Engineered polymeric excipients for enhancing the stability of protein biologics

Abstract

A therapeutic formulation comprising a protein and a polymeric excipient; a polymeric excipient that enhances the stability of therapeutic protein formulations; a method for stabilizing the therapeutic protein formulations; and a method of administering a therapeutic protein formulation to a patient in need thereof by administering to the patient the therapeutic protein formulation.

Description

70668-02 Engineered Polymeric Excipients for Enhancing the Stability of Protein Biologics CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application no.63 / 634,725, which was filed April 16, 2024. The entire contents of the provisional patent application and the appendix thereto are hereby incorporated by reference in their entireties. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CBET-2211843 awarded by the National Science Foundation and CA023168 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present disclosure relates to a polymeric excipient that enhances the stability of therapeutic protein formulations and a therapeutic protein formulation comprising the polymeric excipient. BACKGROUND

[0004] Over the past two decades, there has been a rapid proliferation of monoclonal antibody- based therapies targeting cancer, autoimmune, and degenerative diseases. Despite the well- established biochemical stability of antibodies, they, like all proteins, remain susceptible to denaturation and aggregation. The formation of aggregates, in particular, poses a significant safety risk for patients due to their well-documented immunogenicity. Consequently, there is a need for advancements in formulations, storage conditions, and product purity to improve the long-term colloidal stability of therapeutic antibody solutions. These improvements are crucial not only for reducing health risks for patients but also for minimizing the economic losses experienced by pharmaceutical companies and healthcare systems.70668-01

[0005] Formulations often incorporate excipients such as sugars, salts, and buffers to improve protein stability, which helps to mitigate protein aggregation. However, the amphiphilic nature of antibody proteins has sparked increased interest in the literature regarding protein adsorption from the aqueous bulk solution onto various surfaces / interfaces, including the air-water, solid-water, and oil-water interfaces. Throughout drug product processing, storage, and clinical administration, protein molecules encounter multiple interfaces. It is hypothesized that as proteins adsorb onto interfaces, they may undergo denaturation and unfolding, ultimately leading to aggregate formation in the bulk solution (Trends Biotechnol 2014, 32(7), 372-380). These aggregates, along with sub-visible and visible particles, have the potential to adversely affect biological drug products by limiting shelf life, reducing the effective drug dose, and potentially triggering an immunological response.

[0006] To mitigate surface / interface-induced protein aggregation, significant research has explored the stabilizing ability of surfactants, particularly polysorbates, focusing on the competitive adsorption kinetics between proteins and polysorbates at the air-water interface. One illustrative study investigated the adsorption of polysorbate 80 (also known as Tween 80) in competition with proteins at the air-water interface, utilizing a Wilhelmy Plate tensiometer. This study revealed that the steady-state interfacial behavior was predominantly governed by surfactant adsorption (J. Pharm. Sci. 2009, 98, 9, 3099-3107). Despite the success of polysorbates in outcompeting proteins for surface adsorption, there is a need for investigations into substances with minimal quantities capable of effectively displacing proteins at the interface. Materials that readily adhere to aqueous interfaces, even in small quantities (causing higher surface pressure), would assist in addressing the challenges associated with future requirements for protein drug additives. Polysorbates raise additional concern regarding toxicity and instability. These include oxidative degradation, which produces damaging peroxides, and potential interactions between the excipient and cargo in bulk. Such interactions could compromise the therapeutic efficacy of the formulations (European Journal of Pharmaceutics and Biopharmaceutics 2015, 97, 60-67).

[0007] It is an object of the present disclosure to provide a polymeric excipient that improves the stability of a therapeutic formulation of a protein by suppressing protein aggregation and / or70668-01 denaturation. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description. SUMMARY

[0008] Provided is a therapeutic formulation comprising (i) a protein and (ii) an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA), and poly(poly(ethylene glycol) methacrylate) (PPEGMA). The AB or BAB-type block copolymer can stabilize the therapeutic formulation of protein. In some embodiments, the block copolymer is an excipient.

[0009] The A block polymer can exhibit an equilibrium surface pressure ranging from about 10 mN / m to about 72 mN / m at room temperature. In some embodiments, the A block polymer has a lower critical solution temperature (LCST) ranging from about -20 °C to a physiological body temperature (e.g., 37 °C). The A block polymer can be poly(N-alkyl-substituted acrylamide). In some embodiments, the A block polymer is poly(N-isopropyl acrylamide) (PNIPAM). In some embodiments, the A block polymer is poly(N-methacryloyl-L-alanine methyl ester). In some embodiments, the B block polymer is poly(ethylene glycol) (PEG). In some embodiments, the AB block copolymer is poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PNIPAM-PEG). In some embodiments, the BAB block copolymer is poly(ethylene glycol)-b-poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PEG-PNIPAM-PEG). The A block polymer can be poly(2- alkyl-2-oxazoline). In some embodiments, the A block polymer is selected from poly(2-isopropyl- 2-oxazoline), poly(2-n-propyl-2-oxazoline) and poly(2-cyclopropyl-2-oxazoline).

[0010] The protein can be selected from an antibody, an antibody-drug conjugate, an antibody fragment, an enzyme, an immunogenic protein, a fusion protein, a FC fusion protein, a protein fragment, a polypeptide, a peptide drug, a structural peptide, a lipoprotein, a growth factor and a combination of two or more thereof. In some embodiments, the protein is an antibody. In a therapeutic formulation, the block copolymer can be present in an amount that is effective in70668-01 suppressing the aggregation and / or denaturation of the protein. In therapeutic formulation, the block copolymer can be present in an amount ranging from about 5 × 10-5to about 5 × 105ppm. The amount of the protein present in the therapeutic formulation can be about 5 × 10-5ppm to about 5 × 105ppm.

[0011] Provided is a method for stabilizing a therapeutic formulation comprising a protein, which method comprises adding to the therapeutic formulation in an amount effective to suppress aggregation and / or denaturation of the protein an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA), and poly(poly(ethylene glycol) methacrylate) (PPEGMA), whereupon the therapeutic formulation comprising the protein is stabilized.

[0012] Further provided is a method of administering a therapeutic formulation to a patient in need thereof, which method comprises administering to the patient the therapeutic formulation as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present disclosure will be more readily understood from the detailed description of embodiments presented below considered in conjunction with the attached drawings of which:

[0014] Fig.1 depicts a graphic representation of the effect of the polymeric excipient on protein aggregation.

[0015] Fig.2a depicts surface pressure Π as a function of elapsed time after injecting 1.0 mL of each sample with a concentration of 5.0 mg / mL for immunoglobulin G (IgG), Tween 80, Tween 20, poly(N-isopropyl acrylamide) (PNIPAM), and poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PNIPAM–PEG) into the subphase (to a final concentration of 7.14 x10-3mg / mL in a total of 700 mL subphase).70668-01

[0016] Fig. 2b depicts comparative Π as a function of elapsed time after injecting 1.0 mL of mixtures with a total concentration of 5.0 mg / mL for IgG + Tween 80 (1:1 by weight), IgG + Tween 20 (1:1 by weight), IgG + PNIPAM-PEG (1:1 by weight), Tween 20 + PNIPAM-PEG (1:1 by weight), and Tween 80 + PNIPAM-PEG (1:1 by weight) into the subphase (to a final concentration of 7.14 x 10-3mg / mL in a total of 700 mL subphase).

[0017] Fig. 3a depicts time-resolved dynamic light scattering (DLS) analysis and DLS profiles before and after the experiment for Tween 80 under 55 °C. The intensity values of the histograms have been normalized so that the highest peak intensity is set to 100. All DLS measurements were conducted at an excipient concentration of 2.5 mg / mL, which is well above the known critical micelle concentration (CMC) value for Tween 80 (MW = 1,310 Da): 0.013 mg / mL in pure water at 25 °C.

[0018] Fig. 3b depicts time-resolved dynamic light scattering (DLS) analysis and DLS profiles before and after the experiment for Tween 20 under 55 °C. The intensity values of the histograms have been normalized so that the highest peak intensity is set to 100. All DLS measurements were conducted at an excipient concentration of 2.5 mg / mL, which is well above the known CMC value for Tween 20 (MW = 1,228 Da): 0.060 mg / mL for in pure water at 25 °C.

[0019] Fig. 3c depicts time-resolved dynamic light scattering (DLS) analysis and DLS profiles before and after the experiment for PNIPAM-PEG under 55 °C. The intensity values of the histograms have been normalized so that the highest peak intensity is set to 100. All DLS measurements were conducted at an excipient concentration of 2.5 mg / mL, which is well above the CMC value determined for PNIPAM-PEG (Mn= 11,000 Da): 0.125 ± 0.25 mg / mL for in PBS (1×).

[0020] Fig.3d depicts the relative count ratio (S / S0), i.e., the count rate at time t (S) divided by the count rate at t = 0 (S0), from DLS analysis for Tween 80, Tween 20, and PNIPAM-PEG.

[0021] Fig.4a depicts the relative count ratio (S / S0) from DLS analysis for IgG with and without surface.70668-01

[0022] Fig. 4b depicts the time-resolved DLS analysis of IgG aggregation under accelerated conditions at 55 °C, depicting with surface and without surface. The intensity values of the histograms have been normalized so that the highest peak intensity is set to 100.

