Patent application EP19183033A (EP), filed 2019-06-27
Publication number: EP3757217A1
Title: METHODS FOR PROTEIN PURIFICATION
Abstract: The present invention relates to methods of protein purification, in particular using ion exchange chromatography. Modified proteins and peptide tags suitable for use in purification by ion exchange chromatography are provided, as are related methods.
IPC codes: C07K   1/    18            A I
C12N  15/    62            A I
Claims: number: 1
text: A fusion protein suitable for purification via ion exchange chromatography, which protein comprises
number: 2
text: A fusion protein according to claim 1, wherein the peptide tag is from 2 to 20 amino acids in length, optionally 4 to 12 amino acids in length.
number: 3
text: A fusion protein according to claim 1 or claim 2, wherein the peptide tag comprises or consists of an amino acid sequence of any one of SEQ ID Nos 1 to 9.
number: 4
text: A fusion protein according to any one of claims 1 to 3, further comprising a linker between the protein of interest and the peptide tag, wherein the linker optionally comprises GG, GS, SS or SG.
number: 5
text: A fusion protein according to any one of claims 1 to 4, wherein the protein of interest is an antigenic protein or a carrier protein, wherein the protein of interest is tetanus toxoid (TT), diphtheria toxoid (DT), CRM<197>, AcrA from C. jejuni, protein D from Haemophilus influenzae, exotoxin A of Pseudomonas aeruginosa (EPA), detoxified pneumolysin from Streptococcus. pneumoniae, meningococcal outer membrane protein complex (OMPC), detoxified Hla from S. aureus or ClfA from S. aureus.
number: 6
text: A fusion protein according to claim 5, wherein the protein of interest is EPA, wherein said EPA optionally comprises the amino acid sequence of SEQ ID NO. 10 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 10;
wherein the EPA protein is optionally modified in that
number: 7
text: A fusion protein according to any one of claims 1 to 6, wherein the fusion protein comprises
(i) EPA as defined in any one of claims 9 to 12, and (ii) a peptide tag as defined in any one of claims 1 to 3; wherein the peptide tag optionally comprises or consists of the amino acid sequence of any one of SEQ ID Nos: 6, 8 or 9;
wherein the fusion protein optionally comprises the amino acid sequence of any one of SEQ ID NOs: 12 to 18, optionally SEQ ID NOs: 14, 17 or 18.
number: 8
text: A fusion protein according to any one of claims 1 to 5, wherein the protein of interest is Hla from Staphylococcus aureus, wherein said Hla optionally comprises the amino acid sequence of SEQ ID NO. 19 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 19;
wherein the Hla protein is optionally modified in that
number: 9
text: A fusion protein according to any one of claims 1 to 5 or 8, wherein the fusion protein comprises (i) Hla as defined in claim 8, and (ii) a peptide tag as defined in any one of claims 1 to 6, wherein the peptide tag optionally comprises or consists of the amino acid sequence of any one of SEQ ID Nos: 1-3, optionally the amino acid sequence of SEQ ID No: 1 or SEQ ID NO: 2;
wherein the fusion protein optionally comprises the amino acid sequence of any one of SEQ ID NOs: 21 to 24, optionally SEQ ID NOs: 21 or 23.
number: 10
text: A nucleic acid encoding a fusion protein according to any one of claims 1 to 9, an expression vector comprising said nucleic acid, or a host cell comprising said vector.
number: 11
text: A protein-polysaccharide conjugate comprising a fusion protein according to any one of claims 1 to 9 wherein the protein is conjugated to a polysaccharide, optionally a bacterial polysaccharide, to form a conjugate, optionally a bioconjugate.
number: 12
text: A method of purifying a fusion protein according to any one of claims 1 to 9, or a conjugate of claim 11, the method comprising a step of ion exchange chromatography.
number: 13
text: A method of purifying a protein of interest, the method comprising (i) producing a fusion protein comprising the protein of interest and a peptide tag which binds to an ion exchange resin, and (ii) purifying the fusion protein by ion exchange chromatography, wherein the peptide tag is optionally as defined in any one of claims 1 to 4.
number: 14
text: A fusion protein according to any one of claims 1 to 9, or a method according to claim 12 or claim 13, wherein the ion exchange chromatography is cation exchange chromatography.
Office action: search_report, dated 2020-12-30
Rejection reasons: novelty_epc_54
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Final disposition: pending (2020-12-30)