Random heteropolymer additives for high protein concentration formulations

Abstract

The present application relates to random polymer libraries and particular polymers that can be used to stabilize high concentration protein compositions, such as, for example, high concentration antibody compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 63 / 317,648, filed March 8, 2022, the entire contents of which are incorporated herein by reference for all purposes. [Technical field]

[0002] The present application relates to random polymer libraries and particular polymers that can be used to stabilize high concentration protein compositions, such as, for example, high concentration antibody compositions. [Background technology]

[0003] Many protein compositions, such as pharmaceutical preparations, contain relatively low concentrations of protein active ingredients, which can limit the usefulness of these compositions, for example, by limiting the means by which the protein active ingredients can be administered.For example, many protein therapeutics must be administered intravenously in large volumes of solution, since they cannot be concentrated sufficiently for other modes of administration that require smaller volumes for administration, such as subcutaneous injection, intravitreal injection, intraocular administration, intranasal administration, inhalation, topical administration and other methods.Therefore, there is a continuing need for methods for stabilizing high-concentration protein-containing compositions that allow sufficient shelf life for the composition and are feasible and safe for therapeutic use.

[0004] Some high-concentration protein formulations, even if relatively stable, may have high viscosities that may limit their manufacturability and the type of administration. Certain small molecule additives, such as arginine and other amino acids, may reduce viscosity in individual cases depending on the protein in question, but are not effective for all proteins. Furthermore, such additives may not be safe for all modes of therapeutic administration, and in some cases may be insufficient to stabilize proteins during storage or administration. Small molecule additives, such as amino acids, may also diffuse away from protein species upon administration or dilution of protein formulations, which may reduce protein stability. Therefore, new methods are needed to help reduce the viscosity of high-concentration protein formulations. Furthermore, there is a need to identify new additives that can help limit precipitation of protein formulations and / or do not diffuse away from proteins as quickly as small molecule additives upon dilution or in vivo administration. Summary of the Invention

[0005] The present disclosure describes, inter alia, polymers having a higher molecular weight than conventional small molecule protein formulation stabilizers, such as amino acids and sugars and sugar alcohols, for example polymers having a molecular weight more than 10 times higher than such small molecule additives. Such polymer additives can be used to stabilize high concentration protein formulations, for example, by limiting viscosity and / or inhibiting precipitation. The present disclosure includes multiple embodiments, including but not limited to the following embodiments.

[0006] Embodiment 1 is a random polymer library comprising a mixture of polymers, the library comprising: (a) methyl methacrylate (MMA); (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA); (c) isobutyl methacrylate (IBMA) or butyl methacrylate (BMA); and (d) a compound of formula I: [ka] where R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, and R3 is NH2 or N(CH3)2 or a guanidinium group. The library is a random polymer library containing polymers having a molecular weight of 3 to 20 kDa.

[0007] Embodiment 2 is a random polymer library according to embodiment 1, wherein the library comprises polymers comprising a monomer of formula I, the monomer being dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide.

[0008] Embodiment 3 is a random polymer library according to embodiment 1 or 2, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise at least three monomers, or the random polymer library consists essentially of polymers comprising at least three monomers.

[0009] Embodiment 4 is a random polymer library according to embodiment 1 or 2, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise at least four monomers, or the random polymer library consists essentially of polymers comprising at least four monomers.

[0010] Embodiment 5 is a random polymer library comprising a mixture of polymers, each polymer comprising a mixture of at least three of the monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA) and dimethylamino methacrylate (DMAEMA), the library comprising polymers between 3 and 20 kDa.

[0011] Embodiment 6 is a random polymer library comprising a mixture of polymers, each polymer comprising a mixture of at least three of the monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA) and N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), the library comprising polymers between 3 and 20 kDa.

[0012] Embodiment 7 is a random polymer library according to any one of embodiments 1 to 6, wherein the library comprises polymers of 3 to 15 kDa, 5 to 20 kDa, 5 to 15 kDa, 5 to 10 kDa, 10 to 20 kDa, 7 to 10 kDa, or 10 to 15 kDa.

[0013] Embodiment 8 is the random polymer library according to any one of embodiments 1 to 7, wherein the library comprises OEGMA having a molecular weight of 300 to 1500 g / mol.

[0014] Embodiment 9 is the random polymer library of embodiment 8, wherein the OEGMA has a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol.

[0015] Embodiment 10 is the random polymer library of embodiment 8, wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500).

[0016] Embodiment 11 is the random polymer library according to any one of embodiments 1 to 4 or 7 to 10, wherein the library comprises polymers comprising three types of monomers.

[0017] Embodiment 12 is a random polymer library according to embodiment 11, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise three types of monomers, or the random polymer library consists essentially of polymers comprising three types of monomers.

[0018] Embodiment 13 is the random polymer library of embodiment 11 or 12, wherein the three monomers are MMA, OEGMA, and either IBMA or BMA.

[0019] Embodiment 14 is the random polymer library of embodiment 13, wherein the three monomers are MMA, OEGMA and IBMA.

[0020] Embodiment 15 is the random polymer library of embodiment 13, wherein the MMA, OEGMA, and IBMA or BMA are present in a ratio of (a) 5:3:2, or (b) 3:5:2, or (c) 5:4:1.

[0021] Embodiment 16 is the random polymer library of any one of embodiments 1 to 10, wherein the library comprises polymers that include four types of monomers.

[0022] Embodiment 17 is a random polymer library according to embodiment 16, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise three types of monomers, or the random polymer library consists essentially of polymers comprising three types of monomers.

[0023] Embodiment 18 is the random polymer library of embodiment 16 or 17, wherein the four monomers are MMA, OEGMA, IBMA and DMAEMA.

[0024] Embodiment 19 is the random polymer library of embodiment 18, wherein the polymer library comprises polymers comprising 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, and 5-25% DMAEMA by total weight of polymer.

[0025] Embodiment 20 is the random polymer library of embodiment 18, wherein the polymer library comprises polymers comprising MMA, OEGMA, IBMA, and DMAEMA in the following molar ratios: (a) 5:2.5:2:0.5; (b) 2:5:2:1; (c) 5:2.5:1:1.5; (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; and / or (g) 2:3:1:4.

[0026] Embodiment 21 is the random polymer library of embodiment 18, wherein the MMA, OEGMA, IBMA and DMAEMA are in a molar ratio of 5:2.5:2:0.5, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the library comprises polymers having a molecular weight of 3 to 15 kDa.

[0027] Embodiment 22 is the random polymer library of embodiment 16 or 17, wherein the four monomers are MMA, OEGMA, IBMA and ArgMAm.

[0028] Embodiment 23 is the random polymer library of embodiment 22, wherein the polymer library comprises polymers comprising 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, and 5-25% ArgMAm by total polymer weight.

[0029] Embodiment 24 is the random polymer library of embodiment 22, wherein the polymer library comprises polymers comprising MMA, OEGMA, IBMA, and ArgMAm in the following molar ratios: (a) 5:2.5:2:0.5; (b) 2:5:2:1; (c) 5:2:1:2; (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; and / or (g) 2:3:1:4.

[0030] Embodiment 25 is the random polymer library of embodiment 22, wherein the MMA, OEGMA, IBMA and ArgMAm are in a molar ratio of 5:2:1:2 or 4:4:1:1, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the library comprises polymers having a molecular weight of 3 to 15 kDa.

[0031] Embodiment 26 is a random polymer library comprising a mixture of polymers, the library comprising polymers comprising at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and / or at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), optionally wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and optionally wherein the library comprises polymers having a molecular weight of 3 to 15 kDa.

[0032] Embodiment 27 is a random polymer comprising: (a) methyl methacrylate (MMA); (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA); (c) isobutyl methacrylate (IBMA) or butyl methacrylate (BMA); and (d) a compound of formula I: [ka] where R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, and R3 is NH2 or N(CH3)2 or a guanidinium group. and wherein the polymer is 3 to 20 kDa.

[0033] Embodiment 28 is a method for preparing a polymer comprising the steps of: [ka] 28. The random polymer of embodiment 27, comprising: wherein the monomer is dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide.

[0034] Embodiment 29 is a random polymer comprising a mixture of at least three of the monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA) and dimethylamino methacrylate (DMAEMA), wherein the polymer is 3-20 kDa.

[0035] Embodiment 30 is a random polymer comprising a mixture of at least three of the monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA) and N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), wherein the polymer is 3-20 kDa.

[0036] Embodiment 31 is a random polymer according to any one of embodiments 27 to 30, wherein the polymer is 3-15 kDa, 5-20 kDa, 5-15 kDa, 5-10 kDa, 10-20 kDa, 7-10 kDa or 10-15 kDa.

[0037] Embodiment 32 is the random polymer of any one of embodiments 27 to 31, wherein the polymer comprises OEGMA having a molecular weight of 300 to 1500 g / mol.

[0038] Embodiment 33 is a random polymer according to embodiment 32, wherein the OEGMA has a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol.

[0039] Embodiment 34 is the random polymer of embodiment 33, wherein the OEGMA has a molecular weight of 500 g / mol.

[0040] Embodiment 35 is the random polymer of any one of embodiments 27-28 or 31-34, wherein the polymer comprises three types of monomers.

[0041] Embodiment 36 is a random polymer according to embodiment 35, wherein the three monomers are MMA, OEGMA, and either IBMA or BMA.

[0042] Embodiment 37 is a random polymer according to embodiment 36, wherein the three monomers are MMA, OEGMA and IBMA.

[0043] Embodiment 38 is the random polymer of embodiment 37, wherein the MMA, OEGMA, and IBMA or BMA are present in a ratio of (a) 5:3:2, or (b) 3:5:2, or (c) 5:4:1.

[0044] Embodiment 39 is the random polymer of any one of embodiments 27-34, wherein the polymer comprises four monomers.

[0045] Embodiment 40 is the random polymer of embodiment 39, wherein the four monomers are MMA, OEGMA, IBMA and DMAEMA.

[0046] Embodiment 41 is the random polymer of embodiment 40, wherein the polymer comprises 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, and 5-25% DMAEMA, based on the total weight of the polymer.

[0047] Embodiment 42 is the random polymer of embodiment 40, wherein the polymer comprises MMA, OEGMA, IBMA, and DMAEMA in the following molar ratios: (a) 5:2.5:2:0.5; (b) 2:5:2:1; (c) 5:2.5:1:1.5; (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; or (g) 2:3:1:4.

[0048] Embodiment 43 is the random polymer of embodiment 40, wherein the MMA, OEGMA, IBMA and DMAEMA are in a molar ratio of 5:2.5:2:0.5, the OEGMA is OEGMA 500, and the polymer has a molecular weight of 3 to 15 kDa.

[0049] Embodiment 44 is the random polymer of embodiment 39, wherein the four monomers are MMA, OEGMA, IBMA and ArgMAm.

[0050] Embodiment 45 is a random polymer according to embodiment 44, wherein the polymer comprises 20-50% MMA, 20-50% OEGMA, 5-25% IBMA and 5-25% ArgMAm based on the total weight of the polymer.

[0051] Embodiment 46 is the random polymer of embodiment 44, wherein the polymer comprises MMA, OEGMA, IBMA, and ArgMAm in the following molar ratios: (a) 5:2.5:2:0.5; (b) 2:5:2:1; (c) 5:2.5:1:1.5; (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; or (g) 2:3:1:4.

[0052] Embodiment 47 is the random polymer of embodiment 44, wherein the MMA, OEGMA, IBMA and ArgMAm are in a molar ratio of 5:2:1:2 or 4:4:1:1, the OEGMA is OEGMA 500, and the polymer has a molecular weight of 3 to 15 kDa.

[0053] Embodiment 48 is a random polymer comprising a mixture of at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and / or at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), optionally wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and optionally wherein the polymer has a molecular weight of 3 to 15 kDa.

[0054] Embodiment 49 is a random polymer according to any one of embodiments 27 to 48, wherein the polymer has a molecular weight of 7 to 10 kDa.

[0055] Embodiment 50 is a composition comprising a random polymer according to any one of embodiments 27 to 49 and at least one protein.

[0056] Embodiment 51 is a composition according to embodiment 50, wherein the polymer is at a concentration of 0.01 to 1% w / v, for example 0.01 to 0.1% w / v.

[0057] Embodiment 52 is the composition of embodiment 50 or 51, wherein the protein is an antibody.

[0058] Embodiment 53 is the composition of embodiment 52, wherein the antibody is an IgG antibody, such as a full-length IgG, a bispecific IgG, or the antibody is an antigen-binding fragment, such as a Fab, Fab', (Fab')2, Fv or scFv.

[0059] Embodiment 54 is the composition of any one of embodiments 50 to 53, wherein the composition further comprises at least one buffering agent, such as histidine, phosphate or citrate.

[0060] Embodiment 55 is the composition of any one of embodiments 50 to 54, wherein the composition further comprises at least one surfactant, such as a polysorbate, e.g., polysorbate 20 or polysorbate 80.

[0061] Embodiment 56 is a composition according to any one of embodiments 50 to 55, wherein the antibody has a concentration of 10 mg / mL to 150 mg / mL, for example, 10 to 100 mg / mL, 20 to 100 mg / mL, 20 to 80 mg / mL, 10 to 50 mg / mL, 10 to 25 mg / mL, 25 to 50 mg / mL, 50 to 80 mg / mL, 50 to 100 mg / mL or 70 to 100 mg / mL.

