WO2026006460A2 - Procédés de préparation d'un conjugué d'oxyde de graphène et d'acide hyaluronique - Google Patents

Procédés de préparation d'un conjugué d'oxyde de graphène et d'acide hyaluronique

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Publication number
WO2026006460A2
WO2026006460A2 PCT/US2025/035268 US2025035268W WO2026006460A2 WO 2026006460 A2 WO2026006460 A2 WO 2026006460A2 US 2025035268 W US2025035268 W US 2025035268W WO 2026006460 A2 WO2026006460 A2 WO 2026006460A2
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Prior art keywords
pure
hours
acid
water
modified
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WO2026006460A3 (fr
Inventor
Daniel Holsworth
Wuping Ma
Bing Yang
Jiang Bian
HengWei CHANG
Zhiqiang Fang
Augustine Joseph
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Eluciderm Inc
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Eluciderm Inc
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Publication of WO2026006460A2 publication Critical patent/WO2026006460A2/fr
Publication of WO2026006460A3 publication Critical patent/WO2026006460A3/fr
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B01D15/267—Adsorption chromatography using neutral sorbents, e.g. activated carbon or diatomaceous earth
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase

Definitions

  • a method of purifying a graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution comprises passing the feed solution through a chromatographic process and operating the chromatographic process under conditions effective to purify the GO-HA from one or more impurity compound(s). Also provided is a method of making the GO-HA provided herein. Also provided is a high purity composition comprising the GO-HA provided herein.
  • the Wnt pathway has been shown to play a role in dermal fibrosis and scarring.
  • the Wnt pathway is an evolutionary conserved pathway that regulates crucial aspects of cell fate determination, cell polarity, cell migration, neural patterning, and organogenesis during embryonic development. This pathway plays a role in ensuring proper tissue development in embryos and tissue maintenance in adults. Wnt signaling is involved at the beginning stages of skin development, where following gastrulation, embryonic cells of the ectoderm and the mesoderm differentiate to form the epidermis and dermis, respectively.
  • XAV939 is a small molecule that selectively inhibits Wnt/p-catenin-mediated transcription through TK1 and TK2 inhibition with an IC50 of 11 nM/4nM in cell-free assays, regulates axin levels, and does not affect CRE, NF-KB, or TGF-p.
  • a matrix component comprising graphene oxide (GO) and hyaluronic acid (HA) has been shown as being effective in both providing a supportive matrix for XAV939 and allowing the use of XAV939 as a therapeutic for wound healing in humans and animals (see for example US20210000959) and for increased cartilage regeneration and healing following acute injury, for example (WO/2023/039298).
  • Wu et al. Carbon 69 (2014) 379-389 discloses a conjugate of GO and HA but with a different linker from the GO-HA provided herein.
  • Wu et al. does not provide for chromatographic resolution of their conjugate of GO and HA.
  • CN103030140 discloses a conjugate of GO and HA but with a different linker from the
  • GO-HA provided herein.
  • CN103030140 mentions column separation of their product, a drug- loaded nano-layer composition containing an anti-tumor agent, from organic solvent(s) and from free anti-tumor agent, but does not provide any details of a chromatographic resolution of their conjugate of GO and HA containing an anti-tumor agent.
  • CN102727901 discloses a conjugate of GO and HA but with a different linker from the GO-HA provided herein. CN102727901 does not provide an example of a chromatographic resolution of their conjugate of GO and HA.
  • US20210000959 and WO/2023/039298 provide methods of preparing the GO-HA provided herein.
  • the product of the coupling of Modified GO (graphene oxide modified by reacting with chloroacetic acid) and HA-AD (hyaluronic acid modified by reacting with adipic acid dihydrazide) resulted in a black powder and was of low purity.
  • FIG. 1 shows a solid state 1 H NMR spectra of modified graphene oxide (Modified GO) (500 MHz, KBr pellet) according to one or more embodiments.
  • FIG. 2 shows a solid state 13 C NMR spectra of Modified GO (400 MHz, KBr pellet).
  • FIG. 3 shows a 'H NMR spectra of HA-AD (500 MHz, D 2 O).
  • FIG. 4 shows a 13 C NMR spectra of HA-AD (125.8 MHz, D 2 O).
  • FIG. 5 shows a 13 C NMR spectra of crude GO-HA (125.8 MHz, D 2 O).
  • FIG. 6A shows an SEC-HPLC chromatogram of a first batch of crude GO-HA. Peak 1 is Modified GO, peak 2 is GO-HA, and peak 3 is HA. The SEC-HPLC was scanned on a PDA detector.
  • FIG. 6B shows an SEC-HPLC chromatogram of a second batch of crude GO-HA. Peak 1 is Modified GO, peak 2 is GO-HA, and peak 3 is HA. The SEC-HPLC was scanned on a PDA detector.
  • FIG. 7 shows a 1 H NMR spectra of purified GO-HA (500 MHz, D2O).
  • FIG. 8 shows a 13 C NMR spectra of purified GO-HA (125 MHz, D2O). Peaks labeled with HA are characteristic of the HA portion of GO-HA. Peaks labeled with MGO (Modified GO) are characteristic of the GO-CH 2 C(O)O- portion of GO-HA. Peaks labeled with AD are characteristic of the portion of GO-HA. The peaks labelled HA,
  • MGO, and AD are not, for example, residual material but a part of the GO-HA structure.
  • FIG. 9 shows a reversed phase HPLC chromatogram of crude GO-HA, from the chromatogram overlay of FIG. 9.
  • FIG. 10 shows a reversed phase HPLC chromatogram of purified GO-HA, from the chromatogram overlay of FIG. 9.
  • FIG. 11 shows a reversed phase HPLC chromatogram of a water blank from the chromatogram overlay of FIG. 9.
  • FIG. 12 shows an overlay of GO-HA HPLC chromatograms (reversed phase) before and after purification.
  • the three chromatographs in the overlay include: crude GO-HA (top chromatogram, light gray); purified GO-HA (middle, dark gray); and water blank (bottom, black).
  • FIG. 13 shows a reversed phase UPLC chromatogram of a water blank from the chromatogram overlay of FIG. 13.
  • FIG. 14 shows a reversed phase UPLC chromatogram of purified GO-HA from the chromatogram overlay of FIG. 13.
  • FIG. 15 shows a reversed phase UPLC chromatogram of crude GO-HA from the chromatogram overlay of FIG. 13.
  • FIG. 16 shows an overlay of GO-HA UPLC chromatograms (reversed phase) before and after purification.
  • the three chromatographs in the overlay include: water blank (top chromatogram, light gray); purified GO-HA (middle, dark gray); crude GO-HA (bottom, black).
  • FIG. 17 shows the results of cytotoxicity measurement of GO-HA on myofibroblasts, comparing various levels of purity of GO-HA versus cell viability.
  • FIG. 18 shows the results of human red blood cell hemolysis of GO-HA, comparing various levels of purity of GO-HA at various concentrations versus percent hemolysis.
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • methods for purifying a graphene oxide-hyaluronic acid conjugate as provided herein.
  • methods of making a graphene oxide and hyaluronic acid conjugate as provided herein.
  • high purity compositions of a GO-HA provided herein.
  • a method for purifying a graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution comprising:
  • Step (b) operating the chromatographic process under conditions effective to purify the GO-HA from the one or more impurity compound(s) (in some embodiments, the one or more impurity compound is/are graphene, graphene oxide, Modified GO, hyaluronic acid, and/or HA-AD), producing a purified GO-HA effluent.
  • the one or more impurity compound is/are graphene, graphene oxide, Modified GO, hyaluronic acid, and/or HA-AD
  • a method of making GO-HA comprising:
  • the one or more coupling agent are selected from N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HC1), N-hydroxysuccinimide (NHS), N,N,N',N'-tetramethyl-O-(N- succinimidyl)uronium tetrafluoroborate (TSTU), 2-(lH-benzotriazol-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate (HBTU), and N,N-diisopropylethylamine (DIPEA or Hiinig’s base)) in one or more solvents such as water, wherein the Modified GO, HA-AD, and one or more coupling agent
  • Step (ii) sonicating the mixture after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent;
  • Step (iii) optionally adding additional portions of the one or more coupling agent.
  • Step (iv) adjusting the pH of the mixture from Step (i), (ii), and/or (iii) to about 5, including wherein the pH is adjusted after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent.
  • a high purity composition comprising a graphene oxidehyaluronic acid conjugate (GO-HA) prepared by the method of the first or the second aspect, wherein the GO-HA is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 92% pure for GO-HA, at least about 95% pure for GO-HA, at least about 97% pure for GO-HA, or at least about 99% pure for GO-HA, excluding any solvents, optionally as measured, for example, by SEC-HPLC.
  • GO-HA graphene oxidehyaluronic acid conjugate
  • a high purity composition comprising a graphene oxidehyaluronic acid conjugate (GO-HA), wherein the composition is at least about 85% pure for GOHA, at least about 90% pure for GO-HA, at least about 92% pure for GO-HA, at least about 95% pure for GO-HA, at least about 97% pure for GO-HA, or at least about 99% pure for GO-HA, excluding any solvents, optionally as measured, for example, by SEC-HPLC.
  • GO-HA graphene oxidehyaluronic acid conjugate
  • a pharmaceutical composition comprising a WNT inhibitor; a high purity composition comprising a graphene oxide-hyaluronic acid conjugate (GOHA), wherein the composition is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 92% pure for GO-HA, at least about 95% pure for GO-HA, at least about 97% pure for GO-HA, or at least about 99% pure for GO-HA, excluding any solvents, optionally as measured, for example, by SEC-HPLC; and a pharmaceutically acceptable carrier.
  • a WNT inhibitor a high purity composition comprising a graphene oxide-hyaluronic acid conjugate (GOHA), wherein the composition is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 92% pure for GO-HA, at least about 95% pure for GO-HA, at least about 97% pure for GO-HA, or at least about 99% pure for GO-HA,
  • the WNT inhibitor is named 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), or a tautomer and/or pharmaceutically acceptable salt thereof.
  • a method of treating a disease, disorder, or condition associated with Wnt transcription products or Wnt signaling pathway activity in a mammal in need thereof comprising administering a pharmaceutical composition of the fifth aspect (or any embodiments thereof).
  • the method is for stimulating regeneration of tissue at a wound in the mammal in need thereof.
  • a seventh aspect provided is a method of inducing bacteriostasis associated with Wnt transcription products or Wnt signaling pathway activity in a mammal in need thereof, comprising administering a pharmaceutical composition of the fifth aspect (or any embodiments thereof) to a mammal in need thereof.
