Compositions comprising Anti-PCSK9 antbodies

Abstract

Compositions comprising a monomer and a dimer of an anti-PCSK9 antibody such as evolocumab are described herein. Methods of determining the presence, absence, or amount of a dimer in a composition comprising evolocumab are described herein.

Description

[0001] COMPOSITIONS COMPRISING ANTI-PCSK9 ANTBODIES

[0002] Related Applications

[0003] The present application claims the benefit of U.S. provisional application Nos. 63 / 662,953, filed June 21, 2024, and 63 / 821,975, filed June 11, 2025.

[0004] Sequence Listing

[0005] The present application is being filed with a sequence listing in electronic format. The sequence listing provided as a file titled, "10968-W001-SEC_sequence.xml," created June 11, 2025, and is 3,371 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0006] Field

[0007] The present disclosure relates to post-translational modifications of therapeutic anti- PCSK9 antibodies, and compositions comprising the same.

[0008] Background

[0009] Therapeutic antibodies may comprise post-translational modifications which affect the efficacy or safety of such therapeutic antibodies. Such post-translational modifications may be referred to as "molecular attributes." For the manufacture of therapeutic antibodies, there is a need to identify molecular attributes that may be present, set specifications for molecular attributes to ensure the production of medically useful compositions comprising the therapeutic antibodies.

[0010] Summary

[0011] El. Compositions comprising a first species and a second species are described herein. The first species comprises a monomer of evolocumab (e.g., the evolocumab as depicted by the heavy chain having the amino acid sequence of SEQ ID NO: 1 and the light chain having the amino acid sequence of SEQ ID NO: 2). The second species comprises a dimer of a first evolocumab (also referred to as "the one evolocumab") and a second evolocumab (also referred to as "the other evolocumab"). The dimer comprises a disulfide linkage linking a hinge or Fab region of the first evolocumab to a hinge or Fab region of the second evolocumab.

[0012] E2. For any of the compositions described herein, for example the composition of El, no more than about 2% of the evolocumab of the composition may be comprised by the second species.

[0013] E3. For any of the compositions described herein, for example the composition of El, no more than about 0.3% of the evolocumab of the composition may be comprised by the second species.

[0014] E4. For any of the compositions described herein, for example the composition of El, at least about 98% of the evolocumab of the composition may be comprised by the first species, and no more than about 2% of the evolocumab of the composition may be comprised by the second species.

[0015] E5. For any of the compositions described herein, for example the composition of El, at least about 99.7% of the evolocumab of the composition may be comprised by the first species, and no more than about 0.3% of the evolocumab of the composition may be comprised by the second species.

[0016] E6. For any of the compositions described herein, for example the composition of El, at least about 96% of the evolocumab of the composition may be comprised by the first species, and no more than about 4% of the evolocumab of the composition may be comprised by the second species.

[0017] E7. For any of the compositions described herein, for example the composition of El, at least about 92% of the evolocumab of the composition may be comprised by the first species, and no more than about 8% of the evolocumab of the composition may be comprised by the second species. E8. For any of the compositions described herein, for example the composition of any one of E1-E7, at least 0.1% of the evolocumab the composition may be comprised by the second species. By way of example, for any of the compositions described herein, for example the composition of any one of E1-E7, 0.1%-8%, 0.1%-5%, 0.1%-2%, 0.3%-8%, 0.3%-5%, 0.3%-2%, 0.5%-8%, 0.5%-5%, or 0.5%-2% of the evolocumab of the composition may be comprised by the second species.

[0018] E9. For any of the compositions described herein, for example the composition of any one of E1-E8, at least 0.2% of the evolocumab the composition may be comprised by the second species. By way of example, for any of the compositions described herein, for example the composition of any one of E1-E8, 0.2%-8% or 0.2%-5%, of the evolocumab of the composition may be comprised by the second species.

[0019] E10. For any of the compositions described herein, for example the composition of any one of El, E2, E4, E6, or E6-E9, at least 0.5% of the evolocumab the composition may be comprised by the second species. By way of example, for any of the compositions described herein, for example the composition of any one of El, E2, E4, E6, or E6-E9, 0.5%-8%, or 0.5%-5%, of the evolocumab of the composition may be comprised by the second species.

[0020] Ell. For any of the compositions described herein, for example the composition of El, 0.2% - 2% of the evolocumab of the composition may be comprised by the second species.

[0021] E12. For any of the compositions described herein, for example the composition of El, 0.5% - 2% of the evolocumab of the composition may be comprised by the second species.

[0022] E13. For any of the compositions described herein, for example the composition of any of E1-E12, the second species inhibits binding of PCSK9 to LDLR in an in vitro assay by at least 70% compared to evolocumab monomer. The in vitro assay may comprise huLDLR immobilized on a first bead and a huPCSK9 immobilized on a second bead, wherein binding of huLDLR to huPCSK9 is indicated by proximity of the first bead to the second bead. By way of example, the second species may reduced the quantity of PCSK9 bound to LDLR by at least 70% in this in vitro assay. E14. Described herein is a composition comprising: (i) a monomer of evolocumab, and (ii) a dimer of evolocumab comprising a disulfide linkage between the hinge or Fab region of one evolocumab and the hinge or Fab region of another evolocumab.

[0023] E15. For any of the compositions described herein, for example the composition of E14, no more than about 2% of the evolocumab of the composition may be comprised by (ii) the dimer.

[0024] E16. For any of the compositions described herein, for example the composition of E14, no more than about 0.3% of the evolocumab of the composition may be comprised by (ii) the dimer.

[0025] E17. For any of the compositions described herein, for example the composition of E14, at least about 98% of the evolocumab of the composition may be comprised by (i) the monomer, and no more than about 2% of the evolocumab of the composition may be comprised by (ii) the dimer.

[0026] E18. For any of the compositions described herein, for example the composition of E14, at least about 99.7% of the evolocumab of the composition may be comprised by (i) the monomer, and no more than about 0.3% of the evolocumab of the composition may be comprised by (ii) the dimer.

[0027] E19. For any of the compositions described herein, for example the composition of E14, at least about 96% of the evolocumab of the composition may be comprised by the monomer, and no more than about 4% of the evolocumab of the composition may be comprised by the dimer.

[0028] E20. For any of the compositions described herein, for example the composition of E14, at least about 92% of the evolocumab of the composition may be comprised by the monomer, and no more than about 8% of the evolocumab of the composition may be comprised by the dimer.

[0029] E21. For any of the compositions described herein, for example the composition of any one of E14-E20, at least 0.1% of the evolocumab the composition may be comprised by the dimer. By way of example, for any of the compositions described herein, for example the composition of any one of E14-E20, 0.1%-8%, 0.1%-5%, 0.1%-2%, 0.3%-8%, 0.3%-5%, 0.3%-2%, 0.5%-8%, 0.5%- 5%, or 0.5%-2% of the evolocumab of the composition may be comprised by the dimer.

