Polishing steps used in multi-step protein purification processes

What is this patent about?

'930 is related to the field of biopharmaceutical purification, specifically methods for purifying target molecules, such as recombinant proteins and monoclonal antibodies, from complex biological mixtures. The patent addresses the challenges of removing residual impurities after an initial affinity chromatography step, particularly in preparing the target molecule for subsequent polishing steps.

The underlying idea behind '930 is to use a pH gradient elution in cation-exchange chromatography as a polishing step, followed by filtration through a Q membrane. This approach avoids the high salt concentrations associated with traditional salt gradient elution, which can interfere with downstream processing. The pH gradient allows for efficient separation of the target molecule from impurities while maintaining a low conductivity, and the Q membrane provides further purification by removing residual contaminants.

The claims of '930 focus on a method for removing residual impurities from a target-molecule solution. This involves loading the solution onto a cation-exchange column, eluting the target molecule using a time-dependent pH gradient, and then passing the resulting solution through a Q membrane filter. The Q membrane step is performed after minimal dilution (one-fold or less) and at a relatively high flow rate (400-600 cm/h).

In practice, the process begins with an eluate from an affinity chromatography column, often a Protein A column used to capture antibodies. This eluate, containing the target protein and various impurities, is then loaded onto a cation-exchange column. Instead of using a salt gradient to elute the protein, a pH gradient is applied. This pH gradient is carefully controlled to optimize the separation of the target protein from remaining contaminants like host cell proteins, DNA, and aggregates. The eluate from the cation exchange column is then passed through a Q membrane, which captures negatively charged impurities while allowing the purified target protein to flow through.

The key differentiation from prior approaches lies in the combination of pH gradient elution and Q membrane filtration. Traditional methods often rely on salt gradients, which necessitate dilution steps to reduce conductivity before further processing. By using a pH gradient, '930 minimizes the need for dilution, reducing buffer usage and processing time. Furthermore, the use of a Q membrane provides a high-capacity, single-use polishing step that eliminates the need for cleaning validation and other complexities associated with traditional anion-exchange chromatography columns, leading to a more efficient and cost-effective purification process.