Solid polyacrylamide polymer biopharmaceutical formulations

Abstract

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

Description

Background Technology

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

[0002] Antibodies have shown great promise in modern drug therapy. These biopharmaceuticals tend to aggregate in formulations, thus requiring low concentrations and / or leading to poor stability. Although the aggregation mechanisms between antibodies vary slightly, many antibodies contain hydrophobic plaques and / or can partially denature to expose hydrophobic segments of the molecule, which drive the adsorption of these proteins onto interfaces (e.g., air-water, glass-water, or rubber-water interfaces). The high local concentration of protein molecules at these interfaces, coupled with interface-mediated partial unfolding, can trigger the initial nucleation of aggregation events, creating conditions for further aggregation. This tendency for antibody aggregation at interfaces generally increases with increasing formulation concentration, negatively impacting the overall stability of the formulation.

[0003] Vaccines have become indispensable in human healthcare, significantly reducing the morbidity and mortality rates of common infectious diseases and thus enabling the effective control of many infectious diseases. In recent years, with increased understanding of the mechanisms related to the immune system, vaccine development has achieved remarkable success in routine prevention and treatment, and has begun to be applied to the treatment of HIV, cancer, and other diseases. However, the refrigeration and transportation of vaccine formulations remain a challenge. Freezing and freeze-drying of vaccine formulations can damage the vaccine's structure and / or reduce its immunogenicity.

[0004] Many commercial excipients have been used in an attempt to overcome the challenges associated with biopharmaceutical formulations. These systems are often still limited by their critical micelle concentration, the toxicity that may result from oxidative degradation, and undesirable interactions between the excipient and the supported substance in the bulk phase.

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

[0006] Overview

[0007] This disclosure provides solid compositions comprising a biopharmaceutical agent (such as an antibody or vaccine) and a polyacrylamide-based copolymer. The inventors have demonstrated that specific polyacrylamide-based copolymers can be used as stabilizing excipients or surfactants in pharmaceutical compositions to maintain the stability of the biopharmaceutical agent during and / or after lyophilization. The results presented herein indicate that the polyacrylamide-based copolymers of this disclosure can generally be used to provide significant stability benefits for compositions of a variety of biopharmaceutical agents. Solid compositions (e.g., lyophilized compositions) also provide homogeneous reconstitution of liquid pharmaceutical compositions in pharmaceutically acceptable solutions, provide recovery of the active ingredient, minimize partial loss of potency, and allow for accurate and consistent administration of the active biopharmaceutical agent. In some embodiments, the polyacrylamide-based copolymer stabilizes the composition of the biopharmaceutical agent at concentrations of the biopharmaceutical agent that would be impossible in compositions lacking a polyacrylamide-based copolymer.

[0008] The compositions of this disclosure, comprising polyacrylamide-based copolymers, are readily lyophilized to produce lyophilized compositions having one or more improved properties, such as increased stability and / or reconstitution time. In some embodiments, the compositions of this disclosure can undergo rapid freeze-drying while retaining the bioactivity of the biopharmaceutical reagent. In some embodiments, the compositions of this disclosure can undergo multiple freeze-thaw cycles.

[0009] Some aspects of this disclosure relate to reconstituted liquid compositions produced by dissolving a lyophilized pharmaceutical composition in an aqueous liquid (e.g., sterile water). In some embodiments, the reconstituted composition retains the biopotency and other desired characteristics of the biological pharmaceutical agent (e.g., a vaccine), including viral activity, immunogenicity, and / or stability.

[0010] In some embodiments, the biopharmaceutical agent is an antibody or antibody-drug conjugate (ADC), and the polyacrylamide-based copolymer prevents the antibody from adsorbing onto the interface of the composition, thereby preventing undesirable aggregation events and maintaining the binding activity of the antibody. In some embodiments, the lyophilized compositions of this disclosure are reformulated at antibody or ADC concentrations suitable for subcutaneous (SC) injection into patients, which can be performed under resource-scarce conditions, in contrast to, for example, intravenous administration.

[0011] The lyophilized biopharmaceutical compositions disclosed herein offer advantages over conventional formulations due to their ease of transport, storage, and dispensing.

[0012] Methods of using the biopharmaceutical composition are also provided, including methods of administering the reconstituted composition to human subjects in need by injection, such as subcutaneous injection. Brief description of the attached diagram

[0014] Figure 1 This is a set of schematic diagrams showing an overview of the use of novel copolymer excipients to stabilize high-concentration antibody formulations. (Figure A) Schematic overview of current routes of administration for biologic therapies, with intravenous administration being the most common due to the limited stability of biologics at the high concentrations required for subcutaneous administration, resulting in a high burden on patients. (Figure B) Amphiphilic acrylamide carrier / dopane copolymer (AC / DC) excipients (e.g., poly(acryloylmorpholine-co- N A diagram of (-isopropylacrylamide)(MoNi) is shown, whereby the excipient can be used to stabilize a biopharmaceutical in a formulation. (Figure C) illustrates the use of an AC / DC copolymer as a "drop-in" excipient to stabilize a monoclonal antibody in a formulation. The AC / DC copolymer is amphiphilic and preferentially adsorbs onto the interface, preventing interfacial adsorption of the antibody in the formulation, thereby preventing interface-related destabilization and aggregation events.

[0015] Figure 2 This is a set of schematic diagrams and graphs showing the synthesis and characterization of the MoNi copolymer excipient. (Figure A) Synthetic scheme for poly(acryloylmorpholine-co-N-isopropylacrylamide) (MoNi). (Figure B) Size exclusion chromatography (SEC) characterization of the MoNi-CTA intermediate and (Figure C) MoNi demonstrates that CTA was completely removed in the second step of the synthesis, resulting in a chemically and physically stable copolymer. (Figure D) MoNi... 1 1H-NMR characterization. (Figure E) Comparison of cytotoxicity of MoNi, polysorbate 80 (PS80), and Pluronic L61. (Figure F) DLS characterization of MoNi in solutions at various concentrations showed that the MoNi copolymer did not exhibit critical micelle concentration (CMC) behavior even at high concentrations. (Figure G) Comparison of reported CMC values ​​of PS80 and Pluronic L61.

[0016] Figure 3 This is a set of graphs showing the interfacial interactions of the MoNi polymer excipient in mAb formulations. (Figure A) Comparison of PGT121 (0.2 mg / mL) with and without MoNi (0.1 mg / mL) in time-resolved surface tension measurements (n=3). (Figure B) Interfacial rheological measurements of PGT121 (0.2 mg / mL) with and without MoNi (0.1 mg / mL) (n=3). (Figure C) High shear rate viscosity measurements of high-concentration PGT121 formulations (119 mg / mL) with and without MoNi (10 mg / mL) collected at steady state by viscometer experiments.

[0017] Figure 4 Here is a set of diffusion-ordered (DOSY) nuclear magnetic resonance (NMR) spectra: (Fig. A) MoNi alone (0.1 mg / mL), (Fig. B) PGT121 alone (0.2 mg / mL), and (Fig. C) a co-formulation of PGT121 and MoNi, with concentrations of 0.2 mg / mL and 0.1 mg / mL, respectively.

[0018] Figure 5 This is a set of schematic diagrams and graphs showing the stabilizing effect of MoNi on PGT121. (Figure A) Schematic diagram of accelerated aging, in which the formulation is packaged in glass vials, stirred, and heated. (Figure B) Percentage of monomer composition in the aged samples as determined by GPC. (Figure C) Schematic diagram of epitope binding as determined by ELISA. (Figure D) Half-maximum inhibitory concentration (IC50) of the aged formulations containing and without MoNi. 50 The sample was tested in duplicate.

[0019] Figure 6 This is a set of plots and graphs showing the pharmacokinetics of the MoNi-stabilized PGT121 formulation. (Figure A) Experimental protocol, reporting the injection and blood collection timelines in hFcRn mice for comparing IV administration of a low-concentration PGT121 formulation (5 mg / mL) with SC administration of a high-concentration PGT121 formulation (119 mg / mL) stabilized by MoNi. (Figure B) Serum concentrations of PGT121 over time as determined by ELISA (n = 6 / group). (Figure C) Area under the curve analysis of ELISA data (n = 6 / group).

[0020] Figure 7 (Figures A-C) is a set of graphs showing the change in mouse body weight over time after treatment.

[0021] Figure 8 This is a graph showing the percentage of monomer composition in 55 mg / mL PGT121 aged samples, with or without MoNi, as determined by SEC.

[0022] Figure 9 This is a set of curves showing representative size exclusion chromatograms of PGT121 with and without MoNi after 6 days of stress aging. (Figure A) SEC chromatogram of PGT121 without MoNi. (Figure B) SEC chromatogram of PGT121 with 1 mg / mL MoNi. (Figure C) SEC chromatogram of PGT121 with 10 mg / mL MoNi.

[0023] Figure 10This is a set of photographs showing the appearance of approximately 20 mg / mL IgG formulations F1-F6 reconstituted from an exemplary solid composition containing MoNi in a comparative study of reconstitution time (see Study 1, Example 8, Experimental Section). F1: 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.

[0024] Figure 11 This is a set of photographs showing the appearance of approximately 100 mg / mL IgG formulations F1-F6 reconstituted from an exemplary solid composition containing MoNi in a comparative study of reconstitution time (see Study 2, Example 8, Experimental Section). F1: 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.

[0025] Figure 12 This is a set of photographs showing the appearance of approximately 200 mg / mL IgG formulations F1-F6 reconstituted from an exemplary solid composition containing MoNi in a comparative study of reconstitution time (see Study 2, Example 8, Experimental Section). F1: 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.

[0026] Figure 13 This is a graph showing the relative reconstitution times of high-concentration (200 mg / mL) aqueous formulations of IgG containing the various surfactants described herein (F1 "surfactant-free"; F2 polysorbate 20 ("PS20"); F3: MoNi). Exemplary solid IgG formulations were prepared by lyophilization from histidine buffer solutions of 200 mg / mL IgG containing either surfactant-free polysorbate 20 or 20 mg / mL MoNi.

[0027] Detailed description

[0028] Solid pharmaceutical compositions

[0029] As summarized above, this disclosure relates to solid compositions, such as lyophilized compositions, comprising a biopharmaceutical reagent and a polyacrylamide-based copolymer, prepared by removing the solvent from the corresponding liquid composition. It has been found that when the biopharmaceutical reagent is formulated according to this disclosure, the degradation of the biopharmaceutical reagent is delayed; therefore, such solid formulations are biochemically and physically stable 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 reagent. In some embodiments, the solid composition is a lyophilized composition.

[0030] The storage-stable compositions of this disclosure exhibit less than 10 mol% degradation of the biopharmaceutical formulation's activity after storage at 5°C ± 2°C (e.g., in a sealed container) 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), 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%. It should be understood that stability can be assessed at a variety of suitable storage temperatures.

[0031] In some embodiments, the storage-stabilized compositions of this disclosure exhibit a degradation of less than 10 mol% of the bioactivity of the biopharmaceutical reagent after storage in a sealed container at 25°C ± 2°C 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), for example, a degradation of 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% of the bioactivity of the biopharmaceutical reagent initially present in the composition before storage.

[0032] In some embodiments, the composition is stored in a sealed container. In other embodiments, the composition is in unit dosage form in a container, such as a vial. The bioactivity or various other physical or chemical properties of the biopharmaceutical reagent can be evaluated to determine storage stability.

[0033] 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 composition provides a higher concentration and stability of the biopharmaceutical agent compared to conventional or currently approved formulations. In some embodiments, the biopharmaceutical agent is a therapeutic antibody, and the high concentration is, for example, 5 wt% or more of the composition, such as 7 wt%, 10 wt%, 12 wt%, 15 wt%, 20 wt%, or 25 wt% or more; or 5 wt%, 7 wt%, 10 wt%, or 12 wt% to 15 wt%, 20 wt%, 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 associated 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.

[0034] In some embodiments, the storage-stable solid composition is soluble in water to produce a reconstituted aqueous composition comprising: 0.1 wt% to about 10 wt% of a copolymer; and 0.5 wt% to 25 wt% of a biopharmaceutical reagent.

[0035] The method for preparing the solid composition according to the present disclosure is suitable for use with lyophilization, spray drying, spray freeze-drying, foam pad drying, vacuum drying, vacuum oven drying, freeze concentration or dehydration techniques commonly used in the pharmaceutical industry.

[0036] In some embodiments, the solid compositions of this disclosure are produced by lyophilizing the liquid compositions described herein. The liquid compositions may be lyophilized under standard lyophilization conditions known in the art or under conditions modified thereto. For example, details of the lyophilization technique and other suitable excipients can be found in Cameron et al., “Good Pharmaceutical freeze-drying Practice,” Interpharm, Buffalo Grove (1997).

[0037] In some embodiments, the solid compositions of this disclosure are produced by spray drying of the liquid compositions described herein. The liquid compositions can be spray dried under standard spray drying conditions or modifications thereof known in the art. In some embodiments of this method, the spray drying cycle involves atomizing a biopharmaceutical reagent solution into fine droplets and using a stream of air or inert gas (optionally heated) to evaporate the solvent. In some embodiments, spray drying is a rapid method and is suitable for large-scale production. A preferred spray drying cycle according to this disclosure is a spray drying process in which the liquid composition is pumped into a nozzle in the presence or absence of coaxially supplemented pressurized air (or inert gas) to obtain tiny droplets of the composition. Such droplets can be formed in a large-area ventilated area (temperature between +20°C and +250°C), thereby achieving rapid drying via an evaporation process. For example, a solid composition can be obtained after cyclone separation.

[0038] In some embodiments, the solid compositions of this disclosure are produced by spray freeze-drying of the liquid compositions described herein. The liquid compositions can be spray-dried under standard spray drying conditions or modifications thereof known in the art. In embodiments of this method, the liquid compositions are obtained by pumping them into a nozzle with the coaxial addition or supplementation of pressurized air (or an inert gas). The droplets can be formed in a very cold chamber (between -5°C and -195.8°C), thereby achieving rapid freezing of the droplets. Subsequent drying of such frozen materials can be obtained by a standard freeze-drying process (optionally under vacuum).

[0039] In some embodiments, the solid compositions of this disclosure are produced by aerosol drying of the liquid compositions described herein. Aerosol drying of the liquid compositions can be performed under standard aerosol drying conditions or modifications thereof known in the art.

[0040] In some embodiments, the solid composition of this disclosure is produced by dehydrating the liquid composition described herein. The liquid composition can be dehydrated under standard dehydration drying conditions or modifications thereof known in the art. In some embodiments, the method includes placing the protein solution in a desiccator, which is a sealed container containing a desiccant such as silica gel or other dehydrating agent. The desiccant absorbs moisture from the solution, leaving a solid composition.

[0041] In some embodiments, the solid compositions of this disclosure are produced by freeze-concentration of the liquid compositions described herein. The liquid compositions can be freeze-concentrated under standard conditions or modifications thereof known in the art. Any remaining concentrated protein solution can be dried by additional means, such as air drying, freeze-drying, etc.

[0042] In some embodiments, the solid compositions of this disclosure are produced by drying the liquid compositions described herein in a vacuum oven. The liquid compositions can be vacuum-dried under standard conditions or modifications thereof known in the art. 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 preferred for proteins stable at moderate temperatures.

[0043] In some embodiments, the solid compositions of this disclosure are produced by drying the liquid compositions described herein on a foam pad. The liquid compositions can be foamed and dried under standard conditions or modifications thereof known in the art. This method involves generating foam from a protein solution and drying it on a pad. The foam typically provides a large surface area for drying, and the resulting solid compositions can be readily rehydrated.

[0044] As used herein, the term "liquid composition" refers to a composition in liquid form that comprises a biopharmaceutical reagent and at least one pharmaceutically acceptable excipient, such as a polyacrylamide-based copolymer, and that can be lyophilized to produce the lyophilized pharmaceutical composition described herein.

[0045] As used herein, the terms "solid composition" or "solid pharmaceutical composition" refer to any composition or pharmaceutical composition in solid form. Solid form can be amorphous or crystalline. In some embodiments, the solid composition is in a dried form, prepared from a liquid composition by one of the drying methods described herein (such as lyophilization). The dried solid compositions of the present invention can be reconstituted for injection.

[0046] As used herein, the terms "lyophilized composition" or "lyophilized pharmaceutical composition" refer to any composition or pharmaceutical composition prepared in a dried form by lyophilization. "Lyophilizing" or "lyophilization" has the meaning understood by those skilled in the art, broadly referring to any process of dehydration under vacuum after freezing. Lyophilized compositions can be reconstituted for injection.

[0047] Freeze-drying is a process of removing water from a substance (e.g., a biopharmaceutical reagent) using freezing temperatures and low pressure. In an exemplary freeze-drying process, the material to be freeze-dried is cooled below its triple point, typically between -50°C and -80°C. Once the material is frozen, the ambient pressure is reduced, and sufficient heat is added to cause the ice to sublimate. In a second drying stage, additional heat is added to remove unfrozen water molecules. Upon completion, the freeze-dried material has a residual moisture content of less than 5%, typically less than 3%, and usually in the range of about 0.5% to about 3%.

[0048] A lyophilization method is described in US 7,888,097 (the disclosure of which is incorporated herein by reference). In one aspect, the lyophilized compositions of this disclosure have a residual moisture content of less than 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In another aspect, the lyophilized compositions of this disclosure have a residual moisture content between about 0.5% and about 5%. In another aspect, the lyophilized compositions of this disclosure have a residual moisture content between about 0.5% and about 3%. In another aspect, the lyophilized compositions of this disclosure have a residual moisture content between about 0.5% and about 1%. With respect to residual moisture content, "about" as used herein means a variation of no more than 10% of the cited value.

[0049] A method for lyophilizing the liquid composition of this disclosure may include: (a) providing a container containing a liquid composition having an initial temperature of about 5°C to about -50°C; (b) cooling the composition to a sub-zero temperature below the initial temperature (e.g., -10°C to -50°C); and (c) thoroughly drying the composition. The lyophilization conditions of the formulations disclosed herein, such as temperature and duration, can be adjusted by those skilled in the art taking into account factors affecting the lyophilization parameters (e.g., the type of lyophilizer used, the amount of biopharmaceutical reagent used, and the size of the container used).

[0050] In some embodiments, the steps of (b) cooling the composition and (c) drying the composition are performed simultaneously with the temperature change. For example, the step of (b) cooling the composition may include raising the temperature 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 may include lowering the temperature 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 lowering the temperature of the composition and then raising its temperature.

[0051] Some amorphous products (such as mannitol or glycine) form incompletely crystalline metastable glasses upon initial freezing. These products can benefit from a heat treatment process, also known as annealing. During annealing, the product temperature is cycled (e.g., from -40°C to -20°C, held for several hours, then returned to -40°C; from -50°C to -20°C, held for two (2) hours, then a vacuum is introduced; or from -50°C to -20°C, then returned to -50°C) to achieve more complete crystallization. Annealing has the additional advantage of greater crystal growth and correspondingly shorter drying times. Moisture trapped in the amorphous phase can be further removed during a second drying process.

[0052] In some embodiments, the step of drying the composition (c) is performed in two steps—(i) primary drying and (ii) secondary drying.

