Solid polyacrylamide polymer biopharmaceutical formulations

Abstract

The present disclosure provides storage stable solid compositions including a biopharmaceutical agent and a polyacrylamide-based copolymer. The inventors have demonstrated that particular polyacrylamide-based copolymers can be used as stabilizing excipients in lyophilized formulations of biopharmaceutical agents. Also provided are methods of reconstituting and using the compositions, including methods of administering the reconstituted composition to a human subject in need thereof via injection, e.g., subcutaneous injection.

Description

SOLID POLYACRYLAMIDE POLYMER BIOPHARMACEUTICAL FORMULATIONSBACKGROUND

[0001] Many biopharmaceuticals in pharmaceutical formulations are prone to irreversible aggregation, degradation, and / or inactivation when exposed to temperature fluctuations and / or agitation and require formulation at particular concentrations with excipients, careful storage, and refrigerated transport (the cold chain) to retain activity over its shelf life.Maintaining the integrity of various biopharmaceuticals presents a challenge to the pharmaceutical industry, healthcare providers, and people needing treatment with such biopharmaceuticals worldwide.

[0002] Antibodies present immense promise in modern pharmacotherapy. These biopharmaceuticals tend to aggregate in formulations, thus requiring low concentration drug formulations, and / or resulting in poor stability. While the mechanism of aggregation varies slightly from antibody to antibody, many antibodies contain hydrophobic patches and / or can partially denature to reveal hydrophobic segments of the molecule that drive absorption of these proteins onto interfaces (e.g., air-water, glass-water, or rubber-water interfaces). The high local concentrations of the protein molecules at these interfaces, coupled with interface- mediated partial unfolding, can trigger initial nucleation of aggregation events that make way for further aggregation. This tendency of antibodies to aggregate at interfaces typically increases with formulation concentrations, negatively impacting overall formulation stability.

[0003] Vaccines have become indispensable in human health care, and greatly reduce the incidence and death rate of the common epidemic infectious disease, so that many epidemic infectious diseases are effectively controlled. In recent years, with increased understanding of the immune system-related mechanism, vaccine development has achieved excellent performance in the traditional prevention and treatment field, and has also emerged for use in treating HIV, cancer, and other disorders. However, cold storage and transportation of vaccine formulations remains a challenge. Freezing and freeze-drying of vaccine formulations can damage the structure and / or reduce the immunogenicity of the vaccine.

[0004] Many commercial excipients have been used in trying to overcome challenges associated with formulation of biopharmaceuticals. These systems are still typically limited by their critical micelle concentrations, possible toxicity through oxidative degradation, and undesirable interaction between the excipient and the cargo in the bulk.

[0005] Accordingly, there is a need for biopharmaceutical formulations, including vaccine formulations, with improved stability and reduced costs associated with cold chain transportation.SUMMARY

[0006] The present disclosure provides solid compositions including a biopharmaceutical agent, such as an antibody or vaccine, and a polyacrylamide-based copolymer. The inventors have demonstrated that particular polyacrylamide-based copolymers can be used as stabilizing excipients or surfactants in pharmaceutical compositions to maintain the stability of a biopharmaceutical agent during and / or after lyophilization. The results presented herein indicate that the polyacrylamide-based copolymers of this disclosure can be generally applied to confer a substantial stability benefit to compositions of a variety of biopharmaceutical agents. The solid, e.g., lyophilized, compositions further provide for uniform reconstitution of a liquid pharmaceutical composition in a pharmaceutically acceptable solution, providing for recovery of the active ingredients, minimizing partial loss of potency, and allowing administration of the active ingredient biopharmaceutical agents in an accurate and consistent manner. In some embodiments, the polyacrylamide-based copolymers stabilize compositions of biopharmaceutical agents without altering its pharmacokinetic properties, at concentrations of the biopharmaceutical agents that would not be possible in compositions that lack the polyacrylamide-based copolymers.

[0007] A composition of this disclosure that includes a polyacrylamide-based copolymer can be amenable to lyophilization to produce a lyophilized composition that has one or more improved properties, such as increased stability and / or reconstitution time. In some embodiments, a composition of this disclosure is amenable to rapid freeze-drying treatments with retention of biological activity of the biopharmaceutical agent. In some embodiments, a composition of this disclosure is amenable to multiple freeze-thaw cycles.

[0008] Some aspects of the present disclosure relate to a reconstituted liquid composition produced by dissolving the lyophilized pharmaceutical composition in aqueous liquid, e.g., sterile water. In some embodiments, the reconstituted composition preserves the biological potency and other desired characteristics of a biopharmaceutical agent, e.g., a vaccine, including virus viability, immunogenicity and / or stability.

[0009] In some embodiments, the biopharmaceutical agent is an antibody or antibody drug conjugate (ADC) and the polyacrylamide-based copolymers preclude adsorption of theantibody to the interfaces of the composition, thereby preventing undesirable aggregation events, and maintaining the binding activity of the antibody. In some embodiments, the lyophilized composition of this disclosure is reformulated at an antibody or ADC concentration that provides for subcutaneous (SC) injection to a patient which can be conducted in a low resource setting, in contrast, for example, to IV administration.

[0010] The lyophilized biopharmaceutical compositions of this disclosure can provide advantages over conventional formulations via ease of shipping, storage, and distribution.

[0011] Also provided are methods of using the biopharmaceutical compositions, including methods of administering a reconstituted composition to a human subject in need thereof via injection, e.g., subcutaneously.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a set of schematics showing an overview of stabilization of high- concentration antibody formulations using novel copolymer excipients. (Panel A) Schematic overview of current therapeutic routes of administration for biopharmaceuticals, whereby intravenous administration is common because of limitations on stability of biopharmaceuticals at the high concentrations required for subcutaneous administration, resulting in high patient burden. (Panel B) Graphical representation of amphiphilic acrylamide carrier / dopant co-polymer (AC / DC) excipients, such as poly(acryloylmorpholine- co-7V-isoproyl acrylamide) (MoNi), that can be used to stabilize biopharmaceuticals in formulation. (Panel C) Schematic showing stabilization of monoclonal antibodies in formulation using AC / DC copolymers as a “drop-in” excipient. AC / DC copolymers are amphiphilic and can preferentially absorb to interfaces, precluding antibody interfacial adsorption in formulation and thereby preventing interface-related destabilization and aggregation events.

[0013] FIG. 2 is a set of schematics and graphs showing the synthesis and characterization of MoNi copolymer excipient. (Panel A) Synthetic scheme for the synthesis of poly(acryloylmorpholine-co-N-isopropylacrylamide) (MoNi). Size exclusion chromatography (SEC) characterization of the (Panel B) MoNi-CTA intermediate and (Panel C) MoNi demonstrated that the CTA is completely removed in the second step of the synthesis, yielding a chemically and physically stable copolymer. (Panel D)1H-NMR characterization of MoNi. (Panel E) Comparison of cytotoxicity of MoNi, Polysorbate 80 (PS80), and Pluronic L61. (Panel F) DLS characterization of MoNi in solution at variousconcentrations demonstrated that the MoNi copolymer does not exhibit critical micelle concentration (CMC) behavior even at high concentrations. (Panel G) Comparison of reported CMC values for PS80 and Pluronic L61.

[0014] FIG. 3 is a set of graphs showing interfacial interactions of the MoNi polymer excipient in mAb formulations. (Panel A) Comparison of PGT121 (0.2 mg / mL) with and without MoNi (0.1 mg / mL) in time resolved surface tension measurements (n=3). (Panel B) Interfacial rheology measurements (n=3) of PGT121 (0.2 mg / mL) with and without MoNi (0.1 mg / mL). (Panel C) High shear rate viscosity measurements collected at steady state by viscometry experiments for high-concentration formulations of PGT121 (119 mg / mL) with and without MoNi (10 mg / mL).

[0015] FIG. 4 is a set of diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) spectra of (Panel A) MoNi alone (0.1 mg / mL), (Panel B) PGT121 alone (0.2 mg / mL), and (Panel C) co-formulation of PGT121 and MoNi at 0.2 mg / mL and 0.1 mg / mL, respectively.

[0016] FIG. 5 is a set of schematics and graphs showing stabilization by MoNi of PGT121. (Panel A) Schematic of accelerated aging, whereby formulations are packaged in glass vials, agitated, and heated. (Panel B) Percent monomeric composition of aged samples determined via GPC. (Panel C) Schematic of assaying epitope binding via ELISA. (Panel D) Half maximal inhibitory concentrations (IC50) of aged formulations with and without MoNi. Samples assayed in duplicate.

[0017] FIG. 6 is a set of schematics and graphs showing pharmacokinetics of a MoNi- stabilized PGT121 formulation. (Panel A) Experimental scheme reporting injection dosing and blood collection timeline in hFcRn mice used for comparing an IV administration of a low-concentration PGT121 (5 mg / mL) formulation and a SC administration of a MoNi- stabilized high-concentration PGT121 (119 mg / mL) formulation. (Panel B) Serum concentration of PGT121 over time, as determined by ELISA (n=6 / group). (Panel C) Area under the curve analysis of ELISA data (n=6 / group).

[0018] FIG. 7 (Panels A-C) is a set of graphs showing mouse weight over time posttreatment.

[0019] FIG. 8 is a graph showing percent monomeric composition of aged 55 mg / mL samples of PGT121 with or without MoNi determined via SEC.

[0020] FIG. 9 is a set of graphs showing representative SEC chromatographs of PGT121 with or without MoNi subject to stressed aging conditions over six days. (Panel A) SEC chromatograph of PGT121 with no MoNi. (Panel B) SEC chromatograph of PGT121 with 1 mg / mL MoNi. (Panel C) SEC chromatograph of PGT121 with 10 mg / mL MoNi.

[0021] FIG. 10 is a set of photographs presenting the appearance of reconstituted ~20 mg / mL IgG formulations F1-F6 from a comparative study of reconstitution times of exemplary solid compositions including MoNi (see Study 1 of Example 8 of experimental section). Fl : IgG + trehalose (250 mg) + L-histidine hydrochloride. F2: IgG + trehalose (250 mg) + L-histidine hydrochloride + polysorbate 20 (PS20) (1 mg). F3: IgG + trehalose (250 mg) + L- histidine hydrochloride + MoNi (25 mg). F4: IgG + trehalose (125 mg) + L- histidine hydrochloride + MoNi (25 mg). F5: IgG + MoNi (25 mg) + L-histidine hydrochloride. F6: IgG + L-histidine hydrochloride.

[0022] FIG. 11 is a set of photographs presenting the appearance of reconstituted -100 mg / mL IgG formulations F1-F6 from a comparative study of reconstitution times of exemplary solid compositions including MoNi (see Study 2 of Example 8 of experimental section). Fl: IgG + trehalose (250 mg) + L-histidine hydrochloride. F2: IgG + trehalose (250 mg) + L-histidine hydrochloride + PS20 (1 mg). F3: IgG + trehalose (250 mg) + L-histidine hydrochloride + MoNi (25 mg). F4: IgG + trehalose (125 mg) + L-histidine hydrochloride + MoNi (25 mg). F5: IgG + MoNi (25 mg) + L-histidine hydrochloride. F6: IgG + L-histidine hydrochloride.

[0023] FIG. 12 is a set of photographs presenting the appearance of reconstituted -200 mg / mL IgG formulations F1-F6 from a comparative study of reconstitution times of exemplary solid compositions including MoNi (see Study 2 of Example 8 of experimental section . Fl : IgG + trehalose (250 mg) + L-histidine hydrochloride. F2: IgG + trehalose (250 mg) + L-histidine hydrochloride + PS20 (1 mg). F3: IgG + trehalose (250 mg) + L-histidine hydrochloride + MoNi (25 mg). F4: IgG + trehalose (125 mg) + L-histidine hydrochloride + MoNi (25 mg). F5: IgG + MoNi (25 mg) + L-histidine hydrochloride. F6: IgG + L-histidine hydrochloride.

[0024] FIG. 13 is a graph showing the relative reconstitution times of high concentration (200 mg / mL) IgG aqueous formulations with various surfactants as described herein (Fl “No Surfactant”; F2 polysorbate 20 (“PS20”); F3: MoNi). Exemplary solid IgG formulations wereprepared by lyophilizing from a histidine buffer solution of 200 mg / mL IgG without surfactant, with 0.8 mg / mL of Polysorbate 20, or with 20 mg / mL of MoNi.DETAILED DESCRIPTIONSolid Pharmaceutical Compositions

[0025] As summarized above, this disclosure relates to solid compositions, such as lyophilized compositions, including a biopharmaceutical agent and a polyacrylamide-based copolymer that are produced via solvent removal of a corresponding liquid composition. It was discovered when biopharmaceutical agents are formulated according to the present disclosure that degradation of the biopharmaceutical agents is retarded, and, accordingly, such solid formulations are stable both biochemically and physically and provide more flexible storage conditions and handling. In some embodiments, the solid composition is storage stable and can be reconstituted with a liquid solution that retains the bioactivity of the biopharmaceutical agent. In some embodiments, the solid composition is a lyophilized composition.

[0026] In some embodiments, the storage stable composition of this disclosure demonstrates less than 10 mol % degradation of the bioactivity of the biopharmaceutical agent after storage for 4 weeks or longer (e.g., 6 weeks or longer, 8 weeks or longer, 10 weeks or longer, 12 weeks or longer, 18 weeks or longer, 24 weeks or longer, 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, 7 months or longer, 8 months or longer, 9 months or longer, 10 months or longer, 11 months or longer, 12 months or longer) at 5° C ± 2° C (e.g., in a sealed container), such as less than 9 mol %, less than 8 mol %, less than 7 mol %, less than 6 mol %, less than 5 mol %, less than 4 mol %, less than 3 mol %, less than 2 mol % degradation of bioactivity of the biopharmaceutical agent initially present in the composition prior to storage. It is understood that stability can be assessed at a variety of temperatures suitable for storage.

[0027] In some embodiments, the storage stable composition of this disclosure demonstrates less than 10 mol % degradation of the bioactivity of the biopharmaceutical agent after storage for 1 week or longer (e.g., 2 weeks or longer, 4 weeks or longer, 8 weeks or longer, 12 weeks or longer, 3 months or longer, 6 months or longer) at 25° C ± 2° C in a sealed container, such as less than 9 mol %, less than 8 mol %, less than 7 mol %, less than 6 mol %, less than 5 mol %, less than 4 mol %, less than 3 mol %, less than 2 mol %degradation of the bioactivity of the biopharmaceutical agent initially present in the composition prior to storage.

[0028] In some embodiments, the composition is stored in a sealed container. In certain embodiments, the composition is in a unit dosage form in the container, e.g., a vial. The bioactivity or a variety of other physical or chemical properties of the biopharmaceutical agent can be assessed to determine storage stability.

[0029] In some embodiments, the biopharmaceutical agent is present in a stable solid composition suitable for reconstitution at a high concentration of the biopharmaceutical agent. In some embodiments, the compositions provide a higher concentration and stability of a biopharmaceutical agent as compared to a conventional, or currently approved formulation. In some embodiments, the biopharmaceutical agent is a therapeutic antibody and the high concentration is, e.g., 5 wt% or more of the composition, such as 7, 10, 12, 15, 20, or 25 wt% or more; or 5, 7, 10, or 12 wt% to 15, 20, or 25 wt%, such as 5-25 wt%, 5-20 wt%, 5-15 wt%, 10-25 wt%, 10-20 wt%, or 15-25 wt%. In some embodiments, the biopharmaceutical agent is a therapeutic antibody present in the composition substantially in a monomeric association state. In some embodiments, the composition is formulated for subcutaneous administration of a therapeutically effective amount of the biopharmaceutical agent. In some embodiments, the biopharmaceutical agent is a vaccine.

[0030] In some embodiments, the storage stable solid composition is capable of dissolution with water to produce a reconstituted aqueous composition comprising: 0.1 wt% to about 10 wt% of the copolymer; and 0.5 wt% to 25 wt% of the biopharmaceutical agent.

[0031] The process for preparing the solid composition according to the present disclosure is suitably carried out using lyophilization, spray drying, spray freezing drying, foam mat drying, vacuum drying, vacuum oven drying, freeze concentrating, or desiccation technique commonly used within the pharmaceutical area.

[0032] A solid composition of the present disclosure, in some embodiments, is generated by lyophilizing a liquid composition described herein. The liquid composition can be lyophilized under standard lyophilization conditions known in the art or modifications thereof. For example, the technique of lyophilization, and details of other suitable excipients, may be found in Cameron et al., “Good Pharmaceutical freeze-drying Practice,” Interpharm, Buffalo Grove (1997).

[0033] A solid composition of the present disclosure, in some embodiments, is generated by spray drying a liquid composition described herein. The liquid composition can be spray dried under standard spray drying conditions known in the art or modifications thereof. In certain embodiments of this method, a spray drying cycle is wherein the biopharmaceutical agent solution is atomized into fine droplets, and a stream of air or inert gas (optionally heated) is used to evaporate the solvent. In some embodiments, spray drying is a rapid process and is suitable for large-scale production. A preferred spray dry cycle according to the disclosure is a spray-drying process in which tiny droplets of the composition are obtained by pumping the liquid composition in a spray nozzle, with or without coaxial additional pressurized air (or inert gas). Such droplets may be formed in a largely ventilated area (at a temperature between +20°C and +250°C) so that quick drying by evaporation process is obtained. The solid composition is obtained, e.g., after cyclone separation.

[0034] A solid composition of the present disclosure, in some embodiments, is generated by spray freeze-drying a liquid composition described herein. The liquid composition can be spray dried under standard spray drying conditions known in the art or modifications thereof. In embodiments of this method, tiny droplets of the composition are obtained by pumping the liquid composition in a spray nozzle, with or without coaxial addition of pressurized air (or inert gas). The droplets may be formed in a very cold chamber (between -5°C to -195.8°C) so that quick freezing of the droplet is obtained. Subsequent drying of this frozen material may be obtained following a standard freeze-drying process, optionally under vacuum.

[0035] A solid composition of the present disclosure, in some embodiments, is generated by aerosol drying a liquid composition described herein. The liquid composition can be aerosol dried under standard aerosol drying conditions known in the art or modifications thereof.

[0036] A solid composition of the present disclosure, in some embodiments, is generated by drying by desiccation a liquid composition described herein. The liquid composition can be desiccated under standard desiccation desiccating conditions known in the art or modifications thereof. In some embodiments, this method involves placing the protein solution in a desiccator, which is a sealed container with a drying agent such as silica gel or other desiccants. The drying agent absorbs the moisture from the solution, leaving the solid composition.

[0037] A solid composition of the present disclosure, in some embodiments, is generated by freeze concentrating a liquid composition described herein. The liquid composition can be freeze concentrated under standard conditions known in the art or modifications thereof. The remaining concentrated protein solution may then be dried by additional means like airdrying, lyophilization, or the like.

[0038] A solid composition of the present disclosure, in some embodiments, is generated by drying a liquid composition described herein in a vacuum oven. The liquid composition can be vacuum oven dried under standard conditions known in the art or modifications thereof. In some embodiments, this technique involves placing the liquid composition in a vacuum oven, where reduced pressure facilitates the removal of water at lower temperatures. In some embodiments, this method is preferable for proteins that are stable at moderate temperatures.

[0039] A solid composition of the present disclosure, in some embodiments, is generated by foam mat drying of a liquid composition described herein. The liquid composition can be foamed and dried under standard conditions known in the art or modifications thereof. This method involves creating a foam from the protein solution and drying it on a mat. The foam typically provides a large surface area for drying, and the resulting solid composition can be easily rehydrated.

[0040] The term “liquid composition” as used herein refers to a composition in liquid form that comprises a biopharmaceutical agent and at least one pharmaceutically acceptable excipient, e.g., a polyacrylamide-based copolymer, and that can be lyophilized to generate a lyophilized pharmaceutical composition as described herein.

[0041] The term “solid composition” or “solid pharmaceutical composition” as used herein refers to any composition or pharmaceutical composition in a solid form. The solid form can be an amorphous form or a crystalline form. In some embodiments, the solid composition is in a dry form that is prepared from a liquid composition by one of the drying methods described herein, such as lyophilization. The dried solid compositions of the present disclosure can be reconstituted for injection.

[0042] The term “lyophilized composition” or “lyophilized pharmaceutical composition” as used herein refers to any composition or pharmaceutical composition in dry form that is prepared by lyophilization. “Lyophilizing” or “lyophilization” have the meanings understood by those skilled in the art, referring broadly to any process of freezing followed bydehydration in the frozen state under vacuum. Lyophilized compositions can be reconstituted for injection.

[0043] Lyophilization is a process in which water is removed from a substance (e.g., a biopharmaceutical agent) using freezing temperatures and low pressure. In an exemplary lyophilization, the material to be lyophilized is cooled below its triple point, usually between -50 °C and -80 °C. Once the material is frozen, the surrounding air pressure is reduced, and enough heat is added to sublimate the ice. In a second drying stage, additional heat is added to remove unfrozen water molecules. Upon completion, the lyophilized material has less than 5%, usually less than 3%, and typically in the range of about 0.5% to about 3% residual moisture content.