[0030] E22. For any of the compositions described herein, for example the composition of any one of E14-E20, at least 0.2% of the evolocumab the composition may be comprised by the dimer. By way of example, for any of the compositions described herein, for example the composition of any one of E14-E20, 0.2%-8% of the evolocumab of the composition may be comprised by the dimer. By way of example, for any of the compositions described herein, for example the composition of any one of E14-E20, 0.2%-5% of the evolocumab of the composition may be comprised by the dimer.

[0031] E23. For any of the compositions described herein, for example the composition of any one of E14-E20, at least 0.5% of the evolocumab the composition may be comprised by the dimer. By way of example, for any of the compositions described herein, for example the composition of any one of E14-E20, 0.5%-8% of the evolocumab of the composition may be comprised by the dimer. By way of example, for any of the compositions described herein, for example the composition of any one of E14-E20, 0.5%-5% of the evolocumab of the composition may be comprised by the dimer.

[0032] E24. For any of the compositions described herein, for example the composition of any of E14-E23, (ii) the dimer inhibits binding of PCSK9 to LDLR in an in vitro assay by at least 70% compared to evolocumab monomer. The in vitro assay may comprise huLDLR immobilized on a first bead and a huPCSK9 immobilized on a second bead, wherein binding of huLDLR to huPCSK9 is indicated by proximity of the first bead to the second bead.

[0033] E25. For any of the compositions described herein, for example the composition of any of E1-E24, evolocumab comprises a heavy chain having the amino acid sequence of SEQ ID NO: 1 (EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQAPGQGLEWMGWVSFYNGNTNYAQKLQGRGT MTTDPSTSTAYMELRSLRSDDTAVYYCARGYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK), and a light chain having the amino acid sequence of SEQ ID NO: 2 (ESALTQPASVSGSPGQSITISCTGTSSDVGGYNSVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTA SLTISGLQAEDEADYYCNSYTSTSMVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS).

[0034] E26. For any of the compositions described herein, for example the composition of any of E1-E25, the disulfide linkage comprises a linkage between a light chain of the first evolocumab (or the one evolocumab) and a light chain of the second evolocumab (or the another evolocumab). In various aspects, the disulfide linkage links C214 of the light chain of the first evolocumab (or the one evolocumab) to C214 of the light chain of the second evolocumab (or the another evolocumab).

[0035] E27. For any of the compositions described herein, for example the composition of any of E1-E26, the percentage of evolocumab species (or the percentage of the monomer and the dimer) is as determined by size exclusion chromatography. In various aspects, the size exclusion chromatography comprises UV 280 nm measurement of absorbance, and wherein the percentage of evolocumab species is calculated as a percentage of evolocumab species peak area relative to total peak area.

[0036] E28. For any of the compositions described herein, for example the composition of any of E1-E27, the composition may be for medical use.

[0037] E29. Also described herein is a pharmaceutical composition comprising any of the compositions described herein (for example the composition of any one of E1-E28) and a pharmaceutically acceptable excipient. In various aspects, the pharmaceutical composition comprises 120-140 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5 % (w / v) proline. In various aspects, the pharmaceutical composition comprises 120-160 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5 % (w / v) proline. In various aspects, the pharmaceutical composition comprises 140 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5 % (w / v) proline. In various aspects, the pharmaceutical composition comprises 120 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5 % (w / v) proline.

[0038] E30. Also described herein is an administration device comprising any of the pharmaceutical compositions described herein, for example the pharmaceutical composition of E29. The administration device comprises a unit-dose of 140 mg of evolocumab or 420 mg of evolocumab. In various aspects, the administration device is selected from an autoinjector pen and a syringe.

[0039] E31. A method of lowering LDL-C in a subject is described herein. The method comprises administering any of the compositions described herein, for example the composition of any one of E1-E28, (including, e.g., any of the pharmaceutical compositions described herein, such as the pharmaceutical composition of E29) to the subject. In various aspects of the method, the method comprises administering 140 mg of evolocumab to the subject Q2W; or 420 mg of evolocumab to the subject Q2W; or 420 mg of evolocumab to the subject QM.

[0040] E32. A method of determining the presence, absence, or amount of a species of evolocumab in a composition is described herein. The species (e.g., the shoulder species or second species) comprises a dimer of evolocumab molecules (which may be referred to a dimer of a "first evolocumab" and a "second evolocumab," or a dimer of "one evolocumab" and "another evolocumab") bound to each other by a disulfide linkage between their hinge or Fab regions. The method comprises resolving the composition by size exclusion chromatography (SEC). The method comprises resolving a reference composition comprising (i) a monomer of evolocumab and (ii) a noncovalent dimer of evolocumab by SEC. The method further comprises determining the presence, absence, or amount of said species in the composition, said species having a larger hydrodynamic radius than (i) and a smaller hydrodynamic radius than (ii). In various aspects, the method further comprises obtaining a SEC fraction enriched for the second species relative to (i) and (ii); reducing the SEC fraction; and resolving the reduced fraction by capillary electrophoresis-SDS (CE-SDS).

[0041] E33. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of E32, the method comprises determining that no more than 5%, 2%, 1%, 0.5%, 0.3%, or 0.1%, of the evolocumab of the composition are comprised by the (shoulder) species; and manufacturing the composition for pharmaceutical use.

[0042] E34. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of E32 or E33, the method comprises determining that 0.1%-8%, 0.1%-5%, 0.1%-2%, 0.3%-8%, 0.3%-5%, 0.3%- 2%, 0.5%-8%, 0.5%-5%, or 0.5%-2% of the evolocumab of the composition are comprised by the (shoulder) species; and manufacturing the composition for pharmaceutical use if the amounts of the (shoulder) species are within the specified range.

[0043] E35. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of any one of E32-E34, manufacturing the composition for pharmaceutical use comprises purification of the evolocumab by cation exchange chromatography.

[0044] E36. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of any one of E32-E35, manufacturing the composition for pharmaceutical use comprises placing the composition (or a portion thereof) in an administration device.

[0045] E37. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of any one of E32-E36, the disulfide linkage may comprise a linkage between a light chain of the first evolocumab (or the one evolocumab) and a light chain of the second evolocumab (or the another evolocumab).

[0046] E38. For any of the methods of determining the presence, absence, or amount of a species of evolocumab in a composition described herein, for example the method of any one of E32-E37, the disulfide linkage links C214 of the light chain of the first evolocumab (or the one evolocumab) to C214 of the light chain of the second evolocumab (or the another evolocumab). Brief Description of the Drawings

[0047] FIG. 1A is an annotated chromatogram depicting the main peak fraction, shoulder species fraction, and conventional dimer species fraction of a filtered viral inactivation pool (FVIP) evolocumab composition.

[0048] FIG. IB is a chromatogram depicting the main peak, shoulder species, and conventional dimer species of a drug substance evolocumab composition made according to the new process (and comprising the shoulder species) and a reference standard (or "control") according to a conventional process (in which no shoulder species is detected).