[0053] In some embodiments, primary drying includes maintaining the temperature, or raising or lowering the temperature. 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 lowering the temperature. In some embodiments, secondary drying includes raising the temperature 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, and from -10°C to 10°C.

[0054] In some implementations, a secondary drying process continues until the product has a moisture content acceptable for long-term storage. Depending on the application, the moisture content of a fully dried product is typically between 0.5% and 3%. In most cases, the drier the product, the longer its shelf life. However, some complex biological products may become too dry to achieve optimal storage, and therefore the secondary drying process should be controlled accordingly.

[0055] The secondary drying step may include raising the temperature 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, and from -10°C to 10°C.

[0056] As used herein, the term "reconstituted" or "reconstituted" refers to restoring a substance previously altered for preservation and storage to its original form, such as rehydration, i.e., restoring a previously lyophilized and stored DNA plasmid formulation to a liquid state. The lyophilized compositions of this disclosure can be reconstituted in any aqueous solution that produces a stable solution suitable for drug 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 physiological saline.

[0057] Another attribute of lyophilized products that can affect the quality of biopharmaceutical reagents is the turbidity of the reconstituted composition obtained by dissolving the lyophilized composition. The turbidity of the reconstituted biopharmaceutical reagent may be related to the recovery rate of the active ingredient in the lyophilized composition. Generally, complete dissolution (i.e., low turbidity of the reconstituted composition) is preferred. Incomplete dissolution can lead to waste of the active ingredient in the pharmaceutical composition and clogging of the syringe used to administer the reconstituted composition.

[0058] The turbidity of the reconstituted biopharmaceutical reagent can be measured visually or by measuring absorbance at specific wavelengths (e.g., 450 nm and 650 nm). Absorbance can be measured using equipment available in the art (e.g., Molecular Devices ThermoMAX microplates).

[0059] Reconstitution time is another factor that can be related to drug quality. Generally, a shorter reconstitution time is preferred. If the biopharmaceutical reagent is not completely dissolved, prolonged reconstitution time at the user stage may result in partial loss of efficacy, as in-line filters are typically used during patient administration. The time required for reconstitution of a lyophilized product can be determined, for example, by measuring the turbidity of the reconstituted biopharmaceutical reagent at different time points after reconstitution. For example, turbidity can be measured after 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, or longer.

[0060] Residual moisture after lyophilization can also be important. Various methods known in the art can be used to measure residual moisture in lyophilized compositions. For example, the Karl Fisher coulometric gauging system C20 (Mettler Toledo) can be used for moisture content analysis. Localized pockets of moisture in a lyophilized composition can lead to greater instability and increased product degradation. Therefore, a lower moisture content may be preferred.

[0061] Concentration measurements of biopharmaceutical reagents are performed using various methods known in the art. For example, the ultraviolet absorbance of biopharmaceutical reagents can be measured using Solo-VPE.

[0062] Other components of the compositions of this disclosure will now be described in more detail. It should be understood that the components and amounts of the lyophilized composition may be described based on the lyophilized solid composition, the liquid composition from which the lyophilized composition of this disclosure is derived, or in some cases based on a reconstituted composition 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 volumes and concentrations of biopharmaceutical reagents.

[0063] Characterization of the composition

[0064] This disclosure provides solid pharmaceutical compositions having one or more desired properties as pharmaceutical products. In some embodiments, the solid pharmaceutical composition is a lyophilized pharmaceutical composition. The properties may include the stability and potency of the active ingredient biopharmaceutical reagent under various storage conditions, cake formation, homogeneous reconstitution of the formulation for administration, and less contamination. The various properties provided herein can be used to select a preferred solid composition or to determine ideal storage conditions for the solid composition.

[0065] The stability of biopharmaceutical agents in solid compositions (e.g., lyophilized compositions) 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 the biopharmaceutical agent can be measured under various conditions (e.g., 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 integrity of the biopharmaceutical agent can also be determined before, during, or after storage at different temperatures to select stable solid compositions and determine ideal storage conditions.

[0066] The appearance of the cake can be another important attribute of the dried product. In some embodiments, the dried product is a lyophilized product. Uniform and aesthetically pleasing cakes are generally preferred. An undesirable cake appearance can affect product quality, for example, making it difficult to assess product quality based on visual inspection, or making it difficult to recover the full amount of active ingredient in the container. Furthermore, partial or complete remelting of the cake can lead to instability and degradation of the active ingredient. Remelting is a form of cake collapse caused by a solid-to-liquid transition. That is, incomplete sublimation (from solid to gas) occurs in the vial. These changes can include alterations in the physical form of the biopharmaceutical reagent and the formation of localized moisture pools.

[0067] The appearance of the biscuit can be determined through visual inspection, which may involve taking photographs. Visual inspection can be largely based on historical precedent. A robust visual inspection verification procedure is crucial before judging product quality. Verification procedures can be based on past experience or published information, including FDA visual inspection guidelines for specific drugs.

[0068] In some embodiments, the impurity level at the initial time point after drying can be determined by HPLC or other analytical techniques. In some embodiments, drying is lyophilization. The vials can then be placed in a stability test chamber under different storage conditions. In some embodiments, storage conditions include storage for a period of time in a sealed container at 25°C, 37°C, 40°C, or 60°C, or equivalent temperatures. In some embodiments, storage conditions include storage at specific temperature and relative humidity (RH) (e.g., 25°C / 60% RH or 40°C / 75% RH).

[0069] In some embodiments, the solid composition exhibits higher thermal stability after 14 days or longer at 37°C compared to a dried composition lacking a polyacrylamide-based copolymer. In some embodiments, the lyophilized composition exhibits higher thermal stability after 14 days or longer at 37°C compared to a lyophilized composition lacking a polyacrylamide-based copolymer.

[0070] To determine the formation of impurities and the stability of the biopharmaceutical reagent in the solid composition according to this disclosure, vials are removed from the stability test chamber at different time points (e.g., 45 hours, 1 month, 2 months, etc.) and analyzed using various assays and techniques (e.g., HPLC or other analytical techniques known in the art), such as by potency assay.

[0071] Biological drug reagents

[0072] Biological pharmaceutical reagents can be formulated at a weight percentage and / or volume suitable for injection (e.g., SC or IM) of a unit dose to a patient in need. In some embodiments, the compositions of the present invention are formulated to contain a therapeutically effective amount of the biological pharmaceutical reagent.

[0073] In some embodiments, the biopharmaceutical agent has a high molecular weight (MW), such as a macromolecular biological product. In some embodiments, a high MW biopharmaceutical agent is a biopharmaceutical agent with a MW of 20 kDa or higher (such as 30 kDa or higher, 40 kDa or higher, 50 kDa or higher, 60 kDa or higher, 70 kDa or higher, 80 kDa or higher, 90 kDa or higher, or 100 kDa or higher).

[0074] In some embodiments, the solid composition (e.g., a lyophilized composition) comprises 0.5 wt% to 25 wt%, or 0.5 wt% to 20 wt% of a biopharmaceutical agent, such as 1 wt% to 20 wt% (e.g., 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% to 12 wt%, 2 wt% to 10 wt%, 2 wt% to 8 wt%, 2 wt% to 5 wt%, 3 wt% to 15 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt% to 15 wt%, 5 wt% to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 15 wt%). The composition comprises 7 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, 10 wt% to 15 wt%, or 10 wt% to 12 wt% of a biopharmaceutical agent. In some embodiments, the solid composition comprises 7 wt%, 8 wt%, 9 wt%, or 10 wt% of a biopharmaceutical agent. In such cases, the remainder of the composition may consist of a copolymer based on polyacrylamide and / or one or more optional components (such as stabilizers, e.g., as described herein).

[0075] In some embodiments, the solid composition is soluble in water to produce a reconstituted aqueous composition comprising: 0.1 wt% to about 10 wt% of a copolymer (e.g., as described herein); and 0.5 wt% to 25 wt% of a biopharmaceutical reagent (e.g., as described herein).

[0076] In some embodiments, the solid composition (e.g., a lyophilized composition) comprises 1 wt% to 20 wt% (e.g., 1 wt% to 10 wt%) of a biopharmaceutical agent. In such cases, the remainder of the composition may consist of a copolymer based on polyacrylamide and / or one or more optional components (such as stabilizers (e.g., as described herein)).

[0077] In some implementations, the biopharmaceutical agent is selected from vaccines, gene therapy, cell therapy, and lipid nanoparticles (LNPs) containing nucleic acids (e.g., mRNA).

[0078] In some embodiments, the biopharmaceutical agent is a vaccine composition or an immunogenic composition. The phrase "immunogenic composition" refers to a target antigen or immunogen expressed from a vector, and includes any composition that, upon administration to a subject, elicits an immune response against the target immunogen or target antigen. The phrase "vaccine composition" refers to a composition that induces a protective immune response against a target antigen or provides effective protection against the antigen. This also includes any composition that, upon administration or injection to a subject, elicits a protective immune response against a target antigen or immunogen, or any composition that provides effective protection against an antigen or immunogen expressed from a vector.

[0079] In some embodiments, the biological agent is a vaccine. In some embodiments, the vaccine comprises a live virus or an attenuated virus. In some embodiments, the vaccine is an inactivated virus. In some embodiments, the vaccine is an mRNA vaccine.

[0080] In some embodiments, the biopharmaceutical agent is a composition containing mRNA in a delivery system, such as an LNP containing an mRNA load. Such mRNA compositions have therapeutic applications as viral vaccines, protein replacement therapies, cancer immunotherapy, cell reprogramming, and genome editing.

[0081] In some implementations, the biopharmaceutical agent is a therapeutic cell composition, such as a cancer immunotherapy composition.

[0082] In some implementations, the biopharmaceutical agent is a gene therapy composition.

[0083] In some embodiments, the biopharmaceutical reagent is a polypeptide. In some embodiments, the polypeptide readily aggregates in an aqueous medium. In some embodiments, the polypeptide is a protein. In some embodiments, the polypeptide is a peptide.

[0084] In some implementations, 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.

[0085] In some embodiments, the peptide is a therapeutic protein. In some embodiments, the therapeutic protein is selected from antibodies or fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic proteins.

[0086] In some implementations, the biopharmaceutical reagent is an antibody or antibody fragment.

[0087] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, but are 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.

[0088] In some embodiments, the biopharmaceutical agent is an antibody or a fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is monospecific, i.e., it binds to a single antigen. In some embodiments, the monoclonal antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgM antibody, and an IgA antibody, or any hybrid thereof.

[0089] In some implementations, the antibody is a chimeric antibody. The term "chimeric antibody" refers to an antibody containing one or more regions from one antibody and one or more regions from one or more other antibodies.

[0090] In some implementations, antibodies are multispecific, meaning they bind to multiple antigens, such as bispecific antibodies.

[0091] In some embodiments, the antibody is an antibody fragment. An "antibody fragment" includes a portion of a complete antibody, such as the antigen-binding region or variable region of a complete antibody. Antibody fragments suitable for the compositions of the present invention include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0092] In some embodiments, the biopharmaceutical agent is a chimeric protein. In some embodiments, the chimeric protein is a recombinant fusion protein. A chimeric protein may include two or more domains linked by optional linkers or spacer regions. In some embodiments, the chimeric protein comprises an antibody fragment, such as an Fc fragment or a variant thereof. In some embodiments, the chimeric protein comprises an antibody fragment fused to a protein domain (e.g., a protein domain of a therapeutic target that specifically binds to the biopharmaceutical agent). In some embodiments, the chimeric protein is an Fc fusion protein.

[0093] In some implementations, the antibody is a single-domain antibody, such as a camel antibody or a nanobody.

[0094] In some embodiments, the antibody is an antibody-drug conjugate, for example, an antibody conjugated to one or more heterologous molecules. The heterologous molecule can be a small molecule (e.g., an organic compound with a molecular weight 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 cell inhibitor.

[0095] In some implementations, the antibody is a bispecific antibody-immunoconjugate.

[0096] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody comprises 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 mouse antibody, or a chimeric (mouse / human) antibody.

[0097] Antibodies can target a variety of target proteins, including therapeutic target proteins.

[0098] In some embodiments, the molecular weight of the antibody is from about 100 kDa to about 200 kDa, for example, from about 120 kDa to about 180 kDa. In some embodiments, the molecular weight of the antibody is about 150 kDa. In some embodiments, the molecular weight of the antibody is from 100 kDa to 200 kDa, for example, from 120 kDa to 180 kDa. In some embodiments, the molecular weight of the antibody is 150 kDa. In some embodiments, the molecular weight of the antibody is 100 kDa or less, such as from 40 kDa to 80 kDa, for example, about 50 kDa.

[0099] antibody composition

[0100] This disclosure includes compositions comprising a polyacrylamide-based copolymer and a biopharmaceutical agent as an antibody. Incorporating the polyacrylamide-based copolymer into the composition can prevent or reduce antibody aggregation, thereby maintaining the antibody's biological activity, such as specific binding to antigens.

[0101] As used herein, the term "association state" is used to describe the degree of antibody aggregation when referring to antibodies. For example, a non-aggregated antibody present in a composition may be referred to as a monomeric antibody, or an antibody present in a monomeric associated state. In another instance, an aggregate of two or more antibodies present in a composition may be referred to as an aggregated antibody, or an antibody present in an aggregated associated state.

[0102] The term "substantially monomer-associated" as used when referring to antibody compositions means a composition 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 antibody is present in a monomer-associated state. The term "stability" as used when referring to antibody compositions refers to the ability of a composition to retain at least a portion of the binding activity of the antibody therein over time. For example, in some embodiments, a "stable" composition may retain 70% or more (e.g., 80% or more, 90% or more, or 95% or more) of the binding activity of the antibody therein (e.g., based on a half-maximum inhibitory concentration determined by enzyme-linked immunosorbent assay) after continuous stress aging for 21 days or longer.

[0103] In some embodiments, the solid composition may be reconstituted and adapted for subcutaneous injection, such as aseptic injection. In some embodiments, the reconstituted composition is formulated for subcutaneous injection and has a high concentration of antibody (e.g., 5 wt% or higher as described above) and one or more physical properties suitable for injection under clinically relevant injection conditions (e.g., viscosity of 0.2 Pa·s·m or lower, such as 0.15, 0.1, or 0.05 Pa·s·m).

[0104] In some embodiments, the reconstituted composition has a surface tension of 60 nM / m or less (e.g., 55, 50, 45, 40, 30 or 25 nM / m or less).

[0105] In some embodiments, the reconstituted composition has an interfacial complex viscosity of 0.2 Pa·s·m or less (e.g., 0.15 Pa·s·m or less, or 0.1 or less). Interfacial complex viscosity (also known as interfacial shear viscosity or interfacial complex shear viscosity) is a measure of molecular flow present at the interface between a gas and a liquid or between two immiscible liquids. Figure 3 Figure B shows the interfacial shear viscosity measurements of the exemplary antibody composition compared to the control solution. The exemplary polyacrylamide-based copolymer reduced the interfacial reversion viscosity of the resulting antibody composition to 1 / 3.

[0106] In some embodiments, the reconstituted composition is in 0 s -1 Up to 3,000 s -1 It has a high shear rate viscosity of 6 mPa·s or lower (e.g., 5.5 mPa·s or lower) within the shear rate range. Figure 3 Figure C shows the viscosity profile of the exemplary antibody composition compared to the control solution at a high shear rate representing injection. The exemplary antibody composition comprising a polyacrylamide-based copolymer exhibits bulk solution viscosity at a high shear rate suitable for injection.

[0107] In some embodiments, the reconstituted composition is able to retain 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) of the antibody's specific binding activity to the target epitope or antigen, for example, as assessed in a 21-day continuous stress aging process. In some embodiments, the binding activity is determined by enzyme-linked immunosorbent assay (ELISA).

[0108] peptides

[0109] In some implementations, the biopharmaceutical agent is insulin. The term "insulin" refers to a hormone produced by the β cells in the pancreas that regulates the amount of glucose in the blood. Many eukaryotes, including humans, primates, pigs, cattle, cats, dogs, and rodents, produce insulin. Therefore, unless otherwise stated, "insulin" as used herein includes insulin and its analogues produced by humans, as well as insulin and its analogues produced by other eukaryotes (including but not limited to primates, pigs, cattle, cats, dogs, and rodents), and also includes recombinant, purified, or synthetic insulin or insulin analogues having similar function and structure. Human insulin protein consists of 51 amino acids and has a molecular weight of approximately 5.8 kilodaltons (kDa). Human insulin is a heterodimer composed of an A-chain and a B-chain linked by disulfide bonds.

[0110] Insulin can be isolated from islet extracts of insulin-producing animals or recombinantly expressed in suitable expression systems such as *Escherichia coli*, yeast, insect cells, and mammalian cells (e.g., Chinese hamster ovary (CHO) cells). Insulin is further expressed based on its specific pharmacokinetic and pharmacodynamic (PK / PD) characteristics (e.g., duration of action, observed maximum concentration, etc.). max Insulin can be further characterized as rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin, and premixed insulin, based on factors such as onset time and area under the curve (AUC).

[0111] Insulin also exists in monomeric and oligomeric forms, such as dimers and hexamers. Insulin circulates in plasma in its monomeric form and binds to its receptors even in this monomeric form. Insulin formulations (or insulin analogs) containing protein molecules primarily in monomeric and dimer forms typically exhibit a strong tendency to aggregate and form inactive fibrils. Insulin hexamers are too large to be absorbed; therefore, hexamer insulin formulations must dissociate into dimers or monomers before insulin can be absorbed and exert its effects in the body. The active form of insulin in the bloodstream is the monomeric form.

[0112] In some embodiments, the solid pharmaceutical composition comprises about 0.5 wt% to about 20 wt% of insulin or an analogue thereof, such as about 1 wt% to about 20 wt% of insulin or an analogue 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%, about 2 wt% to about 5 wt%, about 3 wt% to about 15 wt%, about 3 wt% to about 12 wt%, about 3 wt% to about 10 wt%, about 3 wt% to about 8 wt%, about 3 wt% to about 5 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 12 wt%, about 5 The solid composition comprises about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of insulin or its analogues. In some embodiments, the solid composition comprises 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 insulin or its analogues. In some embodiments, the solid composition comprises 7 wt%, 8 wt%, 9 wt%, or 10 wt% of insulin or its analogues.In some embodiments, the lyophilized pharmaceutical composition comprises 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%, about 2 wt% to about 12 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 3 wt% to about 15 wt%, about 3 wt% to about 12 wt%, about 3 wt% to about 10 wt%, about 3 wt% to about 8 wt%, about 3 wt% to about 5 wt%, about 5 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 The lyophilized composition comprises about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of insulin or its analogues. In some embodiments, the lyophilized composition comprises 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 insulin or its analogues. In some embodiments, the lyophilized composition comprises 7 wt%, 8 wt%, 9 wt%, or 10 wt% of insulin or its analogues.

