Ophthalmic formulations and uses thereof

Abstract

This invention relates to an ophthalmic preparation and its use. The ophthalmic preparation comprises: (a) aflibercept, (b) a buffer, (c) a nonionic surfactant, and (d) a tension modifier, wherein the pH of the preparation is from pH 5.0 to pH 6.5. This invention also discloses the use of the ophthalmic preparation in the preparation of a medicament for treating eye conditions or diseases.

Description

An ophthalmic preparation and its uses

[0001] This application is a divisional application of the invention patent application No. 201780069882.9 entitled "Aflibercept Preparation and Its Use", filed on November 20, 2017.

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 497,584, filed November 21, 2016, with the U.S. Patent and Trademark Office. Technical Field

[0003] This invention relates to an ophthalmic preparation and its use, particularly a pharmaceutical preparation of aflibercept fusion protein suitable for ocular administration. Background Technology

[0004] Aflibercept is a recombinant fusion protein comprising two main components: a vascular endothelial growth factor (VEGF) binding moiety derived from the extracellular domains of human VEGF receptors 1 and 2, fused to the Fc region of human IgG1. (Reference: Papadopoulos et al., Modified chimeric polypeptides with improved pharmacokinetic properties, WO 00 / 75319A1; US ​​7070959B2). Structurally, aflibercept is a dimeric glycoprotein with a molecular weight of approximately 96.9 kilodaltons (kDa). It contains approximately 15% glycosylation to yield a total molecular weight of approximately 115 kDa. All five putative N-glycosylation sites on each polypeptide chain predicted by the primary sequence can be occupied by carbohydrates and exhibit some degree of chain heterogeneity, including heterogeneity of terminal sialic acid residues.

[0005] The U.S. Food and Drug Administration (FDA) approved aflibercept in November 2011, and the European Medicines Agency (EMA) approved it in November 2012.

[0006] By product name Aflibercept, produced by Regeneron Pharmaceuticals, Inc., is used as an ophthalmic agent to treat eye conditions or diseases such as macular edema following central retinal vein occlusion (CRVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular (wet) age-related macular degeneration (AMD), vision impairment due to myopic choroidal neovascularization, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascular age-related macular degeneration (AMD).

[0007] Trademark name Ziv-aflibercept from Regeneron Pharmaceuticals, Inc. is being developed as an injectable treatment for metastatic colorectal cancer.

[0008] Known formulations of aflibercept include those described by Furfine et al. (Furfine et al., VEGF Antagonist formulations for intravitreal administration, US8092803; US9580489; EP 2364691B1; WO2007149334A2) and those described by Dix et al. (Dix et al., VEGF Antagonist Formulations, WO2006104852 A2; US8921316; US9636400).

[0009] There is still a need for aflibercept formulations with enhanced stability, and this invention provides such formulations. Summary of the Invention

[0010] This invention relates to an ophthalmic formulation of aflibercept, comprising: (a) aflibercept at a concentration of 5-100 mg / mL; (b) a buffer at a concentration of 5-50 mM; (c) a nonionic surfactant; and (d) a tension modifier selected from the group consisting of polyols and amino acids, or in some embodiments, both polyols and amino acids, wherein the formulation has a final weight molar osmolality of about 300 mOsm / kg (i.e., 300 ± 50 mOsm / kg). The ophthalmic formulation of this invention contains chloride anions (Cl... - The concentration of the aflibercept is less than about 10 mM, and in some embodiments less than about 5 mM or less than about 1 mM; and the pH of the formulation is from about pH 5.0 to about pH 6.5. The ophthalmic formulations of the present invention are suitable for intravitreal or topical administration. The aflibercept-containing ophthalmic formulations of the present invention have stability characteristics, such as significantly reduced aggregation over time, and visual characteristics, which are advantageous or more advantageous than other known aflibercept ophthalmic formulations, such as those containing added sodium chloride. If desired, the formulations of the present invention can also be lyophilized and reconstituted.

[0011] The ophthalmic formulations of the present invention can be used as pharmaceutical ophthalmic agents in methods of treating eye conditions or diseases, such as macular edema following retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular (wet) age-related macular degeneration (AMD), visual impairment due to myopic choroidal neovascularization, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascular age-related macular degeneration (AMD). The ophthalmic formulations of the present invention can be administered via intravitreal injection, or in some cases, via topical application to the eye, as medically appropriate. The formulations of the present invention can be used to treat these eye conditions or diseases and to prepare medicaments for treating these eye conditions and diseases.

[0012] The foregoing summary is not intended to limit every aspect of the invention, and additional aspects are described in other sections such as the detailed description. The entire document is intended to be interconnected as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated even if such combinations are not found in the same sentence, paragraph, or section of the document.

[0013] In addition to the foregoing, as an additional aspect, this invention includes all embodiments of the invention, the scope of which is in any way narrower than the variations defined in the specific paragraphs above. For example, certain aspects of the invention are described as genus, and it should be understood that each member of the genus is individually an aspect of the invention. Furthermore, aspects described as genus or selected members of a genus should be understood to include combinations of two or more members of that genus. Although the applicant has invented the full scope of the invention as set forth herein, the applicant does not intend to claim the subject matter described in the prior art work of others. Therefore, if the patent office or other entity or individual draws the applicant's attention to statutory prior art within the scope of the claims, the applicant reserves the right to exercise the right of amendment under applicable patent law to redefine this subject matter of the claim in order to explicitly exclude such statutory prior art or obvious variations thereof from the scope of the claim. Variations of the invention defined by these amended claims are also intended as aspects of the invention. Attached Figure Description

[0014] Figure 1 shows the results of subvisible particle (insoluble microparticle) analysis performed by small-volume HIAC analysis. The 10 μm particle results show that all samples had low particle levels, except for formulation 7, which showed an increase in particle levels during storage.

[0015] Figure 2 shows the results of sub-visible particle analysis performed by small-volume HIAC analysis. The 25 μm result indicates that all samples have low or undetectable levels of particles. The error bar, representing the standard deviation of three repeated measurements, is greater than the number of observed particles.

[0016] Figure 3 shows the size exclusion high-performance liquid chromatography (HPLC) results of the samples stored at 4 °C. All formulations except Formulation 8 showed similar HMW formation rates.

[0017] Figure 4 shows the size exclusion high-performance liquid chromatography (HPLC) results of the samples stored at 30 °C. All formulations except Formulation 8 showed similar HMW formation rates.

[0018] Figure 5 shows the results of the reduced CE-SDS analysis under 4°C storage conditions. During the 7-week testing period, all formulations exhibited similar levels of purity percentage.

[0019] Figure 6 shows the results of the reduced CE-SDS analysis under 30°C storage conditions. Except for formulation 7, all formulations showed similar levels of purity percentage during storage at 30°C. Formulation 7 showed a continuous decrease in purity percentage over time.

[0020] Figure 7 shows the results of cIEF analysis used to assess the charge distribution of aflibercept during storage at 4 °C. All formulations showed similar levels of basicity percentage over time.

[0021] Figure 8 shows the results of cIEF analysis used to assess the charge distribution of aflibercept during storage at 4 °C. All formulations showed similar levels of acidic substance percentage over time.

[0022] Figure 9 shows the results of cIEF analysis used to assess the charge distribution of aflibercept during storage at 30°C. All formulations showed similar levels of basicity percentage over time.

[0023] Figure 10 shows the results of cIEF analysis used to assess the charge distribution of aflibercept during storage at 30°C. All formulations showed similar levels of acidic substance percentage over time.

[0024] Figure 11 illustrates the formation of HMW in various aflibercept formulations stored at 30°C, as measured by SE-HPLC. (Refer to Table 4 for formulation abbreviations).

[0025] Figure 12 shows the difference between aflibercept recombined at 30°C and commercially available aflibercept. Stability comparison results for (aflibercept; Regeneron Pharmaceuticals, Inc., Tarrytown, NY). The recombinant aflibercept was prepared by Just Biotherapeutics (Seattle, WA) and formulated using 10 mM acetate, 3% (w / v) proline, pH 5.2, and 0.1% (w / v) poloxamer formulation (A52ProP1-0.1).

[0026] Figure 13 shows the effect of 100 mM sodium chloride (“salt”) on the stability of recombinant aflibercept in a formulation (A52ProPl-0.1) with 10 mM acetate, 3% (w / v) proline, pH 5.2, stored at 30 °C, compared to the control (minus any added sodium chloride). Detailed Implementation

[0027] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0028] definition

[0029] Unless otherwise defined herein, scientific and technical terms used in connection with this application should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. Therefore, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. For example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a population of multiple cells.

[0030] This invention relates to aqueous ophthalmic preparations suitable for intravitreal or topical administration to patients, including those commercially also known as... Aflibercept. Aflibercept is an assembly of two identical fusion polypeptide chains having the aflibercept amino acid sequence (SEQ ID NO: 1), and is usually most conveniently produced by recombinant DNA expression technology. The aflibercept amino acid sequence is as follows:

[0031]

[0032] A disulfide bridge is expected to exist between cysteine ​​residues at the following amino acid positions in SEQ ID NO: 1 (the underlined cysteine ​​(C) residue shown in SEQ ID NO: 1 above):

[0033] 30-79 (In-chain)

[0034] 124-185 (in-chain)

[0035] 211-211 (Inter-chain)

[0036] 214-214 (Inter-chain)

[0037] 246-306 (in-chain)

[0038] 352-410 (in-chain).

[0039] The two fusion polypeptide chains of aflibercept are covalently linked by disulfide bonds at amino acid positions 211 and 214 of SEQ ID NO: 1. The fusion protein is typically glycosylated, wherein the N-glycan is covalently linked at asparagine residues (bold / italic asparagine (N) residues shown in SEQ ID NO: 1) at positions 36, 68, 123, 196, and 282 of SEQ ID NO: 1. "Aflibercept" within the scope of this invention also includes embodiments in which one, both, or no fusion polypeptide chains have the amino acid sequence SEQ ID NO: 1 with an additional carboxyl-terminal lysine (K) residue. The concentration of aflibercept in the ophthalmic formulations of this invention is from about 20 mg / mL to about 80 mg / mL, or from about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL is useful in many embodiments of the formulation.

[0040] "Stable" formulations are those in which the proteins in a drug and / or drug product containing aflibercept substantially maintain their physical and / or chemical stability and / or biological activity during processing (e.g., ultrafiltration, permeation, other filtration steps, vial filling), transportation, and / or storage. In summary, the physical, chemical, and biological stability of the proteins in a formulation reflects the "stability" of a protein formulation, such as an aflibercept formulation, which is specific to the storage conditions of the formulated drug product (DP). For example, a drug product expected to be stored at sub-zero temperatures may not show significant changes in chemical, physical, or biological activity, while a drug product expected to be stored at 40°C may show changes in physical, chemical, and biological activity, the extent of which depends on the storage time of the drug or drug product. The formulation of a protein product can also affect the rate of change. For example, aggregate formation is highly influenced by protein concentration, with higher aggregation rates observed with higher protein concentrations. Excipients are also known to affect the stability of drug products; for example, the addition of salt during storage increases the aggregation rate of certain proteins, while other excipients such as sucrose are known to decrease the aggregation rate. Instability is also greatly affected by pH, depending on the type of modification and pH dependence, resulting in both high and low degradation rates.

[0041] Various analytical techniques for measuring protein stability are available in the art and have been reviewed, for example, in Wang, W. (1999), Instability, stabilization and formulation of liquid protein pharmaceuticals, Int J Pharm 185:129-188. Stability can be measured at selected temperatures and for selected time periods. For rapid screening, for example, formulations can be stored at 40°C for 2 weeks to 1 month, during which time stability can be measured. If a formulation needs to be stored at 2–8°C, it should generally be stable at 30°C for at least 1 month, or at 40°C for at least 1 week, and / or at 2–8°C for at least 2 years.

[0042] A protein is considered to “maintain its physical stability” in a pharmaceutical formulation if it shows minimal signs of change in secondary and / or tertiary structure (i.e., intrinsic structure), aggregation and / or precipitation and / or denaturation, as measured by visual inspection of color and / or transparency, or by measurement via UV light scattering, size exclusion high-performance liquid chromatography, or other suitable methods. Physical instability of a protein, i.e., loss of physical stability, can result from oligomerization leading to dimers and higher-order aggregates, sub-visible and visible particle formation, and precipitation. Depending on the type of target degradation product, different techniques can be used to determine the extent of physical degradation. Dimers and higher-order soluble aggregates can be quantified using size exclusion chromatography, while sub-visible particles can be quantified using light scattering, opacity, or other suitable techniques. In one embodiment, protein stability is determined based on the percentage of aflibercept monomeric protein in a solution containing a lower percentage of degradation (e.g., fragmentation) and / or aggregated protein. “Aflibercept monomer” refers to an assembly of two polypeptide chains having the aflibercept amino acid sequence (SEQ ID NO: 1), with or without an additional carboxyl-terminal lysine residue on either polypeptide chain. In the “aflibercept monomer,” as described above, two aflibercept polypeptide chains are assembled via association and disulfide cross-linking of the immunoglobulin Fc domain portions of the sequence. For example, an aqueous formulation comprising a stable protein may comprise (as a percentage of total protein) at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of aflibercept monomer protein. Alternatively, the aqueous formulation of the present invention may comprise (as a percentage of total protein) about 5% aggregates and / or degraded aflibercept protein.

