Method for purifying protein conjugates

Abstract

The present invention relates to the field of producing protein conjugates. The invention relates to a novel method for producing a protein conjugate comprising a protein of interest (POI) bearing a chemical moiety and a payload comprising a complementary chemical moiety. The said method comprises the extracellular expression of the POI, contacting the POI in the cell culture supernatant with the payload, allowing the formation of a covalent bond between the POI and the payload in a cell culture supernatant, thereby creating a protein conjugate, and purifying said conjugate.

Description

METHOD FOR PURIFYING PROTEIN CONJUGATES DescriptionField of the Invention

[0001] The present invention relates to the field of producing protein conjugates.Background Art

[0002] The large-scale, economic purification of proteins is an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors. The culture media (cell culture supernatant) also contains the by-products of the cells themselves.

[0003] The protein of interest must be isolated from the culture supernatant to purity sufficient for use as a human therapeutic. The standards set by health authorities for proteins intended for human administration regarding impurities are very high. Many purification methods for proteins known in the art contain steps requiring the application e.g., of low or high pH, high salt concentration or other extreme conditions that may irreversibly jeopardize the biological activity of the protein to be purified and are therefore not suitable. Thus, separation of the desired protein to sufficient purity poses a formidable challenge.

[0004] Protein conjugates are a class of therapeutics with increased market relevance in recent years. One challenge in the development of protein conjugates is that their manufacture remains complex. After purifying the protein in mostly laborious chromatography steps, a conjugation step is added. After the conjugation, another or multiple purification steps are needed to remove the impurities introduced by the conjugation.

[0005] Click chemistry refers to a group of chemical reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and give high product yields, which makes these reactions ideal tools for targeting and labelling biological targets in vivo. The inverse-electron demand Diels-Alder reaction between the diene tetrazine and dienophiles (iEDDA) shows exceptional kinetics (1 - 106M-1s-1) and is, due to its speed and the metabolic stability of some developed reactants especially suited for such in vivo applications. Because of its specificity, thereaction is a promising tool to optimize the processes of protein conjugates production.

[0006] However, currently used synthetic amino acids based on azide click reactions with dibenzoazacyclooctyne (DBCO) or bicyclononyne (BCN) or synthetic amino acids containing ketones reacting with alkoxyamine are not suited for reactions in the culture supernatant, as they take a long time and lead to various byproducts that are generating impurities that are not desirable in the production process.

[0007] W02014117001A1 discloses a modified amino acid bearing an unsubstituted tetrazine moiety. W02014065860A1 discloses functionalized 1, 2,4,5-tetrazine compounds. WO2022248587A1 relates to novel amino acid derivatives bearing a tetrazine moiety, a process for the preparation thereof and the use of the amino acid derivative in click chemistry or site-specific protein or peptide modification. W02024061950A1 describes a modified peptide or protein comprising one or more bioorthogonal functional groups, wherein the bioorthogonal group can be a diene moiety.

[0008] Therefore, there is still the need for an easy, fast and efficient method for producing and purifying synthetic protein conjugates.Summary of invention

[0009] It is the object of the present invention to provide a fast, reliable and efficient method for producing and purifying synthetic protein conjugates.

[0010] The object is solved by the subject-matter of the present invention.

[0011] According to the invention, there is provided a purification method for synthetic protein conjugates. The synthetic protein conjugate comprises a protein of interest (POI) bearing a reactive moiety covalently bound to a payload.

[0012] One embodiment of the invention relates to a method for producing a protein conjugate comprising the steps of:i. providing one or more cells comprising a nucleic acid encoding a protein of interest (POI) bearing a first chemical moiety;ii. culturing the one or more cells in a cell culture media such that the POI is expressed and excreted from the cells into a culture supernatant;iii. exposing the POI in the supernatant to an excess of a payload bearing a second chemical moiety capable to form a covalent bond with the first chemical moiety of the POI;iv. allowing the formation of a covalent bond between the first chemical moiety of the POI and the second chemical moiety of the payload thereby creating a site-specific protein conjugate; andv. purifying said protein conjugate.