[0049] FIG. 2A is a graph of an SEC-MALS depicting the molecular weight of the shoulder species fraction collected from an evolocumab composition from a new process, and the molecular weight of a reference standard evolocumab produced according to a conventional process in which this shoulder species is not present.

[0050] FIG. 2B is a graph of RP LC-MS data showing the deconvoluted mass of a fraction comprising the shoulder species (shoulder fraction) and a negative control.

[0051] FIG. 3A is a graph of dSEC data showing the main peak fraction, shoulder species fraction, and conventional dimer species fraction of an evolocumab composition.

[0052] FIG. 3B is a graph of NR CE-SDS data for the shoulder species fraction and main peak fraction as described herein.

[0053] FIGs. 4A-4B are graphs of the R PR LC-MS analysis of the shoulder species fraction and reference standard of an evolocumab composition showing deconvoluted mass of the two peaks from shoulder species fraction. FIG. 4A shows UV spectra and FIG. 4B shows the raw mass spectra.

[0054] FIG. 4C is a graph of reduced(R) CE-SDS data showing the main peak fraction and shoulder species fraction of an evolocumab composition.

[0055] FIGs. 5A-5B are graphs of UV spectra (FIG. 5A on the left) of native SEC-MS analysis depicting the main peak fraction, shoulder species fraction, and conventional dimer species fraction of an evolocumab composition, and the raw mass spectra (FIG. 5B on the right) of the first peak of each fraction.

[0056] FIG. 6 is a graph showing reduced peptide mapping results with several highlighted post- translational modifications (PTMs) for the monomer fraction, shoulder species fraction, conventional dimer species fraction, and reference standard of an evolocumab composition.

[0057] Detailed Description

[0058] Described herein is a molecular attribute of a therapeutic anti-PCSK9 antibody or compositions thereof. In particular, the molecular attribute described herein is a "shoulder species" or "second species" or "covalent high molecular weight (HMW) species" that comprises a dimer of a first anti-PCSK9 antibody and a second anti-PCSK9 antibody. A disulfide linkage links a hinge or Fab region of the first anti-PCSK9 antibody to the second anti-PCSK9 antibody. In various aspects, the anti-PCSK9 antibody is evolocumab. The "shoulder species" or second species may also be referred to herein as a dimer of evolocumab, in which the dimer comprises a disulfide linkage between the hinge or Fab region of one evolocumab and the hinge or Fab region of another evolocumab. This shoulder species is an example of a HMW species, and was first identified as a species produced in a new process for manufacturing evolocumab. Typically, HMW species of therapeutic antibodies are not a desired species. HMW species may be considered an impurity in drug product or drug substance. Furthermore, HMW species are conventionally associated with a loss of potency and can be difficult to reverse or dissociate, and so are conventionally considered to be undesirable. Moreover, covalent bounds between molecules comprised in HMW species have the potential to exacerbate the loss of potency and challenges in reversing (or dissociating) the HMW species. Remarkably, despite comprising a dimer of evolocumab covalently bound to each other, this shoulder species was determined to retain an acceptable potency (See Example 8), and at least specified ranges of this shoulder species were determined to be acceptable for inclusion in compositions of evolocumab suitable for medical use. The shoulder species described herein retains a potency for inhibiting the interaction between PCSK9 and LDLR. Accordingly, it is appreciated that evolocumab drug substance and drug product (which drug product is suitable for medical use) may comprise higher amounts of the shoulder species than may be permissible for other HMW species of evolocumab. As there is a tradeoff between extent of removal of HMW species (such as by CEX chromatography) and throughput of production of drug substance and drug substance, it will be appreciated that there is a manufacturing efficiency benefit for identifying and characterizing HMW species (such as the shoulder species) and determining acceptable levels of such HWM species in compositions comprising evolocumab. As such, described herein are compositions of evolocumab comprising specified limits of the shoulder species. The compositions may comprise a first species that is a monomer of evolocumab, and the shoulder species. No more than 2%, 1%, 0.5%, or 0.3% of the evolocumab of the composition may be comprised by the shoulder species. At least 0.1%, 0.2%, or 0.5% of the evolocumab of the composition may be comprised by the shoulder species. For some compositions, 0.1%-2%, 0.1%-l%, 0.1%-0.5%, 0.1%-0.3%, 0.2%-2%, 0.2%-l%, 0.2%-0.5%, 0.5%-l%, or 0.5%-2% of the evolocumab of the composition may be comprised by the shoulder species. All or substantially all of the remainer of the evolocumab in the composition may be a monomer of evolocumab. Such percentages may be as determined by size exclusion chromatography.

[0059] Evolocumab

[0060] An "antibody" refers to an immunoglobulin of any isotype with specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and monoclonal antibodies. An "antibody" as such is a subgenus of an antigen binding protein. For example, human antibodies can be of an IgG isotype, such as the lgG2 subtype. By way of example, evolocumab is an lgG2 antibody. A human IgG antibody generally will comprise two full-length heavy chains and two full-length light chains. It will be noted that common variants of antibodies may exist, such as C-terminal lysine clipping in Fc regions of heavy chains, but such molecules comprising common variants such as C-terminal clipping are still considered to be "antibodies."

[0061] A "light chain" includes a variable region ("VL"), and a constant region ("CL"). Human light chains include kappa chains and lambda chains.

[0062] A "heavy chain" includes a variable region ("VH"), and three constant regions: CHI, CH2, and CH3. A variable region (VH or VL) typically includes "complementary binding regions" ("CDRs") of an antibody. A "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. Antigen binding regions of antibody heavy and light chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three CDRs. The CDRs from the two chains of each heavy chain / light chain pair typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein. From N-terminal to C-terminal, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878- 883.

[0063] By way of example, of evolocumab (commercially available in REPATHA®) is a monoclonal antibody that binds proprotein convertase subtilisin kexin type 9 (PCSK9). The low density lipoprotein receptor (LDLR) on liver cells internalizes low density lipoprotein cholesterol (LDL-C), and thus lowers plasma LDL-C levels in humans. PCSK9 binds to LDLR and mediates the degradation of LDLR. However, evolocumab binds to PCSK9, and inhibits the interaction between PSCK9 and low density lipoprotein receptor (LDLR). By inhibiting this interaction, evolocumab inhibits PCSK9-mediated degradation of LDLR, resulting in more available LDLR on the surface of liver cells, which can internalize LDL-C and lower plasma LDL-C levels. Accordingly, evolocumab is useful for binding to PSCK9 and lowering plasma LDL-C levels.