[0023] Fig. 4c depicts DLS profiles before and after the experiment for IgG with and without surfaces. The intensity values of the histograms have been normalized so that the highest peak intensity is set to 100.

[0024] Fig.5a depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 0.3 mg / mL of Tween 80 and DLS profiles before and after the experiment.

[0025] Fig.5b depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 2.5 mg / mL of Tween 80 and DLS profiles before and after the experiment.

[0026] Fig. 5c depicts the relative count ratio (S / S0) from DLS analysis for IgG with different weight concentrations of Tween 80, ranging from 0.3 to 2.5 mg / mL.

[0027] Fig.6a depicts the relative count ratio (S / S0) from DLS analysis for IgG with two different weight concentrations of Tween 20: 0.3 mg / mL and 2.5 mg / mL.

[0028] Fig.6b depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 0.3 mg / mL of Tween 20 and DLS profiles before and after the experiment.

[0029] Fig.6c depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 2.5 mg / mL of Tween 20 and DLS profiles before and after the experiment.70668-01

[0030] Fig.7a depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 0.3 mg / mL of PNIPAM-PEG and DLS profiles before and after the experiment.

[0031] Fig.7b depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C at a concentration of 2.5 mg / mL of PNIPAM-PEG and DLS profiles before and after the experiment.

[0032] Fig. 7c depicts the relative count ratio (S / S0) from DLS analysis for IgG with different weight concentrations of PNIPAM-PEG, ranging from 0.3 to 2.5 mg / mL.

[0033] Fig.8 depicts degrees of IgG aggregation (^^^^IgGA,2h / ^^^^IgG,0h) estimated under conditions of accelerated IgG aggregation, considering cases with surface, without surface, and with the addition of excipients (Tween 80, Tween 20, and PNIPAM-PEG).

[0034] Fig.9 depicts (a) circular dichroism (CD) spectra for IgG (5.0 mg / mL in PBS (1×) buffer) and excipients (Tween 80 and PNIPAM-PEG, 5.0 mg / mL in PBS (1×) buffer) at 25 °C. (b) CD analysis for IgG (2.5 mg / mL in PBS (1×) buffer) without excipients and with excipients (Tween 80 and PNIPAM-PEG, 2.5 mg / mL in PBS (1×) buffer) was conducted under accelerated aggregation conditions similar to the DLS experiment (55 °C). The curves were shifted by a factor of 10 for clarity. (c) Ellipticity at ^^^^ = 220 nm from CD analysis of different IgG solutions with and without excipients (Tween 80 and PNIPAM-PEG) in (b).

[0035] Fig.10 depicts the relative viscosities of a 2.5 mg / mL PNIPAM-PEG solution in PBS (1×), a 2.5 mg / mL Tween 80 solution in PBS (1×), and a 2.5 mg / mL Tween 20 solution in PBS (1×) at both 25 °C and 55 °C. Relative viscosity is defined as the viscosity of the surfactant solution divided by the viscosity of the medium, which in this case is PBS (1×). These data are further supported by Fig. 11, which shows that PNIPAM-PEG micelle solutions remained optically transparent under all tested concentration and temperature conditions (0.3125 – 2.5 mg / mL, 25 –70668-01 55 °C); micelles likely did not undergo agglomeration or shape transformation at elevated temperatures.

[0036] Fig. 11 shows photographic images of PNIPAM-PEG solutions in PBS (1×) at four different concentrations: 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, and 2.5 mg / mL, taken at both 25 °C and 55 °C after 24-hours equilibration. The PNIPAM-PEG micelle solutions remained optically transparent under all tested concentration and temperature conditions (0.3125 – 2.5 mg / mL, 25 – 55 °C), which indicates that the micelles likely did not undergo agglomeration or shape transformation at elevated temperatures. The solutions of Tween 80 and Tween 20 at the same concentrations also remained clear at both temperatures after 24-hours equilibration period.

[0037] Fig.12 depicts DLS intensity-based size distribution histograms for a 2.5 mg / mL PNIPAM- PEG solution in PBS (1×) at 25 °C before and after filtration through a PTFE filter with a 0.2 μm pore size (N = 5). The average hydrodynamic diameter of the PNIPAM-PEG micelles slightly decreased from 27.36 ± 2.03 (N = 5) before filtration to 21.43 ± 0.29 (N = 5) after filtration.

[0038] Fig. 13 depicts gel permeation chromatography (GPC) traces of PNIPAM-PEG and PNIPAM.

[0039] Fig.14 depicts the DLS intensity correlation function (C(τ)) of PNIPAM-PEG in PBS (1×) solutions at varying concentrations, measured at 25 °C.

[0040] Fig.15a depicts the hydrodynamic diameter (Dh) of PNIPAM-PEG in PBS (1×) solutions at varying concentrations, measured at 25 °C.

[0041] Fig.15b depicts the intensity count rate of PNIPAM-PEG in PBS (1×) solutions at varying concentrations, measured at 25 °C.70668-01

[0042] Fig.16 depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C with Tween 80 concentrations of (a) 0.3 mg / mL, (b) 0.5 mg / mL, (c) 1.5 mg / mL, and (d) 2.5 mg / mL.

[0043] Fig.17 depicts the time-resolved DLS investigation of IgG aggregation under accelerated conditions at 55 °C with PNIPAM-PEG concentrations of (a) 0.3 mg / mL, (b) 0.5 mg / mL, (c) 1.5 mg / mL, and (d) 2.5 mg / mL. DETAILED DESCRIPTION

[0044] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the claimed invention is thereby intended.

[0045] The term "excipient" refers to a substance formulated alongside the active ingredient of a medication. It is a therapeutically inactive substance. Excipients serve various purposes, including long-term stabilization and bulking up solid formulations containing potent active ingredients in small amounts (often referred to as "bulking agents", "fillers", or "diluents").

[0046] The term "therapeutic formulation" refers to a formulation containing a therapeutically effective amount of active ingredients, e.g., proteins, drugs, etc., and an excipient, with or without other optional components.

[0047] The term "antibody" refers to a protein produced by the body's immune system when it detects harmful substances, called antigens. It includes as non-limiting examples monoclonal antibodies (including, for example, full-length antibodies with an immunoglobulin Fc region), single-chain molecules, bi-specific and multi-specific antibodies, diabodies, antibody-drug conjugates, antibody compositions having polyepitopic specificity, and fragments of antibodies (including, for example, Fab, Fv, Fc, and Fab ').70668-01

[0048] The present disclosure is based on the development of a polymeric excipient to enhance protein stability and alleviate aggregation. The aggregation of protein under varied temperature and pH conditions alters the protein structure. The polymeric excipient can reduce the degradation of a therapeutic protein. The block copolymers can act as a polymeric excipient and stabilize therapeutic protein formulations.

[0049] Provided is a therapeutic formulation comprising (i) a protein and (ii) an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA), and poly(poly(ethylene glycol) methacrylate) (PPEGMA).

[0050] The polymeric excipient can comprise a block copolymer, wherein the block copolymer can be an AB-type diblock copolymer or a BAB-type triblock copolymer. The block copolymer can comprise an A block polymer, wherein the A block polymer is selected from poly(N-alkyl- substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and a B block polymer, wherein B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA). The A block polymer can be poly(N-alkyl-substituted acrylamide). In some embodiments, the A block polymer is poly(N-isopropyl acrylamide) (PNIPAM). In some embodiments, the A block polymer is poly(N-methacryloyl-L-alanine methyl ester). In some embodiments, the B block polymer is poly(ethylene glycol) (PEG). In some embodiments, the AB- type block copolymer is poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PNIPAM-PEG). In some embodiments, the BAB-type block copolymer is poly(ethylene glycol)-b-poly(N- isopropyl acrylamide)-b-poly(ethylene glycol) (PEG-PNIPAM-PEG). In some embodiments, the A block polymer can be poly(2-alkyl-2-oxazoline). The poly(2-alkyl-2-oxazoline) can be selected from poly(2-isopropyl-2-oxazoline), poly(2-n-propyl-2-oxazoline) and poly(2-cyclopropyl-2- oxazoline).70668-01

[0051] Examples of poly(N-ester-substituted acrylamide) include, but are not limited to, poly(N- acryloylglycine) (PNAG), poly(N-acryloylglycine methyl ester) (PNAGME), poly(N- acryloylglycine ethyl ester) (PNAGEE) and poly(N-acryloylglycine propyl ester) (PNAGPE).

