Large scale production process for capped and un-capped antibody cysteines and their use in therapeutic protein conjugation

Inventors

Dutta, Kaushik • Gomes, Jose Manuel • Kotch, Frank W. • PATEL, Vimalkumar B. • Prashad, Amarnauth Shastrie • PROCOPIO-MELINO, Renee L. • Zhong, Xiaotian

Assignees

Pfizer Inc

Abstract

Optimizing production of selectively capped, and uncapped, cysteines on antibodies by manipulation of cell growth conditions including the deliberate depletion of cysteine and/or cystine in the cell culture process by way of media components, batch duration, or cell density to achieve efficient production of proteins including antibody-drug-conjugates (ADCs).; conjugating a TNB-capped cysteine-containing protein by reacting it with a reducing agent capable of detaching the TNB-capping moieties from the protein without significantly reducing antibody inter-chain sulfur bonds, and conjugating reduced sulfur bonds on the protein to a payload through a reactive linking moiety.

Core Innovation

The disclosure describes a process for conjugating a 5-thio-2-nitrobenzoic acid (TNB)-capped cysteine-containing protein by manipulating reaction conditions that enable selective conversion of capped cysteine residues to the desired conjugation state. The approach uses tris(3-sulfophenyl) phosphine (TSPP) to react with TNB-capped cysteine residues, followed by removal of excess reducing agent and detached TNB by filtering.

After filtering, the process adjusts temperature and contacts the filtered mixture with dehydroascorbic acid under agitation, and then adds linker-payload to the resulting mixture with further agitation. The disclosed sequence is directed to producing conjugates while controlling the reducing and reoxidation chemistry around the TNB/TSPP and dehydroascorbic acid steps.

The disclosure also addresses production of TNB-capped cysteine-containing proteins at scale by depleting cysteine/cystine in mammalian cell culture to generate predominantly TNB-capped cysteines. It further includes downstream handling of linker-payload conjugation products, including quenching, separation of quenched linker-payload from the conjugate, and purification, and reports performance comparisons using hinge-scrambling assay results to indicate that TNB/TSPP avoids hinge scrambling compared with full TCEP reduction/reoxidation.

Claims Coverage

The partial content provides one independent claim directed to a process for conjugating a TNB-capped cysteine-containing protein, followed by dependent claim features that narrow substrate selection and operational conditions, and add downstream processing options. The inventive features revolve around TSPP reaction with TNB-capped cysteine residues, filtration removal of excess reducing agent/detached TNB, temperature adjustment, dehydroascorbic acid contacting with agitation, and linker-payload addition with agitation.

Across the independent claim and its narrowing dependents, the coverage centers on a TNB-to-conjugation workflow that combines TSPP reaction with capped cysteine residues, filtration removal of excess reducing agent/detached TNB, controlled temperature adjustment, dehydroascorbic acid contacting under agitation, and subsequent linker-payload addition under specified agitation conditions.

Stated Advantages

Documented Applications