[0064] In the context of the present description, each "monomer" of evolocumab refers to a tetramer composed of two identical pairs of polypeptide chains, each pair having one full-length light chain and one full-length heavy chain. Evolocumab comprises a heavy chain having the amino acid sequence of SEQ ID NO: 1

[0065] (EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQAPGQGLEWMGWVSFYNGNTNYAQKLQGRGT MTTDPSTSTAYMELRSLRSDDTAVYYCARGYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK), and a light chain having the amino acid sequence of SEQ ID NO: 2 (ESALTQPASVSGSPGQSITISCTGTSSDVGGYNSVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTA SLTISGLQAEDEADYYCNSYTSTSMVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS).

[0066] Evolocumab may be manufactured using recombinant cells such as Chinese hamster ovary (CHO) cells that are engineered to encode and produce the heavy chain and light chains of evolocumab. In the large-scale manufacture of therapeutic antibodies such as evolocumab, cell culture titers are typically subject to downstream purification processes. The downstream purification processes may remove a diverse population of product- and process-related impurities, including HMW and low molecular weight (LMW) product species, product charge variants, host cell protein and DNA, and adventitious viruses. The shoulder species comprising a dimer of evolocumab as described herein is an example of a HMW species. By way of example, processes useful for making and purifying compositions comprising evolocumab as described herein are described in PCT Pub. Nos. WO 2024 / 263504, WO 2024 / 054414, WO 2024 / 191970, and WO 2024 / 163713. These processes are capable of producing compositions comprising amounts of the second species (or evolocumab dimer) as described herein. For compositions comprising the second species (or evolocumab dimer), including those produced according to methods such as those described in WO 2024 / 263504, WO 2024 / 054414, WO 2024 / 191970, and / or WO 2024 / 163713, chromatography methods as described herein may be used to deplete (or enrich) the second species (or evolocumab dimer) from the composition so that the percentage of evolocumab comprised by the second species is within a specified range.

[0067] Cation-exchange (CEX) chromatography may be used in downstream purification for removal of HMW species, and is useful for its high degree of chromatographic resolution for the product species of interest, robust clearance of host cell impurities and viruses, and reasonably high yields. In bind and elute mode, a chromatography medium having some type of affinity for the product of interest is used. The product binds to the chromatography medium during loading. The bound product is then eluted and collected as the product pool. Certain impurities, such as product related species, may bind to the chromatography medium during loading with greater or lesser affinity than the product of interest. Alternatively, CEX chromatography may be performed in weak partitioning or frontal loading chromatography modes. Both weak partitioning and frontal loading chromatography modes rely on competitive binding to the chromatography medium between the components in the load to separate the product of interest from impurities and contaminants. For example, frontal loading mode is characterized by continuous loading under conditions in which all the components in the load feed initially bind the chromatography medium. In an optimized operation, as loading proceeds, the bound components are displaced in order of increasing affinity for the chromatography medium (i.e., the stationary phase) until the medium is heavily saturated with those components of higher binding affinity. Illustratively, in a CEX operation designed to bind positively charged species, the product of interest (i.e., monomer) and low molecular weight (LMW) product-related impurities (i.e., truncated proteins expressed during culture, degraded proteins, and enzymatically clipped proteins) are usually retained less strongly and are quickly displaced by more positively charged components. The product of interest and any LMW impurities flow through into the effluent, which may be diverted to a downstream unit operation or collected as a product pool. A chromatography operation making use of frontal loading mode can remove HMW product-related species such as dimers, oligomers, and higher-order aggregates at high loading density, along with process-related impurities, such as host cell proteins (HCP), that have a higher binding affinity and are retained by the chromatography medium.

[0068] Accordingly, CEX chromatography may be used to limit the HMW content of evolocumab compositions (such as the shoulder species described herein) in accordance with aspects herein. However, it is contemplated that there is a trade-off between removal of HMW species (such as the shoulder species described herein), and process throughput and efficiency. As such, identifying acceptable contents of HMW species such as the shoulder species is useful for the efficient manufacture of compositions comprising evolocumab suitable for therapeutic use. In some aspects, CEX is used to produce an evolocumab composition comprising no more than 2%, 1%, 0.5%, or 0.3%, of the shoulder species. Shoulder Species

[0069] As described herein the "shoulder species" or "second species" refers to a molecular attribute comprising an anti-PCSK9 antibody, preferably evolocumab. The shoulder species is an example of a HMW species. The shoulder species or second species refers to a dimer of evolocumab comprising a disulfide linkage between the hinge or Fab region of one evolocumab and the hinge or Fab region of another evolocumab. In various aspects, the disulfide linkage comprises a linkage between a light chain of one evolocumab (or the "first evolocumab") and a light chain of another evolocumab (or the "second evolocumab"). In various aspects, the disulfide linkage links C214 of the light chain of the one evolocumab (or the "first evolocumab") to C214 of the light chain of the other evolocumab (or the "second evolocumab").

[0070] Compositions Comprising Evolocumab

[0071] Compositions comprising evolocumab are described herein. The composition comprises a first species comprising a monomer of evolocumab and second species comprising the shoulder species. As described herein, the shoulder species refers to dimer of a first evolocumab and a second evolocumab, in which the dimer comprises a disulfide linkage linking a hinge or Fab region of the first evolocumab to a hinge or Fab region of the second evolocumab. In some aspects, the second (shoulder) species is present in the composition, and no more than about 3%, 2%, 1%, 0.5%, or 0.3% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 98% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab). The composition may be suitable for medical use. By way of example, a composition having suitable potency and safety characteristics may be suitable for medical use. It is contemplated that compositions comprising evolocumab described have a suitable potency for medical use. It is contemplated that compositions comprising evolocumab described have a suitable safety profile for medical use, for example not exhibiting any medically meaningful risk of immunogenicity. As shown in Example 8, the second (shoulder) species is effective in blocking the interaction between PCSK9 and LDLR. Moreover, multiple studies have reported that process-related antibody dimers did not pose an increased risk of immunogenicity in murine models (Bessa et al. (2003), Pharm Res 32: 2344-59 (2015); Kijanka et al. (2020), J Pharm Sci 109: 730-38; and Kijanka et al. (2018), J Pharm Sci 107: 2847-59). Accordingly, the risk of immunogenicity of the covalent dimer (shoulder) species is considered low. In various aspects, at least 86% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.1% - 2% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 98% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.3% - 2% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 98% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.5% - 2% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 96% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.1% - 4% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 96% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.3% - 4% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 96% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.5% - 4% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 92% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.1% - 8% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 92% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.3% - 8% of the evolocumab in the composition is comprised by the second (shoulder) species. In various aspects, at least 92% of the evolocumab of the composition is comprised by the first species (which comprises a monomer of evolocumab), and 0.5% - 8% of the evolocumab in the composition is comprised by the second (shoulder) species. For the compositions described herein, unless stated otherwise, the percentage values of second species (or dimer of evolocumab) and first species (or monomer of evolocumab), and thus the percentage of evolocumab comprised by each species, refer to values as determined by size exclusion chromatography. By way of example, a suitable size exclusion chromatography method may comprise using a SE-HPLC column (5 pm particle size, 7.8 x 300 mm), with 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 as the mobile phase. Samples may be injected neat on the SE-HPLC column, separated isocratically with a flow rate of 0.5 mL / min and run time of 35 min. The eluent may be monitored by UV absorbance at 280 nm. Percentage of each species may be determined by calculating the percentage of each component group's integrated area as compared to the total integrated area. As such, in various aspects, the size exclusion chromatography comprises UV 280 nm measurement of absorbance, and the percentage of evolocumab species is calculated as a percentage of evolocumab species peak area relative to total peak area. It will be appreciated that due to rounding, and / or the possibility of minor peaks being present in the size exclusion chromatography which are different than the first and second species, the percentage of evolocumab comprised by the second species (or dimer of evolocumab) and percentage of evolocumab comprised by the first species (or monomer of evolocumab) may not add up to exactly 100%.