[0062] Embodiment 57 is a method for reducing the viscosity of a protein-containing composition and / or inhibiting precipitation of a protein-containing composition, the method comprising adding a random polymer described in any one of embodiments 27 to 49 to a protein-containing composition, optionally wherein the composition comprises a protein and / or an additive described in any one of embodiments 50 to 56.

[0063] Embodiment 58 is a method for preparing the random polymer library according to any one of embodiments 1 to 26 or the random polymer according to any one of embodiments 27 to 49, the method comprising reversible addition-fragmentation chain transfer (RAFT), free radical polymerization (FRP) or atom transfer radical polymerization (ATRP).

[0064] Embodiment 59 is a method for preparing the random polymer library according to any one of embodiments 1 to 26 or the random polymer according to any one of embodiments 27 to 49, comprising exposing the monomer to LED light and / or heat in the presence of a zinc tetraphenylporphyrin catalyst (ZnTPP) and a chain transfer agent.

[0065] Embodiment 60 is the method of embodiment 59, wherein the chain transfer agent is 2-cyano-2-propylbenzodithioate, 2-cyano-2-propyldodecyltrithiocarbonate, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, or 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA).

[0066] Embodiment 61 is the method of embodiment 59 or 60, wherein the monomer is exposed to LED light in the presence of the ZnTPP and chain transfer agent for 3 to 18 hours.

[0067] Embodiment 62 is the method of any one of embodiments 59-61, wherein the polymerization is terminated by removing the LED light.

[0068] Embodiment 63 is the method of any one of embodiments 58 to 62, further comprising performing at least one filtration, buffer exchange or dialysis step to isolate the polymer.

[0069] Embodiment 64 is a kit comprising a random polymer library described in any one of embodiments 1 to 26 for use in testing polymers contained within the library for their effect on precipitation, viscosity and / or turbidity of a solution containing a protein, the kit optionally further comprising instructions for use.

[0070] Embodiment 65 is the kit of embodiment 64, wherein the library comprises at least 10, at least 20, at least 50, or at least 80 different individual random polymers.

[0071] Embodiment 66 is a method for identifying a random polymer additive for a protein solution, comprising exposing the protein solution to a random polymer library described in any one of embodiments 1 to 26 or a kit described in embodiment 64 or 65, and determining one or more of the viscosity, turbidity, or precipitation of the solution.

[0072] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0073] All references cited herein are hereby incorporated by reference. [Brief description of the drawings]

[0074] [Figure 1] FIG. 1 shows an exemplary method for evaluating a library of polymers for the effect of each polymer on monoclonal antibody (mAb, e.g., mAb G) precipitation.

[0075] [Figure 2A-2B]Figures 2A-B show turbidity measurements (absorbance at 600 nm) of mAb G (80 mg / mL) after overnight incubation with selected polymer additives at 1.6, 6.6, and 33 µM polymer (approximately 0.0014, 0.006, 0.03% polymer) in 15 mM phosphate buffer pH 7.4 containing 100 mM NaCl (Figure 2A) or 150 mM NaCl (Figure 2B).

[0076] [Diagram 3] Figure 3 shows the precipitation kinetics of mAb G (80 mg / mL) in 15 mM phosphate buffer pH 7.4 + 150 mM NaCl with polymer additive. Turbidity was measured using absorbance at 600 nm.

[0077] [Figure 4] Figure 4 shows the precipitation kinetics of mAb G (80 mg / mL) in 15 mM phosphate buffer pH 7.4 + 150 mM NaCl containing polysorbate 20 (PS20). Turbidity was measured using absorbance at 600 nm.

[0078] [Figure 5A-5B] 5A-5B show the change in viscosity of mAb G solution (194 mg / mL pH 5.5 in 30 mM histidine chloride (HisCl)) containing additives Poly1D ("Poly1"), polysorbate 20 (PS20), and Poly1D in combination with PS20 at different additive concentrations. FIG. 5A shows a graph of the change in viscosity with increasing concentrations of either Poly1D or PS20. FIG. 5B shows a bar graph of the change in viscosity at specific concentrations of Poly1D, PS20, or a combination of the two.

[0079] [Figure 6] FIG. 6 shows the viscosity dependence of mAb G (194 mg / mL in HisCl pH 5.5) on shear rate, indicating the addition of surfactant.

[0080] [Figure 7A-7B]Figures 7A-B show processing of samples for turbidity / precipitation assays. Figure 7A shows mAb G (80 mg / mL) after precipitation (A) in 15 mM phosphate buffer pH 7.4 + 150 mM NaCl from a high-throughput screen of mAb G control (left, solid box), increased Poly1E (middle, dashed box) and increased PS20 (right, dotted box). Figure 7B shows mAb G with PS20 (left) or Poly1E (right) after centrifugation.

[0081] [Figure 8] FIG. 8 shows microscale thermophoresis (MST) measurements demonstrating weak binding between Poly1F (15.5 μM) and mAbs A-D (2×10−7 to 1×10−4 M) in HisCl pH 5.5.

[0082] [Figure 9] FIG. 9 shows the average change in viscosity of eight mAbs when incubated with a polymer library containing increasing percentage amounts of DMAEMA.

[0083] [Figure 10] FIG. 10 shows the average change in viscosity of eight mAbs when incubated with a polymer library containing increasing percentage amounts of IBMA in the polymer.

[0084] [Figure 11] FIG. 11 shows the average change in viscosity of eight mAbs when incubated with a polymer library containing increasing percentage amounts of OEGMA.

[0085] [Figure 12] FIG. 12 shows the average change in viscosity of eight mAbs when incubated with Poly1, Poly11, Poly3, Poly5, Poly7 or Poly9, each of which has a molecular weight of 10 kDa.

[0086] [Figure 13A-13B]13A-13B show HPLC traces of (13A) unpurified and (13B) purified Poly 1H confirming removal of DMSO (retention time approximately 7 min).

[0087] [Figure 14A-14B] 14A-14B show exemplary absorbance measurements for the precipitation of an exemplary polymer at 320 nm (14A) and 600 nm (14B).

[0088] [Figure 15] FIG. 15 shows absorbance measurements at 600 nm for the turbidity of an exemplary polymer over time.

[0089] [Figures 16A-16D] 16A-16D show absorbance measurements at 600 nm for the turbidity of an exemplary polymer over time.

[0090] [Figure 17] Figure 17 shows the absorbance maximum (Amax) of exemplary polymers. Dark grey indicates little or no improvement, light grey indicates moderate improvement, and white indicates large improvement in precipitation kinetics attributes compared to the control. Bold indicates polymers with no dark grey classifications and at least two white classifications.

[0091] [Figure 18A-18B] 18A-B show the precipitation of mAB-G with and without an exemplary polymer in 15 mM phosphate buffer pH 7.4 containing 150 mM NaCl at 37° C.

[0092] [Fig. 19A-19H]Figures 19A-H show precipitation evaluation using a low-throughput physiologically relevant model. Solubility of mAb-G (n=3) was evaluated in phosphate buffer pH 7.4 + 150 mM NaCl at 37 °C in Eppondorf tubes by (19A) mAb-G soluble concentration and (19B) mAb-G precipitate weight after drying after separation of solids by centrifugation; **p<0.005 between Poly1 and no additive. Evaluation of mAB-G precipitation (representative of three; 19C-E) and pharmacokinetics (PK; n=3; 19F) in the SCISSOR system. Figure 19G shows Poly1 release from the SCISSOR cartridge (n=3). Figure 19H shows DLS of Poly1 micelles in 20 mM HisCl pH 5.5.

[0093] [Figure 20A-20B] 20A-20B show SCISSOR images (20A) and transmission data (20B) from a 0.2 mL injection of 50 mg / mL mAb-G.

[0094] [Figures 21A-21D] Figures 21A-D show the biophysical characterization of the mAb-G-Poly1 interaction using microscale thermophoresis (MST; 21A), isothermal titration calorimetry (ITC; representative of three; 21B), and autofluorescence quenching (n=3; 21C) and the corresponding Stern-Volmer plot (21D).

[0095] [Figure 22] FIG. 22 shows the cytotoxicity of Poly1 at several concentrations in Jurkat cells using trypan blue to assess cell membrane integrity (n=4).

[0096] [Figure 23] FIG. 23 shows the ITC curve of 1 mM mAb-G containing 100 μM Poly1.

[0097] [Figure 24] FIG. 24 shows 1H-NMR of ArgMAm in DMSO-d6.

[0098] [Diagram 25] FIG. 25 shows 1H-NMR of Poly1 in DMSO-d6.

[0099] [Figure 26] FIG. 26 shows a GPC trace of Poly1.

[0100] [Figure 27] FIG. 27 shows dynamic light scattering (DLS) of Poly 1 micelles in 30 mM HisCl pH 5.5.

[0101] [Fig. 28A-28D] Figures 28A-D show pendant drop tensiometer ST measurements for Poly1 at concentrations of 0.1% (28A), 0.02% (28B) and 0.002% (28C). Figure 28D shows surface tension measurements in mN / m for Poly1 at concentrations of 0.1% (28A), 0.02% (28B) and 0.002% (28C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] I. Definition The present invention may be understood more readily by reference to the following detailed description of specific embodiments and examples included below.

[0103] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0104] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers to more than one preceding independent or dependent claim in the alternative only. Also, terms such as "element" or "component" include both elements and components that contain a single unit and elements and components that contain two or more subunits, unless otherwise specified.

[0105] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0106] Units, prefixes and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Measurements are understood to be approximations taking into account significant digits and errors associated with the measurements.

[0107] As used herein, percentages ("%") are weight to volume ("w / v") percentages unless otherwise specified.

[0108] The present disclosure relates to various protein-containing formulations. Such "formulations" may also be referred to interchangeably herein as "compositions" or "preparations" or "solutions."

[0109] "Polypeptide" or "protein" refers to a sequence of amino acids whose chain length is sufficient to generate a tertiary structure. Thus, proteins herein are distinguished from "peptides," which are short amino acid-based molecules that generally do not have any tertiary structure. Typically, proteins as used herein have a minimum molecular weight of at least about 5-20 kD, alternatively at least about 15-20 kD, preferably at least about 20 kD. Polypeptides or proteins herein include, for example, antibodies.

[0110] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific and multispecific antibodies (including diabodies, one-armed antibodies and single-chain molecules), and antigen-binding fragments (e.g., Fab, F(ab')2, scFv and Fv). The antibodies herein include a set of complementarity-dependent regions (CDRs) located in the heavy (H) and light (L) chain variable domains that jointly recognize a specific antigen. The antibodies herein include at least a portion of the amino acid sequence of the heavy and light chain variable domains sufficient to include a set of CDRs for antigen recognition. In some embodiments, the antibodies include full-length heavy and light chain variable domains. In some embodiments, the antibodies further include heavy and / or light chain constant regions that may or may not be full-length.

[0111] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0112] The term "stabilizer" or "stabilizer" as used herein is a chemical or compound added to a formulation to maintain the formulation in a stable or unaltered state. In some cases, stabilizers may be added to help prevent precipitation, aggregation, oxidation, color change, etc. Depending on the context, stabilizers can include small molecules such as amino acids, sugars and sugar alcohols, as well as certain proteins (e.g., albumin), surfactants, and polymers.

[0113] "Surfactants" are molecules with clearly defined polar and non-polar regions that allow them to aggregate in solution to form micelles. Depending on the nature of the polar regions, surfactants can be nonionic, anionic, cationic and zwitterionic.

[0114] As used herein, "polymer" refers to a molecule that contains linear or branched chains of repeating monomeric elements.

[0115] A "random polymer" is a polymer in which the individual different monomers that make up the polymer chain are not in any particular pre-determined order along the length of the chain. A "random polymer library" or a "mixture of random polymers" refers interchangeably to a mixture of random polymers, for example, of different lengths, different molecular weights and / or different molar ratios of individual monomers. In some embodiments, a random polymer library may contain random polymers with the same set of monomers and the same monomer ratios, but with different chain lengths or molecular weights. In other embodiments, a library may contain random polymers with the same chain length or molecular weight and with the same set of monomers, but with different molar ratios of individual monomers. In some embodiments, a library may contain random polymers with different sets of monomers, but with, for example, the same molar ratios of different monomers and / or the same chain length.

[0116] As used herein, "chain transfer agent" refers to a molecule added to a polymerization reaction that can act to control or retard the growth of polymer chains during polymerization.

[0117] A "buffer" is a substance that may be included in some protein formulations and helps control the pH of the formulation. Examples of buffers used in therapeutic formulations include, for example, phosphate, citrate and histidine. Laboratory formulations often contain buffers such as Tris, HEPES, etc.

[0118] The term "pharmaceutical formulation" or "therapeutic formulation" or "therapeutic preparation" refers to a preparation or composition that contains at least one active ingredient (e.g., a protein) and at least one additional component or excipient substance, and that is in a form that allows the biological activity of the active ingredient to be effective in an animal subject, such as a mammal, and that is "suitable for therapeutic use" or "suitable for pharmaceutical use", meaning that the formulation as a whole is not unacceptably toxic to the subject and does not contain components that are unacceptably toxic to the subject to which the formulation is administered, or concentrations of components that render the components unacceptably toxic to the subject.

