Method of purifying protein

What is this patent about?

’289 is related to the field of protein purification, specifically the removal of host cell or viral DNA from samples containing physiologically active proteins like monoclonal antibodies. In the production of recombinant biologics, stringent regulatory standards require that residual DNA be reduced to extremely low levels (e.g., less than 100 pg per dose) to ensure patient safety. Traditional methods rely on complex, multi-stage chromatographic processes such as anion-exchange or hydroxyapatite chromatography, which are often costly, labor-intensive, and can result in inconsistent yields.

The underlying idea behind ’289 is that contaminant DNA can be effectively precipitated and removed by exploiting the formation of protein-DNA conjugates under specific ionic conditions. The inventors discovered that when a protein sample is maintained in a state of low conductivity and transitioned from an acidic to a neutral pH, the DNA interacts with the protein to form visible particles or cloudiness. By intentionally inducing this precipitation and then physically removing the resulting solids, the DNA concentration in the remaining liquid is drastically reduced without the need for specialized resin-based separation.

The claims of ’289 focus on a specific four-step process for purifying antibody-containing samples using a combination of affinity chromatography and controlled precipitation. The method requires first capturing the antibody on a Protein A or Protein G column and eluting it using an acidic solution with a molarity of 100 mM or less. This low-molarity eluate is then neutralized to a pH between 4 and 8, maintaining the low molarity to trigger the formation of particles, which are subsequently removed to yield a purified product.

In practice, the invention works by taking the acidic output of a standard affinity capture step and carefully adjusting the environment to favor conjugate formation. While the patent notes that a small percentage of the target protein is lost because it is bound within the removed particles, the vast majority—typically around 90%—remains in the solution. The removal of these particles is achieved through simple mechanical means, such as filtration through a 0.22 μm filter or centrifugation, making the process significantly faster than traditional secondary chromatography steps.

This approach differentiates itself from prior art by turning a common problem—protein aggregation or precipitation—into a selective purification tool. Instead of viewing the cloudiness that occurs during neutralization as a failure of the buffer system, the invention uses it as a DNA-scavenging mechanism. By strictly controlling the conductivity and molarity during the pH shift, the method achieves DNA levels as low as 22.5 pg/ml, providing a streamlined and cost-effective alternative to the complex sequences of ion-exchange columns usually required in downstream processing.