WO2023180523A1 - Procédé de purification de protéines de fusion - Google Patents

Procédé de purification de protéines de fusion Download PDF

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Publication number
WO2023180523A1
WO2023180523A1 PCT/EP2023/057627 EP2023057627W WO2023180523A1 WO 2023180523 A1 WO2023180523 A1 WO 2023180523A1 EP 2023057627 W EP2023057627 W EP 2023057627W WO 2023180523 A1 WO2023180523 A1 WO 2023180523A1
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chromatography
protein
antibody
lipocalin
cell culture
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PCT/EP2023/057627
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WO2023180523A9 (fr
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Thibaut ANGEVIN
Daniel VON RÜDEN
Niket BUBNA
Dane GRISMER
David Brown
James Hamlin
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Pieris Pharmaceuticals Gmbh
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Publication of WO2023180523A1 publication Critical patent/WO2023180523A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
    • A61K39/39525—Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • mAbs monoclonal antibodies
  • bispecific biologies e.g., bispecific antibodies or antibody-like bispecific proteins, that recognize two different epitopes on the same or on different antigens have become increasingly attractive for combinatorial therapeutic applications.
  • bispecific biologies e.g., bispecific antibodies or antibody-like bispecific proteins, that recognize two different epitopes on the same or on different antigens have become increasingly attractive for combinatorial therapeutic applications.
  • Fc region Fc region
  • the later category can be further subdivided into bispecifics with asymmetrical or symmetrical architecture: asymmetrical bispecifics usually require the correct pairing of different heavy or light antibody chains, while symmetrical bispecifics may solve this issue by fusing an additional binding site to either a heavy or light antibody chain (for a comprehensive review on bispecific antibodies, see Brinkmann and Kontermann, MAbs, 2017, 9(2), 182-212).
  • the additional binding site is expressed as a single polypeptide chain or as polypeptide chains that do not interfere with the light chain/heavy chain pairing of the backbone antibody.
  • Suitable polypeptide chains are single-chain variable fragments, single-domain antibodies, and other small scaffold proteins, including non-immunoglobulin proteins, such as lipocalin muteins (also referred to as Anticalin® proteins).
  • lipocalin muteins are derived from naturally occurring lipocalins, which are a family of proteins present in a wide range of species, from bacteria to mammals (Akerstrom et al., Biochim Biophys Acta, 2000, 1482, 1-8).
  • Fc-containing bispecific antibodies and antibodylike fusion proteins can be captured by protein A chromatography, but usually have higher levels of aggregation compared to standard mAbs, which often causes problems when a typical mAb purification platform process is applied.
  • high similarities in biochemical properties between monomers and aggregates of such bispecifics often cause issues in subsequent aggregate polishing steps as it is difficult to separate them, entailing reduced yields to achieve acceptable aggregate clearance as well as issues with process robustness.
  • Introducing orthogonal aggregate removal steps in the purification train by distributing the aggregate clearance over two polishing steps may introduce its own technical challenges and facility fit issues.
  • conjugate refers to the joining together of two or more subunits, through all forms of covalent or non-covalent linkage, by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or a cross-linking agent, and non-covalent association.
  • fusion protein or “fusion polypeptide” as used interchangeably herein refers to a protein or polypeptide comprising two or more subunits.
  • a fusion protein comprises at least two subunits, wherein one subunit comprises or is a lipocalin mutein, and wherein another subunit comprises or is an antibody or an antigenbinding fragment thereof.
  • these subunits may be linked by covalent or non-covalent linkage.
  • the fusion protein is a translational fusion between the two or more subunits.
  • the translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit.
  • Both subunits may be interspersed by a nucleotide sequence encoding a linker.
  • the subunits of a fusion protein of the present disclosure may also be linked through chemical conjugation.
  • the subunits forming the fusion protein are typically linked to each other as follows: C-terminus of one subunit to N-terminus of another subunit, or C-terminus of one subunit to C- terminus of another subunit, or N-terminus of one subunit to N-terminus of another subunit, or N-terminus of one subunit to C-terminus of another subunit.
  • the subunits forming the fusion protein may also be linked to each other via one or more amino acid side chains of one or more of the subunits, e.g., through chemical conjugation.
  • the subunits of the fusion protein can be linked in any order and may include more than one of any of the constituent subunits. If one or more of the subunits is part of a protein (complex) that consists of more than one polypeptide chain, the term “fusion protein” may also refer to the polypeptide comprising the fused sequences and all other polypeptide chain(s) of the protein (complex).
  • subunit of a fusion protein/polypeptide disclosed herein refers to a single protein or a separate polypeptide chain, which can form a stable folded structure by itself and defines a unique function of providing a binding motif towards a target.
  • a “linker” that may be comprised by a fusion protein of the present disclosure joins together two or more subunits of a fusion protein as described herein.
  • the linkage can be covalent or non-covalent.
  • a preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids.
  • a preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker comprises one or more amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • Preferred peptide linkers include glycine-serine (Gly/Ser; GS) linkers, glycosylated GS linkers, proline-alanine-serine polymer (PAS) linkers, alpha helical linkers comprising the sequence motif A(EAAAK) X A (SEQ ID NO: 9) or A(EAAAR) X A (SEQ ID NO: 10), wherein x is an integer between (and including) 2 and 6, and hybrid linkers composed of glycine-serine linker sequences and alpha helical linker sequences, such as (G 4 S) x A(EAAAK) y A(G4S)z (SEQ ID NO: 11), (GSG) x A(EAAAK) y A(GSG) z (SEQ ID NO: 12), (G 4 S)xA(EAAAR) y A(G4S) z (SEQ ID NO: 13), or (GSG)xA(EAAAR) y
  • a GS linker is used to join together the subunits of a fusion protein as disclosed herein, wherein, preferably, the GS linker has the general formula (G4S) X (SEQ ID NO: 15) or (GaSGgjx (SEQ ID NO: 16), wherein x is an integer between (and including) 2 and 8.
  • the GS linker comprises or consists of the amino acid sequence as shown in SEQ ID NO: 4.
  • antibody includes whole antibodies or any antigen-binding fragment (or portion) or single chain thereof.
  • a whole antibody refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable domain (VH or HCVR) and a heavy chain constant region (CH).