[0113] In some implementations, insulin or its analogues are selected from lispro insulin, HUMALOG ® (Rapid-acting lispro insulin), glargine insulin, LANTUS ® (Insulin glargine), insulin detemir, LEVEMIR ® (Insulin Detemir), ACTRAPID ® (Rapid-acting human insulin), modern insulin, NOVORAPID ® (Insulin Aspart), VELOSULIN ® (Human insulin), HUMULIN ® M3 (a mixture of soluble insulin and protamine zinc insulin, known as biphasic protamine zinc insulin), HYPURIN ® (Neutral bovine insulin), INSUMAN ® (Recombinant human insulin), INSULATARD ®(Long-acting protamine human zinc insulin), MIXTARD ® 30 (a mixture of 30% soluble insulin and 70% protamine zinc insulin), MIXTARD ® 40 (a mixture of 40% soluble insulin and 60% protamine zinc insulin), MIXTARD ® 50 (a mixture of 50% soluble insulin and 50% protamine zinc insulin), aspart insulin, glutathione insulin, protamine zinc insulin, degludec insulin, icotinamide insulin, long-acting zinc insulin, NOVOLIN ® R (human insulin), HUMULIN ® R (human insulin), HUMULIN ® RU-500 (concentrated regular insulin), NOVOLIN ® N (intermediate-acting human insulin), HUMULIN ® N (intermediate-acting human insulin), RELION ® (NOVOLIN® R, NOVOLIN) ® N and NOVOLIN ® 70 / 30 over-the-counter brands), AFREZZA ® (Rapid-acting inhaled insulin), HUMULIN ® 70 / 30 (a mixture of 70% human protamine zinc insulin suspension and 30% human insulin injection), NOVOLIN ® 70 / 30 (a mixture of 70% NPH human protamine zinc insulin suspension and 30% conventional 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 protamine zinc insulin suspension and 50% human insulin injection), HUMALOG ®Mix 75 / 25 (a mixture of 75% lispro insulin protamine suspension and 25% lispro insulin injection), aspart insulin protamine-aspart insulin, lispro insulin protamine-lispro insulin, human NPH insulin-human regular insulin, degludec insulin-aspart insulin, and combinations thereof. In some embodiments, the insulin or its analogue is human insulin or recombinant human insulin. In some embodiments, the insulin or its analogue is non-human (e.g., primate, porcine, bovine, cat, canine, or rodent) insulin or recombinant non-human insulin. In some embodiments, the insulin or its analogue is purified or synthetic insulin. In some embodiments, the insulin or its analogue is selected from rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin, and premixed insulin. In some embodiments, the insulin or its analogue is lispro insulin. In some embodiments, the insulin or its analogue is aspart insulin. In some embodiments, the insulin or its analogue is recombinant human insulin.

[0114] In some embodiments, the biopharmaceutical reagent is a peptide or peptide analogue. The formulation methods described herein should be particularly useful in preparing storage-stable injectable pharmaceutical compositions of various therapeutic peptides, including but not limited to: glucagon, glucagon-like peptide-1 (GLP-1), GLP-1 receptor agonists, GLP-2, adrenocorticotropic hormone (ACTH), leuprorelin, hirudin, insulin, pramlinide, exendins, exenatide, gastrointestinal inhibitor, calcitonin, calcitonin gene-related peptide, amylin, adrenomedullin, angiotensin, immunogenic peptides (e.g., peptides or peptide complexes derived from viruses, bacteria, or any prokaryotic or eukaryotic organisms and their cells), and analogues thereof.

[0115] In some implementations, the biopharmaceutical agent is a glucagon peptide, a glucagon analog, a glucagon mimic, or a salt thereof.

[0116] Additives

[0117] The solid pharmaceutical compositions disclosed herein may comprise a combination of a biological pharmaceutical agent and one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents may be used to treat a disease or ailment targeted by the biological pharmaceutical agent. In some embodiments, administration of the agent from the pharmaceutical composition may be used to deliver it to cells in a subject's body, thereby exerting its biological or therapeutic effects simultaneously. In some embodiments, the drugs in the lyophilized composition are co-formulated to provide substantially similar pharmacokinetic profiles.

[0118] In some embodiments, one or more adjunctive therapeutic agents are active agents that provide synergistic effects with the biopharmaceutical agent. In some embodiments, the adjunctive therapeutic agent is a small molecule drug.

[0119] In some embodiments, the stable formulations and solid compositions used according to this disclosure include co-formulations or mixtures of biopharmaceutical agents of the type described herein (such as at least one peptide, at least one small molecule, and combinations thereof).

[0120] In some embodiments, the solid composition is co-formulated to comprise a first biopharmaceutical agent as a protein and a second biopharmaceutical agent as a peptide. In some embodiments, the solid composition is co-formulated to comprise both the first and second biopharmaceutical agents as peptides. In some embodiments, the solid composition is co-formulated to comprise a first biopharmaceutical agent and a second therapeutic agent, wherein the first biopharmaceutical agent is a peptide hormone or an analogue thereof, and the second therapeutic agent is a small molecule. In some embodiments, the second therapeutic agent is a steroid.

[0121] The amount of one or more active agents that can be combined with each other and with a carrier material of a solid composition to produce a dosage form will vary depending on the subject in need and the specific method of administration.

[0122] Polyacrylamide-based copolymers

[0123] The term "polyacrylamide-based copolymer" refers to a polymer formed by polymerizing two or more different monomers, wherein at least one monomer has an acrylamide functional group (i.e., an acrylamide monomer). In some embodiments, the polyacrylamide-based copolymer is formed by polymerizing two structurally different acrylamide monomers (two structurally different monomers, each having an acrylamide functional group).

[0124] The resulting copolymers can be alternating copolymers (where monomer types are linked in an alternating manner); random copolymers (where monomer types are linked to each other in a specific pattern within the polymer chain); block copolymers (where polymer blocks of one monomer type are attached to polymer blocks composed of another monomer type); and graft copolymers (where the main polymer chain is composed of one monomer type, and polymer blocks of another monomer type are attached to the main polymer chain as branches (also called side chains). In some embodiments, the polyacrylamide-based copolymers of this disclosure are random copolymers.

[0125] "Acrylamide monomer" refers to the type of monomer that has an acrylamide functional group. The term "acrylamide monomer" includes not only monomeric acrylamide but also its derivatives. Examples of acrylamide monomers include, but are not limited to: acrylamide (AM), N-(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N,N-diethylacrylamide (DEA), N-tert-butylacrylamide (TBA), and N-phenylacrylamide (PHE).

[0126] In some embodiments, the polyacrylamide-based copolymers of the compositions disclosed herein are amphiphilic. In some embodiments, the polyacrylamide-based copolymers are copolymers of two acrylamide monomers (a water-soluble carrier monomer and a functional dopant monomer). In some embodiments, the polyacrylamide-based copolymers are formed by random polymerization of the water-soluble carrier monomer and the functional dopant monomer.

[0127] As defined herein, the term "water-soluble carrier monomer" refers to the type of acrylamide monomer that is a water-soluble monomer in a polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer is the predominant hydrophilic type in the polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer provides hydrophilic side chain groups that impart water solubility to the copolymer.

[0128] In some embodiments, the water-soluble carrier monomer in the polyacrylamide-based copolymer provides an inert barrier at the interface of the aqueous formulation to prevent protein-protein interactions. In some embodiments, the interface is an air-water interface. In some embodiments, the interface is a container wall-water interface, including but not limited to glass-water, rubber-water, plastic-water, or metal-water interfaces. In some embodiments, the interface is an oil-water interface. In some embodiments, the interface is between a liquid and a conduit. In some embodiments, the interface is between a liquid and a conduit. In some embodiments, the container wall-water interface is in a pump system. In some embodiments, the container wall-water interface is in a closed-loop system.

[0129] In some embodiments, the water-soluble carrier monomer is hydrophilic and / or nonionic. Examples of target water-soluble carrier monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), and N-hydroxyethylacrylamide (HEAM).

[0130] As used herein, the term "functional dopant monomer" refers to a class of acrylamide monomers that possess one or more physicochemical properties (e.g., hydrophobicity, charge, etc.) different from those of the water-soluble carrier monomer. In some embodiments, the functional dopant monomer in the polyacrylamide-based copolymer promotes the bonding of the polymer to the composition interface; such interfaces may include, but are not limited to, polymer-air-water 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 component to promote interactions with biomolecules (e.g., proteins, peptides, antibodies, antibody-drug conjugates, nucleic acids, lipid particles, and combinations thereof) (e.g., preventing biomolecule aggregation). Functional dopant monomers can also be further classified according to their chemical composition as hydrogen-bonded monomers, ionic monomers, hydrophobic monomers, and aromatic monomers. Typically, the functional dopant monomer is copolymerized in the copolymer at a lower weight percentage compared to the water-soluble carrier monomer.

[0131] The term "polymerization" refers to the process by 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, such as addition (chain reaction) polymerization, condensation polymerization, ring-opening polymerization, free radical polymerization, controlled free 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 by a free radical generation system. In some embodiments, RAFT polymerization is used to prepare the copolymers of this disclosure.

[0132] 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 the polymer sample by the molecular weight of the monomers. The average molecular weight of a polymer can be expressed as number-average molecular weight (Mn), weight-average molecular weight (Mw), Z-average molecular weight (Mz), or the molecular weight at the peak maximum of a molecular weight distribution curve (Mp). The average molecular weight of a polymer can be determined by various analytical characterization techniques known to those skilled in the art, such as gel permeation chromatography (GPC), static light scattering (SLS) analysis, multi-angle laser light scattering (MALLS) analysis, nuclear magnetic resonance spectroscopy (NMR), intrinsic viscosity (IV), melt flow index (MFI), and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and combinations thereof. The degree of polymerization can also be determined experimentally using suitable analytical methods known in the art, such as nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy.

[0133] The compositions described herein may comprise polyacrylamide-based copolymers comprising:

[0134] Water-soluble carrier monomers selected from N-(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), acrylamide (AM), and combinations thereof; and

[0135] Functional dopant monomers selected from N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N,N-diethylacrylamide (DEA), N-tert-butylacrylamide (TBA), N-phenylacrylamide (PHE) and combinations thereof.

[0136] In some embodiments, the water-soluble carrier monomer of the polyacrylamide-based copolymer is selected from MORPH, MPAM, and combinations thereof. In some embodiments, the water-soluble carrier monomer includes MORPH or MPAM. In some embodiments, the water-soluble carrier monomer is MORPH. In some embodiments, the water-soluble carrier monomer is MPAM.

[0137] In some embodiments, the functional dopant monomer of the polyacrylamide-based 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.

[0138] In some embodiments, the water-soluble carrier monomer is selected from MPAM, 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 MPAM, 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 MPAM, 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 MPAM, 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 MPAM, and the functional dopant monomer includes DEA.

[0139] In some embodiments, the copolymer comprises about 70 wt% to about 98 wt% of a 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 a water-soluble carrier monomer. In some embodiments, the copolymer comprises about 2 wt% to about 30 wt% of a 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 a functional dopant monomer.

[0140] In some embodiments, the copolymer comprises 70 wt% to 98 wt% of a water-soluble carrier monomer, such as 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 a water-soluble carrier monomer. In some embodiments, the copolymer comprises 2 wt% to 30 wt% of functional dopant monomers, such as 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 functional dopant monomers.

[0141] In some embodiments, the copolymer comprises 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 comprises 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 comprises 70 wt% to 85 wt%, 70 wt% to 80 wt%, 74 wt% to 85 wt%, 74 wt% to 80 wt%, or 77 wt% of MORPH. In some embodiments, the copolymer comprises 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.

[0142] In some embodiments, the copolymer comprises 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 comprises 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 comprises 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 comprises 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.

[0143] In some embodiments, the copolymer comprises 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 comprises 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 comprises 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 comprises 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.

[0144] In some embodiments, the degree of polymerization of the copolymer is from about 10 to about 500, for example, from about 10 to about 350, from about 10 to about 200, from about 15 to about 500, from about 15 to about 350, from about 15 to about 200, from about 20 to about 500, from about 20 to about 350, or from about 20 to about 200. In some embodiments, the degree of polymerization of the copolymer is from 10 to 500, for example, from 10 to 350, from 10 to 200, from 15 to 500, from 15 to 350, from 15 to 200, from 20 to 500, from 20 to 350, or from 20 to 200.

[0145] In some embodiments, the number average molecular weight of the copolymer is from about 2,000 g / mol to about 10,000 g / mol, for example, from about 2,000 g / mol to about 7,500 g / mol, from 2,000 g / mol to about 6,000 g / mol, from about 2,000 g / mol to about 5,000 g / mol, from about 3,000 g / mol to about 10,000 g / mol, from about 3,000 g / mol to about 7,500 g / mol, from about 3,000 g / mol to about 6,000 g / mol, or from about 3,000 g / mol to about 5,000 g / mol. In some embodiments, the copolymer has a number-average molecular weight of 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 to 5,000 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.

[0146] In some embodiments, the composition comprises about 0.1 wt% to about 10 wt% of a 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 a copolymer. In some embodiments, the composition comprises about 1 wt% of a copolymer. In some embodiments, the composition comprises 0.1 wt% to 10 wt% of a 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 a copolymer. In some embodiments, the composition comprises 1 wt% of a copolymer.

[0147] Pharmaceutical Composition

[0148] As described above, this disclosure provides compositions of biopharmaceutical reagents formulated with polyacrylamide-based copolymers. The inventors have demonstrated that polyacrylamide-based copolymers can be used to provide stable formulations, such as lyophilized formulations of biopharmaceutical reagents, without altering their pharmacokinetics or bioavailability. Such formulations are applicable to a wide variety of biopharmaceutical reagents.

[0149] In some embodiments, the composition comprises 5 wt% or more of a 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 a biopharmaceutical agent. In some embodiments, the composition comprises 25 wt% or less, 20 wt% or less, or 15 wt% or less of a biopharmaceutical agent.

[0150] In some embodiments, the composition comprises about 2.5 wt% to about 25 wt% of a 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 a biopharmaceutical agent. In some embodiments, the composition comprises about 5.5 wt% or about 12 wt% of a biopharmaceutical agent. In some embodiments, the composition comprises 2.5 wt% to 25 wt% of a 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 a biopharmaceutical agent. In some embodiments, the composition comprises about 5.5 wt% or about 12 wt% of a biopharmaceutical agent, such as 5.5 wt% or 12 wt% of a biopharmaceutical agent.

[0151] In some embodiments, the biopharmaceutical agent is an antibody, and 70% or more of the antibody, such as 75 wt% or more, 80 wt% or more, 85 wt% or more, or 90 wt% or more, is present in the composition in a monomeric state.

[0152] In some embodiments, the composition is a solid composition comprising a biopharmaceutical agent and a polyacrylamide-based copolymer in a specific weight ratio (such as 2:1 or higher). In some embodiments, the solid composition comprises a biopharmaceutical agent and a polyacrylamide-based copolymer in a weight ratio of 5:1 or higher, such as 10:1 or higher. In some embodiments, the weight ratio of the biopharmaceutical agent to the polyacrylamide-based copolymer in the solid composition is in the range of 100:1 to 2:1, such as 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 20:1, or 5:1 and 2:1. In some embodiments, the polyacrylamide-based copolymer is MoNi.

[0153] In some embodiments, the composition is a solid composition comprising 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 comprising a biopharmaceutical agent and a stabilizer in a specific weight ratio (such as 1:2 or higher, or 1:1 or higher). In some embodiments, the weight ratio of the biopharmaceutical agent to the stabilizer in the solid composition is in the range of 20:1 to 1:2, for example, 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.

[0154] In some embodiments, the composition is a solid composition comprising a polyacrylamide-based copolymer and a stabilizer in a specific weight ratio (e.g., 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 weight ratio of the polyacrylamide-based copolymer to the stabilizer in the solid composition is in the range of 1:100 to 1:1, for example, between 1:50 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.

[0155] In some embodiments, the composition is a solid composition comprising a biopharmaceutical agent, a stabilizer, and a polyacrylamide-based copolymer in a specific weight ratio (e.g., a weight ratio in the range of 20:20:1 to 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 weight ratio of the biopharmaceutical agent, stabilizer, and polyacrylamide-based copolymer in the solid composition is in the range of 20:20:1 to 2:2:1, such as 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 and 10:10:1. In some embodiments, the polyacrylamide-based copolymer is MoNi.

[0156] In some embodiments, a biopharmaceutical reagent composition comprising an acrylamide-based copolymer (e.g., as described herein) is a pharmaceutical composition and also comprises pharmaceutically acceptable excipients. The term "pharmaceutical composition" refers to a formulation whose form allows for the administration of the bioactivity of the active ingredient contained therein to a subject and is effective for treatment, and which does not contain any additional components that would have unacceptable toxicity to the subject.

[0157] Pharmaceutical compositions may contain formulation materials used to alter, maintain, or preserve, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. Suitable formulation materials include, but are not limited to, amino acids; antimicrobial agents; antioxidants; buffers; chelating agents; complexing agents; monosaccharides; disaccharides and other carbohydrates; emulsifiers; salt-forming antiions; preservatives; solvents; sugar alcohols; suspending agents; surfactants or wetting agents; stability enhancers; isotonic enhancers; delivery media; diluents; other excipients and / or adjuvants. Neutral buffered saline or saline mixed with the same serum albumin is an example of a suitable diluent. Preservatives may also be added according to appropriate industry standards. Suitable excipient solutions may be used as diluents to formulate the composition into lyophilized products. Suitable components are non-toxic to the recipient at the doses and concentrations used. For more examples of components that can be used in pharmaceutical formulations, see Remington's Pharmaceutical Sciences, 16th edition (1980) and 20th edition (2000), Mack Publishing Company, Easton, PA.

[0158] In some embodiments, the compositions described herein further comprise a surfactant. In some embodiments, the compositions comprise 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 comprises 0.001 wt% to 1 wt%, such as 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 poloxamer. In some embodiments, the surfactant includes polysorbate. In some embodiments, the polysorbate is polysorbate 80 (PS80) or polysorbate 20 (PS20). In some embodiments, the composition comprises about 0.01 wt% of polysorbate 80. In some embodiments, the composition comprises about 0.01 wt% of polysorbate 80, such as 0.01 wt% of polysorbate 80.

[0159] In some embodiments, the compositions described herein further comprise a stabilizer. In some embodiments, the stabilizer comprises a sugar. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the composition comprises about 0.1 wt% to about 50 wt%, such as 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 a stabilizer. In some embodiments, the composition comprises 0.1 wt% to 25 wt%, such as 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 a stabilizer. In some embodiments, the composition comprises 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%, or about 15 wt% to 55 wt% of a stabilizer. In some embodiments, the composition comprises about 9 wt% sucrose. In some embodiments, the composition comprises about 9 wt% sucrose, for example, 9 wt% sucrose. In some embodiments, the composition comprises trehalose.

[0160] The liquid composition also includes a buffer to maintain the pH of the pharmaceutical composition. The buffer may include buffering compounds known in the art, such as TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MPOS, PIPES, carboxylic acid, histidine (e.g., L-histidine hydrochloride), or MES. The buffer may contain citrate, potassium dihydrogen phosphate, boric acid, or diethylbarbituric acid. The buffer may be PBS, HEPES, TRIS, or TRIS / EDTA buffer. The buffer may be other phosphate buffers. Phosphate buffers may contain a mixture of dihydrogen phosphate and dihydrogen phosphate disalt.