[0044] Methods of lyophilizing are described in US 7,888,097, the disclosure of which is incorporated herein by reference. In one aspect, lyophilized compositions of the present disclosure have less than 5%, less about than about 4%, less than about 3%, less than about 2%, or less than about 1% residual moisture. In one aspect, lyophilized compositions of the present disclosure have between about 0.5% and about 5% residual moisture. In one aspect, lyophilized compositions of the present disclosure have between about 0.5% and about 3% residual moisture. In one aspect, lyophilized compositions of the present disclosure have between about 0.5% and about 1% residual moisture. As used herein in regard to residual moisture content, “about” refers to a variation of no more than 10% in the referenced number.

[0045] A method for lyophilizing the liquid composition of the disclosure can include (a) providing a container containing the liquid composition at a starting temperature of about 5 °C to about -50 °C; (b) cooling the composition to subzero temperatures below that of the starting temperature (e.g., -10 °C to -50 °C); and (c) substantially drying the composition. The conditions for lyophilization, e.g., temperature and duration, of the formulation of the disclosure can be adjusted by a person of ordinary skill in the art taking into consideration factors that affect lyophilization parameters, e.g., the type of lyophilization machine used, the amount of biopharmaceutical agent used, and the size of the container used.

[0046] In some embodiments, the steps of (b) cooling the composition and (c) drying the composition are performed while changing temperatures. For example, the step of (b) cooling the composition can include raising temperatures from -50 °C to -40 °C, from -50 °C to -30 °C, from -50 °C to -20 °C, from -50 °C to -10 °C, or from -50 °C to 0 °C. In some embodiments, the step of (b) cooling the composition can include reducing temperatures from-40 °C to -50 °C, from -30 °C to -50 °C, from -20 °C to -50 °C, from -10 °C to -50 °C, from 0 °C to -50 °C, or from 5 °C to -50 °C. In some embodiments, the step of (b) cooling the composition includes reducing the temperature and then raising the temperature of the composition.

[0047] Some amorphous products (such as mannitol or glycine) form a metastable glass with incomplete crystallization when first frozen. These products can benefit from a thermal treatment process, which is also called annealing. During annealing, the product temperature is cycled (for example: from -40 °C to -20 °C for a few hours and then back to -40 °C; from -50 °C to -20 °C, holding for two (2) hours, and then introducing the vacuum; or from -50 °C to -20 °C and then back to -50 °C) to obtain more complete crystallization. Annealing has the added advantage of larger crystal growth and corresponding shorter drying times. Water trapped in the amorphous phase can be further removed during the secondary drying.

[0048] In some embodiments, the step of (c) drying the composition is performed in two steps — (i) primary drying and (ii) secondary drying.

[0049] In some embodiments, primary drying includes maintaining the temperature, or raising or reducing temperatures. In some embodiments, primary drying includes maintaining the temperature at -50 °C, -40 °C, -30 °C, -20 °C, -10 °C, or 0 °C. In some embodiments, secondary drying includes maintaining the temperature, or raising or reducing temperatures. In some embodiments, the secondary drying includes raising temperatures from -50 °C to 20 °C, from -40 °C to 20 °C, from -30 °C to 20 °C, from -20 °C to 20 °C, from -10 °C to 20 °C, from -50 °C to 10 °C, from -40 °C to 10 °C, from -30 °C to 10 °C, from -20 °C to 10 °C, from -10 °C to 10 °C.

[0050] In some embodiments, secondary drying is continued until the product has acceptable moisture content for long term storage. Depending on the application, moisture content in fully dried products is typically from 0.5% to 3%. In most cases, the drier the product, the longer its shelf life will be. However, certain complex biological products can become too dry for optimum storage results and the secondary drying process should be controlled accordingly.

[0051] The step of secondary drying can include raising temperatures from -50 °C to 20 °C, from -40 °C to 20 °C, from -30 °C to 20 °C, from -20 °C to 20 °C, from -10 °C to 20 °C, from -50 °C to 10 °C, from -40 °C to 10 °C, from -30 °C to 10 °C, from -20 °C to 10 °C, from -10 °C to 10 °C.

[0052] The term “reconstituted” or “reconstitution” as used herein refers to the restoration to the original form of a substance previously altered for preservation and storage, such as rehydration, i.e., the restoration to a liquid state of a DNA plasmid formulation that has been previously lyophilized and stored. The lyophilized composition of the present disclosure may be reconstituted in any aqueous solution which produces a stable solution suitable for pharmaceutical administration. Such aqueous solutions include, but are not limited to, sterile water, Tris-EDTA (TE), phosphate-buffered saline (PBS), Tris buffer, histidine (e.g., L-histidine hydrochloride), and normal saline.

[0053] Another attribute of a lyophilized product that can affect the quality of a biopharmaceutical agent is turbidity of a reconstituted composition obtained by dissolving the lyophilized composition. Turbidity of a reconstituted biopharmaceutical agent can correlate with recovery of active ingredients in the lyophilized composition. In general, a complete dissolution, i.e., a low turbidity of the reconstituted composition, is preferred. Incomplete dissolution can result in waste of active ingredients of the pharmaceutical composition and blockage of a syringe for administering the reconstituted composition.

[0054] Turbidity of a reconstituted biopharmaceutical agent can be measured by visual inspections or by measuring light absorbance at certain wavelengths, e.g., at 450 nm and 650 nm. Light absorbance can be measured using a device available in the art, for example, a Molecular Devices ThermoMAX microplate.

[0055] Reconstitution time is another factor that can be related with the quality of a drug product. In general, a short reconstitution time is preferred. Increased time for reconstitution at the user stage may result in partial loss of potency if the biopharmaceutical agent is not completely dissolved, since it is common to use in-line filters during administration to the patient. Time required for reconstitution of the lyophilized product can be determined, for example, by measuring turbidity of the reconstituted biopharmaceutical agent at various time points after reconstitution. For example, turbidity can be measured after 1 min, 5 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 1 hour, 2 hours, or more.

[0056] Residual moisture after lyophilization can be also important. Various methods known in the art can be adopted to measure residual moisture in the lyophilized composition. For example, a Karl Fisher Coulometer C20 (Mettler Toledo) can be used for moisture content analysis. A pocket of moisture in a lyophilized composition can result in greater instability and increased product degradation. Thus, less moisture content can be preferred.

[0057] Concentration measurements of biopharmaceutical agents are conducted by various methods known in the art. For example, a Solo-VPE can be used for measuring UV absorption of a biopharmaceutical agent.

[0058] Further components of the compositions of this disclosure are now described in more detail. It is understood that the components of the lyophilized compositions, and their amounts can be described in terms of the solid composition after lyophilization, or the liquid composition from which the lyophilized compositions of the present disclosure are derived, or in some cases, the reconstituted composition which can be suitable for administration to a subject according to the methods of this disclosure. In some embodiments, the original liquid composition and the reconstituted composition have the same or similar volume and concentration of biopharmaceutical agent.Characterization of the Compositions

[0059] The present disclosure provides a solid pharmaceutical composition having one or more desired properties as a pharmaceutical product. In some embodiments, the solid pharmaceutical composition is a lyophilized pharmaceutical composition. The properties can include stability and potency of the active ingredient biopharmaceutical agent under various storage conditions, cake formation, a uniform reconstitution of the formulation for administration, less contaminations, etc. Various properties provided herein can be used to select a preferred solid composition or to determine ideal storage conditions for the solid composition.

[0060] Stability of biopharmaceutical agents in the solid composition (e.g., lyophilized composition) can be determined based on methods known in the art. In particular, stability can be determined based on the potency or bioactivity of the biopharmaceutical agent. The potency or bioactivity of a biopharmaceutical agent can be measured in various conditions, for example, before, during or after drying, or before, during or after reconstitution of the solid composition, or for example, before, during or after lyophilization, or before, during or after reconstitution of the lyophilized composition. The biopharmaceutical agent integrity can be also determined before, during or after a storage at different temperatures to select a stable solid composition as well as to determine an ideal storage condition.

[0061] Cake appearance can be another important attribute of a dried product. In some embodiments, the dried product is lyophilized product. Uniform and elegant cake appearance is generally preferred. The non-ideal cake appearance can affect product quality, for example,by making it difficult to determine product quality based on visual inspection or making it difficult to recover the full amount of the active ingredient in a container. In addition, partial or complete meltback of the cake can result in instability and degradation of the active ingredient. Meltback is a form of cake collapse and is caused by the change from the solid to liquid state. That is, there is incomplete sublimation (change from the solid to vapor state) in the vial. These changes can involve a change in the physical form of the biopharmaceutical agent and a pocket of moisture.

[0062] Cake appearance can be determined by visual inspection that may involve taking a photograph. Visual inspections can be performed largely based on historical precedent. A robust qualification program for visual inspection is important before judging product quality. The qualification program can be based on past experience or published information, including FDA visual inspection guidelines for particular drug products.

[0063] In some embodiments, after drying, initial time point level of impurities can be determined by HPLC or other analytical technique. In some embodiments, the drying is lyophilization. Afterwards vials can be loaded into stability chambers at different storage conditions. In some embodiments, the storage conditions include storage in a sealed container at 25 °C, 37 °C, 40 °C, or 60 °C, or equivalent for a period of time. In some embodiments, storage conditions include storage at a particular temperature and relative humidity (RH), such as 25 °C / 60% RH or 40 °C / 75% RH.

[0064] In some embodiments, the solid composition demonstrates higher thermostability after 14 days or more at 37 °C as compared to a dried composition lacking the polyacrylamide-based copolymer. In some embodiments, the lyophilized composition demonstrates higher thermostability after 14 days or more at 37 °C as compared to a lyophilized composition lacking the polyacrylamide-based copolymer.

[0065] In order to determine formation of impurities and stability of biopharmaceutical agent in solid compositions according to the present disclosure, vials are taken from stability chambers at various time points such as 45 hours, 1 month, 2 months etc. and analyzed using a variety of assays and techniques, e.g., HPLC or other analytical technique known in the art, for example by potency assay. In order to determine formation of impurities and stability of biopharmaceutical agent in formulations according to the present disclosure, vials are taken from stability chambers at various time points such as 45 hours, 1 month, 2 months etc. andanalyzed use a variety of assays and techniques, e.g., HPLC or other analytical technique, or by potency assay.Biopharmaceutical agents

[0066] The biopharmaceutical agent can be formulated at a weight percent and / or in a volume suitable for injection (e.g., SC or IM) of a unit dose to a patient in need thereof. In some embodiments, the compositions of this disclosure are formulated to contain a therapeutically effective amount of a biopharmaceutical agent.

[0067] In some embodiments, the biopharmaceutical agent has a high MW, e.g., a large biologic. In some embodiments, a high MW biopharmaceutical agent is one having a MW of 20 kDa or more, such as 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more.

[0068] In some embodiments, the solid composition (e.g., lyophilized composition) includes 0.5 wt% to 25 wt%, or 0.5 wt% to 20 wt% of the biopharmaceutical agent, such as 1 wt% to 20 wt% of the biopharmaceutical agent, for example, 1 wt% to 15 wt%, 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 2 wt% to 15 wt%, 2 wt% to12 wt%, 2 wt% to 10 wt%, 2 wt% to 8 wt%, 2 wt% to 5 wt%, 3 wt% to 15 wt%, 3 wt% to12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt% to 15 wt%, 5 wt% to12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 15 wt%, 8 wt% to 12 wt%, 8 wt% to10 wt%, 10 wt% to 15 wt%, or 10 wt% to 12 wt%. In certain embodiments, solid compositions include 7 wt%, 8 wt%, 9 wt%, or 10 wt% of the biopharmaceutical agent. In such cases, the remainder of the composition can be composed of the polyacrylamide-based copolymer, and / or one or more optional components such as a stabilizing agent (e.g., as described herein).

[0069] In some embodiments, the solid composition is capable of dissolution with water to produce a reconstituted aqueous composition comprising: 0.1 wt% to about 10 wt% of the copolymer (e.g., as described herein); and 0.5 wt% to 25 wt% of the biopharmaceutical agent (e.g., as described herein).

[0070] In some embodiments, the solid composition (e.g., lyophilized composition) includes 1 wt% to 20 wt% of the biopharmaceutical agent, for example 1 wt% to 10 wt%. In such cases, the remainder of the composition can be composed of the polyacrylamide-based copolymer, and / or one or more optional components such as a stabilizing agent (e.g., as described herein).

[0071] In some embodiments, the biopharmaceutical agent is selected from a vaccine, gene therapy, cell therapy, and lipid nanoparticles (LNPs) including a nucleic acid (e.g., mRNA).

[0072] In some embodiments, the biopharmaceutical agent is a vaccine composition or immunogenic composition. The phrase “immunogenic composition” refers to the antigen or immunogen of interest expressed from a vector and includes any composition that elicits an immune response against a target immunogen or antigen of interest after administration to a subject. The phrase “vaccine composition” refers to a composition that induces a protective immune response against an antigen of interest or provides effective protection against an antigen. Any composition that elicits a protective immune response against a target antigen or immunogen after administration or injection to a subject, or any composition that provides effective protection against an antigen or immunogen expressed from a vector is also included.

[0073] In some embodiments, the biopharmaceutical agent is a vaccine. In some embodiments, the vaccine includes a live or attenuated virus. In some embodiments, the vaccine is an inactivated virus. In some embodiments, the vaccine is a mRNA vaccine.

[0074] In some embodiments, the biopharmaceutical agent is a composition containing mRNA in a delivery system, such as a LNP containing mRNA cargo. Such mRNA compositions have therapeutic application as viral vaccines, protein replacement therapies, cancer immunotherapies, cellular reprogramming and in genome editing.

[0075] In some embodiments, the biopharmaceutical agent is a therapeutic cellular composition, such as a cancer immunotherapy composition.

[0076] In some embodiments, the biopharmaceutical agent is a gene therapy composition.

[0077] In some embodiments, the biopharmaceutical agent is a polypeptide. In some embodiments, the polypeptide is susceptible to aggregation in an aqueous medium. In some embodiments, the polypeptide is a protein. In some embodiments, the polypeptide is a peptide.

[0078] In some embodiments, the polypeptide is selected from antibodies and fragments thereof, antibody-drug conjugates, chimeric fusion proteins, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof.

[0079] In some embodiments, the polypeptide is a therapeutic protein. In some embodiments, the therapeutic protein is selected from antibody or fragment thereof, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, enzyme, growth factor, hormone, interferon, interleukin, and thrombolytics protein.

[0080] In some embodiments, the biopharmaceutical agent is an antibody or antibody fragment.

[0081] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes, but is not limited to, full length antibodies (e.g., intact immunoglobulins), antibody fragments, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized fully human antibodies, chimeric antibodies, and single domain antibodies.

[0082] In some embodiments, the biopharmaceutical agent is an antibody or fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is monospecific, i.e., binds a single antigen. In some embodiments, the monoclonal antibody is an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgM antibody, and IgA antibody, or any hybrid thereof.

[0083] In some embodiments, the antibody is a chimeric antibody. The term “chimeric antibody” refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.

[0084] In some embodiments, the antibody is multispecific, i.e., binds multiple antigens, e.g., a bispecific antibody.

[0085] In some embodiments, the antibody is an antibody fragment. An “antibody fragment” includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments suitable for use in the present compositions include, for example, Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0086] In some embodiments, the biopharmaceutical agent is a chimeric protein. In some embodiments, the chimeric protein is a recombinant fusion protein. The chimeric protein can include two or more domains connected via optional linkers or spacers. In some embodiments, the chimeric protein comprises an antibody fragment, such as an Fc or fragment or variant thereof. In some embodiments, the chimeric protein comprises anantibody fragment fused to a protein domain, e.g., a protein domain that specifically binds to a therapeutic target of a biopharmaceutical agent. In some embodiments, the chimeric protein is an Fc fusion protein.

[0087] In some embodiments, the antibody is a single domain antibody, e.g., a camelid antibody, or nanobody.

[0088] In some embodiments, the antibody is an antibody-drug conjugate, e.g., an antibody conjugated to one or more heterologous molecule(s). The heterologous molecule can be a small molecule (e.g., an organic compound with a molecular weight of less than 1000, 900, 800, 700, 600, or 500 Daltons). In some embodiments, the heterologous molecule is a cytotoxic agent, a chemotherapeutic agent, or a cytostatic agent.

[0089] In some embodiments, the antibody is a bispecific antibody immunoconjugate.

[0090] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody includes one or more single-chain variable fragments (scFv) of a monoclonal antibody. In some embodiments, the monoclonal antibody is a humanized antibody, a human antibody, a murine antibody, or a chimeric (mouse / human) antibody.

[0091] The antibody can be targeted to a variety of target proteins, e.g., a therapeutic target protein.

[0092] In some embodiments, the antibody has a molecular weight of about 100 kDa to about 200 kDa, for example, about 120 kDa to about 180 kDa. In some embodiments, the antibody has a molecular weight of about 150 kDa. In some embodiments, the antibody has a molecular weight of 100 kDa to 200 kDa, for example, 120 kDa to 180 kDa. In some embodiments, the antibody has a molecular weight of 150 kDa. In some embodiments, the antibody has a molecular weight of 100 kDa or less, such as 40 kDa to 80 kDa, for example, about 50 kDa.Antibody Compositions

[0093] Aspects of this disclosure include compositions including a polyacrylamide-based copolymer and a biopharmaceutical agent that is an antibody. The incorporation of the polyacrylamide-based copolymer into the composition can prevent or reduce aggregation of the antibody, thereby maintaining the biological activity of the antibody, e.g., a specific binding to an antigen.

[0094] As used herein, the term “association state,” used in reference to an antibody, describes the extent of aggregation of the antibody. For example, non-aggregated antibodies present in a composition can be referred to as monomeric antibodies, or antibodies present in a monomeric association state. In another example, aggregates of two or more antibodies present in a composition can be referred to as aggregated antibodies, or antibodies present in an aggregated association state.

[0095] The term “substantially in a monomeric association state,” used in reference to an antibody composition, refers to compositions in which 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 97% or more of the antibodies therein are present in a monomeric association state. The term “stability,” used in reference to an antibody composition, refers to the ability of the composition to retain at least a portion of binding activity of the antibodies therein over time. For example, a “stable” composition can, in some embodiments, retain 70% or more (e.g., 80% or more, 90% or more, or 95% or more) of a binding activity of an antibody therein over 21 days or more of continuous stressed aging (e.g., based upon half-maximal inhibitory concentration as determined by an enzyme-linked immunosorbent assay).

[0096] In some embodiments, the solid compositions can be reconstituted and are suitable for subcutaneous injection, e.g., sterile. In some embodiments, the reconstituted composition is formulated for subcutaneous injection, having a high concentration of antibody (e.g., 5 wt% or more, as described above) and one or more physical properties suitable to be injectable under clinically relevant injection conditions (e.g., a viscosity of 0.2 Pa s m or less, such as 0.15, 0.1, or 0.05 Pa s m).

[0097] In some embodiments, the reconstituted composition has a surface tension of 60 nM / m or less, for example, 55, 50, 45, 40, 30, or 25 nM / m or less.

[0098] In some embodiments, the reconstituted composition has an interfacial complex viscosity of 0.2 Pa s m or less, for example, 0.15 Pa s m or less, or 0.1 or less. Interfacial complex viscosity (also referred to as interfacial shear viscosity or interfacial complex shear viscosity) is a measure of the flow of molecules present at the interface between a gas and a liquid or at the interface between two immiscible liquids. FIG. 3, Panel B, shows interfacial shear viscosity measurements of an exemplary antibody composition versus a control solution. The exemplary polyacrylamide-based copolymer reduced the interfacial complex viscosity of the resulting antibody composition by 3-fold.

[0099] In some embodiments, the reconstituted composition has a high shear rate viscosity of 6 mPa s or less, for example, 5.5 mPa s or less, at shear rates of from 0 s'1to 3,000 s'1. FIG. 3, Panel C, shows a graph of the viscosity of an exemplary antibody composition at high shear rates representative of injection, versus a control solution. The exemplary antibody composition including the polyacrylamide-based copolymer exhibited bulk solution viscosity at high shear rates suitable for injection.

[0100] In some embodiments, the reconstituted composition is capable of retaining 70% or more, for example, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of a specific binding activity of the antibody for a target epitope or antigen, e.g., as assessed over 21 days of continuous stressed aging. In some embodiments, the binding activity is determined by an enzyme-linked immunosorbent assay.Peptides

[0101] In some embodiments, the biopharmaceutical agent is insulin. The term “insulin” refers to a hormone produced by the beta cells in the pancreatic islets that regulates the amount of glucose in the blood. Many eukaryotes, including humans, primates, pigs, cows, cats, dogs, and rodents, produce insulin. Thus, “insulin,” as used herein, includes insulin produced by humans, and analogs thereof, as well as insulin, and analogs thereof, produced by other eukaryotes, including, but not limited to, primates, pigs, cows, cats, dogs, and rodents, and also includes recombinant, purified or synthetic insulin or insulin analogs having similar function and structure, unless otherwise specified. The human insulin protein consists of 51 amino acids, and has a molecular weight of approximately 5.8 kilodalton (kDa). Human insulin is a heterodimer of an A-chain and a B-chain that are connected by disulfide bonds.