  • the heavy chain constant region is comprised of three domains, CHI, CHZ and CHS-
  • Each light chain is comprised of a light chain variable domain (VL or LCVR) and a light chain constant region (CL).
  • the light chain constant region is comprised of one domain, CL.
  • V H and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may optionally mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antigen-binding fragment or “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the VH, VL, CL and CHI domains; (ii) a F(ab')2 fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment consisting of the VH, VL, CL and CHI domains and the region between CHI and CH2 domains; (iv) an Fd fragment consisting of the V H and CHI domains; (v) a single-chain Fv fragment consisting of the VH and VL domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., Nature, 1989, 341, 544-546) consisting of a V H domain; and (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; (viii)
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.
  • “framework” or “FR” refers to the variable domain residues other than the hypervariable region (CDR) residues.
  • “Fragment crystallizable region” or “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof (numbering according to EU index of Kabat; Johnson and Wu, Nucleic Acids Res, 2000, 28, 214-8).
  • the C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies as disclosed herein include human lgG1, lgG2 (lgG2A, lgG2B), lgG3, and lgG4.
  • a human lgG4 backbone is used for an antibody included in a fusion protein as described herein.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • humanized antibody refers to an antibody that consists of the CDRs of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody.
  • a humanized antibody is useful as an effective component in a therapeutic agent due to the reduced antigenicity.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • lipocalin refers to a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical p-pleated sheet supersecondary structural region comprising a plurality of (3-strands (preferably eight p-strands designated A to H) connected pair-wise by a plurality of (preferably four) loops at one end to thereby comprise a ligand-binding pocket and define the entrance to the ligand-binding pocket.
  • the loops comprising the ligand-binding pocket are loops connecting the open ends of 0-strands A and B, C and D, E and F, and G and H, and are designated loops AB, CD, EF, and GH.
  • lipocalin As used herein include, but are not limited to, tear lipocalin, Lipocalin-2 or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D, and Von Ebner's gland protein.
  • NGAL neutrophil gelatinase-associated lipocalin
  • lipocalin mutein refers to a “mutein,” a “mutated” entity (whether protein or nucleic acid), or “mutant” of a wild-type lipocalin, wherein the lipocalin mutein has binding specificity for a target other than the natural target(s) of the respective lipocalin.
  • the present disclosure explicitly encompasses lipocalin muteins having a cylindrical £- pleated sheet supersecondary structural region comprising eight p-strands connected pair-wise by four loops at one end to thereby comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated as compared to the native lipocalin sequence.
  • Proteins falling in the definition of “lipocalin” as used herein include, but are not limited to, human tear lipocalin (Tic, Lcn1), Lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, ai-acid glycoprotein, a-i-microglobulin, complement component 8y, retinol-binding protein, the epididymal retinoic acid-binding protein, glycodelin, odorant-binding protein, prostaglandin D synthase, with human tear lipocalin (Tic, Lcn1) and human neutrophil gelatinase-associated lipocalin (NGAL) being preferred.
  • Tic, Lcn1 human tear lipocalin
  • Lcn2 Lipocalin-2
  • NGAL neutrophil gelatinase-associated lipocalin
  • NGAL neutrophil gelatinase-
  • Lipocalin-2 or “neutrophil gelatinase-associated lipocalin” refers to human Lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and further refers to mature human Lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin.
  • a “mature hNGAL” of the instant disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which is free from the signal peptide.
  • Mature hNGAL is described by residues 21-198 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P80188, and its amino acid sequence is shown in SEQ ID NO: 1.
  • a mutein of hNGAL typically has at least about 60%, preferably at least about 70%, in some cases at least about 80% sequence identity to SEQ ID NO: 1.
  • tissue lipocalin refers to human tear lipocalin (hTIc) and further refers to mature human tear lipocalin.
  • a “mature hTIc” of the instant disclosure refers to the mature form of human tear lipocalin, which is free from the signal peptide. Mature hTIc is described by residues 19-176 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P31025, and its amino acid sequence is shown in SEQ ID NO: 2.
  • a mutein of hTIc typically has at least about 60%, preferably at least about 70%, in some cases at least about 80% sequence identity to SEQ ID NO: 2.
  • CD137 means human CD137 (huCD137).
  • Human CD137 means a full-length protein defined by UniProt Q07011, a fragment thereof, or a variant thereof.
  • CD137 is also known as 4-1 BB, tumor necrosis factor receptor superfamily member 9 (TNFRSF9) or induced by lymphocyte activation (ILA).
  • Human CD137 is encoded by the TNFRSF9 gene.
  • HER2 means human HER2 (huHER2).
  • Human HER2 means a full-length protein defined by UniProt P04626, a fragment thereof, or a variant thereof.
  • HER2 is also known as human epidermal growth factor receptor 2, HER2/neu, receptor tyrosine-protein kinase erbB-2, cluster of differentiation 340 (CD340), protooncogene Neu, ERBB2 (human), Erbb2 (rodent), c-neu, or p185.
  • Human HER2 is encoded by the ERBB2 gene.
  • FAP means human FAP (hFAP).
  • Human FAP means a full-length protein defined by UniProt Q12884, a fragment thereof, or a variant thereof.
  • FAP is also known as prolyl endopeptidase FAP or fibroblast activation protein alpha. Human FAP is encoded by the FAP gene.
  • fragment as used herein in connection with a given protein (e.g., CD137, HER2 or FAP) relates to proteins or peptides derived from the respective full-length mature protein that are N-terminally and/or C-terminally shortened, thus lacking at least one of the N-terminal and/or C-terminal amino acids.
  • Such fragments may include at least 10 or more, such as 20 or 30 or more, consecutive amino acids of the primary sequence of the mature protein and preferably retain the capability of the full-length mature protein to be recognized and/or bound by a lipocalin mutein or an antibody as described herein.
  • variants relate to derivatives of a protein or peptide that include modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Such modifications do, in some embodiments, not reduce the functionality of the protein or peptide, e.g., the capability of the wild-type protein to be recognized and/or bound by a lipocalin mutein or an antibody as described herein.
  • variants include proteins, wherein one or more amino acids have been replaced by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. However, such substitutions may also be conservative,
  • an amino acid residue is replaced with a chemically similar amino acid residue.
  • conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.
  • the term “variant”, as used herein with respect to a given protein e.g., CD137, HER2 or FAP
  • a variant has an amino acid sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with the respective wildtype protein as deposited with UniProt as described herein.