[0119] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability upon storage and administration. Stability can be measured at a selected temperature for a selected period of time. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).

[0120] Increasing the "stability" of a protein-containing formulation can include reducing or preventing the formation of precipitation, protein aggregates or degradation products (compared to untreated protein-containing formulations), reducing oxidation and color changes, and the like.

[0121] The term "aggregate" or "aggregation" as used herein means to come together or gather into a mass or whole, such as, for example, the aggregation of protein molecules. Aggregates may be self-aggregating or may aggregate due to the presence of other factors, such as flocculants, precipitants, agitation, or other means and methods of bringing proteins together. Aggregation may be observed visually, such as when a protein formulation that was clear in solution becomes cloudy or contains precipitate, or may be observed by methods such as size exclusion chromatography (SEC), which separates the proteins in the formulation by size. Aggregates may include dimers, trimers, and multimers of protein species. As used herein, "high molecular weight species" (HMWS) refers to aggregates of proteins that may be observed, for example, by size exclusion chromatography, and that represent at least a dimer of the desired protein molecule, i.e., have at least twice the molecular weight of the desired protein species in the formulation. In the case of a protein species, such as an antibody, that is already a multimer, e.g., a dimer or tetramer, in its normal or desired form, HMWS represents at least a dimer of the normal desired multimeric form of the protein.

[0122] "Isolated" when used to describe various polypeptides and antibodies disclosed herein means a polypeptide or antibody that has been identified, separated and / or recovered from a component of its production environment. In some cases, an isolated polypeptide is also not associated with all other components from its production environment. Contaminating components of the production environment, such as those from transfected recombinant cells, are typically substances that interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0123] In some embodiments herein, the pharmaceutical formulation is "free" of one or more additives or ingredients, such as surfactants or amino acid additives. "Free" in this context means that the excluded ingredient is not present at more than trace levels, for example, due to contaminants or impurities found in other ingredients that have been intentionally added.

[0124] As used herein, the term "consisting essentially of" when referring to a mixture of components of a formulation indicates that components other than those explicitly listed may be present, but that such components are found only in trace amounts or otherwise in amounts low enough that certain fundamental characteristics of the formulation, including protein concentration, such as levels of protein precipitation, turbidity and viscosity, are unchanged.

[0125] II. Exemplary Random Polymer Libraries and Selected Polymers Embodiments herein include random polymer libraries made from a plurality of random polymers. Embodiments herein also include specific random polymers, such as those having a specific relative concentration of a specific set of monomers and a specific average chain length or molecular weight.

[0126] In some cases, the random polymers of the random polymer library comprise a mixture of at least three different monomers, such as acrylate and / or acrylamide monomers. In some cases, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the polymers in the library have at least three monomers, and the remaining polymers in the library have one to two monomers. In some cases, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the polymers in the library have at least four monomers, and the remaining polymers in the library have one to three monomers. In some cases, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the polymers in the library have three monomers, and the remaining polymers in the library have one or two or four or more than four monomers, or a mixture of these possibilities. In some cases, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the polymers in the library have four types of monomers, with the remaining polymers in the library having one, two, or three or more than four types of monomers, or a mixture of these possibilities.

[0127] In some cases, the polymers in the library consist essentially of at least two, at least three, or at least four monomers. In some embodiments, the random polymer library consists essentially of polymers with three or four monomers. In some cases, the library consists essentially of polymers with three monomers. In other cases, the library consists essentially of polymers with four monomers. In such cases, although it is intended that each polymer in the library consists of a specific number of monomers, due to the complexity of chemical synthesis, the library may contain small amounts of polymers with different numbers of monomers.

[0128] In some cases, the random polymer library includes a polymer having a mixture of at least three monomers selected from methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), butyl methacrylate (BMA), dimethylamino methacrylate (DMAEMA), (4-hydroxyphenyl)methacrylamide, 2-hydroxyethyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl)methacrylamide, N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide. In some cases, the random polymer library includes a polymer having a mixture of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and at least three monomers selected from any of the following additional monomers: methyl methacrylate (MMA), isobutyl methacrylate (IBMA), butyl methacrylate (BMA), dimethylamino methacrylate (DMAEMA), (4-hydroxyphenyl) methacrylamide, 2-hydroxyethyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide. In some cases, the library includes a polymer having only one of isobutyl methacrylate (IBMA) and butyl methacrylate (BMA), but not both.In some cases, only one of dimethylamino methacrylate (DMAEMA), (4-hydroxyphenyl)methacrylamide, 2-hydroxyethyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl)methacrylamide, N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), and N-3-(dimethylamino)propyl methacrylamide is selected.

[0129] In some embodiments herein, the random polymer library comprises a polymer comprising three or more monomers selected from (a) methyl methacrylate (MMA), (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA), (c) either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA). In some embodiments herein, the random polymer library comprises a polymer comprising three or more monomers selected from (a) methyl methacrylate (MMA), (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA), (c) either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and (d) at least one compound of formula I: [ka]

[0130] wherein R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, R3 is NH2 or N(CH3)2 or OH, or R1 is O or NH, R2 and R3 together comprise a hydroxyphenyl, such as 4-hydroxyphenyl, or R1 is O or NH, R2 is an N,N-dimethylethylamine group and R3 is a sulfopropyl group. or (a), (b), and (c); or (a), (b), and (d); or (b), (c), and (d); or (a), (c), and (d); or (a)-(d); or (a)-(d), etc., thereby comprising a total of at least three different monomers. In some cases, the library can include a mixture of polymers, each with a different set of monomers from among these options. In other cases, the library can contain polymers each with the same set of monomers, but with different relative concentrations and / or different lengths or molecular weights as determined by the reaction conditions.

[0131] In some embodiments, the present disclosure provides a method for the preparation of a compound of formula I, comprising the steps of: (a) methyl methacrylate (MMA); (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA); (c) isobutyl methacrylate (IBMA) or butyl methacrylate (BMA); and (d) a compound of formula I, [ka]

[0132] where R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, and R3 is NH2 or N(CH3)2 or a guanidinium group (e.g., H2N-C(=NH2 + )-NH2), Random polymer libraries include polymers comprising a mixture of at least three monomers selected from the following, said libraries comprising polymers between 3 and 20 kDa. As noted above, such libraries may include monomers from, for example, (a), (b) and (c); or (a), (b) and (d); or (b), (c) and (d); or (a), (c) and (d); or each of (a)-(d), etc., thereby comprising a total of at least three different monomers. Furthermore, as noted above, in some cases, libraries may include a mixture of polymers, each with a different set of monomers from among these options. In other cases, libraries may contain polymers each with the same set of monomers, but with different relative concentrations and / or different lengths or molecular weights as determined by the reaction conditions. In some cases, the library includes a polymer that includes a monomer of formula I, where the monomer is dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl)methacrylamide, N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide. In some cases, the library includes a polymer that includes at least three of methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), and dimethylamino methacrylate (DMAEMA), or includes four of those monomers. In some cases, the random polymer library comprises at least one monomer of formula I selected from dimethylamino methacrylate (DMAEMA), 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide.

[0133] In any of the above cases, the library may include polymers with three monomers. In other cases, the library includes polymers with four monomers. In still other cases, five monomers are used.

[0134] In some cases, the library includes a polymer of three monomers, the three monomers being MMA, OEGMA, and either IBMA or BMA. In some cases, the three monomers are MMA, OEGMA, and IBMA. In some cases, the library includes a polymer of four monomers: MMA, OEGMA, IBMA, or BMA, and one of DMAEMA, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, or N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm). In some cases, the four monomers are MMA, OEGMA, IBMA, or BMA, and DMAEMA or ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA, or BMA, and DMAEMA. In some cases, the four monomers are MMA, OEGMA, IBMA, or BMA, and ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA, and DMAEMA or ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA, and DMAEMA. In some cases, the four monomers are MMA, OEGMA, IBMA, and ArgMAm. In some cases, the library polymers include a mixture of both IBMA and BMA and MMA and OEGMA. In some cases, the library polymers include a mixture of both IBMA and BMA. In some cases, the library polymers include two or more compounds of formula I, such as both DMAEMA and ArgMAm.

[0135] In other embodiments, the random polymer library includes a polymer having two monomers. In some embodiments, the two monomers are BMA or IBMA and OEGMA. In some cases, the two monomers are IBMA and OEGMA. In some cases, the two monomers are BMA and OEGMA. In other cases, the two monomers are ArgMAm and OEGMA.

[0136] In any of the above cases, in some embodiments, the library includes polymers between 3 and 20 kDa. In some embodiments, the library includes polymers from a library that includes polymers between 3 and 15 kDa, 5 and 20 kDa, 5 and 15 kDa, 5 and 10 kDa, 5 and 8 kDa, 10 and 20 kDa, 7 and 10 kDa, 8 and 10 kDa, 10 and 12 kDa, 12 and 15 kDa, or 10 and 15 kDa. For example, polymer libraries can be created that have polymers of various lengths and, therefore, various molecular weights.

[0137] When OEGMA is present, in some embodiments the library includes OEGMA having a molecular weight of 300-1500 g / mol. In some such embodiments, the OEGMA may have a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol. In some cases, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500). In some cases, random polymer libraries may also be created with the size of the OEGMA as a variable to vary, i.e., libraries may be created with OEGMA of different sizes, but otherwise contain polymers having, for example, the same set of monomers, optionally the same relative concentrations and / or the same approximate chain length.

[0138] In some embodiments, random polymer libraries can be made to include polymers with a range of molar ratios of monomers, or polymers with one particular set of molar ratios, but differing, for example, in length, or the molecular weight of OEGMA (if OEGMA is present), or another modifiable parameter. In some embodiments, the polymers of the library include 20-50% MMA, 20-50% OEGMA, and 5-25% IBMA by total weight of the polymer. In some such cases, the polymer is made from monomers consisting essentially of DMAEMA, IBMA, and OEGMA, for example, 20-50% MMA, 20-50% OEGMA, and 5-25% IBMA by total weight of the polymer. In other cases, at least one additional monomer is also present, for example, DMAEMA or ArgMAm. In some cases, the polymer can include 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, and 5-25% DMAEMA. In some embodiments, the polymers of the library include DMAEMA or ArgMAm, IBMA, OEGMA, and MMA, optionally present in the following molar ratios: (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2.5:1:1.5, (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; (g) 2:3:1:4, or (h) 5:2:1:2. In some embodiments, the MMA, OEGMA, IBMA, and DMAEMA are in a molar ratio of 5:2.5:2:0.5, OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the library includes polymers having molecular weights between 3 and 15 kDa. In some embodiments, the polymer library may be made of MMA, OEGMA and IBMA in a molar ratio of 5:4:1 or 5:3:2 or 3:5:2. Also, such polymer libraries may have a molecular weight range of 3-15 kDa. In some cases, the OEGMA may be OEGMA 500.

[0139] The embodiments herein include random polymers made from a mixture of at least three different monomers, such as acrylate and / or acrylamide monomers. In some cases, the random polymers include a mixture of at least three monomers selected from methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), butyl methacrylate (BMA), dimethylamino methacrylate (DMAEMA), (4-hydroxyphenyl) methacrylamide, 2-hydroxyethyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide. In some cases, the polymer comprises a mixture of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and at least three monomers selected from any of the following additional monomers: methyl methacrylate (MMA), isobutyl methacrylate (IBMA), butyl methacrylate (BMA), dimethylamino methacrylate (DMAEMA), (4-hydroxyphenyl) methacrylamide, 2-hydroxyethyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide. In some cases, the polymer comprises only one of isobutyl methacrylate (IBMA) and butyl methacrylate (BMA), but not both.

[0140] In some embodiments herein, the random polymer comprises (a) methyl methacrylate (MMA), (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA), (c) either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and (d) at least one compound of formula I: [ka]

[0141] wherein R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, R3 is NH2 or N(CH3)2 or OH, or R1 is O or NH, R2 and R3 together comprise a hydroxyphenyl, such as 4-hydroxyphenyl, or R1 is O or NH, R2 is an N,N-dimethylethylamine group and R3 is a sulfopropyl group. Such a polymer may, for example, contain monomers from (a), (b) and (c); or (a), (b) and (d); or (b), (c) and (d); or (a), (c) and (d); or each of (a) through (d), etc., thereby containing a total of at least three different monomers.

[0142] In some embodiments, the present disclosure provides a method for the preparation of a compound of formula I, comprising the steps of: (a) methyl methacrylate (MMA); (b) oligo(ethylene glycol) methyl ether methacrylate (OEGMA); (c) isobutyl methacrylate (IBMA) or butyl methacrylate (BMA); and (d) a compound of formula I, [ka]

[0143] where R1 is O or NH, R2 is methyl, ethyl, propyl or butyl, and R3 is NH2 or N(CH3)2 or a guanidinium group. The library is a random polymer library, comprising polymers comprising at least three monomers selected from: (a), (b) and (c); or (a), (b) and (d); or (b), (c) and (d); or (a), (c) and (d); or (a)-(d), etc., as described above, such polymers may comprise monomers from, for example, (a), (b) and (c); or (a), (c) and (d); or (a)-(d), etc., thereby comprising a total of at least three different monomers. In some cases, the polymer comprises a monomer of formula I, wherein the monomer is dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, or N-3-(dimethylamino)propyl methacrylamide. In some cases, the polymer comprises at least three of methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), dimethylamino methacrylate (DMAEMA) and N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or comprises four of these monomers. In some cases, the random polymer comprises at least one monomer of formula I selected from dimethylamino methacrylate (DMAEMA), 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide.