[0102] Insulin can be isolated from the pancreatic islets extracts of an animal that produces insulin or expressed recombinantly in a suitable expression system such as E. coli, yeast, insect cells, and mammalian cells (e.g., Chinese hamster ovary (CHO) cells).Depending upon their specific pharmacokinetics and pharmacodynamics (PK / PD) properties (e.g., duration of action, maximum concentration observed (Cmax), time-to-onset, area under the curve (AUC)), insulin can be further characterized as a rapid-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, and a pre-mixed insulin.

[0103] Insulin also includes monomeric and oligomeric forms, such as dimeric and hexameric forms. Insulin can exist as a monomer as it circulates in the plasma, and it also binds to its receptor while in a monomeric form. Insulin formulations (or insulin analogformulations) containing a predominance of protein molecules in the form of monomers and dimers ordinarily have a strong tendency to aggregate and form inactive fibrils. Insulin hexamers are too large to be absorbed, and so hexameric insulin formulations must disassemble into dimers or monomers before the insulin can be absorbed and function in the body. The active form of insulin in the blood stream is the monomeric form.

[0104] In some embodiments, the solid pharmaceutical compositions include about 0.5 wt% to about 20 wt% of the insulin or the analog thereof, such as about 1 wt% to about 20 wt% of the insulin or the analog thereof, for example, about 1 wt% to about 15 wt%, about 1 wt% to about 12 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 2 wt% to about 15 wt%, about 2 wt% to about 12 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about2 wt% to about 5 wt%, about 3 wt% to about 15 wt%, about 3 wt% to about 12 wt%, about3 wt% to about 10 wt%, about 3 wt% to about 8 wt%, about 3 wt% to about 5 wt%, about5 wt% to about 15 wt%, about 5 wt% to about 12 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 8 wt% to about 15 wt%, about 8 wt% to about 12 wt%, about 8 wt% to about 10 wt%, about 10 wt% to about 15 wt%, or about 10 wt% to about 12 wt% of the insulin or the analog thereof. In some embodiments, the solid compositions include about7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of the insulin or the analog thereof. In some embodiments, the solid compositions include 1 wt% to 20 wt%, 1 wt% to 15 wt%, 1 wt% to 10 wt%, 1 wt% to 9 wt%, or 1 wt% to 8 wt% of the insulin or the analog thereof. In some embodiments, the solid compositions include 7 wt%, 8 wt%, 9 wt%, or 10 wt% of the insulin or the analog thereof. In some embodiments, the lyophilized pharmaceutical compositions include about 0.5 wt% to about 20 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 2 wt% to about 15 wt%, about2 wt% to about 12 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about2 wt% to about 5 wt%, about 3 wt% to about 15 wt%, about 3 wt% to about 12 wt%, about3 wt% to about 10 wt%, about 3 wt% to about 8 wt%, about 3 wt% to about 5 wt%, about5 wt% to about 15 wt%, about 5 wt% to about 12 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 8 wt% to about 15 wt%, about 8 wt% to about 12 wt%, about8 wt% to about 10 wt%, about 10 wt% to about 15 wt%, or about 10 wt% to about 12 wt% of the insulin or the analog thereof. In some embodiments, the lyophilized compositions include about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of the insulin or the analogthereof. In some embodiments, the lyophilized compositions include 1 wt% to 20 wt%, 1 wt% to 15 wt%, 1 wt% to 10 wt%, 1 wt% to 9 wt%, or 1 wt% to 8 wt% of the insulin or the analog thereof. In some embodiments, the lyophilized compositions include 7 wt%, 8 wt%, 9 wt%, or 10 wt% of the insulin or the analog thereof.

[0105] In some embodiments, the insulin or the analog thereof is selected from insulin lispro, HUMALOG® (fast-acting insulin lispro), insulin glargine, LANTUS® (insulin glargine), insulin detemir, LEVEMIR® (insulin detemir), ACTRAPID® (fast-acting human insulin), modem insulin, NOVORAPID® (insulin aspart), VELOSULIN® (human insulin), HUMULIN® M3 (a mixture of soluble insulin and isophane insulin called biphasic isophane insulin), HYPURIN® (neutral bovine insulin), INSUMAN® (recombinant human insulin), INSULATARD® (long-acting isophane human insulin), MIXTARD® 30 (a mixture of 30% soluble insulin and 70% isophane insulin), MIXTARD® 40 (a mixture of 40% soluble insulin and 60% isophane insulin), MIXTARD® 50 (a mixture of 50% soluble insulin and 50% isophane insulin), insulin aspart, insulin glulisine, insulin isophane, insulin degludec, insulin icodec, insulin zinc extended, NOVOLIN® R (human insulin), HUMULIN® R (human insulin), HUMULIN® R regular U-500 (concentrated regular insulin), NOVOLIN® N (intermediate-acting human insulin), HUMULIN® N (intermediate-acting human insulin), RELION® (over-the-counter brand of NO VOLIN® R, NO VOLIN® N, and NO VOLIN® 70 / 30), AFREZZA® (rapid-acting inhaled insulin), HUMULIN® 70 / 30 (a mixture of 70% human insulin isophane suspension and 30% human insulin injection), NOVOLIN® 70 / 30 (a mixture of 70% NPH, human insulin isophane suspension and 30% regular, human insulin injection), NOVOLOG® 70 / 30 (a mixture of 70% insulin aspart protamine suspension and 30% insulin aspart injection), HUMULIN® 50 / 50 (a mixture of 50% human insulin isophane suspension and 50% human insulin injection), HUMALOG® Mix 75 / 25 (a mixture of 75% insulin lispro protamine suspension and 25% insulin lispro injection), insulin aspart protamine-insulin aspart, insulin lispro protamine-insulin lispro, human insulin NPH-human insulin regular, insulin degludec-insulin aspart, and combinations thereof. In some embodiments, the insulin or the analog thereof is a human insulin or a recombinant human insulin. In some embodiments, the insulin or the analog thereof is a non-human (e.g., primate, pig, cow, cat, dog, or rodent) insulin or a recombinant non-human insulin. In some embodiments, the insulin or the analog thereof is a purified or synthetic insulin. In some embodiments, the insulin or the analog thereof is selected from a rapid-acting insulin, a shortacting insulin, an intermediate-acting insulin, a long-acting insulin, and a pre-mixed insulin.In some embodiments, the insulin or the analog thereof is insulin lispro. In some embodiments, the insulin or the analog thereof is insulin aspart. In some embodiments, the insulin, or an analog thereof, is recombinant human insulin.

[0106] In some embodiments, the biopharmaceutical agent is a peptide or peptide analogue. The formulation approaches described herein should be particularly useful in preparing storage stable injectable pharmaceutical compositions of a variety of therapeutic peptides, including but not limited to glucagon, glucagon-like peptide- 1 (GLP-1), a GLP-1 receptor agonist, GLP-2, adrenocorticotropic hormone (ACTH), leuprolide, hirudin, insulin, pramlintide, exendins, exenatide, gastric inhibitory peptide, calcitonin, calcitonin gene related peptide, amylin, adrenomedullin, angiotensin, an immunogenic peptide (e.g., a peptide or peptide complex derived from a virus, a bacterium, or any prokaryotic or eukaryotic organism or cell thereof), and the like, and analogues thereof.

[0107] In some embodiments, the biopharmaceutical agent is glucagon peptide, glucagon analog, glucagon mimetic, or salt thereof.Additional active agents

[0108] The solid pharmaceutical compositions of the present disclosure can include a combination of biopharmaceutical agent and one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are useful for treating a disease or condition that is targeted by biopharmaceutical agent. In some embodiments, administration of the agents from the pharmaceutical composition can provide for delivery to a cell in the subject's body so as to exert their biological or therapeutic effect at the same time. In some embodiments, the agents in the lyophilized compositions are co-formulated to provide pharmacokinetic profiles that are substantially similar.

[0109] In some embodiments, the one or more additional therapeutic agent is an active agent that provides a synergistic effect with the biopharmaceutical agent. In some embodiments, the additional therapeutic agent is a small molecule drug.

[0110] In some embodiments, the stable formulations and solid compositions used in accordance with the present disclosure include co-formulations or mixtures of the types of biopharmaceutical agent described herein, such as at least one peptide, at least one small molecule, and combinations thereof.

[0111] In some embodiments, the solid compositions are co-formulated to contain a first biopharmaceutical agent that is a protein, and a second biopharmaceutical agent that is a peptide. In some embodiments, the solid compositions are co-formulated to contain first and second biopharmaceutical agents that are peptides. In some embodiments, the solid compositions are co-formulated to contain a first biopharmaceutical agent that is a peptide hormone or analog thereof, and a second therapeutic agent that is a small molecule. In some embodiments, the second therapeutic agent is a steroid.

[0112] The amounts of active agent(s) that may be combined with each other and the carrier materials of the solid compositions to produce a dosage form will vary depending upon the subject in need and the particular mode of administration.Polyacrylamide-based Copolymers

[0113] The term “polyacrylamide-based copolymer” refers to a polymer that is formed from the polymerization of two or more distinct monomers, in which at least one of the monomers possesses an acrylamide functional group (i.e., acrylamide monomer). In some embodiments, the polyacrylamide-based copolymer is formed from the polymerization of two structurally different acrylamide monomers (two structurally different monomers that each possess an acrylamide functional group).

[0114] The resulting copolymer can be an alternating copolymer wherein the monomer species are connected in an alternating fashion; a random copolymer, wherein the monomer species are connected to each other within a polymer chain without a defined pattern; a block copolymer, wherein polymeric blocks of one monomer species are connected to polymeric blocks made up of another monomer species; and graft copolymer, wherein the main polymer chain consists of one monomer species, and polymeric blocks of another monomer species are connected to the main polymer chain as side branches (also referred to as sidechains). In some embodiments, the polyacrylamide-based copolymers of the present disclosure are random copolymers.

[0115] An “acrylamide monomer,” refers to a monomer species that possesses an acrylamide functional group. The term “acrylamide monomer” includes not only monomeric acrylamide, but derivatives of monomeric acrylamide. Examples of acrylamide monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropyl acrylamide (NIP), N,N-diethylacrylamide (DEA), N-tert- butyl acrylamide (TBA), and N-phenyl acrylamide (PHE).

[0116] In some embodiments, the polyacrylamide-based copolymer of the compositions of the present disclosure is amphiphilic. In some embodiments, the polyacrylamide-based copolymers are co-polymers of two acrylamide monomers, a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer is formed via a random polymerization of a water-soluble carrier monomer and a functional dopant monomer.

[0117] As defined herein, the term “water-soluble carrier monomer” refers to an acrylamide monomer species that is the water-soluble monomer species within the polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer is the predominant hydrophilic species within the polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer provides a hydrophilic sidechain group that imparts aqueous solubility to the copolymer.

[0118] In some embodiments, the water-soluble carrier monomer within the polyacrylamide-based copolymer provides an inert barrier at an interface of an aqueous formulation to prevent protein-protein interactions. In some embodiments, the interface is an air-water interface. In some embodiments, the interface is an enclosure-water interface, including, but not limited to, a glass-water interface, a rubber-water interface, a plastic-water interface, or a metal-water interface. In some embodiments, the interface is an oil-water interface. In some embodiments, the interface is an interface between a liquid and tubing. In some embodiments, the interface is an interface between a liquid and a catheter. In some embodiments, the enclosure-water interface is in a pump system. In some embodiments, the enclosure-water interface is in a closed-loop system.

[0119] In some embodiments, the water-soluble carrier monomer is hydrophilic and / or nonionic. Examples of water-soluble carrier monomers of interest include, but are not limited to, acrylamide (AM), N-(3-methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), and N-hydroxy ethyl acrylamide (HEAM).

[0120] The term “functional dopant monomer,” as used herein, refers to an acrylamide monomer species that has one or more physicochemical properties (e.g., hydrophobicity, charge, etc.) different from those of the water-soluble carrier monomer. In someembodiments, the functional dopant monomer within the polyacrylamide-based copolymer promotes association of the polymers to an interface of the composition; such interfaces can include, but are not limited to, polymer-air-water interface interactions, polymer-protein interactions, polymer-peptide interactions, polymer-micelle interactions, polymer-liposome interactions, and polymer-lipid nanoparticle interactions. The functional dopant monomer can act as a stabilizing moiety to facilitate interactions with biomolecules, for example, proteins, peptides, antibodies, antibody-drug conjugates, nucleic acids, lipid particles, and combinations thereof (e.g., to prevent aggregation of the biomolecules). The functional dopant monomers can be further classified into hydrogen-bonding, ionic, hydrophobic, and aromatic monomers based on their chemical composition. Typically, the functional dopant monomers are copolymerized at a lower weight percentage in the co-polymer as compared to the water-soluble carrier monomers.

[0121] The term “polymerization” refers to the process in which monomer molecules undergo a chemical reaction to form polymeric chains or three-dimensional networks. Different types of polymerization reactions are known in the art, for example, addition (chain-reaction) polymerization, condensation polymerization, ring-opening polymerization, free radical polymerization, controlled radical polymerization, atom transfer radical polymerization (ATRP), single-electron transfer living radical polymerization (SET-LRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, nitroxide-mediated polymerization (NMP), and emulsion polymerization. The polymerization reaction can be a vinyl addition polymerization initiated via a free radical generating system. In some embodiments, the copolymers of the present disclosure are prepared using RAFT polymerization.

[0122] The term “degree of polymerization” (DP) refers to the number of monomer units in a polymer. It is calculated by dividing the average molecular weight of a polymer sample by the molecular weight of the monomers. The average molecular weight of a polymer can be represented by the number-averaged molecular weight (Mn), the weight-average molecular weight (Mw), the Z-average molecular weight (Mz) or the molecular weight at the peak maxima of the molecular weight distribution curve (Mp). The average molecular weight of a polymer can be determined by a variety of analytical characterization techniques known to those skilled in the art, for example, gel permeation chromatography (GPC), static light scattering (SLS) analysis, multi-angle laser light scattering (MALLS) analysis, nuclear magnetic resonance spectroscopy (NMR), intrinsic viscometry (IV), melt flow index (MFI),and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), and combinations thereof. Degree of polymerization can also be determined experimentally using suitable analytical methods known in the art, such as nuclear magnetic spectroscopy (NMR), Fourier Transform infrared spectroscopy (FT-IR) and Raman spectroscopy.

[0123] The compositions described herein can include a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from N-(3-methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N- hydroxyethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from A-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), A-isopropyl acrylamide (NIP), N,N- di ethyl acrylamide (DEA), A- / c / 7-butylacrylamide (TBA), A-phenyl acrylamide (PHE), and combinations thereof.

[0124] In some embodiments, the water-soluble carrier monomer of the polyacrylamidebased copolymer is selected from MORPH, MP AM, and combinations thereof. In some embodiments, the water-soluble carrier monomer includes MORPH or MP AM. In some embodiments, the water-soluble carrier monomer is MORPH. In some embodiments, the water-soluble carrier monomer is MP AM.

[0125] In some embodiments, the functional dopant monomer of the polyacrylamidebased copolymer is selected from AMP, TMA, TBA, PHE, and combinations thereof. In some embodiments, the functional dopant monomer includes TRI, PHE, or NIP. In some embodiments, the functional dopant monomer includes DEA, PHE, or NIP. In some embodiments, the functional dopant monomer is NIP. In some embodiments, the functional dopant monomer is PHE. In some embodiments, the functional dopant monomer is DEA.

[0126] In some embodiments, the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof, and the functional dopant monomer is selected from NIP, PHE, and combinations thereof. In some embodiments, the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof, and the functional dopant monomer is selected from DEA, NIP, PHE, and combinations thereof. In some embodiments, the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof, and the functional dopant monomer is selected from AMP, TMA,TBA, PHE, and combinations thereof. In some embodiments, the water-soluble carrier monomer includes MP AM, and the functional dopant monomer includes PHE. In some embodiments, the water-soluble carrier monomer includes MORPH, and the functional dopant monomer includes PHE. In some embodiments, the water-soluble carrier monomer includes MORPH, and the functional dopant monomer includes NIP. In some embodiments, the water-soluble carrier monomer includes MORPH or MP AM, and the functional dopant monomer includes DEA.

[0127] In some embodiments, the copolymer contains about 70 wt% to about 98 wt% of the water-soluble carrier monomer, for example, about 70 wt% to about 95 wt%, about 70 wt% to about 90 wt%, about 75 wt% to about 98 wt%, about 75 wt% to about 95 wt%, about 75 wt% to about 90 wt%, about 80 wt% to about 98 wt%, about 80 wt% to about 95 wt%, about 80 wt% to about 90 wt%, about 83 wt% to about 98 wt%, about 83 wt% to about 95 wt%, or about 83 wt% to about 90 wt% of the water-soluble carrier monomer. In some embodiments, the copolymer contains about 2 wt% to about 30 wt% of the functional dopant monomer, for example, about 2 wt% to about 20 wt%, about 2 wt% to about 17 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 17 wt%, about 10 wt% to about 30 wt%, about 10 wt% to about 20 wt%, or about 10 wt% to about 17 wt% of the functional dopant monomer.

[0128] In some embodiments, the copolymer contains 70 wt% to 98 wt% of the water- soluble carrier monomer, for example, 70 wt% to 95 wt%, 70 wt% to 90 wt%, 75 wt% to 98 wt%, 75 wt% to 95 wt%, 75 wt% to 90 wt%, 80 wt% to 98 wt%, 80 wt% to 95 wt%, 80 wt% to 90 wt%, 83 wt% to 98 wt%, 83 wt% to 95 wt%, or 83 wt% to 90 wt% of the water-soluble carrier monomer. In some embodiments, the copolymer contains 2 wt% to 30 wt% of the functional dopant monomer, for example, 2 wt% to 20 wt%, 2 wt% to 17 wt%, 5 wt% to 30 wt%, 5 wt% to 20 wt%, 5 wt% to 17 wt%, 10 wt% to 30 wt%, 10 wt% to 20 wt%, or 10 wt% to 17 wt% of the functional dopant monomer.

[0129] In some embodiments, the copolymer contains about 70 wt% to about 85 wt%, about 70 wt% to about 80 wt%, about 74 wt% to about 85 wt%, about 74 wt% to about 80 wt%, or about 77 wt% of MORPH. In some embodiments, the copolymer contains about 15 wt% to about 30 wt%, about 15 wt% to about 26 wt%, about 20 wt% to about 30 wt%, about 20 wt% to about 26 wt%, or about 23 wt% of NIP. In some embodiments, the copolymer contains 70 wt% to 85 wt%, 70 wt% to 80 wt%, 74 wt% to 85 wt%, 74 wt% to 80 wt%, or 77wt% of MORPH. In some embodiments, the copolymer contains 15 wt% to 30 wt%, 15 wt% to 26 wt%, 20 wt% to 30 wt%, 20 wt% to 26 wt%, or 23 wt% of NIP.

[0130] In some embodiments, the copolymer contains about 80 wt% to about 99 wt%, about 85 wt% to about 98 wt%, about 88 wt% to about 96 wt%, about 90 wt% to about 95 wt%, or about 94 wt% of MORPH. In some embodiments, the copolymer contains about 2 wt% to about 15 wt%, about 4 wt% to about 15 wt%, about 4 wt% to about 10 wt%, about 5 wt% to about 10 wt%, or about 6 wt% of PHE. In some embodiments, the copolymer contains 80 wt% to 99 wt%, 85 wt% to 98 wt%, 88 wt% to 96 wt%, 90 wt% to 95 wt%, or 94 wt% of MORPH. In some embodiments, the copolymer contains 2 wt% to 15 wt%, 4 wt% to 15 wt%, 4 wt% to 10 wt%, 5 wt% to 10 wt%, or 6 wt% of PHE.

[0131] In some embodiments, the copolymer contains about 80 wt% to about 99 wt%, about 85 wt% to about 98 wt%, about 88 wt% to about 96 wt%, about 90 wt% to about 95 wt%, or about 92 wt% of MORPH. In some embodiments, the copolymer contains about 2 wt% to about 15 wt%, about 4 wt% to about 15 wt%, about 4 wt% to about 10 wt%, about 5 wt% to about 10 wt%, or about 8 wt% of PHE. In some embodiments, the copolymer contains 80 wt% to 99 wt%, 85 wt% to 98 wt%, 88 wt% to 96 wt%, 90 wt% to 95 wt%, or 92 wt% of MORPH. In some embodiments, the copolymer contains 2 wt% to 15 wt%, 4 wt% to 15 wt%, 4 wt% to 10 wt%, 5 wt% to 10 wt%, or 8 wt% of PHE.