[0072] In various aspects of the compositions comprising evolocumab described herein, no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects, the second (shoulder species) is present, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0073] In various aspects of the compositions comprising evolocumab described herein, at least about 98% of the evolocumab of the composition is comprised by the first species, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 98% of the evolocumab of the composition is comprised by the first species, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least about 97% of the evolocumab of the composition is comprised by the first species, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 97% of the evolocumab of the composition is comprised by the first species, and 0.1%-2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 97% of the evolocumab of the composition is comprised by the first species, and 0.2%-2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least about 96% of the evolocumab of the composition is comprised by the first species, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 96% of the evolocumab of the composition is comprised by the first species, and no more than about 2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 96% of the evolocumab of the composition is comprised by the first species, and 0.1%-2% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 96% of the evolocumab of the composition is comprised by the first species, and 0.2%-2% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0074] In various aspects of the compositions comprising evolocumab described herein, at least about 99% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 99% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0075] In various aspects of the compositions comprising evolocumab described herein, at least about 98% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 98% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 98% of the evolocumab of the composition is comprised by the first species, and 0.2%-l% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0076] In various aspects of the compositions comprising evolocumab described herein, at least about 97% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 97% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0077] In various aspects of the compositions comprising evolocumab described herein, at least about 96% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 96% of the evolocumab of the composition is comprised by the first species, and no more than about 1% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0078] In various aspects of the compositions comprising evolocumab described herein, at least about 99.7% of the evolocumab of the composition is comprised by the first species, and no more than about 0.3% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, the second (shoulder species) is present, at least about 99.7% of the evolocumab of the composition is comprised by the first species, and no more than about 0.3% of the evolocumab of the composition is comprised by the second (shoulder) species. In various aspects of the compositions comprising evolocumab described herein, at least 0.1% of the evolocumab the composition is comprised by the second species, for example 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%, including ranges between any two of the listed values, for example 0.1% - 3%, 0.1% - 2%, 0.1% - 1%, 0.2% - 3%, 0.2% - 2%, 0.2% - 1%, 0.3% - 3%, 0.3% - 2%, or 0.3% - 1% is comprised by the second (shoulder) species. For some compositions, 0.1%-2%, 0.1%-l%, 0.1%-0.5%, 0.1%-0.3%, 0.2%-2%, 0.2%-l%, 0.2%-0.5%, 0.3%-2%, 0.3%-l%, 0.3%-0.5%, 0.5%-l%, or 0.5%-2% of the evolocumab of the composition is comprised by the second (shoulder) species.

[0079] In various aspects of the compositions comprising evolocumab described herein, the second (shoulder) species inhibits binding of PCSK9 to LDLR in an in vitro assay by at least 70% compared to evolocumab monomer. That is, the inhibition by the second (shoulder) species in the in vitro assay is compared to the inhibition by the evolocumab monomer under the same in vitro assay conditions, in which the second (shoulder) species inhibits the binding of PCSK9 to LDLR by at least 70% of the value conferred by the evolocumab monomer. An example of a suitable in vitro potency assay is a bead-based Amplified Luminescent Proximity Homogeneous Assay (Alpha) that detects biomolecular interactions as described in Example 8. The in vitro assay may comprise a huLDLR immobilized on a first bead and a huPCSK9 immobilized on a second bead, in which binding of huLDLR to huPCSK9 is indicated by proximity of the first bead to the second bead. The first beads (donor beads) may be coated with a hydrogel that contains phthalocyanine, a photosensitizer and streptavidin bound to biotinylated huLDLR. The second beads (acceptor beads) may be coated with a hydrogel that contains thioxene derivatives as well as nickel chelate bound to His-tagged huPCSK9. By way of illustration, when the huPCSK9 and huLDLR bind to each other, the acceptor beads and the donor are brought into close proximity. When a laser (such as at 570 nm) is applied to this complex, ambient oxygen is converted to singlet oxygen by the donor beads. If the beads are in close proximity, an energy transfer to the acceptor beads occurs, resulting in the production of luminescence. By way of example, a relative potency of at least 70% in such an assay, may identify a functional evolocumab composition.

[0080] Pharmaceutical Compositions In various aspects, a pharmaceutical composition comprises any of the compositions comprising evolocumab described herein. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In various aspects, the pharmaceutical composition comprises 120-140 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5% (w / v) proline. In various aspects, the pharmaceutical composition comprises 140 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5% (w / v) proline.

[0081] In various aspects, an administration device comprises any of the pharmaceutical compositions described herein, for example, any pharmaceutical composition of the preceding paragraph. The administration device may comprise a unit-dose of 140 mg of evolocumab or 420 mg of evolocumab. By way of example, the administration device may be selected from an autoinjector pen or a syringe.

[0082] Medical uses

[0083] Any of the compositions comprising evolocumab described herein, such as any of the pharmaceutical compositions described herein may be suitable for medical use. For example, the composition (e.g., pharmaceutical composition) may be useful for lowering LDL-C. Evolocumab molecule, which inhibits the interaction between PSCK9 and LDLR has been shown to be effective in lowering LDL cholesterol in large-scale clinical studies (See, e.g., Sabatine et al. (2015), New England Journal of Medicine 372: 1500-1509. As such, by way of example, the composition (e.g., pharmaceutical composition) comprising evolocumab as described herein may be useful for treating a cholesterol-related disorder such as hypercholesterolemia. In various aspects, a composition comprising evolocumab as described herein is for medical use. In various aspects, a pharmaceutical composition comprising evolocumab as described herein is for medical use. For example, the composition (e.g., pharmaceutical composition) may be useful for lowering LDL-C. The composition or pharmaceutical composition for medical use may comprise a unit dose of 140 mg or 420 mg of evolocumab.

[0084] In various aspects, a method of lowering LDL-C in a subject comprises administering a composition or pharmaceutical composition as described herein to a subject. The method may comprise administering 140 mg of evolocumab to the subject every two weeks (Q2W); or 420 mg of evolocumab to the subject every two weeks (Q2W); or 420 mg of evolocumab to the subject monthly (QM).