  • the present disclosure provides for high purity pharmaceutical compositions comprising, and methods of making and purifying, graphene oxide-hyaluronic acid conjugate (GO-HA).
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • compositions comprising a WNT inhibitor, GO-HA of high purity, for example as prepared and purified as described herein, and a pharmaceutically acceptable carrier are useful to induce healing of a wound (including a chronic wound, an acute wound, an alkali- burned corneal wound, and an incisional wound — open or closed), healing of a bum (including first, second, and third degree burns), or healing of a lesion (including lesions caused by HPV and/or a virus selected from the Poxviridae family of viruses); treat an inflammatory dermatitis disease (including acne, psoriasis, rosacea, and scleroderma), a cartilage disease, a bone disease, organ fibrosis, or cancer (including melanoma and breast cancer); induce tissue regeneration (including but not limited to regeneration of damaged elastic cartilage); induce bacteriostasis; induce bacterial growth inhibition; maintain bacteriostasis; induce antifungal activity; induce neovascularization (
  • the wound may include but is not limited to one or more selected from the group consisting of a chronic wound, an acute wound, and alkali-burned corneal wound.
  • the inflammatory dermatitis disease may include but is not limited to one or more selected from the group consisting of acne, psoriasis, rosacea, and scleroderma.
  • the cartilage disease may include but is not limited to one or more selected from the group consisting of osteoarthritis, rheumatoid arthritis, internal derangement of the joints, and degenerative cartilage disease.
  • the bone disease may include but is not limited to a bone disease with impaired bone formation, e.g., osteoporosis.
  • the organ fibrosis may include but is not limited to one or more selected from the group consisting of lung fibrosis, heart fibrosis, liver fibrosis, and kidney fibrosis.
  • the cancer may include but is not limited to melanoma, breast cancer, and/or prostate cancer.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se.
  • description referring to “about X” includes description of “X”.
  • the terms “about” and “approximately,” when used in connection with temperatures, doses, amounts, or weight percent of ingredients of a composition or a dosage form mean a dose, amount, or weight percent that is recognized by those of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent.
  • phrases “a” or “an,” as used in herein means one or more, unless context clearly dictates otherwise.
  • “pharmaceutically acceptable carrier” includes one or more ingredients as provided herein.
  • compositions, formulations, and methods of treatment described herein include “comprising,” “consisting of,” and “consisting essentially of’ some or any embodiments.
  • compositions can either comprise the listed components or steps or can “consist essentially of’ the listed components or steps.
  • composition When a composition is described as “consisting essentially of’ the listed components, the composition contains the components listed, and may contain other components which do not substantially affect the basic and novel properties (in one or more embodiments, the condition being treated), but do not contain any other components which substantially affect the basic and novel properties (in one or more embodiments, condition being treated) other than those components expressly listed; or, if the composition does contain extra components other than those listed which substantially affect the basic and novel properties (in one or more embodiments, the condition being treated), the composition does not contain a sufficient concentration or amount of the extra components to substantially affect the basic and novel properties (in one or more embodiments, the condition being treated).
  • the method contains the steps listed, and may contain other steps that do not substantially affect the basic and novel properties (in one or more embodiments, the condition being treated), but the method does not contain any other steps which substantially affect the basic and novel properties (in one or more embodiments, the condition being treated) other than those steps expressly listed.
  • the composition when a composition is described as “consisting essentially of’ a component, the composition may additionally contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents and other such components which do not substantially affect the basic and novel properties (in one or more embodiments, the condition being treated).
  • the “GO-HA” or the “GO-HA as provided herein” refers to the conjugate formed by the reaction of Modified GO (graphene oxide modified by reacting with chloroacetic acid) with HA-AD (hyaluronic acid modified by reacting with adipic acid dihydrazide) to form a conjugate, for example, prepared by the procedure provided in Example 1.
  • the linker is or comprises -CH 2 C(O)NHNHC(O)CH 2 CH 2 CH 2 CH 2 C(O)NHNH- where the HA is linked via a hydrazide bond (-C(O)NHNH-C(O)-, similar in structure to an amide-type bond with another amide-type bond) comprising the terminal NH of the linker and the GO is linked to the CH 2 end of the linker (in some embodiments through an ether bond with phenolic or -OH groups on the GO or through opening of an epoxide on the GO).
  • Modified GO refers to graphene oxide which has been treated with chloroacetic acid to make graphene oxide substituted with -CH 2 C(O)OH groups.
  • the term “pharmaceutically acceptable carrier” includes any and all and/or one or more solvents, co-solvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, excipients, diluents, disintegrants, lubricants, adjuvants, and the like which are not biologically or otherwise undesirable.
  • solvents co-solvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, excipients, diluents, disintegrants, lubricants, adjuvants, and the like which are not biologically or otherwise undesirable.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • regeneration means the renewal or growth of destroyed or devitalized tissue from the remnant tissue. It is a reparative attempt of the body, and in the context of wound represents the migration, differentiation, or replication of cells or transformation of progenitor cells into the appropriate cell types for the respective tissue which may include sebaceous cells, hair follicles, nerve cells, chondrocytes.
  • wound means an injury to tissue or skin caused by scrapes, cuts, abrasion, surgical procedures (e.g., caused by minimally invasive surgery, laparoscopic surgery, robotic surgery, incisional biopsies, general surgery, and cosmetic surgery), denuded skin, bums, ulcers (e.g., diabetic ulcers, ulcers from vascular insufficiency, pressure sores, and bums), or other skin problems (e.g., allergies). Wound may range from superficial (e.g., affecting merely the epidermis) to more traumatic (e.g., lesions which affect layers of skin or tissue at depths which are beneath the epidermis). Wounds may be of any length or shape, e.g., in one or more embodiments, wounds are straight, jagged or curved.
  • a “therapeutically effective amount” or “pharmaceutically effective amount” of a compound as provided herein is one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. “Therapeutically effective amount” is also intended to include one or more of the compositions of the present disclosure so as to result in the increased regeneration of tissue subject to a wound.
  • the combination of compounds is preferably a synergistic combination. Synergy, as described in the art (for example, Chou, 2010, Cane. Res. 70(2):440-446), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds. This amount can further depend upon the patient’s height, weight, sex, age, and medical history.
  • mammal specifically includes humans, cattle, horses, dogs, and cats, but also includes many other mammalian species like pigs, rats, mice, primates (e.g., a monkey such as a cynomolgus monkey, a chimpanzee, and a human). In one or more embodiments, the mammal is a human.
  • subject refers to a mammal, as provided herein, as well as to a cell or biological sample.
  • the term “isolated” with respect to a composition refers to a composition that includes at least 85%, 90%, 95%, 98%, 99% to 100% by weight, of a specified compound, the remainder comprising other chemical species or stereoisomers.
  • IC50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
  • “treating” or “treatment” of any condition or disorder refers, in some or any embodiments, to ameliorating a condition or disorder that exists in a subject.
  • “treating” or “treatment” includes ameliorating one or more physical parameter, which may be indiscernible by the subject.
  • “treating” or “treatment” includes modulating the condition or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • “treating” or “treatment” includes delaying the onset of the condition or disorder.
  • treating includes the reduction or elimination of either the condition or one or more symptoms of the condition, or to retard the progression of the condition or of one or more symptoms of the condition, or to reduce the severity of the condition or of one or more symptoms of the condition.
  • the term “assay %” refers to a measurement of purity determined by an assay. Assay% is calculated as follows: (% area purity by HPLC/100) * (100 - %wt/wt water content - %wt/wt residual solvents - %wt/wt residue on ignition). The amount of purity of the desired compound and the amount of one or more impurities in a sample can be quantitatively analyzed by an assay, for example including but not limited to by an HPLC assay.
  • purity % and “% pure” refer to purity, as determined by HPLC. Chromatographic purity is otherwise known as peak purity. In one or more embodiments, purity % is measured by taking the peak area of DHA77 from the chromatogram over the total area of peaks from the chromatogram, including the area of impurities, but excluding solvents.
  • Embodiment Al Provided is a method of purifying a graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution, comprising passing the feed solution through a chromatographic process and operating the chromatographic process under conditions effective to purify the GO-HA.
  • a method of purifying a graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution comprising passing the feed solution through a chromatographic process and operating the chromatographic process under conditions effective to purify the GO-HA.
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • Embodiment A2 In an embodiment of Embodiment Al, provided is a method of purifying GO-HA comprising a Step (a) passing the feed solution through a chromatographic process comprising a nonpolar stationary phase and a polar mobile phase stream and a Step (b) operating the chromatographic process under conditions effective to purify the GO-HA.
  • Embodiment A3 In an embodiment of Embodiment Al or A2, the feed solution comprises the GO-HA and one or more impurity compound(s). [0065] Embodiment A4. In an embodiment of any one of Embodiments Al to A3, the conditions effective to purify the GO-HA include purification of the GO-HA from the one or more impurity compound(s).
  • Embodiment A5 In an embodiment of any one of Embodiments Al to A4, one or more impurity compound(s) is/are removed and the one or more impurity compound(s) is/are one or more selected from the group consisting of graphene, graphene oxide, Modified graphene oxide (Modified GO), hyaluronic acid, and/or HA-AD. In an embodiment of any one of Embodiments Al to A4, the one or more impurity compound(s) is/are graphene and/or graphene oxide. In an embodiment of any one of Embodiments Al to A4, the one or more impurity compound is Modified GO or hyaluronic acid.
  • the one or more impurity compounds are Modified GO and hyaluronic acid. In an embodiment of any one of Embodiments Al to A4, the one or more impurity compound(s) is/are Modified GO and/or HA-AD. In an embodiment of any one of Embodiments Al to A4, the one or more impurity compound(s) are graphene, graphene oxide, Modified GO, hyaluronic acid, and/or HA-AD.
  • the nonpolar stationary phase is selected from the group consisting of functionalized alumina gel and functionalized silica gel. In an embodiment of any one of Embodiments A2 to A5, the nonpolar stationary phase is a reversed phase gel. In an embodiment of any one of Embodiments A2 to A5, the nonpolar stationary phase is a functionalized alumina gel or functionalized silica gel. In an embodiment of any one of Embodiments A2 to A5, the nonpolar stationary phase is functionalized silica gel, functionalized with C4 or Cl 8. In an embodiment of any one of Embodiments A2 to A5, the nonpolar stationary phase is functionalized silica gel, functionalized with C4. In an embodiment of any one of Embodiments A2 to A5, the nonpolar stationary phase is functionalized silica gel, functionalized with Cl 8.
  • Embodiment A7 In an embodiment of any one of Embodiments A2 to A6, the nonpolar stationary phase has a particle size of about 2 pm to about 300 pm, about 5 pm to about 50 pm, or about 20 pm to about 30 pm.