[0132] In some embodiments, a degree of polymerization of the copolymer is about 10 to about 500, for example, about 10 to about 350, about 10 to about 200, about 15 to about 500, about 15 to about 350, about 15 to about 200, about 20 to about 500, about 20 to about 350, or about 20 to about 200. In some embodiments, a degree of polymerization of the copolymer is 10 to 500, for example, 10 to 350, 10 to 200, 15 to 500, 15 to 350, 15 to 200, 20 to 500, 20 to 350, or 20 to 200.

[0133] In some embodiments, a number-average molecular weight of the copolymer is about 2,000 g / mol to about 10,000 g / mol, for example, about 2,000 g / mol to about 7,500 g / mol, 2,000 g / mol to about 6,000 g / mol, about 2,000 g / mol to about 5,000 g / mol, about 3,000 g / mol to about 10,000 g / mol, about 3,000 g / mol to about 7,500 g / mol, about 3,000 g / mol to about 6,000 g / mol, or about 3,000 g / mol to about 5,000 g / mol. In some embodiments, a number-average molecular weight of the copolymer is 2,000 g / mol to 10,000 g / mol, for example, 2,000 g / mol to 7,500 g / mol, 2,000 g / mol to 6,000 g / mol, 2,000 g / mol to5,000 g / mol, 3,000 g / mol to 10,000 g / mol, 3,000 g / mol to 7,500 g / mol, 3,000 g / mol to 6,000 g / mol, or 3,000 g / mol to 5,000 g / mol.

[0134] In some embodiments, the composition includes about 0.1 wt% to about 10 wt% of the copolymer, for example, about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 2.5 wt%, about 0.25 wt% to about 10 wt%, about 0.25 wt% to about 5 wt%, about 0.25 wt% to about 2.5 wt%, about 0.5 wt% to about 10 wt%, about 0.5 wt% to about 5 wt%, or about 2.5 wt% of the copolymer. In some embodiments, the composition includes about 1 wt% of the copolymer. In some embodiments, the composition includes 0.1 wt% to 10 wt% of the copolymer, for example, 0.1 wt% to 5 wt%, 0.1 wt% to 2.5 wt%, 0.25 wt% to 10 wt%, 0.25 wt% to 5 wt%, 0.25 wt% to 2.5 wt%, 0.5 wt% to 10 wt%, 0.5 wt% to 5 wt%, or 2.5 wt% of the copolymer. In some embodiments, the composition includes 1 wt% of the copolymer.Pharmaceutical Compositions

[0135] As summarized above, this disclosure provides compositions of biopharmaceutical agent formulated with a polyacrylamide-based copolymer. The inventors have demonstrated that polyacrylamide-based copolymers can be used to provide a stable formulation, such as a lyophilized formulation of a biopharmaceutical agent, without altering its pharmacokinetics or bioavailability. This formulation can be applied to a variety of biopharmaceutical agents.

[0136] In some embodiments, the composition includes 5 wt% or more of the biopharmaceutical agent, for example, 7.5 wt% or more, 10 wt% or more, 11 wt% or more, 12 wt% or more, 12.5 wt% or more, 15 wt% or more, 20 wt% or more, or 25 wt% or more of the biopharmaceutical agent. In some embodiments, the composition includes 25 wt% or less, 20 wt% or less, or 15 wt% or less of the biopharmaceutical agent.

[0137] In some embodiments, the composition includes about 2.5 wt% to about 25 wt% of the biopharmaceutical agent, for example, about 2.5 wt% to about 20 wt%, about 2.5 wt% to about 15 wt%, or about 2.5 wt% to about 12.5 wt%, about 4 wt% to about 25 wt%, about 4 wt% to about 20 wt%, about 4 wt% to about 15 wt%, about 4 wt% to about 12.5 wt%, about 8 wt% to about 25 wt%, about 8 wt% to about 20 wt%, about 8 wt% to about 15 wt%, or about 8 wt% to about 12.5 wt% of the biopharmaceutical agent. In some embodiments, the composition includes about 5.5 wt%, or about 12 wt% of the biopharmaceutical agent. In some embodiments, the composition includes 2.5 wt% to 25 wt% of the biopharmaceutical agent, for example, 2.5 wt% to 20 wt%, 2.5 wt% to 15 wt%, or 2.5 wt% to 12.5 wt%, 4 wt%to 25 wt%, 4 wt% to 20 wt%, 4 wt% to 15 wt%, 4 wt% to 12.5 wt%, 8 wt% to 25 wt%, 8 wt% to 20 wt%, 8 wt% to 15 wt%, or 8 wt% to 12.5 wt% of the biopharmaceutical agent. In some embodiments, the composition includes about 5.5 wt% or about 12 wt% of the biopharmaceutical agent, such as 5.5 wt% or 12 wt% of the biopharmaceutical agent.

[0138] In some embodiments, the biopharmaceutical agent is an antibody and 70% or more of the antibody, for example, 75 wt% or more, 80 wt% or more, 85 wt% or more, or 90 wt% or more of the antibody is present in the composition in a monomeric state.

[0139] In some embodiments, the composition is a solid composition including a particular ratio by weight of biopharmaceutical agent to polyacrylamide-based copolymer in the solid composition, such as a ratio by weight of 2: 1 or higher. In some embodiments, the solid composition includes a ratio by weight of 5: 1 or higher, such as 10: 1 or higher of biopharmaceutical agent to polyacrylamide-based copolymer. In some embodiments, the ratio by weight of biopharmaceutical agent to polyacrylamide-based copolymer in the solid composition is in the range between 100: 1 and 2: 1, such as in the range between 90: 1 and 2: 1, 80: 1 and 2: 1, 70: 1 and 2: 1, 60: 1 and 2: 1, 50: 1 and 2: 1, 40: 1 and 2: 1, 30: 1 and 2: 1, 20: 1 and 2:1, 15: 1 and 2: 1, 10: 1 and 2: 1, or 5: 1 and 2: 1. In some embodiments, the polyacrylamide-based copolymer is MoNi.

[0140] In some embodiments, the composition is a solid composition including a stabilizer. In some embodiments, the stabilizer is a sugar. In some embodiments, the stabilizer is trehalose. In some embodiments, the composition is a solid composition including a particular ratio by weight of biopharmaceutical agent to stabilizer in the solid composition, such as a ratio by weight of 1 :2 or higher, or 1 : 1 or higher. In some embodiments, the ratio by weight of biopharmaceutical agent to stabilizer in the solid composition is in the range between 20: 1 and 1 :2, such as in the range between 20: 1 and 1 : 1, 10: 1 and 1 : 1, 9: 1 and 1 : 1, 8: 1 and 1 : 1, 7: 1 and 1 : 1, 6: 1 and 1 : 1, 5: 1 and 1 : 1, 4: 1 and 1 : 1, 3: 1 and 1 : 1, or 2:1 and 1 : 1.

[0141] In some embodiments, the composition is a solid composition including a particular ratio by weight of polyacrylamide-based copolymer to stabilizer in the solid composition, such as a ratio by weight of 1 :5 or higher, such as 1 : 10 or higher. In some embodiments, the stabilizer is a sugar. In some embodiments, the stabilizer is trehalose. In some embodiments, the ratio by weight of polyacrylamide-based copolymer to stabilizer in the solid composition is in the range between 1 : 100 and 1 :1, such as in the range between1 :50 and 1 : 1, 1 :30 and 1 : 1, 1 :20 and 1 : 1, or 1 :20 and 1 :5. In some embodiments, the polyacrylamide-based copolymer is MoNi.

[0142] In some embodiments, the composition is a solid composition including a particular ratio by weight of biopharmaceutical agent to stabilizer to polyacrylamide-based copolymer in the solid composition, such as a ratio by weight in the range between 20:20: 1 and 1 :1 : 1, such as 10: 10: 1. In some embodiments, the stabilizer is a sugar. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, the ratio by weight of biopharmaceutical agent to stabilizer to polyacrylamide-based copolymer in the solid composition is in the range between 20:20: 1 and 2:2: 1, such as in the range between 20: 10: 1 and 1 : 1 : 1, 20:5: 1 and 1 : 1 : 1, 10:20: 1 and 1 : 1 : 1, 5:20: 1 and 1 : 1 : 1, 10: 10:1 and 5:5: 1, or 20:20: 1 to 10: 10: 1. In some embodiments, the polyacrylamide-based copolymer is MoNi.

[0143] In some embodiments, the biopharmaceutical agent composition including the acrylamide-based copolymer (e.g., as described herein) is a pharmaceutical composition, and further includes a pharmaceutically acceptable excipient. The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be administered to, and effective in treating, a subject, and which contains no additional components which are unacceptably toxic to the subject.

[0144] The pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids; antimicrobials; antioxidants; buffers; chelating agents; complexing agents; monosaccharides; disaccharides and other carbohydrates; emulsifying agents; salt-forming counterions; preservatives; solvents; sugar alcohols; suspending agents; surfactants or wetting agents; stability enhancing agents; tonicity enhancing agents; delivery vehicles; diluents; other excipients and / or pharmaceutical adjuvants. Neutral buffered saline or saline mixed with conspecific serum albumin are examples of appropriate diluents. In accordance with appropriate industry standards, preservatives may also be added. The composition may be formulated as a lyophilizate using appropriate excipient solutions as diluents. Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components that may be employed in pharmaceutical formulations are presentedin Remington’s Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed. (2000), Mack Publishing Company, Easton, PA.

[0145] In some embodiments, the compositions described herein further include a surfactant. In some embodiments, the composition includes about 0.001 wt% to about 1 wt%, for example, about 0.001 wt% to about 0.5 wt%, about 0.001 wt% to about 0.1 wt%, about 0.001 wt% to about 0.05 wt%, about 0.005 wt% to about 0.5 wt%, about 0.005 wt% to about 0.1 wt%, about 0.005 wt% to about 0.05 wt%, about 0.01 wt% to about 0.5 wt%, about 0.01 wt% to about 0.1 wt%, or about 0.01 wt% to about 0.05 wt% of a surfactant. In some embodiments, the composition includes 0.001 wt% to 1 wt%, for example, 0.001 wt% to 0.5 wt%, 0.001 wt% to 0.1 wt%, 0.001 wt% to 0.05 wt%, 0.005 wt% to 0.5 wt%, 0.005 wt% to 0.1 wt%, 0.005 wt% to 0.05 wt%, 0.01 wt% to 0.5 wt%, 0.01 wt% to 0.1 wt%, or 0.01 wt% to 0.05 wt% of a surfactant. In some embodiments, the surfactant includes a poloxamer. In some embodiments, the surfactant includes a polysorbate. In some embodiments, the polysorbate is polysorbate 80 (PS80) or polysorbate 20 (PS20). In some embodiments, the composition includes about 0.01 wt% of polysorbate 80. In some embodiments, the composition includes about 0.01 wt% of polysorbate 80, such as 0.01 wt% of polysorbate 80.

[0146] In some embodiments, the compositions described herein further include a stabilizer. In some embodiments, the stabilizer includes a sugar. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the composition includes about 0.1 wt% to about 50 wt%, for example, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 12.5 wt%, about 2 wt% to about 25 wt%, about 2 wt% to about 15 wt%, about 2 wt% to about 12.5 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 15 wt%, or about 5 wt% to about 12.5 wt% of the stabilizer. In some embodiments, the composition includes 0.1 wt% to 25 wt%, for example, 0.1 wt% to 15 wt%, 0.1 wt% to 12.5 wt%, 2 wt% to 25 wt%, 2 wt% to 15 wt%, 2 wt% to 12.5 wt%, 5 wt% to 25 wt%, 5 wt% to 15 wt%, or 5 wt% to 12.5 wt% of the stabilizer. In some embodiments, the composition includes about 15 wt% to 25 wt%, about 15 wt% to 30 wt%, about 15 wt% to 35 wt%, about 15 wt% to 40 wt%, about 15 wt% to 45 wt%, about 15 wt% to 50 wt%, about 15 wt% to 55 wt% of the stabilizer. In some embodiments, the composition includes about 9 wt% of sucrose. In some embodiments, the composition includes about 9 wt% of sucrose, such as 9 wt% of sucrose. In some embodiments, the composition includes trehalose.

[0147] A liquid composition further comprises a buffer to maintain pH of the pharmaceutical composition. The buffer can include a buffer compound known in the art,such as TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MPOS, PIPES, Cacodylate, histidine (e.g., L-histidine hydrochloride), or MES. The buffer can contain citric acid, monopotassium phosphate, boric acid, or diethyl barbituric acid. The buffer can be PBS, HEPES, TRIS or TRISZEDTA buffer. The buffer can be other phosphate buffer. Phosphate buffers can comprise a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate.

[0148] Specifically, the buffer can be potassium phosphate buffer. The potassium phosphate buffer can comprise potassium phosphate at a concentration from 5 mM to 15 mM, from 7.5 mM to 12.5 mM, from 9 mM to 11 mM, or at 10 mM. The buffer contained in the liquid composition can have pH from 7 to 9. In some embodiments, the pH is from 7.0 to 8.5 or 8.0. In some embodiments, the pH is from 7.0 to 8.0. In some embodiments, the liquid composition comprises a 7.5-12 mM potassium phosphate buffer of pH from 7 to 9. In some embodiments, the liquid composition comprises a 7.5-12 mM potassium phosphate buffer of pH from 7.0 to 8.0. In some embodiments, the liquid composition comprises 9-11 mM potassium phosphate buffer of pH from 7.0 to 8.5. In some embodiments, the liquid composition includes 10 mM potassium phosphate buffer at pH 8.0.

[0149] In some embodiments, the liquid composition has a buffer concentration of about 1 mM to about 100 mM, for example, about 1 mM to about 75 mM, about 1 mM to about 50 mM, about 5 mM to about 100 mM, about 5 mM to about 75 mM, about 5 mM to about 50 mM, about 10 mM to about 100 mM, about 10 mM to about 75 mM, or about 10 mM to about 50 mM. In some embodiments, the composition has a buffer concentration of 1 mM to 100 mM, for example, 1 mM to 75 mM, 1 mM to 50 mM, 5 mM to 100 mM, 5 mM to 75 mM, 5 mM to 50 mM, 10 mM to 100 mM, 10 mM to 75 mM, or 10 mM to 50 mM. In some embodiments, the buffer includes phosphate, citrate, acetate, TRIS, succinate, other organic acids, or histidine, or a salt thereof. In some embodiments, the buffer includes one or more phosphate salts. In some embodiments, the buffer includes sodium phosphate. In some embodiments, the composition includes an acetate buffer.

[0150] The term “buffer exchange”, as used herein, refers to a technique wherein a biopharmaceutical agent is transferred from one buffer system to another through filtration and centrifugation processes.

[0151] The liquid composition can further contain a salt. The salt can be NaCl or KC1. In some embodiments, the liquid composition comprises a salt at a concentration greater than0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, greater than 0.4% and 0.5% or at a concentration of 0.45%. In some embodiments, the liquid composition comprises a salt at a concentration from 0.1% to 0.9%, from 0.1% to 0.6%, from 0.25% to 0.75%, from 0.4% to 0.6%, from 0.4% to 0.5% or at a concentration of 0.45%.

[0152] In some embodiments, the liquid composition comprises NaCl at a concentration greater than 0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, greater than 0.4% and 0.5% or at a concentration of 0.45%. In some embodiments, the liquid composition comprises NaCl at a concentration from 0.1% to 0.9%, from 0.1% to 0.6%, from 0.25% to 0.75%, from 0.4% to 0.6%, from 0.4% to 0.5% or at a concentration of 0.45%.

[0153] In some embodiments, the liquid composition comprises KC1 at a concentration greater than 0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, greater than 0.4% and 0.5% or at a concentration of 0.45%. In some embodiments, the liquid composition comprises KC1 at a concentration from 0.1% to 0.9%, from 0.25% to 0.75%, from 0.4% to 0.6%, from 0.4% to 0.5% or at a concentration of 0.45%.

[0154] In some embodiments, the liquid composition is an aqueous formulation. In certain embodiments, such aqueous formulations have a pH of 3 to 7.5, such as a pH of 4 to 6.5.

[0155] The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See for example, Remington’s Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. For example, suitable compositions may be water for injection, physiological saline solution for parenteral administration.

[0156] Another aspect of the present disclosure is a solid pharmaceutical composition in a unit dose. In some aspects of the present disclosure, a lyophilized pharmaceutical composition is in a unit dose form. In some embodiments, the unit dosage form is a vial, ampule, bottle, or pre-filled syringe. In typical embodiments, the unit dosage form is a vial containing an amount of the lyophilized pharmaceutical composition suitable for administration after reconstitution. The administration can include subcutaneous, intradermal,or intramuscular administration using preloaded syringes, auto-injectors, and auto-inject pens, each containing a predetermined amount of the pharmaceutical composition described herein.

[0157] The unit dose in a container can be determined based on various factors, such as an active ingredient (e.g., biopharmaceutical agent), a disease to be treated, a subject, a route and method of administration. The unit dose can be determined based on in vitro or in vivo studies, including clinical trials.

[0158] The unit dose in a container can be sealed and stored for an extended period of time at various temperatures (e.g., room temperature to about -180° C, preferably about 2-8° C to about -80° C, more preferably about -20° C to about -80° C, and most preferably about -20° C).

[0159] The dried compositions, e.g., the lyophilized compositions, stored in the container are preferably stable within a range of from about 2-20° C to about -80° C, for a period of at least 1 month, 3 months, 6 months or 1 year without losing significant activity.

[0160] In some embodiments, the reconstituted pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the reconstituted pharmaceutical composition is prepared according to the reconstitution method described herein. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient can be in the form of a parenterally acceptable aqueous formulation, such as a solution, which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable aqueous formulations using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.Methods

[0161] Aspects of the present disclosure include a method for preparing a solid composition. In some embodiments, a method for preparing a solid composition comprises exposing a liquid biopharmaceutical composition to a drying cycle. In some embodiments, the solid composition is a lyophilized composition. In some embodiments, the drying is selected from lyophilization, spray drying, spray freezing drying, foam mat drying, vacuum oven drying, and desiccation drying. In some embodiments, the liquid biopharmaceutical composition is exposed to a lyophilization cycle in a lyophilizer to produce a lyophilized composition.

[0162] In some embodiments, the lyophilization cycle comprises subjecting the liquid biopharmaceutical composition to a first pressure. In some embodiments, the lyophilization cycle comprises evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy. In some embodiments, the lyophilization cycle comprises cooling the liquid biopharmaceutical composition to freeze any remaining liquid component into a solid.

[0163] In some embodiments, the lyophilization cycle comprises subjecting the liquid biopharmaceutical composition to a first reduced temperature. In some embodiments, the lyophilization cycle further comprises subjecting the liquid biopharmaceutical composition to a first reduced pressure. In some embodiments, the lyophilization cycle further comprises evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy. In some embodiments, the lyophilization cycle further comprises subjecting the liquid biopharmaceutical composition to a second reduced pressure that is less than the first reduced pressure. In some embodiments, the lyophilization cycle further comprises evaporating the remainder of the liquid to produce the solid composition.

[0164] In some embodiments, the drying cycle is a spray drying cycle. In some embodiments, the spray drying cycle comprises atomizing the liquid biopharmaceutical composition. In some embodiments, the spray drying cycle comprises further evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition. In some embodiments, the spray drying cycle further comprises collecting the solid composition.

[0165] In some embodiments, the drying cycle is a spray freeze drying cycle. In some embodiments, a spray freeze drying cycle comprises atomizing the liquid biopharmaceutical composition. In some embodiments, a spray freeze drying cycle further comprises freezing the atomized liquid biopharmaceutical composition. In some embodiments, a spray freeze drying cycle further comprises evaporating at least a portion of a liquid component of the atomized liquid biopharmaceutical composition. In some embodiments, a spray freeze drying cycle further comprises collecting the solid composition.

[0166] In some embodiments, the drying cycle is a foam mat drying cycle. In some embodiments, the foam mat drying cycle comprises foaming the liquid biopharmaceutical composition. In some embodiments, the foam mat drying cycle further comprises spreading the foamed liquid biopharmaceutical composition onto a drying surface. In someembodiments, the foam mat drying cycle further comprises evaporating at least a portion of a liquid component of the foamed liquid biopharmaceutical composition. In some embodiments, the foam mat drying cycle further comprises collecting the solid composition.

[0167] In some embodiments, the drying is a vacuum oven drying cycle. In some embodiments, the vacuum oven drying cycle comprises subjecting the liquid biopharmaceutical composition to reduced pressure. In some embodiments, the vacuum oven drying cycle further comprises evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy. In some embodiments, the vacuum oven drying cycle further comprises returning the pressure to the original pressure to obtain the solid composition.