[0161] Specifically, the buffer solution may be a potassium phosphate buffer. The potassium phosphate buffer may contain potassium phosphate at concentrations of 5 mM to 15 mM, 7.5 mM to 12.5 mM, 9 mM to 11 mM, or 10 mM. The buffer solution contained in the liquid composition may have a pH of 7 to 9. In some embodiments, the pH is 7.0 to 8.5 or 8.0. In some embodiments, the pH is 7.0 to 8.0. In some embodiments, the liquid composition contains 7.5-12 mM potassium phosphate buffer with a pH of 7 to 9. In some embodiments, the liquid composition contains 7.5-12 mM potassium phosphate buffer with a pH of 7.0 to 8.0. In some embodiments, the liquid composition contains 9-11 mM potassium phosphate buffer with a pH of 7.0 to 8.5. In some embodiments, the liquid composition contains 10 mM potassium phosphate buffer with a pH of 8.0.

[0162] In some embodiments, the liquid composition has a buffer concentration of about 1 mM to about 100 mM (e.g., 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 (e.g., 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), and in some embodiments, the buffer comprises phosphate, citrate, acetate, TRIS, succinate, other organic acids, or histidine or salts thereof. In some embodiments, the buffer comprises one or more phosphates. In some embodiments, the buffer comprises sodium phosphate. In some embodiments, the composition comprises an acetate buffer.

[0163] The term “buffer exchange” as used in this article refers to the technique in which biopharmaceutical reagents are transferred from one buffer system to another through filtration and centrifugation processes.

[0164] The liquid composition may also contain a salt. The salt may be NaCl or KCl. In some embodiments, the liquid composition contains a salt 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 less than 0.5%, or a concentration of 0.45%. In some embodiments, the liquid composition contains a salt at a concentration of 0.1% to 0.9%, 0.1% to 0.6%, 0.25% to 0.75%, 0.4% to 0.6%, 0.4% to 0.5%, or a concentration of 0.45%.

[0165] In some embodiments, the liquid composition comprises NaCl at concentrations 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 less than 0.5%, or a concentration of 0.45%. In some embodiments, the liquid composition comprises NaCl at concentrations of 0.1% to 0.9%, 0.1% to 0.6%, 0.25% to 0.75%, 0.4% to 0.6%, 0.4% to 0.5%, or a concentration of 0.45%.

[0166] In some embodiments, the liquid composition comprises KCl at concentrations 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 less than 0.5%, or a concentration of 0.45%. In some embodiments, the liquid composition comprises KCl at concentrations of 0.1% to 0.9%, 0.25% to 0.75%, 0.4% to 0.6%, 0.4% to 0.5%, or a concentration of 0.45%.

[0167] In some embodiments, the liquid composition is an aqueous formulation. In some embodiments, such an aqueous formulation has a pH of 3 to 7.5, for example, a pH of 4 to 6.5.

[0168] The optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, method of delivery, and required dosage. Reference See, for example, Remington's Pharmaceutical Sciences, The same as above. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of peptides. For example, suitable compositions may be water for injection or physiological saline solutions for parenteral administration.

[0169] Another aspect of this disclosure is a solid pharmaceutical composition present in a unit dose. In some aspects of this disclosure, the lyophilized pharmaceutical composition is in unit dosage form. In some embodiments, the unit dosage form is a vial, ampoule, bottle, or pre-filled syringe. In a typical embodiment, the unit dosage form is a vial containing a predetermined amount of the lyophilized pharmaceutical composition suitable for administration after reconstitution. Administration may include subcutaneous, intradermal, or intramuscular administration using a pre-filled syringe, autoinjector, or auto-injection pen, each device containing a predetermined amount of the pharmaceutical composition described herein.

[0170] The unit dose in the container can be determined based on various factors, such as the active ingredient (e.g., a biopharmaceutical agent), the disease to be treated, the subject, the route of administration, and the method of administration. The unit dose can be determined based on in vitro or in vivo studies (including clinical trials).

[0171] The unit dose in the container can be sealed and stored for a long 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).

[0172] The dried composition (e.g., lyophilized composition) stored in the container is preferably stable for at least 1 month, 3 months, 6 months or 1 year in the range of about 2-20°C to about -80°C without losing significant activity.

[0173] 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, skin, or subcutaneous injection, or injection at the site of infection, the active ingredient may be in a parenteral acceptable aqueous formulation, such as a solution, which is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable aqueous formulations using, for example, isotonic carriers (such as sodium chloride injection, Ringer's solution, lactated Ringer's solution). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0174] method

[0175] This disclosure includes methods for preparing solid compositions. In some embodiments, the method for preparing a solid composition includes 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 freeze-drying, foam pad drying, vacuum oven drying, and dehydration drying. In some embodiments, the liquid biopharmaceutical composition is exposed to a lyophilization cycle in a lyophilizer to produce a lyophilized composition.

[0176] In some embodiments, the lyophilization cycle includes subjecting the liquid biopharmaceutical composition to a first pressure. In some embodiments, the lyophilization cycle includes evaporating at least a portion of the liquid component of the liquid biopharmaceutical composition while adding heat. In some embodiments, the lyophilization cycle includes cooling the liquid biopharmaceutical composition to freeze any remaining liquid component into a solid.

[0177] In some embodiments, the lyophilization cycle includes subjecting the liquid biopharmaceutical composition to a first reduced temperature. In some embodiments, the lyophilization cycle further includes subjecting the liquid biopharmaceutical composition to a first reduced pressure. In some embodiments, the lyophilization cycle further includes evaporating at least a portion of the liquid component of the liquid biopharmaceutical composition while adding heat. In some embodiments, the lyophilization cycle further includes subjecting the liquid biopharmaceutical composition to a second reduced pressure below the first reduced pressure. In some embodiments, the lyophilization cycle further includes evaporating the remaining liquid to produce a solid composition.

[0178] In some embodiments, the drying cycle is a spray drying cycle. In some embodiments, the spray drying cycle includes atomizing the liquid biopharmaceutical composition. In some embodiments, the spray drying cycle includes further evaporating at least a portion of the liquid component of the liquid biopharmaceutical composition. In some embodiments, the spray drying cycle also includes collecting the solid composition.

[0179] In some embodiments, the drying cycle is a spray freeze-drying cycle. In some embodiments, the spray freeze-drying cycle includes atomizing the liquid biopharmaceutical composition. In some embodiments, the spray freeze-drying cycle further includes freezing the atomized liquid biopharmaceutical composition. In some embodiments, the spray freeze-drying cycle further includes evaporating at least a portion of the liquid component of the atomized liquid biopharmaceutical composition. In some embodiments, the spray freeze-drying cycle further includes collecting the solid composition.

[0180] In some embodiments, the drying cycle is a foam pad drying cycle. In some embodiments, the foam pad drying cycle includes foaming the liquid biopharmaceutical composition. In some embodiments, the foam pad drying cycle further includes distributing the foamed liquid biopharmaceutical composition onto a drying surface. In some embodiments, the foam pad drying cycle further includes evaporating at least a portion of the liquid component of the foamed liquid biopharmaceutical composition. In some embodiments, the foam pad drying cycle further includes collecting the solid composition.

[0181] In some embodiments, drying is a vacuum oven drying cycle. In some embodiments, the vacuum oven drying cycle includes subjecting the liquid biopharmaceutical composition to a reduced pressure. In some embodiments, the vacuum oven drying cycle further includes evaporating at least a portion of the liquid component of the liquid biopharmaceutical composition while adding heat. In some embodiments, the vacuum oven drying cycle further includes restoring the pressure to the original pressure to obtain a solid composition.

[0182] In some embodiments, drying is a dehydration cycle. In some embodiments, the dehydration drying cycle includes increasing the surface area of ​​the liquid biopharmaceutical composition. In some embodiments, the surface area of ​​the liquid biopharmaceutical composition is increased by pouring it into a tray. In some embodiments, the surface area of ​​the liquid biopharmaceutical composition is increased by pouring it into a shallow tray. In some embodiments, the dehydration drying cycle also includes evaporating at least a portion of the liquid component of the liquid biopharmaceutical composition. In some embodiments, the evaporation of the liquid component is carried out by circulating ambient air above the surface. In some embodiments, the circulating air is optionally dried, heated, or both on the dehydrating material until a solid is obtained.

[0183] For use and administration to subjects, the stable, dried biopharmaceutical compositions provided herein can be reconstituted by rehydration with an aqueous solution. In some embodiments, the stable and lyophilized biopharmaceutical compositions are reconstituted by rehydration with a solvent. Such solvents are typically water, such as softened or distilled water, water for injection, etc., but may also include physiological solutions or buffers (such as phosphate-buffered saline (PBS)) or adjuvants.

[0184] This disclosure includes a method for reconstituted a solid composition, the method comprising adding an aqueous solution for reconstitution to the solid composition. In some embodiments, the method comprises dissolving the solid composition in an aqueous solution for reconstitution. In some embodiments, the solid composition for reconstitution is a lyophilized composition. Reconstitution methods should be controlled in various ways to maintain the stability and activity of the biopharmaceutical reagent upon reconstitution. These methods may also vary depending on the composition of the formulation and the biopharmaceutical reagent used. In some embodiments, the solution for reconstitution is sterile water. In some embodiments, the solution for reconstitution comprises water for injection. In some embodiments, the solution for reconstitution also contains a preservative. In some embodiments, the aqueous solution contains a buffer.

[0185] This disclosure includes reconstituted solutions of biopharmaceutical reagents that retain the biological activity of the biopharmaceutical reagent relative to the mother liquor. In some embodiments, the reconstituted solutions of biopharmaceutical reagents are free of or substantially free of particulate matter. In some embodiments, the method includes separating and / or removing a certain amount of particulate matter from the reconstituted composition. In some embodiments, the method further includes centrifuging the reconstituted composition and optionally decanting a homogeneous solution from the separated particles or particles from the reconstituted composition.

[0186] This disclosure includes providing a method for measuring the reconstitution time of a solid composition. In some embodiments, the method for measuring the reconstitution time of a solid composition of this disclosure includes the steps of: (a) subjecting the solid composition to a lowered first temperature; (b) equilibrating the solid composition to an increased second temperature; (c) dissolving the solid composition in an aqueous solution for reconstitution; (d) stirring the solution for reconstitution until the particulate matter dissolves; and (e) evaluating the time taken for the particulate matter to dissolve. In some embodiments, the method includes 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 an aqueous solution for reconstitution; (d) stirring the solution for reconstitution until the particulate matter dissolves; and (e) evaluating the time taken for the particulate matter to dissolve. In some embodiments, the solid composition is initially cooled to between -50°C and 10°C, for example, between -50°C and -40°C, between -50°C and -30°C, between -50°C and -20°C, between -50°C and -10°C, between -50°C and 0°C, between -50°C and 10°C, between -40°C and -30°C, between -40°C and -20°C, between -40°C and -10°C, between -40°C and 0°C, between -40°C and 10°C, between -30°C and -20°C, between -30°C and -10°C, between -30°C and 0°C, between -30°C and 10°C, between -20°C and -10°C, between -20°C and 0°C, between -20°C and 10°C, between -10°C and 0°C, between -10°C and 10°C, between 0°C and 10°C. In some embodiments, the solid composition is initially cooled to between 2°C and 8°C. In some embodiments, the solid composition is equilibrated to between 20°C and 25°C (e.g., about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C).

[0187] 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 be dissolved in an aqueous solution during reconstitution.

[0188] In some implementations, the reconstitution time is 0 to 30 min, approximately 1 min, approximately 2 min, approximately 3 min, approximately 4 min, approximately 5 min, approximately 6 min, approximately 7 min, approximately 8 min, approximately 9 min, approximately 10 min, approximately 11 min, approximately 12 min, approximately 13 min, approximately 14 min, approximately 15 min, approximately 16 min, approximately 17 min, approximately 18 min, approximately 19 min, approximately 20 min, approximately 21 min, approximately 22 min, approximately 23 min, approximately 24 min, approximately 25 min, approximately 26 min, approximately 27 min, approximately 28 min, approximately 29 min, and approximately 30 min.

[0189] In some embodiments, the formulation reconstituted by this method comprises a therapeutically effective concentration and amount (e.g., as described herein) of a biopharmaceutical agent. In some embodiments, the concentration of the biopharmaceutical agent is 5 wt% or more of the composition, for example, 7 wt%, 10 wt%, 12 wt%, 15 wt%, 20 wt%, or 25 wt% or more; or 5 wt%, 7 wt%, 10 wt%, or 12 wt% to 15 wt%, 20 wt%, 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.

[0190] This disclosure includes methods of using the composition, said methods comprising administering a reconstituted composition of a biopharmaceutical reagent (e.g., as described herein) to a recipient in need. The reconstituted lyophilized compositions of the present invention can be administered by various delivery methods (e.g., orally or via a parenteral route). In some embodiments, administration is by intravenous injection. In some embodiments, administration is by subcutaneous injection. In some embodiments, the administered reconstituted composition comprises a therapeutically effective amount of a targeted therapeutic biopharmaceutical reagent.

[0191] As used herein, the term "subject" refers to a mammalian subject. Exemplary subjects include humans, monkeys, apes, dogs, cats, mice, rats, cows, horses, camels, goats, donkeys, rabbits, and sheep. In some embodiments, the subject is a human. The reconstituted biological pharmaceutical reagent of the present invention can be administered to a mammalian subject to treat a variety of diseases. In some embodiments, the subject suffers from a disease or ailment that can be treated with the biological pharmaceutical reagent described herein.

[0192] The term "treating" (and its variations, such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or ailment in a subject in need. Treatment may be performed during a clinicopathological process. Desired therapeutic effects may include one or more of the following: reducing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the biological pharmaceutical agent is administered to the patient in the form of a reconstituted composition as described herein.

[0193] The term “therapeutic effective amount” or “effective amount” refers to the amount of a biological pharmaceutical agent that, when administered to a subject, is effective in treating a disease or condition. The exact dose or amount will depend on the purpose of treatment and can generally be determined by someone skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0194] The composition, reconstituted for immediate injection, can be administered to the subject via parenteral or mucosal injection (preferably intramuscular or subcutaneous). However, administration of the composition, reconstituted for immediate injection as described above, may also include intranasal, transdermal, topical, and oral administration. The dose per injection can be from about 0.1 ml to about 2.0 ml, preferably about 1.0 ml.

[0195] The actual dosage, rate of administration, and duration of administration depend on the nature and severity of the disease being treated. In a typical implementation, the biological agent is administered in an amount that effectively reduces the symptoms of the target disease. In some implementations, the amount effectively reduces symptoms within one week of administration. In some implementations, the amount effectively reduces symptoms within two, three, or four weeks of administration.

[0196] definition

[0197] In this disclosure, the terms “a,” “an,” and “the” are used to include one or more, unless the context clearly indicates otherwise. Unless otherwise stated, the term “or” is used to mean a non-exclusive “or.” The expression “at least one of A and B” has the same meaning as “A, B, or A and B.” Furthermore, it should be understood that any wording or terminology used in this disclosure that is not otherwise defined is for descriptive purposes only and not for limitation. The use of any section headings is intended to aid in reading the document and should not be construed as limiting; information relating to a section heading may appear within or outside that particular section.

[0198] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly listed as range boundaries, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly listed. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not only about 0.1% to about 5%, but also the individual numerical values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise stated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0199] The term “about” as used in this disclosure may allow for a variation in a value or range of up to 5% of the specified limit of the specified value or range.

[0200] In the methods described in this disclosure, the operations can be performed in any order unless the timing or order of operations is explicitly stated. Furthermore, the specified operations can be performed simultaneously unless explicitly stated in the language of the claims as being performed separately. For example, the claimed operation of performing X and the claimed operation of performing Y can be performed simultaneously in one operation, and the resulting method will fall within the literal scope of the claimed method.

[0201] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques used in conjunction with those described herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this disclosure are generally performed according to conventional methods known in the art and described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terminology used herein in conjunction with analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, as well as their laboratory procedures and techniques, are those well-known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0202] The term "immunoglobulin" refers to a class of structurally related proteins, typically consisting of two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a "complete immunoglobulin," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul, Fundamental Immunology 7th Edition, Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In short, each heavy chain typically contains a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy-chain constant region typically contains three structural domains, abbreviated as C. H1 C H2 and CH3 Each light chain typically contains a light chain variable region (V). L ( ) and the light chain constant region. The light chain constant region typically contains a structural domain, abbreviated as C. L .

[0203] The term "antigen-binding protein" (ABP) refers to a protein containing one or more antigen-binding domains that specifically bind to antigens or epitopes. In some embodiments, the antigen-binding domains bind to antigens or epitopes with similar specificity and affinity to naturally occurring antibodies. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP comprises an antibody fragment.

[0204] The term "antigen-binding domain" refers to the portion of ABP that can specifically bind antigens or epitopes.

[0205] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of naturally occurring antibodies and have a heavy chain containing an Fc region.

[0206] The term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, which, 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 they contain are known in the art. See Schroeder and Cavacini. J. Allergy Clin.Immunol. , 2010, 125:S41-52, incorporated herein 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.

[0207] V H and V L The region can be further subdivided into highly variable regions (“HVR”; also known as “CDR”) interspersed with more conservative regions. The more conservative regions are called framing regions (FR). Each V H and V L It typically contains three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. CDRs participate in antigen binding and affect the antigen specificity and binding affinity of the antibody. Reference See Kabart et al. , Sequences of Proteins of Immunological Interest 5th Edition (1991) Public Health Service, National Institutes of Health, Bethesda, MD It is incorporated into the whole by reference.

[0208] Light chains from any vertebrate species can be classified into one of two types (called Kappa (κ) and Lambda (λ)) based on the sequence of constant structural domains.

[0209] Heavy chains from any vertebrate species can be classified into one of five distinct classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also named α, δ, ε, γ, and µ, respectively. The IgG and IgA classes are further subdivided based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0210] The amino acid sequence boundaries of the CDR can be determined by those skilled in the art using any of many known numbering schemes, including those developed by Kabat et al. (ibid.) (“Kabat” numbering scheme); Al-Lazikani et al. person 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 Plückthun, J. Mol.Biol. , 2001, 309:657-70 (“AHo” numbering scheme) (each of the references mentioned is incorporated in its entirety by reference) those described.

[0211] Table 1 below provides CDR1-L (V L CDR1), CDR2-L (V L CDR2), CDR3-L (V L CDR3), CDR1-H (V H CDR1), CDR2-H (V H CDR2) and CDR3-H (V H The position of CDR3 is determined as per the Kabat and Chothia schemes. For CDR1-H, residue numbering is provided using the Kabat and Chothia numbering schemes.

[0212] CDRs can 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, which is incorporated herein by reference in its entirety.

[0213]

[0214] When using the Kabat numbering convention, the C-terminus of CDR1-H varies between 32 and 34, depending on the length of the CDR.

[0215] When referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is usually used (e.g., as reported by Kabat et al. (ibid.)).

[0216] An "antibody fragment" contains a portion of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0217] The “Fv” fragment is a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.

