Purification of antibody drug conjugates using a sodium phosphate gradient

Inventors

Nadkarni, Durgesh V. • Borgmeyer, Jeffry R. • Meng, He • Jiang, Qingping

Assignees

Pfizer Inc

Abstract

Methods of removing high molecular weight species, in particular aggregates, from antibody drug conjugate preparations, by contacting preparations of the antibody drug conjugate reaction mixture with a hydroxyapatite resin and selectively eluting the ADC from the resin using a gradient comprising sodium phosphate.

Core Innovation

The invention relates to purifying antibody drug conjugate (ADC) monomers from aggregates in an ADC preparation by using hydroxyapatite resin chromatography with sodium phosphate buffer conditions. An ADC preparation is loaded onto a hydroxyapatite resin column, and the column is washed with a sodium phosphate buffer having a concentration ranging from about 5 to about 35 mM.

The method elutes the ADC preparation through the column with a sodium phosphate buffer of gradually increasing concentration. During elution the sodium phosphate concentration is initially about 20-25 mM and is increased to about 140 mM, thereby separating purified ADC monomers from said aggregates.

The disclosed chromatography is supported by buffer condition constraints including a sodium phosphate buffer with a pH from about 6.8 to about 7.5 and, in embodiments, use of about 5-25 column volumes for the sodium phosphate buffer in the elution step. The described results include embodiments achieving low aggregate levels and high monomer purity, with monomer-rich early fractions and aggregates in later fractions during elution under phosphate-gradient conditions.

Claims Coverage

The document provides one independent claim directed to a hydroxyapatite-resin, sodium-phosphate-gradient method for separating ADC monomers from aggregates, with additional dependent claims refining the independent claim by adding quantitative and compositional limitations. Overall, the independent claim covers loading onto hydroxyapatite, washing with low-mM sodium phosphate, and eluting with a gradually increasing sodium phosphate concentration from about 20-25 mM to about 140 mM to separate monomers from aggregates.

Across the independent claim coverage, the core inventive concept is that ADC preparations are fractionated on hydroxyapatite resin using low-phosphate washing followed by phosphate-gradient elution that progressively raises sodium phosphate concentration from about 20-25 mM to about 140 mM, enabling separation of purified ADC monomers from aggregates. Dependent claims further constrain pH, aggregate/monomer levels, elution volume, and certain ADC composition elements, with an optional pre-processing ion or anion exchange chromatography step.

Stated Advantages

Documented Applications