Containers and Methods for Reducing Cavitation of Protein Solutions

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/174,254, filed April 13, 2021, all of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under R21EB026006 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

The efficacy and safety of therapeutic proteins can be severely compromised by their instability upon storage, with potential protein misfolding and aggregation likely to elicit adverse immune responses upon administration to patients. Multiple factors have been suggested to cause these immune responses, but in several cases they have been correlated with the presence of subvisible particulates composed of aggregated protein molecules. Protein aggregation may occur as a result of environmental changes and/or destabilizing conditions such as but not limited to high temperature, high or low ionic strength, adsorption to container and delivery device surfaces, and mechanical agitation/shock. Despite well- controlled production and purification processes, protein particulates in therapeutic protein formulations may still arise from protein adsorption to the surfaces of containers such as vials, delivery pumps, and prefilled syringes, as well as the mechanical stresses resulting from these containers being agitated, dropped, or roughly handled. Even mild mechanical stresses applied to the protein containers may trigger the production of protein aggregates that eventually are injected into patients.

One cause of protein aggregation induced by mechanical shock is the incidence of cavitation (i.e., the formation of a gas bubble within a liquid) at the liquid-solid interface or in the bulk protein solution. Cavitation bubbles form, grow (often to millimeter size), and then collapse, releasing large amounts of energy, causing the formation of strong liquid jets, and developing high transient local temperatures. This energy release is potentially damaging to protein molecules, especially those adsorbed to container surfaces. Furthermore, upon bubble collapse, protein monomers absorbed to the bubble surface condense to form various types of aggregates. In addition to cavitation events, motion of the free liquid interface inside the primary container caused by agitating the protein container can significantly affect the protein aggregation. For a partially-filled vial with protein, liquid motion results in introduction of new air-water interfaces into the solution, causing further protein denaturation.

There is a need in the art for containers comprising a chemical modification that reduces and/or minimizes cavitation of a protein solution stored therein. There is also a need in the art for methods of reducing and/or minimizing cavitation of a protein solution. The present disclosure addresses these unmet needs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention is a method of reducing cavitation of a protein solution in a container, wherein the method comprises: chemically modifying one or more surfaces of the container with a hydrogel coating; and storing the protein solution in the container such that the protein solution is in contact with the hydrogel coating.

In certain embodiments, the container is a glass container.

In certain embodiments, the hydrogel coating is a polyacrylamide coating.

In certain embodiments, the step of chemically modifying one or more surfaces of the glass container with a hydrogel coating comprises at least one of the steps of: reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the surface of the glass container to attach the first polymerizable monomer to the container surface; contacting the attached first polymerizable monomer with a solution comprising a second polymerizable monomer, a third polymerizable monomer, and a photoinitiator; and irradiating the solution to form hydrogel coating on the surface of the glass container.

In certain embodiments, the step of reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the surface of the glass container to attach the first polymerizable monomer to the container surface is preceded by the step of treating the glass container with UV/ozone.

In certain embodiments, at least one applies:

(i) the first polymerizable monomer is selected from an acrylate or methacrylate monomer comprising a trialkoxysilyl functional group;

(ii) the second polymerizable monomer is selected from an acrylic or methacrylic amide; or

(iii) the third polymerizable monomer is selected from a polyethylene glycol diacrylate, a polyethylene glycol dimethacrylate, a polypropylene glycol diacrylate, and a polypropylene glycol dimethacrylate.

In certain embodiments, at least one applies:

(i) the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate;

(ii) the second polymerizable monomer is acrylamide; or

(iii) the third polymerizable monomer is polyethylene glycol diacrylate.

In certain embodiments, the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate, the second polymerizable monomer is acrylamide, the third polymerizable monomer is polyethylene glycol diacrylate, and the hydrogel coating formed on the surface of the glass container is polyacrylamide.

In certain embodiments, the method reduces cavitation induced by mechanical shock to the container.

In certain embodiments, the method reduces protein degradation, protein aggregation, or a combination thereof.

In another aspect, the invention provides a container comprising an interior surface that is at least partially chemically modified with a hydrogel coating, wherein the hydrogel coating reduces cavitation in a protein solution stored within the container.

In certain embodiments, the interior surface of the container is glass.

In certain embodiments, the hydrogel coating is formed by: reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the glass interior surface to attach the first polymerizable monomer to the interior surface; contacting the attached first polymerizable monomer with a solution comprising a second polymerizable monomer, a third polymerizable monomer, and a photoinitiator; and irradiating the solution to form hydrogel coating on the interior surface of the glass container.

In certain embodiments, the interior surface of the glass container is treated with UV/ozone before reacting the interior surface with the first polymerizable monomer.

In certain embodiments, at least one applies:

(i) the first polymerizable monomer is selected from an acrylate or methacrylate monomer comprising a trialkoxysilyl functional group;

(ii) the second polymerizable monomer is selected from an acrylic or methacrylic amide; or

(iii) the third polymerizable monomer is selected from a polyethylene glycol diacrylate, a polyethylene glycol dimethacrylate, a polypropylene glycol diacrylate, and a polypropylene glycol dimethacrylate.

In certain embodiments, at least one applies:

(i) the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate;

(ii) the second polymerizable monomer is acrylamide; or

(iii) the third polymerizable monomer is polyethylene glycol diacrylate.

In certain embodiments, the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate, the second polymerizable monomer is acrylamide, the third polymerizable monomer is polyethylene glycol diacrylate, and the hydrogel coating formed on the surface of the glass container is polyacrylamide.

In certain embodiments, the hydrogel coating reduces cavitation induced by mechanical shock to the container.

In certain embodiments, the hydrogel coating reduces protein degradation, protein aggregation, or a combination thereof.

In certain embodiments, the container is a vial or a syringe.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of exemplary embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, non-limiting embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 depicts hydrogel-coated vials. A Food color was added to the hydrogel solution to help in displaying the deposited hydrogel layer on the interior of the vial.

FIG. 2 is a schematic of the testing setup for analyzing the acoustic cavitation.

FIG. 3 is an oscilloscope recording of voltage in the time domain of an untreated vial tested by ultrasonic treatment with a 28 kHz transducer showing runs without cavitation (top) and with cavitation (bottom).

FIG. 4 is an ATR-FTIR spectra of polyacrylamide hydrogel layer deposited on a glass slide.

FIG. 5 depicts a series of snapshots taken at the time of impact showing the evolution of cavitation bubbles formed in vials containing 0.5 mg/mL intravenous immunoglobulin (IVIg) solution in histidine at pH 6 for an uncoated vial (top) and a hydrogel-coated vial (bottom). The red circles indicate the formed cavitation bubbles in the vial upon impact with the ground.

FIGs. 6A-6F depict snapshots showing the highest number of visible cavitation bubbles in the vials undergoing the mechanical shock within their first, second and third consecutive drops for a vial containing 0.5 mg/mL IVIg solution in histidine at pH 6. FIGs. 6A-6C: Snapshots of an uncoated vial. FIGs. 6D-6F: Snapshots of a hydrogel coated vial.

FIG. 7 A depicts the acoustic intensity as a function of input pressure. FIGs. 7B-7C depict Fourier transforms of data obtained from oscilloscope recordings showing increased frequencies present during cavitation for uncoated vials (FIG. 7B) and hydrogel-coated vials (FIG. 7C) under 28 kHz insonation at the listed input pressure.

FIGs. 8A-8B depict swelling ratio (FIG. 8A) and cavitation threshold energy input (FIG. 8B) variation as a function pH for polyacrylamide coated vials.