[0052] The polymeric excipients can enhance the stability of any therapeutic protein formulation both in solution and at the air-water interface. They can suppress or reduce protein aggregation and / or protein denaturation. The polymeric excipient can also reduce undesired immunological responses of therapeutic drugs and can preserve the protein's structural integrity. The polymeric excipient such as PNIPAM has a strong affinity for the air-water interface. PNIPAM can prevent protein adsorption at the air-water interface. PNIPAM can effectively cover the surface of a protein, thereby reducing protein exposure and providing protection against aggregation triggered at the interface. Thus, PNIPAM can prevent surface-initiated aggregation and maintain the structural integrity of the protein. PNIAPM-PEG consists of two distinct components: the PEG segment acts as an excipient within the bulk solution, while the PNIPAM segment functions as a surface / interface-covering material with a known capability to maintain high surface pressure about 32 mN / m (such as 32 mN / m), even in a dilute state. Langmuir trough experiments involving PNIPAM-PEG, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and a model immunoglobulin G (IgG) compound at adsorption equilibrium states, revealed that PNIPAM-PEG exhibited the highest surface pressure. This observation indicates that PNIPAM has the strongest thermodynamic affinity for the air-water interface, thereby preventing other substances (such as IgG proteins) from reaching the air-water interface. Few water-soluble polymers possess such a strong affinity for the air-water interface. Those that do are anticipated to exhibit lower critical solution temperature (LCST) characteristics in water, given their marginal hydrophilicity, which can be easily disrupted by environmental changes, such as temperature fluctuations. The use of PNIPAM is advantageous because its surface affinity can be concentration-independent, in contrast to polysorbates. For example, as illustrated in Figs. 2a and 2b, at relatively low concentrations (e.g., below their respective critical micelle concentration), PNIPAM-PEG can be significantly more effective in reducing surface tension compared to Tween 20. Also PNIPAM is non-toxic and safe for use in therapeutic formulations.

[0053] The A block polymer can exhibit an equilibrium surface pressure ranging from about 10 mN / m to about 72 mN / m at room temperature, such as about 10 mN / m to 72 mN / m, 10 mN / m to70668-01 about 72 mN / m, or 10 mN / m to 72 mN / m. The A block polymer can have LCST ranging from about -20°C to about 37 °C, a physiological body temperature (e.g., -20 °C to 37 °C). In some embodiments, the A block polymer can have LCST below about room temperature. In some embodiments, the A block polymer can have LCST ranging from about -20 °C to room temperature. In some embodiments, the A block polymer can have LCST ranging from about 0 °C to room temperature. In some embodiments, the A block polymer can have LCST ranging from about 0 °C to about 37 °C. In some embodiments, the A block polymer can have LCST ranging from about room temperature to about 37 °C.

[0054] The protein can be a therapeutic protein. The protein having therapeutic effects can be termed as a therapeutic protein. The therapeutic protein can be selected from antibodies, antibody- drug conjugates, antibody fragments, enzymes, immunogenic proteins, fusion proteins, FC fusion proteins, protein fragments, polypeptides, peptide drugs, structural peptides, lipoproteins, growth factors, and a combination of two or more thereof. In some embodiments, the protein is an antibody, an antibody-drug conjugate, and an antibody fragment. Desirably, the protein is an antibody. Antibodies can be monoclonal antibodies. Examples of monoclonal antibodies include, but are not limited to, immunoglobulins (IgGs), for example, IgG1, IgG2, IgG3, IgG4, and IgGM.

[0055] The block copolymer can be present in an effective amount to suppress protein aggregation and / or protein denaturation. The amount of the protein present in the therapeutic formulation can be ranging from about 5 × 10-5ppm to about 5 × 105ppm. The amount of the block copolymer can be ranging from about 5 × 10-5to about 5 × 105ppm.

[0056] The therapeutic formulation can further comprise any other known drugs. The therapeutic formulation may include other components and / or ingredients, including, but not limited to, other therapeutically active compounds and / or one or more pharmaceutically acceptable carriers, diluents, and the like. The carrier or excipient can vary based on the particular route of administration (see, e.g., Remington’s The Science and Practice of Pharmacy, 23rd ed. (2020)). The therapeutic formulation can be a liquid formulation.

[0057] Provided is a method for stabilizing a therapeutic formulation comprising a protein, which method comprises: adding to the therapeutic formulation in an amount effective to suppress70668-01 aggregation and / or denaturation of the protein an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA), and poly(poly(ethylene glycol) methacrylate) (PPEGMA), whereupon the therapeutic formulation comprising the protein is stabilized.

[0058] The therapeutic formulation can be stable when the aggregation and denaturation of a protein contained therein is prevented, and the protein substantially retains its physical and chemical stability and its therapeutic properties upon storage under varied storage conditions. The formulation can be stored at a temperature from about -200 °C to 60 ° C.

[0059] Provided is a polymeric excipient comprising an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester- substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA), and poly(poly(ethylene glycol) methacrylate) (PPEGMA). The polymeric excipient can be incorporated into any protein drug formulation to enhance the stability of the protein.

[0060] The polymeric excipient can be prepared using reversible addition-fragmentation transfer (RAFT) polymerization. The method comprises: i) combining a solution of a monomer (e.g., N-isopropyl acrylamide (NIPAM)) in a first organic solvent (e.g., 1, 4 dioxane) with a RAFT agent (e.g., 4-cyano-4- [(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid, (CDSP) and an initiator (e.g., azobisisobutyronitrile) to obtain a first solution; ii) subjecting the first solution to a freeze-thaw cycle; and iii) repeating step (ii) to obtain a homopolymer (e.g., poly(N-isopropyl acrylamide) PNIPAM); iv) mixing the RAFT agent with a monomethoxy-monohydroxy-terminated polyethyleneglycol (PEG-OH) in the presence of 4-dimethylaminopyridine and a second organic solvent to obtain a conjugate PEG-RAFT; and70668-01 v) mixing the PEG-RAFT and the homopolymer (PNIPAM) to provide a copolymer e.g., PNIPAM-PEG.

[0061] In some embodiments, the number-average molecular weight (Mn) value of the PNIPAM homopolymer is about 4,500 g / mol. For PNIPAM-PEG, the Mn values are about 6,000 g / mol for the RAFT-synthesized PNIPAM block and about 5,000 g / mol for the PEG block. In some embodiments, the polydispersity indices (PDI) for PNIPAM homopolymer is about 1.12, and for PNIPAM–PEG copolymer is about 1.19. In some embodiments, the critical micellization concentration (CMC) of PNIPAM-PEG in PBS (1×) at 25 °C is about 1.75 ± 0.25 mg / mL.

[0062] Further provided is a method of administering a therapeutic formulation comprising a protein to a patient in need thereof. The method comprises administering to the patient the above- described therapeutic formulation. The formulation can be administered by any suitable route, as is known in the art. Dosages of the formulation can be determined in accordance with methods known in the art.

[0063] PNIPAM-PEG’s effectiveness was evaluated in reducing the aggregation rate of human polyclonal IgG antibodies, using temperature-accelerated DLS-based colloidal stability measurements. Additionally, the relative stability of PNIPAM-PEG to Tween 80 and Tween 20 in dispersing IgG proteins was compared. The findings suggest that PNIPAM-PEG can prevent protein aggregation both in bulk solution and at interfaces. PNIPAM, known to become water- insoluble above its LCST(~ 32 °C), can undergo self-assembly into micelle morphology when integrated into the PNIPAM-PEG polymer. This property can facilitate its easy clearance from the body at physiological temperature (37 °C). Relative viscosity studies of PNIPAM-PEG solutions in PBS (1×) at various concentrations ranging from about 0.31 mg / mL to about 2.5 mg / mL and temperatures ranging from about 25 °C to about 55 °C show that PNIPAM-PEG micelles did not undergo agglomeration or shape transformation at elevated temperatures, and the solutions remained optically transparent. These characteristics enhance the potential applicability of PNIPAM-PEG as a promising solution for addressing protein aggregation concerns in protein biologics formulations.70668-01 EXPERIMENTAL

[0064] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.

[0065] To study protein aggregation, various experimental methods like size exclusion chromatography (SEC), field-flow fractionation (FFF), and analytical ultracentrifugation (AUC) are commonly used for detection and quantification. However, these techniques carry the risk of physically stressing the sample, which could potentially disrupt existing aggregates or induce the formation of new aggregates during analysis. Moreover, these methods may lack the required sensitivity to detect the onset of aggregation at very low concentrations.

[0066] In contrast, light scattering techniques, while they may not provide mass fraction data or consider insoluble proteins, offer the advantage of not subjecting samples to physical stress or inducing aggregation during data collection. (ACS Applied Materials and Interfaces 2020, 12, 8, 9977-9988). The sensitivity of light scattering methods to very small amounts of moderate-size particles is due to the fact that light scattering varies with the sixth power of the particle size. As a result, these techniques can detect aggregation levels well below the detection limit of the methods mentioned above. Moreover, advanced spectroscopic techniques offer real-time insights into molecular interactions and structural changes without requiring extensive sample manipulation.

[0067] Similarly, dynamic light scattering (DLS) emerges as a powerful technique for analyzing colloidal dispersions. Typically used to evaluate the average size of nearly spherical particles in stable, dilute dispersions, DLS provides a fundamental output—the average hydrodynamic diameter (Nanoparticle Research, 2000, 2, 2, 123-131; Langmuir 2015, 31, 1, 3-12 and Journal of Colloid Interface Science, 1998, 207, 1, 150-158). For IgG proteins, this hydrodynamic diameter closely corresponds to the protein’s largest dimension, approximately 11 nm for a monomer. (Biophysics Journal, 2014, 106, 8, 1763-1770) An increased average size reported by DLS indicates the presence of aggregates, demonstrating remarkable sensitivity. Notably, even a single aggregate within the laser focus’s tiny volume can significantly influence the reported size, highlighting the precision of DLS in detecting subtle changes. The application of DLS has been70668-01 extensive in the detailed investigation of aggregate size, providing valuable insights in various contexts (The Journal of Chemical Physics 1988, 88, 6, 4070-4075 and Macromolecules 2003, 36, 6, 2093-2102). However, the precise quantification of aggregate size, especially in the case of IgG, remains a challenging aspect. Nevertheless, it is widely recognized that DLS serves as a valuable indicator of IgG aggregation, contributing to the evolving landscape of analytical techniques in protein characterization.

