WO2026025028A1

WO2026025028A1 - Methods of making ultra-high concentrated protein formulations using lyophilization

Info

Publication number: WO2026025028A1
Application number: PCT/US2025/039251
Authority: WO
Inventors: Xiaolin Tang; Leonid Breydo; Leah STEYN
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2024-07-26
Filing date: 2025-07-25
Publication date: 2026-01-29
Anticipated expiration: 2027-01-26

Abstract

The present inventions provide methods of making ultra-high concentrated proteins for therapeutic and pharmaceutical formulations using lyophilization. The inventions also provide ultra-high concentrated protein formulations for lyophilization and reconstitution liquids for intravenous and subcutaneous administration. The inventions advantageously employ arginine hydrochloride, polysorbates and histidine.

Description

METHODS OF MAKING ULTRA-HIGH CONCENTRATED PROTEIN

FORMULATIONS USING LYOPHILIZATION

This application claims priority to U.S. Application Serial No. 63/678,845, filed August 2, 2024; and U.S. Application Serial No. 63/675,969, filed July 26, 2024. These applications are incorporated by reference in their entirety.

FIELD OF THE INVENTIONS

[0001] The present inventions provide methods of making ultra-high concentrated proteins for therapeutic and pharmaceutical formulations using lyophilization. The inventions also provide ultra-high concentrated protein formulations made using lyophilization and later reconstituted for intravenous and subcutaneous administration.

BACKGROUND OF THE INVENTIONS

[0002] Presently, concentrated protein formulations, particularly ultra-high concentration formulations, are administered intravenously. In the past, subcutaneous injections of such formulations were not suitable because of extracellular structures and high molecular weight aggregates that cause tissue back pressure and injection pain.

[0003] Monoclonal antibody (mAh) therapies have become a vital class of therapeutics in the last decade. Compared to IV, subcutaneous (SC) administration is less invasive, better tolerated by the patients, and SC administration takes only minutes while IV administration takes hours to complete the therapy. Moreover, IV administration often requires a hospital setting, whereas SC administration can be done at a doctor’s office or selfadministered. SC administration thus would increase patient compliance, decrease treatment costs (about 50%), and reduced burden on hospitals. Therefore, high concentration protein formulations are needed to achieve clinical mAh doses at concentrations of 200 mg/mL or more, which are needed for sufficient mAh dosaging.

[0004] In conventional manufacturing, protein formulations are concentrated via ultrafiltration (UF), which removes excess water and creates a higher concentration of a formulation. However, attempts to improve the conventional UF process have been unsuccessful in producing high concentration proteins of about 200 mg/mL or more (Holstein, et al. (2020). Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review. Biotechnology and bioengineering, 117(11), 3591-3606; Binabaji, Elaheh, Ultrafiltration of highly concentrated monoclonal antibody solutions, The Pennsylvania State University, 2015; Binabaji, Elaheh, et al. Ultrafiltration of highly concentrated antibody solutions: Experiments and modeling for the effects of module and buffer conditions, Biotechnology progress 32:3 (2016): 692-701). Moreover, ultra-high concentration protein formulations could not be formulated using the conventional UF methods and there is an unmet need for a process for manufacture ultra-high concentration protein products for therapeutic applications, especially for subcutaneous or intramuscular administration.

SUMMARY OF THE INVENTIONS

[0005] The inventions provide methods of forming concentrated protein formulations comprising the steps of: (a) providing an aqueous solution comprising a protein, a buffer such as histidine buffer, and a sugar at a pH of about 5.0 to about 7.0; (b) lyophilizing the aqueous solution to form a lyophilisate; and (c) reconstituting the lyophilisate using a reconstitution solution comprising arginine hydrochloride and a polysorbate (PS) to form the concentrated protein solution, such as concentrated drug product.

[0006] The inventions also provide ultra-high concentrated protein (UHCP) formulations for lyophilization, comprising: an aqueous solution of protein having a concentration between about 5 to about 700 mg/ml of the protein; a histidine buffer at a pH of between about 5.0 to about 7.0; and a sugar at a concentration of about 0.10% to about 10% (w/v).

[0007] The inventions further provide reconstitution solutions for reconstituting lyophilized ultra-high concentrated protein (UHCP) formulations comprising: (a) arginine hydrochloride solution at a concentration of about 10 to about 300 mM; and (b) the polysorbate at a concentration of about 0.01% to about 0.30% (w/v). Methods for reconstituting using (a) an arginine hydrochloride solution at a concentration of about 10 to about 300 mM; and (b) a polysorbate at a concentration of about 0.01% to about 0.30% (w/v) also are provided. [0008] The concentration of the protein solution such as a drug product can be at least 5 to about 700 mg/ml, about 5 to 50 mg/ml, about 50 to 100 mg/ml, about 100 to 150 mg/ml, about 150 to 200 mg/ml, about 200 to 250 mg/ml, about 250 to 300 mg/ml, about 300 to 350 mg/ml, about 350 to 400 mg/ml, about 400 to 450 mg/ml, about 450 to 500 mg/ml, about 500 to 550 mg/ml, about 550 to 600 mg/ml, about 600 to 650 mg/ml, or about 650 to 700 mg/ml. The protein in the concentrated formulation can be Fc-containing proteins, such as those selected from the group consisting of antibodies, antibody fragments, antibody derivatives and Fc-fusion proteins, such as receptor-Fc-fusion proteins and trap proteins.

[0009] . The concentrated formulation can be a liquid formulation, wherein the liquid formulation can comprise pharmaceutically acceptable excipients including one or more buffers, salts, and sugars. The concentration of histidine buffer can be about 1 to 30 mM, about 1 to 5 mM, about 5 to 10 mM, about 10 to 15 mM, about 15 to 20 mM, about 20 to 25 mM, or about 25 to 30 mM. The sugar can be sucrose, and the concentration of the sugar can be about 0.1% to 10%, about 0.1% to 0.5%, about 0.5% to 1.0%, about 1% to 1.5%, about

1.5% to 2%, about 2% to 3%, about 3% to 4%, about 4% to 5%, about 5% to 6%, about 6% to 7%, about 7% to 8%, about 8% to 9%, or about 9% to 10%. The pH of the aqueous solution can be about 5.0 to 7.0, about 5.5 to 6.0, about 6.0 to 6.5, about 6.5 to 7.0. The concentration of arginine hydrochloride in the reconstitution solution can be about 10 mM to 300 mM, about 10 mM to 50 mM, about 50 mM to 100 mM, about 100 mM to 150 mM, about 150 mM to 200 mM, about 200 mM to 250 mM, or about 250 mM to 300 mM. The polysorbate can be polysorbate 80 (PS 80), polysorbate 20 (PS20), polyethylene glycols (PEGs) such as PEG3350, or any pharmaceutically acceptable surfactants (cosolvents) that protect protein from agitation stress. The concentration of the polysorbate in the reconstitution solution can be about 0.01% to about 0.30% (w/v), about 0.01% to about 0.05% (w/v), about 0.015% to about 0.095% (w/v), about 0.015% to about 0.025% (w/v), about 0.025% to 0.035% (w/v), about 0.035% to 0.045% (w/v), about 0.045% to about 0.055% (w/v), about 0.065% to about 0.075% (w/v), about 0.075% to about 0.085% (w/v), about 0.085% to about 0.095% (w/v), about 0.05% to about 0.10% (w/v), about 0.10% to about 0.15% (w/v), about 0.15% to about 0.20% (w/v), about 0.20% to 0.25% (w/v), or about 0.25% to 0.30% (w/v).

[0010] The lyophilization process for bulk drug substance can be carried out following steps including (i) cooling a solution comprising a protein product below the freezing point of the solution to form a frozen solution; (ii) pressuring the cooled solution of step (i) with a gas; (iii) releasing the pressure of step (ii) to allow nucleation, such as controlled nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) lyophilizing the cooled / frozen solution of step (iii) through primary drying, with or without secondary drying, to form a lyophilized bulk protein product. The gas can be present at about 14 to about 70 psig pressure. Step (iii) of the lyophilization process can be conducted at a temperature of about -2°C to -40°C, about -2°C to -35°C, about -2°C to -30°C, about -2°C to -25°C, about -2°C to -20°C, about -2°C to -15°C, about -2°C to -10°C, about -2°C to -5°C, about -2°C to -4°C, about -2°C to -3°C, about -3°C to -4°C, about -4 °C to -5°C, about -5°C to

-6°C, about -6°C to -7°C, about -7°C to -8°C, about -8°C to -9°C, or about -9°C to -10°C.

[0011] The frozen solution of step (iii) can be under an aggressive lyophilization process through primary drying, with or without secondary drying, at higher primary drying temperature, such as about 0°C to 20°C, thereby forming a lyophilized bulk protein product. The higher primary drying temperature can be about 1°C to 20°C, about 5°C to 20°C, about 10°C to 20°C, about 15°C to 20°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C. The frozen solution of step (iii) also can be under a conservative lyophilization process through primary drying, with or without secondary drying, at lower primary drying temperature, such as about -35 °C to -1°C, thereby forming a lyophilized bulk protein product. The lower primary drying temperature can be about -35°C to -2°C, about -34°C to -2°C, about -32°C to -2°C, about -30°C to -2°C, about -28°C to -2°C, about -26°C to -2°C, about -24°C to -2°C, about -23°C to -2°C, about -

21°C to -2°C, about -20°C to -2°C, about -18°C to -2°C, about -16°C to -2°C, about -14°C to

-2°C, about -12°C to -2°C, about -10°C to -2°C, about -8°C to -2°C, about -6°C to -2°C, about -4°C to -2°C, about -3°C to -2°C, about -35°C, about -30°C, about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, about -3°C, about -2°C, or about -1°C.

[0012] The inventions also provide methods of forming concentrated protein formulations comprising the steps of: (a) providing an aqueous solution comprising a protein, one or more viscosity reducers, a polysorbate, and stabilizer(s) such as a sugar, and a pH of about 5.0 to about 7.0; (b) lyophilizing the aqueous solution to form a lyophilisate; and (c) reconstituting the lyophilisate using water thereby forming the concentrated protein solution, wherein the concentrated protein solution comprises about 50 to about 700 mg/ml of the protein. The concentrated protein solution can be a drug product, and the viscosity reducer can be arginine hydrochloride. The aqueous solution can comprise about 100 mg/ml, about 200 mg/ml, about 100 to about 400 mg/ml, or about 200 to about 350 mg/ml of the protein. The aqueous solution can be self-buffering and does not require an exogenous buffer to maintain a stable pH. The aqueous solution can further comprise a buffer, wherein the buffer can be a histidine buffer. The concentration of the histidine buffer can be about 1 to about 30 mM or about 5 mM or about lOmM. The pH of the aqueous solution can be about 5.5 to about 6.5 or about 6.0. The concentration of the sugar can be about 0.10% to 10% (w/v) or about 0.5 to 1.0% (w/v). The sugar can be sucrose, wherein the concentration of sucrose can be about 0.5% or about 1.0% (w/v). The concentration of arginine hydrochloride can be about 10 to about 300 mM, about 50 to about 200 mM, or about 100 mM or about 200 mM. The polysorbate can be polysorbate 80 (PS80) or polysorbate 20 (PS20), wherein the concentration of the polysorbate can be about 0.01% to about 0.30% (w/v), or about 0.075% (w/v). The protein can be selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc- fusion protein.

[0013] The inventions also provide that the step (b) lyophilizing can comprise the steps of: (i) cooling a solution comprising a protein product below the freezing point of the solution, thereby forming a frozen solution; (ii) pressuring the cooled solution of step (i) with a gas; (iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) lyophilizing the frozen solution of step (iii) through primary drying and with or without secondary drying, thereby forming a lyophilized bulk protein product. The step (b) lyophilizing also can comprise the steps of: (i) cooling a solution comprising a protein product below the freezing point of the solution, thereby forming a frozen solution; (ii) pressuring the cooled solution of step (i) with a gas; (iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) subjecting the frozen solution of step (iii) under an aggressive lyophilization process through primary drying and with or without secondary drying, at higher primary drying temperature, thereby forming a lyophilized bulk protein product. The higher primary drying temperature can be about 1°C to 20°C, about 5°C to 20°C, about 10°C to 20°C, about 15°C to 20°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about

12°C, about 14°C, about 16°C, about 18°C, or about 20°C.

