Cytiva Bioprocess R&d Ab

Ge Healthcare Bioprocess R&d Ab

Ge Healthcare Bio-sciences Ab

Tomas Bjorkman

Mats Ander

Gustav Jose Rodrigo

Jesper Ulf Hansson

Gustav José Rodrigo

Annika Forss

Goran Bauren

Per-Mikael Aberg

Hans Blom

Ronnie Palmgren

Hans J Johansson

Sophia Hober

Bjorn Noren

Anders Ljunglof

Jelena Vasic

Elin Monie

Magnus Bergman

Joakim Galli

Elin Marianne Monie

Karin Torstenson

Magnus Carl Erik Wetterhall

The invention relates to a method of isolating an immunoglobulin, comprising the steps of: a) providing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support: b) contacting a liquid sample comprising an immunoglobulin with the separation matrix; c) washing said separation matrix with a washing liquid; d) eluting the immunoglobulin from the separation matrix with an elution liquid, and e) cleaning the separation matrix with a cleaning liquid, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA).

Separation method

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of an Fc-binding domain ofProtein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO 26 or SEQ ID NO 27, wherein at least the alanine residue at the position corresponding to position 42 in SEQ ID NO:4-7 has been mutated to arginine and/or wherein at least the aspartic acid residue at the position corresponding to position 37 in SEQ ID NO:4-7 has been mutated to glutamic acid.

Mutated immunoglobulin-binding polypeptides

The invention relates to a separation matrix comprising at least 11 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:

Separation matrix

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of a parental Fc-binding domain ofProtein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 22, SEQ ID NO: 51 or SEQ ID NO: 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

The present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix; b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Method of cleaning and/or sanitizing a separation matrix

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; b) permeating the separation matrix with the storage liquid; and c) storing the storage liquid-permeated separation matrix for a storage time of at least days. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Method of storing a separation matrix

Methods that include providing and reacting a solid support and an alkali-stable ligand derived from an immunoglobulin-binding bacterial protein to form a separation matrix having covalently coupled alkali-stable ligands; and washing with a wash solution comprising at least 10 mM of an alkali metal hydroxide.

Method for preparation of a separation matrix

The invention relates to a method of isolating an immunoglobulin, comprising the steps of:

A kappa light chain-binding polypeptide comprising or consisting essentially of one or more mutated binding domains ofProtein L.

Modified kappa light chain-binding polypeptides

The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain ofProtein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, wherein at least the glutamine residue at position 9 has been mutated to an amino acid other than asparagine. The invention also discloses multimers of said polypeptide, as well as separation matrices comprising the multimers or polypeptides.

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of a parental Fc-binding domain ofProtein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

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Uppsala, Sweden

Tomas Bjorkman