[0043] A protein is considered to "maintain its chemical stability" in pharmaceutical formulations if its chemical stability at a given time prevents the formation or disruption of covalent bonds that would alter the primary structure of protein components, such as aflibercept. Changes in primary structure can lead to alterations in the secondary and / or tertiary and / or quaternary structures of a protein, and can result in the formation of aggregates or the reversal of already formed aggregates. Typical chemical modifications can include isomerization, deamidation, N-terminal cyclization, skeletal hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, β-elimination, disulfide formation, disulfide hybridization, disulfide cleavage, and other alterations that result in changes to the primary structure, including the formation of D-amino acids. Chemical instability, i.e., the loss of chemical stability, can be determined using a variety of techniques, including ion-exchange chromatography, capillary isoelectric focusing, peptide digestion analysis, and various types of mass spectrometry. Chemical stability can be assessed by detecting and quantifying the forms of chemical changes in proteins. Chemical alterations can involve size modifications (e.g., clipping), which can be assessed using methods such as size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., due to deamidation), which can be assessed using charge-based methods such as, but not limited to, ion exchange chromatography, capillary isoelectric focusing, or peptide mapping.

[0044] Loss of physical and / or chemical stability can lead to changes in biological activity, such as an increase or decrease in target biological activity, depending on the modification and the modified protein. A protein is considered to "retain its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within approximately 30% of the biological activity exhibited when the drug formulation was prepared. If the activity is less than 70% of its initial value, it is considered to have decreased activity. Bioassays can include in vivo and in vitro-based assays, such as ligand binding, potency, cell proliferation, or other alternative measurements of its biopharmaceutical activity. For example, in vitro ligand binding assays can be used to assess the biological activity of aflibercept, such as inhibition of PGF-bound antiplacental growth factor by ELISA or human umbilical vein endothelial cell (HUVEC) proliferation assays.

[0045] Aflibercept used in this invention is typically produced via recombinant expression techniques. The term “recombinant” means that a material (e.g., a nucleic acid or polypeptide) has been artificially or synthetically altered (i.e., non-naturally) through human intervention. The alteration can be performed on the material within its natural environment or state, or on the material removed from its natural environment or state. For example, a “recombinant nucleic acid” is prepared by means of recombinant nucleic acids, such as during cloning, DNA shuffling, or other well-known molecular biological processes. An example of such a molecular biological process is found in Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). A “recombinant DNA molecule” consists of DNA fragments linked together by such molecular biological techniques. As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules expressed using recombinant DNA molecules. A “recombinant host cell” is a cell containing and / or expressing recombinant nucleic acids. Recombinant DNA molecules that can be used to express aflibercept fusion proteins are described, for example, in Papadopoulos et al., Modified Chimeric Polypeptides with Improved Pharmacokinetic Properties, US 7,070,959 B2 and WO 00 / 75319 A1.

[0046] The term "naturally occurring" refers to a naturally occurring substance when it appears in this specification together with biological materials such as polypeptides, nucleic acids, host cells, etc.

[0047] The term "control sequence" or "control signal" refers to a polynucleotide sequence that can influence the expression and processing of a coding sequence linked to it in a specific host cell. The nature of such a control sequence can be host-dependent. In specific embodiments, control sequences for prokaryotes may include promoters, ribosome-binding sites, and transcription termination sequences. Control sequences for eukaryotes may include promoters containing one or more recognition sites for transcription factors, transcription enhancer sequences or elements, polyadenylation sites, and transcription termination sequences. Control sequences may include leader sequences and / or fusion partner sequences. Promoters and enhancers consist of short DNA arrays that specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987)). Promoter and enhancer elements (similar control elements, i.e., promoters, are also found in prokaryotes) have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses. The specific promoter and enhancer chosen depends on the cell type used to express the target protein. Some eukaryotic promoters and enhancers have a wide host range, while others function in a limited set of cell types (see Voss et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis et al., Science 236:1237 (1987)).

[0048] A promoter is a region of DNA that includes a site to which RNA polymerase binds to initiate transcription of messenger RNA via one or more downstream structural genes. The promoter is located near the transcription start site of a gene, on the same strand of DNA and upstream (towards the 5' region of the sense strand). Promoters are typically about 100-1000 bp in length.

[0049] "Enhancers" are short (50-1500 bp) regions of DNA that can bind to one or more activating proteins (transcription factors) to activate gene transcription.

[0050] As used herein, the terms “operably combined,” “operably ordered,” and “operably linked” refer to the linking of nucleic acid sequences such that the resulting nucleic acid molecule can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linking of amino acid sequences resulting in a functional protein. For example, the linking of a control sequence to a protein-coding sequence in a vector “operably linked” enables the expression of the protein-coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0051] The terms “polypeptide” and “protein” are used interchangeably herein and include a molecular chain of two or more amino acids covalently linked by peptide bonds. These terms do not refer to a specific length of product. Therefore, “peptide” and “oligopeptide” are included within the definition of a polypeptide. This term includes post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Additionally, protein fragments, analogs, mutant or variant proteins, fusion proteins, etc., are included within the meaning of polypeptide. This term also includes molecules that include one or more amino acid analogs or non-standard or non-natural amino acids, such as those recombinantly expressed using known protein engineering techniques. Furthermore, fusion proteins can be derived using well-known organic chemistry techniques as described herein.

[0052] "Variants" of peptides (e.g., immunoglobulins or antibodies) comprise an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted relative to another peptide sequence. Variants include fusion proteins.

[0053] The term "fusion protein," for example, in the case of aflibercept, indicates that the protein comprises polypeptide components derived from more than one parent protein or polypeptide. Typically, fusion proteins are expressed by a "fusion gene," in which the nucleotide sequences encoding a polypeptide sequence from one protein are appended within a frame and optionally separated by a linker from the nucleotide sequences encoding polypeptide sequences from different proteins. The fusion gene can then be expressed as a single protein by a recombinant host cell.

[0054] "Secreted" proteins refer to those proteins that can be directed to the endoplasmic reticulum (ER), secretory vesicles, or extracellular space due to their secreted signal peptide sequences, as well as those proteins that are released into the extracellular space without necessarily containing a signal sequence. If a secreted protein is released into the extracellular space, it can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur through a number of mechanisms, including exocytosis and proteolytic cleavage. In some other embodiments, the target aflibercept fusion protein can be synthesized by the host cell as a secreted protein, which can then be further purified from the extracellular space and / or culture medium.

[0055] As used herein, when referring to proteins produced in host cells using recombinant DNA technology, “soluble” means the protein is present in an aqueous solution; if the protein contains a diarginine signal amino acid sequence, soluble proteins are exported to the periplasmic space of a Gram-negative bacterial host, or secreted into a culture medium by a secretory eukaryotic host cell or by a bacterial host with a suitable gene (e.g., the kil gene). Therefore, a soluble protein is a protein not found in inclusion bodies within the host cell. Alternatively, depending on the context, a soluble protein is a protein not found integrated into the cell membrane, or, in vitro, a protein that is soluble or capable of dissolving in an aqueous buffer solution under physiological conditions without forming significant amounts of insoluble aggregates (i.e., the aggregates formed are less than 10% of the total protein, and typically less than about 5%), when suspended in an aqueous target buffer solution without other proteins under physiological conditions, such buffer does not contain ionic detergents or dissociating agents, such as sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, or lithium perchlorate. Conversely, insoluble proteins are proteins that exist in denatured form within cytoplasmic granules (called inclusion bodies) in the host cell, or again, depending on the context, insoluble proteins are proteins that exist in cell membranes, including but not limited to the cytoplasmic membrane, mitochondrial membrane, chloroplast membrane, endoplasmic reticulum membrane, etc., or form significant amounts of insoluble aggregates in an aqueous buffer solution under physiological conditions (i.e., the aggregates formed are equal to or greater than about 10% of the total protein), when suspended in an aqueous solution of a target buffer solution under physiological conditions, without other proteins (at physiologically compatible temperatures), such that the buffer does not contain ionic detergents or ionizing agents, such as sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, or lithium perchlorate.

[0056] The terms "polynucleotide" or "nucleic acid" encompass both single-stranded and double-stranded nucleotide polymers containing two or more nucleotide residues. The nucleotide residues constituting a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. These modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide bond modifications, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, aniline thiophosphates, aniline phosphates, and aminophosphates.

[0057] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotide residues. In some embodiments, the oligonucleotide is 10 to 60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, used to construct mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides may include labels, including radioactive labels, fluorescent labels, haptens, or antigen labels, for detection and assay. Oligonucleotides can be used as, for example, PCR primers, cloning primers, or hybridization probes.

[0058] As used interchangeably in this document, “polynucleotide sequence”, “nucleotide sequence”, or “nucleic acid sequence” refers to the primary sequence of nucleotide residues in a polynucleotide, including oligonucleotides, DNA and RNA, nucleic acids, or strings representing the primary sequence of nucleotide residues, depending on the context. A given nucleic acid or complementary polynucleotide sequence can be identified based on any specified polynucleotide sequence. This includes DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded, and represents a sense strand or antisense strand. Unless otherwise stated, the left-handed end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-handed direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of nascent RNA transcripts is called the transcription direction; a sequence region on a DNA strand having the same sequence as the RNA transcript (5' to 5' end of the RNA transcript) is called the “upstream sequence”; a sequence region on a DNA strand having the same sequence as the RNA transcript (3' to 3' end of the RNA transcript) is called the “downstream sequence”.

[0059] As used herein, an "isolated nucleic acid molecule" or "isolated nucleic acid sequence" is a nucleic acid molecule that (1) has been identified and isolated from at least one contaminating nucleic acid molecule, which is generally associated with a natural source of the nucleic acid, or (2) has been cloned, amplified, labeled, or otherwise distinguished from background nucleic acids, such that the sequence of the target nucleic acid can be determined. An isolated nucleic acid molecule is not a form or setup found in nature. However, isolated nucleic acid molecules include nucleic acid molecules typically found in cells that express immunoglobulins (e.g., antibodies), wherein, for example, the nucleic acid molecule is located in a different position in the chromosome than in the native cell.

[0060] As used herein, the terms “encoding nucleic acid molecule,” “encoding DNA sequence,” and “encoding DNA” refer to the sequence or order of deoxyribonucleotides along a chain of deoxyribonucleic acid (DNA). This sequence of deoxyribonucleotides determines the sequence of ribonucleotides along the mRNA chain and also determines the amino acid sequence along the polypeptide (protein) chain. Therefore, a DNA sequence encodes both the RNA sequence and the amino acid sequence.

[0061] The term "gene" is broadly used to refer to any nucleic acid associated with a biological function. A gene typically includes a coding sequence and / or regulatory sequences required to express that coding sequence. The term "gene" is also applicable to a specific genomic or recombinant sequence, and the cDNA or mRNA encoded by that sequence. Genes also include non-expressed nucleic acid fragments, which, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include transcriptional control elements to which regulatory proteins (such as transcription factors) bind, causing adjacent or neighboring sequences to be transcribed.

[0062] “Gene expression” or “nucleic acid expression” refers to the transcription of DNA into RNA (optionally including RNA modification, such as splicing), the translation of RNA into a polypeptide (possibly including subsequent post-translational modifications of the polypeptide), or both transcription and translation as indicated by the context.

[0063] Expression cassettes are a hallmark of recombinant expression technologies. An expression cassette includes a gene encoding a target protein, such as a gene encoding an aflibercept fusion protein sequence. A eukaryotic "expression cassette" refers to the portion of an expression vector capable of producing a protein in eukaryotic cells, such as mammalian cells. It includes a promoter that functions in eukaryotic cells for mRNA transcription, one or more genes encoding the target protein, and mRNA termination and processing signals. Expression cassettes may usefully include genes within the coding sequence that can be used as selection markers. In the expression cassette, the promoter operatively links the 5' end to an open reading frame encoding the target exogenous protein; and a polyadenylation site operatively links the 3' end to the open reading frame. Other suitable control sequences may also be included, provided the expression cassette remains operational. The open reading frame may optionally include coding sequences for more than one target protein.

[0064] As used herein, when referring to structural genes, the term "coding region" or "coding sequence" refers to the nucleotide sequence that encodes the amino acids found in the nascent polypeptide due to the translation of the mRNA molecule. In eukaryotes, the coding region is defined by one of three triplets: the 5' triad encoding the initiator methionine "ATG," and the 3' triad specifying the stop codon (i.e., TAA, TAG, TGA).

[0065] Recombinant expression techniques typically involve the use of recombinant expression vectors that contain expression cassettes.