[0168] In some embodiments, the drying is a desiccation drying cycle. In some embodiments, the desiccation drying cycle comprises increasing the surface area of the liquid biopharmaceutical composition. In some embodiments, the surface area of the liquid biopharmaceutical composition is increased by pouring the liquid biopharmaceutical composition into a tray. In some embodiments, the surface area of the liquid biopharmaceutical composition is increased by pouring the liquid biopharmaceutical composition into a shallow tray. In some embodiments, the desiccation drying cycle further comprises evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition. In some embodiments, evaporating of the liquid component is performed by circulating ambient air across the surface. In some embodiments, the circulated air is optionally dried over a desiccating material, heated, or both until the solid is obtained.

[0169] For use and administration to a subject, a stabilized and dried biopharmaceutical agent composition as provided herein can be reconstituted by rehydration with an aqueous solution. In some embodiments, a stabilized and lyophilized biopharmaceutical composition is reconstituted by rehydration with a solvent. Such solvents are typically water, such as demineralized or distilled water, water for injection, etc., but can also include physiological solutions or buffers such as phosphate buffered saline (PBS), or adjuvants.

[0170] Aspects of the present disclosure include a method for reconstituting the solid composition that includes adding an aqueous solution for reconstitution to the solid composition. In some embodiments, the method includes dissolving the solid composition in the aqueous solution for reconstitution. In some embodiments, the solid composition forreconstituting is a lyophilized composition. The methods of reconstituting should be controlled in various ways to maintain biopharmaceutical agent stability and activity upon reconstitution. These methods may also vary depending on the composition of the formulation and the biopharmaceutical agent used. In some embodiments, the solution for reconstitution is sterile water. In some embodiments, the solution for reconstitution includes water for injection. In some embodiments, the solution for reconstitution further includes a preservative. In some embodiments, the aqueous solution includes a buffering agent.

[0171] Aspects of the present disclosure include reconstituted solutions of biopharmaceutical agent that retain the biological activity of the biopharmaceutical agent relative to a parent solution. In some embodiments, the reconstituted solution of biopharmaceutical agent is free or substantially free of particulates. In some embodiments, the method includes isolating and / or removing an amount of particulates in the reconstituted composition. In some embodiments, the method further includes centrifuging the reconstituted composition, and optionally decanting a homogeneous solution from the isolated particles or particulate from the reconstituted composition.

[0172] Aspects of the present disclosure include a method for measuring the reconstitution time of the solid composition is provided. In some embodiments, the method for measuring the reconstitution time of the solid composition of present disclosure comprises the steps of (a) subjecting the solid composition to reduced first temperature; (b) equilibrating the solid composition to elevated second temperature; (c) dissolving the solid composition in the aqueous solution for reconstitution; (d) agitating the solution for reconstitution until the dissolution of particulates; and (e) assessing the time for the dissolution of particulates. In some embodiments, the method comprises the steps of (a) cooling the solid composition to between -50 °C and 10 °C, (b) equilibrating the solid composition to between 20 °C and 25 °C, (c) dissolving the solid composition in the aqueous solution for reconstitution; (d) agitating the solution for reconstitution until the particulates dissolve; and (e) assessing the time to dissolve the particulates. In some embodiments, In some embodiments, the solid composition is initially cooled to between -50 °C and 10 °C, for example, between -50 °C to -40 °C, between from -50 °C to -30 °C, between -50 °C to -20 °C, between -50 °C to -10 °C, between -50 °C to 0 °C, between -50 °C to 10 °C, between -40 °C to -30 °C, between -40 °C to -20 °C, between -40 °C to -10 °C, between -40 °C to 0 °C, between -40 °C to 10 °C, between -30 °C to -20 °C, between -30 °C to -10 °C, between -30 °C to 0 °C, between -30 °C to 10 °C, between -20 °C to -10 °C, between -20 °C to 0 °C, between -20 °C to 10°C, between -10 °C to 0 °C, between -10 °C to 10 °C, between 0 °C to 10 °C. In some embodiments, the solid composition is initially cooled to between 2 °C to 8 °C. In some embodiments, the solid composition is equilibrated to between 20 °C and 25 °C, for example, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C.

[0173] In some embodiments, the solid pharmaceutical composition is a lyophilized pharmaceutical composition. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized cake that, can dissolve in an aqueous solution during reconstitution.

[0174] In some embodiments, the reconstitution time is between 0 and 30 min, about 1 min, about 2 min, about 3 min, about 4 min, about 5 min, about 6 min, about 7 min, about 8 min, about 9 min, about 10 min, about 11 min, about 12 min, about 13 min, about 14 min, about 15 min, about 16 min, about 17 min, about 18 min, about 19 min, about 20 min, about 21 min, about 22 min, about 23 min, about 24 min, about 25 min, about 26 min, about 27 min, about 28 min, about 29 min, about 30 min.

[0175] In some embodiments, the formulation reconstituted by the method includes the biopharmaceutical agent at a therapeutically effective concentration and amount (e.g., as described herein). In some embodiments, the concentration of biopharmaceutical agent is 5 wt% or more of the composition, for example, 7, 10, 12, 15, 20, or 25 wt% or more; or 5, 7, 10, or 12 wt% to 15, 20, or 25 wt%, such as 5-25 wt%, 5-20 wt%, 5-15 wt%, 10-25 wt%, 10- 20 wt%, or 15-25 wt%. In some embodiments, the biopharmaceutical agent is a therapeutic antibody.

[0176] Aspects of this disclosure include methods of using the compositions that include administering a biopharmaceutical agent reconstituted composition (e.g., as described herein) to a subject in need thereof. The reconstituted lyophilized composition of the present disclosure may be administered by various delivery methods, e.g., orally or via parenteral routes. In some embodiments, administration is via intravenous injection. In some embodiments, administration is via subcutaneous injection. In some embodiments, the reconstituted composition administered includes a therapeutically effective amount of a therapeutic biopharmaceutical agent of interest.

[0177] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, apes, dogs, cats, mice, rats, cows, horses, camels, goats, burros, rabbits, and sheep. In certain embodiments, the subject is a human. The reconstitutedbiopharmaceutical agent of the present disclosure can be administered to a mammalian subject to treat various diseases. In some embodiments, the subject has a disease or condition that can be treated with a biopharmaceutical agent as described herein.

[0178] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed during the course of clinical pathology. Desirable effects of treatment can include one or more of reducing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the biopharmaceutical agent is administered to a subject in a reconstituted composition as described herein.

[0179] The term “therapeutically effective amount” or “effective amount” refers to an amount of a biopharmaceutical agent that, when administered to a subject, is effective to treat a disease or disorder. The exact dose or amount will depend on the purpose of the treatment and will typically be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0180] A composition reconstituted for ready injection can be administered to a subject by parenteral or mucosal injection, preferably intramuscular or subcutaneous injection. However, administration of a composition reconstituted for ready injection as described above can also include intranasal, transdermal, topical, and oral administration. The dose per injection may be from about 0.1 ml to about 2.0 ml, preferably about 1.0 ml.

[0181] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. In typical embodiments, the biopharmaceutical agent is administered in an amount effective to reduce symptoms of the disease of interest. In some embodiments, the amount is effective to reduce the symptom within 1 week of administration. In some embodiments, the amount is effective to reduce the symptom within 2 weeks, 3 weeks, or 4 weeks of administration.Definitions

[0182] In this disclosure, the terms “a,” “an,” and “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” has thesame meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed in this disclosure, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0183] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (for example, 1%, 2%, 3%, and 4%) and the subranges (for example, 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0184] The term “about” as used in the present disclosure can allow for a degree of variability in a value or range that is within 5% of a stated value or of a stated limit of a range.

[0185] In the methods described in the present disclosure, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0186] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according toconventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0187] The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, CH2, and CH3. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0188] The term “antigen-binding protein” (ABP) refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP comprises an antibody fragment.

[0189] The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope.

[0190] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region.

[0191] The term “Fc region” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region, or an Fc region modified as described elsewhere in this disclosure.

[0192] The VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0193] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the sequence of its constant domain.

[0194] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, a, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0195] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732- 745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77(“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[0196] Table 1 below provides the positions of CDR1-L (CDR1 of VL), CDR2-L (CDR2 of VL), CDR3-L (CDR3 of VL), CDR1-H (CDR1 of VH), CDR2-H (CDR2 of VH), and CDR3-H (CDR3 of VH), as identified by the Kabat and Chothia schemes. For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0197] CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at www.bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.The C-terminus of CDR1-H, when numbered using the Kabat numbering convention, varies between 32 and 34, depending on the length of the CDR.

[0198] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra).

[0199] An “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0200] “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0201] “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CHI) of theheavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0202] “F(ab’)2” fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds. F(ab’)2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab’) fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0203] “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Pliickthun A. (1994). In some embodiments, n = 1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.

[0204] “ scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, anFc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH -VL or VL - VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0205] The term “single domain antibody” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety.

[0206] A “monospecific ABP” is an ABP that comprises a binding site that specifically binds to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule which, while divalent, recognizes the same epitope at each antigen-binding domain. The binding specificity may be present in any suitable valency.

[0207] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality ofantibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0208] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0209] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321 :522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0210] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies. In some embodiments, rodents are genetically engineered to replace their rodent antibody sequences with human antibodies.

[0211] An “immunoconjugate” is an ABP conjugated to one or more heterologous molecule(s). In some embodiments, the immunoconjugate is an antibody conjugated to one or more heterologous molecule(s), i.e., an antibody-drug conjugate. The heterologous molecule can be a small molecule. In some embodiments, the heterologous molecule is a cytotoxic agent, a chemotherapeutic agent or a cytostatic agent.

[0212] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction.

[0213] A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.

[0214] The term “cytostatic agent” refers to a compound or composition which arrests growth of a cell either in vitro or in vivo. In some embodiments, a cytostatic agent is an agent that reduces the percentage of cells in S phase. In some embodiments, a cytostatic agent reduces the percentage of cells in S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

[0215] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer.

[0216] A “variant” of a polypeptide (e.g., an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to the native polypeptide sequence, and retains essentially the same biological activity as the native polypeptide. The biological activity of the polypeptide can be measured using standard techniques in the art (for example, if the variant is an antibody, its activity may be tested by binding assays, as described herein). Variants of the present disclosure include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0217] A “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0218] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., ABP and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0219] With regard to the binding of an ABP to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule.

[0220] The term “polymer” refers to a substance or material consisting of repeating monomer subunits. The term “co-polymer” refers to a polymer composed of two or more distinct repeating monomer subunits.ADDITIONAL EMBODIMENTS

[0221] The disclosure is further described by the following non-limiting clauses:

[0222] Clause 1. A composition comprising: a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from A-(3-methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), A,A-dimethylacrylamide (DMA), N- hydroxyethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from Y- [tri s(hydroxymethy Ilin ethyl ]acryl ami de (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), A-isopropyl acrylamide (NIP), N-N-di ethyl acrylamide (DEA), A-tert-butylacrylamide (TBA), / ' / -phenyl acrylamide (PEE), and combinations thereof; and a biopharmaceutical agent (e.g., an antibody).

[0223] Clause 2. The composition of clause 1, wherein the water-soluble carrier monomer is selected from MORPH, MP AM, and combinations thereof.

[0224] Clause 3. The composition of clause 1, wherein the water-soluble carrier monomer comprises MORPH.

[0225] Clause 4. The composition of clause 1, wherein the water-soluble carrier monomer comprises MP AM.

[0226] Clause 5. The composition of any one of clauses 1-4, wherein the functional dopant monomer is selected from AMP, TMA, TBA, PEE, and combinations thereof.

[0227] Clause 6. The composition of any one of clauses 1-4, wherein the functional dopant monomer is selected from DEA, PEE, NE, and combinations thereof.

[0228] Clause 7. The composition of any one of clauses 1-4, wherein the functional dopant monomer comprises TRI.

[0229] Clause 8. The composition of any one of clauses 1-4, wherein the functional dopant monomer comprises PEE.

[0230] Clause 9. The composition of any one of clauses 1-4, wherein the functional dopant monomer comprises NE.

[0231] Clause 10. The composition of any one of clauses 1-4, wherein the functional dopant monomer comprises DEA.

[0232] Clause 11. The composition of clause 1, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from NE, PEE, and combinations thereof.

[0233] Clause 12. The composition of clause 1, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; andthe functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

[0234] Clause 13. The composition of clause 1, wherein the water-soluble carrier monomer is MP AM, and the functional dopant monomer is PHE.

[0235] Clause 14. The composition of clause 1, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is PHE.

[0236] Clause 15. The composition of clause 1, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is NIP.

[0237] Clause 16. The composition of any one of clauses 1-15, wherein the copolymer comprises: 70 wt% to 98 wt% of the water-soluble carrier monomer; and 2 wt% to 30 wt% of the functional dopant monomer.

[0238] Clause 17. The composition of any one of clauses 1-15, wherein the copolymer comprises: 80 wt% to 95 wt% of the water-soluble carrier monomer; and 5 wt% to 20 wt% of the functional dopant monomer.

[0239] Clause 18. The composition of any one of clauses 1-15, wherein the copolymer comprises: 83 wt% to 98 wt% of the water-soluble carrier monomer; and 2 wt% to 17 wt% of the functional dopant monomer.

[0240] Clause 19. The composition of clause 1, wherein the copolymer comprises:70 wt% to 85 wt% of MORPH; and 15 wt% to 30 wt% of NIP.

[0241] Clause 20. The composition of clause 1, wherein the copolymer comprises:74 wt% to 80 wt% of MORPH; and 20 wt% to 26 wt% of NIP.

[0242] Clause 21. The composition of clause 1, wherein the copolymer comprises:77 wt% of MORPH; and 23 wt% of NIP.

[0243] Clause 22. The composition of any one of clauses 1-21, wherein a degree of polymerization of the copolymer is 10 to 500.

[0244] Clause 23. The composition of any one of clauses 1-21, wherein a degree of polymerization of the copolymer is 20 to 200.

[0245] Clause 24. The composition of any one of clauses 1-23, wherein a number-average molecular weight of the copolymer is 2,000 g / mol to 10,000 g / mol.

[0246] Clause 25. The composition of any one of clauses 1-23, wherein a number-average molecular weight of the copolymer is 2,000 g / mol to 6,000 g / mol.

[0247] Clause 26. The composition of any one of clauses 1-23, wherein a number-average molecular weight of the copolymer is 3,000 g / mol to 5,000 g / mol.

[0248] Clause 27. The composition of any one of clauses 1-26 comprising 0.1 wt% to 10 wt% of the copolymer.

[0249] Clause 28. The composition of any one of clauses 1-26, comprising 0.5 wt% to 5 wt% of the copolymer.

[0250] Clause 29. The composition of any one of clauses 1-28, comprising 1 wt% of the copolymer.

[0251] Clause 30. The composition of any one of clauses 1-29, wherein the composition further comprises a surfactant.

[0252] Clause 31. The composition of clause 30, comprising 0.001 wt% to 0.5 wt% of the surfactant.

[0253] Clause 32. The composition of clause 30, comprising 0.005 wt% to 0.05 wt% of the surfactant.

[0254] Clause 33. The composition of any one of clauses 30-32, wherein the surfactant comprises a polysorbate.

[0255] Clause 34. The composition of clause 33, wherein the polysorbate is polysorbate 80.

[0256] Clause 35. The composition of clause 34, comprising 0.005 wt% to 0.05 wt% of polysorbate 80.

[0257] Clause 36. The composition of clause 34, comprising 0.01 wt% of polysorbate 80.

[0258] Clause 37. The composition of any one of clauses 1-36, wherein the composition further comprises a stabilizer.

[0259] Clause 38. The composition of clause 37, comprising 0.1 wt% to 25 wt% of the stabilizer.

[0260] Clause 39. The composition of clause 38, comprising 2 wt% to 15 wt% of the stabilizer.

[0261] Clause 40. The composition of any one of clauses 37-39, wherein the stabilizer comprises an oligosaccharide.

[0262] Clause 41. The composition of clause 40, wherein the oligosaccharide is sucrose.

[0263] Clause 42. The composition of clause 40, comprising 2 wt% to 15 wt% of sucrose.

[0264] Clause 43. The composition of clause 40, comprising 9 wt% of sucrose.

[0265] Clause 44. The composition of any one of clauses 1-43, comprising 5 wt% or more of the antibody.

[0266] Clause 45. The composition of clause 44, comprising 10 wt% or more of the antibody.

[0267] Clause 46. The composition of clause 44 or 45, comprising 20 wt% or less of the antibody.

[0268] Clause 47. The composition of any one of clauses 1-43, comprising 2.5 wt% to 20 wt% of the antibody.

[0269] Clause 48. The composition of clause 47, comprising 2.5 wt% to 15 wt% of the antibody.

[0270] Clause 49. The composition of clause 47, comprising 8 wt% to 20 wt% of the antibody.

[0271] Clause 50. The composition of clause 49, comprising 12 wt% to 20 wt% of the antibody.

[0272] Clause 51. The composition of any one of clauses 1-50, wherein the antibody has a molecular weight of 100 kDa to 200 kDa.

[0273] Clause 52. The composition of clause 51, wherein the antibody has a molecular weight of 120 kDa to 180 kDa.

[0274] Clause 53. The composition of any one of clauses 1-50, wherein the antibody has a molecular weight of 50 kDa to 100 kDa.

[0275] Clause 54. The composition of any one of clauses 1-53, wherein the antibody is a monoclonal antibody.

[0276] Clause 55. The composition of any one of clauses 1-53, wherein the antibody is a chimeric antibody.

[0277] Clause 56. The composition of any one of clauses 1-53, wherein the antibody is a bispecific antibody.

[0278] Clause 57. The composition of any one of clauses 1-53, wherein the antibody is a full-length antibody.

[0279] Clause 58. The composition of any one of clauses 1-53, wherein the antibody is an antibody fragment.

[0280] Clause 59. The composition of any one of clauses 1-53, wherein the antibody is an antibody-drug conjugate.

[0281] Clause 60. The composition of clause 1, comprising:0.1 wt% to 10 wt% of a polyacrylamide-based copolymer comprising:70 wt% to 98 wt% of a water-soluble carrier monomer selected from MP AM, MORPH, DMA, HEAM, AM, and combinations thereof; and2 wt% to 30 wt% of a functional dopant monomer selected from TRI, AMP, TMA, NIP, TBA, PHE, and combinations thereof; and5 wt% or more of the antibody.

[0282] Clause 61. The composition of clause 60, comprising 10 wt% or more(e.g., 12 wt % to 20 wt%, such as 12 wt%) of the antibody.

[0283] Clause 62. The composition of clause 60 or 61, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from NIP, PHE, and combinations thereof.

[0284] Clause 63. The composition of any one of clauses 60-62, wherein: the water-soluble carrier monomer is MORPH; and the functional dopant monomer is NIP.

[0285] Clause 64. The composition of clause 63, wherein the composition comprises: 77 wt% of MORPH; and 23 wt% of NIP.

[0286] Clause 65. The composition of any one of clauses 60-64, comprising 1 wt% of the polyacrylamide-based copolymer.

[0287] Clause 66. The composition of any one of clauses 60-65, further comprising 0.001 wt% to 0.5 wt% of a surfactant.

[0288] Clause 67. The composition of any one of clauses 63-66, further comprising 0.1 wt% to 25 wt% of a stabilizer.

[0289] Clause 68. The composition of any one of clauses 1-67, wherein the composition is aqueous.

[0290] Clause 69. The composition of clause 68, wherein the composition has a pH of 3 to 7.5.

[0291] Clause 70. The composition of clause 69, wherein the composition has a pH of 4 to 6.5.

[0292] Clause 71. The composition of clause 69 or 70, further comprising:0.01 wt% to 0.5 wt% of a surfactant (e.g., 0.01 wt% of polysorbate 80);0.1 wt% to 25 wt% of a stabilizer (e.g., 9 wt% of sucrose); and a buffer (e.g., 20 mM acetate buffer).

[0293] Clause 72. The composition of any one of clauses 1-71, wherein at least 70% of the antibody is present in the composition in a monomeric state.

[0294] Clause 73. The composition of clause 72, wherein at least 90% of the antibody is present in the composition in a monomeric state.

[0295] Clause 74. The composition of any one of clauses 1-73, wherein the composition is a liquid having a surface tension of 60 mN / m or less.

[0296] Clause 75. The composition of any one of clauses 1-74, wherein the composition has an interfacial complex viscosity of 0.2 Pa s m or less.

[0297] Clause 76. The composition of any one of clauses 1-75, wherein the composition has a viscosity of 6 mPa s or less at shear rates of 0 s'1to 3,000 s'1.

[0298] Clause 77. The composition of any one of clauses 1-76, wherein the composition is capable of retaining 70% or more of a binding activity of the antibody for atarget antigen over 21 days of continuous stressed aging, based upon half maximal inhibitory concentration as determined by an enzyme-linked immunosorbent assay.

[0299] Clause 78. The composition of any one of clauses 1-77, wherein the composition is formulated for subcutaneous administration.

