be highly sensitive to relatively small changes in the chromatography operating conditions, including changes to buffer solution pH, buffer solution conductivity protein loading, and operating temperature. When performing difficult separations, slight changes to the chemical and physical properties of the anion exchange adsorbent lot may also impact the separation profile. Such lot-to-lot variability may not be readily measurable by the adsorbent manufacturer, since variability can be highly dependent on a specific protein separation. Consequently, manufacturers of chromatographic adsorbents may not be able to control adsorbent lot to lot variability tightly enough to prevent differences from occurring when performing difficult product-related separations at the preparatory scale. In such cases, it is desirable to design a chromatography step with a control strategy which accounts for adsorbent lot to lot variability in the separation performance. In order to avoid the undesired changes to process consistency and product quality, a proper adjustment of the column operating conditions can be implemented, based on the performance of each adsorbent lot or lot mixture. In this work, we describe how the adjustment of the column buffer solution composition can be used as a design space based-control strategy used to ensure consistent process performance and product quality are achieved for an anion exchange chromatography step susceptible to adsorbent lot to lot performance variability. In addition, a "use test" is described that can be employed to determine the optimal buffer solution compositions for different anion exchange adsorbent lots based on the retention volume of the therapeutic protein during a gradient elution.