  • the polar mobile phase stream comprises one or more solvents (A) selected from water and acetonitrile, or selected from water and ethanol; and wherein the polar mobile phase stream optionally comprises an acid.
  • the polar mobile phase stream comprises water; and wherein the polar mobile phase stream optionally comprises an acid.
  • the polar mobile phase stream comprises acetonitrile; and wherein the polar mobile phase stream optionally comprises an acid.
  • the polar mobile phase stream comprises ethanol; and wherein the polar mobile phase stream optionally comprises an acid.
  • Embodiment A9 In an embodiment of any one of Embodiments Al to A7, the chromatographic process is performed over a gradient.
  • the first or initial mobile phase solvent is water which optionally comprises an acid.
  • the second mobile phase solvent is ethanol which optionally comprises an acid and optionally wherein the second mobile phase solvent is 100% and is the final mobile phase solvent at the end of the gradient.
  • the second mobile phase solvent is acetonitrile which optionally comprises an acid and optionally wherein the second mobile phase solvent is 100% and is the final mobile phase solvent at the end of the gradient.
  • the first or initial polar mobile phase solvent further comprises an acid in an amount greater than zero but less than or equal to 5%.
  • the first or initial polar mobile phase solvent comprises trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the first or initial polar mobile phase solvent comprises from 0.1% to 5% trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the first or initial polar mobile phase solvent comprises trifluoroacetic acid. In an embodiment of any one of Embodiments A8 to A10, the first or initial polar mobile phase solvent comprises 0.1% tri fluoroacetic acid.
  • the second polar mobile phase solvent further comprises an acid in an amount greater than zero but less than or equal to 5%.
  • the second polar mobile phase solvent comprises trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the second polar mobile phase solvent comprises from 0.1% to 5% trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the second polar mobile phase solvent comprises trifluoroacetic acid.
  • the second polar mobile phase solvent comprises 0.1% trifluoroacetic acid.
  • the polar mobile phase stream further comprises an acid in an amount greater than zero but less than or equal to 5%.
  • the polar mobile phase stream comprises trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the polar mobile phase stream comprises from 0.1% to 5% trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid.
  • the polar mobile phase stream comprises trifluoroacetic acid.
  • the polar mobile phase stream comprises 0.1% trifluoroacetic acid.
  • Embodiment A13 In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over about 8 minutes to about 24 hours. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over about 8 minutes. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over 8 minutes. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over about 25 minutes. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over 25 minutes. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over about 75 minutes. In an embodiment of any one of Embodiments Al to A12, the chromatographic process is carried out over 75 minutes.
  • the purified GO-HA effluent comprises the GO-HA with at least about 90% purity, or at least about 95% purity.
  • Embodiment A15 In an embodiment of any one of Embodiments Al to A14, the chromatographic process is operated at a rate of from about 1.0 mL/min to about 10 mL/min. In an embodiment of any one of Embodiments Al to A14, the chromatographic process is operated at a rate of about 2.0 mL/min. In an embodiment of any one of Embodiments Al to A14, the chromatographic process is operated at a rate of 2.0 mL/min. In an embodiment of any one of Embodiments Al to A14, the chromatographic process is operated at a rate from about 1.0 mL/min to about 4.0 mL/min. In an embodiment of any one of Embodiments Al to A14, the chromatographic process is operated at a rate from 1.0 mL/min to 4.0 mL/min.
  • Embodiment A16a In an embodiment of any one of Embodiments Al to Al 5, the chromatographic process is operated at a temperature selected from about 10 °C to about 180 °C, selected from about 10 °C to about 160 °C, selected from about 10 °C to about 140 °C, selected from about 10 °C to about 120 °C, selected from about 10 °C to about 100 °C, selected from about 10°C to about 85°C, selected from about 10 °C to about 50°C, selected from about 15 °C to about 40 °C, selected from about 20 °C to about 35 °C, or selected from about 25 °C to about 30°C.
  • the chromatographic process In an embodiment of any one of Embodiments Al to Al 5, the chromatographic process is operated at ambient temperature. In an embodiment of any one of Embodiments Al to Al 5, the chromatographic process is operated at 50 °C.
  • Embodiment A16 In an embodiment of any one of Embodiments Al to A16a, the chromatographic process is operated at a temperature of from about 10 °C to about 180 °C, from about 10 °C to about 160 °C, from about 10 °C to about 140 °C, from about 10 °C to about 120 °C, from about 10 °C to about 100 °C, from about 10°C to about 85°C, from about 10 °C to about 50°C, from about 15 °C to about 40 °C, from about 20 °C to about 35 °C, or from about 25 °C to about 30°C.
  • the method of purifying GO-HA further comprises a Step (al) before Step (a), wherein Step (al) comprises separating solids from a GO-HA feed solution containing one or more solids (in some embodiments by centrifuging and/or filtering) and collecting the supernatant (A) and optionally further comprising washing the solids with one or more solvents (B) (in some embodiments, water), collecting supematant(s) (B), and combining supernatant (A) and supematant(s) (B).
  • Step (al) comprises separating solids from a GO-HA feed solution containing one or more solids (in some embodiments by centrifuging and/or filtering) and collecting the supernatant (A) and optionally further comprising washing the solids with one or more solvents (B) (in some embodiments, water), collecting supematant(s) (B), and combining supernatant (A) and supematant(s) (B).
  • Embodiment A18a In an embodiment of Embodiment Al 7, the method of purifying GO-HA further comprises dialyzing the supernatant (A) and/or the combined supernatant (A) and supernatants (B) against water, optionally for at least about 24 hours, optionally for at least about 48 hours, or optionally for at least about 72 hours to produce a dialysis product. In one or more embodiments, the method includes filtering the dialysis product.
  • the method of purifying GO-HA further comprises dialyzing the supernatant (A) and/or the supernatants (B) against water, optionally for at least about 24 hours, optionally for at least about 48 hours, or optionally for at least about 72 hours to produce a dialysis product.
  • the method includes filtering the dialysis product.
  • the method of purifying GO-HA further comprises concentrating the dialysis product, optionally by lyophilizing the dialysis product to form a concentrated product.
  • the method of purifying GO-HA further comprises adding the concentrated product to one or more solvents (C); optionally wherein the one or more solvents (C) are selected from water and acetonitrile or selected from water and ethanol; and further optionally wherein the wateracetonitrile volume ratio or the acetonitrile:water volume ratio is about 1.5: 1, about 1.2:1, about 1.1 :1, or about 1 : 1 or further optionally wherein the water: ethanol volume ratio or the ethanol : water volume ratio is about 1.5: 1, about 1.2:1, about 1.1 :1, or about 1 : 1; and stirring at ambient temperature for a time period selected from about 18 hours to about 24 hours or about 20 hours to about 22 hours.
  • solvents (C) are selected from water and acetonitrile or selected from water and ethanol
  • the wateracetonitrile volume ratio or the acetonitrile:water volume ratio is about 1.5: 1, about 1.2:1, about 1.1 :1, or about 1 : 1
  • the method of purifying GO-HA further comprises the steps of centrifuging at about 5000 rpm for about 1 h and collecting the supernatant (C) and optionally further comprising washing any solids with the one or more solvents (D) (in some embodiments, a water and acetonitrile mixture; in some embodiments, a water and ethanol mixture), collecting supematant(s) (D), and combining supernatant (C) and supematant(s) (D); optionally concentrating the supernatant (C) and/or the combined supernatant
  • the method of purifying GO-HA further comprises centrifuging at about 5000 rpm for about 1 h and collecting the supernatant (C) and optionally further comprising washing any solids with the one or more solvents (D) (in some embodiments, a water and acetonitrile mixture; in some embodiments, a water and ethanol mixture), collecting supematant(s) (D), and combining supernatants (C) and
  • Embodiment A22 optionally concentrating the supernatant (C) and/or the supernatants (D); filtering the supernatant (C) and/or the supernatants (D) after the concentration step; optionally lyophilizing the supernatant (C) and/or the supernatants (D) after the filtering step.
  • the method of purifying GO-HA further comprises: (1) a Step (a2) before Step (a) of any one of claims 1 to 15; or (2) a Step (a2) after the steps of claim 20, wherein Step (a2) comprises adding the GO-HA to one or more solvents such as water to form a mixture of GO-HA and one or more solvents.
  • the method of purifying GO-HA further comprises: a Step (a3) after Step (a2) and before Step (a), wherein Step (a3) comprises filtering the mixture from Step (a2).
  • Embodiment Bl Provided is a method of making a graphene oxide-hyaluronic acid conjugate (GO-HA), comprising Step (i) contacting Modified GO (graphene oxide modified by reacting with chloroacetic acid), hyaluronic acid linked to adipic acid dihydrazide (HA-AD), and one or more coupling agent in one or more solvents such as water; Step (ii) optionally sonicating the mixture after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent; Step (iii) optionally adding additional portions of the one or more coupling agent; and Step (iv) adjusting the pH of the mixture from Step (i), (ii), and/or (iii) to about 5, including wherein the pH is adjusted after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent.
  • Embodiment B2 In an embodiment of Embodiment Bl, provided is a method of making GO-HA where the one or more coupling agent are selected from: N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HC1), N-hydroxysuccinimide (NHS), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), 2-(lH- benzotriazol-l-yl)-l, l,3,3-tetramethyluronium hexafluorophosphate (HBTU), and N,N- di isopropyl ethyl amine (DIPEA or Hiinig’s base), and where the Modified GO, HA-AD, and one or more coupling agent are added in any order to the one or more solvents; and wherein Step (ii) is selected from:
  • Embodiment B3 In an embodiment of Embodiment Bl or B2, provided is a method of making GO-HA, where in Step (iv) adjusting the pH of the mixture from Step (i), (ii), and/or (iii) to about 5, the pH is adjusted after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent.
  • Embodiment A24 In an embodiment of any one of Embodiments Al to A23, provided is a method of purifying GO-HA that further comprises preparing the GO-HA used in the feed solution, comprising Step (i) contacting Modified GO, hyaluronic acid linked to adipic acid dihydrazide (HA- AD), and one or more coupling agent in one or more solvents such as water; Step (i) contacting Modified GO, hyaluronic acid linked to adipic acid dihydrazide (HA- AD), and one or more coupling agent in one or more solvents such as water; Step (i) contacting Modified GO, hyaluronic acid linked to adipic acid dihydrazide (HA- AD), and one or more coupling agent in one or more solvents such as water; Step (i) contacting Modified GO, hyaluronic acid linked to adipic acid dihydrazide (HA- AD), and one or more coupling agent in one or more
  • Step (ii) optionally sonicating the mixture after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent; Step (iii) optionally adding additional portions of the one or more coupling agent; and Step (iv) adjusting the pH of the mixture from Step (i), (ii), and/or
  • Embodiment A25 In an embodiment of A24, provided is a method of purifying GOHA where the one or more coupling agent are selected from N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC-HC1), N-hydroxysuccinimide (NHS), N,N,N',N'- tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), 2-(lH-benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and N,N-diisopropylethylamine (DIPEA or Hiinig’s base), and where the Modified GO, HA-AD, and one or more coupling agent are added in any order to the one or more solvents; and wherein Step (iii) is not optional.