[0014] The inventions also provide that the step (b) lyophilizing can comprise the steps of: (i) cooling a solution comprising a protein product below the freezing point of the solution, thereby forming a frozen solution; (ii) pressuring the cooled solution of step (i) with a gas; (iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) subjecting the frozen solution of step (iii) under a conservative lyophilization process through primary drying and with or without secondary drying, at lower primary drying temperature, thereby forming a lyophilized bulk protein product. The inventions also provide that during step (ii) the gas can be present at about 14 to about 70 psig pressure, or about 14 to about 42 psig pressure. The lower primary drying temperature can be about -35°C to -2°C, about -34°C to -2°C, about -32°C to -2°C, about - 30°C to -2°C, about -28°C to -2°C, about -26°C to -2°C, about -24°C to -2°C, about -23°C to

-2°C, about -21°C to -2°C, about -20°C to -2°C, about -18°C to -2°C, about -16°C to -2°C, about -14°C to -2°C, about -12°C to -2°C, about -10°C to -2°C, about -8°C to -2°C, about -

6°C to -2°C, about -4°C to -2°C, about -3°C to -2°C, about -35°C, about -30°C, about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, about -3°C, about -2°C, or about -1°C.

[0015] The inventions also provide that step (iii) can be conducted at a temperature of about -2°C to about -20°C, or at about -5°C.

[0016] The inventions also provide ultra-high concentrated protein (UHCP) formulations for lyophilization, comprising: an aqueous solution of protein having a protein, one or more viscosity reducers, a polysorbate, and a sugar at a pH of about 5.0 to about 7.0; wherein the sugar at a concentration of about 0.10% to about 10% (w/v). The aqueous solution can comprise about 100 mg/ml of the protein, about 200 mg/ml of the protein, about 100 to about 400 mg/ml of the protein, or about 200 to about 350 mg/ml of the protein. The inventions also provide that the aqueous solution can be self-buffering and does not require an exogenous buffer to maintain a. stable pH. The aqueous solution further can comprise a buffer, and a pH of between about 5.0 to about 7.0, wherein the buffer can be a histidine buffer, wherein the concentration of histidine buffer can be about 1 to about 30 mM or about

5 mM, wherein the pH of the aqueous solution can be about 5.5 to about 6.5 or about 6.0. The concentration of the sugar can be about 0.5 to about 1.0% (w/v), wherein the sugar can be sucrose, wherein the concentration of sucrose can be about 0.5% or about 1.0% (w/v).

[0017] The protein can be selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc- fusion protein.

[0018] The inventions also provide reconstitution solutions for reconstituting lyophilized ultra-high concentrated protein (UHCP) formulations comprising: (a) arginine hydrochloride solution at a concentration of about 10 to about 300 mM; and (b) the polysorbate at a concentration of about 0.01% to about 0.30% (w/v). The concentration of arginine hydrochloride can be about 100 to about 200 mM, or about 100 mM or about 200 mM. The polysorbate can be polysorbate 80 (PS80) or polysorbate 20 (PS20), wherein the concentration of the PS80 or PS20 can be about 0.01% to about 0.30% (w/v), or about 0.10% or about 0.20% (w/v).

[0019] The inventions also provide that the protein in the UHCP formulation can be selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 is a schematic depiction of a lyophilization process depicting changes in moisture content in a protein product under freeze-drying conditions over time.

[0021] Figure 2 illusttates the effect (flexibility and stability) of aggressive lyophilization cycle on various formulations (Fl, F2, F3, and F4) containing about 100mg/ml dupilumab as a representative mAb drug substance. Fl contains dupilumab about 100mg/mL in H₂O; F2 contains dupilumab about 100mg/mL in 5mM Histidine, pH 6; F3 contains dupilumab about 100mg/mL in 5mM Histidine with 0.5% (w/v) Sucrose, pH 6; and F4 contains dupilumab about 100mg/mL in 5mM Histidine with 1% (w/v) Sucrose, pH 6.

[0022] Figure 3 illustrates the effect (flexibility and stability) of conservative lyophilization cycle on various formulations (Fl, F2, F3, and F4) containing about 100mg/ml dupilumab as a representative mAh drug substance. Fl contains mAh about 100mg/mL in H₂O; F2 contains mAb about 100mg/mL in 5mM Histidine, pH 6; F3 contains mAb about 100mg/mL in 5mM Histidine with 0.5% (w/v) Sucrose, pH 6; and F4 contains mAb about 100mg/mL in 5mM Histidine with 1% (w/v) Sucrose, pH 6.

[0023] Figure 4 illusttates the effect (flexibility and stability) of controlled nucleation with conservative primary drying lyophilization cycle on various formulations (Fl, F2, F3, and F4) containing about 100mg/ml dupilumab as a representative mAb drug substance. Fl contains mAb about 100mg/mL in H₂O; F2 contains mAb about 100mg/mL in 5mM Histidine, pH 6; F3 contains mAb about 100mg/mL in 5mM Histidine with 0.5% (w/v) Sucrose, pH 6; and F4 contains mAh about 100mg/mL in 5mM Histidine with 1% (w/v) Sucrose, pH 6.

[0024] Figure 5 is a Size Exclusion/Ultra-Performance Liquid Chromatography (SE- UPLC) chromatograph showing high molecular weight percentage (%HMW) and low molecular weight percentage (%LMW) protein aggregates resulting from controlled nucleation with pre-lyophilization, aggressive lyophilization, and controlled nucleation with secondary drying lyophilization cycles.

[0025] Figure 6 is a graphical representation of changes in high molecular weight (%) protein aggregates in formulations (Fl, F2, F3, and F4) resulting from different lyophilization processes.

[0026] Figure 7 schematically depicts products of bulk scale lyophilization (105 mL).

[0027] Figure 8 schematically depicts constant mass of a bulk lyophilized cake.

[0028] Figure 9 schematically depicts reconstituted ultra-high concentration protein formulations with calculated volumes of reconstitution liquid for resulting concentrations of 200 mg/ml, 300 mg/ml, and 350 mg/ml, from left to right, respectively.

[0029] Figure 10A is a graph showing the effect of reconstitution liquid compositions on the viscosity of ultra-high concentration protein formulations. Figure 10B is a graph schematically depicting the effect of reconstitution liquid compositions on the viscosity of ultrahigh concentration formulations at 20°C. Figure 10C is a bar graph showing the effect of reconstitution liquid compositions on the viscosity of ultra-high concentration protein formulations F3 and F4.

[0030] Figure 11A is a graph showing the effect of reconstitution liquid compositions and the change in %HMW of ultra-high concentration protein formulations. Figure 11B is a bar graph showing the effect of reconstitution liquid compositions and the change in %HMW of ultra-high concentration protein formulations F3 and F4.

[0031] Figure 12A is a graph showing the stability studies on F3 formulations at different temperatures for up to six months and the effect on protein concentration. Figure 12B is a bar graph showing the stability studies on F3 formulations at different temperatures for up to twelve months and the effect on protein concentration. [0032] Figure 13A is a graph showing the stability studies on F4 formulations at different temperatures for up to six months and the effect on protein concentration. Figure 13B is a bar graph showing the stability studies on F4 formulations at different temperatures for up to twelve months and the effect on protein concentration.

[0033] Figure 14A is a bar graph showing the stability studies on F3 formulations at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 14B is a line graph depicting data from the formulation F3 stability study at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 14C is a bar graph showing the stability studies on F3 formulations at different temperatures for up to 12 months and the change in %HMW protein aggregates.

[0034] Figure ISA is a bar graph showing the stability studies on F4 formulations at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 15B is a line graph depicting data from the formulation F4 stability study at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 15C is a bar graph showing the stability studies on F4 formulations at different temperatures for up to 12 months and the change in %HMW protein aggregates.

[0035] Figure 16A is a graph showing the stability studies on F3 formulations at different temperatures for up to six months and the change in optical density. Figure 16B is a graph showing the stability studies on F3 formulations at different temperatures for up to nine months and the change in optical density.

[0036] Figure 17A is a graph showing the stability studies on F4 formulations at different temperatures for up to six months and the change in optical density. Figure 17B is a graph showing the stability studies on F4 formulations at different temperatures for up to nine months and the change in optical density.

[0037] Figure 18A is a graph showing the stability studies on F3 formulations at different temperatures for up to six months and the effect on pH of the formulation. Figure 18B is a graph showing the stability studies on F3 formulations at different temperatures for up to 12 months and the effect on pH of the formulation.

[0038] Figure 19A is a graph showing the stability studies on F4 formulations at different temperatures for up to six months and the effect on pH of the formulation. Figure 19B is a graph showing the stability studies on F4 formulations at different temperatures for up to 12 months and the effect on pH of the formulation.

[0039] Figure 20A is a bar graph showing the stability studies on F3 formulations at different temperatures for up to six months and the effect on percent composition of ions as determined by Cation Exchange Chromatography (CEX). Figure 20B is a line graph depicting data from the formulation F3 stability study at different temperatures for up to six months and the effect on percent composition of ions as determined by CEX. Figure 20C is a bar graph showing the stability studies on F3 formulations at different temperatures for up to 12 months and the effect on percent composition of ions as determined by Cation Exchange Chromatography (CEX). 40°C data is only shown for 0 and one month.

[0040] Figure 21A is a bar graph showing the stability studies on F4 formulations at different temperatures for up to six months and the effect on percent composition of ions as determined by CEX. Figure 21B is a line graph depicting data from the formulation F4 stability study at different temperatures for up to six months and the effect on percent composition of ions as determined by CEX. Figure 21C is a bar graph showing the stability studies on F4 formulations at different temperatures for up to 12 months and the effect on percent composition of ions as determined by CEX. 40°C data is only shown for 0 and one month.

[0041] Figure 22 is a chart comparing lyophilization cycles. Three different lyophilization cycles were assessed to see which cycle minimized protein aggregation. The chart shows details of the three feasibility studies lyophilization cycles.

[0042] Figure 23 is a bar graph showing the solution viscosity after lyophilization of low concentration EDS (F5) and its reconstitution with WEI (water for injection) to higher protein concentration.

[0043] Figure 24 is a graph showing dupilumab, as a representative mAh, aggregation (%HMW) after lyophilization of low concentration EDS (F5) and its reconstitution to higher protein concentration with WEI.

DETAILED DESCRIPTION OF THE INVENTIONS

DEFINITIONS [0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The term “about” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the invention can perform as intended, such as having a rate, amount, degree, increase, decrease, percentage, value, purity, pH, concentration, molarity, molality, amount of time, presence or absence of a form or variant, temperature, density, viscosity, ionic strength, and/or conductivity, as is apparent from the teachings contained herein. For example, “about” can signify values either above or below the stated value in a range of approximately +/- 10% or more or less depending on the ability to perform. Thus, this term can encompass values beyond those simply resulting from systematic error.

[0046] “Antibodies” (also referred to as "immunoglobulins") are examples of proteins having multiple polypeptide chains and extensive post-translational modifications. The canonical immunoglobulin protein (for example, IgG) comprises four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine disulfide bond, and the two heavy chains are bound to each other via two cysteine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (for example, at asparagine residues) with various polysaccharides, and can differ from species to species, which may affect antigenicity for therapeutic antibodies. Butler and Spearman, "The choice of mammalian cell host and possibilities for glycosylation engineering", Curr. Opin. Biotech. 30:107-112 (2014).

[0047] Antibodies are often used as therapeutic biomolecules. An antibody can include immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CHS. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (ER). Each VH and VL can be composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FRS, CDRS, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3. The term "high affinity" antibody refers to those antibodies having a binding affinity to their target of at least 10-9 M, at least 10-10 M; at least 10-11 M; or at least 10-12 M, as measured by surface plasmon resonance, for example, BIACORE™ or solution-affinity ELISA.