[0085] Methods determining the presence, absence, or amount of a species

[0086] To Applicant's knowledge, prior to the present disclosure, the shoulder species described herein was not known. In view of the present disclosure, methods for determining the presence, absence or amount of the should species are useful, for example to determine whether or not a manufacturing lot comprising evolocumab contains the shoulder species, and whether or not the lot is suitable for release. It will be appreciated that the while conventional dimers comprising evolocumab may have the same or substantially the same molecular weight as the shoulder species, the shoulder species is characterized by a different hydrodynamic radius (detectable by SEC, See, e.g., Example 1), and intermolecular disulfide linkages (identifiable by reduction, and reduced capillary electrophoresis, See, e.g., Example 4).

[0087] In various aspects, a method of determining the presence, absence, or amount of a species in a composition comprising a PCSK9 antibody such as evolocumab is described. The species may be a shoulder species as described herein. As such, the species may comprise a dimer of a first anti-PCSK9 antibody (preferably evolocumab) and a second anti-PCSK9 antibody (preferably evolocumab), in which a hinge or Fab region of the first anti-PCSK9 antibody is bound to a hinger or Fab region of the second anti-PCSK9 antibody by a disulfide linkage. The method may comprise resolving the composition by size exclusion chromatography (SEC). The method may comprise resolving a reference composition comprising (i) a monomer of evolocumab and (ii) a noncovalent dimer of evolocumab by SEC. The method may comprise determining the presence, absence, or amount of said species (such as the shoulder species) in the composition. The species may have a larger hydrodynamic radius than (i) and a smaller hydrodynamic radius than (ii). The SEC may be as described herein.

[0088] In various aspects, the method further comprises obtaining a SEC fraction enriched for the second species relative to (i) and (ii). The method may further comprise reducing the SEC fraction. The method may further comprise resolving the reduced fraction by capillary electrophoresis-SDS (CE-SDS). In various aspects, the method further comprises determining that no more than 2%, 1%, 0.5%, 0.3%, or 0.1%, of the evolocumab of the composition are comprised by the species. The method may further comprise manufacturing the composition for pharmaceutical use. In various aspects, the method comprises determining that the composition comprises the species (or "shoulder" species) within a numerical range of a composition described herein. Optionally, the method further comprises determining that the composition comprises a monomer of evolocumab within a numerical range of a composition described herein. If the composition is determined to have the second species (or shoulder species) within the numerical range described herein, the method may comprise manufacturing the composition for pharmaceutical use. By way of example, manufacturing the composition for pharmaceutical use may comprise purification of the evolocumab by cation exchange chromatography. By way of example, manufacturing the composition for pharmaceutical use may comprise placing the composition in a drug delivery device.

[0089] Examples

[0090] Example 1: Size Exclusion Chromatography identification of the shoulder species

[0091] This example describes a procedure for the purity analysis of evolocumab to determine the size variants in particular high molecular species (HMW), the shoulder species described herein.

[0092] Evolocumab had previously been manufactured according to a process in which the shoulder species was not detectable in the drug substance or drug product by size exclusion chromatography. Following a process change, the shoulder species was detected in drug substance for the first time as described herein. Evolocumab was produced in CHO cell culture. Following harvest, the harvested cell culture fluid was subjected to protein A chromatography, viral inactivation, neutralization, and depth filtration, and CEX chromatography.

[0093] Samples comprising evolocumab were analyzed by size exclusion chromatography as follows. A HPLC column (TSKgel G3000SWxl, 5 pm particle size, 7.8 x 300 mm, (Tosoh Bioscience, 08541)) was used, with 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 as the mobile phase. Samples were injected neat (if less than 10 mg / mL) or diluted to 10 mg / mL with a total load of 100 pg on the SE-HPLC column, separated isocratically with a flow rate of 0.5 mL / min and run time of 35 min. The eluent was monitored by UV absorbance at 280 nm. Peaks eluted include HMW, main peak and LMW. Purity was determined by calculating the percentage of each component group's integrated area as compared to the total integrated area.

[0094] An additional HMW shoulder peak (comprising the shoulder species) was observed in the filtered viral inactivation pool (FIG. 1A). The additional HMW shoulder peak (comprising the shoulder species) was observed in this lot at about 0.3% in the final drug substance (FIG. IB).

[0095] Alternatively an UHPLC size exclusion column, ACQUITY UPLC Protein BEH SEC column, 200 A, 1.7 pm particle size, 4.6 x 150 mm, (Waters Corporation, 186005225) was used with the same mobile phase. Samples were injected neat (total load of 60 pg) on the column, separated isocratically with a flow rate of 0.3 mL / min and run time of 8 min. The eluent was monitored by UV absorbance at 280 nm. Purity was determined by calculating the percentage of each component group's integrated area as compared to the total integrated area. An additional HMW shoulder peak (comprising the shoulder species) was observed in this lot at about 0.3% in the final drug substance.

[0096] This example demonstrated that an additional HMW shoulder peak (the shoulder species) was observed in this lot, with relatively low percentage.

[0097] Example 2: SEC-MALS / RP LC-MS - showed no difference in molecular weight of the shoulder species

[0098] This example describes the use of two procedures for determining the molecular weight of the shoulder species:

[0099] 1. Size exclusion chromatography coupled to multi-angle light scattering (MALS): the SEC method was performed as described in Example 1, except that the SEC HPLC or UHPLC column was used with MALS detector instead of a UV detector. The results are shown in FIG. 2A. 2. Reverse phase liquid chromatography coupled to mass spectrometry (RP LC-MS): samples were separated on a RP column: Waters BioResolve RP mAb Polyphenyl, 2.7 pm, 2.1 X 100 mm (part No. 186008945), and detected by an Agilent 6545XT QTOF MS. The results are shown in FIG. 2B.

[0100] As shown in FIG. 2A and FIG. 2B, the shoulder species has the same molecular weight of an evolocumab dimer. This example demonstrated that the molecular weight of this shoulder species is the same as a conventional dimer.

[0101] Example 3: dSEC + NR CE-SDS - showed that >60% of the species is covalently linked.

[0102] This example describes two orthogonal procedures to analyze the percentage of the covalent HMW shoulder species in the evolocumab composition.

[0103] Denatured SE-HPLC (dSEC) method under non-reduced conditions: Samples comprising evolocumab were denatured in 7 M GdnHCI, 100 mM sodium phosphate pH 5 buffer, and with 0.1 M NEM for alkylation. An Agilent AdvanceBio SEC 2.7 pm 4.6 x 300 mm column (Part No: PL1580-5301) was used with 3 M Guanidine HCI, 100 mM sodium phosphate, pH 6.5 as the mobile phase. Treated samples were injected on the SE-HPLC column, separated isocratically with a flow rate of 0.3 mL / min and run time of 20 min. The peaks were detected by UV absorbance at 220 nm. Covalent HMW was eluted before the main peak. Purity was determined by calculating the percentage of each component group's integrated area as compared to the total integrated area. The results of the dSEC analysis are shown in FIG. 3A.