[0144] In any of the above cases, the polymer may have three monomers. In other cases, the polymer has four monomers. In still other cases, five monomers are used.

[0145] In some cases, the polymer includes three monomers, the three monomers being MMA, OEGMA, and either IBMA or BMA. In some cases, the three monomers are MMA, OEGMA, and IBMA. In some cases, the polymer includes four monomers: MMA, OEGMA, IBMA or BMA, and DMAEMA or ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA or BMA, and DMAEMA. In some cases, the four monomers are MMA, OEGMA, IBMA or BMA, and ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA, and DMAEMA or ArgMAm. In some cases, the four monomers are MMA, OEGMA, IBMA, and DMAEMA. In some cases, the four monomers are MMA, OEGMA, IBMA, and DMAEMA. In some cases, the four monomers are MMA, OEGMA, IBMA, and ArgMAm. In some cases, the polymer includes a mixture of both IBMA and BMA and MMA and OEGMA. In some cases, the polymer includes a mixture of both IBMA and BMA. In some cases, the polymer includes two or more compounds of formula I, such as both DMAEMA and ArgMAm.

[0146] In other embodiments, the random polymer comprises two monomers. In some embodiments, the two monomers are BMA or IBMA and OEGMA. In some cases, the two monomers are IBMA and OEGMA. In some cases, the two monomers are BMA and OEGMA. In other cases, the two monomers are ArgMAm and OEGMA.

[0147] In any of the above cases, in some embodiments the polymer has an average molecular weight of 3-20 kDa, hi some embodiments the polymer has an average molecular weight of 3-15 kDa, 5-20 kDa, 5-15 kDa, 5-10 kDa, 5-8 kDa, 10-20 kDa, 7-10 kDa, 8-10 kDa, 10-12 kDa, 12-15 kDa or 10-15 kDa.

[0148] When OEGMA is present, in some embodiments the polymer comprises OEGMA having a molecular weight of 300 to 1500 g / mol. In some such embodiments, the OEGMA may have a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol. In some cases, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500).

[0149] In some embodiments, the polymer may comprise 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, based on the total weight of the polymer. In some such cases, the polymer may be made from monomers consisting essentially of DMAEMA, IBMA, and OEGMA, e.g., 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, based on the total weight of the polymer. In other cases, at least one additional monomer, e.g., DMAEMA or ArgMAm, is also present. In some cases, the polymer may comprise 20-50% MMA, 20-50% OEGMA, 5-25% IBMA, and 5-25% DMAEMA. In some embodiments, the polymer comprises DMAEMA or ArgMAm, IBMA, OEGMA, and MMA. In some cases, the monomers are present in relative molar ratios of MMA, OEGMA, IBMA, and DMAEMA or ArgMAm as follows: (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2.5:1:1.5, (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; (g) 2:3:1:4; or (h) 5:2:1:2. In some embodiments, the MMA, OEGMA, IBMA, and DMAEMA are in a molar ratio of 5:2.5:2:0.5, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the polymer comprises a molecular weight of 3 to 15 kDa. In some embodiments, the polymer may be made from MMA, OEGMA, and IBMA in a molar ratio of 5:4:1 or 5:3:2 or 3:5:2. Such polymers may also have a molecular weight of 3 to 15 kDa. In some cases, the OEGMA may be OEGMA 500.

[0150] Certain exemplary polymers herein include, for example, Poly1, Poly3, Poly5, Poly7, Poly9, and Poly11, with the following molar ratios: Poly1:5:2.5:2:0.5, Poly3:2:5:2:1, Poly5:5:2.5:1:1.5, Poly11:4:4:1:1, Poly9:3:5:2:0; and (f) Poly7:5:3:2:0 with MMA, OEGMA, IBMA, and / or DMAEMA. In some such cases, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the polymer comprises a molecular weight of 3-15 kDa. In certain embodiments, the library comprises one or more of the above polymers Poly1, Poly3, Poly5, Poly7, Poly9, or Poly11, for example, in a length range corresponding to a molecular weight of 3-15 kDa. Corresponding polymers of different length / molecular weight in the library can be given letter designations A-H, depending on the average molecular weight from 3 to 15 kDa. Thus, for example, Poly1A contains an average molecular weight of about 3 kDa and is made from MMA, OEGMA, IBMA and DMAEMA in the following molar ratios: 5:2.5:2:0.5.

[0151] Further exemplary polymers herein include, for example, Poly20, Poly21 and Poly22 with MMA, OEGMA 500, IBMA and / or ArgMAm in the following molar ratios: Polyy20: 5:2:1:2; Poly21: 0:5:4:1; and Poly22: 4:4:1:1.

[0152] The disclosure herein also encompasses specific polymer libraries that can be used as test additives for specific protein formulations.For example, the disclosure encompasses a product that includes a series of microwell plates or similar containers that contain aliquots of members of the polymer library that can be added to specific protein formulations to test their effects on the turbidity, viscosity and other parameters of the formulation.

[0153] III. Preparation of Polymers and Polymer Libraries The present disclosure also encompasses methods of preparing the polymers or polymer libraries described herein. Acrylate and acrylamide polymers can also be prepared using, for example, reversible addition-fragmentation chain transfer (RAFT), free radical polymerization (FRP) and atom transfer radical polymerization (ATRP). For reviews of RAFT and ATRP methods, respectively, see, for example, S. Perrier, Macromolecules 50:7433-47 (2017); K. Matyjaszewski, Macromolecules 45(10):4015-39 (2012). In some embodiments, the polymers are prepared by a method that includes exposing selected monomers to LED light in the presence of zinc tetraphenylporphyrin catalyst (ZnTPP). In some cases, a chain transfer agent can be added to the reaction mixture, for example, to control the polymerization process and the addition of different monomers. The amount of chain transfer agent and / or catalyst can also help control the average length or average molecular weight of the polymer. Examples of chain transfer agents include 2-cyano-2-propylbenzodithioate, 2-cyano-2-propyldodecyltrithiocarbonate, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid or 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA). (See, e.g., Perrier, cited above.) Exemplary methods for making the polymers and libraries herein are described, for example, in Example 1 below. Methods for producing random polymers and polymer libraries compatible with the polymers and libraries of the present specification include, for example, those described in Gromley, AJ et al., Angew. Chem. Int. Ed. 2018 vol. 57(6), pp. 1557-1562 and Ng. G. et al., Macromolecules 2018 vol. 51(9) pp. 7600-7607.

[0154] IV. Protein Formulations and Their Properties Embodiments herein also include protein-containing formulations that include the polymer additives described herein. In some embodiments, the protein is an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof.

[0155] In some embodiments, the protein, such as an antibody, is present at a concentration of 20 mg / mL to 250 mg / mL, e.g., 20 to 200 mg / mL, 50 to 200 mg / mL, 50 to 150 mg / mL, 20 to 100 mg / mL, 50 to 100 mg / mL, 80 to 120 mg / mL, 100 to 200 mg / mL, or 150 to 200 mg / mL. In some embodiments, the protein formulation also includes at least one buffering agent.

[0156] In some embodiments, the polymer additive herein is present at a concentration of 0.001-1% w / v, such as 0.01-1% w / v or 0.01-0.1% w / v. In some embodiments, the polymer additive is present at 1-100 μM, such as 1-50 μM, 10-100 μM or 10-50 μM.

[0157] In some embodiments, the protein formulation comprises a surfactant such as polysorbate 20 (PS20), polysorbate 80 (PS80), Pluronic, e.g., poloxamer 188 or Pluronic F68, or Brij, as well as a surfactant such as an alkyl glycoside, e.g., octyl maltoside, decyl maltoside, dodecyl maltoside, or octyl glucoside, a cholate surfactant, e.g., CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), SGH (sodium glycocholate hydrate), sodium taurocholate hydrate (STH), sodium cholate hydrate (SCH), SdTH, SdCH, ScdCH, or BigCHAP (N,N'-bis-(3-D-gluconamidopropyl)cholamide). In other embodiments, the protein formulation does not comprise a surfactant. If a surfactant is present, in some embodiments the surfactant is present at a concentration of, for example, 0.001 to 1% w / v, such as 0.01 to 0.1% w / v.

[0158] In some embodiments, the protein formulation includes a stabilizer such as an amino acid, a sugar, a sugar alcohol, or albumin. In some embodiments, such stabilizers are absent (i.e., the formulation does not include an amino acid, a sugar, a sugar alcohol, or albumin as a stabilizer).

[0159] In some formulations, the formulation includes a salt, such as sodium or potassium chloride, acetate, citrate or phosphate, or, for example, arginine succinate, arginine hydrochloride or histidine hydrochloride. In other cases, the formulation does not include such a salt.

[0160] In some embodiments, the formulation does not include any other polymers beyond one or more of the specific polymers described herein.

[0161] In some embodiments, the formulation consists essentially of a protein, a buffer, and a polymer herein. In some embodiments, the formulation consists essentially of a protein, a buffer, a polymer herein, and a surfactant. In some embodiments, the formulation consists essentially of a protein, a buffer, a polymer herein, a surfactant, and a salt. In some embodiments, the formulation consists essentially of a protein, a buffer, a polymer herein, and an amino acid, a sugar, or a sugar alcohol. In some embodiments, the formulation consists essentially of a protein, a buffer, a polymer herein, a surfactant, and an amino acid, a sugar, or a sugar alcohol.

[0162] In some embodiments, the presence of a polymer significantly reduces the turbidity of the formulation as measured at 600 nm compared to an otherwise identical formulation that does not contain the polymer (see Example 2 below). In some embodiments, the presence of a polymer reduces the viscosity of the formulation at 25° C. and a shear rate of 1000 1 / s compared to an otherwise identical formulation that does not contain the polymer (see Example 3 below). In some embodiments, the turbidity and / or viscosity is reduced compared to a formulation in which the polymer is replaced with an equivalent concentration of a surfactant, such as PS20.

[0163] V. Protein Drug Testing In some embodiments, for example, different members of a polymer library with different molecular weights or lengths and different ratios of monomers or different monomer selections can be placed in the wells of a microwell plate filled with a protein solution for testing. Following such an approach, for example, a series of polymers with different ratios of monomers and / or different sets of monomers and different lengths (corresponding to different molecular weights) are obtained. In some cases, branched or linear polymers can also be compared. For example, a 96-well or similar multi-well plate can be used to test multiple polymers for their effect on a particular protein formulation compared to a control formulation.

[0164] In microplates, the effects on protein stability, viscosity, turbidity, precipitation, microscale thermophoresis and surface tension can be studied, for example, for polymers of different composition ratios or different monomers and for polymers of different lengths or molecular weights. Turbidity can be assessed, for example, by absorbance at a light wavelength of 600 nm. Exemplary assays are also described in Example 2 below. For example, viscosity can be measured with a rheometer at a particular temperature and shear rate. (See Example 3 below.) The degree to which a polymer interacts with a protein can also be measured for various polymer or protein concentrations by microscale thermophoresis, as described in Example 4 below. EXAMPLES

[0165] The present disclosure will be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the disclosure.

[0166] Example 1. Materials and Methods A. Material Reagents were purchased from Sigma Aldrich, VWR or Fisher Scientific and used without purification unless otherwise stated. Inhibitors were removed by passing monomer over basic alumina prior to polymerization. mAb-G was expressed in a Chinese hamster ovary (CHO) cell line. Sodium phosphate and histidine chloride buffers were prepared using pharmaceutical grade (USP, NP, EP) chemicals and MilliQ water. mAb-G was concentrated and buffer exchanged using Millipore (Billerica, MA) Amicon Ultra centrifuge tubes (10 or 30 kDa molecular weight cutoff, MWCO). mAb-G solutions were filtered through Corning 0.22 μm polyethersulfone (PES) vacuum filters (Corning, NY) or MilliporeSigma MillexGV 0.22 μm polyvinylidene fluoride (PVDF) filters (Burlington, MA) prior to the experiment. Pierce Bradford assay kits were purchased from ThermoFisher Scientific. Jurkat cells were purchased from ATCC (TIB-152, clone E6-1, lot number 70044353).

[0167] B. Polymer Library Synthesis A library of polymers was synthesized using chain transfer (i.e., polymerization) reactions. Liquid monomer was passed over basic alumina to remove any polymerization inhibitors present in the stock solution (e.g., hydroquinone monomethyl ether (also called p-methoxyphenol or MeHQ)). Stock solutions of monomer, chain transfer agent (CTA), and catalyst were prepared in DMSO. Respective volumes of stock solutions and DMSO were transferred to a 96-well plate and sealed with plate tape.