[0048] The phrase "bispecific antibody" can include an antibody capable of selectively binding two or more epitopes. Bispecific antibodies comprise two different heavy chains, with each heavy chain specifically binding a different epitope — either on two different molecules (for example, antigens) or on the same molecule (for example, on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two, three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (for example, on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.

[0049] The phrase "heavy chain," or "immunoglobulin heavy chain" can include an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified can include a heavy chain variable domain. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain can include a fragment that is capable of specifically recognizing an antigen (for example, recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), that can express and secreting from a cell, and that comprises at least one CDR.

[0050] The phrase "light chain" can include an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified can include human kappa and lambda light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. A full-length light chain can include, from amino terminus to carboxyl terminus, a VL domain that can include FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used with these inventions include those, for example, which do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening for the most employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not interfere with the affinity and/or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen-binding regions of the antigen-binding protein.

[0051] The phrase "variable domain" can include an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" can include an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.

[0052] The phrase "complementarity determining region" ("CDR") can include an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally (that is, in a wild-type organism) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (for example, an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. In some circumstances (for example, for a CDR3), CDRs can be encoded by two or more sequences (for example, germline sequences) that are not contiguous (for example, in a nucleic acid sequence that has not been rearranged) but are contiguous in a B cell nucleic acid sequence, for example, as the result of splicing or connecting the sequences (for example, V-D-J recombination to form a heavy chain CDRS).

[0053] “Antibody derivatives and fragments” include but are not limited to antibody fragments (for example, ScFv-Fc, dAB-Fc, half antibodies, Fab), multi-specifics (for example, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, tri-specific). “Fab” refers to an antibody fragment comprising an antigen binding region. A Fab typically will lack the Fc portion.

[0054] The phrase "Fc-containing protein" can include antibodies, bispecific antibodies, antibody derivatives containing an Fc, antibody fragments containing an Fc, Fc- fusion proteins, receptor Fc-fusion proteins (including trap proteins), immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region. A "functional portion" refers to a CH2 and CHS region that can bind a Fc receptor (for example, an FcyR; or an FcRn, (neonatal Fc receptor), and/or that can participate in the activation of complement. If the CH2 and CHS region contains deletions, substitutions, and/or insertions or other modifications that render it unable to bind any Fc receptor and unable to activate complement, the CH2 and CHS region is not functional. Fc- fusion proteins include, for example, Fc-fusion (N-terminal), Fc-fusion (C-terminal), mono- Fc-fusion and bispecific Fc-fusion proteins.

[0055] “Fc" stands for fragment crystallizable and is often referred to as a fragment constant. Antibodies contain an Fc region that is made up of two identical protein sequences. IgG has heavy chains known as y-chains. IgA has heavy chains known as a-chains; IgM has heavy chains known as μ -chains. IgD has heavy chains known as a-chains. IgE has heavy chains known as e-chains. In nature, Fc regions are the same in all antibodies of a given class and subclass in the same species. Human IgGs have four subclasses and share about 95% homology amongst the subclasses. In each subclass, the Fc sequences are the same. For example, human IgGl antibodies will have the same Fc sequences. Likewise, IgG2 antibodies will have the same Fc sequences; IgG3 antibodies will have the same Fc sequences; and IgG4 antibodies will have the same Fc sequences. Alterations in the Fc region create charge variation. [0056] Fc-containing proteins, such as antibodies, can comprise modifications in immunoglobulin domains, including where the modifications affect one or more effector function of the binding protein (for example, modifications that affect FcyR binding, FcRn binding and thus half-life, and/or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293,

294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324,

326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358,

359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416,

419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0057] For example, and not by way of limitation, the binding protein is an Fc- containing protein (for example, an antibody) and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modification(s)) and have a modification at position 250 (for example, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T), and 256 (for example, S/R/Q/E/D or T); or a modification at 428 and/or 433 (for example, L/R/SI/P/Q or K) and/or 434 (for example, H/F or Y); or a modification at 250 and/or 428; or a modification at 307 or 308 (for example, 308F, V308F), and 434. In another example, the modification can comprise a 428L (for example, M428L) and 434S (for example, N434S) modification; a 428L, 2591 (for example, V259I), and a 308F (for example, V308F) modification; a 433K (for example, H433K) and a 434 (for example, 434Y) modification; a 252, 254, and 256 (for example, 252Y, 254T, and 256E) modification; a 250Q and 428L modification (for example, T250Q and M428L); a 307 and/or 308 modification (for example, 308F or 308P).

[0058] “Fv” stands for fragment variable and is primarily responsible for binding to epitopes.

[0059] As used herein, the expression “formulation” means a combination of at least one active ingredient (e.g., a protein, a drug product, such as polypeptide, antibody, monoclonal antibody, etc. which is capable of exerting a biological effect in a human or nonhuman animal), and at least one inactive ingredient which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term “formulation,” as used herein, means “pharmaceutical” or “therapeutic” formulation unless specifically indicated otherwise. The present invention provides pharmaceutical formulations comprising at least one therapeutic protein. According to the present invention, the therapeutic protein is an antibody, or an antigen-binding fragment thereof. The term “formulation,” as used herein, also means a solid, semisolid, and/or liquid formulation, such as suitable for oral, subcutaneous, and/or intravenous administration.

[0060] The term “lyophilized” or “freeze-dried” can include a state of a substance that has been subjected to a drying procedure through ice sublimation under frozen condition such as lyophilization, where at least 90% of moisture has been removed.

[0061] The term “excipient” can include a non-therapeutic agent added to a pharmaceutical composition to provide a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, amino acids, surfactants or co-solvents, and the like.

[0062] The term “buffer” means a buffering solution or a buffering agent that stabilizes the pH of a solution, A buffer comprises a weak acid and its conjugate base, or a weak base and its conjugate acid. Buffering of a protein solution at or close to the optimal pH helps to ensure proper protein folding and function. The best buffer can be identified, for example, by measuring the thermodynamic stability (DSC), and high molecular weight variants (SEC) and charge variants (CEX) of the protein (e.g., antibody) solution at various pHs following accelerated storage/incubation. Measuring the circular dichroism of the protein (e.g., antibody) solution at various pHs may also assist in identifying a buffer. Circular dichrosim (CD) is one method used to determine structural changes (unfolding) of a protein (S. Beyehok, “Circular dichroism of biological macromolecules.” Science 154(3754): 1288- 99 (1966); Kemmer and Keller, “Nonlinear least-squares data fitting in Excel spreadsheets.” Nat Protoc. 5(21:267-81 (2010)). Many proteins, including antibodies and derivatives thereof, possess the ability to function as buffers (i.e., so called “self-buffering”) and therefore may not require the addition of an exogenous buffer to maintain stable pH (Gokarn et ah, “Selfbuffering antibody formulations,” J Ph arm Sci, 97(8):3051-66 (2008)), Examples of commonly used buffers are listed in Table 1. For a more complete discussion of buffers in biological solutions, see Irwin H. Segel, Biochemical Calculations (2^nd ed. 1976), or Remington, The Science and. Practice of Pharmacy 244 (Paul Beringer ei al. eds., 21^st ed. 2006).

[0063] Table I.

[0064] Various methods of stability testing are known in the art, including Fleischman et al. Shipping-Induced Aggregation in Therapeutic Antibodies: Utilization of a Scale-Down Model to Assess Degradation in Monoclonal Antibodies. J. Pharm. Set. (2017) 106: 994-1000; Ghazvini et al. Evaluating the Role of the Air-Solution Interface on the Mechanism of Sub visible Particle Formation Caused by Mechanical Agitation for an IgGl mAbl. J. Pharm. Set. (2016) 105: 1643-1656; and Torisu et al. Synergistic Effect of Cavitation and Agitation on Protein Aggregation. J. Pharm. Set. (2017) 106: 521-529).

[0065] “Protein,” “polypeptide” or “peptide” refers to sequence(s) of amino acids covalently joined. Polypeptides include natural, semi-synthetic and synthetic proteins and protein fragments. “Protein,” “polypeptide” and “protein” can be used interchangeably. Oligopeptides are considered shorter polypeptides.

[0066] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” can include one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0067] All numerical limits and ranges set forth herein include all numbers or values thereabout or there between of the numbers of the range or limit. The ranges and limits described herein expressly denominate and set forth all integers, decimals and fractional values defined and encompassed by the range or limit. Thus, a recitation of ranges of values herein are merely intended to serve as a way of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

DESCRIPTION

Lyophilization

[0068] Administration of formulations comprising proteins represent significant challenges to pharmaceutical formulators. Proteins possess unique chemical and physical properties that present stability problems. A variety of degradation pathways exist for proteins, implicating both chemical and physical instability. Chemical instability can include deamination, aggregation, clipping of the peptide backbone, and oxidation of methionine residues. Physical instability encompasses many phenomena, including, for example, aggregation and/or precipitation.

[0069] Physical and Chemical stability can be improved by removing water from the proteins and limiting their mobility (Figure 1). Lyophilization (freeze-drying under controlled conditions) is commonly used for long-term storage of proteins. Liquid proteins that are unstable are freeze-dried, or lyophilized. Lyophilization preserves the integrity of the protein for drug delivery. The lyophilized protein cab be resistant to degradation, aggregation, oxidation, and other degenerative processes while in the freeze-dried state. The lyophilized protein can be reconstituted with reconstitution liquids as disclosed herein, and optionally containing a bacteriostatic preservative (e.g., benzyl alcohol) prior to administration.

[0070] The lyophilization of the aqueous solution to form a lyophilisate can be carried out by various processes, including controlled nucleation with conservative primary drying (including freezing, primary drying without secondary drying) (Figure 4), controlled nucleation with secondary drying (including freezing, primary drying and secondary), aggressive lyophilization (including freezing, primary drying and secondary but at higher drying temperatures), aggressive lyophilization with primary drying (including freezing, primary drying without secondary at higher primary drying temperature) (Figure 2), conservative lyophilization (including freezing, primary drying and secondary but at lower drying temperatures), or conservative lyophilization with primary drying (including freezing, primary drying without secondary at lower primary drying temperature) (Figure 3).

[0071] The inventions can involve various lyophilization processes and can utilize several steps including (i) cooling a solution comprising a protein product below the freezing point of the solution; (ii) pressuring the cooled solution of step (i) with a gas; (iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) lyophilizing the cooled/frozen solution of step (iii) to form a frozen protein product. The gas can be present at about 14 to 70 psig, about 14 to 65 psig, about 14 to 60 psig, about 14 to 55 psig, about 14 to 50 psig, about 14 to 45 psig, about 14 to 44 psig, about 14 to 43 psig, about 14 to 42 psig, about 14 to 41 psig, about 14 to 40 psig, about 14 to 35 psig, about 14 to 30 psig, about 14 to 25 psig, about 14 to 20 psig, or about 14 to 15 psig pressure. The step (iii) of the lyophilization process can be conducted at a temperature of about -2°C to -40°C, about -2°C to -35°C, about -2°C to -30°C, about -2°C to -25°C, about -

2°C to -20°C, about -2°C to -15°C, about -2°C to -10°C, about -2°C to -5°C, about -2°C to -

4 °C, about -2°C to -3°C, about -3°C to -4°C, about -4°C to -5°C, about -5°C to -6°C, about -

6°C to -7°C, about -7°C to -8°C, about -8°C to -9°C, or about -9°C to -10°C. The frozen solution of step (iii) can be under an aggressive lyophilization process through primary drying and with or without secondary drying, at higher primary drying temperature, such as above the freezing point (for example, see Table 2 and Figure 22), thereby forming a lyophilized bulk protein product. The higher primary drying temperature can be about 1°C to 20°C, about 5°C to 20°C, about 10°C to 20°C, about 15°C to 20°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about

20°C. The frozen solution of step (iii) also can be under a conservative lyophilization process through primary drying and with or without secondary drying, at lower primary drying temperature, such as below the freezing point (for example, see Table 2 and Figure 22), thereby forming a lyophilized bulk protein product. The lower primary drying temperature can be about -35°C to -2°C, about -34°C to -2°C, about -32°C to -2°C, about -30°C to -2°C, about -28°C to -2°C, about -26°C to -2°C, about -24°C to -2°C, about -23°C to -2°C, about -

[0072] The primary drying can be conducted at a temperature of about 20°C to -50°C, about 15°C to -35°C, about 10°C to -30°C, about 5°C to -25°C, about 2°C to -20°C, about 0°C to -20°C, about 0°C to -10°C, about 0°C to -5°C, about -2°C to -4°C, about -2°C to

-3°C, about -3°C to -4°C, about -4°C to -5°C, about -5°C to -6°C, about -6°C to -7°C, about -

7°C to -8°C, about -8°C to -9°C, about -9°C to -10°C, about 0°C, about -2°C, about -5°C, about -7°C, about-10°C, about -15°C, about -20°C, about -25°C, about -30°C, about -35°C, about -40°C, about -45°C, or about -50°C.