[0104] Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under denaturing but nonreducing conditions (NR CE-SDS): samples comprising evolocumab were denatured by heating at 60 °C in the presence of SDS and N-ethylmaleimide (NEM). The resulting negatively charged SDS- protein complex was electrokinetically injected into a bare-fused silica capillary filled with SDS gel buffer. An electrical voltage is applied across the capillary. Proteins are detected by a photo diode array (PDA) detector at 220 nm. Covalent HMW was migrated as post-peak after the main peak. Purity was determined by calculating the percentage of each component group's corrected area as compared to the total corrected area. The results of the NR CE-SDS are shown in FIG. 3B.

[0105] The dSEC method showed that this shoulder species is 61% covalently-linked. The NR CE- SDS method showed a similar result of the shoulder species being 65% covalently linked. This example demonstrated that over 60% of this dimer is covalently linked.

[0106] Example 4: Reduced RP LC-MS and Reduced CE-SDS

[0107] This example describes two orthogonal procedures to determine whether the covalent linkage in the shoulder species is reducible (linked by disulfide bonds) or not.

[0108] Reduced RP LC-MS (R RP LC-MS): Samples comprising evolocumab were denatured by Guanidine HCI (final concentration 4.8 M) and reduced by Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and separated on RP column: Waters BioResolve RP mAb Polyphenyl, 2.7 pm, 2.1 X 100 mm (part No. 186008945), and detected by QTOF. The results of the R RP LC-MS analysis are shown in FIGs. 4A-B. UV spectra and raw mass spectra are shown in FIG. 4A and FIG. 4B, respectively.

[0109] CE-SDS under denaturing and reducing conditions (R CE-SDS) samples comprising evolocumab were denatured and reduced by heating at 70 °C in the presence of SDS and (3- mercaptoethanol (BME). Treated samples were electrokinetically injected into a bare fused silica capillary filled with SDS gel buffer. An electrical voltage was applied across the capillary. Proteins were detected by a photo diode array (PDA) detector at 220 nm. Purity was evaluated by determining the percent corrected peak area of each component. The results of the R CE-SDS analysis are shown in FIG. 4C.

[0110] It was shown that the shoulder species can be fully reduced (breaking the disulfide bonds) to heavy chain (HC) and light chain (LC), meaning the dimer is linked by disulfide bonds.

[0111] This example demonstrated that the covalent linkage is through disulfide bonds. Example 5: Native SEC-MS + Fabricator digest

[0112] This example describes the procedure to determine the dimerization site.

[0113] Samples comprising evolocumab were digested by Fabricator (IdeS) enzyme, and separated on SEC column: Waters ACQUITY UPLC Protein BEH SEC column, 1.7 pm, 2.1X150 mm (Part No. 186008471), under native conditions and detected by QTOF.

[0114] (Fab' dimer was detected (FIGs. 5A-B) indicating the linkage site occurs at the hinge region and above. UV spectra and raw mass spectra are shown in FIG. 5A and FIG. 5B, respectively.

[0115] This example demonstrated that the dimerization site (linkage) is at the hinge region and above in the Fab.

[0116] Example 6: reduced peptide mapping

[0117] This example describes the procedure to determine the post translational modifications (PTMs).

[0118] Samples comprising evolocumab (conventional dimer, shoulder species and main peak fractions) were denatured and reduced in 6 M Guanidine HCI buffer in the presence of dithiothreitol (DTT), and alkylated by sodium iodoacetate (NalAA). After digestion by trypsin, the reaction was quenched by 10% TFA. Treated samples were separated on RP column: Waters ACQUITY UPLC BEH C8 1.7 pm, 2.1 x 150 mm (part No. 186003377), and detected by UV and mass spectrometry.

[0119] PTMs were compared between this species and regular dimer. The results of the comparisons are shown in FIG. 6. The shoulder species showed similar PTM patterns with slightly higher levels of clips than conventional dimer.

[0120] This example demonstrated that the shoulder species has no significantly different PTMs compared to conventional dimer. These data are consistent with the shoulder species posing a low safety risk. Example 7: Non-reduced peptide mapping

[0121] Shoulder species was observed in a composition comprising evolocumab (FIG IB). Samples (conventional dimer, shoulder species, and main peak fractions) were denatured and digested with Lys-C. Treated samples were separated on a reverse phase (RP) column: Waters ACQUITY UPLC Protein BEH C4 1.7 pm, 2.1 x 150 mm (part No. 186004497), and detected by UV and mass spectrometry. LC-MS / MS data were processed on MassAnalyzer 6.03 to search for disulfide linkages. Major detected disulfide linkages in the dimer and dimer shoulder are shown below. Values shown in Table 1 for the dimer and shoulder represent the abundance ratio of each linkage compared to that in the main peak. A higher ratio indicates an elevated disulfide linkage than the main peak, and a lower ratio indicates a decreased linkage than the main peak. "1:" and "2:" represent the heavy chain and light chain, respectively.

[0122] Table 1

[0123] For both the conventional dimer and the shoulder species, elevated level of linkage #4 is observed. This linkage represents the single hinge peptide 1:C217-K242 with unexpected two intra-chain disulfide bonds. Formation of intra-chain disulfide bonds in the hinge region instead of the expected inter-chain disulfide bonds leaves the hinge cysteines in the other chain free to form complex disulfide network that are too complex to detect by this peptide mapping method. This observation therefore suggests that cysteines near the hinge may be involved in the dimerization of the two species. The effect is stronger in the shoulder species as its level increases by 8-fold compared to the main peak. For the shoulder species, a decrease in the expected inter-chain disulfide linkages in the hinge region (linkage #3) is observed, further supporting the conclusion that hinge cysteines may be involved in the dimer formation. Additionally, a large increase in the LC-LC linkage (#10 - 2:C214 / 2:C214) is observed in the shoulder species (~14 folds compared to the main peak and ~7 folds compared to the conventional dimer). Thus, it is contemplated that the shoulder species comprises an intermolecular LC-LC linkage, involving LC Cys-214.

[0124] For lgG2 molecules it has been previously reported that the two cysteines involved in HC- LC disulfide (HC Cys-129 and LC Cys-214) may also link to the hinge (disulfide heterogeneity) (Figure 8 of Wypych et al. Journal of Biological Chemistry 283: 19164-16205). Therefore, it is reasonable to believe that HC Cys-129, LC Cys-214 and the hinge cysteines can be involved in the formation of the two dimeric species. The dimer formation is unlikely due to a simple intermolecular rearrangement of hinge disulfide bonds, because the product of this type of rearrangement (linkage #11) does not increase in the two dimer species.