[0066] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0067] As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid control sequences necessary for expression of the operatively linked coding sequence in a specific host cell. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region to which they are operatively linked. Nucleic acid sequences necessary for expression in prokaryotes include promoters, optional operon sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Secretion signal peptide sequences may also optionally be encoded by the expression vector and operatively linked to the target coding sequence, such that the expressed polypeptide can be secreted by the recombinant host cell, and the target polypeptide can be more easily isolated from the cell if desired. These techniques are well known in the art (e.g., Goodey, Andrew R et al., Peptide and DNA sequences, U.S. Patent No. 5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Patent Nos. 6,022,952 and 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Patent No. 7,029,909; Ruben et al., 27 human secreted proteins, US 2003 / 0104400 A1). To express a target multi-subunit protein, host cells can be transformed using a suitable number and proportion of individual expression vectors, each containing a coding sequence for each different subunit monomer. In other embodiments, a single expression vector can be used to express different subunits of the target protein.

[0068] Recombinant expression technology typically involves mammalian host cells containing recombinant expression vectors.

[0069] The term "host cell" refers to a cell that has been or is capable of being transformed with nucleic acids and thus expresses the target gene or coding sequence. This term includes the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the target gene is present. In the practice of this invention, any of a large number of available and well-known host cells can be used to obtain aflibercept. The choice of a specific host depends on many factors known in the art. These factors include, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, rate of transformation, recyclability of the peptide, expression characteristics, biosafety, and cost. A balance of these factors must be understood that expression of a particular DNA sequence may not be equally effective for all hosts. Among these general guidelines, useful microbial host cells in cultures include bacteria (such as *Escherichia coli* sp.), yeast (such as *Saccharomyces* sp.) and other fungal cells, algae or algae-like cells, insect cells, plant cells, and mammalian (including human) cells, such as CHO cells and HEK-293 cells. Modifications can also be made at the DNA level. The DNA sequence encoding the peptide can be altered to use codons more compatible with the chosen host cell. For *E. coli*, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or include silencing restriction sites, which can facilitate DNA processing in the chosen host cell. Next, the transformed host cells are cultured and purified. The host cells can be cultured under standard fermentation conditions to express the desired compound. These fermentation conditions are well known in the art.

[0070] Examples of useful mammalian host cell lines include Chinese hamster ovary cells, including CHO-K1 cells (e.g., ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell lines (293 or 293 cell subclones for growth in suspension medium (Graham et al., J. GenVirol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); and monkey kidney cells (CV1 ATCC CCL). 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cancer cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells or FS4 cells; or mammalian myeloma cells.

[0071] The terms “cell,” “cell line,” and “cell culture” are generally used interchangeably, and all of these terms in this document include cell progeny. For example, cells “derived” from CHO cells are cell progeny of Chinese hamster ovary cells, which can be separated from the original primary cell parent by any number of generations, and may also include transformant progeny cells. Transformants and transformed cells include primary test cells and cultures derived from them, regardless of the number of transfers. It should also be understood that all progeny may not be identical in DNA content due to intentional or unintentional mutations. Mutant progeny are included that possess the same function or biological activity as those screened for the original transformed cells.

[0072] The aforementioned nucleic acids or vectors are used to transform or transfect host cells to produce polypeptides (including antigen-binding proteins such as antibodies), and the cells are cultured in appropriately modified conventional nutrient media to induce promoters, select transformants, or amplify genes encoding desired sequences. Additionally, novel vectors and transfected cell lines with multi-copy transcription units separated by selectable markers are particularly suitable for expressing polypeptides, such as antibodies.

[0073] The term "transfection" refers to the uptake of foreign or exogenous DNA by cells, and cells are considered "transfected" when the exogenous DNA has been introduced into the cell membrane. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. These techniques can be used to introduce one or more exogenous DNA motifs into a suitable host cell.

[0074] The term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is considered transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed when new genetic material is introduced through transfection, transduction, or other techniques, genetically modifying the cell from its native state. After transfection or transduction, the transformed DNA can recombine with the cell's DNA by physically integrating into the cell's chromosome, or it can be transiently maintained as an episome element without replication, or it can replicate independently as a plasmid. When the transformed DNA is replicated with cell division, the cell is considered to have been "stable transformed."

[0075] Host cells used to generate the aflibercept fusion peptide usable in this invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM)(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. Furthermore, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO Any culture medium described in 87 / 00195; or U.S. Patent Application No. 30,985, can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), and antibiotics (such as gentamicin).TM The medium may contain drugs, trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or an equivalent energy source, to provide physiological conditions for the host cells in or on the culture medium that promote the expression of the target protein by the host cells; it may also include any other necessary supplements in suitable concentrations known to those skilled in the art. Culture conditions, such as temperature (typically but not necessarily about 37°C), pH (typically but not necessarily about pH 6.5–7.5), oxygenation (oxygenation, dissolved oxygen), etc., are those previously selected for the host cells to express the target protein and will be obvious to those skilled in the art. The culture medium may include an appropriate amount of serum, such as fetal bovine serum (FBS), or preferably, the host cells may be suitable for culture in a serum-free medium. In some embodiments, the aqueous medium is a liquid, such that the host cells are cultured in a cell suspension within the liquid medium. The host cells can be efficiently grown in batch or continuous culture systems.

[0076] In other embodiments, mammalian host cells can be cultured on a solid or semi-solid aqueous culture medium (e.g., containing agar or agarose) to form a culture medium or matrix surface to which the cells adhere and to form an adhesion layer.

[0077] After culturing host cells, recombinant peptides can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the peptide, such as aflibercept, is produced intracellularly, as a first step, particulate debris (host cell fragments or lysed fragments) is removed, for example, by centrifugation or ultrafiltration.

[0078] Target proteins, such as aflibercept, can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably affinity chromatography using the target antigen or protein A or protein G as the affinity ligand. Protein A can be used to purify proteins including peptides based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). For all mouse isotypes and human γ3, protein G is recommended (Guss et al., EMBO J. 5: 1567-1575 (1986)). The most common matrix for affinity ligand attachment is agarose, but other matrices can also be used. Mechanically stable matrices such as controlled-pore glass or poly(divinyl styrene)benzene allow for faster flow rates and shorter processing times compared to using agarose. When the protein contains a CH3 domain, Bakerbond ABX is recommended. TMResin (JTBaker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification, such as ethanol precipitation, reversed-phase HPLC, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, are also possible.

[0079] The phrase "under physiological conditions" for incubation buffers and immunoglobulin or other binding assay reagents refers to incubation under conditions that allow biochemical reactions, such as non-covalent binding reactions, to occur, including temperature, pH, and ionic strength. Typically, the temperature is at room temperature or ambient temperature, up to approximately 37°C, and the pH is 6.5–7.5.

[0080] The term "physiologically acceptable salt" of a composition of substances, such as a salt of a target protein (e.g., a fusion protein or an immunoglobulin such as an antibody, or any other target protein), or a salt of an amino acid (such as, but not limited to, lysine, histidine, or proline salts), refers to any one or more salts known or subsequently found to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: acetates; trifluoroacetates; hydrohalides such as hydrochlorides (e.g., monohydrochloride or dihydrochloride) and hydrobromide; sulfates; citrates; maleates; tartrates; glycolates; gluconates; succinates; methanesulfonates; benzenesulfonates; gallic esters (gallic acid is also known as 3,4,5-trihydroxybenzoic acid), such as salts of pentagalloyl glucose (PGG) and epigallocatechin gallate (EGCG), cholesterol sulfates, dihydroxynaphthyl salts, tannins, and oxalates.

[0081] "Reaction mixture" is an aqueous mixture containing all the necessary reagents and factors that, under physiological conditions of culture, allow the occurrence of the target in vitro biochemical reaction, such as covalent or non-covalent binding reactions.

[0082] A "domain" or "region" of a polynucleotide (used interchangeably herein) is any part of the entire polynucleotide, up to and including the complete polynucleotide, but typically containing less than the complete polynucleotide. Domains may, but do not need to, fold independently of the rest of the polynucleotide chain (e.g., DNA hairpin folding) and / or be associated with a specific biological, biochemical, or structural function or location (such as a coding or regulatory region).

[0083] A protein's "domain" or "region" (used interchangeably in this document) is any part of the whole protein, up to and including the entire protein, but typically containing less than the entire protein. A domain may, but does not need to, fold independently of the rest of the protein chain and / or be associated with a specific biological, biochemical, or structural function or location (e.g., a ligand-binding domain or a cytoplasmic, transmembrane, or extracellular domain).

[0084] Quantification of aflibercept fusion protein is often useful or necessary in tracking protein production or in batch release assays for drugs or pharmaceutical products containing aflibercept. Therefore, antibodies that specifically bind to aflibercept, particularly monoclonal antibodies, can be used for these purposes.

[0085] The term “antibody” or the interchangeable “Ab” is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized, or chimeric antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that can bind to antigens (e.g., Fab, Fab', F(ab')2, Fv, single-chain antibodies, biantibodies), including the aforementioned complementarity-determining regions (CDRs), provided they exhibit the desired biological activity. Multimers or aggregates of intact molecules and / or fragments are considered, including chemically derived antibodies. Any isotype or subclass of antibody is considered, including IgG, IgM, IgD, IgA and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, or any allotype. Different isotypes have different effector functions; for example, the IgG1 and IgG3 isotypes have antibody-dependent cytotoxic (ADCC) activity.

[0086] "Isolated" proteins, such as aflibercept fusion proteins, refer to proteins that have been identified and isolated from one or more components of their natural environment or culture medium in which the fusion protein is secreted by producing cells. In some embodiments, the isolated protein is substantially free of proteins or peptides or other contaminants found in its natural or culture medium environment that could interfere with its therapeutic, diagnostic, preventative, research, or other uses. "Contaminant" components in its natural environment or culture medium are materials that could interfere with the diagnostic or therapeutic uses of the protein (e.g., antibodies) and may include enzymes, hormones, and other protein or non-protein (e.g., polynucleotides, lipids, carbohydrates) solutes. Typically, the "isolated protein" constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. In some embodiments, the target protein, such as aflibercept fusion protein or antibody, is purified (1) to greater than 95% by weight of protein, and most preferably greater than 99% by weight, or (2) to homogeneity, optionally using staining agents such as Coomassie blue or silver staining under reducing or non-reducing conditions by SDS-PAGE or other suitable techniques. Isolated naturally occurring antibodies include in situ antibodies from recombinant cells, as at least one component of the protein's native environment will be absent. However, typically, the isolated target protein (e.g., aflibercept or antibody) is prepared by at least one purification step.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for a small number of potentially naturally occurring mutations. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different epitopes, monoclonal antibodies, as antigen-binding proteins, are highly specific binders targeting a single antigenic site or epitope. Non-limiting examples of monoclonal antibodies include mouse, rabbit, rat, chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments capable of binding antigens (including Fab, Fab', F(ab)2, Fv, single-chain antibodies, biantibodies), giant antibodies, nanobodies, and recombinant peptides containing the aforementioned CDRs (provided they exhibit the desired biological activity), or variants or derivatives thereof.

[0088] The modifier “monoclonal” indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). “Monoclonal antibodies” can also be isolated from phage antibody libraries using techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0089] The term "immunoglobulin" includes intact antibodies containing two dimerized heavy chains (HC), each covalently linked to a light chain (LC); a single undimerized immunoglobulin heavy chain and a covalently linked light chain (HC+LC); or a chimeric immunoglobulin (light chain + heavy chain)-Fc heterotrimer (the so-called "hemibody"). Immunoglobulins are proteins, but not necessarily antigen-binding proteins.

[0090] In an antibody, each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain of approximately 220 amino acids (approximately 25 kDa) and a "heavy" chain of approximately 440 amino acids (approximately 50–70 kDa). The amino-terminal portion of each chain includes a "variable" ("V") region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. The variable regions differ between different antibodies. The constant regions are the same between different antibodies. Within the variable region of each heavy or light chain, there are three hypervariable subregions, which help determine antibody specificity against antigens when the antibody is an antigen-binding protein. The variable domain residues between the hypervariable regions are called framework residues and are often homologous to some extent between different antibodies. Immunoglobulins can be classified into different categories based on the amino acid sequence of their heavy chain constant domain. Human light chains are classified as kappa (.κ.) and lambda (.λ.) light chains. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). "Antibody" also includes recombinant antibodies, as well as glycosylated or non-glycosylated antibodies.

[0091] The term "light chain" or "immunoglobulin light chain" includes the full-length light chain as well as segments thereof with sufficient variable region sequences to confer binding specificity. The full-length light chain includes a variable region domain, V... L and constant region structural domain, C L The variable domain of the light chain is located at the amino terminus of the polypeptide. The light chain includes the κ chain and the λ chain.