  • PRS-343 also known as cinrebafusp alfa, refers to the 4-1BB/HER2-bispecific fusion protein having the amino acid sequences of SEQ ID NOs: 5 and 6. The overall structure of PRS-343 is shown in Figure 1.
  • PRS-347 refers to the 4-1 BB/FAP-bispecific fusion protein having the amino acid sequences of SEQ ID NOs: 7 and 8. The overall structure of PRS-347 is shown in Figure 1.
  • protein A chromatography refers to an affinity chromatography method relying on the reversible and specific binding between an immobilized protein A ligand and the Fc region of antibodies or other Fc-containing molecules. Through such binding, they are retained on a chromatography column comprising a protein A resin and can later be eluted in a purified and concentrated form.
  • a protein of interest e.g., a fusion protein as described herein
  • a low pH buffer typically in the range of pH 2.5-5, e.g., pH 3-3.5
  • a high salt buffer typically 21.5 M NaCI
  • Exemplary protein A chromatography resins that can be used in accordance with the present disclosure include, but are not limited to, AmsphereTM A3 (JSR), MabSelectTM SuReTM LX (Cytiva), nProtein A Sepharose® 4 Fast Flow (Cytiva), and MabSelectTM PrismA (Cytiva).
  • AEX chromatography refers to a form of ion exchange (IEX) chromatography using a positively charged ion exchange resin with an affinity for molecules having net negative surface charges, thereby allowing the separation of molecules based on their net surface charge.
  • mixed mode AEX chromatography is used for purifying a fusion protein as described herein.
  • mixed mode or “multimodal”
  • chromatographic methods that utilize more than one form of interaction between the stationary phase (i.e., the chromatographic resin) and the molecules to be separated/purified.
  • a mixed mode AEX chromatography resin that utilizes both electrostatic and hydrophobic interactions is preferably used.
  • Suitable mixed mode AEX chromatography resins including mixed mode strong AEX chromatography resins and mixed mode weak AEX chromatography resins (classified according to the strength of the electrostatic interaction conferred by them), are known to a person skilled in the art and include, but are not limited to, CaptoTM adhere (Cytiva), CaptoTM adhere ImpRes (Cytiva), and NuviaTM aPrime 4A (Bio-Rad).
  • the AEX chromatography is operated in flow-through mode.
  • CEX chromatography refers to a form of IEX chromatography using a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges, thereby allowing the separation of molecules based on their net surface charge.
  • CEX chromatography resins that can be used according to the present disclosure, including strong CEX chromatography resins, weak CEX chromatography resins, and mixed mode strong or weak CEX chromatography resins, all of which are known to a person skilled in the art.
  • a mixed mode weak CEX chromatography resin is used, including, but not limited to, CaptoTM MMC (Cytiva) and Eshmuno® CMX (Merck).
  • a strong CEX chromatography resin is used, including, but not limited to, CaptoTM S ImpAct (Cytiva), CaptoTM S ImpRes (Cytiva), NuviaTM HR-S (Bio-Rad), Fractogel® EMD SO3- (Merck), Eshmuno® CPX (Merck), PorosTM XS (Thermo Fisher Scientific), and PorosTM 50 HS (Thermo Fisher Scientific).
  • the CEX chromatography is operated in bind-and-elute mode.
  • Hydrophobic interaction chromatography separates molecules based on their hydrophobicity. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin. The interaction between hydrophobic proteins and a HIC resin is greatly influenced by the running buffer. A high salt concentration enhances the interaction. Lowering the salt concentration weakens the interaction.
  • HIC may be performed in flow-through mode or in bind-and-elute mode.
  • HIC resin ligands include, but are not limited to, ether, polypropylene glycol (PPG), phenyl, butyl and hexyl (listed in the order of increasing hydrophobicity).
  • HIC resins include, but are not limited to, TOYOPEARL® Phenyl-600M (Tosoh Bioscience, King of Prussia, PA), Phenyl Sepharose® 6 Fast Flow (Cytiva), Capto Phenyl (Cytiva), TOYOPEARL® Butyl-600M (Tosoh Bioscience, King of Prussia, PA), and Butyl Sepharose® 4 Fast Flow (Cytiva).
  • binding-and-elute mode refers to a chromatographic process in which the molecule to be purified (e.g., a fusion protein as described herein) binds to the chromatographic medium (resin) and is subsequently eluted from the chromatographic medium (resin), e.g., after additional washes of the chromatographic medium (resin) to remove unbound or weaker bound material, such as contaminating molecules.
  • flow-through mode refers to a chromatographic process in which the molecule to be purified (e.g., a fusion protein as described herein) does not bind to the chromatographic medium (resin), i.e., passes through the chromatography column during loading, whereas contaminating molecules bind to the chromatographic medium (resin), i.e., are retained in the chromatography column.
  • the molecule to be purified e.g., a fusion protein as described herein
  • viral filtration refers to a filtration step that removes viruses from a solution, using a membrane barrier to retain viral particles. It is a size-based removal method which uses a specifically designed polymeric membrane to retain viral particles on the surface and within the pores of the membrane.
  • a viral nanofiltration step is used. Suitable viral filtration and nanofiltration membranes are known to a person skilled in the art and include, but are not limited to, PlanovaTM 15N, 20N & 35N (Asahi Kasei), PlanovaTM BioEX (Asahi Kasel), and Viresolve® Pro (Merck).
  • viral inactivation refers to a process step that stops viruses contained in a solution from being infectious and may involve one or more of the following three methods: heat treatment (e.g., pasteurization), low pH treatment, and solvent/detergent treatment.
  • Low pH treatment may include adjustment to a pH of 2 4, preferably a pH ⁇ 3.8, such as a pH of 3.5-3J, followed by incubation for 30-90 minutes and subsequent neutralization using a high concentration buffer.
  • the low pH treatment step may also be integrated with the protein A chromatography step in case elution from the protein A chromatography column is performed at a pH value close to those used for viral inactivation.
  • Viral inactivation by means of solvent/detergent treatment is based on the disruption of the interactions between the molecules in the viral lipid coating, wherein the solvent creates an environment in which the aggregation reaction between the lipid coat and the detergent happens more rapidly.
  • Typical detergents used for solvent/detergent viral inactivation are Triton® X-100 and Tween® 80, a typical solvent is tri-n-butyl phosphate (TnBP).
