The present disclosure relates to methods of re-oxidizing an antigen-binding protein.

The present invention provides a method for making uncapped cysteine protein preparations, including uncapped engineered cysteine antibody preparations. The methods include, inter alia, contacting a reducing agent with engineered cysteine antibody molecules, each of the antibody molecules having at least one capped engineered cysteine residue and at least one interchain disulfide bond and reacting the reducing agent with the antibody molecules under conditions sufficient to uncap engineered cysteine residues and form cap byproducts. The method also includes removing the bap byproduct during the reduction reaction. Substantially all of the interchain disulfide bonds present in the antibody molecules prior to reduction are retained following reduction. Antibody conjugates and methods for preparing antibody conjugates using uncapped antibody preparations are also described.

Methods for the production of high purity recombinant protein such as monoclonal antibodies (mAb) using disulfide bond re-oxidation are provided. In particular, the present disclosure provides methods for converting partial molecules (e.g., antibody fragments) to full molecules (e.g., full antibodies) comprising admixing a starting solution comprising the partial molecules with a redox buffer comprising a redox pair which comprises at least one thiol reducing agent (e.g., cysteine) and at least one thiol oxidizing agent (e.g., cystine), wherein the redox buffer re-oxidizes the partial molecules to full molecules. The disclosed methods can be used, e.g., to prevent or mitigate the formation of partial molecules during protein purification, or to reprocess or rescue a solution comprising partial molecules (e.g., a partially degraded pharmaceutical formulation).

Methods of producing an aqueous formulation of an antigen-binding protein or enhancing re-oxidation of an antigen-binding protein are disclosed. The methods comprise (a) contacting an aqueous solution comprising antigen-binding protein molecules with a charged depth filter under conditions sufficient to enhance re-oxidation of the antigen-binding protein molecules and achieve a decrease in the percentage of reduced antigen-binding protein molecules, compared to the percentage of reduced antigen-binding protein molecules observed prior to step (a); and (b) optionally, measuring the amount or relative amount of reduced antigen-binding protein molecules. Formulations comprising a re-oxidized antigen-binding protein are also described.

Cyclized peptides having a crosslinked structure by one or more intramolecular S—S bonds may be prepared by:

(1-A) as to a completely protected linear peptide having two or more SH groups as functional groups on the peptide, removing protecting groups of all functional groups other than the protected SH groups in the peptide,
(1-B) protecting all SH groups of the linear peptide having two or more SH groups as the functional groups on the peptide by forming a temporary S—S bond,
and
(2) subjecting the peptide obtained by step (1-A) and step (1-B) to a folding step under oxidation and reduction conditions to obtain the cyclized peptide by re-forming an S—S bond in the peptide molecule.

The present invention provides method of increasing the percentage of monomer in a composition of recombinantly expressed antibody molecules characterised in that the antibody molecule comprises at least one Fv with specificity for an antigen of interest comprising one VH and one VL wherein said VH and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprises: a) a conversion step of treating the composition with a denaturant selected from urea and/or Guanidine hydrochloride; b) wherein step a) is performed in the presence of a reducing agent or after treatment with a reducing agent.

The present disclosure provides methods for selectively reducing an antibody, comprising contacting the antibody with a reducing agent selected from the group consisting of 2-[2-(Diphenylphosphino)ethyl]pyridine, 3-(Diphenylphosphino)benzenesulfonic acid, 4-(Diphenylphosphino)benzoic acid, 2-(Diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(Diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, and salts thereof. The present disclosure further provides methods of producing antibody conjugates, such as antibody-drug conjugates.

The present invention provides method of increasing the percentage of monomer in a composition of recombinantly expressed antibody molecules characterised in that the antibody molecule comprises at least one Fv with specificity for an antigen of interest comprising one VH and one VL wherein said VH and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprises: a) a conversion step of treating the composition with a denaturant selected from urea and/or Guanidine hydrochloride; b) wherein step a) is performed in the presence of a reducing agent or after treatment with a reducing agent.

The present invention provides a method for removing cysteine caps from antibodies and re-capping the antibodies with cysteine molecules. The methods include, inter alia, culturing a host cell comprising a protein molecule having at least one capped engineered cysteine residue, and contacting the cell culture with cystine. Dissolved oxygen levels can be manipulated in the cell culture to further enhance the removal and re-capping process.

Disclosed herein are methods that have been developed to control the formation of disulfide bonds between polypeptides of a multimeric protein produced by a bioprocess. Also disclosed are protein solution parameters that allow for controlling the formation of disulfide bonds. In one example, the methods disclosed herein can be used to control the proportion of half antibody molecules in an antibody solution.