[0218] In addition to the variable structural domains of the heavy and light chains, the “Fab” segment also contains the constant structural domain of the light chain and the first constant structural domain (C) of the heavy chain. H1 Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0219] The “F(ab’)2” fragment comprises two Fab’ fragments linked by disulfide bonds near the hinge region. The F(ab’)2 fragment can be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab’) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0220] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V in a single polypeptide chain. H Domain and V L Domain. V H and V L Typically linked via peptide linkers. See Plückthun A. (1994). In some implementations, n = 1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli In Rosenberg M. & Moore GP (editors), The Pharmacology of Monoclonal Antibodies Volume 113 (pp. 269-315). Springer-Verlag, New York (incorporated in its entirety by reference).

[0221] The “scFv-Fc” fragment contains an scFv that is connected to the Fc structure field. For example, the Fc structure field can be connected to the C end of the scFv. The Fc structure field can follow the V...H or V L This depends on the orientation of the variable structural domains in scFv (i.e., V). H -V L or V L -V H Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain contains the IgG4Fc domain.

[0222] The term "single-domain antibody" refers to a molecule in which one variable domain of the antibody specifically binds to the antigen in the absence of another variable domain. Single-domain antibodies and their fragments are described by Arabi Ghahroudi et al. FEBS Letters , 1998, 414:521-526 and Muyldermans et al., Trends in BiochemSci. ,2001, 26:230-245 (each of the references is included in its entirety by way of citation).

[0223] A "monospecific ABP" is an ABP that contains a binding site that specifically binds to a single epitope. An example of a monospecific ABP is the naturally occurring IgG molecule, which, although bivalent, recognizes the same epitope in each antigen-binding domain. Binding specificity can exist at any suitable valence.

[0224] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. A substantially homogeneous group of antibodies contains antibodies that are substantially similar and bind to the same epitopes, aside from variants that may normally occur during the production of monoclonal antibodies. Such variants are typically present only in small quantities. Monoclonal antibodies are usually obtained through a process involving the selection of a single antibody from a variety of antibodies. For example, the selection method may be to choose a unique clone from a library of multiple clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody may be further modified, for example, to increase its affinity for the target ("affinity maturation"), humanize the antibody, increase its yield in cell culture, and / or decrease its immunogenicity in subjects.

[0225] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0226] A “humanized” form of a nonhuman antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman antibody. Humanized antibodies are typically human antibodies (receptor antibodies), in which residues from one or more CDRs are replaced by residues from one or more CDRs from a nonhuman antibody (donor antibody). The donor antibody can be any suitable nonhuman antibody, such as mouse, rat, rabbit, chicken, or nonhuman primate antibodies with the desired specificity, affinity, or biological effect. In some cases, selected frame region residues of the recipient antibody are replaced by corresponding frame region residues from the donor antibody. Humanized antibodies may also contain residues not found in either the recipient or donor antibody. Such modifications can be made to further optimize antibody function. (The text then abruptly shifts to a different topic: "Regarding further...") one For details of the steps, 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 the references is incorporated in its entirety by reference.

[0227] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans or human cells, or an antibody derived from a non-human source using a human antibody library or a human antibody encoding sequence (e.g., obtained from a human source or designed de novo). Human antibodies explicitly exclude humanized antibodies. In some embodiments, rodents are genetically engineered to replace their rodent antibody sequences with human antibodies.

[0228] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules. In some embodiments, the immunoconjugate is an antibody conjugated to one or more heterologous molecules, 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 cell inhibitor.

[0229] As used in this article, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.

[0230] "Chemotherapy agents" are compounds used to treat cancer. Chemotherapy agents include "anti-hormonal agents" or "endocrine therapy agents," which are used to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0231] The term "cell growth inhibitor" refers to a compound or composition that arrests cell growth in vitro or in vivo. In some embodiments, the cell inhibitor is an agent that reduces the percentage of cells in S phase. In some embodiments, the cell inhibitor 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%.

[0232] The term "tumor" refers to all tumor cell growth and proliferation (whether malignant or benign), as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cellular proliferative disorder," "proliferative symptom," and "tumor" are not mutually exclusive herein. The terms "cellular proliferative disorder" and "proliferative symptom" refer to a condition associated with some degree of abnormal cell proliferation. In some implementations, a cellular proliferative disorder is cancer.

[0233] "Variants" of peptides (e.g., antibodies) comprise amino acid sequences in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the natural peptide sequence, and which retain substantially the same biological activity as the natural peptide. The biological activity of the peptide can be measured using standard techniques in the art (e.g., if the variant is an antibody, its activity can be tested by a binding assay, as described herein). Variants disclosed herein include fragments, analogs, recombinant peptides, synthetic peptides, and / or fusion proteins.

[0234] A “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified (e.g., by conjugation, phosphorylation, and glycosylation with another chemical moiety (e.g., polyethylene glycol, albumin (e.g., human serum albumin)). Unless otherwise stated, the term “antibody” includes not only antibodies comprising two full-length heavy chains and two full-length light chains, but also their derivatives, variants, fragments, and mutant proteins, as described below.

[0235] “Affinity” refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., ABP) and its binding partner (e.g., an antigen or epitope). Unless otherwise stated, “affinity” as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., ABP and an antigen or epitope). The affinity of molecule X for its partner Y can be expressed using the dissociation equilibrium constant (K0). D The kinetic components that contribute to the dissociation equilibrium constant will be described in more detail below. Affinity can be measured by methods commonly known in the art, including those described herein. For example, surface plasmon resonance (SPR) techniques (e.g., BIACORE) can be used. ® ) or biological layer interferometry (e.g., FORTEBIO) ®To determine affinity.

[0236] Regarding the binding of ABP to target molecules, the terms "binding," "specific binding," "specifically binding," "specific to," "selectively binding," and "selectively targeting" a specific antigen (e.g., a peptide target) or an epitope on a specific antigen refer to binding that is significantly different from nonspecific or nonselective interactions (e.g., with non-target molecules). For example, specific binding can be measured by measuring the binding to a target molecule and comparing it to the binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics an epitope identified on a target molecule. In this case, if the binding of ABP to the target molecule is competitively inhibited by the control molecule, it indicates specific binding.

[0237] The term "polymer" refers to a substance or material composed of repeating monomer subunits. The term "copolymer" refers to a polymer composed of two or more different repeating monomer subunits.

[0238] Additional Implementation Plan

[0239] This disclosure is further described by the following non-restrictive provisions:

[0240] Clause 1. A composition comprising:

[0241] Polyacrylamide-based copolymers comprising:

[0242] Selected from N -(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH) N , N -Dimethylacrylamide (DMA) N Hydroxyethylacrylamide (HEAM), acrylamide (AM), and water-soluble carrier monomers of combinations thereof; and

[0243] Selected from N -[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA) N - Isopropylacrylamide (NIP), NN-Diethylacrylamide (DEA) N - tert-butylacrylamide (TBA) N 2-Phenylacetamide (PHE) and its combinations as functional dopant monomers; and

[0244] Biological pharmaceutical reagents (e.g., antibodies).

[0245] Clause 2. The composition of Clause 1, wherein the water-soluble carrier monomer is selected from MORPH, MPAM and combinations thereof.

[0246] Clause 3. The composition of Clause 1, wherein the water-soluble carrier monomer comprises MORPH.

[0247] Clause 4. The composition of Clause 1, wherein the water-soluble carrier monomer comprises MPAM.

[0248] Clause 5. A composition of any one of Clauses 1-4, wherein the functional dopant monomer is selected from AMP, TMA, TBA, PHE, and combinations thereof.

[0249] Clause 6. A composition of any one of Clauses 1-4, wherein the functional dopant monomer is selected from DEA, PHE, NIP, and combinations thereof.

[0250] Clause 7. A composition of any one of Clauses 1-4, wherein the functional dopant monomer comprises TRI.

[0251] Clause 8. A composition of any one of Clauses 1-4, wherein the functional dopant monomer comprises PHE.

[0252] Clause 9. A composition of any one of Clauses 1-4, wherein the functional dopant monomer comprises NIP.

[0253] Clause 10. A composition of any one of Clauses 1-4, wherein the functional dopant monomer comprises DEA.

[0254] Clause 11. The composition of Clause 1, wherein:

[0255] Water-soluble carrier monomers are selected from MPAM, MORPH, and combinations thereof; and

[0256] The functional dopant monomers are selected from NIP, PHE and their combinations.

[0257] Clause 12. The composition of Clause 1, wherein:

[0258] Water-soluble carrier monomers are selected from MPAM, MORPH, and combinations thereof; and

[0259] The functional dopant monomers are selected from AMP, TMA, TBA, PHE and combinations thereof.

[0260] Clause 13. The composition of Clause 1, wherein the water-soluble carrier monomer is MPAM and the functional dopant monomer is PHE.

[0261] Clause 14. The composition of Clause 1, wherein the water-soluble carrier monomer is MORPH and the functional dopant monomer is PHE.

[0262] Clause 15. The composition of Clause 1, wherein the water-soluble carrier monomer is MORPH and the functional dopant monomer is NIP.

[0263] Clause 16. A composition of any one of Clauses 1-15, wherein the copolymer comprises: 70 wt% to 98 wt% of a water-soluble carrier monomer; and 2 wt% to 30 wt% of a functional dopant monomer.

[0264] Clause 17. A composition of any one of Clauses 1-15, wherein the copolymer comprises: 80 wt% to 95 wt% of a water-soluble carrier monomer; and 5 wt% to 20 wt% of a functional dopant monomer.

[0265] Clause 18. A composition of any one of Clauses 1-15, wherein the copolymer comprises: 83 wt% to 98 wt% of a water-soluble carrier monomer; and 2 wt% to 17 wt% of a functional dopant monomer.

[0266] Clause 19. The composition of Clause 1, wherein the copolymer comprises:

[0267] 70 wt% to 85 wt% MORPH; and 15 wt% to 30 wt% NIP.

[0268] Clause 20. The composition of Clause 1, wherein the copolymer comprises:

[0269] 74 wt% to 80 wt% MORPH; and 20 wt% to 26 wt% NIP.

[0270] Clause 21. The composition of Clause 1, wherein the copolymer comprises:

[0271] 77 wt% MORPH; and 23 wt% NIP.

[0272] Clause 22. A composition of any one of Clauses 1-21, wherein the degree of polymerization of the copolymer is 10 to 500.

[0273] Clause 23. A composition of any one of Clauses 1-21, wherein the degree of polymerization of the copolymer is 20 to 200.

[0274] Clause 24. The composition of any one of Clauses 1-23, wherein the number average molecular weight of the copolymer is from 2,000 g / mol to 10,000 g / mol.

[0275] Clause 25. The composition of any one of Clauses 1-23, wherein the number average molecular weight of the copolymer is from 2,000 g / mol to 6,000 g / mol.

[0276] Clause 26. The composition of any one of Clauses 1-23, wherein the number average molecular weight of the copolymer is from 3,000 g / mol to 5,000 g / mol.

[0277] Clause 27. A composition comprising any one of Clauses 1-26, comprising 0.1 wt% to 10 wt% of a copolymer.

[0278] Clause 28. A composition comprising any one of Clauses 1-26, comprising 0.5 wt% to 5 wt% of a copolymer.

[0279] Clause 29. A composition comprising any one of Clauses 1-28, wherein the composition comprises 1 wt% of a copolymer.

[0280] Clause 30. A composition of any one of Clauses 1-29, wherein the composition further comprises a surfactant.

[0281] Clause 31. The composition of Clause 30, comprising 0.001 wt% to 0.5 wt% of a surfactant.

[0282] Clause 32. The composition of Clause 30, comprising 0.005 wt% to 0.05 wt% of a surfactant.

[0283] Clause 33. A composition of any one of Clauses 30-32, wherein the surfactant comprises polysorbate.

[0284] Clause 34. The composition of Clause 33, wherein the polysorbate is polysorbate 80.

[0285] Clause 35. The composition of Clause 34, comprising 0.005 wt% to 0.05 wt% of polysorbate 80.

[0286] Clause 36. The composition of Clause 34, comprising 0.01 wt% of polysorbate 80.

[0287] Clause 37. A composition of any one of Clauses 1-36, wherein the composition further comprises a stabilizer.

[0288] Clause 38. The composition of Clause 37, comprising 0.1 wt% to 25 wt% of a stabilizer.

[0289] Clause 39. The composition of Clause 38, comprising 2 wt% to 15 wt% of a stabilizer.

[0290] Clause 40. A composition of any one of Clauses 37-39, wherein the stabilizer comprises an oligosaccharide.

[0291] Clause 41. The composition of Clause 40, wherein the oligosaccharide is sucrose.

[0292] Clause 42. The composition of Clause 40, comprising 2 wt% to 15 wt% sucrose.

[0293] Clause 43. The composition of Clause 40, which contains 9 wt% sucrose.

[0294] Clause 44. A composition comprising any one of Clauses 1-43, wherein the composition comprises 5 wt% or more of antibody.

[0295] Clause 45. The composition of Clause 44, which contains 10 wt% or more of antibody.

[0296] Clause 46. The composition of Clause 44 or 45, which contains 20 wt% or less of antibody.

[0297] Clause 47. A composition comprising any one of Clauses 1-43, comprising 2.5 wt% to 20 wt% of antibody.

[0298] Clause 48. The composition of Clause 47, comprising 2.5 wt% to 15 wt% of antibody.

[0299] Clause 49. The composition of Clause 47, comprising 8 wt% to 20 wt% of antibody.

[0300] The composition of Clause 50 and Clause 49 contains 12 wt% to 20 wt% of antibody.

[0301] Clause 51. A composition of any one of Clauses 1-50, wherein the antibody has a molecular weight of 100 kDa to 200 kDa.

[0302] Clause 52. The composition of Clause 51, wherein the antibody has a molecular weight of 120 kDa to 180 kDa.

[0303] Clause 53. A composition of any one of Clauses 1-50, wherein the antibody has a molecular weight of 50 kDa to 100 kDa.

[0304] Clause 54. A composition of any one of Clauses 1-53, wherein the antibody is a monoclonal antibody.

[0305] Clause 55. A composition of any one of Clauses 1-53, wherein the antibody is a chimeric antibody.

[0306] Clause 56. A composition of any one of Clauses 1-53, wherein the antibody is a bispecific antibody.

[0307] Clause 57. A composition of any one of Clauses 1-53, wherein the antibody is a full-length antibody.

[0308] Clause 58. A composition of any one of Clauses 1-53, wherein the antibody is an antibody fragment.

[0309] Clause 59. A composition of any one of Clauses 1-53, wherein the antibody is an antibody-drug conjugate.

[0310] Clause 60. The composition of Clause 1, comprising:

[0311] 0.1 wt% to 10 wt% of a polyacrylamide-based copolymer comprising:

[0312] 70 wt% to 98 wt% of water-soluble carrier monomers selected from MPAM, MORPH, DMA, HEAM, AM, and combinations thereof; and

[0313] 2 wt% to 30 wt% of functional dopant monomers selected from TRI, AMP, TMA, NIP, TBA, PHE and combinations thereof; and

[0314] 5 wt% or more of antibody.

[0315] Clause 61. The composition of Clause 60, comprising 10 wt% or more (e.g., 12 wt% to 20 wt%, such as 12 wt%) of antibody.

[0316] Clause 62. The composition of Clause 60 or 61, wherein:

[0317] Water-soluble carrier monomers are selected from MPAM, MORPH, and combinations thereof; and

[0318] The functional dopant monomers are selected from NIP, PHE and their combinations.

[0319] Clause 63. A composition of any one of Clauses 60-62, wherein:

[0320] The water-soluble carrier monomer is MORPH; and the functional dopant monomer is NIP.

[0321] Clause 64. The composition of Clause 63, wherein the composition comprises: 77 wt% MORPH; and 23 wt% NIP.

[0322] Clause 65. A composition comprising any one of Clauses 60-64, comprising 1 wt% of a polyacrylamide-based copolymer.

[0323] Clause 66. The composition of any one of Clauses 60-65 further comprises 0.001 wt% to 0.5 wt% of a surfactant.

[0324] Clause 67. A composition of any one of Clauses 63-66, further comprising 0.1 wt% to 25 wt% of a stabilizer.

[0325] Clause 68. A composition of any one of Clauses 1-67, wherein the composition is aqueous.

[0326] Clause 69. The composition of Clause 68, wherein the composition has a pH of 3 to 7.5.

[0327] Clause 70. The composition of Clause 69, wherein the composition has a pH of 4 to 6.5.

[0328] Clause 71. The composition of Clause 69 or 70, further comprising:

[0329] 0.01 wt% to 0.5 wt% of surfactant (e.g., 0.01 wt% of polysorbate 80);

[0330] 0.1 wt% to 25 wt% of stabilizer (e.g., 9 wt% sucrose); and

[0331] Buffer solution (e.g., 20 mM acetate buffer).

[0332] Clause 72. A composition of any one of Clauses 1-71, wherein at least 70% of the antibody is present in the composition in a monomeric state.

[0333] Clause 73. The composition of Clause 72, wherein at least 90% of the antibody is present in the composition in a monomeric state.

[0334] Clause 74. A composition of any one of Clauses 1-73, wherein the composition is a liquid with a surface tension of 60 mN / m or less.

[0335] Clause 75. A composition of any one of Clauses 1-74, wherein the composition has an interfacial complex viscosity of 0.2 Pa·s·m or less.

[0336] Clause 76. A composition of any one of Clauses 1-75, wherein the composition is in 0 s -1 Up to 3,000 s -1 It has a viscosity of 6 mPa·s or less at a shear rate.

[0337] Clause 77. The composition of any one of Clauses 1-76, wherein, based on the half-maximal inhibitory concentration determined by enzyme-linked immunosorbent assay, the composition retains 70% or more of the binding activity of the antibody against the target antigen after 21 days of continuous stress aging.

[0338] Clause 78. A composition of any one of Clauses 1-77, wherein the composition is formulated for subcutaneous administration.

[0339] Clause 79. A composition of any one of Clauses 1-77, wherein the composition comprises a suspension of particles in a liquid carrier.

[0340] Compositions of Clause 80 and Clause 79, wherein the liquid carrier is non-aqueous.

[0341] Clause 81. The composition of Clause 79 or 80, wherein the particles comprise a polyacrylamide-based copolymer and an antibody.

[0342] Clause 82. A method of administering an antibody to a subject in need, comprising injecting the subject with a therapeutically effective amount of a composition according to any one of Clauses 1-81.

[0343] Clause 83. The method of Clause 82, wherein the injection is a subcutaneous injection.

[0344] Clause 84. A composition according to any one of Clauses 1-81, used for subcutaneous administration of a biological agent (e.g., an antibody) to a subject.

[0345] Clause 85. Use of the composition of any one of Clauses 1-81 for the preparation of a medicament for subcutaneous administration of a biological agent (e.g., an antibody) to a subject.

[0346] Clause 86. A storage-stable solid composition comprising:

[0347] Stabilizer;

[0348] Polyacrylamide-based copolymers comprising:

[0349] Water-soluble carrier monomers selected from N-(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), acrylamide (AM), and combinations thereof; and

[0350] Functional dopant monomers selected from N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), NN-diethylacrylamide (DEA), N-tert-butylacrylamide (TBA), N-phenylacrylamide (PHE), and combinations thereof; and

[0351] Biological pharmaceutical reagents.