  • Embodiment A26 In an embodiment of any one of Embodiments A24 to A25, provided is a method of purifying GO-HA, where in Step (iv) adjusting the pH of the mixture from Step (i), (ii), and/or (iii) to about 5, the pH is adjusted after the addition of one or more of the Modified GO, HA-AD, and/or one or more coupling agent.
  • Embodiment AB1 In an embodiment of any one of Embodiments Bl to B3 and A24 to A26, the mixture is stirred for about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours.
  • Embodiment AB2 In an embodiment of any one of Embodiments Bl to B3, A24 to A26, and AB 1, the stoichiometric ratio of Modified GO to HA-AD is about 1 :1, about 1 : 1.1, about 1 : 1.2, or about 1 : 1.3; or is selected from about 1: 1 to about 1 :3; or is greater than about 1 : 1 but less than about 1: 1.4.
  • Embodiment AB3 In an embodiment of any one of Embodiments Bl to B3, A24 to A26, and AB1 to AB 2, the GO-HA in the feed solution has a purity of at least about 70% pure for GO-HA, at least about 75% pure for GO-HA, at least about 80% pure for GO-HA, or at least about 85% pure for GO-HA, optionally as measured, for example, by SEC-HPLC, excluding any solvents. In one or more embodiments of Bl to B3, A24 to A26, and AB1 to AB2, the GO-HA in the feed solution has a purity of at least about 60% pure for GO-HA or at least about 65% pure for GO-HA.
  • Embodiment Cl Provided is a high purity composition comprising a graphene oxidehyaluronic acid conjugate (GO-HA) prepared by the method of any of the foregoing claims, wherein the high purity composition is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 91% pure for GO-HA, at least about 92% pure for GO-HA, at least about 93% pure for GO-HA, at least about 94% pure for GO-HA, at least about 95% pure for GOHA, at least about 96% pure for GO-HA, at least about 97% pure for GO-HA, at least about 98% pure for GO-HA, or at least about 99% pure for GO-HA, optionally as measured, for example, by SEC-HPLC excluding any solvents.
  • GO-HA graphene oxidehyaluronic acid conjugate
  • Embodiment C2 Provided is a high purity composition comprising a graphene oxidehyaluronic acid conjugate (GO-HA), wherein the high purity composition is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 91% pure for GO-HA, at least about 92% pure for GO-HA, at least about 93% pure for GO-HA, at least about 94% pure for GOHA, at least about 95% pure for GO-HA, at least about 96% pure for GO-HA, at least about 97% pure for GO-HA, at least about 98% pure for GO-HA, or at least about 99% pure for GO-HA, optionally as measured, for example, by SEC-HPLC, excluding any solvents.
  • GO-HA graphene oxidehyaluronic acid conjugate
  • Embodiment C3 In an embodiment of Embodiment Cl or C2, provided is a high purity composition where the high purity composition contains less than about 3% or less than about 5% HA linker or HA-AD by weight, optionally as measured by HPLC, excluding any solvents. In an embodiment of Embodiment Cl or C2, provided is a high purity composition where the high purity composition contains less than about 5%, 4%, or 3% HA linker or HA-AD by weight, optionally as measured by HPLC, excluding any solvents.
  • Embodiment C4 In an embodiment of any one of Embodiments Cl to C3, provided is a high purity composition where the composition is at least about 85 assay%, at least about 90 assay%, at least about 91% pure for GO-HA, at least about 92% pure for GO-HA, at least about 93% pure for GO-HA, at least about 94% pure for GO-HA, at least about 95 assay%, at least about 96% pure for GO-HA, at least about 97 assay%, at least about 98% pure for GO-HA, or at least about 99 assay%, as measured by HPLC.
  • a method provided herein is a method of purifying graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution.
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • solvents in some embodiments, water
  • Step (al) further comprises dialyzing the supernatant (A) and/or the supernatants (B) (or dialyzing the supernatant (A) and/or the combined supernatant (A) and supernatants (B)) against water, optionally for at least about 24 hours, optionally for at least about 48 hours, or optionally for at least about 72 hours to produce a dialysis product.
  • the method further comprises filtering the dialysis product.
  • the method further comprises concentrating the dialysis product, optionally by lyophilizing the dialysis product to form a concentrated product.
  • Step (al) further comprises adding the concentrated product to one or more solvents (C); optionally wherein the one or more solvents (C) are selected from water and acetonitrile, and further optionally wherein the water: acetonitrile volume ratio or the acetonitrile:water volume ratio is about 1.5: 1, about 1.2: 1, about 1.1 : 1, or about 1 : 1; and stirring at ambient temperature for a time period selected from about 18 hours to about 24 hours or about 20 hours to about 22 hours.
  • solvents (C) are selected from water and acetonitrile, and further optionally wherein the water: acetonitrile volume ratio or the acetonitrile:water volume ratio is about 1.5: 1, about 1.2: 1, about 1.1 : 1, or about 1 : 1; and stirring at ambient temperature for a time period selected from about 18 hours to about 24 hours or about 20 hours to about 22 hours.
  • Step (al) further comprises adding the concentrated product to one or more solvents (C); optionally wherein the one or more solvents (C) are selected from water and ethanol, and further optionally wherein the waterethanol volume ratio or the ethanol: water volume ratio is about 1.5: 1, about 1.2: 1, about 1.1 : 1, or about 1 : 1; and stirring at ambient temperature for a time period selected from about 18 hours to about 24 hours or about 20 hours to about 22 hours.
  • solvents (C) are selected from water and ethanol, and further optionally wherein the waterethanol volume ratio or the ethanol: water volume ratio is about 1.5: 1, about 1.2: 1, about 1.1 : 1, or about 1 : 1; and stirring at ambient temperature for a time period selected from about 18 hours to about 24 hours or about 20 hours to about 22 hours.
  • Step (al) further comprises the steps of centrifuging at about 5000 rpm for about 1 h and collecting the supernatant (C) and optionally further comprises washing any solids with the one or more solvents (D) (in some embodiments, a water and acetonitrile mixture; in some embodiments, a water and ethanol mixture), collecting supernatant(s) (D), and combining supernatants (C) and (D) (or combining supernatant (C) and supernatant(s) (D)); optionally concentrating the supernatant (C) and/or the supernatants (D) (or the supernatant (C) and/or the combined supernatant (C) and supernatant(s) (D)); filtering the supernatant (C) and/or the supernatants (D) (or the supernatant (C) and/or the combined supernatant (C) and supernatants (D)) after the
  • Method Step (a2) adding the GO-HA to one or more solvents such as water to form a mixture of GO-HA and one or more solvents.
  • Method Step (a3) adding the GO-HA to one or more solvents such as water to form a mixture of GO-HA and one or more solvents.
  • Method Step (a3) filtering the mixture from Step (a2).
  • the one or more impurity compound is/are graphene, graphene oxide, Modified GO, hyaluronic acid, and/or HA-AD).
  • the one or more impurity compound(s) is/are graphene, graphene oxide, Modified GO, hyaluronic acid, and/or HA-AD).
  • a method provided herein is a method of purifying graphene oxide-hyaluronic acid conjugate (GO-HA) from a feed solution.
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • the method comprises Step (a) and Step (b). In one or more embodiments, the method comprises Step (al), Step (a), and Step (b). In one or more embodiments, the method comprises Step (al), Step (a2), Step (a), and Step (b). In one or more embodiments, the method comprises Step (al), Step (a2), Step (a3), Step (a), and Step (b). In one or more embodiments, the method comprises Step (a2), Step (a), and Step (b). In one or more embodiments, the method comprises Step (a2), Step (a3), Step (a), and Step (b).
  • the method comprises Step (i), Step (ii), Step (iii), Step (a), and Step (b). In one or more embodiments, the method comprises Step (i), Step (ii), Step (iii), Step (al), Step (a), and Step (b). In one or more embodiments, the method comprises Step (i), Step (ii), Step (iii), Step (al), Step (a2), Step (a), and Step (b). In one or more embodiments, the method comprises Step (i), Step (ii), Step (iii), Step (al), Step (a2), Step (a3), Step (a), and Step (b).
  • the method comprises Step (i), Step (ii), Step (iii), Step (a2), Step (a), and Step (b). In one or more embodiments, the method comprises Step (i), Step (ii), Step (iii), Step (a2), Step (a3), Step (a), and Step (b).
  • Method of making or preparing graphene oxide-hyaluronic acid conjugate (GO-HA ) comprises Step (i), Step (ii), Step (iii), Step (a2), Step (a3), Step (a), and Step (b).
  • Embodiment DI The covalently-linked graphene oxide and hyaluronic acid is also referred to herein as graphene-oxide hyaluronic acid conjugate, GO-HA conjugate, or GO-HA.
  • the GO-HA can be made according to procedures known to a person of ordinary skill in the art, including those disclosed in US 11,291,730 B2 and US 11,369,685 B2.
  • Graphene oxide (GO) refers to an oxidized form of graphene, which is a single layer form of graphite.
  • GO can be obtained by treating graphite with strong oxidizers.
  • GO contains carbon, oxygen, and hydrogen in various amounts, depending on how it is made. It can be of length of several hundreds of nanometers up to several micrometers, its planar direction, and about 0.7-1.2 nm in thickness.
  • GO can include various oxygen containing moieties, such as epoxide groups, carboxylic acid (-COOH) groups, phenol groups (phenolic-type -OH groups), alcohol groups (other than phenol groups), etc., when prepared using sulfuric acid (e.g. Hummer’s method).
  • GO is a complex molecule and its exact structure is not known and may vary.
  • the GO may have, for example, from 1-3 or 1-4 phenolic-type-OH groups; 0, 1, 2, 3, 4 or 5 acid groups (-COOH); 0-50 (or 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 0-25, 0-20, 0-15, 0-10, 0-5, or 0-4) alcohol groups (other than phenol groups); 0-15, 5-15, or 10-15 epoxide groups; and 0-10, A
  • graphene oxide is modified by reacting with chloroacetic acid, herein referred to as “Modified GO.”
  • hydroxyl groups of the graphene oxide react with the chloroacetic acid, i.e. replacing -OH groups with -OCH2C(O)OH groups.