[0300] Clause 79. The composition of any one of clauses 1-77, wherein the composition comprises a suspension of particles in a liquid carrier.

[0301] Clause 80. The composition of clause 79, wherein the liquid carrier is nonaqueous.

[0302] Clause 81. The composition of clause 79 or 80, wherein the particles comprise the polyacrylamide-based copolymer and the antibody.

[0303] Clause 82. A method of administering an antibody to a subject in need thereof, comprising injecting the subject with a therapeutically effective amount of a composition according to any one of clauses 1-81.

[0304] Clause 83. The method of clause 82, wherein the injection is subcutaneous.

[0305] Clause 84. A composition according to any one of clauses 1-81 for use in subcutaneous administration of a biopharmaceutical agent (e.g., an antibody) to a subject.

[0306] Clause 85. Use of a composition according to any one of clauses 1-81 for the manufacture of a medicament for use in subcutaneous administration of a biopharmaceutical agent (e.g., an antibody) to a subject.

[0307] Clause 86. A storage stable solid composition comprising: a stabilizer; a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from N-(3-methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N- hydroxyethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from N-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2 -methylpropane sulfonic acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide(NIP), N-N-di ethyl acrylamide (DEA), N-tert-butyl acrylamide (TBA), N- phenyl acrylamide (PHE), and combinations thereof; and a biopharmaceutical agent.

[0308] Clause 87. The solid composition of clause 86, wherein the biopharmaceutical agent is not an antibody or antibody fragment.

[0309] Clause 88. The solid composition of clause 86 or 87, wherein the composition is capable of dissolution with water to an aqueous composition comprising:0.1 wt% to about 10 wt% of the copolymer; and0.5 wt% to 25 wt% of the biopharmaceutical agent.

[0310] Clause 89. The solid composition of any one of clauses 86 to 88, wherein the solid composition (e.g., lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition that comprises, prior to drying (e.g., lyophilization), 0.1 mg / mL or more of the polyacrylamide-based copolymer.

[0311] Clause 90. The solid composition of clause 89, wherein the liquid composition comprises 0.3 mg / mL or more of the polyacrylamide-based copolymer.

[0312] Clause 91. The solid composition of clause 90, wherein the liquid composition comprises 1.0 mg / mL or more of the polyacrylamide-based copolymer.

[0313] Clause 92. The solid composition of claim 91, wherein the liquid composition comprises 3 mg / mL or more of the polyacrylamide-based copolymer.

[0314] Clause 93. The solid composition of clause 92, wherein the liquid composition comprises 10 mg / mL or more of the polyacrylamide-based copolymer.

[0315] Clause 94. The solid composition of any one of clauses 86 to 93, wherein the biopharmaceutical agent is selected from a vaccine, gene therapy, cell therapy, and lipid nanoparticle (LNP) comprising a nucleic acid.

[0316] Clause 95. The solid composition of clause 94, wherein the biopharmaceutical agent is a vaccine.

[0317] Clause 96. The solid composition of clause 95, wherein the vaccine comprises a live or attenuated virus.

[0318] Clause 97. The solid composition of clause 96, wherein the vaccine is a mRNA vaccine.

[0319] Clause 98. The solid composition of any one of clauses 86 to 97, wherein the solid composition demonstrates higher thermostability after 14 days at 37 °C as compared to a solid composition lacking the polyacrylamide-based copolymer.

[0320] Clause 99. The solid composition of any one of clauses 86 to 98, wherein the biopharmaceutical agent is a polypeptide.

[0321] Clause 100. The solid composition of clause 99, wherein the polypeptide is a therapeutic protein.

[0322] Clause 101. The solid composition of clause 99 or 100, wherein the polypeptide is selected from antibodies and fragments thereof, antibody-drug conjugates, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof.

[0323] Clause 102. The solid composition of clause 101, wherein the therapeutic protein is selected from antibody or fragment thereof, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, enzyme, growth factor, hormone, interferon, interleukin, and thrombolytics protein.

[0324] Clause 103. The solid composition of clause 102, wherein the therapeutic protein is an enzyme.

[0325] Clause 104. The solid composition of clause 103, wherein the therapeutic protein is an antibody or a fragment thereof.

[0326] Clause 105. The solid composition of clause 104, wherein the therapeutic protein is a monoclonal antibody, a polyclonal antibody, an immunoglobulin G (IgG) antibody, an IgA antibody, an IgM antibody, a Fc fusion protein, or a fragment thereof.

[0327] Clause 106. The solid composition of clause 105, wherein the therapeutic protein is a monoclonal antibody.

[0328] Clause 107. The solid composition of any one of clauses 103-105, wherein the solid composition (e.g., lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition that comprises, prior to drying (e.g., lyophilization), 1.0 mg / mL or more of the polyacrylamide-based copolymer (e.g., 2 mg / mL or more, 3 mg / mL or more, or 4 mg / mL or more).

[0329] Clause 108. The solid composition of clause 107, wherein the liquid composition comprises 10 mg / mL or more of the polyacrylamide-based copolymer.

[0330] Clause 109. The solid composition of any one of clauses 99 to 108, wherein the solid composition demonstrates storage stability as compared to a composition lacking the polyacrylamide-based copolymer.

[0331] Clause 110. The solid composition of any one of clauses 99 to 108, wherein the solid composition demonstrates a shorter reconstitution time as compared to a composition lacking the polyacrylamide-based copolymer.

[0332] Clause 111. The solid composition of any one of clauses 86 to 93, wherein the water-soluble carrier monomer is selected from MORPH, MP AM, and combinations thereof.

[0333] Clause 112. The solid composition of clause 111, wherein the water-soluble carrier monomer comprises MORPH.

[0334] Clause 113. The solid composition of clause 111, wherein the water-soluble carrier monomer comprises MP AM.

[0335] Clause 114. The solid composition of any one of clauses 86 to 113, wherein the functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

[0336] Clause 115. The solid composition of any one of clauses 86 to 114, wherein the functional dopant monomer is selected from DEA, PHE, NIP, and combinations thereof.

[0337] Clause 116. The solid composition of any one of clauses 86 to 114, wherein the functional dopant monomer comprises TRI.

[0338] Clause 117. The solid composition of clause 115, wherein the functional dopant monomer comprises PHE.

[0339] Clause 118. The solid composition of clause 115, wherein the functional dopant monomer comprises NIP.

[0340] Clause 119. The solid composition of clause 115, wherein the functional dopant monomer comprises DEA.

[0341] Clause 120. The solid composition of any one of clauses 86 to 119, wherein:the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from NIP, PHE, and combinations thereof.

[0342] Clause 121. The solid composition of any one of clauses 86 to 120, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

[0343] Clause 122. The solid composition any one of clauses 86 to 121, wherein the water-soluble carrier monomer is MP AM, and the functional dopant monomer is PHE.

[0344] Clause 123. The solid composition any one of clauses 86 to 121, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is PHE.

[0345] Clause 124. The solid composition of any one of clauses 86 to 121, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is NIP.

[0346] Clause 125. The solid composition of any one of clauses 86 to 124, wherein the copolymer comprises:70 wt% to 98 wt% of the water-soluble carrier monomer; and2 wt% to 30 wt% of the functional dopant monomer.

[0347] Clause 126. The solid composition of any one of clauses 86 to 125, wherein the copolymer comprises:80 wt% to 95 wt% of the water-soluble carrier monomer; and5 wt% to 20 wt% of the functional dopant monomer.

[0348] Clause 127. The solid composition of any one of clauses 86 to 126, wherein the copolymer comprises:83 wt% to 98 wt% of the water-soluble carrier monomer; and2 wt% to 17 wt% of the functional dopant monomer.

[0349] Clause 128. The solid composition any one of clauses 86 to 126, wherein the copolymer comprises:70 wt% to 85 wt% of MORPH; and15 wt% to 30 wt% of NIP.

[0350] Clause 129. The solid composition of clause 128, wherein the copolymer comprises:74 wt% to 80 wt% of MORPH; and20 wt% to 26 wt% of NIP.

[0351] Clause 130. The solid composition of clause 129, wherein the copolymer comprises:77 wt% of MORPH; and23 wt% of NIP.

[0352] Clause 131. The solid composition of any one of clauses 86 to 130, wherein a degree of polymerization of the copolymer is 10 to 500.

[0353] Clause 132. The solid composition of any one of clauses 86 to 131, wherein a degree of polymerization of the copolymer is 20 to 200.

[0354] Clause 133. The solid composition of any one of clauses 86 to 132, wherein a number-average molecular weight of the copolymer is 2,000 g / mol to 10,000 g / mol.

[0355] Clause 134. The solid composition of clause 133, wherein a numberaverage molecular weight of the copolymer is 2,000 g / mol to 6,000 g / mol.

[0356] Clause 135. The solid composition of clause 134, wherein a numberaverage molecular weight of the copolymer is 3,000 g / mol to 5,000 g / mol.

[0357] Clause 136. The solid composition of any one of clauses 86 to 135, further comprising one or more additional components selected from a preservative and a surfactant.

[0358] Clause 137. The solid composition of any one of clauses 86 to 136, wherein the stabilizer is a sugar.

[0359] Clause 138. The solid composition of clause 137, wherein the stabilizer sugar is trehalose.

[0360] Clause 139. The solid composition of any one of clauses 136 to 138, wherein the one or more additional components are present in an amount ranging from about 0.1-50 % by weight.

[0361] Clause 140. The solid composition of any one of clauses 136 to 138, wherein the one or more additional components are present in an amount ranging from about 0.001-1.0 % by weight.

[0362] Clause 141. The solid composition of any one of clauses 86 to 140, wherein the solid composition is a lyophilized composition.

[0363] Clause 142. A pharmaceutical unit dosage composition comprising a solid composition according to any one of clauses 86 to 141.

[0364] Clause 143. The pharmaceutical unit dosage composition of clause 142, wherein the composition is contained in a single dose container, vial, or pre-filled syringe.

[0365] Clause 144. A method of reconstituting a solid composition, the method comprising dissolving a solid composition according to any one of clauses 86 to 143 in an aqueous solution to produce a reconstituted composition.

[0366] Clause 145. The method of clause 144, wherein the aqueous solution is sterile water.

[0367] Clause 146. The method of clause 145, wherein the aqueous solution comprises a buffering agent.

[0368] Clause 147. The method of any one of clauses 143 to 145, further comprising centrifuging the reconstituted composition.

[0369] Clause 148. The method of any one of clauses 143 to 146, further comprising removing particulates from the reconstituted composition.

[0370] Clause 149. The method of any one of clauses 143 to 146, further comprising equilibrating the solid composition to room temperature before dissolution.

[0371] Clause 150. The method of any one of clauses 143 to 146, further comprising assessing the reconstitution time for the dissolution of particulates.

[0372] Clause 151. The method of any one of clauses 143 to 150, wherein the solid composition is a fixed dose of the biopharmaceutical agent comprised in a vial.

[0373] Clause 152. The method of any one of clauses 143 to 151, wherein the reconstitution time is less than 30 min.

[0374] Clause 153. A reconstituted composition prepared according to the method of any one of clauses 143 to 152.

[0375] Clause 154. A method of administering a biopharmaceutical agent to a subject in need thereof, the method comprising administering a reconstituted composition according to clause 153 to the subject.

[0376] Clause 155. A method of reconstituting a lyophilized composition, the method comprising dissolving a lyophilized composition according to any one of clauses 86 to 154 in an aqueous solution to produce a reconstituted lyophilizate composition.

[0377] Clause 156. The method of clause 155, wherein the aqueous solution is sterile water.

[0378] Clause 157. The method of clause 156, wherein the aqueous solution comprises a buffering agent.

[0379] Clause 158. The method of any one of clauses 155 to 157, further comprising centrifuging the reconstituted lyophilizate composition.

[0380] Clause 159. The method of any one of clauses 155 to 158, further comprising removing particulates from the reconstituted lyophilizate composition.

[0381] Clause 160. A reconstituted lyophilizate composition prepared according to the method of any one of clauses 155 to 159.

[0382] Clause 161. A method of administering a biopharmaceutical agent to a subject in need thereof, the method comprising administering a reconstituted lyophilizate composition according to clause 160 to the subject.

[0383] Clause 162. A method of preparing a solid composition according to any one of clauses 86 to 161, the method comprising: exposing a liquid biopharmaceutical composition comprising: a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from N-(3- methoxypropyljacrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxy ethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from N-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N- isopropyl acrylamide (NIP), N-N-diethylacrylamide (DEA), N-tert- butyl acrylamide (TBA), N-phenyl acrylamide (PHE), and combinations thereof; and a biopharmaceutical agent; to a drying cycle to produce the solid composition.

[0384] Clause 163. The method of clause 162, wherein the drying is selected from lyophilization, spray drying, spray freezing drying, foam mat drying, vacuum oven drying, and desiccation drying.

[0385] Clause 164. The method of clause 162 or 163, wherein the liquid biopharmaceutical composition is exposed to a lyophilization cycle in a lyophilizer to produce a lyophilized composition.

[0386] Clause 165. The method of clause 164, wherein the lyophilization cycle comprises: subjecting the liquid biopharmaceutical composition to a first pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy; and cooling the liquid biopharmaceutical composition to freeze any remaining liquid component into a solid.

[0387] Clause 166. The method of clause 154 or 165, wherein the lyophilization cycle comprises: subjecting the liquid biopharmaceutical composition to a first reduced temperature; subjecting the liquid biopharmaceutical composition to a first reduced pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy;subjecting the liquid biopharmaceutical composition to a second reduced pressure that is less than the first reduced pressure; and evaporating the remainder of the liquid to produce the solid composition.

[0388] Clause 167. The method of clause 166, wherein the drying cycle is a spray drying cycle comprising: atomizing the liquid biopharmaceutical composition; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition; and collecting the solid composition.

[0389] Clause 168. The method of clause 167, wherein the drying cycle is a spray freeze drying cycle comprising: atomizing the liquid biopharmaceutical composition; freezing the atomized liquid biopharmaceutical composition; evaporating at least a portion of a liquid component of the atomized liquid biopharmaceutical composition; and collecting the solid composition.

[0390] Clause 169. The method of clause 168, wherein the drying cycle is a foam mat drying cycle comprising: foaming the liquid biopharmaceutical composition; spreading the foamed liquid biopharmaceutical composition onto a drying surface; evaporating at least a portion of a liquid component of the foamed liquid biopharmaceutical composition; and collecting the solid composition.

[0391] Clause 170. The method of clause 168, wherein the drying is a vacuum oven drying cycle comprising: subjecting the liquid biopharmaceutical composition to reduced pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy; andreturning the pressure to the original pressure to obtain the solid composition.

[0392] Clause 171. The method of clause 170, wherein the drying is a desiccation drying cycle comprising: increasing the surface area of the liquid biopharmaceutical composition, e.g., by pouring the liquid biopharmaceutical composition into a tray; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition by circulating ambient air across the surface, wherein the circulated air is optionally dried over a desiccating material, heated, or both until the solid is obtained.EXAMPLESGeneral Experimental DetailsMaterials

[0393] Solvents N,N-dimethylformamide (DMF; >99.7%; HPLC grade, Alfa Aeser), ethanol (EtOH; >99.5%; Certified ACS, Acros Organics), acetone (>99.9%; Sigma-Aldrich, HPLC Grade), hexanes (>99.9%; Thermo Fisher Scientific, Certified ACS), ether (anhydrous, >99%; Sigma-Aldrich, Certified ACS), and CDCh (>99.8%; Acros Organics) were used as received. MORPH (>97%; Sigma- Aldrich) was filtered with basic alumina before use. NIP (>99%; Sigma- Aldrich) was used as received. RAFT CTA 2-cyano-2-propyl dodecyl trithiocarbonate (2-CPDT; >97%; Strem Chemicals) was used as received. Initiator 2,2-azobis(2-methyl-propionitrile) (AIBN; >98%; Sigma-Aldrich) was recrystallized from MeOH (>99.9%; Thermo Fisher Scientific, HPLC grade) and dried under vacuum before use. Z group removing agents lauroyl peroxide (LPO; 97%; Sigma-Aldrich) and hydrogen peroxide (H2O2; 30%; Sigma-Aldrich) were used as received. PGT121 monoclonal antibody was provided by Just-Evotec Biologies, Inc. (Seattle, WA, USA) in collaboration with the Bill and Melinda Gates Foundation. PGT121 was supplied at 55.5 mg / mL in 20 mM acetate buffer with 9% (w / v) sucrose, 0.01% PS 80, pH 5.0. 9e9 anti-idiotype monoclonal antibody was synthesized by the Protein Production Facility (PPF) funded by the Bill and Melinda Gates Foundation, using a plasmid provided by the Vaccine Research Center (VIRC), a division of the National Institute of Allergy and Infectious Diseases (NIAID) in the US National Institutes of Health (NIH).Synthesis ofMoNi

[0394] The amphiphilic acrylamide copolymer excipient acryloylmorpholine-co-N- isopropyl acrylamide (MoNi) was prepared according to methods described by Mann et al., Sci. Transl. Med. 12, eaba6676 (2020). Briefly, MORPH (645 mg, 4.57 mmol, 41.5 eq.), NIP (105 mg, 0.93 mmol, 8.5 eq.), 2-CPDT (38 mg, 0.11 mmol, 1 eq.), and AIBN (3.6 mg, 0.02 mmol, 0.2 eq.) were combined and diluted with DMF to a total volume of 2.25 mL [33.3 (w / v) vinyl monomer concentration] in an 8-mL scintillation vial equipped with a PTFE septa. The reaction mixture was sparged with nitrogen gas for 10 min and then heated for 12 hours at 65°C. To remove the Z terminus of the resulting polymer, AIBN (360 mg, 2.2 mmol, 20 eq.) and LPO (88 mg, 0.22 mmol, 2 eq.) were added to the reaction mixture, which was then sparged with nitrogen gas for 10 min and heated for 12 hours at 90°C. Z group removal was confirmed by the ratio of the refractive index to ultraviolet (310 nm) intensity in SEC analysis. Resulting polymers were precipitated three times from ether and dried under vacuum overnight. Resulting composition and molecular weights were determined via1H NMR spectroscopy and SEC with PEG standards.Copolymer molecular weight characterization by SEC

[0395] Mn, Mw, and D for MoNi was determined via SEC implementing PEG standards (American Polymer Standards Corporation) after passing through two SEC columns [inner diameter, 7.8 mm; Mw range, 200 to 600,000 g mol’1; Resolve Mixed Bed Low divinylbenzene (DVB) (Jordi Labs)] in a mobile phase of DMF with 0.1 M LiBr at 35°C and a flow rate of 1.0 mL min1[Dionex UltiMate 3000 pump, degasser, and autosampler (Thermo Fisher Scientific)].Preparation of high-concentration, stabilized PGT121 formulations

[0396] PGT121 monoclonal antibody was concentrated via spin filtration (Coming® Spin-X® UF 5kDa MWCO) at 3030 RPM until desired volume recovery. Concentrated formulations were refrigerated for 12 hours before usage in accelerated aging studies.Dynamic Light Scattering

[0397] DLS measurements ofMoNi, prepared in milliQ water, were taken on a DynaPro Plate Reader II, Wyatt Technology. Average of 5 acquisitions was reported.Concentration determination

[0398] Concentrations of PGT121 formulations were determined by either volume recovery, ELISA, Nanodrop (Thermo Scientific) or aqueous SEC-UV (PBS buffer, 300 ppm sodium azide), depending on the assay. For aqueous SEC-UV, PGT121 aliquots were diluted 133X in MilliQ water and concentration was determined by comparing the area under the curve of the traces obtained from SEC using a Dionex UltiMate 3000 VWD at 280 nm (Thermo Scientific). In these assays, the aged sample was compared to an unaged 55 mg / mL PGT121 solution (n=3). For nanodrop, the protein concentration was compared to a stock standard at 55 mg / mL. For ELISA, concentrations were interpolated by fitting a four- parameter dose-response curve (variable slope) in GraphPad Prism 9. The high-concentration PGT121 formulation was reported as the mean ± standard deviation of the concentration determined by these methods for several preparations of these formulations.Surface Tension

[0399] Time-resolved surface tension of the air-solution interface was measured with a platinum / iridium Wilhelmy plate connected to an electrobalance (KSV Nima, Finland). The Wilhelmy plate was partially immersed in the aqueous solution in a Petri dish, and the surface tension of the interface was recorded for 50 min from the formation of a fresh interface. Equilibrium surface tension values (t = 50 min) were reported as these values more closely describe the environment in a stored vial prior to agitation. Samples were diluted in 20 mM acetate buffer, pH = 5.0, from stock samples to the desired assay concentrations. The experiment was repeated in triplicate.Interfacial Rheology