[0168] The polymers were composed of methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA) and dimethylamino methacrylate (DMAEMA). The monomer composition ranged from 5 to 50 mol% of the polymer, and the monomer molecular weights ranged from 3 to 20 kDa.

[0169] Polymers were prepared with the following molar ratios of monomers: MMA:OEGMA:IBMA:DMAEMA, Poly1 = 5:2.5:2:0.5, Poly3 = 2:5:2:1, Poly5 = 5:2.5:1:1.5, Poly7 = 5:3:2:0, Poly9 = 3:5:2:0, Poly11 = 4:4:1:1. Other monomers used: (4-hydroxyphenyl)methacrylamide (10-20 mol%), 2-hydroxyethyl methacrylate (20-25 mol%), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (zwitterionic monomer, 10-25 mol%), arginine methacrylate, 2-aminoethyl methacrylate hydrochloride.

[0170] In the first polymer library (Example 2 below), polymers were synthesized to obtain a range of lengths (measured as average molecular weight) from 3 to 15 kDa. For example, Poly1, with a molar ratio of MMA:OEGMA:IBMA:DMAEMA of 5:2.5:2:0.5, was made with a range of average molecular weights from minimum to maximum, designated A-H. Thus, for example, Poly1 was made with average molecular weights of 3 kDa (Poly1A), 7 kDa (Poly1D), 8.5 kDa (Poly1E), 10 kDa (Poly1F), Poly1B and Poly1C have molecular weights between those of Poly1A and Poly1D, and Poly1G has a molecular weight higher than that of Poly1F. Similar ranges of average molecular weight polymers were made for each of the polymers Poly3, Poly5, Poly7, Poly9 and Poly11.

[0171] To generate different polymer lengths, the molar ratios of CTA to total monomers were controlled as follows, assuming a monomer to polymer conversion of 85%: A 28:100; B 16:100, C 11:100, D 8:100, E 6:100, F 5:100, G 4:100.

[0172] Individual reactions using different ratios of monomers to produce polymers of different lengths or molecular weights were placed in separate wells of a 96-well plate.

[0173] Synthesis of CN-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm) ArgMAm was synthesized using a slightly modified version of a previously reported protocol (Funhoff et al., 2004). In a round-bottom flask under N2, N-(3-aminopropyl)methacrylamide hydrochloride (0.5 g, 2.8 mmol, 1 mol equiv.), 1H-pyrazole-1-formamidine monohydrochloride (0.41 g, 2.8 mmol, 1 mol equiv.), triethylamine (TEA) (0.9 mL, 6.7 mmol, 2.4 mol equiv.) and the polymerization inhibitor hydroquinone (5 mg) were dissolved in dimethylformamide (DMF) (8 mL). The reaction was stirred at room temperature (21 °C) for 24 h. The solution was poured into 50 mL of cold diethyl ether and the vessel was decanted to remove the oil. The oil was washed twice with 10 mL of acetonitrile and once with 5 mL of TEA, then centrifuged. The resulting solid was washed with 15 mL of dichloromethane (DCM), centrifuged, decanted, and further dried by rotary evaporation to give 147.14 mg (29% yield). 1 H-NMR(400mHz,DMSO-d6)ppm:8.03(t,J=5.6Hz,1H),7.75(t,J=5.9Hz,1H),7.22(s,2H),5.68(p,J=1. 1Hz,1H), 5.33(p,J=1.6Hz,1H),3.20-3.09(m,4H),1.86(dd,J=1.6,0.9Hz,3H),1.64(p,J=6.8Hz,2H).

[0174] D. Synthesis of Polymer Libraries via Photoinduced Electron Transfer Reversible Addition-Fragmentation Chain Transfer (PET RAFT) In the second polymer library (Example 3 below), polymers of the same target length were synthesized using Photoinduced Electron Transfer Reversible Addition-Fragmentation Chain Transfer (PET RAFT). Each polymer in the library is intended to contain the same length, as indicated by the same volumes of chain transfer agent and polymerization catalyst in the polymerization reaction for all polymers in the library.

[0175] Stock solutions of monomer (1 M), chain transfer agent (CTA) (0.2 M) and zinc tetraphenylporphyrin (ZnTPP) catalyst (8 mM) were prepared in dimethyl sulfoxide (DMSO). Respective volumes of stock solutions and DMSO were transferred to a 96-well plate (Table 1) and sealed with plate tape. Polymerization was initiated using a white 5k LED light and allowed to proceed for 3-18 h. Polymerization was terminated by removing the plate from the light source. Polymers were purified by microdialysis in 96 deep-well plates (Thermo Scientific, Pierce 3.5 kDa MWCO) against water for at least 2 days with five changes of dialysate. A representative 1 H-NMR Poly1 (400mHz, DMSO-d6) δppm: 4.15-3.88, 3.82-3.59, 3.57-3.48, 3.48-3.37, 3.29-3.19, 2.26-2.11, 2.11-1.58, 1.52-1.37, 1.37-1.20, 1.20-1.07, 1.02-0.84, 0.84-0.60. Representative M by GPC (polystyrene standard) n Poly1 10.1 kDa, D=1.12.

[0176] A second polymer library of Example 3 was prepared as in Table 1 below: TIFF2025512703000008.tif149170

[0177] Polymerization was initiated using white 5k LED light. The polymerization reaction was allowed to proceed for 3 hours. Polymerization was terminated by removing the 96-well reaction plate from the light source. The polymer was purified by microdialysis in deep 96-well plates against water or buffer for 2 days. The dialysate was changed 5 times.

[0178] Thus, each polymer from the library, corresponding to a particular selection and ratio of monomers and a particular average molecular weight, is present in a specific well of a 96-well plate for subsequent testing.

[0179] E. Nuclear magnetic resonance (NMR) spectroscopy Nuclear magnetic resonance (NMR) spectroscopy was performed on a 400 MHz Bruker NMR instrument. Gel porosity chromatography (GPC) was performed on an Agilent 1200 series equipped with a Wyatt Optilab T-rEX RI detector and a series of three Waters Styragel columns (HR 0.5, HR 2, HR 4) using tetrahydrofuran (THF) and polystyrene fixed standards (Agilent EasiVial PS-M calibration standards) as eluents at 1 mL / min. Protein concentrations were determined by variable pathlength ultraviolet-visible (TUV-vis) spectroscopy using a SoloVPE (C Technologies) connected to a Cary 60 UV-vis (Agilent) at 278 nm using the extinction coefficient. Absorbance precipitation assays and fluorescence measurements were performed on a Biotek Synergy Neo2 multimode plate reader.

[0180] F. Microscale Thermophoresis (MST) Polymer concentration of 15.5 µM and 2 x 10 -7 ~1×10 -4 MST was performed on a Nanotemper Monolith NT.Automated instrument with a Premium Coated capillary at a mAb-G concentration of 100 μM. The change in polymer fluorescence upon heating in the capillary was measured.

[0181] G. Isothermal titration calorimetry (ITC) ITC was performed using 25 injections of 2 μL on a TA instruments Nano ITC with a titration syringe volume of 50 μL and a starting cell volume of 450 μL. mAb-G (100 μM) and Poly1 (500 μM) were in 15 mM phosphate buffer pH 7.4. SCISSOR experiments were performed on a Sirius (now Pion) SCISSOR System.

[0182] H. Dynamic Light Scattering (DLS) DLS was performed on a Wyatt Dynapro plate reader at a polymer concentration of 0.1 w / v% in half-area 96-well plates at 25° C. Live / dead cell counts were measured using a Countess 3 FL (Invitrogen).

[0183] I. Polymer Turbidity / Sedimentation Assay Test sample monoclonal antibody protein (e.g., mAb G) was buffer exchanged into 15 mM sodium phosphate buffer pH 7.4. To a 96-well plate, water (control) or polymer / additive stock solutions were added to yield a final formulation containing 1.6 μM polymer and 80 mg / mL protein and 15 mM sodium phosphate, pH 7.4.

[0184] To serve as control samples, separate samples of protein were diluted in the same volume of phosphate buffer pH 7.4, but with or without 100 mM sodium chloride. Control formulations containing DMSO instead of sodium chloride and with buffer alone were also prepared.

[0185] Samples were pipetted into 96-well plates (total volume = 100 uL). Plates were incubated at 4°C for 18 hours at ambient conditions and turbidity was measured by absorbance at 600 nm. Measurements were recorded over a period of time starting after the incubation or after 24 hours of incubation. After the assay, samples were removed from the plates, centrifuged, and observed for any pellet formation to determine the extent to which the presence of polymer allowed the protein to remain suspended in solution and prevented the protein from precipitating during storage (as without the polymer any pellet would be composed of protein).

[0186] Turbidity measurements obtained using mAb G are shown below in Table 2 (Example 2), where solution turbidity was compared to mAb G in 100 mM NaCl (right-most column showing p-value for mAb G with salt). Samples contained 80 mg / mL mAb G in 15 mM phosphate buffer at pH 7.4 and, where indicated, contained 1.6 μM polymer. Measurements were taken after incubating samples at 4° C. for 18 hours as described above.

[0187] Turbidity measurements using mAb-G are described in Example 3 below.

[0188] In some examples described below, measurements were recorded at 25°C or 37°C instead of 4°C.

[0189] J. Polymer Viscosity Polymer viscosity was measured using a cone-and-plate rheometer with the temperature set at 25° C. Polymer, additive, or water (control) was added to each protein sample to give final additive concentrations ranging from 0.02 and 0.1% (w / v).

[0190] K. Surface tension (ST) A pendant drop tensiometer was used to measure the surface tension of solutions containing polymer or PS20 and a water control. The polymer Poly1F was prepared in water at 0.02% (w / v). PS20 was prepared in water at 0.05% (w / v). The tensiometer measures surface tension (ST) by recording an image of each drop formed from the test solution. The tensiometer software calculates the ST based on the shape of each drop. Each drop was allowed to sit for 30 minutes after formation so that measurements were recorded when the drop was at equilibrium. Measurements were taken in triplicate and the average ST was calculated.

[0191] L. High-throughput Absorbance-based Precipitation Assay mAb-G was buffer exchanged into 15 mM sodium phosphate buffer pH 7.4 and filtered. Water (control) or polymer stock solution (20% w / v in water) was added to a 96-well plate to give a 0.15% (w / v) solution at the time of protein addition. mAb-G was diluted to a final concentration of 80 mg / mL in the well with sodium phosphate buffer pH 7.4 containing NaCl to give a final salt concentration of 150 mM in the well. A no-salt control was prepared similarly. The solution in the tube was mixed thoroughly on a roller and filtered. The solution was pipetted into a 96-well plate containing polymer additive stock solution aliquots or water to a total volume of 100 μL. The plate was sealed, incubated at 25° C., and turbidity was measured by absorbance at 600 nm every 2 hours for 46 hours.

[0192] M. Low-throughput Eppendorf LoBind Tube Precipitation Assay mAb-G was buffer exchanged into 15 mM sodium phosphate buffer pH 7.4 and filtered. mAb-G was diluted to a final concentration of 80 mg / mL in the tubes with sodium phosphate buffer pH 7.4 containing NaCl for a final salt concentration of 150 mM in the wells. A no-salt control was prepared similarly. The solutions in the tubes were mixed thoroughly on a roller. To a series of empty, weighed Eppendorf LoBind tubes, polymer stock or water (controls) and mAb-G solution were added to a total volume of 200 μL. The tubes were incubated at 37°C in an incubator and periodically removed for analysis. The tubes were centrifuged at 3200 x g for 2 min, the supernatant was decanted and lyophilized. Soluble protein concentration was measured by SoloVPE as described in the analytical techniques section, and the precipitates were weighed after lyophilization.

[0193] N. FITC labeling of Poly1 FITC-maleimide was conjugated to the polymer by aminolysis of the trithiocarbonate followed by maleimide-thiol coupling. Poly1 (200 mg / mL, 50 μL) was dissolved in water in an Eppendorf tube. Tris(2-carboxyethyl)phosphine (TCEP) (30.71 mg) dissolved in 100 μL of water was added, followed by 20 uL of ethanolamine. The reaction was mixed on a shaker for 2 hours. FITC-maleimide (3.05 mg, 5 equiv.) was dissolved in 100 μL of 15 mM phosphate pH 7.4 buffer and 80 μL of DMF and added to the reaction mixture. The reaction was mixed for an additional 2 hours, then the polymer was purified by dialysis (Pierce Slide-A-Lyzer dialysis cassette, 3 kDa MWCO) against water for 2 days. FITC (494 nm, 68,000 M -1 cm -1 ) and Poly1 (420 nm, 69,400 M -1 cm -1 The degree of labeling (DOL) was calculated using the molar extinction coefficient of 0.3, a correction factor of 0.3, and the absorbance of the labeled polymer at wavelengths of 494 and 420 nm (Equation 1).

number

[0194] A 494 and A 420 If ε is the absorbance at 494 nm and 420 nm, then ε FITC ε Poly1 are the molar extinction coefficients of FITC and Poly1 at each wavelength. DOL 0.23568

[0195] O.SCISSOR-based solubility and pharmacokinetic (PK) assays SCISSOR experiments were performed as described in the literature (Bown et al., 2018a) with minor modifications. The chamber was filled with 300 mL of SCISSOR buffer containing 6.4 g NaCl, 0.09 g MgCl2·6H2O, 0.4 g KCl, 0.2 g CaCl2 and 2.1 g NaHCO3 per L Milli-Q water equilibrated to 34 °C and maintained at pH 7.4 with CO2. The SCISSOR cartridge was filled with 5 mL of a solution of 6.25 mg / mL 1.38 MDa hyaluronic acid (HA) dissolved in phosphate-buffered saline (PBS) pH 7.4 and kept approximately 1 cm above the bottom of the cartridge holder (above the clip) to allow detection of the lowest transmittance. Using a 1 mL syringe with a 25 G needle, 0.2 mL of mAb-G (80 mg / mL) was injected into the chamber at a constant rate over approximately 20 seconds. The autosampler was set to collect samples at 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360 and 390 min after injection. Standard curves for each method were used to measure the concentrations of mAb-G by Bradford assay or FITC-Poly1 by fluorescence measurement (100 μL samples in black 96-well plates, λ ex = 490 nm, λ em Off-line analysis of collected samples was completed by HPLC using a HPLC-MS / MS HPLC spectrophotometer as described by the manufacturer for the microplate procedure (96-well plate, 150 μL sample, 150 μL reagent, A 595nm ), and a Bradford assay was performed. Photographs of the cartridge through a window in the chamber to visualize any precipitate were also taken during the experiment.