  • Modified GO is a complex molecule and its exact structure is not known.
  • the Modified GO may have, for example, from 1-3 or 1-4 -OCH2C(O)OH groups; 0-4 phenolic-type-OH groups; 0-4 benzylic acid groups or 0, 1, 2, 3, 4 or 5 acid groups (-COOH); 0-50 (or 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 0-25, 0-20, 0-15, 0-10, 0-5, or 0-4) alcohol groups (other than phenol groups); 0-4, 0-15, 5-15, or 10-15 epoxide groups; and 0-10, 0-5, 2-7, or 4-6 ketone groups (
  • the GO in Modified GO has the GO in GO-HA has 1-3 -O-CH2COOH groups, 0-1 phenolic-type-OH groups, 3-5 -COOH groups, 40-50 alcohol groups (other than phenol groups), 10-15 epoxide groups, and 4-6 ketone groups.
  • Modified GO can be made from GO made according to the procedures provided in Rawal et al.
  • Hyaluronic acid is an anionic, highly hydrophilic, non-sulfated glycosaminoglycan, occurring naturally throughout the human body. It can be several thousands of carbohydrate units long and can bind to water giving it a gel of stiff viscous quality.
  • An example structure of HA is provided below.
  • the HA is derivatized with adipic acid dihydrazide (AD), “HA-AD” and an example HA-AD structure is shown below.
  • AD adipic acid dihydrazide
  • HA-AD adipic acid dihydrazide
  • n in the structure this corresponds to the n of the HA described in the preceding paragraph(s).
  • the amount of coupling in a given section of HA-AD and proximity of coupling may vary.
  • the GO-HA conjugate is a conjugate of GO linked to HA via the linker -CH 2 -C(O)-NH-NH-C(O)-(CH 2 ) 4 -C(O)NH-NH- where the HA is linked via a hydrazide bond (-C(O)NHNH-C(O)-, similar in structure to an amide-type bond with another amide-type bond) comprising the terminal NH of the linker and the GO is linked to the CH 2 end of the linker (in some embodiments through an ether bond with phenolic-type -OH on the GO or through opening of an epoxide on the GO).
  • the GO in GO-HA contains carbon, oxygen, and hydrogen in various amounts, depending on how it is made.
  • the GO in GO-HA can include various oxygen containing moieties, such as epoxide groups, carboxylic acid (-COOH) groups, phenol groups (phenolic-type -OH groups), alcohol groups (other than phenol groups), and ketone groups ( ° ), etc.
  • the GO in GO-HA is a complex and its exact structure is not known and may vary.
  • the GO in GO-HA may have, for example, from 1-3 or 1 -4 -CH 2 -C(O)-NH-NH- C(O)-(CH 2 )4-C(O)NH-NH- linker groups; 0-4 phenolic-type-OH groups; 0-4 benzylic acid groups or 0, 1, 2, 3, 4 or 5 acid groups (-COOH); 0-50 (or 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40- 50, 0-25, 0-20, 0-15, 0-10, 0-5, or 0-4) alcohol groups (other than phenol groups); 0-4, 0-15, 5-15, or 10-15 epoxide groups; and 0-10, 0-5, 2-7, or 4-6 ketone groups ( ° ); or any combination thereof.
  • the GO in GO-HA is linked to HA through 1-3 -CH2-C(O)-NH- NH-C(O)-(CH 2 ) 4 -C(O)NH-NH- linker groups, and the GO in GO-HA has 0-2 -O-CH 2 COOH groups, 0-1 phenolic-type-OH groups, 3-5 -COOH groups, 40-50 alcohol groups (other than phenol groups), 10-15 epoxide groups, and 4-6 ketone groups.
  • the Modified GO linked is linked to HA-AD with one or more coupling agent.
  • the coupling agent is selected from N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HC1), N-hydroxysuccinimide (NHS), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluorob orate (TSTU), 2-(lH- benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), and N,N- diisopropylethylamine (DIPEA or Htinig’s base).
  • EDC-HC1 N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • TSTU N,N,N',N'-tetramethyl-O-(N-succinimidy
  • the coupling agent is N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HC1), N-hydroxysuccinimide (NHS), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), and N,N-di isopropyl ethyl amine (DIPEA or Htinig’s base).
  • EDC-HC1 N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • TSTU N,N,N',N'-tetramethyl-O-(N-succin
  • the stoichiometric ratio of Modified GO to HA-AD is about 1:1, about 1:1.1, about 1:1.2, or about 1:1.3. In one or more embodiments, the stoichiometric ratio of Modified GO to HA-AD is from about 1:1 to about 1:3, or is greater than about 1:1 but less than about 1:1.4. In one or more embodiments, the stoichiometric ratio of Modified GO to HA-AD is about 1:1 to about 1:4, or about 1:1.5 to about 1:4, or about 1:1.6 to about 1:4.
  • the stoichiometric ratio of Modified GO to HA-AD is any one of 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1: 2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, or 1:4.0. In one or more embodiments, the stoichiometric ratio of Modified GO to HA-AD is about 1:1 or is 1:1.
  • the weight ratio of GO:HA in the GO-HA conjugate (for example, GO-HA of Embodiment D6), can be from about 1 : 1 to about 1 :20, from about 1 :6 to about 1:10.
  • the weight ratio of G0:HA in the GO-HA conjugate can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1 : 19, or about 1 :20.
  • the yield of the GO-HA conjugate is as high as 99-100%, before purification (mass balance basis, calculated from GO starting material).
  • Purified GO-HA is as high as 99-100%, before purification (mass balance basis, calculated from GO starting material).
  • the purified GO-HA can be characterized by the absence of at least one impurity compound and in conjunction with the purity % determined by assay, for example, by HPLC.
  • at least one impurity compound is removed from the GO-HA to provide a purified GO-HA.
  • the at least one impurity compound can be selected from the group consisting of graphene, graphene oxide, Modified GO, and hyaluronic acid.
  • Other impurity compounds can include components from the method of making, preparing, or purifying GO-HA.
  • the purified GO-HA (for example, with dialysis and chromatographic purification) is at least about 70% purity, at least about 75% purity, at least about 80% purity, at least about 90% purity, at least about 95% purity, at least about 99% purity.
  • the purified GO-HA is at least about 90% purity, at least about 91% purity, at least about 92% purity, at least about 93% purity, at least about 94% purity, at least about 95% purity, at least about 96% purity, at least about 97% purity, at least about 98% purity, or at least about 99% purity.
  • the yield of the purified GO-HA conjugate is from about 30% to about 40%, after purification (mass balance basis, calculated from GO starting material).
  • the yield of the purified GO-HA conjugate can range from about 10% to about 60%, such as from about 20% to about 50%, after purification (mass balance basis, calculated from GO starting material).
  • the average molecular weight (MW) of the purified GO-HA can be, for example, from 1 to 100 kDa, such as from 1 to 90 kDa, 1 to 80 kDa, 1 to 70 kDa, 1 to 60 kDa, 1 to 50 kDa, 1 to 40 kDa, 1 to 30 kDa, 1 to 20 kDa, 1 to 10 kDa, 10 to 100 kDa, 10 to 90 kDa, 10 to 80 kDa, 10 to 70 kDa, 10 to 60 kDa, 10 to 50 kDa, 10 to 40 kDa, 10 to 30 kDa, 10 to 20 kDa, 20 to 100 kDa, 20 to 90 kDa, 20 to 80 kDa, 20 to 70 kDa, 20 to 60 kDa, 20 to 50 kDa, 20 to 40 kDa, 20 to 30 kDa, 30 to 100 kDa,
  • a high purity composition comprising a graphene oxide-hyaluronic acid conjugate (GO-HA) prepared by one or more embodiments of the method.
  • GO-HA graphene oxide-hyaluronic acid conjugate
  • the “high purity composition” comprises GO-HA and the high-purity composition is at least about 85% pure for GO-HA, excluding any solvents.
  • the high purity composition is at least about 90% pure for GO-HA, at least about 91% pure for GO-HA, at least about 92% pure for GO-HA, at least about 93% pure for GO-HA, at least about 94% pure for GO-HA, at least about 95% pure for GO-HA, at least about 96% pure for GOHA, at least about 97% pure for GO-HA, at least about 98% pure for GO-HA, or at least about 99% pure for GO-HA, optionally as measured by SEC-HPLC, excluding any solvents.
  • the “high purity composition” is at least about 85% pure for GO-HA, at least about 90% pure for GO-HA, at least about 91% pure for GO-HA, at least about 92% pure for GO-HA, at least about 93% pure for GO-HA, at least about 94% pure for GOHA, at least about 95% pure for GO-HA, at least about 96% pure for GO-HA, at least about 97% pure for GO-HA, at least about 98% pure for GO-HA, or at least about 99% pure for GO-HA, optionally as measured by SEC-HPLC, excluding any solvents.
  • the GO-HA reaction mixture can be in the form of a heterogeneous mixture, a solid (wet or dry), or an intractable material.
  • the GO-HA reaction mixture is the reaction to produce or make GO-HA itself, without further workup to obtain a solid or intractable material.
  • Components of the GO-HA reaction mixture can include, but are not limited to, graphene, graphene oxide, Modified GO, HA, HA-AD, GO-HA, and salts thereof.
  • Other components of the GO-HA reaction product can include water, HC1, NaOH, EDC, EDC-HC1, NHS, suitable reagents, intermediates, and side products of GO-HA synthesis.
  • the GO-HA reaction mixture can be centrifuged to obtain a supernatant.
  • a suitable non-protic solvent that is miscible with water and a range of organic solvents can be added to the GO-HA containing reaction mixture.
  • An example of a suitable non-protic solvent includes, but is not limited to, acetonitrile.
  • An example of a suitable non-protic solvent includes, but is not limited to, ethanol.
  • a mixture of acetonitrile and water may be used.
  • a mixture of ethanol and water may be used.
  • the mixture is stirred to obtain a supernatant.
  • the mixture can be stirred with a suitable stirring apparatus, including, but not limited to a magnetic stirrer or mechanical stirrer, followed by centrifugation to obtain the supernatant.
  • the mixture is stirred at ambient temperature for about 1 to 6 hours, such as about 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, or about 2 hours.
  • the mixture is centrifuged at about 1,000 to 10,000 rpm, such as from about 2,000 to about 8,000 rpm, from about 3,000 to about 6,000 rpm, from about 4,000 to about 6,000 rpm, or about 5,000 rpm.
  • the mixture is centrifuged at ambient temperature for about 1 to 6 hours, such as about 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, or about 1 hour.
  • the supernatant (A) or the supernatants (B) can be washed with one or more solvents, such as water.