[0092] The term "heavy chain" or "immunoglobulin heavy chain" includes the full-length heavy chain as well as segments thereof with sufficient variable region sequences to confer binding specificity. The full-length heavy chain includes a variable region domain, V... H and three constant region structural domains, C H1 C H2 and C H3 V H The domain is located at the amino terminus of the polypeptide, and C H The domain is located at the carboxyl terminus, where C H3The heavy chain is closest to the carboxyl terminus of the polypeptide. Heavy chains are classified into μ, delta (δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain can be any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. Some of these can be further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Different IgG isotypes can have different effector functions (mediated by the Fc region), such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (Fc. gamma receptors) on the surface of immune effector cells (such as natural killer cells and macrophages), leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering a complement cascade on the cell surface.

[0093] The “Fc region,” or the “Fc domain” or “immunoglobulin Fc domain” used interchangeably in this article, contains two heavy chain segments, which, within the intact antibody, contain the antibody’s C… H1 and C H2 Structural domain. Two heavy chain segments are connected by two or more disulfide bonds and by C... H3 The hydrophobic interactions of the structural domains remain together.

[0094] The term "rescue receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0095] For a detailed description of the structure and production of antibodies, see Roth, DB and Craig, NL, Cell, 94:411-414 (1998), which are incorporated herein by reference in their entirety. In short, the process of generating DNA encoding heavy and light chain immunoglobulin sequences occurs primarily in developing B cells. Prior to rearrangement and linking of various immunoglobulin gene segments, V, D, J, and constant (C) gene segments are typically found relatively close together on a single chromosome. During B cell differentiation, a gene segment from the appropriate family member of each V, D, J gene segment (or only V and J in the case of light chain genes) is recombined to form variable regions for heavy and light chain functional rearrangements. This gene segment rearrangement process appears to be sequential. First, a heavy chain D-to-J linker is prepared, followed by a heavy chain V-to-DJ linker and a light chain V-to-J linker. Besides rearrangements of the V, D, and J segments, other diversity arises in the primary repertoire of immunoglobulin heavy and light chains through variable recombination at the sites of V and J segment connections in the light chain and D and J segment connections in the heavy chain. Such variations in the light chain typically occur within the last codon of the V gene segment and the first codon of the J segment. Similar imprecision in connections occurs at the sites of D and J segments. H On heavy chains of chromosomes between segments, and can extend up to 10 nucleotides. Furthermore, it can be found in D and J. H Between and V H Several nucleotides, not encoded by genomic DNA, are inserted between the D and N gene segments. The addition of these nucleotides is called N-region diversity. The net effect of this rearrangement in the variable region gene segment and the variable recombination that may occur during this connection is the production of a primary antibody library.

[0096] The term "hypervariable" region refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the complementarity-determining region (CDR) or CDR [i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region and residues 31-35 (HI), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 1991, Public Health Service, National Institutes of Health, Bethesda, MD.]. Even a single CDR can recognize and bind to an antigen, although with a lower affinity than the entire antigen-binding site containing all CDRs.

[0097] The alternative definitions of residues from the high-variable "ring" are described by Chothia et al., J.Mol.Biol.196:901-917 (1987) as residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region.

[0098] "Frame" or "FR" residues are those variable region residues other than hypervariable region residues.

[0099] An "antibody fragment" comprises a portion of a full-length antibody, preferably the antigen-binding region or variable region of the full-length antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (Zapata et al., Protein Eng., 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0100] Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment containing constant regions. The Fab fragment contains all variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fc fragment displays carbohydrates and is responsible for many antibody effector functions (such as complement binding and cell receptor binding), distinguishing one class of antibodies from another.

[0101] The F(ab')2 fragment produced by pepsin treatment contains antibody V. H and V L Two “single-chain Fv” or “scFv” antibody fragments containing domains, wherein these domains are present within a single polypeptide chain. The Fab fragment differs from the Fab' fragment in that it includes additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Preferably, the Fv polypeptide also contains V... H and V L The polypeptide linkers between the domains allow Fv to form the desired structures for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0102] A "Fab fragment" consists of a light chain and a heavy chain, along with a CH1 region and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0103] The “Fab” fragment contains a light chain and a portion of a heavy chain, the latter containing V. H Domain and C H1 Domains and C H1 and C H2 The regions between the structural domains allow interchain disulfide bonds to form between the two heavy chains of the two Fab' segments to form the F(ab')2 molecule.

[0104] The “F(ab')2 segment” contains two light chains and two heavy chains, with the heavy chains containing C. H1 and C H2 Part of the constant region between the structural domains allows for the formation of interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments, which are held together by disulfide bonds between the two heavy chains.

[0105] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. This region consists of a dimer of a tightly bound, non-covalently bound heavy chain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to form a V H V L The surface of the dimer defines the antigen-binding site. A single variable domain (or only half of the Fv containing three CDRs that are specific to the antigen) has the ability to recognize and bind the antigen, although its affinity is lower than that of the entire binding site.

[0106] A "single-chain antibody" is an Fv molecule in which the variable regions of the heavy and light chains are linked by flexible linkers to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patents Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference in their entirety.

[0107] The "single-chain Fv" or "scFv" antibody fragment contains the antibody's V. H and V L Domains, wherein these domains are present in a single polypeptide chain and optionally contain V H and V LThe peptide linker between the domains enables Fv to form the structure required for antigen binding (Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Nati. Acad. Sci. USA 85:5879-5883, 1988). The “Fd” fragment is composed of V H and C H1 Composition of structural domains.

[0108] The term "dual antibody" refers to a small antibody fragment with two antigen-binding sites, containing antibodies linked to the same polypeptide chain (V). H V L The light chain variable structural domain (V) in ) L The heavy chain variable structural domain (V) H By using a linker that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain on another strand, creating two antigen-binding sites. Biantibodies are described more comprehensively, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0109] "Domain antibodies" are immunoglobulin fragments containing only heavy chain variable regions or light chain variable regions. In some cases, two or more V... H The region is covalently linked to the peptide linker to generate a bivalent domain antibody. The two V's of the bivalent domain antibody... H The region can target the same or different antigens.

[0110] The term “antigen-binding protein” (ABP) includes aflibercept, or antibodies or antibody fragments as defined herein, as well as recombinant peptides or other compounds containing sequences derived from a CDR having the desired antigen-binding properties, such that they specifically bind to the target antigen.

[0111] Typically, when an antigen-binding protein exhibits a significantly higher affinity for a specific antigen compared to its affinity for other unrelated proteins under similar binding assay conditions, and is therefore able to distinguish that antigen, the antigen-binding protein (e.g., aflibercept, an antibody, or an antibody fragment) "specifically binds" to the target antigen. Typically, when the dissociation constant (K...)... D ) is 10 -8 When M is low or below, antigen-binding proteins are said to "specifically bind" to their target antigens. When K is low... D 10 -9 When M is lower, antigen-binding proteins specifically bind to antigens with "high affinity," and when K... D10 -10 When M or lower, it has "very high affinity".

[0112] An "antigen-binding region" or "antigen-binding site" refers to the portion of a protein that specifically binds to a particular antigen. For example, the portion of an antigen-binding protein containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen is called an "antigen-binding region." Antigen-binding regions typically include one or more "complementary binding regions" ("CDRs"). Some antigen-binding regions also include one or more "framework regions" ("FRs"). A "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. A "framework region" helps maintain the correct conformation of the CDR to facilitate binding between the antigen-binding region and the antigen. In conventional antibodies, the CDR is embedded within the framework of the heavy and light chain variable regions, which constitute the regions responsible for antigen binding and recognition. The variable region of an immunoglobulin antigen-binding protein contains at least three heavy or light chain CDRs, referencing above (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md; see also Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 877-883), within the frame region (designated frame regions 1-4, FR1, FR2, FR3 and FR4, Kabat et al., 1991, ibid.; see also Chothia and Lesk, 1987, ibid.).

[0113] The term "antigen" refers to a molecule or part of a molecule that can be bound by selective binders such as antigen-binding proteins (including, for example, aflibercept, or antibodies or immunofunctional fragments of antibodies) and can also be used in animals to produce antibodies that can bind to that antigen. An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0114] The term "epitope" is a portion of a molecule that is bound by an antigen-binding protein (e.g., aflibercept or an antibody). This term includes any determinant capable of specifically binding antigen-binding proteins, such as antibodies or T-cell receptors. Epitopes can be continuous or discontinuous (e.g., in a single-chain polypeptide, amino acid residues that are not sequentially connected in the polypeptide sequence but are bound by an antigen-binding protein in the context of the molecule). In some embodiments, epitopes can be mimics because they contain a three-dimensional structure that resembles an epitope used to generate an antigen-binding protein, but does not contain or only contains some amino acid residues found in an epitope used to generate an antigen-binding protein. In most cases, epitopes are present on proteins, but in some cases they may be present on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of molecules (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) and can have specific three-dimensional structural features and / or specific charge features. Typically, antibodies specific to a particular target antigen will preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0115] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) must be handled using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described below: Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined using standard methods commonly used to compare the similarity of amino acid positions of two polypeptides. Using computer programs such as BLAST or FASTA, two polypeptide or polynucleotide sequences are aligned to achieve optimal residue matching (either along the full length of one or both sequences, or along predetermined portions of one or both sequences). The programs provide default open and default empty penalties, as well as scoring matrices such as PAM 250 [standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978)] which can be used in conjunction with the computer program.For example, the identity percentage can be calculated as: the total number of identical matches multiplied by 100, then divided by the sum of the length of the longer sequence within the matching range and the number of gaps introduced to align the two sequences. When calculating the identity percentage, the sequences compared result in the maximum possible match between them.

[0116] The GCG package is a computer program used to determine the percentage of identity. This package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two peptides or two polynucleotides to determine the percentage of sequence identity. Sequences are aligned to achieve the best possible match of their respective amino acids or nucleotides (e.g., a “match span” determined by the algorithm). A vacancy opening penalty (calculated as 3 times the diagonal mean, where the “diagonal mean” is the average of the diagonals of the comparison matrix used; the “diagonal” is a fraction or number assigned to each perfect amino acid match by a specific comparison matrix) and a vacancy extension penalty (typically 1 / 10 of the vacancy opening penalty), along with comparison matrices such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In some implementations, the algorithm also uses a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5: 345-352 for PAM250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 for the BLOSUM 62 comparison matrix).

[0117] Recommended parameters for determining the percentage of identity of a peptide or nucleotide sequence using the GAP procedure include the following:

[0118] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48: 443-453;

[0119] Comparison matrix: BLOSUM 62, from Henikoff et al., 1992, ibid.;

[0120] Open shot penalty: 12 (but no penalty for open shots at the end of the game)

[0121] Open space length penalty: 4

[0122] Similarity threshold: 0.

[0123] Some alignment schemes used to align two amino acid sequences can produce short region matches between only two sequences, and this smaller alignment region can have very high sequence identity even if there is no obvious relationship between the two full-length sequences. Therefore, if needed, the selected alignment method (GAP procedure) can be modified to produce alignments of at least 50 consecutive amino acids across the target peptide.

[0124] When used in conjunction with a target protein, the term "modification" includes, but is not limited to, changes in one or more amino acids (including substitution, insertion, or deletion); chemical modification; covalent modification by conjugation of therapeutic or diagnostic agents; labeling (e.g., with radionuclides or various enzymes); covalent polymer attachment such as PEGylation (derivative with polyethylene glycol); and insertion or substitution of non-natural amino acids by chemical synthesis. Proteins can be "engineered" or modified to improve target affinity, selectivity, stability, and / or manufacturability before the coding sequence of the "engineered" protein is included in an expression cassette, using methods known to those skilled in the art.

[0125] When used in conjunction with target proteins (such as aflibercept or antibodies), the term "derivative" refers to a covalently modified protein that is inserted into or substituted by conjugation to a therapeutic or diagnostic agent, labeling (e.g., with a radionuclide or various enzymes), covalent polymer attachment such as PEGylation (with polyethylene glycol derivatization), or chemical synthesis of non-natural amino acids.

[0126] Within the scope of this invention, aflibercept protein can be a therapeutic protein or "biological agent" used to treat diseases, including but not limited to human diseases or conditions, such as eye diseases or conditions. "Treatment" or "treating" is an intervention undertaken to prevent the development of a disease or to alter its pathology. Therefore, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those requiring treatment include those already suffering from a condition and those requiring prevention of a condition. "Treatment" includes any indication of successful improvement in an injury, pathology, or condition, including any objective or subjective parameters such as symptom reduction; relief; weakening or making the injury, pathology, or condition more tolerable for the patient; slowing the rate of degeneration or decline; making the endpoint of degeneration less debilitating; and improving the patient's physical and mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of a physical examination performed by a physician, such as an ophthalmologist or other healthcare provider, or self-reported by the patient.

[0127] An "effective amount" of a therapeutic agent is generally sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or repair damage caused by or associated with an eye condition or disease. In some embodiments, the effective amount is a therapeutically effective amount or a preventatively effective amount. "Therapeutic effective dose" is an amount sufficient to treat a disease state (e.g., macular edema following central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular (wet)-related macular degeneration (AMD), visual impairment due to myopic choroidal neovascularization, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascular age-related macular degeneration (AMD), transplant rejection or GVHD, inflammation, multiple sclerosis, cancer, cardiovascular disease, diabetes, neuropathy, pain) or symptoms, particularly those related to the disease state, or otherwise prevents, inhibits, delays, or reverses the progression of the disease state or any other adverse symptoms related to the disease (i.e., provides "therapeutic efficacy"). "Prophylactic effective dose" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended preventive effect. A complete therapeutic or preventive effect does not necessarily occur with a single dose and may occur after a series of doses. Therefore, therapeutic or preventive effective doses may be administered in a single or multiple-dose manner.