[0196] P. Autofluorescence 100 μL of 1 mg / mL mAb-G stock and 100 μL of various concentrations (5-100 mol equivalents) of polymer stock, both in 15 mM phosphate buffer pH 7.4, were mixed in a 96-well plate (n=3 for each polymer concentration). Polymer without buffer and buffer only controls were also included and used to subtract as background. The plate was briefly centrifuged (20 s, approx. 150×g) and 50 μL of each solution was transferred to a black 384-well plate. The plate was briefly centrifuged (20 s, approx. 150×g) and fluorescence was measured (λ ex = 295 nm, λ em = 340 nm). In addition, absorbance was measured at 295 nm and 340 nm. Correction and control experiments were included as described in the literature (van de Weert & Stella, 2011a). In summary, absorbance measurements were used to correct for the inner filter effect (Equation 2).

number

[0197] In the formula, F obs and F corr are the measured and corrected fluorescence, respectively, and A ex and A em is the absorbance value at the excitation and emission wavelengths, and d ex and d em is the path length (cm). The path length in the well (0.2 cm) was estimated using the well dimensions and the volume of the sample in the well.

[0198] Using the Stern-Volmer equation (Equation 3), the quenching constant k q It was also confirmed that the fluorescence decrease was not due to collisional quenching by calculating

number

[0199] where F is the fluorescence at a given polymer concentration [L] and F ois the fluorescence without polymer, and τ is the fluorescence lifetime of tryptophan (1-10 ns). The quenching constant is 1.02×10 12 M -1 s -1 and 2×10 for the collisional quenching process. 10 M -1 s -1 It was bigger.

[0200] Q. Cell viability in Jurkat cells by trypan blue assay Jurkat cells were passaged four times in RPMI 1640 medium (10% FBS, 1% PenStrep) by incubation at 37°C, 95% humidity and 5% CO2. Cells were centrifuged and 4.4 x 10 6 The cells were suspended at 8 × 10 cells / mL in a sterile 24-well plate. 5 100 μL of cell suspension was added to each well for a final cell concentration of cells / mL. Poly1 was diluted in medium and sterile filtered. 400 μL of Poly1 solution or medium was added to each well (n=4). Cells were incubated for 24 hours and cell viability was assessed by trypan blue assay. Trypan blue was added 1:1 to the suspended cells and cells were counted for % live / dead. Cell viability was calculated by normalizing to controls without additive.

[0201] Example 2. Libraries containing polymers of different lengths Several monomers were selected to target various intermolecular interactions (see Figure 1). Two hydrophobic monomers, methyl methacrylate (MMA) and isobutyl methacrylate (IBMA), were selected to impart hydrophobic interactions with slightly different molecular weights. Two hydrophilic monomers were selected: oligo(ethylene glycol) methyl ether methacrylate 500 Da (OEGMA) as a hydrogen bond acceptor and to convey water solubility. 500) was selected as the positively charged monomer. To target interactions with the negative patches in the mAb, dimethylaminoethyl methacrylate (DMAEMA) was selected as the positively charged monomer. From these monomers, polymers Poly1, Poly3, Poly5, Poly7 and Poly9 were prepared, each with different lengths, designated by letters after the polymer name (e.g., Poly1A-G, Poly3A-G, etc.), with the letters ranging from shortest to longest.

[0202] A. Polymer turbidity Polymer turbidity was measured as described above in Example 1. As shown in Table 2, the absorbance at 600 nm (A) was increased by either removing the salt or replacing it with DMSO, or by the addition of certain polymer species. 600 ) was significantly reduced compared to protein in 100 mM NaCl. Items with an asterisk (*) are samples that were statistically significantly different from the relevant control as well as the positive control, specifically Poly1D, Poly1F and Poly7D. TIFF2025512703000012.tif175170

[0203] Further data are presented in Figures 2A-4 and 7A-B. Figures 2A-2B show the absorbance at 600 nm in formulations containing 80 mg / mL mAb G and 15 mM phosphate buffer at pH 7.4, and either 100 mM NaCl (Figure 2A) or 150 mM NaCl (Figure 2B), in the presence of increasing concentrations of the Poly7 species Poly7D (average 7 kDa), as well as two different molecular weight Poly1 species, Poly1D (average 7 kDa), Poly1F (average 10 kDa), over a range of polymer concentrations from 0 to 0.4 mM (specifically, 0, 1.6, 6.6, or 33 μM; at polymer concentrations of approximately 0.0014, 0.006, or 0.03% w / v). As shown in the figure panels, all polymers reduced the absorbance at 600 nm in the 100 mM NaCl formulation, but in the 150 mM NaCl formulation, only the two Poly1 species reduced the absorbance at 600 nm.

[0204] Figure 3 shows the precipitation kinetics of the above formulation with 150 mM NaCl and three different concentrations of Poly1E (average 12 kDa). All three concentrations of Poly1E were able to prevent a significant increase in absorbance at 600 nm over incubation times of up to 32 h at 23 °C. Figure 4 shows the precipitation kinetics of the above formulation in the presence of several different concentrations of polysorbate 20 (PS20) instead of a polymer additive. As shown in Figures 3-4, the presence of Poly1E resulted in a lower increase in absorbance over time compared to the increase in absorbance of solutions containing PS20 or control solutions without additives. Figure 7A shows images of aliquots of mAb G formulations containing 80 mg / mL mAb G, 15 mM phosphate buffer at pH 7.4, 150 mM NaCl, and no additional additives (left), with increasing concentrations of Poly1E (middle), and with increasing concentrations of PS20 (right). Figure 7B shows aliquots of formulations containing PS20 (left) or Poly1E (right) additives after centrifugation to separate the precipitating agent. As can be seen in Figure 7B, the formulation containing PS20 has significantly more insoluble precipitate after centrifugation than the formulation containing Poly1E.

[0205] B. Polymer Viscosity Polymer viscosity was measured as described in Example 1 above. Viscosity data is shown in Figures 5A-6. Figure 5A shows the change in viscosity (in cP) for mAb G solutions at 195 mg / mL of mAb G in 15 mM histidine chloride buffer at pH 5.5, 25°C with either Poly1D or PS20 additives in formulations with increasing concentrations from 0 to 0.1% w / v. Single point viscosity was measured at a shear rate of 1000 1 / s. As shown in Figure 5A, Poly1D was more effective than PS20 in reducing viscosity. Figure 5B shows the extent of viscosity reduction upon addition of 0.02% w / v Poly1D alone, 0.05% w / v PS20 alone, or a combination of both 0.02% Poly1D and 0.05% PS20. The change is similar for each polymer alone, but the addition of the two significantly reduced the viscosity further.

[0206] Figure 6 shows the viscosity dependence on shear rate for 194 mg / mL mAb G in 15 mM histidine chloride buffer at pH 5.5, 25 °C for shear rate gradients from 10 to 10,000 1 / s to evaluate the effect of shear rate on viscosity. As shown in the figure, in the absence of either PS20 or Poly1D, the mAb G formulation shows a decrease in viscosity upon increasing the shear rate between approximately 10 and 100 1 / s. The presence of either PS20 or Poly1D removes this dependence, indicating that both of these additives are surface active. In the next set of experiments, a shear rate of 1,000 1 / s was chosen.

[0207] The effect of additives on the viscosity of various protein formulation samples is shown in Tables 3 and 4 and Figures 9-12. The concentrations of each of the sample monoclonal antibody proteins mAbs G, B, C and E and the Fab fragment Fab G ranged from 194 to 207 mg / mL in 15 mM histidine chloride pH 5.5. The concentration of each polymer from the library was 33 μM (i.e., 0.03%).

[0208] The antibody species tested are as follows: mAb G, mAb B, mAb C and mAb E are monoclonal IgG antibodies, mAb D is a bispecific scFv monoclonal antibody, and Fab G is a Fab fragment.

[0209] Viscosity was measured in the library in 30 mM HisCl with and without 0.15% (w / v) polymer. In Figures 9-12, the average relative % change in viscosity with polymer (% change normalized by the initial viscosity without additive) is plotted by polymer composition (for several monomers) and polymer identity.

[0210] mAbs are classified as electrostatic or hydrophobic based on the mechanism that causes high viscosity. For solutions of mAb G, mAb E and mAb C, hydrophobic protein-protein interactions are believed to cause the change in viscosity, e.g., as shown in Table 3. For solutions of mAb B and Fab G, electrostatic interactions are believed to cause the change in viscosity, e.g., as shown in Table 4. In general, the table shows that the decrease in viscosity in the presence of polymer additives is greater when the viscosity is caused by hydrophobic interactions. TIFF2025512703000013.tif197170

[0211] C. Microscale Thermophoresis (MST) Microscale thermophoresis (MST), a method used to calculate the percentage of protein bound to a polymer, was used to measure the molecular interactions between the polymer and several proteins. MST was performed as described in Example 1 above. As shown in Figure 10, four different proteins in formulations containing Poly1F exhibited different degrees of molecular interactions with increasing polymer concentration.

[0212] Example 3. A library containing a series of polymers of the same length and varying monomer content A second polymer library was prepared as described above in Example 1 in 96-well plates using photoinduced electron transfer reversible addition-fragmentation chain transfer (PET RAFT) (Table 1; Figure 1). This technique has been used previously to prepare polymers in a high-throughput manner without the rigorous degassing required for other controlled radical polymerization techniques, and can be achieved at room temperature by photocatalyst-mediated visible light-initiated polymerization (Gormley et al., 2018; Ng et al., 2018).

[0213] Similarly, three hydrophilic monomers were selected, one neutral and two charged. Oligo(ethylene glycol) methyl ether methacrylate 500 Da (OEGMA) was used as a hydrogen bond acceptor and to convey water solubility.500 ) were selected. To promote electrostatic interactions with negative patches on the mAb surface and / or to increase electrostatic repulsion between mAbs, dimethylaminoethyl methacrylate (DMAEMA) and the arginine mimetic monomer N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm) were selected as positively charged monomers at pH 7-4 (Funhoff et al., 2004; Laaser et al., 2015; Lee et al., 2011). In addition, ArgMAm was selected as an arginine mimetic because arginine is a commonly used additive as a buffer component and arginine reduces viscosity when added as an additive in many protein formulations (Shukla & Trout, 2010; Sudrik et al., 2017). The library was developed to target the solubility of the final polymer in aqueous solutions with 2-4 monomers included in the composition while also including MMA, OEGMA, and DMAEMA. 500 The study was designed to investigate the composition of IBMA and DMAEMA or ArgMAm. Polymerization was confirmed by GPC using three randomly selected polymers. The molecular weight of the polymers described herein is approximately 10 kDa M n (Representative Poly1 M n 10.1kDa, D=1.12, Poly7 M n 11.3kDa, D=1.22, Poly22 M n The molecular weight was 12.3 kDa, D = 1.11). The polymer was purified by microdialysis in the same 96-well format against water for 3 days, and removal of DMSO and reactant impurities was confirmed by HPLC (Figure 13A-B).

[0214] A. Polymer turbidity A high-throughput turbidity screening assay based on visible light absorbance (above in Example 1) was used to investigate the effect of polymers from the second library on the precipitation of mAb-G. In preliminary experiments, the best sensitivity to precipitation was observed by measuring absorbance at 600 nm, which minimized the background from the protein (Figures 14A-B). Since the protein does not precipitate at low ionic strength at pH 7.4, the protein was buffer exchanged into 15 mM phosphate buffer pH 7.4. 150 mM sodium chloride (NaCl) and 0.15% heteropolymer additive stock solution were added. The absorbance was monitored at 600 nm to quantitate the precipitation of mAb. Plots of absorbance over incubation time are shown in Figures 15 and 16A-D. Three attributes of precipitation kinetics were determined: the time to onset of precipitation (t onset ), total area under the curve (AUC) and maximum absorbance (A max ) were quantified. Qualitative ranking compared to controls revealed promising polymer candidates (Figure 17). Several polymers with two or more white or no light gray attributes were selected as leads for further analysis.