[0125] Example 8: Shoulder species showed potency in receptor- ligand blocking assay

[0126] Fraction enriched for the shoulder species (>80%) was evaluated in a receptor- ligand potency assay. An AlphaScreen® assay to measure PCSK9-LDLR receptor- ligand blocking utilized a bead-based Amplified Luminescent Proximity Homogeneous Assay (Alpha) that detects biomolecular interactions. Donor beads were coated with a hydrogel that contains phthalocyanine, a photosensitizer and streptavidin. Acceptor beads were coated with a hydrogel that contains thioxene derivatives as well as nickel chelate. Biotinylated huLDLR(EGFa:EGFb)huFc, his-tagged human PCSK9 (huPCSK9:8xHis), and buffer (25 mM Hepes, 0.1M NaCI, 0.2% BSA, pH 7.4) were combined with donor beads, acceptor beads, and test sample (or positive or negative control) in wells of 96-well microwell plates and incubated at 23 °C to 27 °C for 1 to 2 hours. After the excitation signal was applied to the assay plates the emission signal was read at 570 nm. Test sample activity is determined by comparing the test sample response to that of a Reference Standard. The results of this analysis are shown in Table 2. The relative potency of the reference standard was 97%.

[0127] Table 2

[0128] The shoulder species had a relative potency of 85%, showing that the shoulder species was functionally active. The shoulder species was contemplated to retain a suitable potency for inhibiting the interaction between PCSK9 and LDLR. Accordingly, the shoulder species was determined to have suitable potency for medical use.

Claims

What is claimed:

1. A composition comprising a first species and a second species, the first species comprising a monomer of evolocumab, and the second species comprising a dimer of a first evolocumab and a second evolocumab, said dimer comprising a disulfide linkage linking a hinge or Fab region of the first evolocumab to a hinge or Fab region of the second evolocumab.

2. The composition of claim 1, wherein no more than about 2% of the evolocumab of the composition is comprised by the second species.

3. The composition of claim 1, wherein no more than about 0.3% of the evolocumab of the composition is comprised by the second species.

4. The composition of claim 1, wherein at least about 98% of the evolocumab of the composition is comprised by the first species, and wherein no more than about 2% of the evolocumab of the composition is comprised by the second species.

5. The composition of claim 1, wherein at least about 99.7% of the evolocumab of the composition is comprised by the first species, and wherein no more than about 0.3% of the evolocumab of the composition is comprised by the second species.

6. The composition of claim 1, wherein at least about 96% of the evolocumab of the composition is comprised by the first species, and wherein no more than about 4% of the evolocumab of the composition is comprised by the second species.

7. The composition of claim 1, wherein at least about 92% of the evolocumab of the composition is comprised by the first species, and wherein no more than about 8% of the evolocumab of the composition is comprised by the second species.

8. The composition of any one of the preceding claims, wherein at least 0.1% of the evolocumab of the composition is comprised by the second species.

9. The composition of any one of the preceding claims, wherein at least 0.2% of the evolocumab in the composition is comprised by the second species.

10. The composition of any one of the preceding claims, wherein at least 0.5% of the evolocumab the composition is comprised by the second species.

11. The composition of any one of claims 1-9, wherein 0.2% - 2% of the evolocumab of the composition is comprised by the second species.

12. The composition of any one of claims 1-10, wherein 0.5% - 2% of the evolocumab of the composition is comprised by the second species.

13. The composition of any one of the preceding claims, wherein the second species inhibits binding of PCSK9 to LDLR in an in vitro assay by at least 70% compared to evolocumab monomer, the in vitro assay comprising a huLDLR immobilized on a first bead and a huPCSK9 immobilized on a second bead, wherein binding of huLDLR to huPCSK9 is indicated by proximity of the first bead to the second bead.

14. The composition of any one of the preceding claims, wherein evolocumab comprises a heavy chain having the amino acid sequence of SEQ ID NO: 1, and a light chain having the amino acid sequence of SEQ ID NO: 2.

15. The composition of any one of the preceding claims, wherein the disulfide linkage comprises a linkage between a light chain of the first evolocumab and a light chain of the second evolocumab.

16. The composition of claim 15, wherein the disulfide linkage links C214 of the light chain of the first evolocumab to C214 of the light chain of the second evolocumab.

17. The composition of any one of the preceding claims, wherein the percentage of evolocumab species is as determined by size exclusion chromatography.

18. The composition of claim 17, wherein the size exclusion chromatography comprises UV 280 nm measurement of absorbance, and wherein the percentage of evolocumab species is calculated as a percentage of evolocumab species peak area relative to total peak area.

19. The composition of any one of claims 1-18, wherein the composition is for medical use.

20. A pharmaceutical composition comprising the composition of any one of the preceding claims and a pharmaceutically acceptable excipient.

21. The pharmaceutical composition of claim 20, comprising: 120-140 mg / ml of evolocumab, 20 mM acetate, 0.01% (w / v) polysorbate 80, and 2.5 % (w / v) proline.

22. An administration device comprising the pharmaceutical composition of claim 20 or 21, wherein the administration device comprises a unit-dose of 140 mg of evolocumab or 420 mg of evolocumab, optionally wherein the administration device is selected from an autoinjector pen and a syringe.

23. A method of lowering LDL-C in a subject, the method comprising administering the composition or pharmaceutical composition of any one of the preceding claims to the subject.

24. The method of claim 23, comprising administering 140 mg of evolocumab to the subject Q2W; or 420 mg of evolocumab to the subject Q2W; or 420 mg of evolocumab to the subject QM.

25. A method of determining the presence, absence, or amount of a species in a composition comprising evolocumab, said species comprising a dimer of a first evolocumab and a second evolocumab, wherein a hinge or Fab region of the first evolocumab is bound to a hinger or Fab region of the second evolocumab by a disulfide linkage, the method comprising: resolving the composition by size exclusion chromatography (SEC); and resolving a reference composition comprising (i) a monomer of evolocumab and (ii) a noncovalent dimer of evolocumab by SEC; determining the presence, absence, or amount of said species in the composition, said species having a larger hydrodynamic radius than (i) and a smaller hydrodynamic radius than (ii).

26. The method of claim 25, further comprising:obtaining a SEC fraction enriched for the second species relative to (i) and (ii); reducing said SEC fraction; and resolving said reduced fraction by capillary electrophoresis-SDS (CE-SDS).

27. The method of claim 25 or 26, comprising: determining that no more than 2%, 1%, 0.5%, 0.3%, or 0.1%, or 0.1%-8%, 0.1%-5%, 0.1%-2%, 0.3%-8%, 0.3%-5%, 0.3%-2%, 0.5%-8%, 0.5%-5%, or 0.5%-2% of the evolocumab of the composition are comprised by the species; and manufacturing the composition for pharmaceutical use.

28. The method of claim 27, wherein manufacturing the composition for pharmaceutical use comprises purification of the evolocumab by cation exchange chromatography.

29. The method of any one of claims 27-28, wherein manufacturing the composition for pharmaceutical use comprises placing at least a portion of the composition in an administration device.

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