[0068] The Brookhaven DLS instrument was used to evaluate the stability of diverse protein dispersions. This system integrates DLS with a programmable temperature ramping platform, allowing the sample to reach thermal equilibrium before data acquisition at a specified temperature, followed by the temperature ramp. Particle size distribution estimation is conducted at each temperature interval, and an increase in particle size indicates potential protein aggregation. This comprehensive analysis can be completed within a few hours, effectively highlighting differences in stability among protein dispersions with distinct aggregation points. The aggregation point is identified by the temperature at which the average protein cluster size undergoes a rapid increase (Biochemistry 2003, 42, 48, 14234-14241). Materials

[0069] Polysorbate 80 (Tween 80), Polysorbate 20 (Tween 20), N-isopropylacrylamide (NIPAM), 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDSP), azobisisobutyronitrile (AIBN), monomethoxy-monohydroxy-terminated PEG (mPEG–OH), dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), dichloromethane (DCM), and lyophilized polyclonal Immunoglobulin G (IgG) (MW = 150 kDa) were obtained from commercial vendors. Milli-Q®water was used for the initial reconstitution of the IgG protein to its original concentration, and phosphate-buffered saline (PBS) tablets or powder were used to prepare PBS buffer (pH 7.4) for diluting the sample solutions. PNIPAM-PEG synthesis

[0070] PNIPAM was synthesized via Reversible Addition Fragmentation Transfer (RAFT) polymerization. N-isopropylacrylamide (NIPAM) was recrystallized in a 50 / 50 (v / v) heptane / toluene solvent mixture before polymerization. Subsequently, NIPAM (2.0 g) was70668-01 dissolved in 1,4-dioxane (6.0 g) and combined with 4-cyano-4-[(dodecylsulfa nylthiocarbonyl)sulfanyl] pentanoic acid (CDSP, 0.148 g) as the RAFT agent and azobisisobutyronitrile (AIBN, 0.012g) as the initiator. The solution underwent three freeze-thaw cycles and was purged with nitrogen. Polymerization was conducted at 70 °C for 3 hours. The resulting products were precipitated twice in hexane and then subsequently dried under vacuum. A PEG macroinitiator was utilized to synthesize PNIPAM-PEG. Initially, the RAFT agent was conjugated to purified monomethoxy-monohydroxy-terminated PEG (mPEG–OH, Mn= 5,000 g / mol) through Steglich esterification (Angewandte Chemie International Edition in English 1978, 17, 7, 522-524). The reaction involved mixing PEG–OH (4 g, 0.8 mmol), the RAFT agent (645 mg, 1.6 mmol), and 4-dimethylaminopyridine (DMAP, 19.36 mg, 0.16 mmol) in 12 mL dichloromethane (DCM). The mixture was stirred magnetically at 0 °C. Simultaneously, a separately prepared solution of dicyclohexylcarbodiimide (330 mg, 1.6 mmol) in dichloromethane (8 mL) was added drop-wise to the above mixture. The reaction proceeded for 5 min at 0 °C and then for 3 hours at 20 °C to yield PEG–RAFT. The synthesized PEG–RAFT product was initially filtered through filter paper to remove the insoluble urea byproduct and further purified by precipitation in hexane twice. The RAFT polymerization reaction was then conducted. Time-Dependent Surface Pressure Measurements

[0071] Time-dependent surface pressures were monitored using the KSV 5000 or NIMA Langmuir trough, which has dimensions of 51.8 × 15.0 cm2without barrier coverage. Milli-Q®water was used as the subphase solvent, with a surface tension of 72 mN / m at 25 °C. The Wilhelmy plate employed was a Whatman CHR1 chromatography paper plate with a dry width of 1 cm. To ensure accuracy, the true perimeter of the water-saturated filter paper was calibrated using the known value of the air-water surface tension at 25 °C. Before each measurement, the trough underwent meticulous cleaning, including three washes with acetone, followed by rinsing with Milli-Q®water. Using a pipette with its tip-tilted at 45° angle, solution samples, including IgG, Tween 80, Tween 20, and PEG–PNIPAM, were introduced under a 3 cm depth from the air-water interface. The samples were directed toward the perimeter of the Wilhelmy plate at a rate of 0.2 mL / s.70668-01 Dynamic Light Scattering (DLS) Analysis

[0072] Size distributions, serving as indicators of both monomers and aggregates within the dispersion, were evaluated using DLS with a Brookhaven ZetaPALS instrument. This analysis utilized a laser with a wavelength of 659 nm to measure scattering intensities at a 90° incidence angle. Prior to measurement, the sample underwent filtration with a PTFE 0.2 μm pore size filter. The temperature was incrementally raised to 55 °C, providing a suitable range for an accelerated antibody aggregation test based on the experimental time (2 hours). Data was collected over time to document variations in size distributions, revealing the presence of both unimers and aggregates in the dispersion. DLS results (Figs.3A – 7c and Figs.14, 16, and 17) were obtained by analyzing raw intensity correlation data using the temperature-dependent viscosities of water as the medium's viscosities at various temperatures. All tested surfactant concentration and temperature conditions (0.3125 mg / mL – 2.5 mg / mL, 25 °C – 55 °C), the relative viscosities of the surfactant solutions (PNIPAM-PEG, Tween 80, or Tween 20) remained close to unity, as demonstrated in Figs 10 and 11. To facilitate quantitative analysis, all DLS samples were filtered through a PTFE filter with a 0.2 μm pore size unless specified otherwise. As illustrated in Fig.12, this filtration step removed larger particles, which, based on calculations from Eqs. (1) – (4) are estimated to represent a negligible fraction (approximately 28 ppm by weight) of the total dispersed polymer mass in the case of the 2.5 mg / mL PNIPAM-PEG in PBS (1×). For other samples, including Tween 80, Tween 20, and IgG solutions, the filtration had negligible impact on the solution composition. Circular Dichroism (CD) Analysis

[0073] CD measurements were performed using a Jasco J-1500 CD Spectrophotometer to examine the structural components and deformation of proteins. CD spectra were recorded in the wavelength range of 190 to 300^nm with a resolution of 1 nm and a scan speed of 100 nm / min. Quartz cuvettes with a path length of 0.1^cm were utilized for these measurements. To facilitate accelerated aggregation analysis for IgG proteins, the temperature was ramped to 55 °C (10 °C / min) for the quartz cuvette. This temperature selection was made to align with the identical temperature range used in the DLS case for accelerated antibody aggregation, considering the experimental time of 2 hours. Also, for consistency with DLS measurements, all samples were70668-01 filtered using a PTFE filter with a 0.2 μm pore size prior to measurement. The filtration procedures and medium types used in various experiments are summarized in Table 1 below. Table 1: Filtration procedures and medium types used in various experiments. ExperimentFiltration of thesample Medium usedccccaAp g . μ p e, unless specified otherwise. b The Langmuir trough was filled with Milli-Q water, and the respective sample solution to be injected was also prepared in Milli-Q water. c It was confirmed that the dissolution of PNIPAM-PEG, Tween 80, or Tween 20 does not alter the pH of the medium, which remains at 7.4. As indicated in the table, most experiments, with the exception of surface pressure measurements, were conducted using samples prepared in 1× PBS buffer (pH 7.4) to maintain a consistent pH. It was confirmed that the dissolution of PNIPAM-PEG, Tween 80, or Tween 20 does not alter the pH of the medium. For convenience, surface pressure measurements were performed using Milli- Q®water as the subphase medium. The DLS size characteristics of PNIPAM-PEG, Tween 80, and Tween 20 micelles were consistent whether using PBS or Milli-Q water.

[0074] Statistical Analysis The experiments underwent analysis using one-way analysis of variance (ANOVA) to assess the differences in the results obtained from time-resolved DLS and CD analyses (N = 3). This statistical method was chosen to ascertain whether there were significant differences among the various experimental conditions. The obtained p-values from the ANOVA were significantly70668-01 lower than 0.05, indicating that the differences observed in the experiments were statistically significant and unlikely to have occurred by chance. A p-value below 0.05 is commonly used as a threshold to reject the null hypothesis, suggesting no difference between different conditions.

[0075] Results and Discussion Polymer Molecular Characteristics

[0076] Methodically synthesized a PNIPAM–PEG material with a narrow molecular weight distribution using controlled Reversible Addition–Fragmentation (Chain) Transfer (RAFT) polymerization. The synthesis process involved employing a pre-made monomethoxy- monohydroxy-terminated PEG (mPEG–OH) as a precisely defined precursor, ensuring accuracy in molecular design. A PNIPAM homopolymer was also synthesized using RAFT.