FIGs. 9A-9C depict protein aggregation and particulate formation of an IVIg solution undergoing mechanical stress via 10 min of tumbling or 1 h of plate shaking in uncoated and hydrogel-coated vials, as measured by fluorescence emission of bis-ANS (FIG. 9A), particle concentration (FIG. 9B), and the monomer percentage of remaining soluble IVIg (FIG. 9C) undergoing tumbling and shaking mechanical stresses. Error bars in FIGs. 9A-9C represent standard deviation (n = 3).

FIGs. 10A-10C depict protein aggregation and particulate formation of an IVIg solution undergoing mechanical stress via tumbler in uncoated and hydrogel-coated vials, as measured by fluorescence emission of bis-ANS (FIG. 10A), particle concentration (FIG.

10B), and the monomer percentage of remaining soluble IVIg (FIG. IOC) undergoing tumbling mechanical stress as a function of tumbling time. Error bars in FIGs. 10A-10C represent standard deviation (n = 3).

FIGs. 1 lA-1 IB depict contour plots of the ConvNet-derived FIM image embedding for samples exposed to different stress conditions in uncoated vials (FIG. 11 A) or hydrogel- coated vials (FIG. 1 IB). The contour plot indicates the fraction of flow imaging microscopy (FIM) images that embedded in that region of the embedding space as indicated by the associated bars. Also shown are sample FIM images that mapped to the circled regions of the embedding space.

FIGs. 12A-12B depict contour plots of the ConvNet-derived FIM image embedding and sample FIM images for samples tumbled for different lengths of time in uncoated vials (FIG. 12A) or hydrogel-coated vials (FIG. 12B). Contour plots are interpreted as described for FIGs. 11A-11B.

FIG. 13 depicts particle formation in tumbled buffer solutions for 5 min in uncoated and hydrogel-coated glass vials as compared to quiescent (No Tumb) control buffer solutions. Error bars represent standard deviation (n = 3).

FIG. 14 depicts contour plots of the ConvNet-derived FIM image embedding and sample FIM images that resulted from tumbling buffer solution in acrylamide-coated vials. Contour plots are interpreted as described for FIGs. 11 A-l IB.

DETAILED DESCRIPTION OF THE DISCLOSURE

In one aspect, one non-limiting strategy to suppress the cavitation at the solid-liquid interface is to employ hydrophilic surface chemistry to the interior of the vial. Lower contact angles of liquids on hydrophilic surfaces result in a higher energy barrier to bubble nucleation therefore a lower likelihood of cavitation inception. However, hydrophilicity alone is insufficient to prevent cavitation caused by prolonged and successive mechanical stresses. Instead, surface roughness inherent to glass vial surfaces allows heterogeneous nucleation of gas bubbles, which in turn facilitate cavitation and subsequently protein damage and aggregation. Such nuclei may persist within the micro/nanoscale structures found on glass surfaces due to contact line pinning and expand to form cavitation bubbles when mechanical shocks are imposed on the container. Based on these observations, a cavitation-resistant surface would have low energy at the solution interface, low roughness, and potentially a fluid-like structure to prevent stable contact line pinning.

The present disclosure provides, in one aspect, hydrogel coatings to protect the protein formulation against mechanical stresses responsible for subsequent protein aggregation and particle formation. Hydrogels, three-dimensional hydrophilic polymeric networks, are capable of absorbing large amounts of water or biological fluids. Their unique properties such as high-water content, flexibility, biocompatibility and resemblance to living tissue opens up many opportunities for applications in biomedical areas. In this disclosure, because of their high water content, the water-gel interface is a wide transition region rather than an abrupt interface, and above 30% water content the tension at the water-gel is near zero. In addition, swelling causes the gel to thrust out potential crevices, reducing the incidence of wrinkles and other surface inhomogeneities. Therefore, upon tuning the swelling ratio of the hydrogel coatings, one may possibly control the degree of surface crevices and inhomogeneities. The swelling ratio of polyacrylamide hydrogels can be tuned by varying the pH of solutions that come in contact with hydrogel.

In one aspect, the present disclosure provides polymerizable monomers which can be reacted under irradiation to form hydrophilic hydrogels. In other aspects, the present disclosure provides containers having one or more surfaces that are chemically modified with a hydrogel coating. In some embodiments, the container is a glass or plastic container. In some embodiments, the container is used to store and/or transfer a protein solution. In some embodiments, the container is a vial or syringe used to store and/or transfer a protein solution. In yet another aspect, the present disclosure relates to a method of preventing cavitation of a protein solution in a container. In some embodiments, the method comprises chemically modifying one or more surfaces of the container with a hydrogel coating; and storing the protein solution in the container such that the protein solution is in contact with the hydrogel coating.

Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

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

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

The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

The term "acyl" as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

The term "alkyl" as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH ₃ ) ₂ , -C(CH ₃ )=CH ₂ , -C(CH ₃ )=CH(CH ₃ ), -C(CH ₂ CH ₃ )=CH ₂ , cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

The term "alkoxy" as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

The term "alkynyl" as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - CºCH, -CºC(CH ₃ ), -CºC(CH ₂ CH ₃ ), -CH ₂ CºCH, -CH ₂ CºC(CH ₃ ), and -CH ₂ CºC(CH ₂ CH ₃ ) among others.

The term "amine" as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group) ₃ wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NEh, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

The term "amino group" as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR ₃ ⁺ , wherein each R is independently selected, and protonated forms of each, except for -NRf. which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

The term "aminoalkyl" as used herein refers to amine connected to an alkyl group, as defined herein. The amine group can appear at any suitable position in the alkyl chain, such as at the terminus of the alkyl chain or anywhere within the alkyl chain.

The term "aralkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

The term "aryl" as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

As used herein, the term "C6-10- Ce-io biaryl" means a Ce-io aryl moiety covalently bonded through a single bond to another Ce-io aryl moiety. The Ce-io aryl moiety can be any of the suitable aryl groups described herein. Non-limiting example of a C6-10- Ce-io biaryl include biphenyl and binaphthyl. The term "cycloalkyl" as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bomyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

The terms "halo," "halogen," or "halide" group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

The term "haloalkyl" group, as used herein, includes mono-halo alkyl groups, poly halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.

The term "heteroaryl" as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N,

O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1 -naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2 -thienyl, 3 -thienyl), furyl (2 -fury 1, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2 -thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),

6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl,

3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl,

4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5 -benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5 -benzothiazolyl, 6-benzothiazolyl,

7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f|azepin-l-yl, 5H-dibenz[b,f|azepine-2-yl, 5H-dibenz[b,f|azepine-3-yl, 5H-dibenz[b,f|azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11 -dihy dro-5H-dibenz[b,f| azepine (10,11 -dihy dro-5H-dibenz[b,f| azepine- 1 -y 1,

10,1 l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-3-yl,

10,1 l-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

The term "heteroarylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

As used herein, the term "Ce-io-5-6 membered heterobiaryl" means a Ce-io aryl moiety covalently bonded through a single bond to a 5- or 6-membered heteroaryl moiety. The Ce-io aryl moiety and the 5-6-membered heteroaryl moiety can be any of the suitable aryl and heteroaryl groups described herein. Non-limiting examples of a C6-IO-5-6 membered heterobiaryl include: When the Ce-io-5-6 membered heterobiaryl is listed as a substituent ( e.g ., as an "R" group), the Ce-io-5-6 membered heterobiaryl is bonded to the rest of the molecule through the Ce-io moiety.

As used herein, the term "5-6 membered- Ce-io heterobiaryl " is the same as a C6-IO-5- 6 membered heterobiaryl, except that when the 5-6 membered- Ce-io heterobiaryl is listed as a substituent (e.g., as an "R" group), the 5-6 membered- Ce-io heterobiaryl is bonded to the rest of the molecule through the 5-6-membered heteroaryl moiety.

The term "heterocyclyl" as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise, a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.