  • the wash can include rinsing the supernatant(s) with water and decanting off the water.
  • the wash can include mixing the supernatant(s) with water, stirring with a suitable stirring apparatus, and decanting or filtering off the water.
  • the supernatant (A) and/or the supernatants (B) can be dialyzed to form a dialysis product.
  • the dialysis product comprises GO-HA.
  • the dialysis product can be a crude GO-HA.
  • the dialysis product can be filtered and further concentrated resulting in a GO-HA (crude) solid.
  • the dialysis membrane can be equilibrated in water and rinsed with DI water before use, to remove any preservatives that may be present in the packaging solution.
  • the supernatant (A) and/or supernatants (B) (or the supernatant (A) and/or the combined supernatant (A) and supernatants (B)) are loaded in the dialysis membrane and dialyzed from between 1 day to 2 weeks.
  • the supernatant (A) and/or supernatants (B) are dialyzed from 1 day to 13 days, 1 day to 12 days, 1 day to 11 days, 1 day to 10 days, 1 day to 9 days, 1 day to 8 days, 1 day to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 2 to 13 days, 2 to 12 days, 2 to 11 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, about 3 days, or 3 days.
  • the supernatant (A) and/or supernatants (B) are dialyzed for at least about 24 hours, at least about 48 hours, or at least about 72 hours. In one or more embodiments, the supernatant (A) or supernatants (B) are dialyzed for at least about 72 hours.
  • the dialysis apparatus can be any suitable (chemical) dialysis apparatus, such as tangential flow fdtration (TFF), a dialysis bag, or a dialysis tube.
  • the dialysis apparatus molecular weight cut-off can range from about 1 kDa to about 50 kDa.
  • the MWCO of the dialysis apparatus is from about 1 kDa to about 50 kDa.
  • the MWCO of the dialysis apparatus can be from about 3.5 kDa to about 15 kDa.
  • the MWCO of the dialysis apparatus is from about 3.5 kDa, about 8 kDa, about 10 kDa, about 1 kDa, about 20 kDa, about 25 kDa, about 30 kDa, or about 50 kDa. In one or more embodiments, the MWCO of the dialysis apparatus is 3.5 kDa, 8 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, or 50 kDa.
  • the dialysis apparatus may have any suitable width, volume, or length.
  • dialysis tubing with a 45 mm nominal flat width and a 29 mm diameter can be used to produce a 2.5 gram sample of dialysis product after removal of water.
  • the dialysis product is an aqueous solution (dialysis solution) before removal of water.
  • the dialysis product can be fdtered, for example, with a 0.3 to 0.5 micron filter, such as a 0.35 to 0.45 micron filter, or a 0.40 to 0.45 micron filter, or a 0.40 micron filter.
  • the filter membrane may be polyvinylidene fluoride (PVDF).
  • the dialysis product can be concentrated to form a concentrated product, GO-HA (crude) solid.
  • Suitable methods of removing water include, but are not limited to, lyophilizing, drying on a rotary evaporator, azeotroping off water and washing, or increasing surface area to solution ratio and drying at ambient temperature.
  • the concentrated product can be further dried under vacuum or under air to yield the crude GO-HA product.
  • the purity of the crude GO-HA with dialysis is from about 60% purity to about 70% purity of GO-HA in the total crude GO-HA.
  • the crude GO-HA can have a purity of greater than or equal to about 60% purity and less than 70% purity (content of GO-HA in the total crude GO-HA with dialysis and without further chromatographic purification).
  • the crude GO-HA can have a purity of from about 60% to about 68%, from about 60% to about 66%, from about 60% to about 64%, from about 60% to about 62%, from about 65% to less than 70%, from about 65% to about 69%, from about 65% to about 68%, from about 65% to about 67%, from about 65% to about 66% purity (content of GO-HA in the total crude GO-HA with dialysis and without further chromatographic purification).
  • the concentrated product can be worked up again. In one or more embodiments, multiple concentrated products are combined and are worked up an additional time.
  • the concentrated product is added to one or more solvents (C).
  • the one or more solvents (C) are selected from water and acetonitrile.
  • the one or more solvents (C) are water and acetonitrile, where the water: acetonitrile volume ratio or the acetonitrile: water volume ratio is about 1.5:1, about 1.2:1, about 1.1 : 1, or about 1 : 1.
  • the one or more solvents (C) are selected from water and ethanol.
  • the one or more solvents (C) are water and ethanol, where the water: ethanol volume ratio or the ethanol :water volume ratio is about 1.5: 1, about 1.2: 1, about 1.1: 1, or about 1 : 1.
  • the mixture is stirred at ambient temperature for about 1 to 3 days.
  • the mixture can be stirred from about 18 hours to about 72 hours, from about 18 hours to about 48 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, or from about 20 hours to about 22 hours.
  • the additional workup includes a process similar to the previously performed centrifugation step.
  • the work up includes centrifuging the mixture of concentrated product and one or more solvents (C). For example, the centrifuging can proceed at about 5000 rpm for about 1 hour.
  • the additional work up includes collecting the supernatant (C).
  • the solids can be further washed with one or more solvents (D) and supernatant (C) and supernatant(s) (D) can be combined.
  • the one or more solvents (D) is a water and acetonitrile mixture.
  • the one or more solvents (D) is a water and ethanol mixture.
  • the additional work up can include concentrating the supernatant (C) and/or the supernatants (D) (or the supernatant (C) and/or the combined supernatant (C) and supernatants (D)), as previously described.
  • the additional work up can include filtering the supernatant (C) and/or the supernatants (D) (or the supernatant (C) and/or the combined supernatant (C) and supernatants (D)).
  • the additional work up can include removing water from the supernatant (C) and/or the supernatants (D) (or the supernatant (C) and/or the combined supernatant (C) and supernatants (D)) after the filtering step, for example, by lyophilizing.
  • Crude GO-HA may range in color from black, such as fluffy black flakes, to gray, off-white, or white with faint black specs. In some instances, crude GO-HA can appear to be fluffy white flakes.
  • the purity of the crude GO-HA is from about 60% purity to about 89% purity of GO-HA in the total crude GO-HA.
  • the crude GO-HA can have a purity of greater than or equal to about 60% purity and less than 90% purity (content of GO-HA in the total crude GO-HA).
  • the crude GO-HA can have a purity of from about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, from about 60% to about 65%, from about 65% to less than 90%, from about 65% to about 85%, from about 65% to about 80%, from about 65% to about 75%, from about 65% to about 70%, from about 70% to less than 90%, from about 70% to about 85%, from about 70% to about 80%, from about 70% to about 75%, from about 75% to less than 90%, from about 75% to about 85%, from about 75% to about 80%, from about 80% to less than 90%, from about 80% to about 85% purity (content of GO-HA in the total crude GO-HA).
  • a chromatographic process can be used to improve purity of GOHA to 70% or greater.
  • a chromatographic process is used to improve purity of GO-HA to 90% purity or greater.
  • the chromatographic process includes a nonpolar stationary phase, a polar mobile phase stream and a feed stream.
  • the feed stream is a feed solution comprising GO-HA and one or more impurity compound(s) introduced into the chromatographic process.
  • the chromatographic process is a preparatory HPLC (prep- HPLC) or preparatory UPLC (prep-UPLC) process.
  • the feed solution is at least about 60% pure to less than 90% pure.
  • the feed solution is at least about 61% pure to less than 90% pure, at least about 62% pure to less than 90% pure, at least about 63% pure to less than 90% pure, at least about 64% pure to less than 90% pure, at least about 65% pure to less than 90% pure, at least about 66% pure to less than 90% pure, at least about 67% pure to less than 90% pure, at least about 68% pure to less than 90% pure, at least about 69% pure to less than 90% pure, at least about 70% pure to less than 90% pure, at least about 71% pure to less than 90% pure, at least about 72% pure to less than 90% pure, at least about 73% pure to less than 90% pure, at least about 74% pure to less than 90% pure, at least about 75% pure to less than 90% pure, at least about 76% pure to less than 90% pure, at least about 77% pure to less than 90% pure, at least about 78% pure to less than 90% pure, at least about 79% pure to
  • the GO-HA in the feed solution has a purity of at least about 60%.
  • the GO-HA in the feed solution has a purity of at least about 61% pure, at least about 62% pure, at least about 63% pure, at least about 64% pure, at least about 65% pure, at least about 66% pure, at least about 67% pure, at least about 68% pure, at least about 69% pure, at least about 70% pure, at least about 71% pure, at least about 72% pure, at least about 73% pure, at least about 74% pure, at least about 75% pure, at least about 76% pure, at least about 77% pure, at least about 78% pure, at least about 79% pure, at least about 80% pure, at least about 81% pure, at least about 82% pure, at least about 83% pure, at least about 84% pure, at least about 85% pure, at least about 86% pure, at least about 87% pure, at least about 88% pure, or at least about 89% pure.
  • the method comprises passing the feed solution through the chromatographic process.
  • components and compounds present in the feed solution (GO-HA and one or more impurity compound(s)) compete with components in the polar mobile phase stream for interaction at the surface of the nonpolar stationary phase.
  • the most hydrophilic compounds elute out of the column, such as with the eluent front.
  • Hydrophobic (carbon containing) components and compounds compete successfully with the polar mobile phase stream for access to the nonpolar stationary phase surface.
  • the method includes operating the chromatographic process under conditions effective to purify the GO-HA from at least one impurity compound.
  • the operating includes control of operation processes including, but not limited to, polar mobile phase stream gradient and flow rate, column pressure, column temperature, injection volume, and run time. Operating the chromatographic process results in a purified GO-HA stream.
  • At least one impurity compound elutes faster or slower than the GO-HA from the chromatographic process, and is thus removed from the feed stream.
  • the purified GO-HA stream includes GO-HA and at least one less impurity compound compared to the feed solution, thereby increasing the purity of GO-HA (content of GO-HA compared to the total purified GO-HA).
  • purified GO-HA consists of GO-HA.
  • purified GO-HA has one or more of the following removed graphene, GO, Modified GO, HA, and HA-AD, compared to the crude GO-HA. In other words, a purified GO-HA effluent is produced, comprising purified GO-HA.
  • the polar mobile phase stream comprises one or more solvents (A) selected from water and acetonitrile or selected from water and ethanol.
  • the polar mobile phase stream can comprise water which optionally comprises an acid.
  • the polar mobile phase stream can comprise acetonitrile which optionally comprises an acid.
  • the polar mobile phase stream can comprise ethanol which optionally comprises an acid.
  • the polar mobile phase stream can comprise water, acetonitrile, and an acid.
  • the polar mobile phase stream can comprise water, ethanol, and an acid.