[0077] Number-average molecular weights and Polydispersity indices The number-average molecular weights (Mn) of the resulting polymers, namely PNIPAM and PNIPAM–PEG, were determined using1H NMR spectroscopy. The analysis provided Mnvalues, with the PNIPAM homopolymera molecular weight of 4,500 g / mol and PNIPAM– PEG displaying individual Mnvalues of 6,000 g / mol for the RAFT-synthesized PNIPAM block and 5,000 g / mol for the precursor PEG block. The polydispersity indices (PDI) of both PNIPAM and PNIPAM–PEG were determined through gel permeation chromatography (GPC) measurement (Fig. 13). The obtained values, PDI = 1.12 for PNIPAM and PDI = 1.19 for PNIPAM–PEG, underscore the controlled nature of the RAFT polymerization process.

[0078] Hydrodynamic diameter DLS was employed to assess the hydrodynamic diameter (Dh) of PNIPAM-PEG in PBS (1×) solutions at room temperature (25 °C) across varying concentrations (Fig. 15a). A discernible signal for the hydrodynamic diameter of PNIPAM–PEG was not detected until the concentration reached 0.15 mg / mL at 25 °C. This suggests that at lower concentrations (e.g., 0 mg / mL – 0.1 mg / mL), PNIPAM–PEG existed in a fully dissolved state without forming micelle structures. At concentrations ≥ 0.15 mg / mL, PNIPAM-PEG aggregated to form nano-sized particles (micelles) within the solution. The hydrodynamic diameter of PNIPAM-PEG micelles remained approximately constant at ~ 18 nm across the range of polymer concentrations examined. Based70668-01 on these results, it can be concluded that the critical micellization concentration (CMC) of PNIPAM-PEG in PBS (1×) at 25 °C is approximately 1.75 ± 0.25 mg / mL. PNIPAM is noted for exhibiting lower critical solution temperature (LCST) behavior in aqueous solutions with an LCST of ~ 32 °C in pure water (Polymer Chemistry 2016, 7(21), 3509-3519), and its LCST is known to decrease in saline mediums such as PBS (Journal of Physical Chem. 2010, 114, 16594-16604). The observations of micelle formation by PNIPAM-PEG in PBS solutions at room temperature were aligned with these reports. Surface Pressure Measurements Using the Langmuir Trough

[0079] The highest surface pressure exhibited by the surfactant at the air-water interface is fundamentally linked to the surfactant’s thermodynamic affinity for the interface. Surfactant molecules typically adsorb at the air-water interface, forming monolayers. A high surface pressure of the surfactant indicates that the surfactant molecules exhibit a strong tendency to arrange themselves into tightly packed and stable monolayers at the air-water interface, minimizing their unfavorable interactions with water.

[0080] To compare the thermodynamic preferential adsorption at the air-water interface, samples of PNIPAM, PNIPAM–PEG, Tween 80, Tween 20, and IgG human plasma were measured using a Langmuir trough containing 700 mL of Milli-Q-purified water in the subphase (Figs.2a-2b).1 mL of each sample with a concentration 5.0 mg / mL was injected into the middle of the subphase (700 mL), situated 3 cm beneath the air-water interface. The surface pressure (Π) was recorded as a function of time. Here, the surface pressure is defined as Π ≡ γo– γ, where γois the surface tension of the clean air-water interface, and γ is the surface tension of a surfactant / protein-coated air-water interface. When the samples are individually injected (Fig. 2a), each sample reaches a different value of surface pressure called the equilibrium surface pressure (Πe) in increasing order of IgG (~ 1 mN / m),Tween 80 (~ 14 mN / m), Tween 20 (~ 15 mN / m, still rising), PNIPAM–PEG (~ 30 mN / m) and PNIPAM (~ 31 mN / m). It is noteworthy that, even though PNIPAM is conjugated with PEG to form a diblock copolymer, PNIPAM–PEG shows an almost equivalent Πeto PNIPAM due to its strong adsorption tendency toward the air-water interface.70668-01

[0081] The samples were simultaneously injected to conduct a comparative analysis of surface preference (Fig.2b). Upon injection of IgG along with an equal weight concentration of either of the three excipients (Tween 80, Tween 20, or PNIPAM–PEG), a slight depression in surface pressure was initially observed (at times < ~ 30 min) relative to the respective equilibrium surface pressures (Πe) for pure Tween 80, Tween 20, and PNIPAM–PEG. This suggests that surface coverage occurs under competition between IgG and the other excipients during the early stage. However, in the cases of IgG + Tween 80 and IgG + PNIPAM–PEG, the surface pressure (Π) eventually (i.e., by the end of the experimental period (~ 120 min)) converges to the Πeof Tween 80 and PNIPAM–PEG, indicating complete coverage of excipients over IgG. In the case of IgG + Tween 20, the surface pressure at 120 min was higher than that observed with pure Tween 20 at 30 min (Fig.2b), as Π is still increasing even at the 30-min mark, indicating insufficient time to reach Πefor Tween 20. To better understand the surface preference between the excipients Tween 80 (or Tween 20) and PNIPAM–PEG, they were simultaneously injected. Initially, there was an increase in Π around 5 min, coinciding with the initiation of the Π increase for Tween 80 (or Tween 20). However, after approximately 7 min, Π begins to rise above the Tween 80 (or Tween 20) curve, mirroring the PNIPAM–PEG curve. This suggests that PNIPAM–PEG demonstrates a stronger surface preference than Tween 80 (or Tween 20) and effectively displaces the surface coverage initially occupied by Tween 80 (or Tween 20). Accelerated Aggregation Analysis Using Time-Dependent DLS

[0082] Elevating sample temperature has been utilized to assess the stability of protein dispersions (Journal of Pharmaceutical Science 2014, 103, 3, 828-839, and Journal of Pharmaceutical Science 2011, 100, 10, 4234-4243). However, these methods often lack continuous measurements, which compromises accuracy and detailed analysis within short time periods. A DLS-based technique was employed to evaluate protein stability against aggregation in order to study accelerated aggregation. This involved setting up a time-dependent experiment at the accelerated aggregation point of 55 °C. Above this temperature, rapid aggregation occurs, presenting challenges in determining the extent of aggregation. The samples were maintained at this temperature while continuously collecting DLS data approximately every 2 min for a total of 60 measurements over the full experiment duration (t) of approximately 2 hours.70668-01

[0083] The stability of the excipients Tween 80 and PNIPAM-PEG during DLS analysis with an open surface was evaluated (Figs. 3a and 3c). By presenting the time-resolved DLS data, which illustrates the dispersion profile over time, this method provides a clear depiction of dispersion stability. Each excipient was dissolved in PBS (1×) buffer at a concentration of 2.5 mg / mL. The DLS intensity profiles of Tween 80 and PNIPAM-PEG showed distinctive peaks at approximately 11 nm and 30 nm, respectively, at 25 °C. The DLS profiles were normalized to the maximum peak to achieve a maximum intensity of 100 after normalization. The time-dependent DLS profiles of Tween 80 and PNIPAM-PEG exhibited a consistent profile, maintaining the peak position without any indication of aggregate peaks during the analysis.

[0084] After the time-dependent experiments, the DLS profile remained similar to that before the experiment. This suggests that the excipient is unaffected by the elevated temperature during the experimental period, retaining its original structure. The count rate (S) data for each experiment showed slight increases, with relative ratios (S / S0) between before (t = 0) and after (t = 120 min) of 1.05 and 1.16 for Tween 80 and PNIPAM-PEG, respectively. The DLS analysis for accelerated aggregation of IgG proteins was conducted without excipient, maintaining an identical concentration of 2.5 mg / mL in PBS (1×) buffer with an open surface (Fig.4b). Similar to Tween 80, the DLS intensity profile exhibited one characteristic peak at approximately 11 nm at 25 °C. In contrast to the sole Tween 80 excipient system, during the time-dependent experiment at 55 °C, the characteristic peak from the DLS profile of IgG began to divide around 30 min and completely separated into a size of about 53 nm at 50 min into the experiment. This indicates that the IgG protein aggregates at 55 °C, and its aggregate manifests as larger signals than non-aggregated proteins in the DLS profile. The aggregate size distribution does not appear to undergo any dramatic changes, as the aggregation size relatively maintained its size. After the experiment, the DLS profile showed two distinct bimodal peaks compared to the original one-peak profile. The relative intensity (^^^^50nm) of the peak from the larger size (~ 50 nm) after the experiment (Fig.4b) was higher than that of the smaller peak (^^^^11nm) with the intensity ratio (^^^^50nm,2h / ^^^^11nm,2h= 3.13) between the two peaks, being the largest at the end (~ 2 hours) of the experiment.

[0085] To investigate the influence of the surface (i.e., the air-water interface) on the aggregation of IgG proteins, DLS experiments were conducted under the same conditions but without exposure70668-01 to a surface by completely sealing the sufficiently loaded solution in the cuvette. The sample was prepared with an upward meniscus, capped without any space (trapped air) in the cuvette, and any overflowed sample was carefully removed. By plotting the time-dependent DLS data for different IgG conditions, this method allows for a clear presentation and comparison of differences in the aggregation rate. During the experiment, the second peak indicative of aggregates (~ 50 nm) becomes more pronounced as time progresses under the constant temperature of 55 °C. The results revealed that the divergence in the DLS profile occurs at approximately 55 min for IgG without a surface, which is more delayed compared to the case with a surface. After the experiment at ~ 2 hours (Fig.4c), ^^^^50nmexhibited a lower value than ^^^^11nm(^^^^50nm,2h / ^^^^11nm,2h= 0.62), indicating a reverse trend compared to the open surface case. This suggests that milder aggregation occurred in the absence of a surface compared to the system with a surface. In the case of IgG with a surface (Fig.4b), the relative count ratio (S / S0) (Fig.4a) exhibited a large value of 2.55, attributed to the substantial size of the IgG aggregates. Conversely, S / S0 displayed a lower value (~ 1.63) than in the open surface case. This disparity between the two DLS experiments, with and without a surface, supports the notion that the surface may serve as the initiation site for the aggregation of IgG proteins.