The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X ¹ , X ² , and X ³ are independently selected from noble gases" would include the scenario where, for example, X ¹ , X ² , and X ³ are all the same, wherein X ¹ , X ² , and X ³ are all different, wherein X ¹ and X ² are the same but X ³ is different, and other analogous permutations.

The term "monovalent" as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

The term "organic group" as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, 0C(0)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R) ₂ , SR, SOR, SO2R, S0 ₂ N(R) ₂ , SO3R, C(0)R, C(0)C(0)R, C(0)CH ₂ C(0)R, C(S)R, C(0)0R, 0C(0)R, C(0)N(R) ₂ , 0C(0)N(R) ₂ , C(S)N(R) ₂ , (CH ₂ )O- ₂ N(R)C(0)R, (CH ₂ )O- ₂ N(R)N(R) ₂ , N(R)N(R)C(0)R, N(R)N(R)C(0)0R, N(R)N(R)CON(R) ₂ , N(R)S0 ₂ R, N(R)S0 ₂ N(R) ₂ , N(R)C(0)0R, N(R)C(0)R, N(R)C(S)R, N(R)C(0)N(R) ₂ , N(R)C(S)N(R) ₂ , N(COR)COR, N(OR)R, C(=NH)N(R) ₂ , C(0)N(0R)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

The terms "patient," "subject," or "individual" are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the

The term "solvent" as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

The term "substituted" as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, 0C(0)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R) ₂ , SR, SOR, SO2R, S0 ₂ N(R) ₂ , SO3R, C(0)R, C(0)C(0)R, C(0)CH ₂ C(0)R, C(S)R, C(0)0R, 0C(0)R, C(0)N(R) ₂ , 0C(0)N(R) ₂ , C(S)N(R) ₂ , (CH ₂ )O- ₂ N(R)C(0)R, (CH ₂ )O-2N(R)N(R) ₂ , N(R)N(R)C(0)R, N(R)N(R)C(0)0R, N(R)N(R)C0N(R) ₂ , N(R)S0 ₂ R, N(R)S0 ₂ N(R) ₂ , N(R)C(0)0R, N(R)C(0)R, N(R)C(S)R, N(R)C(0)N(R) ₂ , N(R)C(S)N(R) ₂ , N(COR)COR, N(OR)R, C(=NH)N(R) ₂ , C(0)N(0R)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (Ci- Cioo)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

The term "thioalkyl" as used herein refers to a sulfur atom connected to an alkyl group, as defined herein. The alkyl group in the thioalkyl can be straight chained or branched. Examples of linear thioalkyl groups include but are not limited to thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiohexyl, and the like. Examples of branched alkoxy include but are not limited to iso-thiopropyl, sec-thiobutyl, tert-thiobutyl, iso- thiopentyl, iso-thiohexyl, and the like. The sulfur atom can appear at any suitable position in the alkyl chain, such as at the terminus of the alkyl chain or anywhere within the alkyl chain.

Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Compounds and Compositions

In one aspect, the present disclosure relates to polymerizable monomers and compositions comprising the polymerizable monomers. The polymerizable monomers can be any polymerizable monomers known to a person of skill in the art. In certain embodiments, the polymerizable monomers comprise a first, second, and third polymerizable monomer.

In certain embodiments, the first polymerizable monomer is biomacromolecule or a derivative thereof. Exemplary biomacromolecules include, but are not limited to, alginate, chitosan, gelatin, and derivatives thereof. In some embodiments, the biomacromolecule is substituted with one or more substituents. In certain embodiments, the biomacromolecule is substituted with one or more functional groups selected from an amine, a thiol, an alkyne, an acrylate, a methacrylate, a trialkoxysilane, a silane, and combinations thereof. In certain embodiments, the biomacromolecule is substituted with a halogen containing silane functional group. Exemplary halogen containing silanes include, but are not limited to, trichlorosilyl, trifluorosilyl, and tribromosilyl. In yet other embodiments, the biomacromolecule is substituted with a trialkoxysilane functional group. In certain embodiments, the trialkoxysilane is trimethoxysilyl. In other embodiments, the first polymerizable monomer is an acrylate or methacrylate. Exemplary acrylates or methacrylates include but are not limited to, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, propyl methacrylate, ethylene glycol acrylate, ethylene glycol methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, sulfobetaine acrylate, and sulfobetaine methacrylate, wherein the acrylate or methacrylate may be optionally substituted with one or more substituents. In certain embodiments, the acrylate or methacrylate is substituted with one or more functional groups selected from an amine, a thiol, an alkyne, a trialkoxysilane, a silane, and combinations thereof. In some embodiments, the amine or thiol functional group reacts with the carboxylic acid group of the acrylate or methacrylate monomer. In one embodiment, the acrylate or methacrylate is substituted with a halogen containing silane functional group. Exemplary halogen containing silanes include, but are not limited to, trichlorosilyl, trifluorosilyl, and tribromosilyl. In another embodiment, the acrylate or methacrylate is substituted with a trialkoxysilane functional group. In one embodiment, the trialkoxysilane is trimethoxysilyl. In certain embodiments, the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate.

In certain embodiments, the second polymerizable monomer is an acrylic or methacrylic amide. In some embodiments, the second polymerizable monomer is acrylamide.

In certain embodiments, the third polymerizable monomer is selected from a polyethylene glycol diacrylate, a polyethylene glycol dimethacrylate, a polypropylene glycol diacrylate, and a polypropylene glycol dimethacrylate. In some embodiments, the third polymerizable monomer comprises polyethylene glycol diacrylate (PEGDA).

In some embodiments, the first, second, or third monomer can be substituted with any substituent known to a person of skill in the art. Exemplary substituents are described elsewhere herein.

In another aspect, the present disclosure relates to a composition comprising one or more of the polymerizable monomers. In certain embodiments, the composition comprises a photoinitiator. The photoinitiator can be any photoinitiator known to a person of skill in the art. In certain embodiments, the photoinitiator is a UV active photoinitiator. In certain embodiments, UV active photoinitiator is Irgacure 2959. In some embodiments, the composition comprises a solvent. In certain embodiments, the composition comprises an organic solvent. In certain embodiments, the composition comprises an aqueous solvent. Exemplary aqueous solvents include, but are not limited to, distilled water, deionized water, tap water, and saline. In certain embodiments, the solvent is deionized water.

In certain embodiments, the composition comprises the first, second, and third polymerizable monomers. In other embodiments, the composition comprises the second and third polymerizable monomers. In certain embodiments, the composition comprises between about 5 wt% - 60 wt%, about 5 wt% - 50 wt%, about 5 wt% - 40 wt%, about 10 wt% - 35 wt%, about 15 wt% - 30 wt%, or about 15 wt% - 25 wt% of the second polymerizable monomer relative to a solvent. In some embodiments, the composition comprises about 20 wt% of the second polymerizable monomer relative to a solvent. In some embodiments, the composition comprises about 20 wt% of acrylamide relative to a deionized water solvent. In certain embodiments, the composition comprises between about 0.1 wt% - 30 wt%, about 0.1 wt% - 25 wt%, about 0.1 wt% - 20 wt%, about 0.1 wt% - 15 wt%, about 0.1 wt% - 10 wt%, about 0.1 wt% - 5 wt%, about 1 wt% - 5 wt%, or about 1 wt% - 3 wt% of the third polymerizable monomer relative to the second polymerizable monomer. In certain embodiments, the composition comprises about 2 wt% of the third polymerizable monomer relative to the second polymerizable monomer. In certain embodiments, the composition comprises about 2 wt% PEGDA relative to acrylamide. In certain embodiments, the composition comprises between about 0.01 wt% - 20 wt%, about 0.01 wt% - 15 wt%, about 0.01 wt% - 10 wt%, about 0.01 wt% - 5 wt%, about 0.1 wt% - 5 wt%, about 0.1 wt% - 2 wt% of photoinitiator relative to the second polymerizable monomer. In some embodiments, the composition comprises about 1 wt% of photoinitiator relative to the second polymerizable monomer. In some embodiments, the composition comprises about 1 wt% of Irgacure 2959 relative to acrylamide.