[0128] Cloned DNA

[0129] DNA cloning is performed using standard techniques (see, for example, Sambrook et al. (1989), Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, which is incorporated herein by reference). For instance, a cDNA library can be constructed by reverse transcription of polyA+ mRNA, preferably membrane-associated mRNA, and screened using probes specific to human immunoglobulin polypeptide gene sequences. However, in one implementation, polymerase chain reaction (PCR) is used to amplify cDNA (or a portion of full-length cDNA) encoding a fragment of the target immunoglobulin gene (e.g., a variable fragment of the light or heavy chain). The amplified sequence can be readily cloned into any suitable vector, such as an expression vector, a minigene vector, or a phage display vector. It should be understood that the specific cloning method used is not important, as long as the sequence of certain portions of the target polypeptide (e.g., the aflibercept fusion polypeptide sequence) can be determined.

[0130] One source of antibody nucleic acids is a hybridoma produced by obtaining B cells from an animal immunized with the target antigen and fusing the B cells into immortalized cells. Alternatively, nucleic acids can be isolated from the B cells (or the entire spleen) of an immunized animal. Another source of nucleic acids encoding antibodies is a library of such nucleic acids, for example, produced via phage display technology. Polynucleotides encoding target peptides, such as variable region peptides with the desired binding characteristics, can be identified using standard techniques such as panning.

[0131] DNA sequencing was performed using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which are incorporated herein by reference). By comparing the sequence of the cloned nucleic acid with publicly available gene and cDNA sequences, technicians will be able to readily identify the region based on the sequencing data. One source of gene sequence information is the National Center for Biotechnology Information, the National Library of Medicine, and the National Institutes of Health (Bethesda, MD).

[0132] The isolated DNA can be operatively ligated to a control sequence or placed in an expression vector, which is then transfected into host cells that do not produce immunoglobulins to direct the synthesis of monoclonal antibodies in recombinant host cells. Recombinant antibody production is well known in the art.

[0133] Operable ligation occurs when a nucleic acid is functionally related to another nucleic acid sequence. For example, if it is expressed as a pre-protein involved in polypeptide secretion, the DNA for the pre-sequence or secretory leader is operably ligated to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably ligated to the coding sequence; or if a ribosome binding site is located to facilitate translation, the ribosome binding site is operably ligated to the coding sequence. Generally, operable ligation means that the ligated DNA sequences are contiguous, and in the case of a secretory leader, they are contiguous and in the reading phase. However, enhancers do not need to be contiguous. Ligation is accomplished by ligating at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to standard practice.

[0134] Many vectors are known in the art. Vector components may include one or more of the following: signal sequences (e.g., that can direct the secretion of expressed proteins); origin of replication; one or more selective marker genes (e.g., that can confer resistance to antibiotics or other drugs, complement auxotrophy, or provide key nutrients not available in the culture medium); enhancer elements; promoters; and transcription termination sequences, all of which are well known in the art.

[0135] Purity of water and other components The water and all other ingredients used in the preparation of the ophthalmic formulations of the present invention preferably meet the applicable legal or pharmacopoeia standards required for such pharmaceutical compositions and drugs in the target jurisdiction, such as the United States Pharmacopeia (USP), the European Pharmacopeia, the Japanese Pharmacopeia, or the Chinese Pharmacopeia. For example, according to the USP, water for injection is used as an excipient in the production of parenteral preparations and other formulations where the endotoxin content of the product must be controlled; and in other pharmaceutical applications, such as the cleaning of certain equipment and parenteral product-contact parts; the minimum quality of the water source or supply used to produce water for injection is drinking water, as defined by the U.S. Environmental Protection Agency (EPA), the EU, Japan, or the WHO.

[0136] Before being administered to a patient, the formulations of the present invention shall meet the applicable legal or pharmacopoeia standards for such pharmaceutical compositions and drugs in the target jurisdiction regarding sterility, absence of endotoxins or viral contaminants, etc.

[0137] Buffer system

[0138] The ophthalmic formulations of the present invention comprise buffers in the concentration range of about 5 to 50 mM. Suitable buffer systems for use in the ophthalmic formulations of the present invention may be selected from phosphate buffers, histidine buffers, acetate buffers, succinate buffers, citrate buffers, glutamate buffers, and lactate buffers, or the buffer may be a combination of two or more of these buffer systems. Some useful embodiments of the present invention have buffer concentrations of about 5 mM to about 20 mM, and other embodiments have buffer concentrations of about 5 to about 10 mM. If a histidine buffer is selected, a histidine concentration of about 5-20 mM is preferred.

[0139] Nonionic surfactants

[0140] The ophthalmic formulation of the present invention comprises a nonionic surfactant, preferably at a concentration of about 0.001% (w / v) to about 5.0% (w / v). In some embodiments, the concentration of the nonionic surfactant is about 0.001% (w / v) to about 2.0% (w / v), or about 0.001% (w / v) to about 1.0% (w / v), or about 0.001% (w / v) to about 0.10% (w / v), or about 0.001% (w / v) to about 0.01% (w / v). A useful nonionic surfactant may be a polysorbate (e.g., polysorbate 20 or polysorbate 80). 35 (i.e., polyethylene glycol dodecyl ether), poloxamer (i.e., polyethylene glycol-polypropylene glycol; polyoxyethylene-polyoxypropylene block copolymer; poly(ethylene oxide-co-polyoxypropylene)), such as poloxamer 188 (i.e., Pluronic F68), or Triton TM X-100 (i.e., 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol)). For the purposes of carrying out this invention, it also includes, in the "nonionic surfactant" an alkyl sugar or alkyl glycoside (e.g., by Aegis Therapeutics, LLC under the trade name...). Sales; see, for example, Maggio, Stabilizing Alkylglycoside Compositions and Methods Thereof, US 8,133,863 B2).

[0141] Tension regulator

[0142] The ophthalmic formulation of the present invention includes a tension modifier, such that the formulation has a final molal osmotic pressure concentration of about 300 mOsm / kg (i.e., 300 ± 50 mOsm / kg), and a chloride anion concentration of less than about 10 mM, preferably less than about 5 mM, and more preferably less than about 1 mM. Molal osmotic pressure concentration is a measure of the number of dissolved particles per unit volume of water. In a solution, the fewer the number of solute particles proportional to the number of units of water (solvent), the lower the solution concentration and the lower the permeability. If a semipermeable membrane (a membrane that allows only solvent molecules to pass through) is used to separate solutions of different solute concentrations, a phenomenon called osmosis occurs, in which solvent molecules cross the membrane from a lower concentration to a higher concentration to establish a concentration equilibrium. The pressure driving this movement is called osmotic pressure and is determined by the number of solute “particles” in the solution. Solutions containing the same concentration of particles and therefore exerting equal osmotic pressures are called isotonic. For example, the osmotic pressure inside a red blood cell (rbc) is equal to that of the surrounding solution, preventing it from contracting or expanding. If RBC is placed in water, it will break down because water itself is hypotonic. If RBC is placed in a high-salt solution, i.e., greater than 0.9% (w / v) sodium chloride, it will shrink because the solution is hypertonic. In both cases, the RBC is damaged. The same thing can happen to any biological cell, such as those in the eye. Applying hypotonic or hypertonic solutions to the eye will cause damage, thus necessitating the use of isotonic solutions for medications intended for the eye. In practice, a 0.9% (w / v) sodium chloride solution is isotonic and has a concentration of 270-300 mOsm / kg. All solutions are compared to this standard, and those falling within the extended range of 250-350 mOsm / kg are considered isotonic. Excipients used to stabilize proteins are added at concentrations necessary to produce isotonic solutions. For example, disaccharides such as sucrose and trehalose are isotonic at a concentration of 9.25%, monosaccharides such as glucose and mannose are isotonic at a concentration of 5%, and amino acids such as proline are isotonic at a concentration of approximately 3%. The weight-molar osmotic concentration can be determined theoretically or experimentally. Theoretical calculations can be performed based on the following equation:

[0143] Weight molality osmotic pressure concentration = (g compound / 100mL solution) * (E value of compound).

[0144] The E value of a compound is determined by the following equation:

[0145] E value = (MW NaCl / i value NaCl) * (i value compound / MW compound).

[0146] The i-value is the number of ions in a compound with an 80% theoretical dissociation rate. For a non-dissociating compound, i.e., sucrose, the i-value is 1. For a compound that dissociates into two ions, i.e., NaCl, the i-value is 1.8, and for a compound that dissociates into three ions, the i-value is 2.6. Since molality is a colligative property of a solution, the freezing point depression or vapor pressure reduction due to the addition of a solute is directly related to the total number of solute molecules in the liquid. Each of these principles has been utilized in the art to develop useful instruments for measuring molality. Any one or two types of instruments can be used for biological samples. For example, a solution containing 10 mM sodium phosphate, 40 mM NaCl, 5% (w / v) sucrose, and 0.03% (w / v) polysorbate 20 has a theoretical molality of 263 mOsm / kg. In our laboratory, the actual measured value, as measured by freezing point depression, is 270 mOsm / kg. This demonstrates that the experimental values ​​closely match the theoretical values, further proving that, for the purposes of this invention, theoretical or experimental values ​​can be used to determine whether a solution is suitable for intravitreal injection based on its weight molar osmotic pressure concentration.

[0147] Useful tension modifiers can be polyols or amino acids. Examples of useful polyol tension modifiers include sucrose, trehalose, sorbitol, mannitol, and glycerol. Typically, polyol tension modifiers are present in concentrations ranging from 5-10% (w / v), depending on buffer concentrations and other formulation excipients. Amino acid tension modifiers are present in concentrations ranging from 2-4% (w / v), depending on buffer concentrations and other tension modifiers used. In the ophthalmic formulations of this invention, amino acid tension modifiers may be in L-amino acid and / or D-amino acid forms, if pharmaceutically acceptable. The term "amino acid tension modifier" also includes pharmaceutically acceptable amino acid salt forms.

[0148] Other stabilizers

[0149] In some embodiments of the ophthalmic formulation of the present invention, the tension modifier is a polyol, such as sucrose, trehalose, sorbitol, mannitol, or glycerol, and the formulation also contains additional amino acid stabilizers. These additional amino acid stabilizers may be, for example, proline, arginine, methionine, glycine, or lysine. The additional amino acid stabilizers may be L-amino acids or D-amino acids, or in salt form, provided that the amino acid is pharmaceutically acceptable and in a pharmaceutically acceptable form, such as a pharmaceutically acceptable salt form. (Reference, for example, Falconer et al., Stabilization of amonoclonal antibody during purification and formulation by addition of basicamino acid excipients, J Chem Technol Biotechnol (2011) 86:942-948; Platts et al., Control of Globular Protein Thermal Stability in Aqueous Formulations by the Positively Charged Amino Acid Excipients, Journal of Pharmaceutical Sciences105(2016)3532-3536;Wang,W.,Instability,stabilization,and formulation ofliquid protein pharmaceuticals,International Journal of Pharmaceutics 185(1999)129-188;Yin et al.,Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-StimulatingFactor and Human Parathyroid Hormone Fragment 13-34,Pharmaceutical Research (2004) 21(12): 2377-2383; Lam et al.,用于预防重组单克隆抗体HER2中甲硫氨酸氧化的抗氧化剂,《药物科学杂志》86(11):1250 - 1255(1997);Levine等人,蛋白质中甲硫氨酸残基作为内源性抗氧化剂,《美国国家科学院院刊》93(26):15036 - 15040(1996);Maeder等人,一种用于皮下给药的新型20%脯氨酸稳定化多克隆免疫球蛋白长达24个月的局部耐受性和稳定性,《生物制品》39:43 - 49(2011);Cramer等人,一种新型液体10%多克隆免疫球蛋白产品(IgProlO)超过36个月的稳定性。 Stabilized with L-proline, Vox Sanguinis (2009) 96, 219-225; Bolli et al., L-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (WIG) solutions, Biologicals 38 (2010) 150-157; Truong, Combination of D-Amino Acids and Lipoteichoic Acid, EP2545909 A1; Stroppolo et al., Pharmaceutical compositions containing the salts of S(+)-2-(4-isobutylphenyl)propionic acid with basic amino acids, US5510385). The concentration of additional amino acid stabilizers in combination with polyols is usefully about 0.01-3% (w / v), depending on the concentration of the polyol in combination with the amino acids. However, if methionine is combined with a polyol tension modifier, methionine can be used as a scavenger of reactive oxygen species at low concentrations (i.e., 10 mM or lower).