  • TnBP tri-n-butyl phosphate
  • Other suitable solvents and detergents are known to a person skilled in the art. In some embodiments of the method disclosed herein, Tween® 80 is used as detergent, and TnBP is used as solvent.
  • ultrafiltration/diafiltration refers to a filtration step which comprises (i) ultrafiltration (UF) separating molecules in solution based on the membrane pose size or molecular weight cutoff (MWCO) and being used for concentrating a given molecule of interest (e.g., a fusion protein as described herein), and (ii) diafiltration (DF) primarily used for buffer exchange.
  • UF/DF membranes include, but are not limited to, cellulose acetate, polyvinylidene fluoride (PVDF), and polyether sulfone (PES).
  • UF/DF membranes include, but are not limited to, Ultracell® (Merck), Biomax® (Merck), and OmegaTM (Pall).
  • Ultracell® Merck
  • Biomax® Merck
  • OmegaTM OmegaTM
  • UF and DF typically use tangential flow filtration.
  • a UF/DF membrane with a 30 kDa MWCO is used.
  • cell culture refers to a process by which cells are grown under controlled conditions, e.g., in a bioreactor, to produce a biomolecule of interest, e.g., a fusion protein as described herein.
  • a typical cell culture comprises four main phases: a lag phase (cells do not divide; cells are adjusting to the culture conditions and preparing for the cell division), a log phase (also referred to as logarithmic phase or exponential phase; cells actively proliferate, and the cell density increases exponentially), a stationary or plateau phase (cell proliferation slows down due to a growth-limiting factor, such as the depletion of an essential nutrient and/or the formation of an inhibitory product, resulting in a situation in which growth rate and death rate are equal) and a death or decline phase (cell death predominates at this phase and the number of viable cells reduces).
  • a lag phase cells do not divide; cells are adjusting to the culture conditions and preparing for the cell division
  • a log phase also referred to as logarithmic phase or exponential phase; cells actively proliferate, and the cell density increases exponentially
  • a stationary or plateau phase cell proliferation slows down due to a growth-limiting factor, such as the depletion of an essential nutrient and/or the
  • Suitable cells for the production of a fusion protein as described herein can be prokaryotic (e.g., bacterial) cells, such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic cells, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, or mammalian cells, such as immortalized mammalian cell lines (e.g., HeLa cells, Chinese hamster ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, NSO cells, or SP2/0 cells).
  • prokaryotic e.g., bacterial
  • E. coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • eukaryotic cells such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells
  • mammalian cells such as immortalized mammalian cell lines (e.g., HeLa cells,
  • fed-batch refers to a process in which one or more nutrients (substrates) are fed (supplied) to the bioreactor during cell cultivation (e.g., to prevent nutrient depletion) and in which the product(s) remain in the bioreactor until the end of the run.
  • N-1 perfusion refers to the intensification of cell growth in a seed step prior to the production bioreactor (N) by cell retention combined with media exchange.
  • Respective means and methods are known to the skilled person and are described, for example, in Stepper et al. (Bioprocess Biosyst Eng, 2020, 43, 1431-1443), Kloth et al. (Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology, 2009, 1-30) and Voisard et al. (Biotechnol Bioeng, 2003, 82(7), 751-765), which are incorporated herein by reference in their entirety.
  • binding affinity describes the ability of a biomolecule (e.g., a polypeptide or a protein, such as an antibody or lipocalin mutein) to bind a selected target (and form a complex). Binding affinity is measured by a number of methods known to those skilled in the art including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), quartz crystal microbalance (QCM), bio-layer interferometry (BLI), and surface plasmon resonance (SPR).
  • ITC isothermal titration calorimetry
  • QCM quartz crystal microbalance
  • BLI bio-layer interferometry
  • SPR surface plasmon resonance
  • Binding affinity is thereby reported as a value of dissociation constant (Ko), half maximal effective concentration (ECso), or half maximal inhibitory concentration (IC50) measured using such methods.
  • Ko dissociation constant
  • ECso half maximal effective concentration
  • IC50 half maximal inhibitory concentration
  • binding affinity e.g., fluorescence titration, competitive ELISA (also called competition ELISA), and surface plasmon resonance
  • binding affinity reported by a KD, ECSO, or IC50 value may vary within a certain experimental range, depending on the method and experimental setup.
  • binding specificity relates to the ability of a biomolecule to discriminate between the desired target and one or more reference targets. It is understood that such specificity is not an absolute but a relative property and can be determined, for example, by means of SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans.
  • mutant and wild-type e.g., in connection with an amino acid or nucleotide sequence, generally refer to something that is naturally occurring (i.e., it can be derived/isolated from nature).
  • sequence identity denotes a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.
  • sequence homology or “homology” has its usual meaning, and a homologous amino acid includes identical amino acids as well as amino acids which are regarded to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or a polypeptide of the disclosure.
  • BLAST Altschul et al., Nucleic Acids Res, 1997, 25, 3389-3402
  • BLAST2 Altschul et al., J Mol Biol, 1990, 215, 403-410
  • Smith- Waterman Smith and Waterman, J Mol Biol, 1981 , 147, 195- 197
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., Nucleic Acids Res, 1997, 25, 3389-3402).
  • the percentage of homology is based on the alignment of the entire protein or polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10 ⁇ 3 ) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.
  • Gaps are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements (substitutions), may have a lower degree of sequence identity.
  • pharmaceutically acceptable refers to the non-toxicity of a material which, in certain exemplary embodiments, does also not interact with the action of the active agent(s) of the pharmaceutical composition.
  • carrier refers to an organic or inorganic component of natural origin or synthetic nature, in which the active agent(s) of a pharmaceutical composition is/are provided in order to facilitate, enhance or enable its/their application.
  • carrier may include one or more solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject.
  • excipient is intended to include all substances which may be present in a pharmaceutical composition and which are not pharmaceutically active ingredients, such as salts, binders, filters, lubricants, thickeners, surfactants, preservatives, emulsifiers, or buffer substances.
  • the term “about”, “approximately” or “similar to” means within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes the concrete number, i.e., “about 20” includes the number of 20. The term “at least about” as used herein includes the concrete number, i.e., “at least about 20” includes 20.