[0086] To estimate the aggregation ratio from the DLS data, utilized the relative intensities of the characteristic peaks at 11 nm and 50 nm from the DLS intensity profile to infer the relative weight concentration of the aggregated IgG proteins (Journal of Applied Physics, 1951, 22, 10, 1242- 1246). ^^^^11nm,2h = ^^^^IgG,2h ∙ ^^^^IgG,2h (1)Here, ^^^^11nm,2hand ^^^^50nm,2hrepresent the relative intensities of characteristic peaks around 11 nm and 50 nm from the DLS intensity profile, respectively, with the intensities normalized by the maximum intensity. ^^^^^^^^,yand ^^^^^^^^,^^^^denote the proportionality (optical) constants for DLS intensity under present experimental conditions and the weight concentration for substance x (size or aggregation state) at time point y (h). Maintaining the total weight balance between IgG proteins before and after aggregation (denoted as the subscripts “IgG” and “IgGA”, respectively), we express it as:70668-01 ^^^^IgG,0h = ^^^^IgG,2h + ^^^^IgGA,2h (3)

[0087] As the inteower-law dependence with an exponent of 3, the correlation between non-aggregated and aggregated IgG (IgGA) can be described by: ^^^^ 3IgGA,2h / ^^^^IgG,2h = (^^^^h,IgGA,2h / ^^^^h,IgG,2h) (4)where the DhisDLS measurement. Using these equations, a preliminary estimate of the aggregated IgG protein amount can be made (Table 2).

[0088] Table 2. Quantification of aggregation from time-resolved DLS analysis during accelerated aggregation of IgG under different surface conditions. Four unknowns calculated from Eqs. (1), (2), (3), and (4) are marked in bold typeface. Initial IgG weight ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^

[0089] In the case of IgG proteins with an open surface, the estimated concentration of IgGA was approximately 0.0805 mg / mL compared to the original amount (2.5 mg / mL). However, in the case without a surface, it was found to be 0.0164 mg / mL, significantly lower than the previous case. This suggests that the surface has a substantial impact on the aggregation of IgG proteins. Figs.5a and 5b illustrate the DLS experiment profiles for IgG with the typical excipient of Tween 80 under identical conditions. In comparison to IgG without the excipient, the characteristic peak in the DLS profile of IgG began to diverge later, around ~ 37 min, and eventually completely separated into a similar size of about 53 nm at 50 min during the experiment. The appearance of the split peak at70668-01 53 nm indicates aggregated IgG proteins, as Tween 80 alone does not demonstrate a higher peak in the DLS profile during its individual experiment under identical conditions (Fig.3a).

[0090] The intensity of ^^^^50nmafter the experiment (Fig.5a) remained higher than ^^^^11nm, although ^^^^11nmwas slightly higher than in the excipient-free case. This indicates that Tween 80 mitigates the aggregation of IgG proteins, although there is still a significant level of aggregation evident in the DLS profile. Upon increasing the concentration of Tween 80 to 2.5 mg / mL, the characteristic peak in the DLS experiment profiles for IgG with the excipient Tween 80 divided at a delayed time point, around 50 min. Additionally, the intensity of aggregated IgG (^^^^50nm) was lower than that of ^^^^11nm, indicating a more pronounced attenuation of IgG protein aggregation with an increased amount of Tween 80 in the solution. The relative count ratio over time (Fig. 5c) corroborates these experimental findings, showing greater increases in the count ratio with decreased amounts of Tween 80. Specifically, the final S / S0for Tween 80 at 0.3 mg / mL was 2.32, whereas for Tween 80 at 2.5 mg / mL, it exhibited a lower value of 1.81. It suggests that at a lower concentration of Tween 80 (0.3 mg / mL), there was more pronounced IgG aggregation, resulting in a greater increase in the scattering intensity (i.e., the count rate) during the experiment compared to the higher concentration of Tween 80 (2.5 mg / mL).

[0091] For quantitative analysis, employed the peak intensities corresponding to each size point (11 and 50 nm) derived from the DLS profiles to examine both initial and final conditions: ^^^^11nm,0h = ^^^^IgG,0h ∙ ^^^^IgG,0h + ^^^^T,0h ∙ ^^^^T,0h (5)Here, the subscript letter “T” denotes Tween 80. The same mass balance equation (Eq.3) for IgG applicable, provided the additional condition that Tween 80 remains inert throughout the DLS analysis: ^^^^T,0h = ^^^^T,2h (8)70668-01

[0092] Furthermore, alongside the size correlation of DLS intensity (Eq. 4), constant proportionality factors for non-aggregated IgG and Tween 80 can be assumed regardless of time and temperature during the experiments: ^^^^IgG,0h / ^^^^^^^^,0h = ^^^^IgG,2h / ^^^^T,2h (9)^^^^ = ^^^^ ∙ ^^^^ (10)Here, ^^^^ (≅ 1.2, presents the ratio between the proportionality constants for non-aggregated IgG and Tween 80. Upon computation of these equations, the results (Table 3) indicated that ^^^^IgGA,2hunder conditions of Tween 80 ranging from 0.3 to 2.5 mg / mL was estimated to be between 0.0691 and 0.0321 ± 0.0020 mg / mL.

[0093] Table 3. Quantification of aggregation from time-resolved DLS analysis during accelerated aggregation of IgG under various weight concentrations of Tween 80 (^^^^IgG,0h= 2.5 mg / mL). Eight unknowns calculated from Eqs. (5), (6), (7), (3), (8), (4), (9), and (10) are marked in bold typeface. Excipient concentra ^^^^ ^^^^ ^^^^ ^^^^^1 7 3 3

[0094] This range is lower than the case of IgG with an open surface but no other excipient (0.00816 ± 0.0016 mg / mL). Notably, ^^^^IgGA,2h(0.0160 ± 0.0012 mg / mL) in the case of IgG without a surface is even lower than the Tween 802.5 mg / mL case, indicating that Tween 80 is insufficient to cover the solution’s surface adequately to protect IgG from aggregation. The same measurements were also conducted using Tween 20, as shown in Figs.6b and 6c. The data were analyzed similarly to the Tween 80 case described above. As summarized in Table 4, Tween 20 was found to be more effective than Tween 80 at suppressing IgG aggregation at the lower70668-01 concentration tested (0.3 mg / mL), which was expected given its higher surface pressures relative to Tween 80 (Figs.2a and 2b and Table 1). Additionally, the effectiveness of Tween 20 showed less dependence on concentration when comparing 0.3 mg / mL and 2.5 mg / mL. Table 4. Quantification of aggregation from time-resolved DLS analysis during accelerated aggregation of IgG under various weight concentrations of Tween 20 (^^^^IgG,0h= 2.5 mg / mL). Eight unknowns calculated from Eqs. (5), (6), (7), (3), (8), (4), (9), and (10) are marked in bold typeface. Excipient concentration ^^^^ ^^^^^^^^^^^^^^^^,^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^,^^^^^^^^^^^^^^^^^^^^^^,^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^3 4

[0095] The same experimental procedure was conducted for IgG proteins with the addition of the PNIPAM-PEG excipient (Figs. 7a and 7b). Since PNIPAM-PEG itself forms larger particles (micelles), exhibiting a characteristic peak at a larger size (Fig. 3c), the DLS intensity profile displays two peaks from the onset of the experiment. For PNIPAM-PEG at the lowest concentration of 0.3 mg / mL, the peak intensity (^^^^50nm) at ~ 50 nm is lower than that at the smaller- size position (11 nm). As the experiment progresses up to 2 h, ^^^^50nmincreases compared to ^^^^11nm(Fig.7a). This suggests that the peak intensity at larger positions is attributed to aggregated IgG, the amount of which increases during the experiment, in addition to the pristine PNIPAM-PEG. With the weight concentration of PNIPAM-PEG increasing to 2.5 mg / mL, ^^^^11nmis much lower than ^^^^50nmdue to the increased amount of PNIPAM-PEG. Throughout the time-dependent experiment, the peak intensities of the DLS profile undergo slight changes between before and after the experiment, indicating a minor degree of aggregation of IgG proteins compared to other cases. The relative count ratio over time (Fig. 7c) corroborates these findings, showing that the increase in PNIPAM-PEG amount from 0.3 to 2.5 mg / mL in the IgG solution attenuates S / S0 from 2.12 to 1.51.70668-01

[0096] A comparable quantitative assessment was undertaken utilizing the peak intensities at each size point (11 and 50 nm) extracted from the DLS profiles to analyze both initial and final conditions: ^^^^11nm,0h = ^^^^IgG,0h ∙ ^^^^IgG,0h (11)^^^^ = ^^^^ ∙ ^^^^ (12)Here, the. IgG (Eq.3) was applied, assuming that PNIPAM-PEG remains intact during the DLS analysis: ^^^^P,0h = ^^^^P,2h (15)

[0097] Consider ng e s ze- n ens y corre a on ( q. ), was assume a he proportionality factors between non-aggregated IgG and PNIPAM-PEG were equal, given their consistent characteristics during the experiments: ^^^^IgG,0h / ^^^^P,0h = ^^^^IgG,2h / ^^^^P,2h (16)From these equations, the results obtained (Table 5) forIgGA,2hunder different PNIPAM-PEG concentrations varying from 0.3 to 2.5 mg / mL ranged from 0.0255 to 0.0088 ± 0.0005 mg / mL. Notably, this range is lower than the corresponding values with Tween 80.