  • the polar mobile phase stream can comprise water, acetonitrile, ethanol, and an acid.
  • the acid can be an organic acid, including but not limited to trifluoroacetic acid, hydrochloric acid, formic acid, and acetic acid.
  • the acid can be in an amount greater than 0 but less than or equal to 5%, or from 0.1% to 5%.
  • the polar mobile phase stream can be selected from one or more solvents selected from: an acid, such as 0.1% trifluoroacetic acid, in water; an acid, such as 0.1% trifluoroacetic acid, in acetonitrile; and an acid, such as 0.1% trifluoroacetic acid, in water and acetonitrile.
  • the polar mobile phase stream can be selected from one or more solvents selected from: an acid, such as 0.1% trifluoroacetic acid, in water; an acid, such as 0.1% trifluoroacetic acid, in ethanol; and an acid, such as 0.1% trifluoroacetic acid, in water and ethanol.
  • the nonpolar stationary phase can be functionalized alumina gel or functionalized silica gel. In one or more embodiments, the nonpolar stationary phase can be functionalized alumina gel or functionalized silica gel.
  • the nonpolar stationary phase can be a reversed phase gel. In one or more embodiments, functionalized alumina gel or functionalized silica gel is functionalized with C4 or Cl 8; in some embodiments, with C4, and in some embodiments, with C18.
  • the nonpolar stationary phase has a particle size of about 2 pm to about 300 pm.
  • the nonpolar stationary phase particle has a particle size of about 2 pm to about 250 pm, about 2 pm to about 200 pm, about 2 pm to about 150 pm, about 2 pm to about 100 pm, about 2 pm to about 50 pm, about 2 pm to about 40 pm, about 2 pm to about 30 pm, about 5 pm to about 300 pm, about 5 pm to about 250 pm, about 5 pm to about 200 pm, about 5 pm to about 150 pm, about 5 pm to about 100 pm, about 5 pm to about 50 pm, about 5 pm to about 40 pm, about 5 pm to about 30 pm, about 10 pm to about 300 pm, about 10 pm to about 250 pm, about 10 pm to about 200 pm, about 10 pm to about 150 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 10 pm to about 40 pm, about 10 pm to about 30 pm, about 15 pm to about 300 pm, about 15 pm to about 250 pm, about 15 pm to about 200 pm, about 15
  • operating the chromatographic process includes introducing the polar mobile phase stream after injection of the feed solution (feed stream).
  • the polar mobile phase stream can be isocratic, or it can be a gradient. In some embodiments, the polar mobile phase stream can be isocratic, or it can be a gradient.
  • a first mobile phase solvent may be water or 0.1% trifluoroacetic acid in water
  • a second mobile phase solvent may be acetonitrile or 0.1% trifluoroacetic acid in acetonitrile
  • a first mobile phase solvent may be water or 0.1% trifluoroacetic acid in water
  • a second mobile phase solvent may be ethanol or 0.1% trifluoroacetic acid in ethanol.
  • the second mobile phase solvent (Mobile Phase B) competes with the carbonaceous compounds and components of the feed stream for access to the surface of the nonpolar stationary phase.
  • the first mobile phase solvent may be an initial mobile phase solvent.
  • the second mobile phase solvent may be a subsequent mobile phase solvent.
  • the polar mobile phase stream may include 100% (by volume) of a first mobile phase solvent.
  • the polar mobile phase stream may include 100% (by volume) of a second mobile phase solvent, and so on for more than a second mobile phase solvent (a third, a fourth, etc.).
  • the polar mobile phase stream may include any volumetrically allowed % (by volume) of the combined mobile phase solvents.
  • the polar mobile phase stream is isocratic
  • one or more solvents can be included as the polar mobile phase stream.
  • the polar mobile phase stream can include from 1% to 30% (by volume) of acid in acetonitrile, such as 0.1% trifluoroacetic acid in acetonitrile, and the remainder of the polar mobile phase stream can be acid in water, such as 0.1% trifluoroacetic acid in water.
  • the polar mobile phase stream can include about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% (by volume) of acid in acetonitrile, such as 0.1% tri fluoroacetic acid in acetonitrile, and the remainder of the polar mobile phase stream can be acid in water, such as 0.1% trifluoroacetic acid in water.
  • the acid may be selected from trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid (0.1% to 5%).
  • the polar mobile phase stream can include from 1% to 30% (by volume) of acid in ethanol, such as 0.1% trifluoroacetic acid in ethanol, and the remainder of the polar mobile phase stream can be acid in water, such as 0.1% trifluoroacetic acid in water.
  • the polar mobile phase stream can include about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% (by volume) of acid in ethanol, such as 0.1% trifluoroacetic acid in ethanol, and the remainder of the polar mobile phase stream can be acid in water, such as 0.1% trifluoroacetic acid in water.
  • the acid may be selected from trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid (0.1% to 5%).
  • the chromatographic process is carried out over about 0.05 hour to about 12 hours.
  • the chromatographic process can be carried out over about 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 3 hours, or 1 hour.
  • the chromatographic process can be carried out over about 5 minutes, about 8 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes.
  • the chromatographic process can be carried out over 1 minute, 2 minutes 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, or 25 minutes.
  • the chromatographic process is operated at a rate of about 0.1 mL/min to about 10 mL/min.
  • the chromatographic process can be operated at a rate of about 0.1 mL/min, 0.5 mL/min, 1.0 mL/min, 1.5 mL/min, 2.0 mL/min, 2.5 mL/min, 3.0 mL/min, 3.5 mL/min, 4.0 mL/min, 4.5 mL/min, 5.0 mL/min, 5.5 mL/min, 6.0 mL/min, 6.5 mL/min, 7.0 mL/min, 7.5 mL/min, 8.0 mL/min, 8.5 mL/min, 9.0 mL/min, 9.5 mL/min, or 10.0 mL/min.
  • the chromatographic process is operated at a temperature of selected from about 10 °C to about 180 °C, selected from about 10 °C to about 160 °C, selected from about 10 °C to about 140 °C, selected from about 10 °C to about 120 °C, or selected from about 10 °C to about 100 °C.
  • the chromatographic process is operated at a temperature of from about 10 °C to about 180 °C, from about 10 °C to about 160 °C, from about 10 °C to about 140 °C, from about 10 °C to about 120 °C, or from about 10 °C to about 100 °C.
  • the chromatographic process is operated at a temperature of about 10°C to about 85°C.
  • the chromatographic process can be operated at a temperature of about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, or about 80 °C.
  • the chromatographic process can be operated at ambient temperature.
  • a procedure of preparing GO-HA is disclosed in United States Patent Nos. 11,291,730 B2 and 11,369,685 B2. The procedure of preparing GO-HA is described in Example 1.
  • GO-HA was produced in 3 synthetic steps (steps A-C).
  • Step A production of Modified GO (MGO)
  • Graphene oxide (GO) dispersion in water was mixed with chloroacetic acid under basic conditions to provide modified GO, the product of Step A.
  • the reaction to produce Modified GO from GO is shown in Schemes la and lb and described as follows.
  • Schemes la and lb are schemes with illustrative structures.
  • Modified GO is a complex molecule. Its exact structure is not known and may vary (for example, in the position and number of -CH2C(O)OH group(s)).
  • the structures of Modified GO herein and below are provided for illustrative purposes.
  • Graphene oxide (6 g, 600 mL of 10 mg/mL graphene oxide dispersion in deionized (DI) water, CAS No.: 2432843-15-5) was placed in a 5 L beaker. DI water (1.8 L) was added to the 5 L beaker. The mixture was stirred for 10-15 minutes (min) at room temperature using an overhead stirrer. Sodium hydroxide (pellets, 14.6 g, 0.365 mol, source: Chem Impex, Wood Dale, IL, USA) was added to the mixture in 3 portions over a period of 30 min while stirring. The mixture was stirred further at room temperature for 60-70 min.
  • DI deionized
  • step A product, Modified GO, about 6-8 g was used as-is in the subsequent step without further purification.
  • Table 2 Specifications for chloroacetic acid
  • Table 3 Specifications for Modified GO (step A product)
  • Step B Preparation of hyaluronic acid - adipic acid dihydrazide (HA-AD)
  • HA Hyaluronic acid
  • AD adipic acid dihydrazide
  • Scheme 2 provides illustrative structures. For example, the amount of coupling in a given section of HA-AD and proximity of coupling can be variable.
  • n of HA represents an integer sufficient to provide HA from about 10 kDa to about 30 kDa
  • n of HA- AD represents the same integer carried over from HA in Step B.
  • suitable HA used are as follows.
  • Hyaluronic acid of molecular weight 10K was obtained from Creative PEGWorks (Chapel Hill, NC, USA).
  • Hyaluronic acid of molecular weight 10-20 kDa was obtained from Stanford Chemicals (Lake Forest, CA, USA).
  • Hyaluronic acid (HA) (16.0 g, 1.60 mmol) was dissolved in DI water (4 L) in a 5 L beaker at room temperature with magnetic stirring.
  • adipic acid dihydrazide (AD) (8.62 g, 49.5 mmol, source: Oakwood Chemical, Estill, SC, USA) was added to the solution and stirred for 10-15 min. Then the pH of the mixture was adjusted from pH 5-6 to pH 4.7-4.8 with 1 N HC1 (approximately 10-20 mL) while stirring.
  • AD adipic acid dihydrazide
  • N,N,N',N'- tetramethyl-O-(N-succinimidyl)uronium tetrafluorob orate (TSTU) 2 mmol
  • 2-(lH-benzotriazol-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) 2 mmol
  • DIPEA Hiinig’s base N,N- diisopropylethylamine
  • the dialyzed solution was then lyophilized (Labconco FreeZone 8 L, -50 °C) to yield HA-AD (step B product) as a white cotton-like powder (14 g, 88% weight yield).
  • the specifications for and properties of hyaluronic acid, adipic acid dihydrazide, and HA-AD are listed in Table 4, Table 5, Table 6, respectively.
  • Step C Preparation of GO-HA
  • Modified GO product of step A
  • HA-AD product of step B
  • the reaction to form GO-HA from Modified GO and HA-AD is shown in Schemes 3a and 3b.
  • Schemes 3a and 3b is an illustrative scheme.
  • the exact structure of Modified GO is not known and may vary (for example, the position and number of -CH2C(O)OH groups and coupling).
  • the structures of Modified GO herein and below are provided for illustrative purposes.
  • Modified GO product of step A, 6 g, obtained as described in above step A experimental procedure
  • DI water 1.2 L
  • HA-AD step B product, 4.0 g, 2.7 weight equivalent
  • the pH of the mixture was adjusted to 5 with IN HCl/IN NaOH (typically pH was found to be 3-4 before adjustment, and about 5 mL of IN NaOH was added), followed by sonication for 5-10 min (ultrasonicator, 40% power level, Sonomechanics LSP-600 probe).