[0400] Interfacial shear rheology was measured using a Discovery HR-3 rheometer (TA Instruments) with an interfacial geometry comprising a Du Nouy ring made of platinum / iridium wires (CSC Scientific, Fairfax, VA, catalog no. 70542000). Before each experiment, the Du Nouy ring was rinsed with ethanol and water and flame treated to remove organic contaminants. The solution chamber consisted of a double-wall Couette flow cell with an internal Teflon cylinder and an external glass beaker. A time sweep was performed at a strain of 1% (within the linear regime) and a frequency of 0.05 Hz (sufficiently low such that the effects due to instrument inertia will not be significant). Interfacial complex shear viscosity was measured for 30 min. Samples were diluted in 20 mM acetate buffer, pH = 5.0,from stock samples to the desired assay concentrations. The experiment was repeated in triplicate.PGT121 ELISA

[0401] The capture antibody, 9e9, was coated at 2 pg / mL in phosphate buffered saline (PBS) (25 pg per well) on Coming 96-well high binding flat-bottom half-area microplates (Fisher Scientific) and incubated at 4°C overnight. The blocking buffer used was 2% non-fat dry milk (NFDM) in PBS, the assay buffer used was 2% bovine serum albumin (BSA) in PBS, and the wash buffer was PBS-T (0.05% Tween 20). Plates were washed twice and blocked with 125 pL of blocking buffer and incubated for 1 hr at room temperature. Sample dilutions and PGT121 standards were prepared in assay buffer. After blocking, assay buffer was removed from the plate before adding 50 pL of each sample and incubating for 1 hr at room temperature. Plates were washed five times before adding 50 pL per well of the secondary antibody, Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fey fragment specific (Jackson Immunoresearch, 109-036-008, RRID: AB\_2337591) was diluted 1 : 5000 in assay buffer. After incubating for 1 hr at room temperature, plates were washed 10 times and then developed by adding 50 pL of TMB ELISA Substrate (High Sensitivity) (Abeam, ab 171523). Development was stopped after 2.5 min by adding 1 N hydrochloric acid. PGT121 concentration was measured by absorbance intensity at 450 nm using a Synergy Hl Hybrid Multi-Mode Plate Reader (BioTek). Each plate contained a 15-point standard curve assayed in duplicate which was used to interpolate PGT121 concentrations by fitting a four-parameter dose-response curve (variable slope) in GraphPad Prism 9. Stressed aging samples were diluted 1 : 10000 and then serially diluted by 4x to create a 7-point curve assayed in duplicate for each sample. IC50 values were determined by fitting a four-parameter dose-response curve (variable slope) in GraphPad Prism 9. Mean value and standard deviation were reported.In vivo pharmacokinetic study

[0402] Animal studies were conducted in accordance with the guidelines for care and use of laboratory animals under protocols approved by the Stanford Institutional Animal Care and Use Committee (IACUC). Eight week old female B6.Cg-FcgrttmlDcrPrkdcscldTg(FCGRT)32Dcr / DcrJ (The Jackson Laboratory, Stock No. 018441) mice were administered PGT121 antibody (1.5 mg / mouse) via IP or SC injection under brief isoflurane anesthesia (n=6 / group). The polymer-stabilized high-concentration SC formulation wasprepared with 102 mg / mL PGT121 (as determined by ELISA) and 1 wt% MoNi (15 pL injection volume), and the low concentration IP formulation was prepared at 5 mg / mL (300 pL injection volume) in the same buffer in which the antibody was provided. Blood samples were collected at 24 hours, 48 hours, and then at days 4, 7, 10, 14, and 21 post-injection for analysis of PGT121 serum concentration via ELISA.Viscometry

[0403] A Rheosense m-VROC viscometer with a low viscosity chip was used to measure the viscosity at high shear rates representative of injection. Samples were measured from low to high shear rates using a Hamilton syringe. Each data point was collected at steady state.In vitro stability assay

[0404] 150 pL of a PGT121 solution was diluted with 7.5 pL of MoNi in the formulation buffer at 21 mg / mL to reach a final excipient concentration of 0.1 wt.%. These formulations were agitated at 200 rpm at 50°C. 5 mL aliquots were removed every 24 hours.Diffusion-Ordered Spectroscopy

[0405] 'H two-dimensional DOSY spectra were recorded at a PGT121 concentration of 0.2 mg / mL diluted from 20 mM acetate buffer with 9% (w / v) sucrose, 0.01% PS 80, pH 5.0 in D2O (Acros Organics) and dialyzed with 2000 Da MWCO Slide-A-Lyzer dialysis cassettes (Thermo Scientific) against D2O for 24 hours prior to use. MoNi was prepared directly in D2O at 0.1 mg / mL.

[0406] A Varian Inova 600 MHz NMR instrument was used to acquire the data. Magnetic field strengths ranging from 2 to 57 GThe DOSY time and gradient pulse were set at 66.5 ms (A) and 2 ms (5), respectively. All NMR data were processed using MestReNova 11.0.4 software.Statistics

[0407] All results were expressed as mean ± standard deviation (SD) unless specified otherwise and analyzed using Graphpad Prism 9.1 (GraphPad Software Inc., USA).

[0408] FIG. 1 is a set of schematics showing an overview of stabilization of high- concentration antibody formulations using novel copolymer excipients. (Panel A) Schematic overview of current therapeutic routes of administration for biopharmaceuticals, wherebyintravenous administration is common because of limitations on stability of biopharmaceuticals at the high concentrations required for subcutaneous administration, resulting in high patient burden. (Panel B) Graphical representation of amphiphilic acrylamide carrier / dopant co-polymer (AC / DC) excipients, such as poly(acryloylmorpholine- co-A-isoproyl acrylamide) (MoNi), that can be used to stabilize biopharmaceuticals in formulation. (Panel C) Schematic showing stabilization of monoclonal antibodies in formulation using AC / DC copolymers as a “drop-in” excipient. AC / DC copolymers are amphiphilic and can preferentially absorb to interfaces, precluding antibody interfacial adsorption in formulation and thereby preventing interface-related destabilization and aggregation events.Example 1. Selective Adsorption of Copolymer to Interfaces in mAb Formulations

[0409] To synthesize MoNi, reversible addition fragmentation transfer (RAFT) controlled radical polymerization was employed. The polymer was generated with a reactive trithiocarb onate chain transfer agent (CTA) attached at the Z terminus of the polymer following polymerization (denoted MoNi -CTA). The CTA moiety was removed from the MoNi -CTA copolymer to produce the MoNi excipient before utilization in subsequent assays to ensure both chemical stability and biological inertness (FIG. 2, Panel A-Panel D).

[0410] Reported values for the concentration whereby 50% of cells are killed in vitro, a parameter called the LCso, were compared, and showed that MoNi is biocompatible and over 100-fold less cytotoxic than common commercial surfactant excipients such as Polysorbate 80 (PS80) and Pluronic L61 (FIG. 2, Panel E). Maikawa et al., Biomacromolecules 22(8):3386-95 (2021); Arechabala et al., Journal of Applied Toxicology 19(3): 163-65 (1999); Demina et al., Biomacromolecules 15(7):2672-81 (2104).

[0411] The MoNi copolymer excipient was synthesized at molecular weights well below the glomerular filtration threshold to ensure that they are rapidly excreted following administration in the body without bioaccumulation. Further, determination of MoNi’s critical micelle concentration (CMC) was attempted by performing dynamic light scattering (DLS) analysis on a dilution series of MoNi from 100 to 1 mg / mL (FIG. 2, Panel F). In these assays, the MoNi excipient did not form micelles or aggregates within the concentration range assessed. This behavior was unique compared to the common commercial surfactant excipients PS80 and Pluronic L61, which exhibit CMC values well below 1 mg / mL (FIG. 2, Panel G). Chou et al., J. Pharm. Sci. 94(6): 1368-81 (2005); Carvalho et al., Internationaljournal of pharmaceutics 602: 120635 (2021). Notably, most surfactants are toxic in their micellular state, suggesting that the significantly lower cytotoxicity of MoNi may relate to the fact that it does not exhibit self-assembly into micelles within formulation-relevant concentrations, contrasting its commercial counterparts. Demina et al., Biomacromolecules 15(7):2672-81 (2104).

[0412] Whether MoNi preferentially adsorbs to the air-water interface was tested. Time- resolved surface tension experiments were conducted with formulations of the anti-HIV bnAb PGT121, a promising candidate for passive immunization against HIV targeting the well-conserved V1 / V2 glycan on the gpl20 surface glycoprotein. Patel et al., J. Pharm. Sci. 107(12): 2969-82 (2018); Stephenson et al., Nature Medicine 27: 1718-24 (2021). Formulations of PGT121 (20 mM acetate buffer, pH~5.0) with MoNi (0.01 wt%) exhibited lower surface tension values compared to PGT121 alone (53 mN / m and 62 mN / m, respectively; FIG. 3, Panel A). The lower surface tension observed for the PGT121 formulation comprising MoNi suggested that there were more species packed at the interface in this formulation than in the formulation with only PGT121, indicating that MoNi was preferentially adsorbing to the interface at higher levels. Notably, the surface tension of both a formulation of MoNi only in buffer and a formulation of PGT121 and MoNi were found to be identical (FIG. 3, Panel A), indicating that the air- water interface was indeed dominated by MoNi at these concentrations. These results suggested that MoNi preferentially adsorbed to the air-water interface, precluding mAb adsorption.

[0413] To further characterize the effects of MoNi on the formulation of PGT121 the interfacial viscoelasticity of these formulations was probed via interfacial shear rheology measurements. These measurements revealed that addition of MoNi to PGT121 formulations reduced the interfacial complex viscosity by 3-fold (from ~0.3 Pa s m to -0.1 Pa s mJ; FIG.3, Panel B). The high interfacial complex viscosity of the PGT121 formulation without MoNi suggested that protein interactions at the interface were indeed leading to the formation of a gel-like skin at the interface. Consequently, the significant reduction in interfacial complex viscosity upon addition of MoNi indicated that the copolymer excipient reduced interfacial interactions of PGT121, commensurate with the surface tension measurements discussed above that showed that the mAbs were precluded from adsorbing to the interfaces.

[0414] Whether MoNi interacted with the mAbs in the bulk was also determined using diffusion ordered spectroscopy (DOSY), a well-established NMR technique that provides direct evaluation of the diffusion behavior of different species in solution. As shown in FIG.4, Panel A-Panel C, PGT121 mAb and MoNi exhibited distinct diffusion behavior, suggesting that the two species did not interact in the bulk. These results further indicate that MoNi acts to stabilize protein cargo by preventing interfacial aggregation.

[0415] Whether formulations with mAb concentrations greater than 100 mg / mL were easily injectable under clinically relevant injection conditions was determined. Hernandez et s ., Macromol. Biosci. 21 :2000295 (2021). The viscosity of high-concentration PGT 121 (119 ± 7 mg / mL) solutions with and without MoNi (10 mg / mL) was at shear rates that are typically achieved during injection through a standard syringe and needle geometry and flow rate. Addition of MoNi at this concentration did not alter the injection viscosity of the formulation, and both formulations exhibited sufficiently low viscosities to be easily injected (FIG. 3, Panel C).Example 2. Copolymer Stabilization of mAb Formulations in Stressed- Aging Assays

[0416] Accelerated aging assays were conducted. Initially, a stock PGT121 formulation at 55.5 mg / mL (20 nM acetate, 9wt% sucrose, 0.01 wt% PS80, pH —5.0) with MoNi (1 mg / mL, and 10 mg / mg) were compared. These formulations, including a control containing only the stock formulation of PGT121, were packaged in glass vials and subjected to accelerated aging (constant agitation at 50°C). The monomeric mAb content in the formulations was monitored over 1 week (7 days) with size exclusion chromatography (SEC). While formulations containing MoNi at either concentration resulted in greater preservation of monomeric PGT121 over 7 days than in the control formulation without MoNi, the sample containing 10 mg / mL MoNi exhibited the best stability. (FIG. 8). Next, a stock PGT121 formulation at 55.5 mg / mL (20 mM acetate, 9 wt% sucrose, 0.01 wt% PS80, pH~5.0) was compared to a formulation concentrated more than 2-fold at 119 ± 7 mg / mL (denoted herein as high concentration). These formulations with and without MoNi (10 mg / mL) were then packaged in glass vials and subjected to accelerated aging (constant agitation at 50°C; FIG. 5, Panel A). The monomeric mAb content in these formulations was monitored over four weeks with size exclusion chromatography (SEC; FIG. 5, Panel B). Within 5 days, both the stock and high-concentration PGT121 formulations had lost more than 50% of their monomeric mAb content, indicating significant aggregation of the protein. The loss of monomeric PGT121 and formation of aggregates over 6 days is shown in representative SEC chromatograms of FIG. 9, panels A-C, where panel A is the representative SEC chromatogram for PGT 121 with no MoNi; panel B is the representativeSEC chromatogram for PGT121 with 1 mg / mL MoNi; and panel C is the representative SEC chromatogram for PGT121 with 10 mg / mL MoNi.

[0417] By day 7, both formulations had macroscopically aggregated, becoming visibly opaque. In contrast, the stock PGT121 formulation including MoNi maintained greater than 95% monomeric mAb content through three weeks of continuous stressed aging (FIG. 5, Panel B). While the high-concentration sample comprising MoNi exhibited a small initial decrease in monomer mAb content over the first week of stressed aging, likely arising from the concentration process, this formulation maintained near 70% monomer mAb content over the same three-week period (FIG. 5, Panel B).

[0418] Whether binding epitope activity (as a proxy for therapeutic efficacy) was preserved through continuous stressed aging was determined. An enzyme-linked immunosorbent assay (ELISA) was used to determine whether samples subjected to stressed aging retained binding activity, and thus conformational fidelity, in both their Fab and Fc domains (FIG. 5, Panel C). Half maximal inhibitory concentrations (IC50) were used to compare the functional potency of the high-concentration formulations with and without MoNi (10 mg / mL) subjected to stressed aging. In the absence of MoNi, the PGT121 mAbs rapidly lost functionality, losing more than 65% of their potency after just five days of stressed aging (FIG. 5, Panel D). In contrast, the addition of MoNi significantly enhanced formulation stability and the PGT121 mAbs retained more than 75% of their original potency through 21 days of continuous stressed aging.

[0419] Taken together, these results indicate that the addition of the MoNi excipient to high-concentration mAb formulations conferred a substantial stability benefit by precluding mAb adsorption to the interfaces, thereby preventing aggregation events and maintaining binding activity under accelerated aging conditions.Example 3. Pharmacokinetic Profile of Copolymer-Stabilized High-Concentration PFT121 Formulation

[0420] Whether MoNi functions as an inactive ingredient was determined through a pharmacokinetic study in rodents. 1.5 mg of PGT121 was administered to transgenic SCID mice with humanized FcRn receptors (n=6 / group; B6.Cg-FcgrttmlDcrPrkdcscldTg[FCGRT]32Dcr / DcrJ; Jackson Labs No. 018441) via either SC injection of 15 pL of MoNi -stabilized high-concentration PGT121 (119 mg / mL) or IP injection of 300 pL of lowconcentration PGT121 (5 mg / mL). Serum was collected for three weeks post administration to be analyzed for systemic PGT121 concentration via ELISA (FIG. 6, Panel A - Panel B).

[0421] Quantification of serum concentration over time as well as total PGT121 exposure (area-under-the-curve; AUC) demonstrated comparable pharmacokinetics and bioavailability between the two routes of administration and formulations (FIG. 6, Panel C). No significant therapeutic difference was observed in terms of serum titer or bioavailability of PGT121 when administered in a MoNi-stabilized, high-concentration formulation. Moreover, no acute weight loss (a common metric for treatment related toxicity) was observed, further showing MoNi’s biocompatibility and tolerability (FIG. 7, panels A-C).

[0422] The addition of MoNi to high-concentration PGT121 mAb formulations significantly improved their stability without impacting pharmacokinetics, as would be required for development of clinically relevant SC formulations. These results demonstrated MoNi’s remarkable usefulness as an excipient to address biopharmaceutical formulation challenges.Example 4. Lyophilization and Reconstitution of Liquid Composition

[0423] Lyophilization procedure: (a) loading a container with the liquid composition at a starting temperature of about 5 °C to about -50 °C; (b) cooling the composition to subzero temperatures (e.g., -10 °C to -50 °C ); and (c) substantially drying the composition. The conditions for lyophilization, e.g., temperature and duration, of the composition can be adjusted taking into consideration factors that affect lyophilization parameters, e.g., the type of lyophilization machine used, the amount of biopharmaceutical agent used, and the size of the container used.

[0424] The lyophilized composition is reconstituted using a target volume of liquid solution, e.g., filtered water for injection (WFI), or an aqueous buffer solution. Various methods for reconstitution are employed, such as the swirling methods, methods using a mechanical orbital shaker or methods of keeping the container remained stationary.

[0425] Lyophilized compositions of the present disclosure can have reduced reconstitution times relative to compositions that lack the polyacrylamide-based copolymer.

[0426] Reconstitution times are assessed as follows: Samples of the liquid composition are prepared and then stirred using a magnetic stirrer and protected from atmosphere. The solution is then transferred to a volumetric flask and water for injection (WFI) is added tomake up to target volume, e.g., 50 mL. The solution is mixed to ensure homogeneity, filtered through a 0.2 pm filter, transferred to vials and lyophilized.

[0427] Samples are reconstituted using a target volume of filtered water for injection (WFI), restoppered, and resealed. Various methods for reconstitution are employed, such as the swirling methods, methods using a mechanical orbital shaker or methods of keeping the vial remained stationary.

[0428] Reconstitution can be assessed using various methods, including light absorbance of the reconstituted composition at suitable wavelength. A reconstituted composition can have a light absorbance, e.g., at 450 nm, of 0.002 or less, such as 0.001 or less.

[0429] Reconstitution time is measured after lyophilization (initial) and after stability testing (e.g., storage stability or stability to stress conditions), wherein vials were reconstituted with target volume of WFI.Example 5. Assessment of Lyophilized Composition

[0430] The lyophilized compositions are exposed to varying stress conditions to assess their relative stability. To thermally stress the samples, compositions are stored at 5 °C, 25 °C, or 40 °C for up to 10 weeks. For the assessment of stability after reconstitution, samples are reconstituted in a biosafety cabinet using a target volume of filtered water for injection (WFI), restoppered, and resealed. Following reconstitution, samples are stored at 25 °C for 3 or 7 days. Various conditions tested in this experiment are summarized below in Table 2.Table 2

[0431] The following assays can be performed to analyze stability of various formulations in different conditions. (1) Visual Inspection: Visual inspection is performed against a dark and white background. Digital photographs are acquired. (2) UV Spectrophotometry. (3) Bioassay to assess potency of active ingredient.Example 6. Freeze-thaw stability assessment of exemplary antibody composition

[0432] Formulations of this disclosure including two different monoclonal antibody APIs are subjected to five freeze-thaw cycles, including freezing at -70 °C, followed by thawing of the formulation to room temperature. Samples of the formulate are assessed before and after subjection to the freeze-thaw cycles for amount of particulates in the formulation (e.g., >0.2 pm in particle size). The number of particles in each sample is assessed.

[0433] Formulations for testing include:1. control formulation with no surfactant or copolymer,2. formulation with polysorbate 80 surfactant,3. formulation with 2 mg / mL MoNi copolymer, and4. formulation with 8 mg / mL MoNi copolymer.Example 7. Thermostability assessment of exemplary vaccine composition

[0434] Lyophilized live-attenuated vaccine is formulated with various concentrations of MoNi copolymer and subjected to thermal stress to assess stability. Samples of vaccine formulated with no MoNi, 0.01 mg / mL MoNi, 0.1 mg / mL MoNi, 1 mg / mL MoNi, or 10 mg / mL MoNi copolymer are prepared and lyophilized. To thermally stress the samples, the sample compositions are stored at, e.g., 25 °C, or 40 °C for, e.g., 2 weeks or more, 4 weeks or more, or 6 weeks or more, up to 10 weeks, or at 5 °C for 3 months, 6 months or 12 months or more.

[0435] The stability of the samples is assessed after reconstitution, by assaying the potency, viability and / or infectivity of the vaccine.Example 8. Reconstitution of lyophilized formulations of IgG

[0436] To access whether the formulations of this disclosure can be reconstituted, selected formulations are first lyophilized, followed by analysis of lyophilized cake reconstitution, and analysis of reconstituted formulations.

[0437] The objective of the experiment is to select and prepare lyophilized formulations of IgG and MoNi, sufficient to yield 20 mg / mL, 100 mg / mL, and 200 mg / mL after reconstitution in select vehicles.Pre-Lyophilization Procedure

[0438] Pre-lyophilization procedure is performed to assess suitability of the process for buffer exchange of IgG and design compounding process. Pre-lyophilization analysis is conducted on the formulations to assess a) visual appearance; b) measurement of IgG concentration by Solo-VPE; and optionally c) IgG integrity by SEC.