[0013] A further embodiment relates to the method as described herein, wherein said first chemical moiety or said second chemical moiety are independently from one another a dienophile moiety or a diene moiety.

[0014] According to one embodiment of the invention, the dienophile moiety is a trans-cyclooctene dienophile such as for example, bicyclononyne (BCN) or cyclooctene.

[0015] According to one embodiment of the invention, the diene moiety is a tetrazine moiety.

[0016] A further embodiment relates to the method as described herein, wherein POI comprises a diene moiety.

[0017] A further embodiment relates to the method as described herein, wherein the payload comprises a dienophile moiety.

[0018] A further embodiment relates to the method as described herein, wherein the one or more cells are mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2.

[0019] A further embodiment relates to the method as described herein, wherein the payload further comprises a pharmacologically or biologically active agent.

[0020] A further embodiment relates to the method as described herein, wherein the payload is present at least with a 2-fold molar excess over the POI.

[0021] A further embodiment relates to the method as described herein, wherein the payload and the POI are reacted for at least 0.5h.

[0022] A further embodiment relates to the method as described herein, wherein the protein conjugate is purified by supernatant clarification and / or chromatographic techniques. Suitable chromatographic techniques are for example, affinity chromatography, size exclusion chromatography hydrophobic interaction chromatography, ion exchange chromatography and mixed-mode chromatography.Brief description of drawings

[0023] Fig. 1 depicts an SDS-PAGE gel of reaction samples of Trastuzumab with synthetic tetrazine-carrying amino acid incorporated with three BCN-derivatized toxic payloads. The lanes show reaction with no payload, BCN-DM1, BCN-MMAE and BCN-DXd. The upper gel shows a fluorescent image of the samples.Trastuzumab samples which reacted with the payload where not derivatized with the fluorescent dye, thus showing full antibody-drug-conjugate formation in Chinese hamster ovary cell culture supernatant. mAb, monoclonal antibody.Description of Embodiments

[0024] The present invention provides a simplified method for producing and purifying protein conjugates.

[0025] Proteins bearing one or more synthetic amino acids with a chemical moiety are useful conjugation partners for linking one or more payloads resulting in synthetic protein conjugates. For example, a tetrazine moiety present in a protein represents a very specific handle to generate a site-specific reaction for conjugation. The tetrazine moiety may react for example with BCN- or transcyclooctene moiety comprised in a molecule. These reactions are very specific and fast and work with little excess of the molecule that should be attached to the protein of interest (POI) .

[0026] The herein disclosed method enables production of synthetic protein conjugates while they still reside in the supernatant of their cellular expression system. The synthetic protein conjugate comprises the protein of interest (POI) and a payload, wherein the POI may bear a diene moiety which is capable to form a covalent bond with a complementary reactive group present in the payload molecule thereby generating a protein conjugate. The payload is introduced to the cell culture supernatant right after the POI’s production. No purification of the POI is needed. The protein conjugate can be formed in the cell culture supernatant by simply adding the payload to the cell culture supernatant and allowing the POI to react with the payload.

[0027] The present invention relates to the field of protein purification. Protein purification methods known in the art mostly rely on differences in the molecular properties of size, charge and solubility between the protein to be purified and undesired protein contaminants. Methods based on these parameters include several types of chromatography, e.g., affinity chromatography, size exclusionchromatography, ion exchange chromatography, or differential precipitation and the like.

[0028] Methods for purification of protein conjugates known to a skilled person follow protocols involving laborious chromatography steps. For a protein conjugate comprising a protein and a further molecule, conventionally, the protein is expressed in a suitable host cell and purified. Then, the purified protein is contacted with a molecule which should be attached to the purified protein in order to conjugate the protein to the molecule. Additional purification steps are needed to remove the impurities which may be introduced by the conjugation step.

[0029] Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in a cell line (the host cells) in vitro such that it expresses large amounts of a recombinant gene. For production of a peptide or protein of interest (POI), a nucleic acid encoding the POI is introduced into the host cell. The person skilled in the art is familiar with standard methods for incorporating genes into a host cell system for protein production, e.g., through vectors such as plasmids. According to the present invention, the POI comprises a chemical moiety which is capable to link to a chemical counterpart.