  • A-(3-Dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (EDC-HC1, 5.0 g, 26.08 mmol, 3.3 weight equivalent) and N-hydroxysuccinimide (NHS, 2.5 g, 21.72 mmol, 1.7 weight equivalent (weight equivalent)) were added to the mixture, under mechanical stirring, at room temperature and stirred further for 5-10 min.
  • the pH of the mixture was adjusted to 5 with IN HCl/IN NaOH (typically pH was found to be 5-6 before adjustment, and a few drops of IN HC1 were added; after stirring, the pH dropped below 5 and so a few drops of IN NaOH were added), followed by sonication for 30 min (ultrasonicator, 40% power level, Sonomechanics LSP- 600 probe).
  • Additional EDC-HC1 (5 g, 26.08 mmol, 3.3 weight equivalent) and NHS (2.5 g, 21.72 mmol, 1.7 weight equivalent) were added to the mixture, under mechanical stirring, at room temperature and stirred further for 5-10 min.
  • the pH of the mixture was adjusted to 5 with IN HC1/1N NaOH.
  • the reaction mixture was allowed to stir at room temperature for 20-24 h.
  • Example 2 Purification of GO-HA
  • Prep-HPLC purified GO-HA samples were tested for purity by HPLC (amount of GO-HA within the sample).
  • a first purification step resulted in purified GO-HA from about 70% to about 90% purity.
  • a second purification step resulted in purified GO-HA greater than 90% purity (such as 95%, 96%, 97%, 98%, 99%, or up to 99.8-100% purity).
  • the second purification step provided purified GO-HA in a range of about 15.4% to about 15.7% yield.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • SEC-HPLC samples were prepared as follows. Crude GO-HA was dissolved in 2-6 mg/mL of DI water. The mixture was vortexed and sonicated for 15 min, with visible particles remaining in the mixture. The supernatant was filtered with a 0.45 pm PVDF syringe filter and injected in the SEC-HPLC system for analysis. Results of the SEC-HPLC analysis are shown in FIGS. 6A-6B
  • FIG. 6A shows an SEC-HPLC chromatogram of a batch of crude GO-HA (4.06 mg/mL in water) at 254 nm wavelength having three peaks.
  • Peak 1 Modified GO: 9.220 min RT, 1447.5 area, 18.9 height, 1.1152 width, 60.012 area %, 0.769 symmetry.
  • Peak 2 GO-HA: 12.349 min RT, 411 area, 15.6 height, 0.4043 width, 17.041 area %, 0.754 symmetry.
  • FIG. 6B shows an SEC-HPLC chromatogram of a different batch of crude GO-HA (4.14 mg/mL) at 254 nm wavelength having three peaks.
  • Peak 1 Modified GO: 9.11 min RT, 1539 area, 21.1 height, 1.0595 width, 26.840 area %, 0.774 symmetry. Shoulder: 11.257 min RT, 165.8 area, 2.9 height, 0.7695 width, 2.892 area %, 2.808 symmetry.
  • Peak 3 Peak 3 (HA): 13.946 min RT, 463.7 area, 4.9 height, 1.3216 width, 8.087 area %, 0.288 symmetry.
  • the components of crude GO-HA can vary from one batch to another.
  • the retention time (RT) of the crude GO-HA ranged from 8 to 20 minutes (concentration of about 4 mg/mL) in the SEC-HPLC tests. Each peak was scanned on the PDA detector from 0-950 nm. Peak 1 (RT about 9.2 min) was found to be Modified GO. Peak 2 (RT about 12.3 min) was found to be GO-HA. Peak 3 (RT about 14.0) was found to be HA.
  • the crude GO-HA comprised at least Modified GO, GO-HA, and HA.
  • Solvents tested included N,N-dimethyl formamide (DMF), tetrahydrofuran (THF), acetonitrile (ACN), acetone, methanol, acetone:water mixture, and methanol: water mixture, each in a separate vial.
  • DMF N,N-dimethyl formamide
  • THF tetrahydrofuran
  • ACN acetonitrile
  • acetone methanol
  • acetone:water mixture acetone:water mixture
  • methanol water mixture
  • FIG. 7 purified GO-HA 'H NMR peak areas indicated at the bottom of the spectra (hash marks at bottom), from 0 PPM: 1.669, 1.806, 1.448, 6.025, 17.029, 12.653, 4.343, 3.307, 4.513, 32.005, 28.652, 2.250, 9.054, 0.803, 1.190, and 0.998.
  • FIG. 8 purified GO-HA 13 C NMR chemical shifts indicated at the top of the spectra (hash marks at top), from 0 PPM: 14.395, 22.342, 24.271, 32.969, 33.394, 34.933, 35.095, 35.160, 35.273, 35.377, 35.903, 36.376, 42.640, 54.295, 54.408, 60.453, 60.497, 68.057, 68.085, 68.151, 68.213, 72.069, 72.184, 73.432, 73.458, 73.504, 73.618, 73.683, 75.320, 80.062, 82.294, 101.094, 102.831, 102.922, 102.958, 115.151, 117.473, 162.832, 163.109, 171.165, 173.706, 173.801, 173.897, 174.460, 174.622, and 174.699.
  • FIGS. 9-12 Results of the HPLC analysis (reversed phase) are shown in FIGS. 9-12.
  • FIG. 9 shows an overlay of three RP-HPLC chromatograms: the top chromatogram (light gray line) is the crude GO-HA; the middle chromatogram (gray line) is the purified GO-HA; the bottom chromatogram (black line) is the blank, water. These chromatograms are separated out in FIGS. 10-12.
  • FIG. 10 shows the top chromatogram from the FIG. 9 overlay: crude GO-HA.
  • FIG. 11 shows the middle chromatogram from the FIG. 9 overlay: purified GO-HA.
  • FIG. 12 shows the bottom chromatogram from the FIG. 9 overlay: blank (water), for reference.
  • the retention time (RT) of the purified GO-HA chromatographic peak was from 6.5 to
  • HPLC purity % represents purity of the GO-HA peak without precipitate (ppt), also called chromatographic purity or peak purity.
  • HPLC assay % represents the amount of GO-HA in the sample compared to total components, in other words, assay % is the purity of the crude GO-HA sample as determined by HPLC assay.
  • FIGS. 13-16 Results of the UPLC analysis (reversed phase) are shown in FIGS. 13-16.
  • FIG. 13 shows an overlay of three RP-UPLC chromatograms: the top chromatogram (light gray line) is the blank, water; the middle chromatogram (gray line) is the purified GO-HA; the bottom chromatogram (black) is the crude GO-HA. These chromatograms are separated out in FIGS. 14-16.
  • FIG. 14 shows the top chromatogram from the FIG. 13 overlay: blank (water), for reference.
  • FIG. 15 shows the middle chromatogram from the FIG. 13 overlay: purified GO-HA.
  • FIG. 16 shows the bottom chromatogram from the FIG. 13 overlay: crude GO-HA.
  • the retention time (RT) of the purified GO-HA chromatographic peak was from 2.9 to 4.2 minutes (concentration of 1 mg/mL) in the reversed phase UPLC tests.
  • UPLC purity % represents purity of the GO-HA peak without precipitate (ppt), also called chromatographic purity or peak purity.
  • UPLC assay % represents the amount of GO-HA in the sample compared to total components, in other words, assay % is the purity of the crude GO-HA sample as determined by UPLC assay.
  • Example 5 Weight ratios of GO:HA-AD
  • Ratios of Modified G0:HA-AD were tested at 1 : 1.6, 1 :2, 1 :2.9, 1 :4, 1: 10, and 0:2 in the reaction described in Steps A-C (Example 1).
  • the ratio of 0:2 indicated absence of Modified GO in the reaction, for example, to study the formation of a potential impurity.
  • GO-HA obtained from each batch was analyzed by 1 H NMR, EA (elemental analysis), HPLC, and solubility tests.
  • a cell viability assay was performed with a Sigma-Aldrich (St. Louis, MO, USA) Cell Counting Kit 8 (WST-8 / CCK8) (ab228554).
  • WST-8 / CCK8 Cell Counting Kit 8
  • 5,000 myofibroblasts were seeded per well in a 96 well plate and incubated for 24 hours at 37°C, 5% CO2 incubator. Each well was treated with 1 mg/mL concentration of GO-HA and was incubated for 24 hours at 37°C, 5% CO2. 10 pl of WST-8 / CCK8 solution was added to directly each well of the 96 well plate without premixing the components. The plate was protected from light and was incubated for 4 hours at 37°C.
  • the kit used a water-soluble tetrazolium salt that quantified the number of live cells (myofibroblasts) by producing an orange formazan dye upon bio-reduction in the presence of an electron carrier.
  • WST-8 / CCK8 tetrazolium salt was reduced by cellular dehydrogenases to an orange formazan product that was soluble in tissue culture medium.
  • the amount of formazan produced was directly proportional to the number of living cells (myofibroblasts) and was measured by absorbance at 460 nm. Results are shown in FIG. 17.
  • the % myofibroblast viability ranged from about 77.5% to about 80% viability.
  • 70% pure GO-HA purified GO-HA
  • the % myofibroblast viability ranged from about 80% to about 82.5% viability.
  • 97% pure GO-HA purified GO-HA
  • the % myofibroblast viability ranged from about 85% to about 90% viability.
  • Example 7 Stability studies of GO-HA
  • Additional studies can include accelerated stability studies (6 months, 40 °C/75% RH) and long-term stability studies (3 years, 25°C/60% RH), as shown in Table 14. Specifications for GO-HA stability studies are shown in Table 13.

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Abstract

L'invention concerne un procédé de purification d'un conjugué oxyde de graphène-acide hyaluronique (GO-HA), tel que défini dans la description, à partir d'une solution d'alimentation. Le procédé consiste à faire passer la solution d'alimentation par un processus chromatographique et à faire fonctionner le processus chromatographique dans des conditions efficaces pour purifier le GO-HA, tel que défini dans la description, à partir d'un ou de plusieurs composés d'impureté. L'invention concerne également un procédé de production du conjugué GO-HA selon l'invention. L'invention concerne en outre une composition de haute pureté comportant le GO-HA selon l'invention.
PCT/US2025/035268 2024-06-25 2025-06-25 Procédés de préparation d'un conjugué d'oxyde de graphène et d'acide hyaluronique Pending WO2026006460A2 (fr)

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CN102727901A (zh) 2012-07-12 2012-10-17 上海师范大学 一种氧化石墨烯/透明质酸纳米药物载体材料及其制备方法和应用
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