In yet another aspect, the present disclosure relates to a hydrogel formed via irradiation of a composition comprising the first, second, and third polymerizable monomers and further comprising a photoinitiator. In some embodiments, the first polymerizable monomer is attached to a glass or plastic surface via the reaction of a functional group on the first polymerizable monomer with the glass or plastic surface. In certain embodiments, the first polymerizable monomer comprises a trialkoxysilyl functional group which reacts with the glass surface. In certain embodiments, the first polymerizable monomer is 3- (trimethoxysilyl)propyl methacrylate which reacts with the glass surface. In certain embodiments, the hydrogel is formed from a composition which further comprises a solvent. In some embodiments wherein the first polymerizable monomer is attached to a glass or plastic surface, the hydrogel is formed via UV irradiation. In some embodiments, the hydrogel is formed via UV irradiation of a composition comprising 3-(trimethoxysilyl)propyl methacrylate, acrylamide, PEGDA, and Irgacure 2959. In some embodiments wherein the first polymerizable monomer is attached to a glass or plastic surface, the composition comprises a solvent, the second polymerizable monomer, and the third polymerizable monomer. In some embodiments, the first polymerizable monomer is attached to a glass or plastic surface and the hydrogel is formed on the glass or plastic surface by contacting the surface with a solution comprising the second and third monomers as well as the photoinitiator while exposing the solution to UV irradiation. Therefore, in some embodiments, the hydrogel forms via a reaction between the second and third polymerizable monomers while the first polymerizable monomer acts to attach the hydrogel to the glass or plastic surface. In certain embodiments, the hydrogel forms between acrylamide and PEGDA while 3-(trimethoxysilyl)propyl methacrylate acts to attach the hydrogel to the glass or plastic surface.

In certain embodiments, the hydrogel formed from the irradiation of the composition described elsewhere is not a hydrogel which has high adhesion. In certain embodiments, the hydrogel is not poly(dimethylaminoethyl methacrylate).

Articles In another aspect, the present disclosure relates to an article wherein one or more surfaces of the article is coated with a hydrogel of the instant disclosure. In certain embodiments, the article is an article which is used for the transfer and/or storage of a protein solution. In certain embodiments, the one or more surfaces of the article that are coated with a hydrogel are the surfaces that are in contact or are expected to come into contact with the protein solution. In some embodiments, the article for storage of a protein solution is a container such as a vial. In some embodiments, the article for transfer of a protein solution is a syringe. In some embodiments, the container and/or syringe are made of glass or plastic.

In some embodiments, the article is treated before coating with the hydrogel in order to improve the adherence of the hydrogel to the article and/or to improve the quality of the hydrogel coating. In certain embodiments, the article is a glass article. In certain embodiments, the glass article is sonicated in a mixture of alcohol and water before coating with the hydrogel. In some embodiments, the glass article is sonicated in a mixture of ethanol and water. In certain embodiments, the sonicated glass article is dried and then exposed to UV/ozone. In some embodiments, the article is a container comprising an interior surface which is coated with the hydrogel. In certain embodiments, the container comprises a glass interior surface which is coated with the hydrogel using a method disclosed elsewhere herein.

The one or more surfaces of the article can be coated with any hydrophilic hydrogel known to a person of skill in the art. In certain embodiments, the hydrogel coating reduces or prevents aggregation of the protein solution. In certain embodiments, the hydrogel coating reduces or prevents mechanical shock induced aggregation of the protein solution. In some embodiments, the mechanical shock is tumbling and/or shaking of the protein solution during storage or transfer. In some embodiments, the hydrogel coating reduces or prevents cavitation of the protein solution. Although not wishing to be limited by theory, it is believed that cavitation damages protein molecules and/or promotes protein aggregation as cavitation bubbles form, grow (often to millimeter size), and then collapse, releasing large amounts of energy, causing the formation of strong liquid jets, and developing high transient local temperatures. In some embodiments, cavitation events occur following mechanical shock to an article which contains a protein solution, such as a vial or syringe. Although not wishing to be limited by theory, it is believed that the hydrophilic hydrogel coating on the article results in a higher energy barrier to bubble nucleation and therefore a lower likelihood of cavitation inception due to lower contact angles of liquids on hydrophilic surfaces. However, it is further believed that hydrophilicity alone is insufficient to prevent cavitation and that the roughness of the article surface which contacts the protein solution is also important in reducing or preventing cavitation and/or protein aggregation. While not wishing to be limited by theory, it is believed that surface roughness allows heterogeneous nucleation of gas bubbles, which in turn facilitate cavitation and subsequently protein damage and aggregation. Such nuclei may persist within the micro/nanoscale structures found on surfaces due to contact line pinning and expand to form cavitation bubbles when mechanical shocks are imposed on the container. Therefore, in some embodiments, the hydrogel coating should would have a low energy at the solution interface, low roughness, and potentially a fluid-like structure to prevent stable contact line pinning.

In some embodiments, the thickness of the hydrogel coating is between about 0.1 mm to 10 mm, about 0.1 mm to 8 mm, about 0.1 mm to 6 mm, about 2 mm to 6 mm, or about 2 mm to 4 mm. In certain embodiments, the thickness of the hydrogel coating is about 3 ± 1 mm. In some embodiments, the water contact angle on the hydrogel coating is between about 5° to 60°, about 10° to 55°, about 15° to 50°, about 20° to 45°, about 25° to 40°, or about 27° to 35°. In certain embodiments, the water contact angle on the hydrogel coating is about 32 ± 1°.

Methods

In another aspect, the present disclosure relates to a method of reducing cavitation of a protein solution in a container, the method comprising: chemically modifying one or more surfaces of the container with a hydrogel coating; and storing the protein solution in the container such that the protein solution is in contact with the hydrogel coating.

In some embodiments, the disclosed method reduces cavitation of a protein solution to the extent that the disclosed method prevents cavitation of the protein solution. The protein can be any protein known to a person of skill in the art. In certain embodiments, the protein is an antibody. In some embodiments, the antibody is immunoglobulin. The solution can have any concentration of protein known to a person of skill in the art. The protein solution can comprise any solvent known to a person of skill in the art to be suitable for protein storage and/or transfer and the solution can have any pH known to a person of skill in the art to be suitable for protein storage and/or transfer. The container can be any container used to store a protein solution. In some embodiments, the container is a vial or syringe. In some embodiments, the container is a glass or plastic vial or syringe. In certain embodiments, the protein solution is stored in the container for an extended period of time before use. In other embodiments, the protein solution is stored in the container for a short period of time, such as when the protein solution is transferred to (i.e. stored in) a syringe.

In some embodiments, the method reduces degradation of the protein in the protein solution by reducing cavitation of the protein solution. In some embodiments, the method reduces aggregation of the protein in the protein solution by reducing cavitation of the protein solution. In some embodiments, the method reduces both degradation and aggregation of the protein in the protein solution.

In certain embodiments, the container is a glass container. In some embodiments, the step of chemically modifying one or more surfaces of the glass container with a hydrogel coating comprises the steps of: reacting a first polymerizable monomer that comprises an amine, a thiol, a silane, or a trialkoxysilyl functional group with the surface of the container to attach the first polymerizable monomer to the container surface; contacting the attached first polymerizable monomer with a solution comprising a second polymerizable monomer, a third polymerizable monomer, and a photoinitiator; and irradiating the solution. In one embodiment, the silane functional group is a halogen containing silane functional group. Exemplary halogen containing functional groups are described elsewhere herein. In one embodiment, the functional group that reacts with the surface of the container is a trialkoxysilyl functional group. In one embodiment, the functional group that reacts with the surface of the container is trimethoxysilyl.