[0352] Clause 87. Solid compositions of Clause 86, wherein the biopharmaceutical reagent is not an antibody or antibody fragment.

[0353] Clause 88. A solid composition of Clause 86 or 87, wherein the composition is soluble in water to form an aqueous composition comprising:

[0354] 0.1 wt% to about 10 wt% of copolymers; and

[0355] Biopharmaceutical reagents ranging from 0.5 wt% to 25 wt%.

[0356] Clause 89. A solid composition of any one of Clauses 86 to 88, wherein the solid composition (e.g., a lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition, said liquid composition containing 0.1 mg / mL or more of a polyacrylamide-based copolymer prior to drying (e.g., lyophilizing).

[0357] Clause 90. Solid compositions of Clause 89, wherein the liquid composition comprises 0.3 mg / mL or more of a polyacrylamide-based copolymer.

[0358] Clause 91. The solid composition of Clause 90, wherein the liquid composition comprises 1.0 mg / mL or more of a polyacrylamide-based copolymer.

[0359] Clause 92. The solid composition of claim 91, wherein the liquid composition comprises 3 mg / mL or more of a polyacrylamide-based copolymer.

[0360] Clause 93. The solid composition of Clause 92, wherein the liquid composition comprises 10 mg / mL or more of a polyacrylamide-based copolymer.

[0361] Clause 94. A solid composition of any one of Clauses 86 to 93, wherein the biopharmaceutical reagent is selected from vaccines, gene therapy, cell therapy and lipid nanoparticles (LNPs) containing nucleic acids.

[0362] Clause 95. The solid composition of Clause 94, wherein the biological pharmaceutical reagent is a vaccine.

[0363] Clause 96. Solid compositions of Clause 95, wherein the vaccine contains live or attenuated virus.

[0364] Clause 97. The solid composition of Clause 96, wherein the vaccine is an mRNA vaccine.

[0365] Clause 98. A solid composition of any one of Clauses 86 to 97, wherein the solid composition exhibits greater thermal stability after 14 days at 37°C compared with a solid composition lacking a polyacrylamide-based copolymer.

[0366] Clause 99. A solid composition of any one of Clauses 86 to 98, wherein the biopharmaceutical reagent is a polypeptide.

[0367] Clause 100. Clause 99 solid compositions wherein the polypeptide is a therapeutic protein.

[0368] Clause 101. A 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.

[0369] Clause 102. The solid composition of Clause 101, wherein the therapeutic protein is selected from antibodies or fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic proteins.

[0370] Clause 103. The solid composition of Clause 102, wherein the therapeutic protein is an enzyme.

[0371] Clause 104. A solid composition of Clause 103, wherein the therapeutic protein is an antibody or a fragment thereof.

[0372] 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, an Fc fusion protein, or a fragment thereof.

[0373] Clause 106. The solid composition of Clause 105, wherein the therapeutic protein is a monoclonal antibody.

[0374] Clause 107. A solid composition of any one of Clauses 103-105, wherein the solid composition (e.g., a lyophilized composition) is obtained by drying (e.g., lyophilizing) a liquid composition, said liquid composition comprising 1.0 mg / mL or more (e.g., 2 mg / mL or more, 3 mg / mL or more, or 4 mg / mL or more) of a polyacrylamide-based copolymer prior to drying (e.g., lyophilizing).

[0375] Clause 108. The solid composition of Clause 107, wherein the liquid composition comprises 10 mg / mL or more of a polyacrylamide-based copolymer.

[0376] Clause 109. A solid composition of any one of Clauses 99 to 108, wherein the solid composition exhibits storage stability compared to a composition lacking a polyacrylamide-based copolymer.

[0377] Clause 110. A solid composition of any one of Clauses 99 to 108, wherein the solid composition exhibits a shorter reconstitution time compared to a composition lacking a polyacrylamide-based copolymer.

[0378] Clause 111. A solid composition of any one of Clauses 86 to 93, wherein the water-soluble carrier monomer is selected from MORPH, MPAM, and combinations thereof.

[0379] Clause 112. The solid composition of Clause 111, wherein the water-soluble carrier monomer comprises MORPH.

[0380] Clause 113. The solid composition of Clause 111, wherein the water-soluble carrier monomer comprises MPAM.

[0381] Clause 114. A 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.

[0382] Clause 115. A solid composition of any one of Clauses 86 to 114, wherein the functional dopant monomer is selected from DEA, PHE, NIP and combinations thereof.

[0383] Clause 116. A solid composition of any one of Clauses 86 to 114, wherein the functional dopant monomer comprises TRI.

[0384] Clause 117. Solid compositions of Clause 115, wherein the functional dopant monomer comprises PHE.

[0385] Clause 118. Solid compositions of Clause 115, wherein the functional dopant monomer comprises NIP.

[0386] Clause 119. Solid compositions of Clause 115, wherein the functional dopant monomer comprises DEA.

[0387] Clause 120. A solid composition of any one of Clauses 86 to 119, wherein:

[0388] Water-soluble carrier monomers are selected from MPAM, MORPH, and combinations thereof; and

[0389] The functional dopant monomers are selected from NIP, PHE and their combinations.

[0390] Clause 121. A solid composition of any one of Clauses 86 to 120, wherein:

[0391] Water-soluble carrier monomers are selected from MPAM, MORPH, and combinations thereof; and

[0392] The functional dopant monomers are selected from AMP, TMA, TBA, PHE and combinations thereof.

[0393] Clause 122. A solid composition of any one of Clauses 86 to 121, wherein the water-soluble carrier monomer is MPAM and the functional dopant monomer is PHE.

[0394] Clause 123. A solid composition of any one of Clauses 86 to 121, wherein the water-soluble carrier monomer is MORPH and the functional dopant monomer is PHE.

[0395] Clause 124. A 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.

[0396] Clause 125. A solid composition of any one of Clauses 86 to 124, wherein the copolymer comprises:

[0397] 70 wt% to 98 wt% water-soluble carrier monomers; and

[0398] 2 wt% to 30 wt% of functional dopant monomers.

[0399] Clause 126. A solid composition of any one of Clauses 86 to 125, wherein the copolymer comprises:

[0400] 80 wt% to 95 wt% water-soluble carrier monomer; and

[0401] 5 wt% to 20 wt% of functional dopant monomers.

[0402] Clause 127. A solid composition of any one of Clauses 86 to 126, wherein the copolymer comprises:

[0403] 83 wt% to 98 wt% of water-soluble carrier monomers; and

[0404] 2 wt% to 17 wt% of functional dopant monomers.

[0405] Clause 128. A solid composition of any one of Clauses 86 to 126, wherein the copolymer comprises:

[0406] 70 wt% to 85 wt% MORPH; and

[0407] 15 wt% to 30 wt% NIP.

[0408] Clause 129. The solid composition of Clause 128, wherein the copolymer comprises:

[0409] 74 wt% to 80 wt% MORPH; and

[0410] 20 wt% to 26 wt% NIP.

[0411] Clause 130. The solid composition of Clause 129, wherein the copolymer comprises:

[0412] 77 wt% MORPH; and

[0413] 23 wt% NIP.

[0414] Clause 131. A solid composition of any one of Clauses 86 to 130, wherein the degree of polymerization of the copolymer is 10 to 500.

[0415] Clause 132. A solid composition of any one of Clauses 86 to 131, wherein the degree of polymerization of the copolymer is 20 to 200.

[0416] Clause 133. A solid composition of any one of Clauses 86 to 132, wherein the number average molecular weight of the copolymer is from 2,000 g / mol to 10,000 g / mol.

[0417] Clause 134. The solid composition of Clause 133, wherein the number average molecular weight of the copolymer is from 2,000 g / mol to 6,000 g / mol.

[0418] Clause 135. The solid composition of Clause 134, wherein the number average molecular weight of the copolymer is from 3,000 g / mol to 5,000 g / mol.

[0419] Clause 136. A solid composition of any one of Clauses 86 to 135, further comprising one or more additional components selected from preservatives and surfactants.

[0420] Clause 137. A solid composition of any one of Clauses 86 to 136, wherein the stabilizer is sugar.

[0421] Clause 138. The solid composition of Clause 137, wherein the stabilizer sugar is trehalose.

[0422] Clause 139. A solid composition of any one of Clauses 136 to 138, wherein one or more additional components are present in an amount of about 0.1-50% by weight.

[0423] Clause 140. A solid composition of any one of Clauses 136 to 138, wherein one or more additional components are present in an amount of about 0.001 to 1.0% by weight.

[0424] Clause 141. A solid composition of any one of Clauses 86 to 140, wherein the solid composition is a lyophilized composition.

[0425] Clause 142. A pharmaceutical unit dose composition comprising the solid composition of any one of Clauses 86 to 141.

[0426] Clause 143. The unit dose composition of the drug as described in Clause 142, wherein the composition is contained in a single-dose container, vial, or pre-filled syringe.

[0427] Clause 144. A method for reconstituted 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.

[0428] Clause 145. The method of Clause 144, wherein the aqueous solution is sterile water.

[0429] Clause 146. The method of Clause 145, wherein the aqueous solution contains a buffer.

[0430] Clause 147. The method of any one of Clauses 143 to 145, further comprising the composition reconstituted by centrifugation.

[0431] Clause 148. The method of any one of Clauses 143 to 146, further comprising removing particles from the reconstituted composition.

[0432] Clause 149. The method of any one of Clauses 143 to 146, further comprising equilibrating the solid composition to room temperature before dissolution.

[0433] Clause 150. The method of any one of Clauses 143 to 146, which further includes assessing the resolution time of particulate matter dissolution.

[0434] Clause 151. The method of any one of Clauses 143 to 150, wherein the solid composition is a fixed dose of a biological pharmaceutical reagent contained in a vial.

[0435] Clause 152. The method of any one of Clauses 143 to 151, wherein the reconstitution time is less than 30 min.

[0436] Clause 153. A reconstituted composition prepared according to any one of Clauses 143 to 152.

[0437] Clause 154. A method of administering a biological pharmaceutical agent to a subject in need, the method comprising administering to the subject a reconstituted composition according to Clause 153.

[0438] Clause 155. A method for reconstituted lyophilized composition, the method comprising dissolving a lyophilized composition of any one of Clauses 86 to 154 in an aqueous solution to produce a reconstituted lyophilized composition.

[0439] Clause 156. The method of Clause 155, wherein the aqueous solution is sterile water.

[0440] Clause 157. The method of Clause 156, wherein the aqueous solution contains a buffer.

[0441] Clause 158. The method of any one of Clauses 155 to 157, which further includes the lyophilized composition reconstituted by centrifugation.

[0442] Clause 159. The method of any one of Clauses 155 to 158, further comprising removing particles from the reconstituted lyophilized composition.

[0443] Clause 160. A reconstituted lyophilized composition prepared according to any one of Clauses 155 to 159.

[0444] Clause 161. A method of administering a biological pharmaceutical agent to a subject in need, the method comprising administering to the subject a reconstituted lyophilized composition according to Clause 160.

[0445] Clause 162. A method for preparing a solid composition according to any one of Clauses 86 to 161, said method comprising:

[0446] A liquid biopharmaceutical composition is exposed to a drying cycle to produce a solid composition, said liquid biopharmaceutical composition comprising:

[0447] Polyacrylamide-based copolymers comprising:

[0448] Water-soluble carrier monomers selected from N-(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), acrylamide (AM), and combinations thereof; and

[0449] Functional dopant monomers selected from N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), NN-diethylacrylamide (DEA), N-tert-butylacrylamide (TBA), N-phenylacrylamide (PHE), and combinations thereof; and

[0450] Biological pharmaceutical reagents.

[0451] Clause 163. The method of Clause 162, wherein the drying is selected from freeze drying, spray drying, spray freeze drying, foam pad drying, vacuum oven drying and dehydration drying.

[0452] Clause 164. The method of Clause 162 or 163, wherein a liquid biopharmaceutical composition is exposed to a lyophilization cycle in a lyophilizer to produce a lyophilized composition.

[0453] Clause 165. The method of Clause 164, wherein the freeze-drying cycle includes:

[0454] To subject the liquid biopharmaceutical composition to initial pressure;

[0455] Evaporation of at least a portion of the liquid component of the liquid biopharmaceutical composition while adding heat energy; and

[0456] Cooling liquid biopharmaceutical compositions to freeze any remaining liquid components into solids.

[0457] Clause 166. The method of Clause 154 or 165, wherein the freeze-drying cycle includes:

[0458] The liquid biopharmaceutical composition is placed at a first reduced temperature;

[0459] The liquid biopharmaceutical composition is subjected to a first reduced pressure;

[0460] At least a portion of the liquid component of the liquid biopharmaceutical composition is evaporated while heat energy is added;

[0461] Subjecting the liquid biopharmaceutical composition to a second reduced pressure below the first reduced pressure; and

[0462] The remaining liquid is evaporated to produce a solid composition.

[0463] Clause 167. The method of Clause 166, wherein the drying cycle is a spray drying cycle, comprises:

[0464] Nebulized liquid biological drug composition;

[0465] At least a portion of the liquid component of the evaporated liquid biopharmaceutical composition; and

[0466] Collect the solid composition.

[0467] Clause 168. The method of Clause 167, wherein the drying cycle is a spray freeze-drying cycle, comprises:

[0468] Nebulized liquid biological drug composition;

[0469] Frozen, atomized liquid biopharmaceutical compositions;

[0470] Evaporation of at least a portion of the liquid component of an atomized liquid biopharmaceutical composition; and

[0471] Collect the solid composition.

[0472] Clause 169. The method of Clause 168, wherein the drying cycle is a foam pad drying cycle, includes:

[0473] To foam the liquid biological drug composition;

[0474] Spread the foamed liquid biopharmaceutical composition onto a dry surface;

[0475] Evaporation of at least a portion of the liquid component of a foamed liquid biopharmaceutical composition; and

[0476] Collect the solid composition.

[0477] Clause 170. The method of Clause 168, wherein drying is a vacuum oven drying cycle, comprises:

[0478] To subject the liquid biopharmaceutical composition to reduced pressure;

[0479] Evaporation of at least a portion of the liquid component of the liquid biopharmaceutical composition while adding heat energy; and

[0480] The pressure is restored to the initial pressure to obtain a solid composition.

[0481] Clause 171. The method of Clause 170, wherein drying is a dehydration drying cycle, comprising:

[0482] Increase the surface area of ​​the liquid biopharmaceutical composition, for example, by pouring the liquid biopharmaceutical composition into a tray;

[0483] At least a portion of the liquid component of the liquid biopharmaceutical composition is evaporated by circulating ambient air above a surface, wherein the circulating air is optionally dried, heated, or both on a dehydrating material until a solid is obtained.

[0484] Example

[0485] General experimental details

[0486] material

[0487] Solvents N,N-dimethylformamide (DMF; >99.7%; HPLC grade, Alfa Aeser), ethanol (EtOH; >99.5%; ACS certified, Acros Organics), acetone (>99.9%; Sigma-Aldrich, HPLC grade), hexane (>99.9%; ThermoFisher Scientific, ACS certified), diethyl ether (anhydrous, >99%; Sigma-Aldrich, ACS certified), and CDCl3 (>99.8%; Acros Organics) are used as accepted. MORPH (>97%; Sigma-Aldrich) is filtered with alkaline alumina prior to use. NIP (>99%; Sigma-Aldrich) is used as accepted. RAFT CTA 2-cyano-2-propyldodecyl trithiocarbonate (2-CPDT; >97%; Strem Chemicals) is used as accepted. The initiator 2,2-azobis(2-methylpropionitrile) (AIBN; >98%; Sigma-Aldrich) was recrystallized from methanol (>99.9%; Thermo Fisher Scientific, HPLC grade) and dried under vacuum before use. Z-group removers lauroyl peroxide (LPO; 97%; Sigma-Aldrich) and hydrogen peroxide (H2O2; 30%; Sigma-Aldrich) were used as received. The PGT121 monoclonal antibody was provided by Just-Evotec Biologics, Inc. (Seattle, WA, USA) in collaboration with the Bill & Melinda Gates Foundation. PGT121.9e9 anti-idiotype monoclonal antibody was provided at a concentration of 55.5 mg / mL in 20 mM acetate buffer containing 9% (w / v) sucrose, 0.01% PS 80, and pH 5.0. It was synthesized at the Protein Production Facility (PPF), funded by the Bill & Melinda Gates Foundation, using plasmids provided by the Vaccine Research Center (VIRC) of the National Institute of Allergy and Infectious Diseases (NIAID), a division of the National Institutes of Health (NIH).

[0488] Synthesis of MoNi

[0489] According to Mann et al., Sci.Transl.Med.12. The method described in eaba6676 (2020) for preparing the amphiphilic acrylamide copolymer excipient acryloylmorpholine-co-N-isopropylacrylamide (MoNi) involves mixing MORPH (645 mg, 4.57 mmol, 41.5 equivalents), NIP (105 mg, 0.93 mmol, 8.5 equivalents), 2-CPDT (38 mg, 0.11 mmol, 1 equivalent), and AIBN (3.6 mg, 0.02 mmol, 0.2 equivalents) and diluting with DMF to a total volume of 2.25 mL [33.3% (w / v) vinyl monomer concentration], and placing the mixture in an 8-mL scintillation vial equipped with a PTFE diaphragm. The reaction mixture was then purged with nitrogen for 10 min and heated at 65 °C for 12 h. To remove the Z-terminus of the resulting polymer, AIBN (360 mg, 2.2 mmol, 20 equivalents) and LPO (88 mg, 0.22 mmol, 2 equivalents) were added to the reaction mixture, followed by nitrogen injection for 10 min and heating at 90 °C for 12 h. The removal of the Z-terminus was confirmed by the ratio of refractive index to UV (310 nm) intensity in SEC analysis. The resulting polymer was precipitated three times from diethyl ether and dried under vacuum overnight. 1 The composition and molecular weight were determined by 1H NMR spectroscopy and SEC using PEG standards.

[0490] Characterization of copolymer molecular weight by SEC

[0491] MoNi's M n M w The determination of PEG and Ð was performed using SEC of PEG standards (American Polymer Standards Corporation): 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)], mobile phase was DMF containing 0.1 M LiBr, temperature was 35 °C, flow rate was 1.0 mL / min -1 [Dionex UltiMate 3000 pump, degasser and autosampler (Thermo Fisher Scientific)].

[0492] Preparation of high-concentration stable PGT121 formulation

[0493] The PGT121 monoclonal antibody was concentrated at 3030 RPM using a rotary filtration system (Corning® Spin-X® UF 5kDa MWCO) until the desired volumetric recovery was achieved. The concentrated formulation was refrigerated for 12 hours before use in accelerated aging studies.

[0494] Dynamic light scattering

[0495] DLS measurements of MoNi prepared in milliQ water were performed on a DynaPro Plate Reader II (Wyatt Technology). The average value of five acquisitions is reported.

