US 2020/0246423 Al  22  Aug. 6, 2020  [0127]  Buffers maintain pH in a desired range, e.g.,  between pH 4 and pH 9. Buffers may also serve to stabilize  aflibercept by a variety of other mechanisms, meaning they  may be used outside of the nominal buffer capacity range  indicated by their respective pKa values. Examples of suit› able buffers include acetate (e.g., at pH 4 to 5.5), citrate (e.g.,  at pH 5 to 6.5), histidine ( e.g., at pH 5 to 7), phosphate ( e.g.,  at about pH 6 and 8), Tris (e.g., at pH 7 to 8), and glycine  (e.g., at pH 8 to 9). Specific embodiments include, without  limitation, sodium or potassium phosphate, sodium or potas› sium citrate, ammonium acetate, tris-(hydroxymethyl)› aminomethane (tris), various forms of acetate and dietha› nolamine. Other suitable buffers include succinate, histidine,  tartrate, bicarbonate, borate, imidazole, and maleate. In one  embodiment, the concentration of the buffer in the formu› lation is between about 1 mM to about IM ( e.g., about 1 mM  to about 950 mM, about 1 mM to about 900 mM, about 1  mM to about 850 mM, about 1 mM to about 800 mM, about  1 mM to about 750 mM, about 1 mM to about 700 mM,  about 1 mM to about 650 mM, about 1 mM to about 600  mM, about 1 mM to about 550 mM, about 1 mM to about  500 mM, about 1 mM to about 450 mM, about 1 mM to  [0119]  Numerous embodiments of the present invention  are explained in a greater detail below.  [0118]  When pharmaceutical compositions containing  aflibercept are stored, the activity of aflibercept can be lost  or decreased due to aggregation and/or degradation. Thus,  the present invention provides aqueous formulations of  aflibercept that allow stable storage of aflibercept, so that  aflibercept is stable over the course of storage either in liquid  or frozen states. The provided formulations do not require  any extra steps such as rehydrating.  [0126]  The formulations of the invention may include  buffers, tonicity modifiers, excipients, pharmaceutically  acceptable carriers and other commonly used inactive ingre› dients of the pharmaceutical compositions. However, for› mulations that indicate that an ingredient is excluded ( e.g.  buffer-free formulations) do not include such ingredient.  [0117]  Substances defined or recited separately from  "organic co-solvent" in U.S. Pat. No. 9,580,489 are  excluded from term "organic co-solvent" in the present  application. For example, U.S. Pat. No. 9,580,489 lists types  of substances, and gives examples thereof, as distinct from  an "organic co-solvent." See, colunm 2, lines 25-38. In  particular, U.S. Pat. No. 9,580,489 provides the following  examples of substances that are not organic co-solvents: a  tonicity agent for example sodium chloride or potassium  chloride; a stabilizing agent for example sucrose, sorbitol,  glycerol, trehalose, or mannitol; and a buffering agent for  example phosphate buffer. Examples of compounds that are  not an "organic co-solvent," as used herein, include salt ( e.g.  sodium chloride, magnesium chloride), sugar (e.g. dextran  or other polysaccharides), polyol (e.g. sorbitol, glycerol,  mannitol), and amino acid (e.g. arginine, glycine, praline).  Additional examples of compounds that are not an organic  co-solvent include block copolymers, alkyl saccharides  (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl› pyranoside, N-octyl-glucopyranoside, Intravailfi, and those  described in U.S. Pat. Nos. 9,446,134; 8,846,044, 8,226,949;  7,998,927; 8,084,022; 7,425,542; and 10,046,025), and  FM! 000, or any combination thereof.  [0125]  B. pH/Buffer Selection for Aflibercept Formula› tions  [0116]  The term "organic co-solvent" is used herein in the  same sense as it is used in U.S. Pat. No. 9,580,489. See, for  example, colunm 2, lines 25-38. U.S. Pat. No. 9,580,489  states "organic co-solvent may be polysorbate, for example,  polysorbate 20 or polysorbate 80, polyethylene glycol  (PEG), for example, PEG 3350, or propylene glycol, or a  combination thereof." An organic co-solvent is an organic  (i.e., carbon-based) substance that is added to composition  primarily to keep the ingredients of the composition in  solution (i.e. dissolved). This includes preventing precipita› tion, aggregation, and adsorption of composition ingredi› ents.  [0124]  For example, aflibercept can be purified using, for  example, hydroxyapatite chromatography, gel electrophore› sis, dialysis, ion exchange chromatography and affinity  chromatography, and any combination of known or yet to be  discovered purification techniques, including but not limited  to Protein A chromatography, fractionation on an ion-ex› change column, hydrophobic interaction chromatography  (HIC), ethanol precipitation, reverse phase HPLC, chroma› tography on silica, chromatography on heparin SEPHARO› SETfi, an anion or cation exchange resin chromatography  (such as a polyaspartic acid colunm), chromatofocusing,  SDS-PAGE, and ammonium sulfate precipitation.  [0123]  Purification of the expressed aflibercept can be  performed by standard methods known in the art. When  aflibercept is produced intracellularly by host cells, the host  cells are lysed and particulate debris is removed, for  example, by centrifugation or ultrafiltration. When afliber› cept is secreted into the medium by host cells, supematants  from such expression systems can be first concentrated using  standard polypeptide concentration filters. Protease inhibi› tors can also be added to inhibit proteolysis and antibiotics  can be included to prevent the growth of microorganisms.  [0122]  Aflibercept can also be produced by purifying  and/or diluting from commercially-available Eyleafi and/or  Zaltrapfi preparations using standard methods  [0121]  Aflibercept suitable for storage in one of the pres› ent pharmaceutical compositions or formulations can be  produced by standard methods known in the art. For  example, U.S. Pat. No. 9,580,480 and WO 2006/104852  describe various methods that a skilled artisan could use to  prepare aflibercept protein for use in the formulations of the  present invention. These methods are incorporated by ref› erence herein.  [0120]  A. Aflibercept  [0115]  As used herein, "purified" means that a molecule is  present in a sample at a concentration of at least 95% by  weight, or at least 98% by weight of the sample in which it  is contained.  [0114]  As used herein, the terms "buffer-free" or "free of  buffer" are synonymous and intend to exclude components  traditionally used to introduce buffer capacity in the formu› lation in addition to any buffering capacity present in the  formulation absent the buffer component. For example,  proteins are known to provide buffering capacity to a for› mulation. Hence, these terms do not mean a formulation  cannot have any buffering capacity. In one embodiment, a  buffer-free protein formulation is self-buffering.  a weak acid and a conjugate salt or a weak base and a  conjugate salt ( e.g sodium acetate). Buffers suitable for use  in connection with this invention may have a pH in the range  from about 4.0 to about 9.0; from about pH 4.0 to about 7.0;  or from about pH 4.5 to about 6.5. A pH of any point in  between the above ranges is also contemplated. 