In some embodiments, the glass container is treated with UV/ozone before reaction with the first polymerizable monomer. Exemplary first polymerizable monomers are described elsewhere herein. In certain embodiments, the one or more surfaces of the glass container are reacted with the first polymerizable monomer by contacting the UV/ozone exposed surface of the glass container with a solution of the first polymerizable monomer. In some embodiments, the UV/ozone exposed surface of the glass container is contacted with the solution of the first polymerizable monomer at room temperature for about 24 hours. In some embodiments, the reaction of the first polymerizable monomer that comprises a trialkoxysilyl functional group with the surface of the container attaches the first polymerizable monomer to the container surface via a silane bridge. In certain embodiments, one or more surfaces of the glass container are reacted with an acrylate or methacrylate polymerizable monomer that comprises a trialkoxysilyl functional group. In some embodiments, the acrylate or methacrylate polymerizable monomer is 3- (trimethoxysilyl)propyl methacrylate.

The attached first polymerizable monomer is then contacted with a solution comprising any second polymerizable monomer and any third polymerizable monomer described elsewhere herein. The solution further comprises any UV or visible light active photoinitiator known to a person of skill in the art. In some embodiments, the second polymerizable monomer is acrylamide and the third polymerizable monomer is PEGDA. In some embodiments, the photoinitiator is Irgacure 2959.

In some embodiments, the solution comprising the second polymerizable monomer, the third polymerizable monomer, and the photoinitiator is irradiated with UV or visible light while it is in contact with the surface of the glass container. In certain embodiments, the UV or visible light irradiation promotes a reaction between the first polymerizable monomer attached to the surface of the glass container and the second and third polymerizable monomers to form a cured hydrogel on the glass surface. In certain embodiments, when a cylindrical surface such as the interior of a vial or syringe is contacted with the solution, the vial or syringe is filled with the solution and the vial or syringe is turned as the solution is irradiated. In some embodiments, the second polymerizable monomer is acrylamide and the third polymerizable monomer is PEGDA. In some embodiments wherein the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate, the second polymerizable monomer is acrylamide, and the third polymerizable monomer is PEGDA, the cured hydrogel is polyacrylamide.

In some embodiments, the hydrogel coating is a hydrophilic coating. In some embodiments, the hydrogel coating reduces the roughness of the container surface. In some embodiments, the swelling ratio of the hydrogel coating can be changed by changing the pH of the protein solution stored in contact with the hydrogel coating. In certain embodiments, the pH of the protein solution stored in contact with the hydrogel coating is selected such that the hydrogel coating has a low swelling ratio. In certain embodiments the pH of the protein solution stored in contact with the hydrogel coating is about 6. In certain embodiments, storing a protein solution with a pH of about 6 in contact with the hydrogel coating results in a low swelling ratio for the coating.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.

EXPERIMENTAL EXAMPLES

The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1: Hydrogel Coatings Reduce Protein Aggregation Caused by Mechanical

Stress due to Suppression of Cavitation on Glass Surfaces

The present disclosure demonstrates that the surface structure of containers such as glass vials can be engineered to control and mitigate the effects of mechanically-induced cavitation on protein aggregation and particle formation. Here, the stability of a therapeutic protein, intravenous immunoglobulin (IVIg), was analyzed as a model for other therapeutic antibodies. Protein aggregation, particle formation, and protein loss were assessed using fluorescence-based assays, flow imaging microscopy (FIM), and size exclusion high performance liquid chromatography (SEC-HPLC). Furthermore, the resistance of synthetic hydrogel coatings to cavitation was tested by ultrasonic treatment with a 28 kHz transducer, where the acoustic response of cavitation was monitored using a hydrophone. Convolutional neural network (ConvNet)-based machine learning algorithms were employed to compare the morphology of IVIg aggregates formed under different mechanical stresses in coated and uncoated vials. Finally, the effect of solution pH on cavitation thresholds was studied in hydrogel-coated vials. The knowledge gained from this work will lead to the development of novel polymer gel coatings that reduce cavitation-induced protein aggregation, particularly under prolonged and successive mechanical stresses.

Materials and Methods Materials

IVIg (GammaGard, Illinois) solutions were formulated at 0.5 mg/mL concentration by diluting 100 mg/mL into 20 mM histidine (Sigma- Aldrich, Missouri) at pH 6. Nominal 5 mL borosilicate glass vials (DWK Life Sciences, Milville, NJ) and butyl rubber stoppers (Fisher, Waltham, MA) were tripled-washed using ethanol and deionized (DI) water and air-dried prior to use. Metal caps (Wheaton, Millville, NJ) were used to seal the vials. DI water, ethanol, and histidine buffer were filtered through a 0.2 pm PVDF syringe filter (Millipore, Burlington, MA) prior to use. 3-(Trimethoxysilyl) propyl methacrylate, 4,4'-dianilino-l,l'- binaphthyl-5,5'-disulfonic acid (bis-ANS), sodium sulfate, sodium phosphate monobasic, and sodium azide were purchased from Sigma Aldrich (St. Louis, MO). Sodium phosphate dibasic was purchased from (Fisher Scientific, Waltham, MA). Acrylamide and Irgacure 2959 were purchased from TCI Chemicals America. Polyethylene(glycol) diacrylate (PEGDA; Mw = 400) was purchased from PolySciences, Inc (Warrington, PA).

Hydrogel synthesis and vial coating

Prior to hydrogel synthesis, the vials were bath sonicated in ethanol/DI water (1:1, v/v) for 15 min and air-dried at room temperature overnight. To functionalize with acrylate groups, the vials were treated with UV/ozone for 1 h and then filled completely with a solution of 95% ethanol (pH was adjusted to 5 using acetic acid) with 0.5 % v/v of 3- (trimethoxysilyl)propyl methacrylate and incubated quiescently at room temperature for 24 h. The vials were then triple-washed with filtered ethanol, DI water, and isopropyl alcohol, then the vials were kept on the bench and air-dried at room temperature.

To synthesize hydrogels, the acrylated-functionalized vials were filled completely with monomer solutions containing 20 wt% acrylamide relative to DI water, 2 wt% PEGDA relative to acrylamide, and 1 wt% of Irgacure 2959 relative to acrylamide. The glass vials containing the hydrogel solution were exposed to 365 nm UV light while rotating using a benchmark laboratory mixer for 7 min to ahain a thin and uniform layer of hydrogel coating (FIG. 1). The hydrogel-coated vials were then rinsed with a 70% filtered ethanol solution to remove any uncured hydrogel solution from the vials.

Characterization of chemical bonding using Fourier Transform Infrared (FTIR)

Spectroscopy FTIR was used to identify chemical bonding on hydrogel-coated glass slides using a Thermo Fisher Scientific Nicolet 6700 FTIR with an attenuated total reflection (ATR) accessory, acquiring 400 scans at a resolution of 4 cm ¹ . A plain glass slide was used to capture the background.