[0215] To mitigate artifacts from evaporation observed in plate-based UV-vis assays at temperatures above 25 °C, characterization of precipitation under relevant temperature conditions was completed by a lower throughput, higher volume method in Eppendorf tubes (data not shown). mAb-G, NaCl solution and polymer solution formulated in 15 mM phosphate buffer pH 7.4 were added to a series of tubes, and the concentration of mAb-G in the supernatant after centrifugation and the weight of the dry pellet were measured at predetermined time intervals. Surprisingly, most of the polymers selected from the screening did not improve the solubility of mAb-G (Figure 18A-B). In one case, at higher concentrations of polymer (Poly11), precipitation was faster than without additive. Although changes in temperature and polymer concentration could have played a significant role in how additives affected the precipitation kinetics of mAb-G at 37 °C, it had three "white" attributes at 25 °C (i.e., values ​​in the white box in Figure 17 indicating a large improvement in precipitation kinetics). While 1% Poly1 did not improve precipitation (Figure 18A-B), 0.15% Poly1, when added at 0.15%, improved precipitation by a statistically significant amount compared to no additive during the first 48 hours of the experiment (Figure 19A). Higher concentrations of polymer could result in steric crowding by the mAb, overpowering any beneficial effects. Additionally, both Poly22 and Poly11 are less hydrophobic than Poly1, which may contribute to the difference in their effects on mAb-G precipitation compared to Poly1. Given these results, 0.15% Poly1 was selected for continued evaluation.

[0216] B. Polymer Viscosity It was expected that the in vivo protein concentration upon subcutaneous (sc) injection would gradually decrease as the mAb diffused away from the injection site. It was anticipated that the addition of polymer could prevent injection site reactions induced by protein precipitation if the protein concentration fell below the solubility limit prior to the time to precipitation. To more fully evaluate precipitation in vitro and to assess the effect of varying mAb-G concentrations as a result of diffusion, the SCISSOR system (as described in Example 1), an instrument developed for the in vitro evaluation of subcutaneous mAb pharmacokinetics (PK), was used. The system uses a sample cartridge containing hyaluronic acid (HA) in a buffer solution, with a dialysis membrane conditioned to mimic mAb subcutaneous PK in humans (Bown et al., 2018a; Kinnunen et al., 2015). The pH and temperature of the device are also monitored and controlled. The system can measure the in-line percent transmittance of the cartridge throughout the experiment, and aliquots from the bulk reservoir are automatically removed for offline analysis. Interestingly, during the first experiment, mAb-G precipitation was observed immediately upon injection, unlike previous in vitro experiments where mAb-G precipitation was observed on the order of hours. This may be due to steric crowding and increased viscosity of the HA matrix used. We calibrated the injection volume by injecting 50 mg / mL mAb-G, which we observed from in vivo experiments was the highest concentration that did not induce injection site inflammation by mAb-G in vivo (data not shown) (Figure 20A-B). Future experiments to investigate the effect of cartridge matrix on precipitation may improve this model for evaluation of precipitation, as cartridge conditions were developed to correlate with mAb PK in humans and do not necessarily reflect the physiological composition of the subcutaneous space.

[0217] First, the precipitation and PK of additive-free mAb-G were measured by in-line transmittance, offline protein concentration quantification by Bradford assay, and recording images of the cartridge through the cell window. When mAb-G was injected into the cartridge, the protein precipitated, as shown by the transmittance from the bottom channel (channel 4) decreasing to 60% within 5 min (Figure 19C) and the image of the cartridge (Figure 19D). The extent of precipitation decreased when mAb-G was formulated with 0.15% Poly11, as seen in the cartridge image (Figure 19D). The transmittance data from channel 4, which increased to approximately 80% within 5 min (Figure 19C), confirms the reduced extent of precipitation. The release of mAb-G from the cartridge measured by Bradford assay showed similar initial release rates with and without Poly1 (Figure 19F), and the total release of mAb-G was slightly less when Poly1 was present. Note that the total amount of mAb-G released was only statistically significantly different (p<0.05) after approximately 270 min. This difference could be due to protein-polymer interactions that could alter the diffusion of mAb-G and its interaction with the HA matrix. Additionally, the PK of the polymer alone used a polymer fluorescently labeled with FITC. The polymer disappeared faster than mAb-G, as expected due to its lower molecular weight (10 kDa) (Figure 19G). However, the rate of release also suggests that interactions occur between the slightly positively charged polymer and the negatively charged HA matrix. The higher error of the replicates could result from the heterogeneity of the micelles formed from the self-assembly of the random heteropolymer due to its amphiphilic nature, as shown in DLS (Figure 27).

[0218] Example 4. Properties of Poly1 polymer A. Poly1 as a surfactant The mechanism by which the polymer improves colloidal stability was also investigated. Because the polymer contains a mixture of hydrophobic and hydrophilic monomers, it was hypothesized that the polymer has the potential to act as a surfactant and form micelles. Surface tensions (ST) measured by pendant drop tensiometer (as described in Example 1 above) were consistent with this hypothesis. The critical micelle concentration (CMC) of Poly1 was found to be 0.002-0.02% by measuring the ST at several polymer concentrations (Figure 28A-D). Poly1 above its CMC reduced the ST of water from 72 to 40.3 mN / m, which was similar to the ST reduction observed using polysorbate 20 (PS20) above its CMC (Mittal, 1972) (Table 4). Interestingly, Poly1 altered the precipitation kinetics in a plate-based absorbance assay, whereas PS20 did not (Figure 16A-D). Thus, we hypothesized that an additional mechanism was at work in this system. TIFF2025512703000014.tif33170

[0219] B. Microscale Thermophoresis (MST) Poly1 was evaluated for interaction with mAb-G through intermolecular interactions including electrostatic interactions, hydrogen bonds and hydrophobic / van der Waals interactions. Several methods were used to evaluate the binding of Poly1 to proteins. First, microscale thermophoresis (MST; as described in Example 1 above) was first used to evaluate the binding of Poly1 to mAb-G due to its sensitivity to changes in interactions and the small volume required (Figure 21A) (Jerabek-Willemsen et al., 2011). A constant Poly1 concentration with increasing amounts of protein was used to obtain an estimated dissociation constant (K) of greater than 0.5 mM. d ) were calculated. For protein solubilities and viscosities above 1 mM, complete binding curves with true quantification of binding were not achieved.

[0220] C. Isothermal titration calorimetry (ITC) Next, isothermal titration calorimetry (ITC; as described in Example 1 above) was also used to evaluate the enthalpy of binding interaction (Figure 21B). Viscosities above 1 mM for mAb-G and above 100 μM for Poly1 prevented the generation of a complete ITC binding curve (Figure 23). However, the millimolar concentrations used showed an order of magnitude of interaction. For redundancy, the enthalpy of binding was determined by measuring the enthalpy of demicellization and the critical micelle concentration (CMC) (Bam et al., 1998). The total heat of demicellization, polymer dilution and protein-polymer binding was determined from the heat released during titration of the polymer into buffer solution and into mAb-G solution. The CMC estimated by this technique was 0.01 mM (0.01%), which is consistent with the results obtained from ST measurements (Table 4). The chosen concentrations that alter the precipitation kinetics of mAB-G are above the CMC to ensure sufficient polymer concentration in the bulk relative to the surface. The enthalpy of binding of polymer to mAb-G, approximately -0.18 kcal / mol, calculated from the change in the initial slope of titrating the polymer into mAb-G, indicates a weak interaction between the two species. The endotherm of binding indicates entropy-driven binding, likely due to hydrophobic interactions and the release of bound "structured" water (Ladbury & Chowdhry, 1996). As expected, autofluorescence decreased with increasing amounts of polymer. Molar equivalents of polymer to achieve this decrease, on the order of 10-100, further indicated the weak nature of this interaction. To ensure that the decrease in fluorescence was due to binding and not due to artifacts from inner filter effects or collisional quenching (van de Weert & Stella, 2011a), control experiments were performed as detailed in Example 1. Assuming 1:1 binding, the association constant (K a ) was calculated (Figure 21D) and the corresponding K d was estimated to be approximately 0.9 mM, the same order of magnitude as the MST results.

[0221] D. Poly1 autofluorescence Finally, autofluorescence quenching (as described in Example 1 above) was used to qualitatively corroborate the findings of the other two techniques (Figure 21C-D). Autotryptophan fluorescence was measured in the presence of increasing concentrations of Poly1 (Figure 21C). As expected, autofluorescence decreased with increasing amounts of Poly1. The molar equivalents of Poly1 required to achieve this decrease were on the order of 10-100, again indicating the weak nature of this interaction. To ensure that the decrease in fluorescence was due to binding and not due to artifacts from inner filter effects or collisional quenching as described in the publication by van de Weert and Stella, control experiments were included as detailed in Methods and Materials (van de Weert & Stella, 2011b). Assuming 1:1 binding, the association constant (K a ) was calculated (Figure 21D) and the corresponding K d was estimated to be approximately 0.9 mM, the same order of magnitude as the MST results.

[0222] Summary of the interaction of E.Poly1 with mAB-G From biophysical studies, it is possible that the inclusion of Poly1 altered mAb-G precipitation via binding equilibrium. Similar studies investigating viscosity reduction via reversible PEGylation of high concentration mAb formulations also demonstrated that PEG conjugation with many proteins within a short period of time disrupted mAb self-interactions (Gong et al., 2019). PEG conjugation with many proteins within a short period of time disrupted mAb self-interactions (Gong et al., 2019). Similar to the need for increasing concentrations to measure binding in this report, binding experiments using surface plasmon resonance (SPR) required high concentrations (100 mM) of mPEG-phenylglyoxal to generate association / dissociation curves. For the random heteropolymers described herein, molecular weight, strength of binding, number of binding sites and additional mechanisms such as micellization also contribute to the mechanism of precipitation inhibition.

[0223] F. Poly1 cytotoxicity The polymer additives described in the examples herein may be used in pharmaceutical formulations, such as pharmaceutical proteins, including antibodies, in some cases, and subcutaneous (sc) formulations that may require high protein concentrations. Poly1 toxicity was therefore investigated in vitro. Since Poly1 is a surfactant, the membrane integrity of the cells was used as a measure of cytotoxicity by trypan blue assay. Jurkat cells were used to model toxicity to immune cells and for ease of experimentation as a suspension cell line. Addition of Poly1 at all tested concentrations did not significantly alter cell viability (Figure 22). Polymer immunogenicity, biocompatibility and bioaccumulation are also considerations for the development of new polymer additives for pharmaceutical formulations. Immune responses to polymers are generally believed to be driven by flexibility and regular repeating structures. The polymers described herein contain a relatively random distribution of monomers, making them advantageous over other polymers. Anti-PEG antibodies have been observed in some products and bioconjugates containing PEG (Kozma et al., 2020; Zhang et al., 2016). However, OEGMA 500 The length of the PEG side chains of the monomer (500 Da) is below the ~750 Da required to conjugate / generate anti-PEG antibodies (Huckaby et al., 2020). Finally, SCISSOR experiments showed that the polymer is likely cleared from SC tissue into the circulation at a rate similar to or slightly faster than mAb-G. Furthermore, the polymer reported here is ~10 kDa, which is smaller than the ~30 kDa molecular weight limit of renal clearance for PEG (Bertrand & Leroux, 2012). Thus, the polymer is expected to be excretable.

[0224] Consideration As described in the Examples herein, a library of random heteropolymers was synthesized and screened for the ability of the polymers in the library to improve the solubility of monoclonal antibodies. The polymers were synthesized and purified in a high-throughput fashion. A plate-based absorbance turbidity assay was used to screen the polymers for their effect on the solubility of monoclonal antibody mAb-G, which precipitates at physiological pH and salt. Selected polymers were further studied for their solubilizing effect on mAb-G under more appropriate conditions (temperature, in the presence of HA as a simulated matrix). Mechanistic studies showed that the polymers could act as surfactants and weakly interact with mAb-G to alter the kinetics of precipitation. In summary, random heteropolymers are a promising new class of additives that may improve the safety and ease of formulating poorly soluble proteins at high concentrations, especially for subcutaneous administration.

[0225] The changes in precipitation kinetics in the presence of various polymers obtained from the initial solubility screen at 25 °C (Figure 15) did not fully correspond to the results from the follow-up study at 37 °C (Figures 18A-B and 19A-B). For example, Poly22 and Poly11 showed slower t onset and smaller AUC, and Poly22 has a lower AUC than Poly1. max At 37°C, only Poly1 extended the time to precipitation (Figs. 19A-B). Furthermore, only 1% Poly11 exacerbated the precipitation of mAb-G compared to 1% Poly1, Poly22, and Poly7, which were the same as the control (Fig. 17). Of these polymers, Poly11 and Poly22 were the least hydrophobic, with relatively high mole percent mOEGMA. 500Poly1 has DMAEMA and Poly22 incorporates ArgMAm instead of DMAEMA. These results, taken together with the ITC experiments (Figure 21B), indicate that hydrophobic interactions between the polymer and mAb are likely important and contribute to the structure-property correlation that portrays Poly1 as the best polymer for improving mAb-G colloidal stability. The temperature dependence of the mAb-G precipitation time scale, with the slowest kinetics at roughly room temperature (25°C) and faster kinetics occurring at higher (37°C) temperatures, also indicates the importance of hydrophobic interactions. Underlying this change may be increased structural flexibility exposing more hydrophobic residues coupled with increased desolvation resulting in a larger entropy increase at higher temperatures (Chen et al., 2003) and an increase in the strength of hydrophobic interactions (Baldwin, 1986). Additionally, differences in diffusion rates and / or solubility of aggregates may contribute to this temperature dependence (Wang & Roberts, 2018). Overall, the kinetics of the precipitation process and how the random heteropolymers affect the initial stages / seeding of precipitation appear to be important in this system.