  • Figure 1 shows the overall structure of preferred lipocalin mutein-antibody fusion proteins that can be manufactured/purified according to the methods disclosed herein.
  • Each of the heavy chains of an antibody is fused at its C-terminus via a peptide linker to the N- terminus of a lipocalin mutein.
  • Figure 3 shows a preferred sequence of purification steps according to the methods disclosed herein.
  • a viral inactivation step is included prior to (e.g., solvent/detergent viral inactivation) or after (e.g., low pH viral inactivation) the protein A chromatography step.
  • Figure 4 shows the determination of dynamic binding capacity (DBC) for the bispecific fusion proteins PRS-343 and PRS-347 using different protein A chromatography resins (MSSLX - MabSelectTM SuReTM LX; AA3 - AmsphereTM A3).
  • Protein A chromatography included post-load washes to reduce impurities and elution of the fusion proteins using low pH glycine or acetate buffers.
  • Each fusion protein achieved a load factor of greater than 30 g/l prior to the 10% breakthrough threshold being met.
  • FIG. 5 shows the analysis of the mixed mode anion exchange (AEX) chromatography step for the bispecific fusion proteins PRS-343 and PRS-347 in terms of removal of impurities and yields across different production scales.
  • AEX chromatography was performed in flow-through mode to reduce impurities, including residual host cell protein (rHCP) and high molecular weight species (HMWS).
  • the AEX chromatography step achieved greater than one log clearance of rHCP as well as significant clearance of HMWS across all productions scales for both fusion proteins, as determined by size exclusion chromatography (SEC) (A). Consistent yields of both fusion proteins were also observed across all production scales (B).
  • SEC size exclusion chromatography
  • Figure 6 shows the analysis of hydrophobicity of PRS-343 and PRS-347 by hydrophobic interaction chromatography (HIC) (A) and the removal of rHCP and HMWS impurities in a polishing step across different production scales.
  • HIC hydrophobic interaction chromatography
  • CEX cation exchange
  • Figure 7 shows successful removal of impurities, rHCP (A) and HMWS (B), by completing all purification steps in series in a 3 L production scale: protein A chromatography, mixed mode AEX chromatography and CEX chromatography. BDS refers to “bulk drug substance”. rHCP and HMWS levels, respectively, are plotted chronologically to demonstrate the clearance of these impurities throughout the process.
  • Figure 8 shows successful removal of impurities, rHCP (A) and HMWS (B), by completing the purification steps of protein A chromatography, mixed mode AEX chromatography, and CEX chromatography for PRS-343 in series across different production scales (3 L, 200 L or 1000 L).
  • BDS refers to “bulk drug substance”.
  • rHCP and HMWS levels, respectively, are ploted chronologically to demonstrate the clearance of these impurities throughout the process. Consistent cumulative chromatography yields above 50% were also observed across all production scales (C).
  • the present disclosure relates to a method of purifying a fusion protein comprising (i) a lipocalin mutein and (ii) an antibody or an antigen-binding fragment thereof, said method comprising, in the following order, the steps of:
  • the mixed mode AEX chromatography is performed in flow- through mode. In some embodiments, a mixed mode strong AEX chromatography resin is used. In some embodiments, a mixed mode weak AEX chromatography resin is used.
  • the CEX chromatography in step (c) is performed in bind- and-elute mode.
  • a mixed mode weak CEX chromatography resin is used.
  • a strong CEX chromatography resin is used.
  • the HIC in step (c) is performed in flow-through mode.
  • the method further comprises one or more viral inactivation and/or viral filtration steps.
  • the method comprises, prior to step (a) or between steps (a) and (b), a low pH viral inactivation step or a solvent/detergent viral inactivation step. In some embodiments, the method comprises, prior to step (a), a solvent/detergent viral inactivation step. In some other embodiments, the method comprises, between steps (a) and (b), a low pH viral inactivation step.
  • the method further comprises, subsequent to step (c), the step of:
  • the method comprises, subsequent to step (c) or, optionally, between step (c) and step (d), a viral filtration step.
  • the method comprises, in the following order, the steps of: (a) solvent/detergent viral inactivation; (b) protein A chromatography; (c) mixed mode AEX chromatography; (d) CEX chromatography or HIC; (e) viral filtration; and (f) ultraflltration/diafiltration.
  • the method comprises, in the following order, the steps of: (a) protein A chromatography; (b) low pH viral inactivation; (c) mixed mode AEX chromatography; (d) CEX chromatography or HIC; (e) viral filtration; and (f) ultraflltration/diafiltration.
  • the method comprises, in the following order, the steps of: (a) solvent/detergent viral inactivation; (b) protein A chromatography; (c) mixed mode AEX chromatography; (d) CEX chromatography; (e) viral filtration; and (f) ultraflltration/diafiltration.
  • the fusion protein comprises a lipocalin mutein that is linked to the C-terminus of an antibody heavy chain (HC), the N-terminus of the HC, the C- terminus of an antibody light chain (LC), and/or the N-terminus of the LC.
  • a lipocalin mutein can be fused at its N-terminus and/or its C-terminus to an antibody or antigen-binding fragment thereof, e.g though via a peptide linker, such as a GS linker.
  • the GS linker comprises or consists of the amino acid sequence as shown in SEQ ID NO: 4.
  • the fusion protein comprises an antibody and two copies of the lipocalin mutein, wherein each of the heavy chains of the antibody is fused at its C-terminus to the N-terminus of a lipocalin mutein, e.g., as essentially shown in Figure 1.
  • each of the heavy chains of the antibody is fused at its C-terminus to the N- terminus of a lipocalin mutein via a peptide linker, such as a GS linker.
  • the GS linker comprises or consists of the amino acid sequence as shown in SEQ ID NO: 4.
  • the lipocalin mutein is specific for CD137.
  • the lipocalin mutein is capable of binding CD137 with a binding affinity measured by a KD of about 300 nM or lower, about 200 nM or lower, about 100 nM or lower, about 75 nM or lower, about 50 nM or lower, about 25 nM or lower, about 10 nM or lower, or about 5 nM or lower, for example, as determined by surface plasmon resonance (SPR) analysis.
  • the lipocalin mutein is capable of activating downstream signaling pathways of CD137 by binding to CD137.