[0150] Exemplary formulations of the present invention

[0151] Exemplary ophthalmic formulations of the present invention include those in which the buffer is a phosphate buffer. In one such embodiment, (a) the aflibercept concentration is 20-80 mg / mL; (b) the phosphate buffer concentration is about 10 mM; (c) the nonionic surfactant is polysorbate 20 at a concentration of about 0.03% (w / v); and (d) the tension modifier is:

[0152] (i) sucrose or trehalose at a concentration of about 9% (w / v), or

[0153] (ii) a concentration of about 3% (w / v) proline; (e) a concentration of chloride anion less than about 1 mM; and the pH of the formulation is about pH 6.0 to about pH 6.5. In some preferred embodiments of the formulation, the tension modifier is about 9% (w / v) sucrose or trehalose, having an aflibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL. In other preferred embodiments, the tension modifier is about 3% (w / v) proline, having an aflibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL.

[0154] Exemplary ophthalmic formulations of the present invention further include those in which the buffer is a histidine buffer at a concentration of 5-20 mM. In one such embodiment, (a) the concentration of aflibercept is 20-80 mg / mL; (b) the concentration of the histidine buffer is about 10 mM; (c) the nonionic surfactant is polysorbate 20 at a concentration of about 0.03% (w / v); and (d) the tension modifier is:

[0155] (i) sucrose or trehalose at a concentration of about 9% (w / v), or

[0156] (ii) a concentration of about 3% (w / v) of proline; (e) a concentration of chloride anion less than about 10 mM, or more preferably less than about 5 mM; and the pH of the formulation is from about pH 5.5 to about pH 6.5, or in some embodiments from about pH 6.0 to about pH 6.5. In some preferred embodiments of the formulation, the tension modifier is sucrose or trehalose at a concentration of about 9% (w / v), having an aflibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL. In other preferred embodiments, the tension modifier is proline at a concentration of about 3% (w / v), having an aflibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL.

[0157] Other exemplary ophthalmic formulations of the present invention include those in which the buffer is an acetate buffer. In one such embodiment, (a) the concentration of aflibercept is 20-80 mg / mL; (b) the acetate buffer is about 10 mM; (c) the nonionic surfactant is polysorbate 20, or polysorbate 80, or poloxamer, such as poloxamer 188, at a concentration of about 0.01% (w / v), about 0.03% (w / v), about 0.1% (w / v), or about 1% (w / v); and (d) the tension modifier is:

[0158] (i) sucrose or trehalose at a concentration of about 9% (w / v), or

[0159] (ii) a concentration of about 3% (w / v) proline; (e) a concentration of chloride anion less than about 1 mM; and the pH of the formulation is from about pH 5.0 to about pH 5.5. In some preferred embodiments of the formulation, the tension modifier is sucrose or trehalose at a concentration of about 9% (w / v) and has a flibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL. In other preferred embodiments, the tension modifier is proline at a concentration of about 3% (w / v) and has a flibercept concentration of about 30 mg / mL to about 50 mg / mL; for example, a concentration of about 40 mg / mL.

[0160] For further explanation, the present invention includes embodiments numbered as follows:

[0161] Implementation method 1: An ophthalmic preparation comprising:

[0162] (a) Aflibercept, at a concentration of 5-100 mg / mL;

[0163] (b) Buffers with a concentration of 5-50 mM;

[0164] (c) Nonionic surfactants;

[0165] (d) A tension modifier selected from the group consisting of polyols and amino acids, wherein the formulation has a final weight molar osmolality concentration of approximately 300 mOsm / kg, and

[0166] (e) wherein the concentration of chloride anions is less than about 10 mM; and

[0167] The pH of the formulation is approximately pH 5.0 to approximately pH 6.5.

[0168] Implementation Method 2: The ophthalmic preparation of Implementation Method 1, wherein the concentration of chloride anion is less than about 5 mM.

[0169] Implementation Method 3: The ophthalmic preparations of Implementation Methods 1-2, wherein the concentration of chloride anions is less than about 1 mM.

[0170] Implementation method 4: Ophthalmic preparations of implementation methods 1-3, wherein the buffer is a phosphate buffer.

[0171] Implementation Method 5: Ophthalmic preparations of Implementation Methods 1-3, wherein the buffer is a histidine buffer with a concentration of 5-20 mM.

[0172] Implementation method 6: Ophthalmic preparations of implementation methods 1-3, wherein the buffer is an acetate buffer.

[0173] Implementation Method 7: Ophthalmic preparations of Implementation Methods 1-3, wherein the buffer is selected from phosphates, histidines, acetates, succinates, citrates, glutamates, and lactates; or combinations of two or more of these.

[0174] Implementation method 8: Ophthalmic preparations of implementation methods 1-7, wherein the buffer concentration is 5-20 mM.

[0175] Embodiment 9: Ophthalmic preparations of Embodiments 1-8, wherein the nonionic surfactant is selected from the group consisting of: polysorbate (e.g., polysorbate 20 or polysorbate 80), polyethylene glycol dodecyl ether (i.e., ... 35) Poloxamer (e.g., Poloxamer 188), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (i.e., Triton) TM X-100), alkyl sugars and alkyl glycosides.

[0176] Implementation method 10: Ophthalmic preparations of implementation methods 1-9, wherein the nonionic surfactant is poloxamer 188.

[0177] Implementation Method 11: Ophthalmic preparations of Implementation Methods 1-10, wherein the tension modifier is a polyol selected from sucrose, trehalose, sorbitol, mannitol and glycerol.

[0178] Implementation method 12: Ophthalmic preparations of implementation methods 1-11, wherein the tension modulator is sucrose.

[0179] Implementation method 13: Ophthalmic preparations of implementation methods 1-11, wherein the tension modulator is trehalose.

[0180] Implementation method 14: The ophthalmic preparation of implementation method 11 further includes an additional amino acid stabilizer.

[0181] Implementation Method 15: The ophthalmic preparation of Implementation Method 14, wherein the additional amino acid stabilizer is selected from the group consisting of: proline, arginine, methionine, glycine and lysine.

[0182] Implementation Method 16: Ophthalmic preparations of Implementation Methods 1-15, wherein the tension modulator is an amino acid selected from proline, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine and lysine.

[0183] Implementation method 17: Ophthalmic preparations of implementation methods 1-16, wherein the tension modulator is proline.

[0184] Implementation method 18: The ophthalmic preparation of implementation method 4, wherein:

[0185] (a) The concentration of aflibercept is 20-80 mg / mL;

[0186] (b) The concentration of the phosphate buffer is approximately 10 mM.

[0187] (c) The nonionic surfactant is polysorbate or poloxamer.

[0188] (d) The tension modifier is (i) sucrose or trehalose at a concentration of about 9% (w / v) or (ii) proline at a concentration of about 3% (w / v);

[0189] (e) The concentration of chloride anions is less than about 1 mM;

[0190] Furthermore, the pH of ophthalmic preparations is approximately pH 6.0 to approximately pH 6.5.

[0191] Implementation method 19: The ophthalmic preparation of implementation method 18, wherein the tension modulator is sucrose or trehalose at a concentration of about 9% (w / v).

[0192] Implementation Method 20: The ophthalmic preparation of Implementation Method 18, wherein the tension modulator is proline at a concentration of about 3% (w / v).

[0193] Implementation Method 21: The ophthalmic preparation of Implementation Method 5, wherein:

[0194] (a) The concentration of aflibercept is 20-80 mg / mL;

[0195] (b) The histidine buffer is approximately 10 mM;

[0196] (c) The nonionic surfactant is polysorbate or poloxamer;

[0197] (d) The tension modifier is (i) trehalose at a concentration of about 9% (w / v) or (ii) proline at a concentration of about 3% (w / v);

[0198] Furthermore, the pH of ophthalmic preparations is approximately pH 5.5 to approximately pH 6.5.

[0199] Implementation Method 22: The ophthalmic preparation of Implementation Method 21, wherein the tension modulator is trehalose at a concentration of about 9% (w / v).

[0200] Implementation Method 23: The ophthalmic preparation of Implementation Method 21, wherein the tension modulator concentration is about 3% (w / v) of proline.

[0201] Implementation Method 24: The ophthalmic preparation of Implementation Method 6, wherein:

[0202] (a) Aflibercept concentration is 20-80 mg / mL;

[0203] (b) The acetate buffer is approximately 10 mM;

[0204] (c) The nonionic surfactant is polysorbate or poloxamer;

[0205] (d) The tension modifier is (i) sucrose or trehalose at a concentration of about 9% (w / v) or (ii) proline at a concentration of about 3% (w / v);

[0206] (e) The concentration of chloride anions is less than about 1 mM;

[0207] Furthermore, the pH of ophthalmic preparations is approximately pH 5.0 to approximately pH 5.5.

[0208] Implementation Method 25: Use of any of the formulations of Implementation Methods 1-24 for the treatment of eye conditions or diseases.

[0209] Implementation 26: Use of Implementation 25, wherein the ocular condition or disease is selected from macular edema following retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular (wet) age-related macular degeneration (AMD), visual impairment due to myopic choroidal neovascularization, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascular age-related macular degeneration (AMD).

[0210] Implementation Method 27: Use of Implementation Methods 25-26, wherein the ophthalmic preparation is administered to a patient suffering from an eye condition or disease via intravitreal injection.

[0211] The following working examples are illustrative and should not be construed in any way as limiting the scope of the invention.

[0212] Example

[0213] Example 1. Stability Study

[0214] Material A VEGF-specific aflibercept fusion protein antagonist was produced using industry-standard recombinant expression technology and purification methods. The purified drug was buffer-exchanged with specific formulation buffers using a Millipore Corporation laboratory-scale tangential flow filtration system. The protein concentration was adjusted to the final target concentration by dilution with the formulation buffers. Water used for preparing all formulations was... Millipore Corporation's water purification system includes an ion exchange column. Water purity is monitored by measuring conductivity, with a value greater than 18.2 MΩcm⁻¹. It is acceptable. All excipients, buffers, and other components used to prepare the formulation buffer are USP grade or equivalent.

[0215] method

[0216] Titration An acid-base conjugate prepared at equimolar concentrations was mixed together in an appropriate molar ratio to obtain the desired formulation pH of the histidine-phosphate buffer system. An acetate formulation was prepared by adding glacial acetic acid to Milli-Q purified water and then titrating with sodium hydroxide to achieve the desired final pH.

[0217] Appearance Qualitative visual appearance testing was conducted to assess protein particles or environmental contaminants in the pharmaceutical product. Aliquots (1.1 mL) of the protein sample were placed in pre-sterilized Type I glass vials and sealed with a 13 mm suffix. Under ambient light, the samples were gently rotated and visually inspected for 5 seconds to detect visible particles. For all tested samples, the number and description of any detected particles were recorded.

[0218] color Qualitative visual color assessment was performed to monitor the color of the pharmaceutical product during its stabilization period. Commercially available EP 2.2.2 tan standards (BY1-BY7) from Ricca Chemicals were aliquoted into pre-sterilized Type 1 glass vials. Aliquots (1.1 mL) of the pharmaceutical product were placed in each vial and compared with an equal volume of each tan standard to determine the coloring level. All samples were tested against a white background, and the coloring level of each sample was recorded.

[0219] Subvisible particle analysis with reduced volumetric optical obstruction Sub-visible particles were measured using a HIAC counter equipped with an HRLD-150 sensor (Beckman Corporation) via photoobscuration. The sensor was calibrated using polystyrene beads in the 1–100 μm range. Prior to analysis, the system was flushed through with MilliQ water until a clean baseline was achieved, and the system's suitability was confirmed using a standard solution of 15 μm polystyrene beads. Replicated samples were combined in glass vials to a final volume of 1.1 mL and degassed under vacuum at 75 Torr for 2 hours. For each sample, the instrument was set to aspirate five 0.2 mL aliquots and measure particles larger than 10 μm and 25 μm. The average of the last three measurements is reported.

[0220] Size exclusion chromatographySize exclusion high-performance liquid chromatography (SE-FIPLC) was performed using a Waters XBridge Protein BEH SEC 200A column. Separation was performed under natural conditions using phosphate and sodium chloride run buffers. Peak elution was detected by UV absorbance, and the consolidated purity results are reported as the percentage of peak area relative to the total corrected area for higher molecular weight (HMW) fractions, major fractions (monomers), and lower molecular weight (LMW) fractions.

[0221] CZE chloride ion analysis Chloride ion analysis using the Microsolv Technologies CElixirOA pH 5.4 kit was performed on a Beckman PA 800 capillary electrophoresis system. Samples and standards were prepared according to the manufacturer's instructions. Injection was performed for 10 seconds at 1 psi in a bare molten capillary with an effective length of 50.2 cm. Samples were monitored using a photodiode array detector (PDA). Analysis of the standard curve was used to quantify the concentration of chloride anions in each sample.

[0222] Reducing capillary electrophoresis-sodium dodecyl sulfate (rCE SDS) Protein samples were denatured by heating in SDS at 70°C for 10 minutes and then reduced with β-mercaptoethanol. The samples were then electro-injected into 30.2 cm bare fused silica capillaries filled with SDS gel buffer. A voltage of -15 kV was applied through the capillaries, which separated the substances by their size differences. Proteins were detected using a photodiode array detector. Purity was assessed by determining the percentage of peak area for each component.