[0073] The secondary drying can be conducted at a temperature of about 10°C to 50°C, about 15°C to 45°C, about 10°C to 40°C, about 5°C to 35°C, about 10°C to 30°C, about 20°C to 25°C, about 20°C to 35°C, about 25°C to 30°C, about 30°C to 35°C, about

35°C to 40°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C or about 50°C.

Protein Concentration

[0074] The concentrated protein formulation of the present inventions can comprise a protein concentration from about 5+0.25 mg/mL to about 700+50.0 mg/mL, about 50 to 500+37.5 mg/mL, or about 50 to 200+37.5 mg/mL. The concentrated protein formulation of the present inventions can comprise a protein concentration from about 5 to 50 mg/ml, about 50 to 75 mg/ml, about 75 to 100 mg/ml, about 100 to 150 mg/ml, about 150 to 200 mg/ml, about 200 to 250 mg/ml, about 250 to 300 mg/ml, about 300 to 350 mg/ml, about 350 to 400 mg/ml, about 400 to 450 mg/ml, about 450 to 500 mg/ml, about 500 to 550 mg/ml, about 550 to 600 mg/ml, about 600 to 650 mg/ml, or about 650 to 700 mg/ml. Proteins can include antibodies such as monoclonal antibodies, antibody fragments and derivatives, Fc-fusion proteins and the like. For example, the antibody concentration in the concentrated protein formulation of the present inventions can be about 5 mg/mL+2.5 mg/mL, about 5 mg/mL, about 10 mg/mL+5.0 mg/mL, about 10 mg/mL, about 255 mg/mL+5.0 mg/mL, about 25 mg/mL, about 50 mg/mL+7.5 mg/mL, about 50 mg/mL, about 100 mg/mL+15 mg/mL, about 100 mg/mL, about 150 mg/mL+22.5 mg/mL, about 150 mg/mL, about 175 mg/mL+26.25 mg/mL, about 175 mg/mL, about 200 mg/mL+30 mg/mL, about 200 mg/mL, about 250+37.5 mg/mL, about 250 mg/mL, about 300+50.0 mg/mL, or about 300 mg/mL. [0075] The inventions are amenable to use with a wide variety of Fc-containing proteins and other proteins. The inventions can be employed in the production of biological and pharmaceutical products. For example, for antibodies, the inventions are amendable for research and production use for diagnostics and therapeutics based upon all major antibody classes, namely IgG, IgA, IgM, IgD and IgE. IgG is a preferred class, such as IgGl (including IgG Iλ and IgG IK), IgG2 and IgG4. Exemplary antibodies to be produced according to the inventions include Alirocumab, Atoltivimab, Maftivimab, Odesivimab, Odesivivmab-ebgn, Casirivimab, Imdevimab, Cemiplimab, Cemplimab-rwlc, Dupilumab, Evinacumab, Evinacumab-dgnb, Fasimumab, Nesvacumab, Trevogrumab, Rinucumab and Sarilumab. Antibodies can include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. The antibody can be an IgGl antibody. The antibody can be an IgG2 antibody. The antibody can be an IgG3 antibody. The antibody can be an IgG4 antibody. The antibody can be a chimeric IgG2/IgG4 antibody. The antibody can be a chimeric IgG2/IgGl antibody. The antibody can be a chimeric IgG2/IgGl/IgG4 antibody.

[0076] In addition to the antibodies described in the Examples and Figures, the antibody can be selected from the group consisting of an anti-Programmed Cell Death 1 antibody (e.g., an anti-PDl antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), an anti-Programmed Cell Death Ligand- 1 (e.g., an anti-PD-Ll antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580A1), an anti-D114 antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti- Angiopoetin-Like 3 antibody (e.g. an antiAngPtl3 antibody as described in U.S. Pat. No. 9,018,356), an anti-platelet derived growth factor receptor antibody (e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti- Prolactin Receptor antibody (e.g. anti-PRLR antibody as described in U.S. Pat. No. 9,302,015), an anti-Complement 5 antibody (e.g. an 25 anti-C5 antibody as described in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNF antibody, an anti- epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No. US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1), an anti-Growth and Differentiation Factor-8 antibody (e.g., an anti- GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g., anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1), an anti-VEGF antibody, an anti-ILlR antibody, an interleukin four receptor antibody (e.g., an antiIL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g. an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880), an anti-ILl antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti- IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g. anti- IL33 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0271658A1 or US2014/0271642A1), an anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271653A1), an anti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US2015/0266966A1, and in U.S. Application No. 62/222,605), an anti- Cluster of differentiation 20 (e.g., an anti-CD20 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US2015/0266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti- Cluster of Differentiation 48 (e.g. anti-CD48 antibody as described in U.S. Pat. No. 9,228,014), an anti-Fel dl antibody (e.g. as described in U.S. Pat. No. 9,079,948), an anti-Middle East Respiratory Syndrome virus (e.g. an anti- MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015/0337029A1), an antiEbola virus antibody (e.g., as described in U.S. Pat. Appln. Pub. No. US2016/0215040), an anti-Zika virus antibody, an anti-Lymphocyte Activation Gene 3 antibody (e.g., an anti- LAG3 antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g., an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody. The bispecific antibody can be selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Mucl6 bispecific antibody), and an anti-CD3 x anti- Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody). See also U.S. Patent Publication No. US 2019/0285580 Al. [0077] The inventions also are amenable to the production of other molecules, including fusion proteins. Preferred fusion proteins include Receptor-Fc-fusion proteins, such as Trap proteins, the protein of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein). The Fc-fusion protein can be a receptor Fc- fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety. The Fc moiety can comprise a hinge region followed by a CH2 and CH3 domain of an IgG. The receptor Fc-fusion protein can contain two or more distinct receptor chains that bind to either a single ligand or multiple ligands. For example, an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the I1-1R1 extracellular region fused to Fc of hlgGl; see U.S. Pat. No. 6,927,044, or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc of hlgGl; see U.S. Pat. Nos. 7,087,411 and 7,279,159). The Fc-fusion protein can be a ScFv-Fc-fusion protein, which contains one or more of one or more antigen binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.

[0078] Other proteins lacking Fc portions, such as recombinantly produced enzymes and mini-traps, also can be made according to the inventions. Mini-traps are trap proteins that use a multimerizing component (MC) instead of an Fc portion and are disclosed in U.S. Patent Nos. 7,279,159 and 7,087,411. Derivatives, components, domains, chains, and fragments of the above also are included.

Buffers and pH

[0079] The concentrated protein formulations of the present inventions can include pharmaceutically acceptable buffering agents. The buffering agents can include, without limitation, phosphate buffers, histidine buffers, sodium citrate buffers, HEPES buffers, Tris buffers, Bicine buffers, glycine buffers, N-glycylglycine buffers, sodium acetate buffers, sodium carbonate buffers, glycyl glycine buffers, lysine buffers, arginine buffers, sodium phosphate buffers, and/or mixtures thereof. The buffering agent can be a Histidine buffer, a Phosphate buffer (e.g., a sodium phosphate buffer) or a Tris buffer.

[0080] The concentrated protein formulation of the present inventions can comprise about 1 to about 30, about 1 mM to about 20 mM, about 5 mM to about 25 mM, about 5 to about 15, about 7 mM to about 13 mM, or about 8 mM to about 12 mM of a buffering agent. The formulation can comprise about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about

18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM of a buffering agent.

[0081] The concentrated protein formulations of the present inventions can comprise about 1 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or about 10 mM+1 mM Histidine buffer. The formulation can comprise about 1 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or about 10 mM±l mM

Histidine buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about

21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM Histidine buffer.

[0082] The concentrated protein formulations of the present inventions can comprise about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM+1 mM Tris buffer. The formulation can comprise about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 8 mM to about 12 mM, or about 10 mM+1 mM sodium phosphate buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about

14 mM, or about 15 mM Tris buffer. The formulation can comprise about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM sodium phosphate buffer. The formulation also can comprise about 10 mM+2 mM Tris buffer. The formulation can comprise about 10 mM+2 mM sodium phosphate buffer.

[0083] Additionally, as explained above, many proteins, including antibodies and. derivatives thereof, possess the ability to function as buffers (“self-buffering’’) and therefore may not require the addition of an exogenous buffer to maintain stable pH.

[0084] The concentrated protein formulations of the present inventions have a physiologically compatible pH. The concentrated protein formulations are provided that contain a buffering agent suitable to maintain the formulation at pH between about 5.0 and about 7.0. The pH of the concentrated protein formulation of the present inventions can be about 5.0 to about 7.0, about 5.5 to about 6.5, about 5.6 to about 7.0, about 5.7 to about 7.5, about 5.8 to about 7.0, about 5.9 to about 7.0, about 5.0 to about 7.0, about 6.5 to about 7.0, or about 6.9 to about 7.0. The pH of the formulation can be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about

6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. The pH of the formulation of the present inventions preferably can be about 6.0+0.1, about 6.0+0.05, about 5.9+0.1, or about 5.9+0.05.

Stabilizers

[0085] The concentrated protein formulation of the present inventions can include one or more sugars and other pharmaceutically acceptable stabilizers.

[0086] Inclusion of one of more sugars and other pharmaceutically acceptable stabilizers (e.g., at between about 1% to about 10%) improves the stability of the liquid formulations of the present inventions. The concentrated protein formulation of the present inventions can contain from about 1% to about 10% of one or more sugars. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, mannitol, sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethylcellulose can be used in the formulation. The sugar can be sucrose, trehalose, or a combination thereof.