[0226] The polymer additives described in the examples herein may be used in pharmaceutical formulations, such as pharmaceutical proteins, including antibodies, in some cases, and subcutaneous (sc) formulations, which may require high protein concentrations. Polymer immunogenicity, biocompatibility, and bioaccumulation are each considerations for the development of new polymer additives for pharmaceutical formulations. Immune responses to polymers are generally believed to be driven by flexibility and regular repeating structures. The polymers described herein contain a relatively random distribution of monomers, making them more advantageous than other polymers.

[0227] Another aspect of pharmaceutical formulation development, such as for subcutaneous antibody formulations, is ensuring product quality during manufacturing, storage, and administration of these therapeutics. Precipitation observed during any of these stages, including upon injection, can limit the development of mAbs, especially if the mAb is intended for subcutaneous administration or must be formulated at high concentrations. For the mAbs described herein, the relatively high salt and pH under physiologically relevant conditions resulted in an increase in non-covalent intermolecular interactions that led to precipitation (Pindrus et al., 2015). The observable difference in the precipitation kinetics of mAb-G in simulated subcutaneous matrices indicates that random heteropolymer additives may enable the delivery of poorly soluble mAbs via subcutaneous injection. 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Claims

1. 1. A random polymer library comprising a mixture of polymers, said library comprising: a. methyl methacrylate (MMA), b. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA), c. isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and d. Compound of Formula I 【Chemistry 1】 (In the formula, R １ is O or NH, and R ２ is methyl, ethyl, propyl or butyl, R ３ is NH ２ or N(CH ３ ) ２ or a guanidinium group) 1. A random polymer library comprising a polymer comprising at least three monomers selected from the group consisting of:

2. 2. The random polymer library of claim 1, wherein the library comprises polymers comprising monomers of Formula I, wherein the monomers are dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl)methacrylamide, N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propylmethacrylamide.

3. 2. The random polymer library of claim 1, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise at least three types of monomers, or the random polymer library consists essentially of polymers comprising at least three types of monomers.

4. 2. The random polymer library of claim 1, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise at least four types of monomers, or the random polymer library consists essentially of polymers comprising at least four types of monomers.

5. A random polymer library comprising a mixture of polymers, each polymer comprising a mixture of at least three of the following monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), and dimethylamino methacrylate (DMAEMA), said library comprising polymers between 3 and 20 kDa.

6. A random polymer library comprising a mixture of polymers, each polymer comprising a mixture of at least three of the following monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), and N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), said library comprising polymers between 3 and 20 kDa.

7. 2. The random polymer library of claim 1, wherein the library comprises polymers of 3-15 kDa, 5-20 kDa, 5-15 kDa, 5-10 kDa, 10-20 kDa, 7-10 kDa, or 10-15 kDa.

8. the library comprises OEGMA having a molecular weight of 300 to 1500 g / mol, and optionally a) the OEGMA has a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol; b) The random polymer library of claim 1, wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500).

9. The random polymer library of claim 1 , wherein the library comprises polymers comprising three types of monomers.

10. 10. The random polymer library of claim 9, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise three types of monomers, or the random polymer library consists essentially of polymers comprising three types of monomers.

11. the three monomers being MMA, OEGMA, and either IBMA or BMA, and optionally a) the three monomers are MMA, OEGMA and IBMA; 10. The random polymer library of claim 9, wherein b) the MMA, OEGMA, and IBMA or BMA are present in a ratio of (a) 5:3:2, or (b) 3:5:2, or (c) 5:4:

1.

12. The random polymer library of claim 1 , wherein the library comprises polymers containing four types of monomers.

13. 13. The random polymer library of claim 12, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the polymers in the library comprise three types of monomers, or the random polymer library consists essentially of polymers comprising three types of monomers.

14. the four monomers being MMA, OEGMA, IBMA and DMAEMA, and optionally a) the polymer library comprises polymers comprising 20-50% MMA, 20-50% OEGMA, 5-25% IBMA and 5-25% DMAEMA based on the total weight of the polymer; b) the polymer library comprises polymers comprising MMA, OEGMA, IBMA, and DMAEMA in the molar ratios of (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2.5:1:1.5, (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; and / or (g) 2:3:1:4; c) The random polymer library of claim 12, wherein the MMA, OEGMA, IBMA, and DMAEMA are in a molar ratio of 5:2.5:2:0.5, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the library comprises polymers having a molecular weight of 3 to 15 kDa.

15. the four monomers are MMA, OEGMA, IBMA and ArgMAm, and optionally a) the polymer library comprises polymers comprising 20-50% MMA, 20-50% OEGMA, 5-25% IBMA and 5-25% ArgMAm by total weight of polymer; b) the polymer library comprises polymers comprising MMA, OEGMA, IBMA, and ArgMAm in the molar ratios of (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2:1:2, (d) 4:4:1:1; (e) 3:4:1:2; (f) 2:4:1:3; and / or (g) 2:3:1:4; c) The random polymer library of claim 12, wherein the MMA, OEGMA, IBMA, and ArgMAm are in a molar ratio of 5:2:1:2 or 4:4:1:1, the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and the library comprises polymers having a molecular weight of 3 to 15 kDa.

16. 1. A random polymer library comprising a mixture of polymers, the library comprising polymers comprising at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and / or at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), optionally wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and optionally wherein the library comprises polymers having a molecular weight of 3 to 15 kDa.

17. A random polymer, a. methyl methacrylate (MMA), b. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA), c. isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and d. Compound of Formula I 【Chemistry 2】 (In the formula, R １ is O or NH, and R ２ is methyl, ethyl, propyl or butyl, R ３ is NH ２ or N(CH ３ ) ２ or a guanidinium group) A random polymer comprising a mixture of at least three monomers selected from:

18. The polymer comprises a monomer of formula I 【Transformation 3】 18. The random polymer of claim 17, comprising: dimethylamino methacrylate (DMAEMA), 2-aminoethyl methacrylate, arginine methacrylate, arginine methacrylamide, N-(3-aminopropyl) methacrylamide, N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), or N-3-(dimethylamino)propyl methacrylamide.

19. A random polymer comprising a mixture of at least three of the following monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), and dimethylamino methacrylate (DMAEMA), wherein the polymer is 3 to 20 kDa.

20. A random polymer comprising a mixture of at least three of the following monomers: methyl methacrylate (MMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA), isobutyl methacrylate (IBMA), and N-(3-methacrylamidopropyl) guanidinium chloride (ArgMAm), wherein the polymer is 3 to 20 kDa.

21. 18. The random polymer of claim 17, wherein the polymer is 3-15 kDa, 5-20 kDa, 5-15 kDa, 5-10 kDa, 10-20 kDa, 7-10 kDa, or 10-15 kDa.

22. the polymer comprises OEGMA having a molecular weight of 300 to 1500 g / mol, and optionally a) the OEGMA has a molecular weight of 300, 500, 750, 950, 1000, 1200 or 1500 g / mol; b) The random polymer of claim 17, wherein the OEGMA has a molecular weight of 500 g / mol.

23. 20. The random polymer of claim 17, wherein the polymer comprises three types of monomers.

24. 24. The random polymer of claim 23, wherein the three monomers are MMA, OEGMA, and either IBMA or BMA, and optionally the three monomers are MMA, OEGMA, and IBMA, and optionally the MMA, OEGMA, and IBMA or BMA are present in a ratio of (a) 5:3:2, or (b) 3:5:2, or (c) 5:4:

1.

25. 20. The random polymer of claim 17, wherein the polymer comprises four types of monomers.

26. the four monomers being MMA, OEGMA, IBMA and DMAEMA, and optionally a) the polymer comprises, based on the total weight of the polymer, 20-50% MMA, 20-50% OEGMA, 5-25% IBMA and 5-25% DMAEMA; b) the polymer comprises MMA, OEGMA, IBMA, and DMAEMA in the molar ratios of (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2.5:1:1.5, (d) 4:4:1:1, (e) 3:4:1:2; (f) 2:4:1:3; or (g) 2:3:1:4; c) The random polymer of claim 25, wherein the MMA, OEGMA, IBMA and DMAEMA are in a molar ratio of 5:2.5:2:0.5, the OEGMA is OEGMA 500, and the polymer has a molecular weight of 3 to 15 kDa.

27. the four monomers are MMA, OEGMA, IBMA and ArgMAm, and optionally a) the polymer comprises, based on the total weight of the polymer, 20-50% MMA, 20-50% OEGMA, 5-25% IBMA and 5-25% ArgMAm; b) the polymer comprises MMA, OEGMA, IBMA, and ArgMAm in the molar ratios (a) 5:2.5:2:0.5, (b) 2:5:2:1, (c) 5:2.5:1:1.5, (d) 4:4:1:1, (e) 3:4:1:2; (f) 2:4:1:3; or (g) 2:3:1:4; c) The random polymer of claim 25, wherein the MMA, OEGMA, IBMA and ArgMAm are in a molar ratio of 5:2:1:2 or 4:4:1:1, the OEGMA is OEGMA 500, and the polymer has a molecular weight of 3 to 15 kDa.

28. A random polymer comprising a mixture of at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and either isobutyl methacrylate (IBMA) or butyl methacrylate (BMA), and / or at least two monomers selected from oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and N-(3-methacrylamidopropyl)guanidinium chloride (ArgMAm), optionally wherein the OEGMA has a molecular weight of 500 g / mol (i.e., OEGMA 500), and optionally wherein the polymer has a molecular weight of 3 to 15 kDa.

29. 18. The random polymer of claim 17, wherein the polymer has a molecular weight of 7 to 10 kDa.

30. A composition comprising the random polymer of any one of claims 17 to 29 and at least one protein.

31. 31. The composition of claim 30, wherein the polymer is at a concentration of 0.01 to 1% w / v, such as 0.01 to 0.1% w / v.

32. The composition of claim 30, wherein the protein is an antibody, optionally wherein the antibody is an IgG antibody, such as a full-length IgG, a bispecific IgG, or wherein the antibody is an antigen-binding fragment, such as a Fab, Fab', (Fab')2, Fv, or scFv.

33. The composition comprises: a) at least one buffering agent, such as histidine, phosphate, or citrate, and / or 31. The composition of claim 30, further comprising b) at least one surfactant such as a polysorbate, e.g., polysorbate 20 or polysorbate 80.

34. 31. The composition of claim 30, wherein the antibody is at a concentration of 10 mg / mL to 150 mg / mL, e.g., 10-100 mg / mL, 20-100 mg / mL, 20-80 mg / mL, 10-50 mg / mL, 10-25 mg / mL, 25-50 mg / mL, 50-80 mg / mL, 50-100 mg / mL or 70-100 mg / mL.

35. 30. A method for reducing the viscosity of a protein-containing composition and / or inhibiting precipitation of a protein-containing composition, said method comprising adding a random polymer according to any one of claims 17 to 29 to a protein-containing composition, optionally wherein said composition comprises a protein and / or an additive.

36. 30. A method for preparing the random polymer library of any one of claims 1 to 16 or the random polymer of any one of claims 17 to 29, wherein the method comprises reversible addition-fragmentation chain transfer (RAFT), free radical polymerization (FRP) or atom transfer radical polymerization (ATRP).

37. A method for preparing the random polymer library of any one of claims 1 to 16 or the random polymer of any one of claims 17 to 29, comprising exposing the monomers to LED light and / or heat in the presence of a zinc tetraphenylporphyrin catalyst (ZnTPP) and a chain transfer agent.

38. 38. The method of claim 37, wherein the chain transfer agent is 2-cyano-2-propylbenzodithioate, 2-cyano-2-propyldodecyltrithiocarbonate, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, or 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA).

39. 38. The method of claim 37, wherein the monomer is exposed to LED light in the presence of the ZnTPP and chain transfer agent for 3 to 18 hours.

40. 38. The method of claim 37, wherein the polymerization is terminated by removing the LED light.

41. 37. The method of claim 36, further comprising performing at least one filtration, buffer exchange, or dialysis step to isolate the polymer.

42. 17. A kit comprising the random polymer library of any one of claims 1 to 16 for use in testing polymers contained within the library for their effect on precipitation, viscosity and / or turbidity of a solution containing a protein, the kit optionally further comprising instructions for use, and optionally the library comprising at least 10, at least 20, at least 50 or at least 80 different individual random polymers.

43. 17. A method for identifying a random polymer additive for a protein solution, comprising exposing the protein solution to a random polymer library of any one of claims 1 to 16 and determining one or more of the viscosity, turbidity or precipitation of the solution.

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