Acoustic cavitation experiments

Acoustic cavitation studies in uncoated and coated vials were performed using a home-built ultrasonic analysis setup (FIG. 2). Coated and hydrogel-coated vials were filled with filtered water and placed on a 28 kHz transducer (NBL45282H-A3, KEMET) which was connected to a 30 MHz Function/ Arbitrary Waveform Generator (Agilent Technologies) via an AG Series Amplifier (T&C Power Conversion, Inc.) set at 100% output. A thin layer of ultrasound gel was applied between the vial bottom and the transducer. Input values ranging from 100 mV to 250 mV in increments of 10 mV were sent to the amplifier to provide between 3.8 and 9 V to the transducer. The input pressure conversion was performed offline using a pre-recorded calibration curve. A capsule hydrophone (HGL-0400, Onda Corp.) was placed in the vial. The vial and the hydrophone were centered over the transducer. The hydrophone was connected to an oscilloscope by BNC cables. The hydrophone measured voltage readings over time that were saved on the Tektronix TDS2012C oscilloscope. Voltages were converted to pressure using hydrophone intensity. Peak pressures from each experiment were obtained for further analysis. The captured pressure was then converted to acoustic intensity by considering the density of the propagating medium (water) and the velocity of sound in water. The cavitation threshold is defined as the minimum energy input pressure above which fluctuation in hydrophone signal voltage was observed (FIG. 3).

Swelling ratio

Hydrogel films were synthesized in plastic petri dishes and peeled off once the hydrogel was completely cured. The hydrogel films were cut into small squares (with an edge of ~ 1 cm). The hydrogel squares were immersed in solutions with different pH values, ranging from 2 to 10, for 48 h. The experiments were performed for three squares per pH value. Each hydrogel square was weighted to obtain the "Wet" weight. After that, the swollen hydrogels were lyophilized for 72 h to let the hydrogels become completely dried and the "Dry" weight of hydrogel squares were then obtained. The mass swelling ratio was calculated as the ratio of "Dry" weight to "Wet" weight for each hydrogel squares. Application of different types of mechanical stresses

Different types of mechanical stresses were applied to IVIg samples in uncoated and hydrogel-coated vials using:

(a) An electric tumbler: The vials were enclosed in a small plastic box and placed in an electric tumbler (runs at 60 rpm; dropping the samples from a height of approximately 0.4 to 0.6 m each time). The samples were dropped continuously for different times at room temperature.

(b) A plate shaker: The vials were taped together and placed in a horizontal orientation on a plate shaker agitating at speed 600 rpm for 1 h at room temperature.

(c) Drop test tower: In order to visualize the cavitation occurrence and bubble formation, uncoated and hydrogel-coated glass vials containing IVIg solutions were dropped from 0.5 m above the ground using a Lansmont Model 15D drop tower system. The videos of the impact were captured using a high-speed camera (iX Cameras, Essex, UK) at a rate of 15,000 frames per second.

Characterization of protein aggregation using an extrinsic fluorescence assay

4,4'-dianilino-l,r-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence dye was used for detecting IVIg aggregation. Fluorescence intensity measurements were made by adding 20 pL of 25 mM bis-ANS to 180 pL of the studied solutions in black 96-well plates (Greiner Bio-One, Kremsmunster, Upper Austria) immediately before measuring fluorescence using a plate reader (Tecan, Mannedorf, Switzerland) with the excitation at 390 nm and emission at 490 nm.

Characterization of particle formation and morphology using flow imaging microscopy analysis

Flow imaging microscopy (FIM) (FlowCAM®, Scarborough, ME) was used to measure the concentration of formed particles in protein solution. 150 pL of each sample was injected into a FC80 flow cell at a flow rate of 0.05 mL/min, and the lOx objective was used to observe and count sub-visible particles of size > 2 pm. RGB color images of particles larger than 2 pm in diameter were recorded and analyzed.

Size exclusion high performance liquid chromatography (SEC-HPLC) An SE-HPLC system (Agilent, Santa Clara, CA) was used to determine the concentration of soluble IVIg and protein aggregate levels within the IVIg solutions before and after applying the mechanical stress. IVIg solutions were first centrifuged for 20 min at 20,000g and 20 °C to remove insoluble particles prior to analysis. The mobile phase contained 100 mM sodium sulfate, 50 mM sodium phosphate dibasic, 50 mM sodium phosphate monobasic, and 0.05% sodium azide. For each sample, 100 pL of the supernatant was injected into G3000SWXL column (Tosoh Bioscience, King of Prussia, PA) at a rate of 0.6 mL/min, and protein was detected by absorbance at 280 nm. To quantify the soluble protein aggregate levels, the total area under monomer peak of the UV absorbance at 280 nm for stressed samples was compared with that for unstressed protein sample.

Particle comparison algorithm

A convolutional neural network (ConvNet) algorithm described in more detail elsewhere (Daniels, A. L. et ak, "Machine Learning & Statistical Analyses for Extracting and Characterizing "Fingerprints" of Antibody Aggregation at Container Interfaces from Flow Microscopy Images," Biotechnol. Bioeng., 2020, 117:3322-3335) was used to compare protein aggregates formed in uncoated and hydrogel-coated vials during tumbling stress. Briefly, a ConvNet was trained using a triplet loss approach to map raw FIM images onto two-dimensional representations ("embeddings") that efficiently capture information in the raw FIM image and group images formed by the same stress and coating together. This ConvNet was trained using 16,000 FIM images from uncoated and coated vials filled with IVIg after 10 minutes of tumbling, 4,500 FIM images formed in uncoated vials after 60 minutes of shaking, and 2,500 FIM images from coated vials after 60 minutes of shaking.

The trained ConvNet was then used to analyze the remaining images from these samples as well as those generated after tumbling uncoated and coated vials filled with IVIg solution after 0, 1, 4, and 7 minutes of tumbling from a separate experiment. FIM images obtained after tumbling buffer solution without protein in both vial types were also analyzed. The resulting 2D embeddings were then compared against each other to determine the impact of the hydrogel coating on particles formed after shaking or tumbling stress was applied. These comparisons were also performed using the previously-described goodness-of-fit hypothesis testing strategy in which 10,000 sets of 20 FIM images of particles generated under one "test" condition were compared against those generated under a second "baseline" condition. This hypothesis test checks if the embeddings from the test condition were consistent with an estimated probability density function (PDF) of embeddings from the baseline condition. Each test was performed at a 5% Type I (false positive) error rate. The rejection rate or the fraction of these 10,000 image sets that were not consistent with those from the uncoated vial was used as a measure of similarity between the particles produced under the two conditions with higher rejection rates indicating more dissimilar particle populations.

Results

Fabrication of hydrogel-coated vials

As an initial step, the hydrogel coating was applied to the interior of 5 mL borosilicate glass vials. Polyacrylamide was chosen as the test hydrogel due to its nontoxicity, stability, ubiquity in biotechnological applications, simple synthesis, and being a non-resorbable sterile watery gel. In order to ensure an effective hydrogel coating, the hydrogel layer was anchored to the glass surface by a silane bridge, which ensures that the hydrogel does not delaminate from the surface during post-synthesis swelling (Velankar, S. S. et al., "Swelling-Induced Delamination Causes Folding of Surface-Tethered Polymer Gels," ACS Appl. Mater. Interfaces, 2012, 4:24-29). The polyacrylamide coatings were then fabricated onto the pre functionalized glass vials through photopolymerization (see Methods). Once synthesized, a thin layer (~3±1 mm) of polyacrylamide hydrogel was deposited on the interior walls of glass vials, which was observed and measured through the optical microscope. The water contact angle on dry polyacrylamide hydrogels was 32±1°. The gel structure was characterized using FTIR-ATR to confirm the incorporation of expected functional groups, including with bands corresponding to VN-H (asym, sym), vc=o, 5N-H and VC-N (FIG. 4).