[0496] Concentration determination

[0497] Depending on the assay method, the concentration of PGT121 formulations was determined by volumetric recovery, ELISA, Nanodrop (Thermo Scientific), or aqueous SEC-UV (PBS buffer, 300 ppm sodium azide). For aqueous SEC-UV, PGT121 aliquots were diluted 133-fold in MilliQ water, and the concentration was determined by comparing the area under the curve of the trace obtained from the SEC at 280 nm using a Dionex UltiMate 3000 VWD (Thermo Scientific). In these assays, aged samples were compared to unaged 55 mg / mL PGT121 solutions (n=3). For nanodrops, protein concentrations were compared to 55 mg / mL stock solution standards. For ELISA, concentrations were calculated by interpolation by fitting a four-parameter dose-response curve (variable slope) in GraphPad Prism 9. High concentrations of PGT121 formulations were reported as the mean ± standard deviation of concentrations determined by these methods for several preparations of these formulations.

[0498] Surface tension

[0499] Time-resolved surface tension at the air-solution interface was measured using a platinum / iridium Wilhelmy plate connected to an electroequilibration (KSV Nima, Finland). The Wilhelmy plate was partially immersed in an aqueous solution in a petri dish, and the interfacial surface tension was recorded for 50 min from the start of new interface formation. Equilibrium surface tension values ​​(t = 50 min) are reported because these values ​​more closely describe the environment in the storage vials before stirring. Samples were diluted from stock samples to the desired assay concentration in 20 mM acetate buffer (pH = 5.0). The experiment was repeated three times.

[0500] Interfacial rheology

[0501] Interfacial shear rheology was measured using a Discovery HR-3 rheometer (TA Instruments) with an interfacial geometry comprising a Du Noüy ring made of platinum / iridium wire (CSC Scientific, Fairfax, VA, catalog no. 70542000). Prior to each experiment, the Du Noüy ring was rinsed with ethanol and water and flame-treated to remove organic contaminants. The solution chamber consisted of a double-walled Couette flow cell with an inner Teflon cylinder and an outer glass beaker. Time scans were performed at 1% strain (within the linear region) and a frequency of 0.05 Hz (low enough to negligible instrument inertial effects). Interfacial composite shear viscosity was measured for 30 min. The sample was diluted from the stock sample to the desired assay concentration in 20 mM acetate buffer at pH 5.0. The experiment was repeated three times.

[0502] PGT121 ELISA

[0503] Capture antibody 9e9 was coated at a concentration of 2 μg / mL (in phosphate-buffered saline (PBS)) (25 μg per well) onto Corning 96-well high-binding flat-bottom half-area microplates (Fisher Scientific) and incubated overnight at 4°C. The blocking buffer used was 2% nonfat milk powder (NFDM) in PBS, the assay buffer was 2% bovine serum albumin (BSA) in PBS, and the wash buffer was PBS-T (0.05% Tween 20). The plates were washed twice, blocked with 125 μL of blocking buffer, and incubated at room temperature for 1 hour. Sample diluents and PGT121 standards were prepared in assay buffer. After blocking, the assay buffer was removed from the plates before adding 50 μL of each sample and incubating at room temperature for 1 hour. The plate was washed 5 times, and then 50 μL of secondary antibody (horseradish peroxidase AffiniPure F(ab'')2 fragment goat anti-human IgG (specific to the Fcγ fragment)) was added to each well and diluted 1:5000 in assay buffer. After incubation at room temperature for 1 hour, the plate was washed 10 times, and then developed by adding 50 μL of TMB ELISA substrate (high sensitivity) (Abcam, ab171523). Development was stopped by adding 1N hydrochloric acid after 2.5 min. The PGT121 concentration was measured by absorbance at 450 nm using a Synergy H1 hybrid multimode plate reader (BioTek). Each plate contained a 15-point standard curve measured in duplicate wells. This standard curve was used to interpolate and calculate PGT121 concentration by fitting a four-parameter dose-response curve (variable slope) in a GraphPad Prism 9. Stress-induced aging samples were diluted 1:10000 and then serially diluted 4-fold to generate 7-point curves (measured in duplicate wells for each sample). IC50 was determined by fitting a four-parameter dose-response curve (variable slope) in a GraphPad Prism 9. 50 Values. The mean and standard deviation are reported.

[0504] In vivo pharmacokinetic study

[0505] Animal studies were conducted in accordance with the laboratory animal care and use guidelines approved by the Stanford University Animal Care and Use Committee (IACUC). Eight-week-old female B6.Cg-Fcgrt were administered via intraperitoneal injection (IP) or subcutaneous injection (SC) under brief isoflurane anesthesia. tm1Dcr Prkdc scidTg(FCGRT)32Dcr / DcrJ (The Jackson Laboratory, strain 018441) mice were administered PGT121 antibody (1.5 mg / mouse) (n=6 / group). A polymer-stable high-concentration SC formulation was prepared with 102 mg / mL PGT121 (as determined by ELISA) and 1 wt% MoNi (15 μL injection volume), while a low-concentration IP formulation was prepared at 5 mg / mL (300 μL injection volume) in the same buffer containing the antibody. Blood samples were collected at 24 hours, 48 ​​hours, and then on days 4, 7, 10, 14, and 21 post-injection for analysis of serum PGT121 concentrations by ELISA.

[0506] Viscosimetry

[0507] Viscosities at high shear rates, representing the injection, were measured using a Rheosense m-VROC viscometer equipped with a low-viscosity chip. Samples were measured from low to high shear rates using a Hamilton syringe. Each data point was collected under steady-state conditions.

[0508] In vitro stability determination

[0509] Dilute 150 µL of PGT121 solution with 7.5 μL of MoNi in formulation buffer at a concentration of 21 mg / mL to achieve a final excipient concentration of 0.1 wt.%. Take 5 mL aliquots of these formulations every 24 hours while stirring at 200 rpm at 50 °C.

[0510] Diffusion ordered spectroscopy

[0511] Record at a concentration of 0.2 mg / mL PGT121 (this concentration was obtained by diluting 20 mM acetate buffer containing 9% (w / v) sucrose and 0.01% PS 80 in D2O (Acros Organics) at pH 5.0). 1 Two-dimensional DOSY spectra were obtained, and D2O was dialyzed for 24 hours using a 2000 Da MWCO Slide-A-Lyzer dialysis kit (Thermo Scientific) before use. MoNi was prepared directly in D2O at a concentration of 0.1 mg / mL.

[0512] Data were acquired using a Varian Inova 600 MHz NMR spectrometer. Magnetic field strength ranged from 2 to 57 G cm⁻¹. -1The DOSY time and gradient pulse were set to 66.5 ms (Δ) and 2 ms (δ), respectively. All NMR data were processed using MestReNova 11.0.4 software.

[0513] Statistics

[0514] Unless otherwise stated, all results are expressed as mean ± standard deviation (SD) and were analyzed using GraphpadPrism 9.1 (GraphPad Software Inc., USA).

[0515] Figure 1 This is a set of schematic diagrams showing an overview of the use of novel copolymer excipients to stabilize high-concentration antibody formulations. (Figure A) Schematic overview of current routes of administration for biologic therapies, with intravenous administration being the most common due to the limited stability of biologics at the high concentrations required for subcutaneous administration, resulting in a high burden on patients. (Figure B) Amphiphilic acrylamide carrier / dopane copolymer (AC / DC) excipients (e.g., poly(acryloylmorpholine-co- N A diagram of (-isopropylacrylamide)(MoNi) is shown, whereby the excipient can be used to stabilize a biopharmaceutical in a formulation. (Figure C) illustrates the use of an AC / DC copolymer as a "drop-in" excipient to stabilize a monoclonal antibody in a formulation. The AC / DC copolymer is amphiphilic and preferentially adsorbs onto the interface, preventing interfacial adsorption of the antibody in the formulation, thereby preventing interface-related destabilization and aggregation events.

[0516] Example 1. Selective adsorption of copolymers to the interface in mAb formulations

[0517] To synthesize MoNi, reversible addition-fracture transfer (RAFT) controlled radical polymerization was employed. This yielded a polymer (denoted as MoNi-CTA) with a reactive trithiocarbonate chain transfer agent (CTA) attached post-polymerization to the Z-terminus of the polymer. Prior to use in subsequent assays, the CTA moiety was removed from the MoNi-CTA copolymer to yield the MoNi excipient, ensuring chemical stability and bioinertness. Figure 2 (Figures A–D).

[0518] The concentration that killed 50% of cells in vitro (referred to as LC) was compared. 50 The reported values ​​of the parameters were used to demonstrate that MoNi is biocompatible, with cytotoxicity less than 1 / 100 of that of common commercial surfactant excipients such as polysorbate 80 (PS80) and Pluronic L61. Figure 2 (Figure E). Maikawa et al. Biomacromolecules22(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).

[0519] The MoNi copolymer excipient was synthesized at a molecular weight well below the glomerular filtration threshold to ensure rapid excretion after administration in vivo without bioaccumulation. Furthermore, dynamic light scattering (DLS) analysis was performed on a series of MoNi dilutions from 100 to 1 mg / mL to attempt to determine the critical micelle concentration (CMC) of MoNi. Figure 2 (Figure F). In these assays, the MoNi excipient did not form micelles or aggregates within the evaluated concentration range. This behavior is unique compared to common commercial surfactant excipients PS80 and Pluronic L61, both of which exhibit CMC values ​​well below 1 mg / mL ( Figure 2 (G diagram). Chou et al. J. Pharm.Sci. 94(6):1368–81 (2005); Carvalho et al., International journal of pharmaceutics 602:120635 (2021). It is noteworthy that most surfactants are toxic in their micellar state, suggesting that MoNi's significantly lower cytotoxicity may be related to the fact that it does not exhibit self-assembly into micelles at formulation-related concentrations compared to its commercial counterparts. Demina et al., Biomacromolecules 15(7):2672–81 (2104).

[0520] Whether MoNi preferentially adsorbs to the air-water interface was tested. Time-resolved surface tension experiments were performed using an anti-HIV bnAb PGT121 formulation (a promising candidate for HIV passive immunization that targets the highly conserved V1 / V2 glycans on the gp120 surface glycoprotein). Patel et al. J. Pharm.Sci. 107(12):2969–82 (2018); Stephenson et al., Nature Medicine 27:1718–24 (2021). Compared with PGT121 alone, the PGT121 formulation with MoNi (0.01 wt%) (20 mM acetate buffer, pH ~5.0) exhibited lower surface tension values ​​(53 mN / m and 62 mN / m, respectively); Figure 3(Figure A). The lower surface tension observed in the PGT121 formulation containing MoNi indicates that more species accumulate at the interface in this formulation compared to the formulation containing PGT121 alone, suggesting that MoNi preferentially adsorbs to the interface at a higher level. Notably, the surface tension of the formulation containing only MoNi and the formulation containing both PGT121 and MoNi was found to be the same in the buffer solution. Figure 3 Figure A shows that at these concentrations, the air-water interface is indeed dominated by MoNi. These results indicate that MoNi preferentially adsorbs to the air-water interface, thereby preventing mAb adsorption.

[0521] To further characterize the effect of MoNi on the PGT121 formulation, interfacial viscoelasticity of these formulations was probed by interfacial shear rheological measurements. These measurements revealed that the addition of MoNi to the PGT121 formulation reduced the interfacial complex viscosity by 1 / 3 (from about 0.3 Pa·s·m to about 0.1 Pa·s·m). 1 ; Figure 3 (Figure B). The high interfacial recurrent viscosity of the MoNi-free PGT121 formulation indicates that protein interactions at the interface do indeed lead to the formation of a gel-like surface layer at the interface. Therefore, the significant reduction in interfacial recurrent viscosity after the addition of MoNi indicates that the copolymer excipient reduces the interfacial interactions of PGT121, consistent with the surface tension measurements above (showing that mAb is prevented from adsorbing to the interface).

[0522] Diffusion ordering spectroscopy (DOSY) (a mature NMR technique that can directly assess the diffusion behavior of different substances in solution) was also used to determine whether MoNi interacted with mAb in the bulk. Figure 4 As shown in Figures A-C, PGT121mAb and MoNi exhibit different diffusion behaviors, indicating that these two species do not interact in the bulk. These results further suggest that MoNi stabilizes the protein load by preventing interfacial aggregation.

[0523] The ease of injection of mAb formulations at concentrations greater than 100 mg / mL under clinically relevant injection conditions was determined. Hernandez et al., Macromol.Biosci .21:2000295 (2021). The viscosities of high-concentration PGT121 solutions (119 ± 7 mg / mL) with and without MoNi (10 mg / mL) are at shear rates typically achieved during injection using standard syringe and needle geometry and flow rates. Adding this concentration of MoNi does not alter the injectable viscosity of the formulation, and both formulations exhibit sufficiently low viscosity for easy injection. Figure 3 (Figure C).

[0524] Example 2. Copolymer stability of mAb formulations in stress aging assays

[0525] Accelerated aging assays were performed. Initially, a stock formulation of PGT121 at 55.5 mg / mL (20 nM acetate, 9 wt% sucrose, 0.01 wt% PS80, pH ~5.0) was compared with MoNi (1 mg / mL and 10 mg / mL). These formulations, including a control containing only the PGT121 stock formulation, were packaged in glass vials and subjected to accelerated aging (continuous stirring at 50°C). The monomer mAb content in the formulations was monitored by size exclusion chromatography (SEC) for more than 1 week (7 days). Although formulations containing either concentration of MoNi retained more PGT121 monomers within 7 days than the control formulation without MoNi, the sample containing 10 mg / mL MoNi showed the best stability. Figure 8 Next, a stock formulation of PGT121 at 55.5 mg / mL (20 mM acetate, 9 wt% sucrose, 0.01 wt% PS80, pH ~5.0) was compared with a formulation at more than twice the concentration of 119 ± 7 mg / mL (referred to herein as high concentration). These formulations, with and without MoNi (10 mg / mL), were then packaged in glass vials and subjected to accelerated aging (continuous stirring at 50°C). Figure 5 (Figure A). Size exclusion chromatography (SEC) was used. Figure 5 (Figure B) The monomer mAb content in these formulations was monitored over 4 weeks. Within 5 days, both the PGT121 stock formulation and the high-concentration PGT121 formulation lost more than 50% of their monomer mAb content, indicating significant protein aggregation. The monomer PGT121 loss and aggregate formation within 6 days are shown in Figure B. Figure 9 The representative SEC chromatograms (Figures A-C) are shown, where Figure A is a representative SEC chromatogram of PGT121 without MoNi; Figure B is a representative SEC chromatogram of PGT121 containing 1 mg / mL MoNi; and Figure C is a representative SEC chromatogram of PGT121 containing 10 mg / mL MoNi.

[0526] By day 7, both formulations exhibited macroscopic aggregation and became noticeably opaque. In contrast, the MoNi-containing PGT121 stock formulation maintained over 95% of its monomer mAb content during three weeks of continuous stress aging. Figure 5 (Figure B). Although the high-concentration sample containing MoNi showed a slight initial decrease in monomer mAb content during the first week of stress aging (likely due to the concentration process), the formulation maintained nearly 70% of the monomer mAb content over the same three-week period. Figure 5 (Figure B).

[0527] The study determined whether epitope binding activity (as a surrogate indicator of therapeutic efficacy) was preserved during continuous stress aging. Enzyme-linked immunosorbent assay (ELISA) was used to determine whether stress-aged samples retained binding activity in their Fab and Fc domains, thereby preserving conformational fidelity. Figure 5 (Figure C). Using half-maximum inhibition concentration (IC50) 50 The functional potency of high-concentration formulations containing and without MoNi (10 mg / mL) subjected to stress aging was compared. In the absence of MoNi, PGT121 mAb rapidly lost function, losing more than 65% of its potency after only five days of stress aging. Figure 5 (Figure D). In contrast, the addition of MoNi significantly enhanced formulation stability, with PGT121 mAb retaining more than 75% of its original potency during 21 days of continuous stress aging.

[0528] In summary, these results demonstrate that the addition of MoNi excipients to high-concentration mAb formulations provides significant stability benefits by preventing aggregation events and maintaining binding activity under accelerated aging conditions by inhibiting mAb adsorption to the interface.

[0529] Example 3. Pharmacokinetic profile of a copolymer-stabilized high-concentration PFT121 formulation

[0530] Pharmacokinetic studies in rodents determined whether MoNi functions as an inactive component. Transgenic SCID mice (n=6 / group; B6.Cg-Fcgrt) with humanized FcRn receptors were injected with either 15 μL of MoNi-stabilized high-concentration PGT121 (119 mg / mL) via subcutaneous injection (SC) or 300 μL of low-concentration PGT121 via intraperitoneal injection (IP). tm1Dcr Prkdc scid Tg[FCGRT]32Dcr / DcrJ; Jackson Labs strain number 018441) was administered 1.5 mg of PGT121. Serum was collected within three weeks after administration, and systemic PGT121 concentration was analyzed by ELISA. Figure 6 (Figures A–B)

[0531] Quantitative analysis of serum concentrations over time and total PGT121 exposure (area under the curve; AUC) showed that the pharmacokinetic and bioavailability of the two administration routes and formulations were comparable. Figure 6(Figure C). No significant therapeutic differences in serum titers or bioavailability of PGT121 were observed when administered in a stable, high-concentration formulation of MoNi. Furthermore, no acute weight loss (a common indicator of treatment-related toxicity) was observed, further demonstrating the biocompatibility and tolerability of MoNi. Figure 7 (Figures A-C)

[0532] The addition of MoNi to high-concentration PGT121 mAb formulations significantly improved their stability without affecting pharmacokinetics, which is essential for the development of clinically relevant SC formulations. These results demonstrate the significant usefulness of MoNi as an excipient in addressing the challenges of biopharmaceutical formulation.

[0533] Example 4. Lyophilization and reconstitution of liquid compositions

[0534] The freeze-drying process involves: (a) loading the container with the liquid composition at an initial temperature of about 5°C to about -50°C; (b) cooling the composition to a sub-zero temperature (e.g., -10°C to -50°C); and (c) thoroughly drying the composition. The conditions for freeze-drying the composition (e.g., temperature and duration) may be adjusted taking into account factors that affect the freeze-drying parameters (e.g., the type of freeze-drying machine used).

[0535] 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 reconstitution methods are employed, such as vortexing, using a mechanical orbital oscillator, or keeping the container stationary.

[0536] Compared to compositions lacking polyacrylamide-based copolymers, the lyophilized compositions of this disclosure can have reduced reconstitution time.

[0537] The reconstitution time was evaluated as follows: A sample of the liquid composition was prepared, then stirred using a magnetic stirrer and kept aeration. The solution was then transferred to a volumetric flask, and water for injection (WFI) was added to the target volume (e.g., 50 ml). The solution was mixed to ensure homogeneity, filtered through a 0.2 μm filter, transferred to a vial, and lyophilized.

[0538] Reconstitute the sample using the target volume of filtered water for injection (WFI), restop, and reseal. Various reconstitution methods can be employed, such as vortexing, using a mechanical orbital oscillator, or keeping the vial stationary.

[0539] Reconstitution can be evaluated using various methods, including the absorbance of the reconstituted composition at a suitable wavelength. The reconstituted composition may, for example, have an absorbance of 0.002 or less at 450 nm, such as 0.001 or less.