  • Suitable lipocalin muteins specific for CD137 that may be used in a fusion protein as described herein are disclosed in, e.g., WO 2016/177762, WO 2016/177802, WO 2020/025659, and WO 2020/173897, which are incorporated herein by reference in their entirety.
  • the lipocalin mutein is a mutein of mature human neutrophil gelatinase-associated lipocalin (hNGAL).
  • the lipocalin mutein comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the lipocalin mutein comprises the following set of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL as shown in SEQ ID NO: 1: Leu 36 Gin; Ala 40 ⁇ He; He 41 ⁇ Arg; Gin 49 ⁇ lie; Tyr 52 ⁇ Met; Ser 68 ⁇ Met; Arg 72 ⁇ Asp; Lys 73 ⁇ Asp; Trp 79 ⁇ Asp; Arg 81 ⁇ Trp; Asn 96 ⁇ ys; Tyr 100 ⁇ Phe; Leu 103 ⁇ His; Tyr 106 Ser; Lys 125 ⁇ Phe; Ser 127 ⁇ Phe; Tyr 132 Glu; and Lys 134 -» Tyr.
  • the lipocalin mutein comprises the following set of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL as shown in SEQ ID NO: 1: Gin 28 His; Leu 36 ⁇ Gin; Ala 40 ⁇ He; He 41 Arg; Gin 49 lie; Tyr 52 ⁇ Met; Asn 65 ⁇ Asp; Ser 68 ⁇ Met; Leu 70 ⁇ Lys; Arg 72 ⁇ Asp; Lys 73 ⁇ Asp; Asp 77 ⁇ Met; Trp 79 ⁇ Asp; Arg 81 Trp; Cys 87 ⁇ Ser; Asn 96 ⁇ Lys; Tyr 100 ⁇ Phe; Leu 103
  • the antibody or antigen-binding fragment thereof is specific for an antigen expressed on the surface or in the microenvironment of a cell, e.g., a tumor cell.
  • the antibody is a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof is specific for HER2.
  • the antibody is trastuzumab or pertuzumab.
  • the fusion polypeptide comprises an antibody, wherein the binding of the Fc region of the antibody to Fc receptor-positive cell may be reduced or fully suppressed by protein engineering. This may be achieved, for example, by switching from an lgG1 backbone to an lgG4 backbone, as lgG4 is known to display reduced Fc-gamma receptor interactions compared to lgG1.
  • the antibody as used in a fusion protein as disclosed herein has an lgG4 backbone.
  • mutations may be introduced into the lgG4 backbone, such as F234A and L235A.
  • a S228P mutation may be introduced into the lgG4 backbone to minimize the exchange of lgG4 half-antibody.
  • an additional N297A mutation may be present in the immunoglobulin heavy chain of the fusion polypeptide in order to remove the natural glycosylation motif.
  • the lgG4 backbone has one or more of the following mutations: S228P, N297A, F234A, and L235A (numbering according to EU index of Kabat).
  • the lgG4 backbone has the following mutations: S228P, F234A, and L235A (numbering according to EU index of Kabat).
  • Exemplary fusion proteins that may be purified with the method as disclosed herein are described in, e.g., WO 2016/177802, WO 2020/025659, and WO 2020/173897, which are incorporated herein by reference in their entirety.
  • the fusion protein comprises the amino acid sequences of SEQ ID NOs: 5 and 6 or amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 5 and 6.
  • the fusion protein comprises two chains having the amino acid sequence of SEQ ID NO: 5 and two chains having the amino acid sequence of SEQ ID NO: 6.
  • the fusion protein is cinrebafusp alfa (PRS-343).
  • the fusion protein prior to its purification, is produced in a cell culture.
  • the cell culture is a mammalian cell culture or a bacterial cell culture.
  • the cell culture is a Chinese hamster ovary (CHO) cell culture.
  • the cell culture is an E. coli cell culture.
  • the cell culture is a fed-batch culture, optionally a fed-batch culture with N-1 perfusion.
  • the cell culture comprises a temperature shift to a lower temperature during the exponential growth phase.
  • the method further comprises the step of formulating the purified fusion protein by mixing it with one or more pharmaceutically acceptable carriers and/or excipients.
  • a fusion protein comprising the amino acid sequences of SEQ ID NOs: 1 and 8 or amino acid sequences having at least 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs: 7 and 8, said fusion protein having binding specificity for CD137 and FAP.
  • the fusion protein comprises two chains having the amino acid sequence of SEQ ID NO: 7 and two chains having the amino acid sequence of SEQ ID NO: 8.
  • Example 1 Production of lipocalin mutein-antibody fusion proteins in CHO cells
  • CHO cell lines stably expressing the lipocalin mutein-antibody fusion protein PRS-343 (SEQ ID NOs: 5 and 6) or PRS-347 (SEQ ID NOs: 7 and 8) were obtained from Selexis (Geneva, Switzerland). Vial thaw was performed in non-baffled shake flasks (Corning Life Sciences, NY) containing BalanCD Growth A medium (Irvine Scientific, CA) supplemented with L-glutamine and cultivated in an incubator shaker (Multitron, Infers HT) set at 36.5-37.0°C, 5% CO2, and 80% relative humidity. Cells were passaged every 3-4 days prior to fed-batch production.
  • pH was maintained between 6.80-7.00 and then from pH 6.80- 7.20 during exponential and stationary growth phases (typically on day 7).
  • Offline pH and gases were measured using an ABL80 Flex blood gas analyzer (Radiometer, CA); cell count and cell viability were measured using a Vi-CELL XR automated cell counter (Beckman Coulter, CA); metabolites, including glucose, lactate, ammonia, glutamine and glutamate, were measured using a Nova Bioprofile 400 (Nova Biomedical, MA) and CEDEX Bio HT analyzer (Roche, Switzerland). Production cultures were harvested upon cultivation for 14 days using POD depth filters (MilliporeSigma, MA) and 0.22-micron PES filters (MilliporeSigma, MA). Cell culture supernatant and clarified harvest samples were analyzed for titer using an HPLC method. In- process quality attributes were assessed by partial purification using a PreDictor plate.
  • inoculum was transferred to fresh BalanCD Growth A medium supplemented with L-glutamine at a fixed target viable cell density to initiate production.
  • Cells were cultured in a bioreactor with temperature, pH and dissolved oxygen control.