Visualizing cavitation bubble formation in dropped vials

The cavitation-suppressing effects of hydrogel coatings on glass vials was immediately evident by high-speed video analysis of dropped vials (FIG. 5). The vials with and without polyacrylamide coatings were dropped using a drop test tower from a height of 0.5 m relative to ground, and the movies of the vials at impact were recorded and analyzed. This study was performed for up to three drops, as the probability of cavitation on dropping increases with successive drops. The violent fluid motion caused by the ground impacting the vial and introducing air bubbles into the solution. These bubbles can be trapped on the surface and create new cavitation nuclei cites for the subsequent drops. FIGs. 1A-1F show the still images from movies recorded at impact for three consecutive drops of uncoated and hydrogel-coated vials. For uncoated vials, the number of bubbles were increased for the subsequent drops (FIGs. 6A-6C). Interestingly, for hydrogel-coated vials not only was there was no cavitation on the first drop (FIG. ID), very few bubbles were formed upon repeated drops compared to the bubble formation in the uncoated vials (FIG. IE, FIG. IF). Although not wishing to be limited by theory, it is believed that, due to the high hydration of hydrogel layer, confinement of bubbles on the surface has significantly reduced, which results in prolonged protection against mechanical shocks applied in rapid succession.

Analysis of cavitation on hydrogel-coated glass surfaces using ultrasonic treatment

Next, the effect of container surface structure on cavitation threshold, or the energy required to induce cavitation, was determined. For this aim, uncoated and hydrogel-coated vials were filled with filtered water, and a 28 kHz transducer coupled with ultrasound gel was placed below the vial with a capsule hydrophone inserted inside the vial. The pressure of the acoustic wave was gradually increased until the signal recorded on the oscilloscope showed evidence of cavitation (FIG. 3). Uncoated vials possessed significantly lower cavitation threshold pressures as compared to hydrogel-coated vials (FIG. 7A). For uncoated vials, cavitation events observed at input pressure of ~21 MPa, while the cavitation was not incepted up to input pressure of 31 MPa meniscus with air. FIGs. 7B and 7C show that greater acoustic energy was required to initiate cavitation when the vial was coated with a polyacrylamide hydrogel.

Furthermore, it was demonstrated that the cavitation threshold for hydrogel-coated vials could be controlled by tuning the swelling ratio (FIGs. 8A-8B). The smallest swelling ratio was obtained at low pH values (pH = 2). By increasing the pH up to 6, there was no significant change in swelling ratio values, and no significant change in cavitation threshold was observed. When the pH was increased from 6 to 10, the swelling ratio drastically increased. A similar trend was observed for cavitation thresholds as the pH varied between 2 and 10.

Analysis of protein particulates caused by application of mechanical stress to uncoated and hydrogel-coated vials containing protein solution

In order to explore how modification of the vial surfaces using a hydrogel coating influenced the extent of mechanical shock-induced protein aggregation, two different mechanical stresses (tumbling and shaking) were applied to the uncoated and hydrogel- coated vials containing the IVIg solutions. After application of each mechanical stress, samples were analyzed for soluble aggregates and particle levels, which were compared to those obtained in quiescent control samples. Bis-ANS fluorescence emission intensity indicated that the hydrogel coating significantly suppressed protein aggregation for both tumbling and shaking mechanical stresses (FIG. 9A). In addition, the number of particles generated in the hydrogel coated vials in tumbled and shaken samples were lower than those in uncoated vials, as measured by FIM (FIG. 9B). The higher protein aggregation but lower particle formation induced by shaking as compared to tumbling could be attributed to the larger formed particles during shaking studies. The remaining soluble IVIg was measured by SEC-HPLC analyses of IVIg solution in uncoated and hydrogel coated vials after tumbling and shaking (FIG. 9C). After 10 min of tumbling, about 8% and 4% of monomer were lost for uncoated and hydrogel-coated vials, respectively. After 1 h of horizontal shaking, about 6% and 2% of monomer were lost for uncoated and hydrogel-coated vials, respectively.

As the presence of a hydrogel coating reduced protein aggregation in both tumbled and shaken vials, the kinetics of protein aggregate formation were examined more closely.

For this aim, IVIg solutions in uncoated and hydrogel-coated vials were tumbled for various times. For each tumbling experiment, uncoated and hydrogel coated vials were tested simultaneously to reduce experimental variation. Bis-ANS fluorescence emission intensity indicated that the presence of hydrogel layer led to the strong reduction of IVIg aggregate formation (FIG. 10A). In addition, based on flow imaging microscopy analysis, the greatest number of particles were generated in uncoated vials compared to the hydrogel-coated vials for each tumbling time (FIG. 10B). Interestingly, the hydrogel coating reduced the effect of repeated tumbling, whereas repeated tumbling had an additive effect of aggregate formation in uncoated vials. The remaining soluble IVIg in solution was measured by SEC-HPLC analyses of IVIg solution in uncoated and hydrogel coated vials prior to and after tumbling (FIG. IOC). After 7 min of tumbling, about 5% and 2% of monomer were lost for uncoated and hydrogel-coated vials, respectively

Particle comparison algorithm

Machine learning techniques were applied to distinguish differences in protein aggregate morphology for hydrogel-coated and uncoated vials. ConvNets were used to analyze FIM images of aggregates generated in uncoated and coated vials upon exposure to different mechanical stresses. FIGs. 11 A-l IB shows the contour plots of the ConvNet- derived FIM image embeddings for these samples as well as sample images from each sample that map to the circled regions of the embedding space. While the unstressed samples in each vial type (FIGs. 11 A-l IB, "reference," left column) appeared to contain similar images, the particle populations generated after mechanical agitation depended strongly on whether the vial was hydrogel-coated or not (FIGs. 11A-11B, "shaking" and "tumbling").

The coating effects were the most pronounced for tumbling stress. The uncoated vials produced a poly disperse particle population after tumbling stress including several bubble- like particles. In contrast, the hydrogel-coated vials instead produced a particle population that qualitatively resembles that present in the unstressed sample. This behavior is reflected in the rejection rates: the rejection rate between the unstressed and tumbled samples was 100% in uncoated vials but only 39% in the hydrogel -coated vials. This behavior was not observed for particles generated by shaking stress as both vial coating resulted in embedding populations distinct from that present in the unstressed sample. Shaking stress generated many large aggregates in the uncoated vials and a poly disperse particle population with few bubbles in the hydrogel-coated vials.

FIGs. 12A-12B shows contour plots of the FIM image embeddings from samples tumbled in either uncoated or hydrogel-coated vials after 1, 4, and 7 min tumbling time. Interestingly, these tumbled samples appeared to exhibit a different particle morphology than the tumbled samples shown in FIGs. 1 lA-1 IB wherein the unstressed samples exhibited a different embedding distribution and the tumbled samples in the uncoated vials contained a larger fraction of large aggregates. Despite this difference, the embedding plots in FIGs. 12A-12B also suggested that vial coatings impacted the particle populations generated after different durations of tumbling stress. While increasing tumbling time resulted in a major embedding shift relative to the unstressed samples in the uncoated vials, the tumbled coated vials all exhibited similar particle populations to that present in the reference sample.

Furthermore, to see if any observed particles came from the surface treatments, uncoated and hydrogel-coated vials containing buffer without protein were tumbled for 7 min (FIG. 13). There was no significant particle generation in hydrogel-coated vials as compared to uncoated ones. The trained ConvNet was used to analyze FIM images obtained from tumbling buffer solutions in both vial types (FIG. 14). FIG. 14 shows the distribution of FIM image embeddings obtained after tumbling buffer solution in the hydrogel-coated vials. An insufficient number of particles were obtained from the uncoated vials to be effectively plotted in this fashion. Tumbling buffer in the hydrogel-coated vials did produce small lightly colored particles similar to those observed from both the unstressed formulations and the tumbled samples plotted in FIGs. 11 A-l IB.