[0030] Mammalian cell systems are considered one of the best for protein expression due to their enhanced protein folding, ultra-high yields, and ability to perform human-like glycosylation, which is vital for therapeutic proteins such as antibodies. Typical mammalian cell lines used for recombinant protein production include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, fibroblast-like cells e.g. monkey kidney (COS) cells, baby hamster kidney (BHK) cells, or immortal carcinoma cells e.g. human hepatic adenocarcinoma such as SK-Hep or HepG2 cells. In general, cell lines used for recombinant protein production are cultured in cell culture media. The cell culture supernatant comprises media component for growth of cells, as well as metabolic by-products of the cells. Excretion of protein from cells into culture supernatant can be e.g. achieved through genetic modification of the host cells, a routinely performed and well-established method the person skilled in the art is familiar with.

[0031] The production of synthetic protein conjugates involves the formation of a bond between the protein of interest and a payload. Some embodiments of theinvention relate to the formation of a covalent bond between the protein of interest and the payload to create a synthetic protein conjugate.

[0032] In chemistry the Diels-Alder reaction is an important reaction in which a conjugated diene reacts with a dienophile (a substituted alkene), producing a substituted cyclohexene derivative. This reaction has two partners reacting with each other: the diene and the dienophile.

[0033] A diene is an unsaturated hydrocarbon consisting of two double bonds between carbon atoms. A diene is also known as diolefin or alkadiene. It is a covalent compound containing two alkene units. Dienes usually exist as subunits of more complex organic molecules. Moreover, dienes can be found in naturally occurring compounds as well as in synthetic chemicals. These chemicals are useful in organic synthesis reactions.

[0034] A dienophile is an organic compound that readily reacts with a diene. A dienophile is commonly used in the Diels-Alder reaction that involves the reaction between a conjugated diene and a substituted alkene, wherein the substituted alkene acts as the dienophile.

[0035] A proper dienophile typically bears one or two of the following functional groups: CHO, COR, COOR, ON, C=C, Ph, or halogen. Moreover, the diene has to be highly electron-rich.

[0036] According to the invention, the peptide or protein of interest may be modified by one or more chemical moiety. The peptide or protein of interest thus, may either bear a diene or a dienophile. For example, the protein may be modified by incorporating a diene moiety, e.g., an amino acid bearing a tetrazine moiety. Methods for producing such modified amino acids are described in WO2022248587A1.

[0037] Preferably, the tetrazine moiety is incorporated at predefined sites of the peptide or protein. The accordingly modified peptide or protein may comprise one single amino acid or multiple amino acids bearing the tetrazine moiety. Having amino acids bearing a tetrazine moiety at predefined sites provides the ability to produce a precisely defined protein conjugate.

[0038] According to the invention, the PCI is exposed to an excess of a payload in the supernatant to support conjugate bond formation. The payload comprises a chemical moiety such as for example, a diene or a dienophile which is complementary to the chemical moiety present in the modified peptide or proteinof interest. Thus, in cases wherein the peptide or protein of interest is modified with a diene, the payload is modified with a dienophile. In case wherein the peptide or protein of interest is modified with a dienophile, then the payload is modified with a diene.

[0039] Synthetic amino acids with a chemical moiety are specifically useful to generate synthetic proteins with site-specific reaction handles for protein conjugation. For example, a tetrazine moiety present in the POI can react with BCN- or trans-cyclo-octene containing payload molecules. These reactions are very specific and fast and work with little excess of the payload molecule that should be attached to the protein of interest. Due to high specificity of this reaction, no purification of the POI is required. The conjugation of the POI to the payload can be immediately follow the expression of the POI in the cell culture supernatant.

[0040] According to the invention, the payload can be a detectable label or a therapeutic agent. The payload may be a therapeutic agent (e.g., a drug, a radiotherapeutic atom, etc.), a detectable label (e.g., fluorescent, radioactive, radiopaque, etc.), or any other agent.