[0223] Non-reducing capillary electrophoresis-sodium dodecyl sulfate (nrCE SDS) The sample was denatured at low pH by heating at 60°C for 5 minutes in the presence of SDS and N-ethylmaleimide (NEM). The resulting negatively charged SDS-protein complex was electro-injected into a 30.2 cm bare fused silica capillary filled with SDS gel buffer. A voltage of -15 kV was applied through the capillary, and the substances were separated by their size differences. The protein substances were detected by a photodiode array (PDA) detector as the protein passed through the detection window. Purity was assessed by determining the percentage of peak area for each component.

[0224] Capillary isoelectric focusingProteins are separated using capillary isoelectric focusing (cIEF) based on differences in isoelectric point (pI). A neutral-coated capillary is filled with an amphoteric solution, and a voltage is applied to focus the amphoteric substances into a pH gradient. Proteins are focused onto a portion of this pH gradient, where the pH equals their isoelectric point. The proteins are chemically unfixed and are detected by UV absorbance (280 nm) as they pass through the detection window. Purity is assessed by determining the percentage of peak area for each component.

[0225] Effectiveness assessment The potency of aflibercept was evaluated in a cell-based VEGF-A165-dependent proliferation assay. Human umbilical vein endothelial cells were seeded in 96-well plates containing different concentrations of the drug product and 100 ng / mL VEGF-A165 in the absence of growth factors. The assay plates were incubated at 37°C and 5% CO2 for approximately 3 days, followed by the addition of a fluorescence activity reagent. Dose-response curves were generated by plotting the relationship between drug product concentration and fluorescence, and then fitting the results using a 4-parameter equation. Relative potency was measured by evaluating the offset along the x-axis between the test sample and a reference standard.

[0226] Measurement of osmotic concentration by weight molar The weight-molar osmolality of the samples was measured using an Advanced Instruments, Inc. freezing point osmoremeter. Prior to sample analysis, a concentration of 290 mOsm / kg Clinitrol was used. TM The instrument calibration was checked using a 290 reference solution (Fisher Scientific). 20 μL of isomeric sample from the sample was transferred to a sample tube and placed in the instrument for freezing point analysis. Each sample was analyzed in triplicate, and the results were reported as the average of these values.

[0227] Example 2. Stability Study

[0228] Stability analysis of aflibercept was performed to understand the importance of chloride ions, pH, buffer, and stabilizer type to molecular stability. Table 1 summarizes the composition of each aflibercept (40 mg / mL) formulation and the abbreviations (i.e., formulation numbers) used in the results in Example 2 and Figures 1-10 of this paper. The amino acids used were L-isomers.

[0229] Table 1 lists the formulations studied. The concentration of aflibercept was 40 mg / mL.

[0230] Table 1

[0231]

[0232]

[0233] Chloride ion detection Chloride levels were measured by capillary region electrophoresis. Formulation buffers and formulated drug products were tested to determine the chloride levels present in each sample. Drug product samples formulated with sodium chloride or histidine were expected to have chloride levels present in both the buffer and drug product samples. As shown in Table 2, the formulation buffers with added sodium chloride had the expected chloride levels, while the histidine formulations without added sodium chloride showed low chloride levels, a result attributed to the histidine monohydrochloride used during buffer preparation. All formulated drug product samples showed slightly higher chloride levels than the corresponding buffer samples. This result may be due to residual chloride from product purification.

[0234] Table 2 shows the results of capillary region electrophoresis of chloride ions from formulation buffer and prepared aflibercept drug product samples. Compared with formulation buffer alone, residual chloride ions from the purification process resulted in a slight increase in chloride ion levels in the drug product samples.

[0235] Table 2

[0236]

[0237] osmotic concentration by weight The gravimetric osmolality of the drug product solutions was measured to ensure that co-concentration or exclusion of formulation components during drug product preparation did not result in unacceptable gravimetric osmolality levels for intravitreal injection. The gravimetric osmolality results for the eight drug product formulations evaluated in this study showed gravimetric osmolality levels within the acceptable range of 250–350 mOsm / kg. These results are summarized in Table 3.

[0238] Table 3 shows the weight molality of eight aflibercept formulation samples measured by a freezing point osmoremeter, indicating that all samples were within acceptable ranges.

[0239] Table 3

[0240] Formulation No. Weight Molar Osmotic Concentration (mOsm / kg) 1 2 5 8 2 3 2 6 3 2 7 7 4 3 0 2 5 3 2 0 6 2 9 7 7 3 3 0 8 3 3 2 surface

[0241] Visual assessment: inspection of visible particles and color Visual assessments of the drug product samples were performed to determine the presence or absence of environmental or product-related particles and to evaluate product color during stability studies. Visual particle counts were examined for stability samples stored at 4°C for 7 weeks and at 30°C for 4 weeks, and all samples passed the assessment.

[0242] The color of aflibercept drug product stability samples was examined by comparing the formulated protein with the commercially available amber-yellow pharmacopoeia color standard (EP 2.2.2). The results of this assessment determined that the color of the drug product did not change during storage, regardless of formulation or temperature.

[0243] Subvisible particle test: HIAC: Subvisible particle analysis was performed on samples stable at 4 °C. Figures 1 and 2 show the results for 10 μm and 25 μm particle sizes, respectively. The error bars in Figures 1 and 2 represent the standard deviation calculated from the three readings. The measured subvisible particle levels are significantly lower than those in the USP monograph. <789> The levels specified in {Particulate Matter In Ophthalmic Solutions} were as follows. Except for formulation 7 (which had a sub-visible particle level that increased over time), ten (10) μm particles were observed for all formulations stored at 4 °C. When stored at 4 °C, the 25 μm particle level shown in Figure 2 was undetectable or at a very low level. It should be noted that the error bars for the 25 μm results are larger than the reported particle number, and therefore no trend can be derived from these early time-point data.

[0244] SE-HPLC Size exclusion high-performance liquid chromatography (SE-HPLC) analysis was used to assess the levels of high molecular weight substances (HMW) in the stable samples throughout the study period. SE-HPLC was performed at all time points for all temperature conditions. Results from storage conditions at 4 °C and 30 °C are shown in Figures 3 and 4, respectively. After 7 weeks of storage at 4 °C, formulations 1-7 had HMW levels that were difficult to distinguish from each other, with equal pH values, but differences in buffer type, tension modifier, and the presence of sodium chloride. Surprisingly, formulation 8, with a significantly lower pH than the other formulations, also had significantly reduced aggregate levels. The trend observed at 4 °C was comparable to the trend detected after 4 weeks of storage at 30 °C.

[0245] HMW data measured by SE-HPLC suggest that sodium chloride is not necessary for stabilizing aflibercept during long-term storage for any of the formulations studied, which is surprisingly contrary to the teachings of Dix et al. (Refer to US 8,921,316, column 6, lines 62-65; stating that "although either NaCl or sucrose can be used as a stabilizer, the atomization of NaCl and sucrose has been established to stabilize the fusion protein more effectively than either individual stabilizer alone," which contradicts our experimental results reported in this paper.) Histidine and acetate buffer systems are viable alternatives to phosphate, depending on the desired pH. Using buffers with a pH range of around 5 significantly reduces aggregate levels. In addition to sucrose, tension regulators proline and trehalose can be used to achieve appropriate weight molar osmotic pressure concentrations (~300 mOsm / kg) while maintaining or enhancing the molecular stability of aflibercept.

[0246] Reduced capillary electrophoresis of sodium dodecyl sulfate (rCE-SDS) The rCE-SDS method was used to monitor product degradation over time, such as the cleavage of the amino acid backbone. Results are reported as a purity percentage, i.e., the total percentage of heavy and light chains. Results at 4°C are summarized in Figure 5, and data at 30°C are shown in Figure 6. Regardless of storage temperature, all formulations studied, except for Formulation 7, exhibited similar purity levels throughout the test time points. Numerical differences observed between formulations are within the assay variability. Formulation 7 showed increased product degradation at elevated temperatures above 30°C, resulting in a decrease in purity percentage over time.

[0247] Capillary isoelectric focusing (cIEF) Capillary isoelectric focusing (cIEF) was used to monitor changes in the distribution of charge variants over time. To evaluate different formulations, the percentage levels of basic and acidic substances were plotted against time. Increases or decreases in these substances would indicate changes in protein charge, possibly due to chemical modifications of the peptide backbone. As shown in Figures 7, 8, 9, and 10, no changes in the distribution of charge variants were observed over time. These data indicate that no detectable chemical modifications affecting the charge of the aflibercept protein occurred.

[0248] Example 3. Stability Study

[0249] Further stability tests of aflibercept were performed in various embodiments of the formulations of this invention. Table 4 lists the tested aflibercept (40 mg / mL) formulations and their abbreviations. The amino acids used were L-isomers.

[0250] Table 4 lists the formulations of aflibercept (40 mg / mL) tested, their weight molality osmolality, and the relevant abbreviations used in Tables 5-15 of this document.

[0251] Table 4

[0252]

[0253]

[0254] Material A VEGF-specific aflibercept fusion protein antagonist was produced using industry-standard recombinant expression technology and purification methods. The purified drug was buffer-exchanged with specific formulation buffers using a Millipore Corporation lab-scale or bench-scale tangential flow filtration system. Protein concentrations were adjusted to the final target concentrations by dilution with the formulation buffers. Water used for preparing all formulations was... Millipore Corporation's water purification system includes an ion exchange chamber. Water purity is monitored by measuring conductivity, with a value greater than 18.2 MΩcm. -1 It is acceptable. All excipients, buffers, and other components used to prepare the formulation buffer are USP grade or equivalent.

[0255] method

[0256] Titration An acid-base conjugate prepared at equimolar concentrations was mixed together in an appropriate molar ratio to obtain the desired formulation pH of the histidine-phosphate buffer system. The acetate formulation was prepared using the conjugation method or by adding glacial acetic acid to Milli-Q purified water and then titrating with sodium hydroxide to achieve the final desired pH.

[0257] Multi-attribute method (MAM) based on mass spectrometryStability testing involved denaturing the sample with 6.8 M guanidine, reducing it with 10 mM dimercaptothreitol (DTT), and alkylating it with 20 mM iodoacetic acid. Excess reagents were removed using a size exclusion-based desalting column. Trypsin was added at a 1:10 enzyme-to-substrate ratio, and the sample was digested at 37 °C for 30 min. Peptides were separated by RP-HPLC using a formic acid / acetonitrile (FA / ACN) gradient along a C18 column, and the separation was monitored by mass spectrometry using a Thermo Fisher Q-Exactive mass spectrometer. Various peptides were identified and quantified using Genedata's Expressionist software.

[0258] Other analytical methods are as described in Example 1 above.

[0259] Effect of pH on the stability of aflibercept The effect of pH on the formation rate of high molecular weight particles, as measured by SE-HPLC at 4°C and 30°C (see Figure 11), and the formation of subvisible particles, as measured by HIAC at 4°C, was investigated using aflibercept produced at different production scales in three (3) different batches. Aflibercept produced at the three different scales showed similar product characteristics, including similar glycosylation levels across all batches. Aflibercept was then formulated with 10 mM acetate, 3% (w / v) proline, and 0.1% (w / v) poloxamer 188 to three (3) different pH values ​​(see Table 4, regarding formulation abbreviations). As shown in Tables 5 and 6 and Figure 11, the HMW formation rate of aflibercept, measured by SE-HPLC with 10 mM acetate, 3% (w / v) proline, and 0.1% (w / v) poloxamer 188, was not affected by pH and was consistently lower than the rate of change observed in the same batch of aflibercept with 10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, and 0.03% (w / v) polysorbate 20 at pH 6.2. Subvisible particle formation, measured by HIAC, was also examined during storage at 4°C, and no significant changes were observed in aflibercept in any formulation during storage (Table 7).

[0260] Table 5 shows the formation rate of HMW at 4°C as measured by SE-HPLC; *The value in parentheses refers to the pH of the solution being measured.

[0261] Table 5

[0262]

[0263]

[0264] Table 6 shows the formation rate of HMW at 30°C as measured by SE-HPLC; *The value in parentheses refers to the pH of the solution being measured.

[0265] Table 6

[0266]

[0267] Table 7 shows the subvisible particle formation at 4°C as measured by small-volume HIAC analysis; *values ​​in parentheses refer to the pH of the solution being measured.