[0087] The sugars can be used individually or in combination. The sugar, or a combination thereof, can be present in the formulation or composition at a concentration of about 0.10% to about 1.0% (w/v), about 0.20% to about 1.0% (w/v), about 0.30% to about

1.0% (w/v), about 0.40% to about 1.0% (w/v), about 0.50% to about 1.0% (w/v), about

0.60% to about 1.0% (w/v), about 0.70% to about 1.0% (w/v), about 0.80% to about 1.0%

(w/v), about 0.90% to about 1.0% (w/v), about 1.0% to about 10% (w/v), about 2% (w/v) to about 8% (w/v), about 2.5% to about 7.5% (w/v), about 3% (w/v) to about 7% (w/v), or about

4% to about 6% (w/v). The formulation of the present inventions can comprise about 1.0% (w/v), about 1.1% (w/v), about 1.2% (w/v), about 1.3% (w/v), about 1.4% (w/v), about 1.5%

(w/v), about 1.6% (w/v), about 1.7% (w/v), about 1.8% (w/v), about 1.9% (w/v), about 2.0%

(w/v), about 2.1% (w/v), about 2.2% (w/v), about 2.3% (w/v), about 2.4% (w/v), about 2.5%

(w/v), about 2.6% (w/v), about 2.7% (w/v), about 2.8% (w/v), about 2.9% (w/v), about 3.0% (w/v), about 3.1% (w/v), about 3.2% (w/v), about 3.3% (w/v), about 3.4% (w/v), about 3.5%

(w/v), about 3.6% (w/v), about 3.7% (w/v), about 3.8% (w/v), about 3.9% (w/v), about 4.0%

(w/v), about 4.1% (w/v), about 4.2% (w/v), about 4.3% (w/v), about 4.4% (w/v), about 4.5%

(w/v), about 4.6% (w/v), about 4.7% (w/v), about 4.8% (w/v), about 4.9% (w/v), about 5.0%

(w/v), about 5.1% (w/v), about 5.2% (w/v), about 5.3% (w/v), about 5.4% (w/v), about 5.5%

(w/v), about 5.6% (w/v), about 5.7% (w/v), about 5.8% (w/v), about 5.9% (w/v), about 6.0%

(w/v), about 6.1% (w/v), about 6.2% (w/v), about 6.3% (w/v), about 6.4% (w/v), about 6.5%

(w/v), about 6.6% (w/v), about 6.7% (w/v), about 6.8% (w/v), about 6.9% (w/v), about 7.0%

(w/v), about 7.1% (w/v), about 7.2% (w/v), about 7.3% (w/v), about 7.4% (w/v), about 7.5%

(w/v), about 7.6% (w/v), about 7.7% (w/v), about 7.8% (w/v), about 7.9% (w/v), about 8.0%

(w/v), about 8.1% (w/v), about 8.2% (w/v), about 8.3% (w/v), about 8.4% (w/v), about 8.5%

(w/v), about 8.6% (w/v), about 8.7% (w/v), about 8.8% (w/v), about 8.9% (w/v), about 9.0%

(w/v), about 9.1% (w/v), about 9.2% (w/v), about 9.3% (w/v), about 9.4% (w/v), about 9.5%

(w/v), about 9.6% (w/v), about 9.7% (w/v), about 9.8% (w/v), about 19.9% (w/v), or about

10% (w/v) sugar.

[0088] The concentrated protein formulation of the present inventions can include about 0.10% to about 1.0% (w/v), about 0.20% to about 1.0% (w/v), about 0.30% to about

(w/v), about 0.90% to about 1.0% (w/v), about 1.0% to about 10% (w/v), sucrose. The concentrated protein formulation can contain about 0.5% w/v±0.1% or about 1.0% w/v±0.1% w/v sucrose.

Salts

[0089] The concentrated protein formulation and/or the reconstitution liquid of the present inventions can include one or more pharmaceutically acceptable salts.

[0090] The pharmaceutically acceptable salts can include, but are not limited to, metal salts such as sodium, potassium and cesium salts; alkaline earth metal salts such as calcium and magnesium salts; organic amine salts such as triethylamine, guanidine and N- substituted guanidine salts, acetamidine and N-substituted acetamidine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine salts. Pharmaceutically acceptable salts (of basic nitrogen centers) can include, but are not limited to inorganic acid salts such as the hydrochloride, hydrobromide, sulfate, phosphate; organic acid salts such as trifluoroacetate and maleate salts; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphor sulfonate and naphthalenesulfonate; amino acid salts such as arginate, alaninate, asparginate and glutamate; and carbohydrate salts such as gluconate and galacturonate. Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sodium salts, ammonium salts, potassium salts, calcium salts, and magnesium salts (e.g., sodium, ammonium, potassium, calcium, and magnesium chloride; sodium, ammonium, potassium, calcium and magnesium acetate; sodium, ammonium, potassium, calcium and magnesium citrate; sodium, ammonium, potassium, calcium and magnesium phosphate; sodium, ammonium, potassium, calcium and magnesium fluoride; sodium, ammonium, potassium, calcium and magnesium bromide; and sodium, ammonium, potassium, calcium and magnesium iodide). The pharmaceutically acceptable salt can be sodium chloride or arginine hydrochloride (L-arginine hydrochloride).

[0091] The concentrated protein formulation and/or the reconstitution liquid of the present inventions can also include one or more pharmaceutically viscosity reducers such as sodium chloride, lysin, proline, and the like.

[0092] The protein formulation and/or the reconstitution liquid of the present inventions can comprise about 10 mM to about 300 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 100 mM to about 200 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, 150 mM to about 200 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, about 250 mM to about 300 mM, about

75 mM to about 100 mM, about 175 mM to about 200 mM, about 175 mM to about 225 mM, about 200 mM to about 225 mM, about 225 mM to about 275 mM, about 275 mM to about

300 mM, or about 175 mM to about 275 mM of a pharmaceutically acceptable salt. The protein formulation of the present inventions can comprise about 0 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, or about 150 mM of a pharmaceutically acceptable salt. The protein formulation and/or the reconstitution liquid of the present inventions can comprise about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about

67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about

86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, 106 mM, about 107 mM, about 108 mM, about 109 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about

170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about

260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, or about 300 mM, of a pharmaceutically acceptable salt.

[0093] The protein formulation and/or the reconstitution liquid of the present inventions can comprise about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about

250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM of arginine hydrochloride. The protein formulation and/or the reconstitution liquid of the present inventions can comprise about 100 mM+5 mM, about 110 mM+5 mM, about 120 mM+5 mM, about 130 mM+5 mM, about 140 mM+5 mM, about 150 mM+5 mM, about 160 mM+5 mM, about 170 mM+5 mM, about 180 mM+5 mM, about 190 mM+5 mM, about 200 mM+5 mM, or about 210 mM+5 mM arginine hydrochloride.

Surfactants

[0094] The concentrated protein formulations and/or the reconstitution liquid of the present inventions can include one or more surfactants. [0095] The concentrated protein formulations and/or the reconstitution liquid of the present inventions can contain a stabilizing concentration of a pharmaceutically acceptable non-ionic surfactant. Pharmaceutically acceptable non-ionic surfactants that can be used in the concentrated protein formulations and/or the reconstitution liquid discussed herein can include, without limitation, polysorbate 80 (Tween 80; PS80), polysorbate 20 (Tween 20; PS20), and various poloxamers (e.g., poloxamer 188), PEGs (e.g., PEG 3350), or mixtures thereof.

[0096] The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise from about 0.01% (w/v) to about 0.30% (w/v) non-ionic surfactant. The concentrated protein formulations and/or the reconstitution liquid can comprise about 0.01% to 0.30% (w/v), about 0.01% to 0.05% (w/v), about 0.05% to 0.10% (w/v), about 0.10% to 0.15% (w/v), about 0.15% to 0.20% (w/v), about 0.20% to 0.25%

(w/v), or about 0.25% to 0.30% (w/v) non-ionic surfactant. The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise about

0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), about 0.10% (w/v), about 0.11% (w/v), about 0.12% (w/v), about 0.13% (w/v), about 0.14%

(w/v), about 0.15% (w/v), about 0.16% (w/v), about 0.17% (w/v), about 0.18% (w/v), about

0.19% (w/v), about 0.20% (w/v), about 0.21% (w/v), about 0.22% (w/v), about 0.23% (w/v), about 0.24% (w/v), about 0.25% (w/v), about 0.26% (w/v), about 0.27% (w/v), about 0.28%

(w/v), about 0.29% (w/v), or about 0.30% (w/v) non-ionic surfactant. The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.20% w/v±0.01% w/v non-ionic surfactant.

[0097] The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.01% (w/v), about 0.03% (w/v), about 0.015% to about 0.095% (w/v), about 0.015% to about 0.025% (w/v), about 0.025% to 0.035% (w/v), about 0.035% to 0.045% (w/v), about 0.045% to about 0.055% (w/v), about 0.065% to about

0.075% (w/v), about 0.075% to about 0.085% (w/v), about 0.085% to about 0.095% (w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v), about 0.08% (w/v), about 0.09%

(w/v), about 0.10% (w/v), about 0.11% (w/v), about 0.12% (w/v), about 0.13% (w/v), about

0.14% (w/v), about 0.15% (w/v), about 0.16% (w/v), about 0.17% (w/v), about 0.18% (w/v), about 0.19% (w/v), about 0.20% (w/v), about 0.21% (w/v), about 0.22% (w/v), about 0.23%

(w/v), about 0.24% (w/v), about 0.25% (w/v), about 0.26% (w/v), about 0.27% (w/v), about 0.28% (w/v), about 0.29% (w/v), or about 0.30% (w/v) polysorbate 80 (PS80) or polysorbate 20 (PS20). The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.20% w/v±0.01% w/v polysorbate 80 (PS80) or polysorbate 20 (PS20).

[0098] The concentrated protein formulations and/or the reconstitution liquid of the present inventions can comprise 0.01% to 0.30% (w/v), about 0.01% to 0.05% (w/v), about 0.05% to 0.10% (w/v), about 0.10% to 0.15% (w/v), about 0.15% to 0.20% (w/v), about

0.20% to 0.25% (w/v), or about 0.25% to 0.30% (w/v) polysorbate 80 or polysorbate 20. The protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.01% (w/v), about 0.03% (w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07%

(w/v), about 0.08% (w/v), about 0.09% (w/v), about 0.10% (w/v), about 0.11% (w/v), about

0.12% (w/v), about 0.13% (w/v), about 0.14% (w/v), about 0.15% (w/v), about 0.16% (w/v), about 0.17% (w/v), about 0.18% (w/v), about 0.19% (w/v), about 0.20% (w/v), about 0.21%

(w/v), about 0.22% (w/v), about 0.23% (w/v), about 0.24% (w/v), about 0.25% (w/v), about

0.26% (w/v), about 0.27% (w/v), about 0.28% (w/v), about 0.29% (w/v), or about 0.30% (w/v) polysorbate 80. The pharmaceutical compositions and/or the reconstitution liquid of the present inventions can comprise about 0.001% (w/v) to about 0.01% (w/v) poloxamer 188. The protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.001% (w/v), about 0.0015% (w/v), about 0.002% (w/v), about 0.0025% (w/v), about 0.003% (w/v), about 0.0035% (w/v), about 0.004% (w/v), about 0.0045% (w/v), about 0.005% (w/v), about 0.0055% (w/v), about 0.006% (w/v), about 0.0065% (w/v), about 0.007% (w/v), about 0.0075% (w/v), about 0.008% (w/v), about 0.0085% (w/v), about 0.009% (w/v), about 0.0095% (w/v), or about 0.01% (w/v) poloxamer 188. The protein formulations and/or the reconstitution liquid of the present inventions can comprise about 0.005% w/v±0.001% w/v polysorbate 80 or about 0.005% w/v±0.001% w/v poloxamer 188.

[0099] The inventions are further described by the following examples, which do not limit the inventions in any manner. The order of performance of the below experiments and/or examples or example steps can be altered or combined as determined by the person of skill in the art in view of the teachings and data contained herein.

EXAMPLES

Experimental Approaches: [00100] Lyophilization was conducted on bulk proteins at normal concentrations with minimal excipients. Lyophilized proteins were reconstituted with excipient buffer or water to obtain ultra-high concentrated protein (UHCP) formulations. Feasibility testing was conducted on the UHCP formulations to ensure less than a 1 % increase in protein aggregation, which is to make sure < 1 %HMW protein aggregation in the final product.

[00101] Further, feasibility studies were conducted to evaluate UHCP formulation stability as follows:

(i) Bulk lyophilization was done using one-liter UHCP of optimal formulations;

(ii) Lyophilized UHCP formulations were reconstituted using reconstitution liquid; and

(iii) Long term stability studies were conducted on lyophilized bulk UHCP formulations.

Assessing Feasibility of Lyophilized Formulations:

[00102] Using dialyzed dupilumab as a representative mAb drug substance (DS) as model protein, four UHCP formulations, Pl, P2, P3 and P4 were prepared to assess multiple combinations for flexibility and stability. Composition of the formulation P1-P4 are as follows:

[00103] F1: dupilumab about 100mg/mL in H₂O;

[00104] F2: dupilumab about 100mg/mL, 5mM Histidine, pH 6.0;

[00105] F3: dupilumab about 100mg/mL, 5mM Histidine, 0.5% (w/v) Sucrose, pH 6.0; and

[00106] F4: dupilumab about 100mg/mL, 5mM Histidine, 1% (w/v) Sucrose, pH 6.0.