[0098] Table 5. Quantification of aggregation from time-resolved DLS analysis during accelerated aggregation of IgG under various weight concentrations of PNIPAM-PEG (^^^^IgG,0h= 2.5 mg / mL). Eight unknowns were calculated from Eqs. (11), (12), (13), (14), (3), (15), (4), and (16) are marked in bold typeface. Excipient ^^^^ ^^^^ ^^^^ ^^^^^^^570668-01 75.59 179.29 0.0163 7099 0.5 40.00 128.072.484 .4 ±12.04± 0.002 ± 0.500 ± ± ± 117 1 2 24 11 47 6 9 .6g , g ,ted in Fig.8, a notable observation emerges. It was observed that in the absence of added excipients, IgG aggregation was reduced by a factor 4.7 when the air-water interface was removed. This result indicates that the air-water interface has a more pronounced impact on IgG aggregation compared to the container surfaces. Instances where PNIPAM-PEG concentrations exceed 1.5 mg / mL exhibit a lower ^^^^IgGA,2h / ^^^^IgG,0h(0.0043) compared to the case with IgG lacking a surface (0.0064 ± 0.0005), a trend not observed in the case of Tween 80 and Tween 20, even at 2.5 mg / mL (^^^^IgGA,2h / ^^^^IgG,0h≅ 0.0128± 0.0008 for Tween 80, and ^^^^IgGA,2h / ^^^^IgG,0h≅ 0.0144 for Tween 20). This suggests that PNIPAM-PEG can effectively cover the surface of the IgG solution, providing protection against aggregation triggered at the interface. The data summarized in Fig. 8 also indicate that while PNIPAM-PEG is highly effective at suppressing surface-induced aggregation of IgG, its suppressive effect on bulk IgG aggregation appears to be minimal. This supports the notion that PNIPAM-PEG does not in fact interact with IgG molecules in the bulk phase. This finding has important implications for enhancing the stability of IgG proteins, showcasing PNIPAM-PEG as a promising excipient in formulations aimed at mitigating aggregation-related challenges. The ability of PNIPAM-PEG to effectively cover the solution's surface points towards its potential in preventing surface-initiated aggregation and maintaining the structural integrity of antibody proteins, marking a significant stride in the development of protein biologic formulations with enhanced stability. Circular Dichroism (CD) Analysis

[0100] IgG proteins are part of a class characterized by a common subunit structure, consisting of structurally independent domains with continuous polypeptide chain stretches. As aggregation can be associated with alterations in secondary and tertiary structure, CD measurement has proven valuable for monitoring conformational changes during protein aggregation (Acta Pharmaceutica70668-01 Sinica B 2022, 12, 11, 4249-4261). This technique offers a straightforward and reliable means of rapidly determining protein structure or tracking conformational changes, particularly under varied temperature and pH conditions, as has been extensively studied in these contexts (Analytical Chemistry 2014, 86, 23, 11606-11613).

[0101] Initially, reference CD spectra were obtained for IgG and excipients (Tween 80 and PNIPAM-PEG) at 25 °C (Fig. 9a). IgG displayed a characteristic negative band around the wavelength (^^^^) ~ 220 nm and a zero intensity point at ^^^^ ~ 206 nm, indicative of a high content of ^^^^-sheet structure, along with broad positive bands in the near-UV region between 260 and 300 nm (Analytical Biochemistry, 1978, 91, 1, 13-31). In contrast, excipients (Tween 80 and PNIPAM- PEG) exhibited no typical signals in the CD spectra, suggesting that these excipients do not contribute to the CD data during the IgG aggregation experiment. CD analysis for IgG was conducted without excipients following the accelerated aggregation experiment under identical conditions (55 °C) as the DLS experiment (Fig.9b). The characteristic negative band at ^^^^ ~ 220 nm shifted in the negative direction during the experiment, indicating an increase in ^^^^-helix and random coil contents. This observed induction of ^^^^ -helix with time may be attributed to peptide units entering a dehydrated (hydrophobic) environment upon IgG aggregation (General Subjects 2001, 1526, 1, 61-69).

[0102] In the case of dissolved native proteins, it is more favorable for the peptide units at the aqueous periphery to form hydrogen bonds with water molecules rather than with each other. However, denaturation can disrupt this equilibrium, exposing part of the hydrophobic interior of IgG to the solution, thereby promoting aggregate formation. This exposure may trigger the formation of ^^^^-helices at the interfaces between the building blocks of the aggregate, facilitated by hydrogen bonds between peptide units in the polypeptide chain. Experimental observations supporting this notion show that ^^^^-helixes are induced in proteins that adsorb at hydrophobic surfaces (Colloid Interface Sci.1996, 180, 2, 632-633 and Biochimica et Biophysica Acta (BBA) - General Subjects 1998, 1425, 1, 1-12). This phenomenon significantly influences the course of aggregation in IgG proteins, despite the preservation of a substantial fraction, approximately 50%, of ordered secondary structural elements (^^^^-sheets, ^^^^-helices, and ^^^^-turn conformations), even when IgG is fully denatured (Biochemistry 1988, 27, 5, 1670-1677).70668-01

[0103] Consistent negative shifts at ^^^^ ~ 220 nm were observed across all cases with excipients, suggesting that some degree of aggregation occurs even in the presence of excipients in the IgG solution. However, the magnitudes of the negative shifts (Fig.9c) during the experiments varied, following the order IgG > IgG + Tween 80 > IgG + PNIPAM-PEG with the corresponding negative slopes of -0.0170 > -0.0147 > -0.0122 °·cm2·mg-1, respectively. This observation is in line with the results from the accelerated DLS experiment. The implications of these findings suggest that even with the presence of excipients like Tween 80 and PNIPAM-PEG, some degree of aggregation occurs, albeit to varying extents. The negative shifts observed in the CD spectra act as markers for conformational changes in IgG during the aggregation process. Notably, the greater negative shift observed in the case of IgG alone compared to the IgG + Tween 80 and IgG + PNIPAM-PEG conditions signifies a higher degree of denaturation in the absence of these stabilizing excipients. The intermediate shift observed in IgG + Tween 80 suggests that Tween 80 provides some stability to IgG’s secondary structures, while the minimal shift in IgG + PNIPAM- PEG indicates that PNIPAM-PEG offers greater protection of IgG proteins against denaturation compared to Tween 80. These insights underscore the crucial role of excipients, especially PNIPAM-PEG, in mitigating the aggregation of IgG proteins and preserving their structural integrity. The integration of CD spectroscopy and DLS experiments offers a comprehensive understanding of how excipients influence protein stability, offering valuable insights for optimizing formulations in biopharmaceutical applications. Ramifications across LCST Polymers

[0104] The primary characteristic of PNIPAM that renders it particularly suitable for protein stabilization is its remarkable affinity for the air-water interface. This attribute allows PNIPAM to effectively cover the interface, thereby reducing protein exposure. Few water-soluble polymers possess such a strong affinity for the air-water interface. Those that do are anticipated to exhibit LCST characteristics in water, given their marginal hydrophilicity, which can be easily disrupted by environmental changes, such as temperature fluctuations. Table 6 below provides a concise list of polymers meeting these criteria, specifically having an LCST below physiological body temperature (~ 37 °C) and Πeabove ~ 15 mN / m, which corresponds to the value for Tween 80. These polymers, whether in their homopolymer form or as PEGylated block copolymers can hold significant promise for protein stabilization applications. PEGylation, particularly beneficial for70668-01 polymers with an LCST < 37 °C, allows the polymer’s PEGylated versions to form micelles at physiological temperatures, thereby minimizing undesired protein interactions within the body and facilitating their clearance even at large doses.

[0105] Among all these candidate polymers, PNIPAM stands out due to its strongest affinity for the air-water interface (the highest Πevalue), making it potentially the most effective in preventing protein adsorption at the air-water interface. While Tween 20 also demonstrates strong surface affinity (Table 6), direct side-by-side comparative tests (Figs.2a and 2b) indicate that it does not surpass the surface affinity of PNIPAM. Additionally, PNIPAM is advantageous because its surface affinity is nearly concentration-independent, in contrast to polysorbates. For example, as illustrated in Figs.2a and 2b, at relatively low concentrations (e.g., below their respective CMCs), PNIPAM-PEG is significantly more effective in reducing surface tension compared to Tween 20. PNIPAM is non-toxic and safe for use in therapeutic formulations.