[0268] Table 7

[0269]

[0270] Stability of aflibercept in proline and arginine formulations containing different nonionic surfactants The stability of aflibercept in formulations containing proline or arginine as tension modifiers in the presence of either poloxamer 188 or polysorbate 80 was investigated (see Tables 8 and 9, respectively; see Table 4 for formulation abbreviations). When aflibercept was formulated with either polysorbate 80 or poloxamer 188, compared with 10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, pH 6.2, the formation of HMW at 30°C was significantly reduced, as measured by SE-HPLC. Compared to aflibercept in formulations containing 10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, and pH 6.2, aflibercept in formulations containing 10 mM phosphate, 3% (w / v) proline, and pH 6.2 showed similar HMW formation rates as measured by SE-HPLC (each formulated with polysorbate 80 or poloxamer 188 as a nonionic surfactant). Conversely, replacing proline with arginine as a tension modifier in the acetate formulation at pH 5.2 increased the HMW formation rate as measured by SE-HPLC by at least 10-fold. However, regardless of the type of nonionic surfactant present, arginine showed similar stability to aflibercept in the 10 mM phosphate buffer formulation at pH 6.2, containing 10 mM phosphate, 3% (w / v) proline, and pH 6.2.

[0271] Table 8 shows the HMW formation rate at 30°C as measured by SE-HPLC.

[0272] Poloxamer 188 (0.1% (w / v)) was present as a nonionic surfactant in each of the tested formulations.

[0273] Table 8

[0274]

[0275] Table 9 shows the HMW formation rate at 30 °C as measured by SE-HPLC.

[0276] In each of the tested formulations, polysorbate 80 (0.03% (w / v)) was present as a nonionic surfactant.

[0277] Table 9

[0278]

[0279] The stability of aflibercept and the effect of surfactant concentration after simulated shipping The stability of aflibercept in formulations with various surfactants and concentrations at 10 mM acetate, 3% (w / v) proline, and pH 5.2 was characterized after simulated transport. The simulated transport protocol was designed to account for multiple transport modes that could potentially damage the product and affect the stability profile. As shown in Table 10, the HMW formation rate of aflibercept in the 10 mM acetate, 3% (w / v) proline, pH 5.2 formulation stored at 4 °C, as measured by SE-HPLC, was independent of the type or concentration of surfactant and significantly lower than that observed in the 10 mM phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, pH 6.2 formulation. Consistent with previous results, the stability of aflibercept in the 10 mM phosphate, 3% (w / v) proline, pH 6.2 formulation was similar to that in the 10 mM phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, pH 6.2 formulation. Furthermore, although the HMW formation rate was faster at 30°C than at 4°C, the stability order remained unchanged, with 10 mM acetate, 3% (w / v) proline, and pH 5.2 being the most stable (Table 11). After simulated transport, the subvisible particle count, as measured by HIAC, increased to a similar degree in all formulations (Table 12, comparison column: week 0 control to week 0). The subvisible particle count, as measured by HIAC, did not appear to be formulation-dependent. For subsets of formulations and storage temperatures, product potency was evaluated at the 13-week time point (see Table 13). No potency difference was detected between the 10 mM acetate, 3% (w / v) proline, and pH 5.2 formulation and the 10 mM phosphate, 3% (w / v) proline, and pH 6.2 formulation compared to the 10 mM phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, and pH 6.2 formulation. These results indicate that various proline formulations stabilize proteins and maintain their functional activity.

[0280] Multi-attribute method (MAM) analysis based on mass spectrometry:MAM analysis was performed to assess changes in post-translational modification levels regarding properties such as isomerization of aspartic residues, deamidation of asparagine, and oxidation of methionine. Samples from three formulations were evaluated after 3 months of storage at 4°C, 30°C, and 40°C, compared to a -70°C control sample. In this analysis, 10 mM acetate, 3% (w / v) proline, 0.1% (w / v) poloxamer 188, pH 5.2 and 10 mM acetate, 3% (w / v) proline, 0.03% (w / v) polysorbate 80, pH 5.2 were compared to a commercially available aflibercept formulation (10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.2).

[0281] Equivalent levels of methionine oxidation were observed between the -70°C control and the 4°C stability samples of the three formulations. Higher levels of oxidation were observed in all three formulations after storage at 30°C and 40°C, with the lowest oxidation level reported at 10 mM acetate, 3% (w / v) proline, 0.1% (w / v) poloxamer 188, pH 5.2. Two formulations with higher levels of methionine oxidation contained polysorbates, an excipient known to promote protein oxidation (Kerwin, Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways, Journal of Pharmaceutical Sciences, (2008) 97, 8: 2924-2934).

[0282] The deamidation levels of the three formulations were evaluated after 3 months of storage at 4°C, 30°C, and 40°C. No detectable differences were observed between samples stored at 4°C, but samples stored at elevated temperatures showed significantly increased deamidation levels. For example, the aflibercept formulation at 40°C (10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.2) showed 7.6% deamidation at the N84 position, while the formulations of 10 mM acetate, 3% (w / v) proline, 0.1% (w / v) poloxamer 188, pH 5.2 and 10 mM acetate, 3% (w / v) proline, 0.03% (w / v) polysorbate 80, pH 5.2 showed 2.8% and 3.0% deamidation at the N84 position, respectively. Of all five deamidation sites detected, the 10 mM acetate, 3% (w / v) proline, pH 5.2 formulation exhibited significantly less deamidation after elevated temperature storage than the pH 6.2 phosphate formulation.

[0283] The levels of isomerized aspartic acid residues were also assessed during the MAM analysis. Six (6) aspartic acid residues are readily isomerized. Regardless of temperature or formulation, the measured differences in % isomerization levels were within the margin of error of the measurement technique, and therefore no conclusions could be drawn from these data.

[0284] In summary, compared to commercially available aflibercept formulations with 10 mM sodium phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.2, MAM data indicate that the formulation with 10 mM acetate, 3% (w / v) proline, 0.1% (w / v) poloxamer 188, pH 5.2 has a lower or equivalent rate of post-translational modification formation.

[0285] Table 10 shows the HMW formation rate at 4°C as measured by SE-HPLC; *Control refers to materials maintained at 4°C during transport studies and not subjected to simulated transport treatment. (For formulation abbreviations, see Table 4).

[0286] Table 10

[0287]

[0288] Table 11 shows the HMW formation rate at 30°C as measured by SE-HPLC; *Control refers to materials maintained at 4°C during transport studies and not subjected to simulated transport treatment. (See Table 4 for formulation abbreviations).

[0289] Table 11

[0290]

[0291]

[0292] Table 12 shows the subvisible particle formation rate at 4°C as measured by HIAC; *Control refers to materials maintained at 4°C during transport studies and not subjected to simulated transport treatment. (For formulation abbreviations, see Table 4).

[0293] Table 12

[0294]

[0295] Table 13 shows the relative effectiveness; *NT - all temperatures were not tested for effectiveness; otherwise n=3.

[0296] Table 13

[0297]

[0298] With commercial purchase In comparison, the recombinant aflibercept produced in the acetate buffer formulation of the present invention... The stability of recombinant aflibercept produced at 30°C in a formulation containing 10 mM acetate, 3% (w / v) proline, pH 5.2, and 0.1% (w / v) poloxamer was compared with that of [other products / treatments]. (Aflibercept; Regeneron Pharmaceuticals, Inc., Tarrytown, NY; see Table 14) were compared. Unless otherwise specified herein, all aflibercept used in the experiments described herein was recombined by Just Biotherapeutics, Inc. (Seattle, WA) for these studies and formulated as described in Tables 1 and 4 herein. The pharmaceutical product was purchased from the European market and remained stable in its own container. Samples were taken from the vials at specified time points and analyzed by SE-HPLC to characterize the amount of HMW present. The data in Table 14 indicate that aflibercept manufactured by Just Biotherapeutics and formulated as specified has a higher HMW content than... Aflibercept in pharmaceutical products has a lower % HMW and a lower HMW substance formation rate.

[0299] Table 14 shows the embodiments of the formulations of the present invention and commercially available aflibercept formulations. Comparison of HMW formation rates at 30°C (SE-HPLC). (See Table 4 for formulation abbreviations).

[0300] Table 14

[0301]

[0302] Effect of salt on the stability of aflibercept formulations The effect of salt on the stability of aflibercept in a formulation containing 10 mM acetate, 3% (w / v) proline, and pH 5.2 during storage at 30 °C was investigated. As shown in Table 15, the addition of 100 mM sodium chloride to the formulation increased the rate of HMW formation as measured by SE-HPLC by 3.8 times.

[0303] Table 15 shows a comparison of the HMW formation rate (SE-HPLC) at 30°C for embodiments of the formulations of the present invention with or without 100 mM sodium chloride.

[0304] Table 15

[0305]

[0306] Stability comparison between commercially available aflibercept and the recombinant aflibercept used in these experiments As (ziv-aflibercept; Regeneron Pharmaceuticals, Inc., Tarrytown, NY) Commercially available aflibercept was reprocessed to remove commercial formulation components (except for ziv-aflibercept) and reformulated into a formulation (P62NaSuT) of 10 mM phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.2. The commercially available reprocessed aflibercept and recombinant aflibercept (manufactured by Just Biotherapeutics, Inc. and formulated in 10 mM phosphate, 40 mM sodium chloride, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.2) used in these studies were incubated at 30°C and HMW formation was monitored. As shown in Table 16, from commercially available... The purified aflibercept formed HMWs at a slightly faster rate than those observed with aflibercept produced by Just Biotherapeutics. These results indicate that there is no substantial difference in the intrinsic aggregation rate of commercially available aflibercept fusion proteins compared to the aflibercept protein used in the experiments described herein.

[0307] Table 16 shows the comparison between commercially available Ziv-Aflibercept and recombinant aflibercept (Just Biotherapeutics, Inc.). HMW formation at 30°C (SE-HPLC).

[0308] Table 16

[0309]

[0310]

[0311] Example 4. Tolerance studies of multiple placebo formulations administered intravitreal in rabbits

[0312] To determine the tolerability of some embodiments of the formulation of the present invention intended for use with aflibercept drug products, a placebo formulation was administered as a single dose to rabbits at Charles River Laboratories, Inc., 640 N. Elizabeth Street, Spencerville, OH 45887, United States of America, as shown in Table 17.

[0313] Table 17. Placebo formulations used as a single-dose test in male rabbits.

[0314]

[0315] The following parameters and endpoints were evaluated according to the study design: clinical signs, weight, weight gain, food consumption, and ophthalmology.

[0316] In the rabbits tested in this study, there were no early deaths, no treatment-related clinical signs, and no effects on body weight, weight gain, food consumption, or any ophthalmic aspect were observed. In summary, all placebo formulations administered via intravitreal injection were well tolerated in rabbits.

Claims

1. An ophthalmic preparation comprising: (a) Aflibercept, at a concentration of 5-100 mg / mL; (b) a buffer, at a concentration of 5-50 mM, wherein the buffer is a histidine buffer; (c) a nonionic surfactant selected from the group consisting of polysorbate, polyethylene glycol dodecyl ether, poloxamer, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, alkyl sugars and alkyl glycosides, at a concentration of 0.001% (w / v) to 5% (w / v); (d) a tension regulator, wherein the tension regulator is an amino acid selected from proline, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine and lysine, at a concentration of 2-4% (w / v), wherein the formulation has a final weight molar osmolality of 300 ± 50 mOsm / kg; (e) a chloride anion concentration of less than 10 mM; and wherein the pH of the formulation is from pH 5.0 to pH 5.

0. 6.

5.

2. An ophthalmic preparation comprising: (a) Aflibercept, at a concentration of 5-100 mg / mL; (b) a buffer, at a concentration of 5-50 mM, wherein the buffer is an acetate buffer; (c) a nonionic surfactant selected from the group consisting of polysorbate, polyethylene glycol dodecyl ether, poloxamer, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, alkyl sugars and alkyl glycosides, at a concentration of 0.001% (w / v) to 5% (w / v); (d) a tension regulator, wherein the tension regulator is an amino acid selected from proline, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine and lysine, at a concentration of 2-4% (w / v), wherein the formulation has a final weight molar osmolality of 300 ± 50 mOsm / kg; (e) a chloride anion concentration of less than 10 mM; and wherein the pH of the formulation is pH 5.0 to pH 6.

5.

3. The ophthalmic preparation according to claim 1, wherein, The concentration of the buffer is 5-20 mM.

4. The ophthalmic preparation according to claim 1 or 2, wherein, The nonionic surfactant is poloxamer 188.

5. The ophthalmic preparation according to claim 1 or 2, wherein, The tension regulator is proline.

6. The ophthalmic preparation according to claim 1 or 2, wherein: (a) The concentration of aflibercept is 20-80 mg / mL; (b) The concentration of the buffer is 10 mM; (c) The nonionic surfactant is polysorbate or poloxamer; (d) The tension modifier is proline at a concentration of 3% (w / v); (e) The concentration of the chloride anion is less than 10 mM; and the pH of the formulation is pH 5.0 to pH 6.

5.

7. Use of an ophthalmic preparation according to any one of claims 1-6 for the treatment of ophthalmic conditions or diseases; wherein, The eye conditions or diseases mentioned are selected from the group consisting of: macular edema after retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascularized wet age-related macular degeneration (AMD), visual impairment due to myopic choroidal neovascularization, diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, and neovascularized age-related macular degeneration (AMD).

8. The use according to claim 7, wherein, The ophthalmic preparation is administered via intravitreal injection.

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