[0540] Reconstitution time was measured after lyophilization (initial) and after stability testing (e.g., storage stability or stability under stress conditions), with vials reconstituted with the target volume of WFI.

[0541] Example 5. Evaluation of the lyophilized composition

[0542] The lyophilized compositions were exposed to different stress conditions to assess their relative stability. To impose thermal stress on the samples, the compositions were stored at 5°C, 25°C, or 40°C for up to 10 weeks. To assess stability after reconstitution, the samples were reconstituted in a biosafety cabinet using the target volume of filtered water for injection (WFI), re-stopped, and resealed. After reconstitution, the samples were stored at 25°C for 3 or 7 days. The various conditions tested in this experiment are summarized in Table 2 below.

[0543] Table 2

[0544]

[0545] The following assays can be performed to analyze the stability of various formulations under different conditions: (1) Visual inspection: Visual inspection is performed against a black and white background. Digital photographs are obtained. (2) Ultraviolet spectrophotometry. (3) Bioassays to assess the potency of active ingredients.

[0546] Example 6. Freeze-thaw stability assessment of exemplary antibody compositions

[0547] The formulation of this disclosure, containing two different monoclonal antibody APIs, was subjected to five freeze-thaw cycles, including freezing at -70°C and then thawing the formulation to room temperature. The amount of particulate matter (e.g., particle size > 0.2 μm) in the formulation samples was assessed before and after the freeze-thaw cycles. The number of particles in each sample was evaluated.

[0548] The formulations used for testing include:

[0549] 1. A control formulation that does not contain surfactants or copolymers.

[0550] 2. Formulations containing polysorbate 80 surfactant,

[0551] 3. Formulations containing 2 mg / mL MoNi copolymer, and

[0552] 4. Formulations containing 8 mg / mL MoNi copolymer.

[0553] Example 7. Thermal stability assessment of an exemplary vaccine composition

[0554] Lyophilized live attenuated vaccines were formulated with different concentrations of MoNi copolymer and subjected to heat stress to assess stability. Vaccine samples formulated without MoNi copolymer, and vaccine samples formulated with 0.01 mg / mL MoNi copolymer, 0.1 mg / mL MoNi copolymer, 1 mg / mL MoNi copolymer, or 10 mg / mL MoNi copolymer were prepared and lyophilized. To subject the samples to heat stress, the sample compositions were stored at, for example, 25°C or 40°C for, for example, 2 weeks or longer, 4 weeks or longer, or 6 weeks or longer, up to 10 weeks, or at 5°C for, for, 3 months, 6 months, or 12 months or longer.

[0555] The stability of the sample is assessed after reconstitution by measuring the efficacy, viability, and / or infectivity of the vaccine.

[0556] Example 8. Reconstitution of IgG lyophilized formulation

[0557] To assess whether the formulations disclosed herein can be reconstituted, the selected formulations were first lyophilized, and then the reconstitution of the lyophilized cakes was analyzed, along with the analysis of the reconstituted formulations.

[0558] The aim of the experiment was to select and prepare lyophilized formulations of IgG and MoNi that, after reconstitution with a selected medium, were sufficient to produce concentrations of 20 mg / mL, 100 mg / mL, and 200 mg / mL.

[0559] Procedure before lyophilization

[0560] Perform pre-lyophilization procedures to assess the suitability of the process for IgG buffer exchange and design a formulation. Perform pre-lyophilization analyses on the formulation to assess a) visual appearance; b) IgG concentration measured by Solo-VPE; and optionally c) IgG integrity assessed by SEC.

[0561] The pre-freeze-drying process includes:

[0562] 1. Test the ability to concentrate IgG using an Amicon centrifugal filter and exchange it for 5 mM L-histidine hydrochloride buffer; and

[0563] 2. Samples were taken during the concentration process and the aggregation was analyzed using the SEC method, with a target final protein concentration of 100 mg / mL.

[0564] Preparation of lyophilized formulation

[0565] Seven formulations for lyophilization were prepared according to the following procedure:

[0566] 1. Use 50 cm 2The appropriate amount of IgG was buffer-exchanged with 5 mM L-histidine hydrochloride buffer (pH 6) via a membrane (i.e., Millipore Pellicon XL) using TFF. Six diafiltration volumes were exchanged to remove >99% of the original formulation components upon initial concentration to the previously determined target. IgG concentration was measured using a Solo-VPE via UV.

[0567] 2. Using appropriate spiking solutions and volumes (as determined based on IgG concentration and necessary dilution factors), dilute the concentrated IgG in L-histidine hydrochloride into the following formulations. Prepare a total of 25 mL (or 1.25 g IgG) for each formulation. The compositions of the evaluated formulations are shown in Table 3.

[0568] Table 3

[0569]

[0570] Lyophilization and reconstitution

[0571] The five formulations shown in Table 3 were freeze-dried, and then the reconstitution of the above formulations at different concentrations was evaluated.

[0572] Perform freeze-drying according to the following procedure:

[0573] 1. Five formulations were aseptically filtered using a 0.2 M syringe membrane, and 5 mL (250 mg IgG) of each formulation was filled into 4 × 20 mL serum vials; and

[0574] 2. Use PLS conservative lyophilization cycle to lyophilize 1-5 of each formulation in 4 vials (Table 3).

[0575] Visually evaluate the freeze-dried cake blocks according to the following procedure:

[0576] 1. Observe and record the color, appearance, cracks, uniformity, and shrinkage of the freeze-dried cake pieces in all vials of the five freeze-dried formulations;

[0577] 2. If one or more lyophilized solid dosage forms are not in cake form, a reconstitution study is not performed on the sample.

[0578] 3. If none of the samples are in a pie shape, suspend the project.

[0579] According to the procedure in Table 4, three reconstitution experiments were performed in 20 mL vials at 200 mg / mL (a total of 18 reconstitution experiments).

[0580] Table 4

[0581]

[0582] Reconstitute according to the following procedure:

[0583] 1. Before starting the study, remove all vials from 2-8°C and allow them to equilibrate to room temperature;

[0584] 2. Add the specified volume of WFI to each vial of lyophilized formulation F1 through F6. Manually rotate the vial until all powder dissolves and a clear solution is obtained. Record the time required for the cake to dissolve and observe visually.

[0585] Optional analysis of reconstituted formulations was performed to assess the impact of lyophilization and reconstitution on the IgG integrity of consensus-based formulations.

[0586] SoloVPE was used to assess IgG concentration via UV (IgG concentration was measured using the IgG extinction coefficient provided or in the literature).

[0587] Aggregation / fragmentation was assessed using the SEC method, and the integrity of IgG was evaluated. Aggregation / fragmentation was compared with IgG samples before lyophilization and freshly prepared samples (as controls).

[0588] Results

[0589] The purpose of this experiment was to freeze-dry 250 mg IgG and 250 mg trehalose in a formulation containing control, 0.8 mg / mL polysorbate 20 (PS20), and 20 mg / mL MoNi in L-histidine hydrochloride buffer, and to measure the time required for the resulting freeze-dried cake to completely dissolve in a 200 mg / mL aqueous IgG formulation.

[0590] The results of reconstitution study 1 (approximately 20 mg / mL IgG, with the addition of 12 mL WFI) are shown in Table 5 below. Figure 10 A set of photographs is shown, which demonstrate the appearance of formulations F1-F6 in reconstitution study 1.

[0591] Table 5

[0592]

[0593] The presence of trehalose in the formulation promoted rapid dissolution. MoNi was found to assist in dissolution.

[0594] F5 has a shorter reconstitution time than F6. The cakes in F1-F4 show instantaneous dissolution.

[0595] The results of reconstitution study 2 (approximately 100 mg / mL IgG, with the addition of 2.5 mL WFI) are shown in Table 6 below. Figure 11 A set of photographs is shown, which demonstrate the appearance of formulations F1-F6 in reconstitution study 2.

[0596] Table 6

[0597]

[0598] In the reconstitution study 2, the solution with rich foam made it difficult to clearly determine the dissolution end point. In this experiment, there was no obvious difference between formulations F1 and F3, but compared with F1, the bubbles in the solution of F3 dissipated faster. The dissolution time of the cake of formulation F6 (without trehalose and MoNi) was the longest.

[0599] The results of reconstitution study 3 (about 200 mg / mL IgG, adding 1.2 mL WFI) are shown in Table 7 below. Figure 12 A set of photos are shown, which show the appearance of formulations F1 - F6 in reconstitution study 3.

[0600] Table 7

[0601]

[0602] Formulation F3 dissolved the fastest. The reconstitution time of formulation F1 was prolonged because a small amount of powder adhered to the bottom, which required a longer time to dissolve. The relative reconstitution times of the formulations F3 < F4 and F1 < F4 indicate that both MoNi and trehalose can drive the dissolution rate. The dissolution time of formulation F6 was longer than that of F5. Compared with formulation F6, formulation F5 formed a significantly clearer solution.

[0603] Table 8 summarizes the reconstitution times at each concentration.

[0604] Table 8

[0605]

[0606] The relationship between the properties of the surfactant and the reconstitution time of the 200 mg / mL IgG formulation is described in Figure 13 in. Figure 13This is a graph showing the relative reconstitution times of certain high-concentration aqueous IgG formulations with different surfactants, as shown in the last column of Tables 7 and 8 (F1: "surfactant-free"; F2: polysorbate 20 ("PS20"); F3: "MoNi"). Exemplary solid IgG formulations were prepared by lyophilization from a histidine buffer solution containing 200 mg / mL IgG (surfactant-free, containing 0.8 mg / mL polysorbate 20, or containing 20 mg / mL MoNi). Reconstitution time was measured as the time required for the resulting lyophilized cake to completely dissolve into an aqueous formulation of 200 mg / mL IgG. All formulations F1-F6 formed nearly clear solutions, characterized by a very slight tint under light and the absence of visible particles at all concentrations. Formulation F6 was slightly turbid at high concentrations.

[0607] The lyophilized cakes of IgG formulations were successfully reconstituted using WFI. Formulations F1-F6 all produced nearly clear solutions (note: F6 was slightly turbid at high concentrations), with foam but no visible particles. For formulations containing MoNi / PS20, the foam completely dissipated over time during standing.

[0608] For all formulations F1-F6, the reconstitution time increased with increasing concentration. Formulation F6 showed the longest reconstitution time and formed a slightly turbid solution (the turbidity appeared to decrease partially over storage time), indicating that MoNi has a significant effect on dissolution.

[0609] At lower concentrations (20 mg / mL IgG), the effect of MoNi on reconstitution time is difficult to determine because it dissolves very quickly (1-2 minutes) in formulations containing trehalose (with and without MoNi).

[0610] At 100 mg / mL and 200 mg / mL, the reconstitution time of formulation F3 was shorter than that of F4, and the reconstitution time of formulation F1 was shorter than that of F4.

[0611] At higher concentrations (200 mg / mL), formulation F3 showed the fastest dissolution. In the case of formulation F1, the prolonged time could be attributed to a small amount of powder adhering to the bottom of the vial, requiring a longer dissolution time. Furthermore, significant foaming made it difficult to accurately determine the dissolution endpoint. Repeated readings were necessary to determine whether the difference was within the expected range of variation between vials or a true difference in the nature of the cake. Compared to IgG alone (formulation F6), IgG + MoNi (formulation F5) showed significantly faster dissolution and formed a clearer solution at higher concentrations. Trehalose was also found to be a driving force for dissolution (compare F4 vs. F5), with the enhancement of dissolution depending on the amount of trehalose (compare F3 vs. F4). However, MoNi was found to be a dissolution enhancer when combined with trehalose, particularly when compared to another surfactant, PS20 (compare F3 vs. F2), or without a surfactant (compare F3 vs. F1).

[0612] In summary, MoNi reduced the reconstitution time of high-concentration IgG antibody formulations, while surfactants such as polysorbate 20 had almost no effect. The effect of MoNi on accelerating the reconstitution of other lyophilized mAb formulations was evaluated.

[0613] By incorporating references

[0614] All publications, patents, patent applications and other documents cited in this application, including published International Applications WO 2021 / 211976, PCT / US2023 / 023257, PCT / US2023 / 023212, PCT / US2023 / 027945 and U.S. Provisional Applications 63 / 590,460, 63 / 601,888 and 63 / 644,982, are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application or other document is individually indicated as incorporated by reference for all purposes.

Claims

1. A storage-stable solid composition comprising: Stabilizer; Polyacrylamide-based copolymers comprising: Selected from N -(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH) N , N -Dimethylacrylamide (DMA) N Hydroxyethylacrylamide (HEAM), acrylamide (AM), and water-soluble carrier monomers of combinations thereof; and Selected from N -[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA) N - Isopropylacrylamide (NIP), NN-Diethylacrylamide (DEA) N - tert-butylacrylamide (TBA) N 2-Phenylacetamide (PHE) and its combinations as functional dopant monomers; and Biological pharmaceutical reagents.

2. The solid composition according to claim 1, wherein the biopharmaceutical reagent is not an antibody or antibody fragment.

3. The solid composition according to claim 1 or 2, wherein the composition is soluble in water to form an aqueous composition, the aqueous composition comprising: 0.1 wt% to about 10 wt% of the copolymer; and The biopharmaceutical reagent is present in amounts ranging from 0.5 wt% to 25 wt%.

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

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 according to any one of claims 1 to 8, wherein the biopharmaceutical reagent is selected from vaccines, gene therapy, cell therapy, and lipid nanoparticles (LNPs) containing nucleic acids.

10. The solid composition according to claim 9, wherein the biopharmaceutical reagent is a vaccine.

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

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

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

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

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

16. The solid composition according to 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 antibodies or fragments thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic proteins.

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

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 according to any one of claims 14 to 23, wherein the solid composition exhibits storage stability compared to compositions lacking the polyacrylamide-based copolymer.

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

26. The solid composition according to any one of claims 1 to 14, wherein the water-soluble carrier monomer is selected from MORPH, MPAM, 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 MPAM.

29. The solid composition according to 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 according to 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 according to 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 according to any one of claims 1 to 25, wherein: The water-soluble carrier monomer is selected from MPAM, MORPH, and combinations thereof; and The functional dopant monomers are selected from NIP, PHE, and combinations thereof.

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

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

38. The solid composition according to 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 according to 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 according to any one of claims 1 to 39, 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.

41. The solid composition according to any one of claims 1 to 40, wherein the copolymer comprises: 80 wt% to 95 wt% of the water-soluble carrier monomer; and The functional dopant monomer is 5 wt% to 20 wt%.

42. The solid composition according to any one of claims 1 to 40, wherein the copolymer comprises: 83 wt% to 98 wt% of the water-soluble carrier monomer; and The functional dopant monomer is 2 wt% to 17 wt%.

43. The solid composition according to any one of claims 1 to 25, wherein the copolymer comprises: 70 wt% to 85 wt% MORPH; and 15 wt% to 30 wt% NIP.

44. The solid composition of claim 43, wherein the copolymer comprises: 74 wt% to 80 wt% MORPH; and 20 wt% to 26 wt% NIP.

45. The solid composition of claim 44, wherein the copolymer comprises: 77 wt% MORPH; and 23 wt% NIP.

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

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

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

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

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

51. The solid composition according to any one of claims 1 to 50, further comprising one or more additional components selected from preservatives and surfactants.

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

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

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

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

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

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

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

59. A method for reconstituted solid composition, the method comprising dissolving the 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 buffer.

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

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

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

65. The method according to any one of claims 59 to 64, further comprising evaluating the resolution time of particulate matter dissolution.

66. The method according to any one of claims 59 to 65, wherein the solid composition is a fixed dose of a biological pharmaceutical reagent contained in a vial.

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

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

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

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

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

74. The method according to any one of claims 70 to 73, further comprising removing particulate matter from the reconstituted lyophilized composition.

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

76. A method of administering a biological pharmaceutical agent to a subject in need, the method comprising administering to the subject a reconstituted lyophilized composition according to claim 75.

77. A method for preparing a solid composition according to any one of claims 1 to 56, the method comprising: The liquid biopharmaceutical composition is exposed to a drying cycle to produce the solid composition, the liquid biopharmaceutical composition comprising: Polyacrylamide-based copolymers comprising: Selected from N -(3-methoxypropyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH) N , N -Dimethylacrylamide (DMA) N Hydroxyethylacrylamide (HEAM), acrylamide (AM), and water-soluble carrier monomers of combinations thereof; and Selected from N -[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA) N - Isopropylacrylamide (NIP), NN-Diethylacrylamide (DEA) N - tert-butylacrylamide (TBA) N 2-Phenylacetamide (PHE) and its combinations as functional dopant monomers; and Biological pharmaceutical reagents.

78. The method of claim 77, wherein the drying is selected from freeze drying, spray drying, spray freeze drying, foam pad drying, vacuum furnace drying, and dehydration 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 freeze-drying cycle comprises: The liquid biopharmaceutical composition was subjected to a first pressure; At least a portion of the liquid components of the liquid biopharmaceutical composition are evaporated while heat is added; as well as The liquid biopharmaceutical composition is cooled to freeze any remaining liquid components into a solid.

81. The method according to claim 79 or 80, wherein the freeze-drying cycle comprises: The liquid biopharmaceutical composition is placed at a first reduced temperature; The liquid biopharmaceutical composition is subjected to a first reduced pressure; At least a portion of the liquid components of the liquid biopharmaceutical composition are evaporated while heat is added; The liquid biopharmaceutical composition is subjected to a second reduced pressure, lower than the first reduced pressure. as well as The remaining liquid is evaporated to produce the solid composition.

82. The method of claim 77, wherein the drying cycle is a spray drying cycle, comprising: Atomize the liquid biopharmaceutical composition; Evaporate at least a portion of the liquid component of the liquid biopharmaceutical composition; as well as Collect the solid composition.

83. The method of claim 77, wherein the drying cycle is a spray freeze-drying cycle, comprising: Atomize the liquid biopharmaceutical composition; Frozen, atomized liquid biopharmaceutical compositions; Evaporation of at least a portion of the liquid component of the atomized liquid biopharmaceutical composition; as well as Collect the solid composition.

84. The method of claim 77, wherein the drying cycle is a foam pad drying cycle, comprising: The liquid biopharmaceutical composition is foamed; Spread the foamed liquid biopharmaceutical composition onto a dry surface; Evaporation of at least a portion of the liquid component of the foamed liquid biopharmaceutical composition; and Collect the solid composition.

85. The method of claim 77, wherein the drying is a vacuum oven drying cycle, comprising: The liquid biopharmaceutical composition is subjected to reduced pressure; At least a portion of the liquid components of the liquid biopharmaceutical composition are evaporated while heat is added; as well as The pressure is restored to the initial pressure to obtain the solid composition.

86. The method of claim 77, wherein the drying is a dehydration drying cycle comprising: Increase the surface area of ​​the liquid biopharmaceutical composition, for example, by pouring the liquid biopharmaceutical composition into a tray; At least a portion of the liquid component of the liquid biopharmaceutical composition is evaporated by circulating ambient air above the surface, wherein the circulating air is optionally dried, heated, or both on a dehydrating material until a solid is obtained.

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