[0106] Table 6. Characteristics of lower critical solution temperature (LCST) and equilibrium surface pressure (Πe) for polysorbates (e.g., Tween 80), PNIPAM, and other candidate polymers LCST Polymer Πe (mN / m) 0 s70668-01

[0107] In an endeavor to enhance antibody stability and alleviate aggregation, conducted an experimental study highlighting the distinctive role of the PNIPAM-PEG excipient. Synthesized via RAFT polymerization, PNIPAM-PEG exhibited a narrow molecular weight distribution alongside robust protective properties. Through a combination of DLS and CD spectroscopy, examined how excipients, including Tween 80 and PNIPAM-PEG, influence IgG protein aggregation. Accelerated DLS experiments unveiled differing aggregation levels under various conditions. Particularly noteworthy was the superior performance of the PNIPAM-PEG excipient, effectively protecting against IgG aggregation and outperforming the traditional excipient, Tween 80 and Tween 20. It highlighted the excipient's exceptional efficacy in preserving IgG stability, surpassing the conventional excipient Tween 80 under high-temperature conditions. This was evident in the sustained stability and minimal aggregation observed in the IgG + PNIPAM-PEG system, even under accelerated conditions. CD spectroscopy results further substantiated these findings, offering insights into conformational changes linked with aggregation. Consistent negative shifts at ^^^^ ~ 220 nm indicated alterations in secondary structure, with the magnitude of shifts reflecting the degree of aggregation. The sequence of shifts—IgG > IgG + Tween 80 > IgG + PNIPAM-PEG—paralleled the DLS results, underscoring the protective effect of PNIPAM- PEG. These findings bear significant implications for biopharmaceutical applications, underscoring the pivotal role of excipients, particularly PNIPAM-PEG, in preserving antibody structural integrity. PNIPAM-PEG’s ability to efficiently shield IgG from aggregation, even under accelerated conditions, positions it as a promising excipient for enhancing antibody stability, and facilitating the development of more stable and effective biopharmaceuticals.

[0108] While specific embodiments of the subject invention have been disclosed herein, the above specification is illustrative and not restrictive. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Many variations of the invention will become apparent to those of skilled art upon review of this specification. Unless otherwise indicated, all numbers expressing reaction conditions, quantities of ingredients, and so forth, as used in this specification and the claims, are to be understood as being modified in all instances by the term “ about. ” Accordingly, unless indicated to the contrary, the numerical70668-01 parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure.

[0109] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0110] The term "alkyl" refers to substituted and unsubstituted straight-chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms (e.g., C1-C20), 1 to 12 carbons (e.g.C1-C12), 1 to 8 carbon atoms (e.g.C1-C8), or, in some embodiments, from 1 to 6 carbon atoms (e.g.C1-C6). Examples of straight-chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. The term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups, as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0111] The term "about," when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., + / - 5 % to 15% of the recited value, such as within 10%, within 5%, or within 1% of a stated value or stated limit of a range) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term "substantially" can allow for a degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.

[0112] In addition, any of the embodiments described in the following clause list are considered to be part of the invention.70668-01 A. A therapeutic formulation comprising (i) a protein and (ii) an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2- oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA). B. The therapeutic formulation of clause A, wherein the A block polymer exhibits an equilibrium surface pressure ranging from about 10 mN / m to about 72 mN / m at room temperature. C. The therapeutic formulation of clause A or B, wherein the A block polymer has a lower critical solution temperature (LCST) ranging from about -20 °C to about 37 °C. D. The therapeutic formulation of any one of clauses A-C, wherein the A block polymer is poly (N-isopropyl acrylamide). E. The therapeutic formulation of clause A, wherein the B block polymer is poly(ethylene glycol) (PEG). F.The therapeutic formulation of any one of clauses A-E, wherein the AB-type block copolymer is poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PNIPAM-PEG). G. The therapeutic formulation of any one of clauses A-E, wherein the BAB-type block copolymer is poly(ethylene glycol)-b-poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PEG-PNIPAM-PEG). H.The therapeutic formulation of any one of clauses A-C, wherein the A block polymer is poly (N-methacryloyl-L-alanine methyl ester).70668-01 I. The therapeutic formulation of clause A, wherein the A block polymer is selected from poly(2-isopropyl-2-oxazoline), poly(2-n-propyl-2-oxazoline), and poly(2-cyclopropyl-2- oxazoline). J. The therapeutic formulation of clause A, wherein the protein is selected from an antibody, an antibody-drug conjugate, an antibody fragment, an enzyme, an immunogenic protein, a fusion protein, a FC fusion protein, a protein fragment, a polypeptide, a peptide drug, a structural peptide, a lipoprotein, a growth factor and a combination two or more thereof. K. The therapeutic formulation of clause A, wherein the protein is an antibody. L.The therapeutic formulation of clause A, J, or K, wherein the protein is present in an amount of about 5 × 10-5ppm to about 5 × 105ppm. M. The therapeutic formulation of any one of clauses A-L, wherein the block copolymer is present in an effective amount to suppress an aggregation and / or a denaturation of the protein. N. The therapeutic formulation of clause M, wherein the block copolymer is present in an amount ranging from about 5 × 10-5ppm to about 5 × 105ppm. O. A method for stabilizing a therapeutic formulation comprising a protein, which method comprises: adding to the therapeutic formulation in an amount effective to suppress aggregation and / or denaturation of the protein an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester- substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide), and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA), whereupon the therapeutic formulation comprising the protein is stabilized. P. A method of administering a therapeutic formulation to a patient in need thereof, which method comprises administering to the patient the therapeutic formulation of any one of clauses A-N.70668-01

[0113] The invention illustratively described herein may be suitably practiced in the absence of any element(s) or limitation(s), which is / are not specifically disclosed herein. Thus, for example, each instance herein of any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms "a," "an," or "the" are used to include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods and / or steps of the type, which are described herein and / or which will become apparent to those ordinarily skilled in the art upon reading the disclosure. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. In addition, the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid the reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. The terms "including" and "having" are defined as comprising (i.e., open language).

[0114] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0115] It is intended that the scope of the present methods and apparatuses be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

Claims

70668-01 WE CLAIM:

1. A therapeutic formulation comprising (i) a protein and (ii) an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester-substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide) and poly(2-alkyl-2- oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA).

2. The therapeutic formulation of claim 1, wherein the A block polymer exhibits an equilibrium surface pressure ranging from about 10 mN / m to about 72 mN / m at room temperature.

3. The therapeutic formulation of claim 1 or 2, wherein the A block polymer has a lower critical solution temperature (LCST) ranging from about -20 °C to about 37 °C.

4. The therapeutic formulation of any one of claims 1-3, wherein the A block polymer is poly (N-isopropyl acrylamide).

5. The therapeutic formulation of claim 1, wherein the B block polymer is poly(ethylene glycol) (PEG).

6. The therapeutic formulation of any one of claims 1-5, wherein the AB-type block copolymer is poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PNIPAM-PEG).

7. The therapeutic formulation of any one of claims 1-5, wherein the BAB-type block copolymer is poly(ethylene glycol)-b-poly(N-isopropyl acrylamide)-b-poly(ethylene glycol) (PEG- PNIPAM-PEG).

8. The therapeutic formulation of any one of claims 1-3, wherein the A block polymer is poly (N-methacryloyl-L-alanine methyl ester).70668-01 9. The therapeutic formulation of claim 1, wherein the A block polymer is selected from poly(2- isopropyl-2-oxazoline), poly(2-n-propyl-2-oxazoline), and poly(2-cyclopropyl-2-oxazoline).

10. The therapeutic formulation of claim 1, wherein the protein is selected from an antibody, an antibody-drug conjugate, an antibody fragment, an enzyme, an immunogenic protein, a fusion protein, a FC fusion protein, a protein fragment, a polypeptide, a peptide drug, a structural peptide, a lipoprotein, a growth factor and a combination two or more thereof.

11. The therapeutic formulation of claim 1, wherein the protein is an antibody.

12. The therapeutic formulation of claim 1, 10, or 11 wherein the protein is present in an amount of about 5 × 10-5ppm to about 5 × 105ppm.

13. The therapeutic formulation of any one of claims 1-12, wherein the block copolymer is present in an effective amount to suppress an aggregation and / or a denaturation of the protein.

14. The therapeutic formulation of claim 13, wherein the block copolymer is present in an amount ranging from about 5 × 10-5ppm to about 5 × 105ppm.

15. A method for stabilizing a therapeutic formulation comprising a protein, which method comprises: adding to the therapeutic formulation in an amount effective to suppress aggregation and / or denaturation of the protein an AB or BAB-type block copolymer, wherein the A block polymer is selected from poly(N-alkyl-substituted acrylamide), poly(N-ester- substituted acrylamide), poly(N-alkyl-substituted methacrylamide), poly(N-ester-substituted methacrylamide), and poly(2-alkyl-2-oxazoline) and wherein the B block polymer is selected from poly(ethylene glycol) (PEG), poly(poly(ethylene glycol) acrylate) (PPEGA) and poly(poly(ethylene glycol) methacrylate) (PPEGMA), whereupon the therapeutic formulation comprising the protein is stabilized.

16. A method of administering a therapeutic formulation to a patient in need thereof, which method comprises administering to the patient the therapeutic formulation of any one of claims 1-14.

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