[00107] Formulations F1-F4 use dupilumab as a representative mAb with minimal excipients prior to lyophilization. Excipients required for the final formulation were added during reconstitution. An alternative approach is to lyophilize dupilumab in the presence of the excipients required for the final formulation and reconstitute with lower volume of water. [00108] This approach was assessed with formulation F5. In this formulation both arginine and PS-80 were added prior to lyophilization. Pre-lyo formulation F5 was 100 mg/mL dupilumab, 5 mM histidine, 1% sucrose, 50 mM arginine hydrochloride, 0.075% PS- 80, pH 6.0. After lyophilization dupilumab was reconstituted with water to achieve different protein concentrations. In this case concentrations of all excipients after lyophilization are proportional to the final dupilumab concentration. Formulation F5 can be reconstituted with water for injection (WFI) (without any excipients).

[00109] The UHCP formulations, Fl, F2, F3 and F4 were subject to different lyophilization cycles, including aggressive lyophilization cycle (Figure 2), conservative lyophilization cycle (Figure 3), and controlled nucleation with conservative primary drying only cycle (Figure 4). Lyophilized cakes were reconstituted with H₂O to obtain a final concentration of 100 mg/mL, product quality of samples was subsequently assessed by pH, Optical Density (OD), size exclusion chromatography (SEC) and compared with prelyophilized samples.

[00110] Fl employs dupilumab as a representative mAh and water and is very flexible because no excipients are added in the formulation, excipients can be added later in the process. However, because no excipients are present in Fl, the mAbs are not very stable and expected to aggregate easily. F4 which has the most excipients is expected to be stable and form minimal aggregates, but the many excipients present in F4 limit the flexibility of the formulation later in production. After the formulations were prepared, each one was vacuum filtered through a 0.22-micron sterile filter to eliminate bacteria and plastic particles. 2mL of each formulation was transferred into the corresponding polycarbonate (PC) vials, and one capped vial was saved in 5 °C as an experimental control. Three different lyophilization cycles were assessed to see which cycle minimized protein aggregation (Table 2 and Figure 22).

[00111] Table 2: Details of feasibility studies lyophilization cycles.

*Pirani/CM pressure differential 10 mTorr was taken as the end point for primary drying.

[00112] In the controlled nucleation cycle, the chamber was pressurized which causes the products to freeze uniformly. Samples in the aggressive, conservative, and controlled nucleation lyophilization cycles were removed after primary drying to evaluate the effects of omitting secondary drying on protein aggregation. Samples were run in the lyophilization machine uncapped with thermocouples (TCs) to measure the temperature of the formulations throughout the lyophilization cycle.

[00113] The lyophilized cakes were reconstructed with MQ water to reach a target ending concentration of 100 mg/mL. After reconstitution, the quantitative amount of degradation was determined by evaluating optical density (OD), pH, and aggregation of the samples. Slight changes in pH can denature proteins, so a difference in pH from prelyophilization to post-lyophilization indicates protein instability. Protein aggregation is measured using size-exclusion chromatography (SEC) in which aggregates are separated from other proteins by a chromatography column filled with small beads. For assessing the feasibility of the four formulations, the goal was to have less than a 1% increase in High Molecular Weight (HMW) or protein aggregation from pre-lyophilization to postlyophilization. The two formulations that exhibited the least product loss and the lyophilization cycle that minimized the protein aggregation were chosen for the next phase of the study.

Creating Ultra-high Concentration Formulations in Bulk

[00114] In the feasibility study, only 2 mF of Fl to F4 were lyophilized in each PC vial; however, the bulk study investigated lyophilization on a much larger scale with 105 mF of the two chosen formulations per container. The process for dialyzing the protein as well as preparing and filtering the formulation were the same as listed above including saving pre-lyophilization formulations as controls. 105 mF of both formulations respectively were prepared and transferred to a 1 -Liter PC container for lyophilization. Postlyophilization, the protein cakes in the two 1 -Filer containers were crushed up and then moisture content was measured for the formulations to further ensure the stability of the product and success of the lyophilization cycle. To reach ultra-high protein concentrations, a constant mass of lyophilized cake was measured and then reconstituted with variable amounts of water to reach different ending concentrations. To calculate the amount of water necessary to reach ending concentration targets of 200, 300 and 350 mg/mE, the density of the target ending concentrations needed to be calculated. The following formula calculates density from protein concentrations:protein density

[00115] After the density of the different protein concentrations was determined, the following formula was used to calculate the needed volume of water for reconstitution:

[00116] An amount of 0.4 grams of the two chosen lyophilized formulations were measured out into smaller vials and then reconstituted with the calculated reconstitution liquid volume to get to target protein concentrations of 200, 250, 300, and 350 mg/mL. The lyophilized cakes were repositioned with 100mM Arginine-HCl or 200mM Arg-HCl and 0.2% Polysorbate-80 to reduce viscosity and protein aggregation even further. Viscosity and %HMW were determined as measurements of product quality for all the reconstituted samples.

Long Term Stability of Lyophilized Products

[00117] A long-term stability study was conducted on lyophilized samples from the two selected formulations from the feasibility study. Measuring the stability of the formulations over time indicates how viable the proteins will be after storage in various real- world conditions. The 30 vials per formulation were prepared, filtered, and lyophilized (in addition to a pre-lyophilization control). Post-lyophilization cakes of both formulations were stored at -30°C, 5 °C, 25°C, and 40°C to replicate different storage conditions. -30°C and 5°C represent storage in frozen and chilled environments. Although samples stored at -30°C and 5 °C are expected to be the most stable, products that maintain quality at room temperature minimize transport costs and therefore lower the cost of drugs themselves. Samples stored at 25°C represent room temperature, or more specifically, room temperature in hospitals. Samples stored at 40°C represent transport conditions in trucks as well as a stress study which indicates how proteins will degrade over shorter time intervals. Sample controls for pre-lyophilization and TO (reconstituted straight after lyophilization) were assessed for concentration, pH, SEC, Cation Exchange Chromatography, OD, sub-visible particles, and visible particles. Cation Exchange Chromatography (CEX) separates protein molecules based on their charge and indicates what percentage of the formulation is acidic, neutral, or basic. Mean Fluorescence Intensity (MFI) shows the morphology of sub-visible particles in a formulation by using a camera that snaps images while samples are run through flow cytometry. Qualitative observations of visible particles were recorded from photographs of samples in front of white and black backgrounds. Samples in the stability study were reconstituted with 1.9 mL of MQ water at 1, 3, 6, 9, 12, and 24 months and then evaluated for concentration, pH, SEC, CEX, OD, sub-visible particles, and visible particles. Assessing Feasibility of Lyophilized Formulations

[00118] Several methods can be used to measure protein aggregation. Protein aggregation can be primarily measured through high molecular weight percentage (%HMW) using size exclusion chromatography with ultra-high performance liquid chromatography (SEC-UPLC) and optical density at 405 nm (OD405 or turbidity). SEC and OD405 can be the primary methods of measurement because they accounted for both soluble and insoluble protein aggregates. SEC can account for soluble protein aggregates but is not able detect the insoluble aggregates. OD405 can be used to account for the insoluble aggregates. This is because insoluble aggregates cause light scattering resulting in increased absorbance at all wavelengths. For stability studies the number of insoluble particles in solution also can be measured using micro-flow imaging (MFI) at some of the time points. This can be used as an additional measure of insoluble protein aggregates.

[00119] Lyophilized cakes were reconstituted with H₂O to reach an ending concentration of 100mg/mL, product quality of samples then assessed. pH, OD, and size exclusion chromatography (SEC) were compared to pre-lyophilized samples.

[00120] The percent high molecular weight (%HMW) and the percent low molecular weight (%LMW) protein aggregates resulting from controlled nucleation with prelyophilization, aggressive lyophilization, and controlled nucleation with secondary drying lyophilization cycles were determined by using size Exclusion-Ultra-Performance Liquid Chromatography (SE-UPLC) (Figure 5).

[00121] The target for a stable formulation in the feasibility was a less than 1 % increase in protein aggregation from pre-lyophilization to post-lyophilization. The optimal lyophilization cycle was found to be the controlled nucleation conservative cycle without secondary drying, and the optimal formulation with minimal stabilizer and negligible HMW increase was the F3 formulation. Formulation F4 also met the HMW criteria but was less flexible for final drug product formulation compared with formulation F3 (see Figure 6). When analyzing SEC results to determine protein aggregation, controlled nucleation drastically decreased %AHMW, and F2, F3, and F4 met the target of a less than 1% increase in protein aggregation from pre-lyophilization to post-lyophilization (Figure 6). pH stayed constant in both pre-lyophilization and post-lyophilization formulations and optical density increase is correlated with HMW, the main degradation pathway. Because F3 and F4 in a controlled nucleation cycle had a negligible increase in protein aggregation, formulations F3 and F4 were used in the bulk lyophilization study with controlled nucleation. The changes in high molecular weight (HMW) protein aggregates in UHCP formulations (Fl, F2, F3, and F4) resulting from different lyophilization processes are shown in graph (Figure 6). Results of F3 and F4 in controlled nucleation cycle after conservative primary drying indicates: o Negligible increase in protein aggregation (HMW) in F3 and F4; o pH and OD increase is correlated with HMW, the main degradation pathway; and o F2, F3, and F4 met target <1% increase in HMW.

Reconstitution Liquids (Solutions) and UHCP Formulations:

[00122] Figure 7 schematically depicts products of bulk scale lyophilization of 105 mL in glass containers. Figure 8 schematically depicts constant mass of a bulk lyophilized cakes. Figure 9 schematically depicts reconstituted ultra-high concentration protein (UHCP) formulations with calculated volumes of reconstitution liquid for resulting concentrations of 200 mg/ml, 300 mg/ml, and 350 mg/ml, from left to right, respectively.

[00123] Reconstitution liquids (solutions) used included:

. 100 mM Arginine-HCl; and

• 200 mM Arginine-HCl containing 0.2% PS 80

[00124] Reconstituted final concentrations of the UHCP formulations were:

200 mg/mL, 250 mg/mL, 300 mg/mL, and 350 mg/mL.

Creating Ultra-high Concentration Formulations in Bulk

[00125] After running successfully reconstituting samples to 200, 250, 300, and 350 mg/mL, reconstituted and pre-lyophilization samples were run through SEC to assess protein degradation and the viscosity of reconstituted samples was evaluated. Samples that were reconstituted with 200mM Arg-HCl and 0.2% PS-80 showed a notable decrease in viscosity compared with samples reconstituted with 100mM Arg-HCl (see for example, Figure 10A, 10B and 10C). Figure 10A illustrates the effect of reconstitution liquid compositions on the viscosity of ultra-high concentration protein formulations. Samples reconstituted with 200 mM Arginine-HCl and 0.2% PS 80 showed notable decrease in viscosity compared to the samples reconstituted with 100 mM Arginine-HCl. Figure 10B illustrates the effect of reconstitution liquid compositions on the viscosity of ultra-high concentration protein formulations at 20°C. Samples reconstituted with 200 mM Arginine- HC1, and 0.2% PS-80 showed notable decrease in viscosity compared to the samples reconstituted with 100 mM Arginine-HCl. Figure 10C illustrates the effect of reconstitution liquid compositions on the viscosity of ultra-high concentration protein formulations F3 and F4. Therefore, the addition of more concentrated Arg-HCl and polysorbates (such as PS-80) significantly decreased the viscosity of reconstituted samples and therefore were successful in improving the product quality of the drug product. The viscosity of the reconstituted drug product is not an obstacle with subcutaneous drug delivery as all reconstituted drug products have viscosities under 1000 cp that can be delivered with novel technologies as discussed herein.