  • Chemically defined feed media (Cell Boost 7A and Cell Boost 7B) and CHO bioreactor feed supplement (C1615) were added to the bioreactor culture on specified days; cultures were sampled daily to test for cell growth, viability, and concentration of selected metabolites.
  • Bioreactor temperature was shifted down to 33°C during exponential growth phase to reduce growth rate, to promote product formation, and to ensure high cell viability.
  • VDCs Viable cell densities
  • Figure 2A Viable cell densities
  • Figure 2B Lactate is an important indicator of culture health, including pH.
  • Both cell lines showed a similar trend of lactate accumulation during the growth phase and consumption in the stationary culture phase, albeit with minor differences in peak levels and rate of consumption (Figure 2C).
  • Specific productivity ranged from 10-43 pg/cell/day, and was highest for PRS-347 (Figure 2D).
  • PRS-347 cell line For the PRS-347 cell line, an identical process was used except for the dissolved oxygen set point, which was changed from 30% to 40% to accommodate a new standard procedure during process development. Peak cell density for the PRS-347 cell line was similar to the PRS-343 cell line, peaking between 20-30 x 10 6 cells/ml. No glucose depletion was observed and, as the PRS-343 cell line, the PRS-347 cell line showed complete lactate metabolism within the 14-day fed-batch production phase. Specific productivities were consistent across tested bioreactor scales, ranging from 35-43 pg/cell/day.
  • Example 2 Establishment of a platform process for the purification of lipocalin mutein-antibody fusion proteins
  • Resins tested and/or incorporated into the final purification processes included AmsphereTM A3 (JSR), MabSelectTM SuReTM LX (Cytiva), CaptoTM adhere (Cytiva), CaptoTM S ImpAct (Cytiva), CaptoTM MMC (Cytiva), NuviaTM HR-S (Bio-Rad) and TOYOPEARL® Phenyl-600M (Tosoh Bioscience, King of Prussia, PA). Resins were packed into Vantage columns (Millipore Sigma) to a bed height of 18-22 cm, and an internal diameter of 1.1 -4.4 cm.
  • Hydrophobicity was assessed via hydrophobic interaction chromatography (HIC). Proteins were initially loaded onto the HIC column in 50% ammonium sulfate to promote binding. A gradient elution was then performed using decreasing salt concentration until the product eluted. Proteins with higher hydrophobicity elute later during the gradient (higher retention time).
  • HIC hydrophobic interaction chromatography
  • Protein A affinity high performance liquid chromatography was utilized to assess protein concentration in cell culture samples.
  • a POROSTM A 20 pm column (Applied Biosystems, Waltham MA) was used with mobile phases consisting of 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 and 50 mM glycine hydrochloride, 150 mM sodium chloride, pH 3.0.
  • Sample injection volumes were adjusted to achieve an injection load of 90 pg.
  • the flow rate for the method was 1.0 ml/min, with a total run time of 4.5 minutes.
  • Column temperature was set to 22°C.
  • Absorbance was monitored at 280 nm, and peaks were integrated between 2.2-2.8 minutes. Product concentration was then determined by extrapolating peak areas from a standard curve of known column loadings.
  • Residual host cell protein present was measured using the 3G CHO HCP ELISA kit (Cat. NO. #F550 Cygnus Technologies, Southport, NC) according to the manufacturer’s instructions. Dilutions of fusion protein-containing samples were diluted as needed by kit diluent and added to well plates pre-coated with anti-CHO antibodies. An anti-CHO antibody conjugated to horseradish peroxidase (HRP) was then added to the wells, and the plate was incubated for 2 hours at 25°C with gentle shaking. After using the provided wash solution for a total of 4 washes, a substrate was added to the wells, and the reaction was measured spectrophotometrically at 450 and 650 nm.
  • HRP horseradish peroxidase
  • rHCP residual HCP
  • Protein concentration was measured using UV/Vis spectroscopy (Agilent 8453) and SoloVPE (C Technologies IN-VPE-SOLO5). Samples within a range of 0.5-25 mg/ml were added to a small vessel (OC0009-1-P50), and a new fibrette was installed. Samples were measured at 280 nm with an extinction coefficient of 1.3. Concentrations were calculated using a standard curve. For UV/Vis spectroscopy, samples were diluted to 0.5-0.9 mg/ml with a buffer consisting of 20 mM Histidine, 250 mM Sorbitol, 0.01% (w/v) PS80, pH 6.3. The UV/Vis was blanked with the same buffer. Samples were scanned in cuvettes with absorbance readings of 280 and 320 nm. Concentrations were calculated using Beer’s Law with an extinction coefficient of 1.3.
  • the protein A capture step required litle development beyond the determination of dynamic binding capacity (DBC) of the resin to set a maximum resin loading limit.
  • DBC dynamic binding capacity
  • AEX chromatography using a mixed mode AEX resin (here: CaptoTM adhere, Cytiva) was evaluated for suitability as a flow-through polishing step.
  • the mixed mode AEX resin was determined to be suitable in flow-through mode after a pH screen was conducted to determine the optimal operating pH.
  • Samples were collected before and after the step to evaluate various aspects of product quality, including residual host cell protein (rHCP) and size exclusion chromatography HPLC to assess %HMWS, a measure of product aggregation.
  • rHCP residual host cell protein
  • HPLC size exclusion chromatography
  • the mixed mode AEX chromatography step achieved greater than one log clearance of the rHCP remaining after the capture step as well as substantial clearance of HMWS ( Figure 5A), while ensuring high yields of the bispecific (>80%) (Figure 5B) across all production scales.
  • PRS-343 was scaled-up to the 200 L and 1000 L production scale and achieved comparable clearance and yield.
  • HIC in flow-through mode e.g., using a Phenyl-600M resin
  • HIC in flow-through mode may be used as an alternative second polishing step. This was confirmed with another lipocalin mutein-antibody fusion protein (data not shown).
  • FIG. 7A and Figure 7B show rHCP and HMWS levels, respectively, ploted chronologically to demonstrate the clearance of these impurities throughout the process.
  • the clearance of rHCP exhibits a similar trend for each molecule, with the protein A chromatography and flow-through AEX chromatography steps clearing the majority of the rHCP and the final polishing step (e.g., CEX chromatography in bind-and-elute mode) further reducing the levels to achieve a high level of purity.
  • the AEX chromatography step provided clearance to less than 1% HMWS for PRS-343.