[0439] The pre-lyophilization procedure includes:1. testing ability to concentrate and exchange IgG into 5 mM L-histidine hydrochloride buffering agent using Amicon centrifugal filters; and2. sampling and analyzing using SEC method for aggregation during the concentration, targeting final protein concentration of 100 mg / mL.Lyophilization Formulations Preparation

[0440] Seven formulations for lyophilization are prepared according to the following procedure:1. An appropriate amount of IgG is buffer exchanged via TFF, using a 50 cm2membrane (i.e. Millipore Pellicon XL) against 5 mM L-histidine hydrochloride buffering agent, pH 6. Upon initial concentration to the previously determined target, 6 diavolumes are exchanged to remove >99% of the original formulation components. Measurement of IgG concentration is performed via UV using Solo-VPE;2. The concentrated IgG in L-histidine hydrochloride is diluted into the following formulations using appropriate spiking solutions and volumes as determined by the IgG concentration and necessary dilution factors. A total of 25 mL each (or 1.25 g IgG) is prepared. The composition of the assessed formulations is depicted in Table 3.Table 3Lyophilization and Reconstitution

[0441] Five formulations presented in Table 3 are lyophilized, followed by assessing reconstitution of the above formulations at different concentrations.

[0442] Lyophilization is conducted according to the following procedure:1. Sterile filtering 5 formulations using 0.2 pM syringe membranes and fill 4 x 20 mL serum vials with 5 mL (250 mg IgG) each; and2. Lyophilizing 4 vials of each Formulations 1-5 (Table 3) using the PLS conservative lyophilization cycle.

[0443] Lyophilized cakes are visually evaluated according to the procedure below:1. Observing and recording the color, appearance, cracks, uniformity, shrinkage of the lyophilized cakes in all vials of the 5 lyophilized formulations;2. On a condition that a lyophilized solid formulation(s) is not cake-like, the reconstitution study is not performed for that sample.3. On a condition that all samples are not cake-like, the project is paused.

[0444] Three reconstitution experiments are conducted in 20 mL vials at 200 mg / mL (18 total reconstitutions) according to the procedures in Table 4.Table 4

[0445] Reconstitution is conducted according to the following procedure:1. All the vials are taken out of 2-8 °C and equilibrated to room temperature prior to start of study;2. Stipulated volume of WFI is added to one vial each of lyophilized formulations Fl to F6. The vial is manually swirled till all the powder dissolved and a clear solution is obtained. The time to dissolve the cake is recorded and visual observations are made.

[0446] Analysis of reconstituted formulations is optionally performed to assess the impact of lyophilization and reconstitution on IgG integrity for mutually agreed upon formulations.

[0447] IgG concentration is assessed by UV using SoloVPE by measuring the IgG concentration using either provided or literature extinction coefficient of IgG.

[0448] Aggregation / fragmentation is assessed by SEC by assessing the integrity of IgG using a SEC method and comparing the aggregation / fragmentation to pre-lyophilization and freshly prepared IgG sample as a control.Results

[0449] The objective of the experiment was to lyophilize formulations of 250 mg IgG and 250 mg trehalose in a L-histidine hydrochloride buffer with a control, 0.8 mg / mL polysorbate 20 (PS20), and 20 mg / mL MoNi, and measure the time to compete dissolution of resulting lyophilized cakes into a 200 mg / mL IgG aqueous formulation.

[0450] The results of the Reconstitution Study 1 (~20 mg / mL IgG, 12 mL WFI addition) are presented in Table 5 below. FIG. 10 presents a set of photographs presenting the appearance of the formulations F1-F6 in the Reconstitution Study 1.Table 5

[0451] The presence of trehalose in the formulation provided for quick dissolution. MoNi was found to assist in dissolution.

[0452] Reconstitution time of F5 was shorter than that of F6. The cakes in F1-F4 showed instantaneous dissolution.

[0453] The results of the Reconstitution Study 2 (-100 mg / mL IgG, 2.5 mL WFI addition) are presented in Table 6 below. FIG. 11 presents a set of photographs presenting the appearance of the formulations F1-F6 in the Reconstitution Study 2.Table 6

[0454] Very foamy solutions in the Reconstitution Study 2 made it difficult to clearly determine end point of dissolution. In this experiment, a clear distinction was not made between the formulations Fl and F3, however bubbles in solution dissipated quicker in F3 compared to Fl. The cake of Formulation F6 (without trehalose and MoNi) took the longest time to dissolve.

[0455] The results of the Reconstitution Study 3 (-200 mg / mL IgG, 1.2 mL WFI addition) are presented in Table 7 below. FIG. 12 presents a set of photographs presenting the appearance of the formulations F1-F6 in the Reconstitution Study 3.Table 7

[0456] Formulation F3 dissolved the fastest. An extended reconstitution time in case of formulation Fl is due to a tiny amount of powder stuck to the bottom, which took longer to dissolve. The relative reconstitution times of formulations F3<F4 and F1<F4 indicates that both MoNi and trehalose can drive dissolution rate. Formulation F6 took longer to dissolve than F5. Formulation F5 formed a visibly clearer solution as compared to formulation F6.

[0457] The reconstitution times at each concentration are summarized in Table 8.Table 8

[0458] The relationship between the nature of the surfactant and the reconstitution time of 200 mg / mL of IgG formulation is depicted in FIG. 13. FIG. 13 is a graph showing the relative reconstitution times of certain high concentration IgG aqueous formulations with various surfactants as shown in Table 7 and the last column of Table 8 (Fl : “No Surfactant”; F2: polysorbate 20 (“PS20”); F3: “MoNi”). Exemplary solid IgG formulations were prepared by lyophilizing from a histidine buffer solution of 200 mg / mL IgG without surfactant, with 0.8 mg / mL of polysorbate 20, or with 20 mg / mL of MoNi. Reconstitution time was measured as time to complete dissolution of resulting lyophilized cakes into 200 mg / mL IgG aqueous formulations. All the formulations F1-F6 formed almost clear solutions, characterized by a very slight hue under light free of visible particles at all concentrations. Formulation F6 appeared slightly hazy at high concentrations.

[0459] The lyophilized cakes of IgG formulations were successfully reconstituted using WFI. All the formulations F1-F6 resulted in almost clear solutions (note: F6 appeared slightly hazy at high concentrations) with foam, free of any visible particles. The foam dissipated completely over time on standing for formulations containing MoNi / PS20.

[0460] The reconstitution time increased with increasing the concentration for all the formulations F1-F6. Formulation F6 showed the highest reconstitution time and formed slightly hazy solutions (haziness appeared to decrease partially over storage time), indicating that MoNi has substantial effect on dissolution.

[0461] At lower concentration (20 mg / mL IgG), it is difficult to delineate the effect of MoNi on reconstitution time, since the dissolution is very fast (1-2 mins) in formulations containing trehalose with and without MoNi.

[0462] At 100 and 200 mg / mL, the reconstitution time of formulation F3 was shorter than that of F4, and the reconstitution time of formulation Fl was shorter than that F4.

[0463] At higher concentration (200 mg / mL), formulation F3 showed the fastest dissolution. Extended time in case of formulation Fl can be assigned to a tiny amount of powder stuck to the bottom of the vial, which took longer to dissolve. Furthermore, extensive foaming made it difficult to accurately determine the endpoint of dissolution. Replicate readings are needed to determine if the difference is within expected vial to vial variability or there is true difference in the cake nature. Compared to IgG alone (formulation F6), IgG + MoNi (formulation F5) showed significantly faster dissolution and formed a clearer solution at high concentrations. Trehalose was also found to be a driver for dissolution (compare F4 to F5) with dissolution enhancement depending on the amount of trehalose (compare F3 to F4). However, MoNi was found to be an enhancer of dissolution in combination with trehalose, particularly when compared to another surfactant, PS20 (compare F3 to F2), or to no surfactant (compare F3 to Fl).

[0464] In summary, MoNi reduces the reconstitution time of high concentration formulations of IgG antibodies while a surfactant such as polysorbate 20 has little impact. MoNi is assessed for acceleration of reconstitution of other lyophilized mAb formulations.INCORPORATION BY REFERENCE

[0465] All publications, patents, patent applications, and other documents cited in this application, including published International Application No. WO 2021 / 211976,International Application No. PCT / US2023 / 023257, International Application No. PCT / US2023 / 023212, International Application No. PCT / US2023 / 027945, and U.S. Provisional Application Nos. 63 / 590,460, 63 / 601,888, and 63 / 644,982 are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

Claims

CLAIMS1. A storage stable solid composition comprising: a stabilizer; a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from N-(3- methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), A-hydroxyethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from A- [tri s(hydroxymethy Ilin ethyl ]acryl ami de (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), A-isopropyl acrylamide (NIP), N-N-di ethyl acrylamide (DEA), A-terLbutylacrylamide (TBA), A- phenyl acrylamide (PHE), and combinations thereof; and a biopharmaceutical agent.

2. The solid composition of claim 1, wherein the biopharmaceutical agent is not an antibody or antibody fragment.

3. The solid composition of claim 1 or 2, wherein the composition is capable of dissolution with water to an aqueous composition comprising:0.1 wt% to about 10 wt% of the copolymer; and0.5 wt% to 25 wt% of the biopharmaceutical agent.

4. The solid composition of any one of claims 1 to 3, wherein the solid composition (e.g., lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition that comprises, prior to drying (e.g., lyophilization), 0.1 mg / mL or more of the polyacrylamide-based copolymer.

5. The solid composition of claim 4, wherein the liquid composition comprises 0.3 mg / mL or more of the polyacrylamide-based copolymer.

6. The solid composition of claim 5, wherein the liquid composition comprises 1.0 mg / mL or more of the polyacrylamide-based copolymer.

7. The solid composition of claim 6, wherein the liquid composition comprises 3 mg / mL or more of the polyacrylamide-based copolymer.

8. The solid composition of claim 7, wherein the liquid composition comprises 10 mg / mL or more of the polyacrylamide-based copolymer.

9. The solid composition of any one of claims 1 to 8, wherein the biopharmaceutical agent is selected from a vaccine, gene therapy, cell therapy, and lipid nanoparticle (LNP) comprising a nucleic acid.

10. The solid composition of claim 9, wherein the biopharmaceutical agent is a vaccine.

11. The solid composition of claim 10, wherein the vaccine comprises a live or attenuated virus.

12. The solid composition of claim 10, wherein the vaccine is a mRNA vaccine.

13. The solid composition of any one of claims 1 to 12, wherein the solid composition demonstrates higher thermostability after 14 days at 37 °C as compared to a solid composition lacking the polyacrylamide-based copolymer.

14. The solid composition of any one of claims 1 to 8, wherein the biopharmaceutical agent is a polypeptide.

15. The solid composition of claim 14, wherein the polypeptide is a therapeutic protein.

16. The solid composition of claim 14 or 15, wherein the polypeptide is selected from antibodies and fragments thereof, antibody-drug conjugates, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof.

17. The solid composition of claim 15, wherein the therapeutic protein is selected from antibody or fragment thereof, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, enzyme, growth factor, hormone, interferon, interleukin, and thrombolytics protein.

18. The solid composition of claim 17, wherein the therapeutic protein is an enzyme.

19. The solid composition of claim 17, wherein the therapeutic protein is an antibody or a fragment thereof.

20. The solid composition of claim 19, wherein the therapeutic protein is a monoclonal antibody, a polyclonal antibody, an immunoglobulin G (IgG) antibody, an IgA antibody, an IgM antibody, a Fc fusion protein, or a fragment thereof.

21. The solid composition of claim 20, wherein the therapeutic protein is a monoclonal antibody.

22. The solid composition of any one of claims 19 to 21, wherein the solid composition (e.g., lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition that comprises, prior to drying (e.g., lyophilization), 1.0 mg / mL or more of the polyacrylamide-based copolymer (e.g., 2 mg / mL or more, 3 mg / mL or more, or 4 mg / mL or more).

23. The solid composition of claim 22, wherein the liquid composition comprises 10 mg / mL or more of the polyacrylamide-based copolymer.

24. The solid composition of any one of claims 14 to 23, wherein the solid composition demonstrates storage stability as compared to a composition lacking the polyacrylamidebased copolymer.

25. The solid composition of any one of claims 14 to 23, wherein the solid composition demonstrates a shorter reconstitution time as compared to a composition lacking the polyacrylamide-based copolymer.

26. The solid composition of any one of claims 1 to 14, wherein the water-soluble carrier monomer is selected from MORPH, MP AM, and combinations thereof.

27. The solid composition of claim 26, wherein the water-soluble carrier monomer comprises MORPH.

28. The solid composition of claim 26, wherein the water-soluble carrier monomer comprises MP AM.

29. The solid composition of any one of claims 1 to 28, wherein the functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

30. The solid composition of any one of claims 1 to 28, wherein the functional dopant monomer is selected from DEA, PHE, NIP, and combinations thereof.

31. The solid composition of any one of claims 1 to 28, wherein the functional dopant monomer comprises TRI.

32. The solid composition of claim 30, wherein the functional dopant monomer comprises PHE.

33. The solid composition of claim 30, wherein the functional dopant monomer comprises NIP.

34. The solid composition of claim 30, wherein the functional dopant monomer comprises DEA.

35. The solid composition of any one of claims 1 to 25, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from NIP, PHE, and combinations thereof.

36. The solid composition of any one of claims 1 to 25, wherein: the water-soluble carrier monomer is selected from MP AM, MORPH, and combinations thereof; and the functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

37. The solid composition any one of claims 1 to 25, wherein the water-soluble carrier monomer is MP AM, and the functional dopant monomer is PHE.

38. The solid composition any one of claims 1 to 25, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is PHE.

39. The solid composition of any one of claims 1 to 25, wherein the water-soluble carrier monomer is MORPH, and the functional dopant monomer is NIP.

40. The solid composition of any one of claims 1 to 39, wherein the copolymer comprises:70 wt% to 98 wt% of the water-soluble carrier monomer; and2 wt% to 30 wt% of the functional dopant monomer.

41. The solid composition of any one of claims 1 to 40, wherein the copolymer comprises:80 wt% to 95 wt% of the water-soluble carrier monomer; and5 wt% to 20 wt% of the functional dopant monomer.

42. The solid composition of any one of claims 1 to 40, wherein the copolymer comprises:83 wt% to 98 wt% of the water-soluble carrier monomer; and2 wt% to 17 wt% of the functional dopant monomer.

43. The solid composition any one of claims 1 to 25, wherein the copolymer comprises:70 wt% to 85 wt% of MORPH; and15 wt% to 30 wt% of NIP.

44. The solid composition of claim 43, wherein the copolymer comprises:74 wt% to 80 wt% of MORPH; and20 wt% to 26 wt% of NIP.

45. The solid composition of claim 44, wherein the copolymer comprises:77 wt% of MORPH; and23 wt% of NIP.

46. The solid composition of any one of claims 1 to 45, wherein a degree of polymerization of the copolymer is 10 to 500.

47. The solid composition of any one of claims 1 to 46, wherein a degree of polymerization of the copolymer is 20 to 200.

48. The solid composition of any one of claims 1 to 47, wherein a number-average molecular weight of the copolymer is 2,000 g / mol to 10,000 g / mol.

49. The solid composition of claim 48, wherein a number-average molecular weight of the copolymer is 2,000 g / mol to 6,000 g / mol.

50. The solid composition of claim 49, wherein a number-average molecular weight of the copolymer is 3,000 g / mol to 5,000 g / mol.

51. The solid composition of any one of claims 1 to 50, further comprising one or more additional components selected from a preservative and a surfactant.

52. The solid composition of any one of claims 1 to 51, wherein the stabilizer is a sugar.

53. The solid composition of claim 52, wherein the stabilizer sugar is trehalose.

54. The solid composition of any one of claims 51 to 53, wherein the one or more additional components are present in an amount ranging from about 0.1-50 % by weight.

55. The solid composition of any one of claims 51 to 53, wherein the one or more additional components are present in an amount ranging from about 0.001-1.0 % by weight.

56. The solid composition of any one of claims 1 to 55, wherein the solid composition is a lyophilized composition.

57. A pharmaceutical unit dosage composition comprising a solid composition according to any one of claims 1 to 56.

58. The pharmaceutical unit dosage composition of claim 57, wherein the composition is contained in a single dose container, vial, or pre-filled syringe.

59. A method of reconstituting a solid composition, the method comprising dissolving a solid composition according to any one of claims 1 to 56 in an aqueous solution to produce a reconstituted composition.

60. The method of claim 59, wherein the aqueous solution is sterile water.

61. The method of claim 59, wherein the aqueous solution comprises a buffering agent.

62. The method of any one of claims 59 to 61, further comprising centrifuging the reconstituted composition.

63. The method of any one of claims 59 to 62, further comprising removing particulates from the reconstituted composition.

64. The method of any one of claims 59 to 63, further comprising equilibrating the solid composition to room temperature before dissolution.

65. The method of any one of claims 59 to 64, further comprising assessing the reconstitution time for the dissolution of particulates.

66. The method of any one of claims 59 to 65, wherein the solid composition is a fixed dose of the biopharmaceutical agent comprised in a vial.

67. The method of any one of claims 59 to 66, wherein the reconstitution time is less than 30 min.

68. A reconstituted composition prepared according to the method of any one of claims 59 to 67.

69. A method of administering a biopharmaceutical agent to a subject in need thereof, the method comprising administering a reconstituted composition according to claim 64 to the subject.

70. A method of reconstituting a lyophilized composition, the method comprising dissolving a lyophilized composition according to any one of claims 1 to 56 in an aqueous solution to produce a reconstituted lyophilizate composition.

71. The method of claim 70, wherein the aqueous solution is sterile water.

72. The method of claim 70, wherein the aqueous solution comprises a buffering agent.

73. The method of any one of claims 70 to 72, further comprising centrifuging the reconstituted lyophilizate composition.

74. The method of any one of claims 70 to 73, further comprising removing particulates from the reconstituted lyophilizate composition.

75. A reconstituted lyophilizate composition prepared according to the method of any one of claims 70 to 74.

76. A method of administering a biopharmaceutical agent to a subject in need thereof, the method comprising administering a reconstituted lyophilizate composition according to claim 75 to the subject.

77. A method of preparing a solid composition according to any one of claims 1 to 56, the method comprising: exposing a liquid biopharmaceutical composition comprising: a polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from N-(3- methoxypropyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), A-hydroxyethyl acrylamide (HEAM), acrylamide (AM), and combinations thereof; and a functional dopant monomer selected from A-[tri s(hydroxy methyl )- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N- isopropyl acrylamide (NIP), N-N-diethylacrylamide (DEA), N-tert- butyl acrylamide (TBA), A-phenylacrylamide (PHE), and combinations thereof; and a biopharmaceutical agent; to a drying cycle to produce the solid composition.

78. The method of claim 77, wherein the drying is selected from lyophilization, spray drying, spray freezing drying, foam mat drying, vacuum oven drying, and desiccation drying.

79. The method of claim 77 or 78, wherein the liquid biopharmaceutical composition is exposed to a lyophilization cycle in a lyophilizer to produce a lyophilized composition.

80. The method of claim 79, wherein the lyophilization cycle comprises: subjecting the liquid biopharmaceutical composition to a first pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy; and cooling the liquid biopharmaceutical composition to freeze any remaining liquid component into a solid.

81. The method of claim 79 or 80, wherein the lyophilization cycle comprises: subjecting the liquid biopharmaceutical composition to a first reduced temperature; subjecting the liquid biopharmaceutical composition to a first reduced pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy;subjecting the liquid biopharmaceutical composition to a second reduced pressure that is less than the first reduced pressure; and evaporating the remainder of the liquid to produce the solid composition.

82. The method of claim 77, wherein the drying cycle is a spray drying cycle comprising: atomizing the liquid biopharmaceutical composition; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition; and collecting the solid composition.

83. The method of claim 77, wherein the drying cycle is a spray freeze drying cycle comprising: atomizing the liquid biopharmaceutical composition; freezing the atomized liquid biopharmaceutical composition; evaporating at least a portion of a liquid component of the atomized liquid biopharmaceutical composition; and collecting the solid composition.

84. The method of claim 77, wherein the drying cycle is a foam mat drying cycle comprising: foaming the liquid biopharmaceutical composition; spreading the foamed liquid biopharmaceutical composition onto a drying surface; evaporating at least a portion of a liquid component of the foamed liquid biopharmaceutical composition; and collecting the solid composition.

85. The method of claim 77, wherein the drying is a vacuum oven drying cycle comprising: subjecting the liquid biopharmaceutical composition to reduced pressure; evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition while adding thermal energy; and returning the pressure to the original pressure to obtain the solid composition.

86. The method of claim 77, wherein the drying is a desiccation drying cycle comprising: increasing the surface area of the liquid biopharmaceutical composition, e.g., by pouring the liquid biopharmaceutical composition into a tray;evaporating at least a portion of a liquid component of the liquid biopharmaceutical composition by circulating ambient air across the surface, wherein the circulated air is optionally dried over a desiccating material, heated, or both until the solid is obtained.

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