[00126] Compared to samples reconstituted with 100mM Arg-HCl, samples reconstituted with the addition of 200mM Arg-HCl and 0.2% PS-80 showed no decrease in %AHMW (Figures 11A-11B). The standard for protein aggregation in pharmaceuticals is that %AHMW must be less than 2-3%, so for the 350 mg/mL formulation, the proteins are too aggregated for use in drug delivery. Bar graphs in Figure 11A illustrate the effect of reconstitution liquid compositions and the change in %HMW of ultra-high concentration protein formulations. Figure 11B illustrates the effect of reconstitution liquid compositions and the change in %HMW of ultra-high concentration protein formulations F3 and F4. Results show that 200 mg/mL, 250 mg/mL, and 300 mg/mL UHCP samples meet the threshold of <%HMW for protein aggregation in the UHCP formulations.

Stability Studies on Bulk Lyophilized UHCP Formulations:

[00127] Thirty 2mL vials of F3 and thirty 2mL vials of F4 samples were formulated and lyophilized. Samples were stored at -30°C, 5 °C, 25 °C, and 40°C to mimic storage in real-world conditions. Samples were reconstitution after 1, 3, 6, 9, 12, and 24 months. Protein aggregation was primarily measured through high molecular weight percentage (%HMW) using size exclusion chromatography (SEC). Cation Exchange Chromatography (CEX), OD, pH, sub-visible particles, and visible particles also were determined. The above testing criteria allowed determination of how long the lyophilized cakes can be stored in manufacturing, hospitals and during transportations.

[00128] After reconstituting lyophilized stability study (SS) formulas, protein concentration and pH of each of the samples were measured as controls. Protein concentration and pH were constant which minimizes the possible error of SS samples being different (Tables 2 and 3, Figures 12A-12B, 13A-13B, 18A-18B and 19A-19B).

[00129] Table 3: Concentration of F3 and F4 Stability Study Formulations

(mg/mL).

[00130] Table 4: pH of F3 and F4 Stability Study Formulations.

[00131] Bar graphs in Figure 12A show the results of the stability studies on F3 formulations at different temperatures for up to six months and the effect on protein concentration. Figure 12B illustrates the results of the stability studies on F3 formulations at different temperatures for up to twelve months and the effect on protein concentration.

[00132] Bar graphs in Figure 13 A show the results of the stability studies on F4 formulations at different temperatures for up to six months and the effect on protein concentration. Figure 13B illustrates the results of the stability studies on F4 formulations at different temperatures for up to 12 months and the effect on protein concentration.

[00133] For long term stability study for lyophilized bulk drug substances, 30 2 ml vials containing F3 sample and 30 2 ml vials containing F4 sample were stored at -30°C, 5°C, 25°C, and 40°C to mimic the storage in real-world conditions. Samples were reconstitution after 1, 3, 6, 9, 12, and 24 months. pH, SEC, Cation Exchange Chromatography (CEX), sub-visible particles, visible particles were assessed at each time point.

[00134] After 6 months at -30°C, both F3 and F4 were stable with a negligible increase in protein aggregation, turbidity, and acidity. Lyophilized cakes at chilled temperatures demonstrated long-term stability (Figures 14A, 14B, 14C, ISA, 15B, and 15C). For samples, stored at 5°C and 25°C, F4 was more stable than F3. Samples stored at 40°C after 6 months were degraded and unstable.

[00135] Bar graphs in Figure 14A show the results of the stability studies on F3 formulations at different temperatures for up to six months and the change in %HMW protein aggregates. Line graphs in Figure 14B show data from the formulation F3 stability study at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 14C illustrates the results of the stability studies on F3 formulations at different temperatures for up to 12 months and the change in %HMW protein aggregates.

[00136] Bar graphs in Figure ISA show the results of the stability studies on F4 formulations at different temperatures for up to six months and the change in %HMW protein aggregates. Line graphs in Figure 15B show data from the formulation F4 stability study at different temperatures for up to six months and the change in %HMW protein aggregates. Figure 15C illustrates the results of the stability studies on F4 formulations at different temperatures for up to 12 months and the change in %HMW protein aggregates.

[00137] After running pre-lyophilization (PL) and reconstituted samples from the stability study through optical density to measure turbidity, SEC to measure protein aggregation, and CEX to measure acidity, the difference in OD, HMW, and CEX results was calculated (Table 5, Figures 16A-16B and 17A-17B).

[00138] Table 5: AOptical Density of F3 and F4 Stability Study Formulations

[00139] Bar graphs in Figure 16A show the results of the stability studies on F3 formulations at different temperatures for up to six months and the change in optical density. Figure 16B illustrates the results of the stability studies on F3 formulations at different temperatures for up to nine months and the change in optical density. [00140] Bar graphs in Figure 17A show the results of the stability studies on F4 formulations at different temperatures for up to six months and the change in optical density. Figure 17B illustrates the results of the stability studies on F4 formulations at different temperatures for up to nine months and the change in optical density.

[00141] Bar graphs in Figure 18 A show the results of the stability studies on F3 formulations at different temperatures for up to six months and the effect on pH of the formulation. Figure 18B illustrates the results of the stability studies on F3 formulations at different temperatures for up to 12 months and the effect on pH of the formulation.

[00142] Bar graphs in Figure 19 A show the results of the stability studies on F4 formulations at different temperatures for up to six months and the effect on pH of the formulation. Figure 19B illustrates the results of the stability studies on F4 formulations at different temperatures for up to 12 months and the effect on pH of the formulation.

[00143] The Cation Exchange Chromatography (CEX) data is not the most precise measurement and has a percent variability of multiple points, so the only significant increase in acidity was seen in the reconstituted drug products stored at 40°C which had a drastic increase in % acidity (see for example, Figures 20A, 20B, 20C, 21A, 21B, and 21C).

[00144] Bar graphs in Figure 20 A show the results of the stability studies on F3 formulations at different temperatures for up to six months and the effect on percent composition of ions as determined by Cation Exchange Chromatography (CEX). Figure 20C illustrates the results of the stability studies on F3 formulations at different temperatures for up to 12 months and the effect on percent composition of ions as determined by Cation Exchange Chromatography (CEX).

[00145] Bar graphs in Figure 21A show the results of the stability studies on F4 formulations at different temperatures for up to six months and the effect on percent composition of ions as determined by CEX. Figure 21C illustrates the results of the stability studies on F4 formulations at different temperatures for up to 12 months and the effect on percent composition of ions as determined by CEX.

[00146] Bar graphs in Figure 23 show the solution viscosity after lyophilization of low concentration EDS (F5) and its reconstitution to higher protein concentration. Prior to lyophilization F5 contains dupilumab (mAh) at about 100 mg/mL in 5 mM histidine with 1% sucrose, 50 mM arginine hydrochloride and 0.075% PS-80 at pH 6.0.

After lyophilization F5 was reconstituted with water to different protein concentrations.

[00147] Bar graphs in Figure 24 show dupilumab (mAb) aggregation (%HMW) after lyophilization of low concentration EDS (F5) and its reconstitution to higher protein concentration.

[00148] Analyses of the SEC, CEX, pH, OD, protein concentration, sub- visible, and visible particles data indicate:

• No changes for protein concentration and pH, shows controls with protein concentration and pH (Figures 18A-18B and 19A-19B);

• No visible particles;

• UHCP formulations stored at -30°C, 5°C, and 25°C are stable at least for 6 months: No notable increase observed in %HMW (Figures 14A, 14B, 14C, ISA, 15B, and ISC), CEX (Figures 20A, 20B, 20C, 21A, 21B, and 21C), or

OD (Figures 16A-16B and 17A-17B); and

• UHCP formulations stored at 40°C degrade with increased %HMW, with increased acidic species, and with increased optical density, as degradation pathways for bulk lyophilized formulations (Figures 14A, 14B, 14C, ISA, 15B, and 15C).

[00149] Studies disclosed herein effectively created a manufacturing process for ultra-high concentration proteins (UHCP) formulations , including dupilumab as a representative mAb formulations using lyophilization. The findings of the inventions fulfill: o Combating manufacturing limits with conventional ultrafiltration (UF) process; o Breaking the innate limit of concentration necessitated by conventional manufacturing, for example for formulations containing greater than 200 mg/mL proteins; and o Stabilizing the UHCP product cakes at chilled temperatures for long term storage.

[00150] The UHCP formulations of the inventions enable subcutaneous (SC) drug delivery, which is advantageous to intravenous (IV) administration The SC administration, which takes minutes instead of hours to administer, is preferred by the patients and hospital administrators. The UHCP formulations of the inventions vastly open future paths for mAh and protein therapeutics by:

• Facilitating convenient therapy treatments with less frequent doses, which is less invasive, more cost effective, and relieves burden on hospital systems; and

• Providing more protein therapeutics and treatments to be delivered subcutaneously as opposed to IV.

Claims

WHAT IS CLAIMED IS

1. A method of forming a concentrated protein formulation, wherein the method comprises the steps of

(a) providing an aqueous solution comprising a protein, and a sugar, and a pH of about 5.0 to about 7.0;

(b) lyophilizing the aqueous solution to form a lyophilisate; and

(c) reconstituting the lyophilisate using a reconstitution solution comprising one or more viscosity reducers and a polysorbate thereby forming the concentrated protein solution, wherein the concentrated protein solution comprises about 50 to about 700 mg/ml of the protein.

2. The method according to claim 1 , wherein the concentrated protein solution is a drug product, and the viscosity reducer is arginine hydrochloride.

3. The method according to claim 1 , wherein the aqueous solution comprises about 100 mg/ml of the protein.

4. The method according to claim 1 , wherein the aqueous solution comprises about 200 mg/ml of the protein.

5. The method according to claim 1 , wherein the aqueous solution comprises about 100 to about 400 mg/ml of the protein.

6. The method according to claim 1 , wherein the aqueous solution comprises about 200 to about 350 mg/ml of the protein.

7. The method according to claim 1, wherein the aqueous solution is self -buffering and does not require an exogenous buffer to maintain a stable pH.

8. The method according to claim 1, wherein the aqueous solution further comprises a buffer.

9. The method according to claim 8, wherein the buffer is a histidine buffer.

10. The method according to claim 9, wherein the concentration of the histidine buffer is about 1 to about 30 mM.

11. The method according to claim 9, wherein the concentration of the histidine buffer is about 5 mM or about lOmM.

12. The method according to claim 1 , wherein the pH of the aqueous solution is about 5.5 to about 6.5.

13. The method according to claim 1 , wherein the pH of the aqueous solution is about 6.0.

14. The method according to claim 1 , wherein the concentration of the sugar is about 0.10% to 10% (w/v).

15. The method according to claim 1 , wherein the concentration of the sugar is about 0.5 to 1.0% (w/v).

16. The method according to claim 1, wherein the sugar is sucrose.

17. The method according to claim 16, wherein the concentration of sucrose is about 0.5% or about 1.0% (w/v).

18. The method according to claim 1 , wherein the concentration of arginine hydrochloride is about 10 to about 300 mM.

19. The method according to claim 1 , wherein the concentration of arginine hydrochloride is about 50 to about 200 mM.

20. The method according to claim 1 , wherein the concentration of arginine hydrochloride is about 100 mM or about 200 mM.

21. The method according to claim 1, wherein the polysorbate is polysorbate 80 (PS20) or polysorbate 20 (PS20).

22. The method according to claim 1 , wherein the concentration of the polysorbate is about 0.01% to about 0.30% (w/v).

23. The method according to claim 1, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

24. The method according to claim 1 , wherein the step (b) lyophilizing comprises the steps of: (i) cooling a solution comprising a protein product below the freezing point of the solution, thereby forming a frozen solution;

(ii) pressuring the cooled solution of step (i) with a gas;

(iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and

(iv) lyophilizing the frozen solution of step (iii) through primary drying and with or without secondary drying, thereby forming a lyophilized bulk protein product.

25. The method according to claim 1 , wherein the step (b) lyophilizing comprises the steps of:

(i) cooling a solution comprising a protein product below the freezing point of the solution, thereby forming a frozen solution;

(ii) pressuring the cooled solution of step (i) with a gas;

(iv) subjecting the frozen solution of step (iii) under an aggressive lyophilization process through primary drying and with or without secondary drying, at higher primary drying temperature, thereby forming a lyophilized bulk protein product.

26. The method according to claim 23, wherein the higher primary drying temperature can be about 1°C to 20°C, about 5 °C to 20°C, about 10°C to 20°C, about 15 °C to 20°C, about 2 °C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C.