[0041] According to one embodiment of the invention, the payload is present in at least a 2-fold molar excess in the cell supernatant over the POI to promote bond formation between the POI and the payload. The payload may be present in at least a 3-fold molar excess, or in at least a 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or in at least a 10-fold molar excess in the cell supernatant over the POI.

[0042] In some embodiments, the POI and the payload are reacted for at least 0.25 h to allow conjugation. In some embodiments, the POI and the payload are reacted for at least 0.5 h, or at least 0.75 h, or at least 1 h to allow conjugation.

[0043] One advantage of incorporation of a tetrazine moiety into a protein of interest is that it permits a range of extremely useful payload compounds such as labels or pharmaceutically active substances to be easily and specifically attached to the tetrazine moiety. The compounds and methods described herein include the use of Diels-Alder pairs that include a diene and a dienophile.

[0044] The conjugate generated in the cell culture supernatant may then be purified by conventional purification methods such as for example, supernatant clarification and / or chromatographic techniquesExamples

[0045] The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.Example 1 - Synthetic protein conjugate comprising an antibody

[0046] Trastuzumab with Urea-Tetrazine-Lysine synthetic amino acid incorporated into the heavy chain was exposed to BCN-derived payloads BCN-DM1, BCN-MMAE and BCN-Dxd (all BroadPharm) in Chinese hamster ovary cell culture supernatant. The synthetic Trastuzumab was present at 85 mg / L concentration while the payload was added at six-fold molar excess.

[0047] After a reaction time of one hour Sulfo-Cy5-TCO dye (BroadPharm) was added at 10 pM concentration. The samples were added to SDS running buffer (lx), denatured for 10 min at 95C and applied to an SDS-PAGE gel. The gel was run at 110V for 45 min. The gel was imaged using an Invitrogen iBright system.

[0048] Where the payload reacted with synthetic Trastuzumab, barely any fluorescence was visible at the heavy chain, e.g. for DM1 and Dxd (Fig. 1). Fig. 1 shows an SDS-PAGE gel of a protein conjugate comprising Trastuzumab bound to BCN-derived payloads.

Claims

Claims1. A method for producing a protein conjugate comprising a protein of interest (POI) comprising the steps of:a. providing one or more cells comprising a nucleic acid encoding the POI bearing a first chemical moiety;b. culturing the one or more cells in a cell culture media such that the POI is expressed and excreted from the cells into a culture supernatant;c. exposing the POI in the supernatant to an excess of a payload bearing a second chemical moiety capable to form a covalent bond with the first chemical moiety;d. allowing the formation of a covalent bond between the POI and the payload thereby creating a site-specific protein conjugate; ande. purifying said protein conjugate.

2. The method according to claim 1, wherein said first chemical moiety and said second chemical moiety are independently from one another a dienophile moiety or a diene moiety.

3. The method according to claim 2, wherein the dienophile moiety is a transcyclooctene dienophile.

4. The method according to claim 3, wherein the trans-cyclooctene dienophile is bicyclononyne (BCN) or cyclooctene.

5. The method according to claim 2, wherein the diene moiety is a tetrazine moiety.

6. The method according to claim 1 or 2, wherein the POI comprises a diene moiety.

7. The method according to claim 1 or 2, wherein the payload comprises a dienophile moiety.

8. The method according to any one of claims 1 or 7, wherein the one or more cells are mammalian cells.

9. The method according to claim 8, wherein the mammalian cell is selected from the group consisting of CHO, COS, HEK 293, BHK, SK-Hep, and HepG2 cells.

10. The method according to any one of claims 1 to 9, wherein the payload further comprises pharmacologically or biologically active agents.

11. The method according to any one of claims 1 to 10, wherein the payload is provided in a molar ratio of at least 2:1 with respect to the POI.

12. The method according to any one of claims 1 to 11, wherein the payload and the POI are reacted for at least 0.25 h.

13. The method according to any one of claims 1 to 12, wherein the protein conjugate is purified by supernatant clarification and / or chromatographic techniques.

14. The method according to claim 13, wherein the chromatographic technique is selected from the group consisting of affinity chromatography, size exclusion chromatography hydrophobic interaction chromatography, ion exchange chromatography and mixed-mode chromatography.

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