US 2020/0246423 Al  40  Aug. 6, 2020  [0167]  Salts, e.g., NaCl, are often part of protein (e.g.,  antibody) formulations. For instance, salts such as NaCl,  Na$_{2}$SO$_{4}$ , CaCl$_{2}$ , MgCl$_{2}$ , and KC!, together with specific  buffer combinations as described above, can confer particu› larly advantageous stability properties. Such salts may be  used with as well as without a sugar added ( e.g., sucrose and  NaCl) or a polyol added (e.g., mannitol and NaCl). For  example, concentrations as low as 20 to 50 mM can provide  decreased levels of proteolysis (such as hinge region hydro› lysis), oxidation, deamidation, or other chemical instabili› ties. Furthermore, low levels of salt ( <50 mM) may lead to  improved colloidal stability. Likewise, it is known that high  [0166]  III. Salts  [0165]  In some embodiments, combinations of stabilizers  may act in concert to stabilize aflibercept. In one embodi› ment, an aflibercept formulation comprises a sugar and a  polyol. In another example, amino acids, such as praline,  serine, or glutamate may be used together with a sugar to  achieve a stability profile better than either excipient could  provide on its own. In one embodiment, the ratio of a sugar  to a polyol ( or amino acid) in the formulation is between 5: 1  and 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1,  about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5). In  another embodiment, a formulation with a combination of  stabilizers has better stability than a formulation with just  one of the stabilizers. In another embodiment, a formulation  with a combination of stabilizers has better stability than a  formulation with just one of the stabilizers where the for› mulation with the combination of stabilizers has a lower  total amount of stabilizers than the formulation with the  single stabilizer (e.g. a formulation with 100 mM of sucrose  and 50 mM arginine has better stability than a formulation  with 200 mM sucrose).  [0174]  Polymers,  such  as  dextrans,  starches  (e.g.,  hydroxyl ethyl starch (HETA)), poly(ethylene glycols  (PEGs), e.g., PEG-3350 or PEG-4000, may also provide  stabilization to aflibercept, presumably by being excluded  from the surface of the protein due to steric effects arising  from their higher molecular weight. In particular, hydro› philic polymers, such as polyethylene glycols (PEGs), poly› saccharides, and inert proteins, and may be used to stabilize  proteins and enhance protein assembly. Examples include  dextran, hydroxyl ethyl starch (HETA), PEG-4000, and  gelatin. Additionally, non-polar moieties on certain poly› mers such as PEGs and Pluronics can decrease water surface  tension rendering them as surfactants that suppress surface  adsorption induced aggregation. In one embodiment, the  concentration of polymer is between 0.01% and 40% (e.g.,  about 0.01 % to about 35%, about 0.01 % to about 30%, about  0.01 % to about 25%, about 0.01 % to about 20%, about  0.01% to about 18%, about 0.01% to about 16%, about  0.01% to about 14%, about 0.01% to about 12%, about  0.01 % to about 10%, about 0.01 % to about 8%, about 0.01 %  to about 6%, about 0.01 % to about 5%, about 0.01 % to about  4.5%, about 0.01 % to about 4.0%, about 0.01 % to about  3.5%, about 0.01 % to about 3.0%, about 0.01 % to about  2.5%, about 0.01 % to about 2.0%, about 0.01 % to about  1.5%, about 0.01 % to about 1.0%, about 0.01 % to about  0.5%, about 0.01 % to about 0.1 %, about 0.01 % to about  0.05%, about 0.05% to about 40%, about 0.05% to about  35%, about 0.05% to about 30%, about 0.05% to about 25%,  about 0.05% to about 20%, about 0.05% to about 18%, about  0.05% to about 16%, about 0.05% to about 14%, about  0.05% to about 12%, about 0.05% to about 10%, about  0.05% to about 8%, about 0.05% to about 6%, about 0.05%  to about 5%, about 0.05% to about 4.5%, about 0.05% to  about 4.0%, about 0.05% to about 3.5%, about 0.05% to  [0173]  V. Polymers  [0172]  As above, optimal formulation employing such  surfactants may also employ specific buffers at specific pH  ranges, and may optionally include other tonicity modifier  such as NaCl, polyol, or amino acid(s).  [0164]  Aflibercept Stabilized with Combinations of Sta› bilizers  [0171]  In the embodiments of the invention where organic  co-solvents are excluded, surfactants cannot be present.  [0170]  Surfactants can confer protection against agitation  and freeze/thaw damage, as well as stabilizing a formulation  during storage. Surfactants which may be employed with  aflibercept formulations of the present invention include  Tweenfi-80 (polysorbate 80, PS 80), Tweenfi-20 (polysor› bate 20, PS 20), SDS, polysorbate, polyoxyethylene copo› lymer, Brij 35, Triton X-10, poloxamer 188 (Pluronic F-68),  Pluronic F127, and Maltosides, e.g., n-Decyl-~-D-maltopy› ranoside (DM), n-Dodecyl-~-D-maltopyranoside (DDM),  and 6-Cyclohexyl-1-hexyl-~-D-maltopyranoside (Cymal- 6). In certain embodiments, surfactants that may be particu› larly advantageous include PS 20, PS 40, PS 60, and PS 80  at different concentrations, as well as DMM and poloxamer  188 (Pluronic F-68). Other zwitterionic or nonionic surfac› tants may be used as well.  [0169]  IV. Surfactants  [0168]  In specific embodiments, sodium chloride may be  replaced with Na$_{2}$SO$_{4}$ , KC!, MgCl 2 , CaCl 2 , MgSO 4 , ZnCl 2 ,  or other physiologically-acceptable salts.  concentrations of certain salts (up to 400 mM or more) can  increase conformational stability. The specific effects of  NaCl and other salts on aflibercept is dependent on pH,  buffer composition, and stress condition.  200 mM, about 140 mM to about 180 mM, about 140 mM  to about 160 mM, about 160 mM to about 500 mM, about  160 mM to about 450 mM, about 160 mM to about 400 mM,  about 160 mM to about 350 mM, about 160 mM to about  300 mM, about 160 mM to about 250 mM, about 160 mM  to about 200 mM, about 160 mM to about 180 mM, about  180 mM to about 500 mM, about 180 mM to about 450 mM,  about 180 mM to about 400 mM, about 180 mM to about  350 mM, about 180 mM to about 300 mM, about 180 mM  to about 250 mM, about 180 mM to about 200 mM, about  200 mM to about 500 mM, about 200 mM to about 450 mM,  about 200 mM to about 400 mM, about 200 mM to about  350 mM, about 200 mM to about 300 mM, about 200 mM  to about 250 mM, about 250 mM to about 500 mM, about  250 mM to about 450 mM, about 250 mM to about 400 mM,  about 250 mM to about 350 mM, about 250 mM to about  300 mM, about 300 mM to about 500 mM, about 300 mM  to about 450 mM, about 300 mM to about 400 mM, about  300 mM to about 350 mM, about 350 mM to about 500 mM,  about 350 mM to about 450 mM, about 350 mM to about  400 mM, about 400 mM to about 500 mM, about 400 mM  to about 450 mM, or about 450 mM to about 500 mM). In  another embodiment, the concentration of the amino acid(s)  is between about 10 mM and about 350 mM; in related  embodiments, the concentration of the amino acid(s) is  about 50 mM, 100 mM, 150 mM, 200 mM, 220 mM, 240  mM, 260 mM, 280 mM, 300 mM, 320 mM and 340 mM, for  example, 50-100 mM, 100-150 mM, 150-200 mM, 200-300  mM, 200-250 mM, 250-300 mM, and 300-350 mM. 