27. The method according to claim 1 , wherein the step (b) lyophilizing comprises the steps of:

(ii) pressuring the cooled solution of step (i) with a gas;

(iii) releasing the pressure of step (ii) to allow nucleation, thereby resulting ice nuclei to form in the cooled solution; and (iv) subjecting the frozen solution of step (iii) under a conservative lyophilization process through primary drying and with or without secondary drying, at lower primary drying temperature, thereby forming a lyophilized bulk protein product.

28. The method according to any one of claims 24-27, wherein during step (ii) the gas is present at about 14 to about 70 psig pressure.

29. The method according to any one of claims 24-27, wherein during step (ii) the gas is present at about 14 to about 42 psig pressure.

30. The method according to any one of claims 24-27, the lower primary drying temperature is about -35°C to -2°C, about -34°C to -2°C, about -32°C to -2°C, about -30°C to -2°C, about -28°C to -2°C, about -26°C to -2°C, about -24°C to -2°C, about -23°C to -2°C, about -21°C to -2°C, about -20°C to -2°C, about -18°C to -2°C, about -16°C to -2°C, about -

14°C to -2°C, about -12°C to -2°C, about -10°C to -2°C, about -8°C to -2°C, about -6°C to -

2°C, about -4 °C to -2°C, about -3°C to -2°C, about -35°C, about -30°C, about -25°C, about -

20°C, about -15°C, about -10°C, about -5°C, about -3°C, about -2°C, or about -1°C.

31. The method according to any one of claims 24-27, wherein step (iii) is conducted at a temperature of about -2°C to about -20°C.

32. The method according to any one of claims 24-27, wherein step (iii) is conducted at a temperature of about -5 °C.

33. An ultra-high concentrated protein (UHCP) formulation for lyophilization, comprising:

(a) an aqueous solution of protein having a concentration between about 50 to about

700 mg/ml of the protein; and

(b) a sugar at a concentration of about 0.10% to about 10% (w/v).

34. The UHCP formulation according to claim 33, wherein the aqueous solution comprises about 100 mg/ml of the protein.

35. The UHCP formulation according to claim 33, wherein the aqueous solution comprises about 200 mg/ml of the protein.

36. The UHCP formulation according to claim 33, wherein the aqueous solution comprises about 100 to about 400 mg/ml of the protein.

37. The UHCP formulation according to claim 33, wherein the aqueous solution comprises about 200 to about 350 mg/ml of the protein.

38. The UHCP formulation according to claim 33, wherein the aqueous solution is self-buffering and does not require an exogenous buffer to maintain a stable pH.

39. The UHCP formulation according to claim 33, wherein the aqueous solution further comprises a buffer, and a pH of between about 5.0 to about 7.0.

40. The UHCP formulation according to claim 39, wherein the buffer is a histidine buffer.

41. The UHCP formulation according to claim 39, wherein the concentration of histidine buffer is about 1 to about 30 mM.

42. The UHCP formulation according to claim 39, wherein the concentration of histidine buffer is about 5 mM.

43. The UHCP formulation according to claim 33, wherein the pH of the aqueous solution is about 5.5 to about 6.5.

44. The UHCP formulation according to claim 33, wherein the pH of the aqueous solution is about 6.0.

45. The UHCP formulation according to claim 33, wherein the concentration of the sugar is about 0.5 to about 1.0% (w/v).

46. The UHCP formulation according to claim 33, wherein the sugar is sucrose.

47. The UHCP formulation according to claim 46, wherein the concentration of sucrose is about 0.5% or about 1.0% (w/v).

48. The UHCP formulation according to claim 33, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

49. A reconstitution solution for reconstituting a lyophilized ultra-high concentrated protein (UHCP) formulation comprising:

(a) arginine hydrochloride solution at a concentration of about 10 to about 300 mM; and

(b) the polysorbate at a concentration of about 0.01% to about 0.30% (w/v).

50. The reconstitution solution according to claim 49, wherein the concentration of arginine hydrochloride is about 100 to about 200 mM.

51. The reconstitution solution according to claim 49, wherein the concentration of arginine hydrochloride is about 100 mM or about 200 mM.

52. The reconstitution solution according to claim 49, wherein the polysorbate is polysorbate 80 (PS80) or polysorbate 20 (PS20).

53. The reconstitution solution according to claim 52, wherein the concentration of the PS 80 or PS 20 is about 0.01% to about 0.30% (w/v).

54. The reconstitution solution according to claim 52, wherein the concentration of the PS80 or PS20 is about 0.10% or about 0.20% (w/v).

55. The reconstitution solution according to claim 49, wherein the protein in the UHCP formulation is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

56. A method of forming a concentrated protein formulation, wherein the method comprises the steps of

(a) providing an aqueous solution comprising a protein, one or more viscosity reducers, a polysorbate, and a sugar, and a pH of about 5.0 to about 7.0;

(b) lyophilizing the aqueous solution to form a lyophilisate; and

(c) reconstituting the lyophilisate using water thereby forming the concentrated protein solution, wherein the concentrated protein solution comprises about 50 to about 700 mg/ml of the protein.

57. The method according to claim 56, wherein the concentrated protein solution is a drug product, and the viscosity reducer is arginine hydrochloride.

58. The method according to claim 56, wherein the aqueous solution comprises about 100 mg/ml of the protein.

59. The method according to claim 56, wherein the aqueous solution comprises about 200 mg/ml of the protein.

60. The method according to claim 56, wherein the aqueous solution comprises about 100 to about 400 mg/ml of the protein.

61. The method according to claim 56, wherein the aqueous solution comprises about 200 to about 350 mg/ml of the protein.

62. The method according to claim 56, wherein the aqueous solution is self-buffering and does not require an exogenous buffer to maintain a stable pH.

63. The method according to claim 56, wherein the aqueous solution further comprises a buffer.

64. The method according to claim 63, wherein the buffer is a histidine buffer.

65. The method according to claim 64, wherein the concentration of the histidine buffer is about 1 to about 30 mM.

66. The method according to claim 64, wherein the concentration of the histidine buffer is about 5 mM or about lOmM.

67. The method according to claim 56, wherein the pH of the aqueous solution is about 5.5 to about 6.5.

68. The method according to claim 56, wherein the pH of the aqueous solution is about 6.0.

69. The method according to claim 56, wherein the concentration of the sugar is about 0.10% to 10% (w/v).

70. The method according to claim 56, wherein the concentration of the sugar is about 0.5 to 1.0% (w/v).

71. The method according to claim 56, wherein the sugar is sucrose.

72. The method according to claim 71 , wherein the concentration of sucrose is about 0.5% or about 1.0% (w/v).

73. The method according to claim 56, wherein the concentration of arginine hydrochloride is about 10 to about 300 mM.

74. The method according to claim 56, wherein the concentration of arginine hydrochloride is about 50 to about 200 mM.

75. The method according to claim 56, wherein the concentration of arginine hydrochloride is about 100 mM or about 200 mM.

76. The method according to claim 56, wherein the polysorbate is polysorbate 80 (PS80) or polysorbate 20 (PS20).

77. The method according to claim 56, wherein the concentration of the polysorbate is about 0.01% to about 0.30% (w/v).

78. The method according to claim 56, wherein the concentration of the polysorbate is about 0.075% (w/v).

79. The method according to claim 56, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

80. The method according to claim 56, wherein the step (b) lyophilizing comprises the steps of:

(ii) pressuring the cooled solution of step (i) with a gas;

81. The method according to claim 56, wherein the step (b) lyophilizing comprises the steps of:

(ii) pressuring the cooled solution of step (i) with a gas;

82. The method according to claim 79, wherein the higher primary drying temperature can be about 1°C to 20°C, about 5 °C to 20°C, about 10°C to 20°C, about 15 °C to 20°C, about 2 °C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C.

83. The method according to claim 56, wherein the step (b) lyophilizing comprises the steps of:

(ii) pressuring the cooled solution of step (i) with a gas;

(iv) subjecting the frozen solution of step (iii) under a conservative lyophilization process through primary drying and with or without secondary drying, at lower primary drying temperature, thereby forming a lyophilized bulk protein product.

84. The method according to any one of claims 80-83, wherein during step (ii) the gas is present at about 14 to about 70 psig pressure.

85. The method according to any one of claims 80-83, wherein during step (ii) the gas is present at about 14 to about 42 psig pressure.

86. The method according to any one of claims 80-83, the lower primary drying temperature is about -35°C to -2°C, about -34°C to -2°C, about -32°C to -2°C, about -30°C to -2°C, about -28°C to -2°C, about -26°C to -2°C, about -24°C to -2°C, about -23°C to -2°C, about -21°C to -2°C, about -20°C to -2°C, about -18°C to -2°C, about -16°C to -2°C, about -

87. The method according to any one of claims 80-83, wherein step (iii) is conducted at a temperature of about -2°C to about -20°C.

88. The method according to any one of claims 80-83, wherein step (iii) is conducted at a temperature of about -5 °C.

89. An ultra-high concentrated protein (UHCP) formulation for lyophilization, comprising an aqueous solution of protein having a protein, one or more viscosity reducers, a polysorbate, and a sugar at a pH of about 5.0 to about 7.0; wherein the sugar at a concentration of about 0.10% to about 10% (w/v).

90. The UHCP formulation according to claim 89, wherein the aqueous solution comprises about 100 mg/ml of the protein.

91. The UHCP formulation according to claim 89, wherein the aqueous solution comprises about 200 mg/ml of the protein.

92. The UHCP formulation according to claim 89, wherein the aqueous solution comprises about 100 to about 400 mg/ml of the protein.

93. The UHCP formulation according to claim 89, wherein the aqueous solution comprises about 200 to about 350 mg/ml of the protein.

94. The UHCP formulation according to claim 89, wherein the aqueous solution is self-buffering and does not require an exogenous buffer to maintain a stable pH.

95. The UHCP formulation according to claim 89, wherein the aqueous solution further comprises a buffer, and a pH of between about 5.0 to about 7.0.

96. The UHCP formulation according to claim 95, wherein the buffer is a histidine buffer.

97. The UHCP formulation according to claim 96, wherein the concentration of histidine buffer is about 1 to about 30 mM.

98. The UHCP formulation according to claim 96, wherein the concentration of histidine buffer is about 5 mM.

99. The UHCP formulation according to claim 89, wherein the pH of the aqueous solution is about 5.5 to about 6.5.

100. The UHCP formulation according to claim 89, wherein the pH of the aqueous solution is about 6.0.

101. The UHCP formulation according to claim 89, wherein the concentration of the sugar is about 0.5 to about 1.0% (w/v).

102. The UHCP formulation according to claim 89, wherein the sugar is sucrose.

103. The UHCP formulation according to claim 102, wherein the concentration of sucrose is about 0.5% or about 1.0% (w/v).

104. The UHCP formulation according to claim 89, wherein the protein is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.

105. A reconstitution solution for reconstituting a lyophilized ultra-high concentrated protein (UHCP) formulation according to claim 89 comprising:

(b) the polysorbate at a concentration of about 0.01% to about 0.30% (w/v).

106. The reconstitution solution according to claim 105, wherein the concentration of arginine hydrochloride is about 100 to about 200 mM.

107. The reconstitution solution according to claim 105, wherein the concentration of arginine hydrochloride is about 100 mM or about 200 mM.

108. The reconstitution solution according to claim 105, wherein the polysorbate is polysorbate 80 (PS80) or polysorbate 20 (PS20).

109. The reconstitution solution according to claim 108, wherein the concentration of the PS 80 or PS 20 is about 0.01% to about 0.30% (w/v).

110. The reconstitution solution according to claim 108, wherein the concentration of the PS80 or PS20 is about 0.10% or about 0.20% (w/v).

111. The reconstitution solution according to claim 105, wherein the protein in the UHCP formulation is selected from the group consisting of an antibody, antibody derivative, antibody fragment, a monoclonal antibody, an Fc-containing protein, and an Fc-fusion protein.