Selected Discussion Efficacious and safe protein formulations can be compromised due to the tendency of therapeutic proteins to aggregate under various stress conditions. These stress conditions (e.g., freeze-thaw, heat, shaking, shear stress, surface adsorption) may result in irreversible drug loss, and provoke adverse immune responses. Therefore, there is an important need for design and development of approaches and tools to mitigate the effects of these stressing conditions. Protein aggregation resulting from adsorption to containers and delivery devices as well as protein damage due to mechanical shocks can be mediated by the container type and surface chemistry. In certain embodiments, the surface defects found in typical glass containers are ideal sites for cavitation, and they can trap and pin bubble nuclei, even on surfaces with high wettability.

Herein, in a non-limiting example, it was shown that acrylamide hydrogel coatings, when applied to pharmaceutical containers, minimize the formation of aggregates and improve therapeutic protein formulation stability and patient safety. Acrylamide hydrogel coatings have shown promising results in increasing the acoustic cavitation threshold. In addition, very few bubbles were observed in subsequent drops of hydrogel-coated vials containing IVIg solutions. Two mechanical stresses (tumbling and shaking) were chosen as those mostly mimic the common stress conditions the pharmaceutical containers may experience during shipping and handling and administration to patients. During tumbling stress, the vials were repeatedly exposed to high g-force events that likely induced cavitation in the uncoated vials. The acrylamide coating reduced cavitation events that contributed to particle formation in the uncoated vials. Hydrogel-coated vials also reduced the protein aggregation and particle formation as compared to uncoated vials for shaking studies.

Without wishing to be limited by any theory, this reduction perhaps is due to the lower shear stresses the protein solution experiences at hydrogel interface as compared to a glass interface.

The hydrogel coating was found to significantly impact the particles formed by both mechanical stresses. During tumbling stress, hydrogel-coated vials produced particle morphologies that appeared similar to those in the unstressed samples and did not appreciably change with increasing tumbling time. The uncoated vials instead exhibited much more dramatic changes in particle morphology with increased tumbling time. While the hydrogel coating did contribute a small number of particles to the formulation during tumbling stress (FIG. 14), these results suggest that the acrylamide coating overall helped protect the formulation against tumbling-induced aggregation and particle formation. In contrast, shaking the IVIg solution in the hydrogel-coated vials did result in a particle population that differed from that present in the unstressed samples, suggesting that the coating was less protective against shaking stress. However, the hydrogel coating still resulted in a distinct particle population from that generated by the uncoated vials, suggesting that the coating did offer some protection against shaking stress.

In conclusion, the present disclosure provides a new interfacial coating that mitigates the effects of mechanical stresses on protein aggregation and particle formation. The inventive hydrogel coatings have shown to significantly increase the cavitation threshold tested by ultrasonic treatment. Further, the hydrogel coatings created a proper protection for protein aggregation even for repeated drops. Protein aggregation and particle formation was suppressed by employing the hydrogel layers to glass containers. More interestingly, hydrogel-coated vials produced particle morphologies that appeared similar to those in the unstressed samples and did not appreciably change with increasing tumbling time. Finally, it was demonstrated that, by tuning the swelling ratio, the cavitation threshold in hydrogel- coated vials can be controlled. These studies and new chemistries offer strategies that will impact the design and manufacturing of a variety of containers and protein delivery systems, leading to safer, more stable formulations of therapeutic proteins.

Enumerated Embodiments

The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

Embodiment 1 provides a method of reducing cavitation of a protein solution in a container, the method comprising at least one of the following: chemically modifying one or more surfaces of the container with a hydrogel coating; and storing the protein solution in the container such that the protein solution is in contact with the hydrogel coating.

Embodiment 2 provides the method of embodiment 1, wherein the container is a glass container.

Embodiment 3 provides the method of embodiments 1-2, wherein the hydrogel coating is a polyacrylamide coating.

Embodiment 4 provides the method of embodiments 1-3, wherein the step of chemically modifying one or more surfaces of the glass container with a hydrogel coating comprises at least one of the steps of: reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the surface of the glass container to attach the first polymerizable monomer to the container surface; contacting the attached first polymerizable monomer with a solution comprising a second polymerizable monomer, a third polymerizable monomer, and a photoinitiator; and irradiating the solution to form hydrogel coating on the surface of the glass container.

Embodiment 5 provides the method of embodiments 1-4, wherein the step of reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the surface of the glass container to attach the first polymerizable monomer to the container surface is preceded by the step of treating the glass container with UV/ozone.

Embodiment 6 provides the method of embodiments 1-5, wherein at least one applies:

(i) the first polymerizable monomer is selected from an acrylate or methacrylate monomer comprising a trialkoxysilyl functional group;

(ii) the second polymerizable monomer is selected from an acrylic or methacrylic amide; or

(iii) the third polymerizable monomer is selected from a polyethylene glycol diacrylate, a polyethylene glycol dimethacrylate, a polypropylene glycol diacrylate, and a polypropylene glycol dimethacrylate.

Embodiment 7 provides the method of embodiments 1-6, wherein at least one applies:

(i) the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate;

(ii) the second polymerizable monomer is acrylamide; or

(iii) the third polymerizable monomer is polyethylene glycol diacrylate.

Embodiment 8 provides the method of embodiments 1-7, wherein the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate, the second polymerizable monomer is acrylamide, the third polymerizable monomer is polyethylene glycol diacrylate, and the hydrogel coating formed on the surface of the glass container is polyacrylamide.

Embodiment 9 provides the method of embodiments 1-8, wherein the method reduces cavitation induced by mechanical shock to the container.

Embodiment 10 provides the method of embodiments 1 -9, wherein the method reduces protein degradation, protein aggregation, or a combination thereof.

Embodiment 11 provides a container comprising an interior surface that is at least partially chemically modified with a hydrogel coating, wherein the hydrogel coating reduces cavitation in a protein solution stored within the container.

Embodiment 12 provides the container of embodiment 11, wherein the interior surface of the container is glass.

Embodiment 13 provides the container of embodiments 11-12, wherein the hydrogel coating is formed by at least one of the following: reacting a first polymerizable monomer that comprises a trialkoxysilyl functional group with the glass interior surface to attach the first polymerizable monomer to the interior surface; contacting the attached first polymerizable monomer with a solution comprising a second polymerizable monomer, a third polymerizable monomer, and a photoinitiator; and irradiating the solution to form hydrogel coating on the interior surface of the glass container.

Embodiment 14 provides the container of embodiments 11-13, wherein the interior surface of the glass container is treated with UV/ozone before reacting the interior surface with the first polymerizable monomer.

Embodiment 15 provides the container of embodiments 11-14, wherein at least one applies:

(i) the first polymerizable monomer is selected from an acrylate or methacrylate monomer comprising a trialkoxysilyl functional group;

(ii) the second polymerizable monomer is selected from an acrylic or methacrylic amide; or

(iii) the third polymerizable monomer is selected from a polyethylene glycol diacrylate, a polyethylene glycol dimethacrylate, a polypropylene glycol diacrylate, and a polypropylene glycol dimethacrylate.

Embodiment 16 provides the container of embodiments 11-15, wherein at least one applies:

(i) the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate;

(ii) the second polymerizable monomer is acrylamide; or

(iii) the third polymerizable monomer is polyethylene glycol diacrylate.

Embodiment 17 provides the container of embodiments 11-16, wherein the first polymerizable monomer is 3-(trimethoxysilyl)propyl methacrylate, the second polymerizable monomer is acrylamide, the third polymerizable monomer is polyethylene glycol diacrylate, and the hydrogel coating formed on the surface of the glass container is polyacrylamide.

Embodiment 18 provides the container of embodiments 11-17, wherein the hydrogel coating reduces cavitation induced by mechanical shock to the container.

Embodiment 19 provides the container of embodiments 11-18, wherein the hydrogel coating reduces protein degradation, protein aggregation, or a combination thereof.

Embodiment 20 provides the container of embodiments 11-19, wherein the container is a vial or a syringe. Other Embodiments

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.