  • PRS-347 exhibited a higher level of HMWS after protein A chromatography, but it was ultimately cleared to less than 1%.
  • PRS-343 was chosen for scale-up to confirm the robustness of the process at various scales approaching an intended manufacturing scale. Impurity clearance ( Figure 8A, Figure 8B) and process yield ( Figure 8C) trended well across the three tested production scales.

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Abstract

La présente divulgation concerne un procédé de purification de protéines de fusion comprenant une mutéine de lipocaline et un anticorps ou un fragment de liaison à l'antigène de celle-ci.
PCT/EP2023/057627 2022-03-24 2023-03-24 Procédé de purification de protéines de fusion WO2023180523A1 (fr)

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3917527A (en) 1973-04-17 1975-11-04 Yeda Res & Dev Hydrophobic chromatography
US4000098A (en) 1974-08-16 1976-12-28 Palo Alto Medical Research Foundation Separation of proteins by hydrophobic adsorption
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
WO2016177802A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Polypeptide de fusion anticancéreux
WO2016177762A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Protéines spécifiques de cd137
WO2020025659A1 (fr) 2018-07-31 2020-02-06 Pieris Pharmaceuticals Gmbh Nouvelle protéine de fusion spécifique à cd137 et pd-l1
WO2020173897A1 (fr) 2019-02-26 2020-09-03 Pieris Pharmaceuticals Gmbh Nouvelles protéines de fusion spécifique à cd137 et gpc3
WO2020183332A1 (fr) * 2019-03-11 2020-09-17 Intas Pharmaceuticals Ltd. Purification d'adalimumab à l'aide d'une chromatographie en tandem
WO2020208049A1 (fr) * 2019-04-12 2020-10-15 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à un antigène comprenant des mutéines de lipocalines
WO2020252072A1 (fr) * 2019-06-10 2020-12-17 Millennium Pharmaceuticals, Inc. Procédés de purification d'anticorps et compositions associées
EP3916001A1 (fr) * 2019-01-23 2021-12-01 Daiichi Sankyo Company, Limited Procédé de purification d'anticorps comprenant une étape utilisant un matériau de carbone activé

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3917527A (en) 1973-04-17 1975-11-04 Yeda Res & Dev Hydrophobic chromatography
US4000098A (en) 1974-08-16 1976-12-28 Palo Alto Medical Research Foundation Separation of proteins by hydrophobic adsorption
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US7250297B1 (en) 1997-09-26 2007-07-31 Pieris Ag Anticalins
WO2016177762A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Protéines spécifiques de cd137
WO2016177802A1 (fr) 2015-05-04 2016-11-10 Pieris Pharmaceuticals Gmbh Polypeptide de fusion anticancéreux
WO2020025659A1 (fr) 2018-07-31 2020-02-06 Pieris Pharmaceuticals Gmbh Nouvelle protéine de fusion spécifique à cd137 et pd-l1
EP3916001A1 (fr) * 2019-01-23 2021-12-01 Daiichi Sankyo Company, Limited Procédé de purification d'anticorps comprenant une étape utilisant un matériau de carbone activé
WO2020173897A1 (fr) 2019-02-26 2020-09-03 Pieris Pharmaceuticals Gmbh Nouvelles protéines de fusion spécifique à cd137 et gpc3
WO2020183332A1 (fr) * 2019-03-11 2020-09-17 Intas Pharmaceuticals Ltd. Purification d'adalimumab à l'aide d'une chromatographie en tandem
WO2020208049A1 (fr) * 2019-04-12 2020-10-15 F. Hoffmann-La Roche Ag Molécules bispécifiques de liaison à un antigène comprenant des mutéines de lipocalines
WO2020252072A1 (fr) * 2019-06-10 2020-12-17 Millennium Pharmaceuticals, Inc. Procédés de purification d'anticorps et compositions associées

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"SWISS-PROT", Database accession no. P31025
"UniProt", Database accession no. Q12884
ABHINAV A. SHUKLA ET AL: "Evolving trends in mAb production processes : SHUKLA et al.", BIOENGINEERING & TRANSLATIONAL MEDICINE, vol. 2, no. 1, 1 March 2017 (2017-03-01), pages 58 - 69, XP055562281, ISSN: 2380-6761, DOI: 10.1002/btm2.10061 *
AKERSTROM ET AL., BIOCHIM BIOPHYS ACTA, vol. 1482, 2000, pages 337 - 24
ALTSCHUL ET AL., J MOL BIOL, vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 3389 - 3402
BRINKMANNKONTERMANN, MABS, vol. 9, no. 2, 2017, pages 182 - 212
CASSIA ANDRADE ET AL: "An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process", BIOTECHNOLOGY PROGRESS, AMERICAN CHEMICAL SOCIETY, HOBOKEN, USA, vol. 35, no. 1, 17 October 2018 (2018-10-17), pages n/a, XP072300182, ISSN: 8756-7938, DOI: 10.1002/BTPR.2720 *
FLOWER, BIOCHEM J, vol. 318, 1996, pages 1 - 14
HOLLIGER ET AL., PROC NATL ACAD SCI U S A, vol. 90, no. 14, 1993, pages 6444 - 6448
JOHNSONWU, NUCLEIC ACIDS RES, vol. 28, 2000, pages 214 - 8
KLOTH ET AL., ENCYCLOPEDIA OF INDUSTRIAL BIOTECHNOLOGY: BIOPROCESS, BIOSEPARATION, AND CELL TECHNOLOGY, 2009, pages 1 - 30
QUEIROZ ET AL., J BIOTECHNOL., vol. 87, no. 2, 2001, pages 143 - 159
SCHIEFNERSKERRA, ACC CHEM RES., vol. 48, no. 4, 2015, pages 976 - 985
SCHLEHUBERSKERRA, DRUG DISCOV TODAY, vol. 10, no. 1, 2005, pages 23 - 33
SHUKLA ET AL., BIOENG TRANSL. MED., vol. 2, no. 1, 2017, pages 58 - 69
SMITHWATERMAN, J MOL BIOL, vol. 147, 1981, pages 195 - 197
STEPPER ET AL., BIOPROCESS BIOSYST ENG, vol. 43, 2020, pages 1431 - 1443
VOISARD ET AL., BIOTECHNOL BIOENG, vol. 82, no. 7, 2003, pages 751 - 765
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546

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