EP4598563A2 - Zusammensetzungen mit in fluorierten lösungsmitteln gelösten proteinen sowie verfahren zur herstellung und verwendung davon - Google Patents

Zusammensetzungen mit in fluorierten lösungsmitteln gelösten proteinen sowie verfahren zur herstellung und verwendung davon

Info

Publication number
EP4598563A2
EP4598563A2 EP23895200.6A EP23895200A EP4598563A2 EP 4598563 A2 EP4598563 A2 EP 4598563A2 EP 23895200 A EP23895200 A EP 23895200A EP 4598563 A2 EP4598563 A2 EP 4598563A2
Authority
EP
European Patent Office
Prior art keywords
kda
protein
compound
interest
dispersant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23895200.6A
Other languages
English (en)
French (fr)
Inventor
Scott MEDINA
Harminder Singh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Penn State Research Foundation
Original Assignee
Penn State Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Penn State Research Foundation filed Critical Penn State Research Foundation
Publication of EP4598563A2 publication Critical patent/EP4598563A2/de
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a composition comprising a fluorinated solvent, a dispersant compound, and a protein of interest, wherein: the dispersant compound interacts with the protein of interest; and the protein of interest is dissolved in the fluorinated solvent.
  • the fluorinated solvent is selected from the group consisting of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, Attorney Docket No.148411.002502 PATENT perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, and perfluorodecalin.
  • the fluorinated solvent is perfluorohexane.
  • the fluorinated solvent is perfluorooctane.
  • the dispersant compound is perfluorinated.
  • the dispersant compound comprises Formula II: (Formula II), wherein x is 6, 7, 8, 9, 10, 12, or 16. In some embodiments, x is 7 (perfluoronanonic acid). In some embodiments, the dispersant compound comprises Formula III: (Formula III). In some embodiments, the dispersant compound comprises Formula V: (Formula V), wherein R is OH or COH. In the dispersant compound comprises Formula VI: (Formula VI), wherein y is 7. In some compound comprises Formula VIII: (Formula VIII), wherein R 1 is C 6 F 13 , C 8 F 17 , or R 2 is CN, H, or COOH. In some embodiments, R1 is C6F13 and R2 is H.
  • the dispersant compound interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone and amino acid side chains of the protein of interest.
  • the protein of interest comprises a non-native secondary structure.
  • the fluorinated solvent is perfluorohexane and the dispersant compound is perfluoronanonic acid.
  • the fluorinated solvent is perfluorooctane and the dispersant compound comprises Formula VIII: (Formula VIII), wherein R1 is C6F13 and R2 is
  • a method of dissolving a protein of interest in a fluorinated solvent comprising: contacting the protein of interest with a dispersant compound and dissolving the protein of interest and dispersant compound in the fluorinated solvent.
  • the method of dissolving a protein of interest in a fluorinated solvent comprises a fluorinated solvent selected from the group consisting of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, and perfluorodecalin.
  • the fluorinated solvent is perfluorohexane.
  • the fluorinated solvent is perfluorooctane.
  • the method of dissolving a protein of interest in a fluorinated solvent comprises a dispersant compound, wherein the dispersant compound is perfluorinated.
  • the dispersant compound comprises Formula II: (Formula II), wherein x is 6, 7, 8, 9, 10, 12, x is 7 (perfluoronanonic acid).
  • the dispersant compound comprises Formula III: Attorney Docket No.148411.002502 PATENT (Formula III).
  • the comprises Formula V: (Formula V), wherein R is OH or COH.
  • the dispersant compound comprises Formula VI: (Formula VI), wherein y is 7.
  • the dispersant compound comprises Formula VIII: (Formula VIII), wherein R 1 is C 6 F 13 , C 8 F 17 , or R 2 is CN, H, or COOH. In some embodiments, R1 is C6F13 and R2 is H.
  • the method of dissolving a protein of interest in a fluorinated solvent comprises a dispersant compound, wherein the dispersant compound interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone and amino acid side chains of the protein of interest.
  • the protein of interest comprises a non-native secondary structure.
  • FIGs.2A-2B depicts the structure of fluorochemical compounds 3-7 (FIG.2A) and compounds 8-12 (FIG.2B), which were based off Formula II with 0, 1, 3, 6, 7, 8, 9, 10, 12, or 16 repeats (indicated as ‘x’ in Formula II) for each compound 3-12, respectively.
  • the compounds were screened for protein binding avidity.
  • FIG.3 depicts the structure of fluorochemical compound 13, which was based on the Formula III.
  • the compound was screened for protein binding avidity.
  • FIG.4 depicts the structure of fluorochemical compound 14, which was based on the Formula IV.
  • the structure of the bound FMOC group is shown at the bottom of the figure.
  • the compound was screened for protein binding avidity.
  • FIGs.8A-8G depict fluorochemical-mediated dispersion of test proteins hemoglobin (Hb, FIG.8A), green fluorescent protein (GFP, FIG.8B), bovine serum albumin (BSA, FIG. 8C), ⁇ -Galactosidase ( ⁇ -Gal, FIG.8D), rabbit serum immunoglobulin G (IgG, FIG.8E), bovine apo-transferrin (TFN, FIG.8F), and urease (URE, FIG.8G) into perfluorohexane. Results are displayed as percent soluble protein relative to initial loading. The compound ‘0’ indicates a control sample absent of an additive.
  • FIGs.9A-9C depict the relationship between protein dispersion efficiency (FIG.9A) and length of perfluoroalkyl tail of tested perfluorinated carboxylic acids (compounds 3-12), as well as predicted logP (FIG.9B) and pKa (FIG.9C) of all relevant fluorochemical compounds.
  • FIGs.10A-10F depict the comparison of PFNA-mediated fluorous phase seperation (dispersion) and protein macromolecular properties, including molecular weight (FIG.10A), solvent-accessible surface area (FIG.10B) (SASA), hydrodynamic radius (FIG.10C), isoelectric point (pI) (FIG.10D), ⁇ -helix content (FIG.10E), and ⁇ -sheet content (FIG.10F). Dashed lines in FIG.10C and FIG.10D represent polynomial regression analyses, with a goodness-of-fit (R 2 ) of 0.80 and 0.60, respectively.
  • R 2 goodness-of-fit
  • FIGs.12B-12E depicts the relative frequency of hydrogen bonding between PFNA’s carboxylic acid (FIG.12B) and fluorine atoms (FIG.12C) with each protein’s solvent shell water molecules (H2O), backbone nitrogen and oxygen (NB, OB) and solvent accessible amino acid side chains capable of forming hydrogen bonds.
  • FIG.12D relative frequency of PFNA hydrophobic contacts with protein solvent accessible amino acids.
  • FIG.13 depicts a model of a PFNA ligand docked with GFP (PDB structure 3UFZ).
  • FIG.14H Change in molar Attorney Docket No.148411.002502 PATENT ellipticity at 216 nm ( ⁇ 216) of PFNA-treated proteins, relative to native controls, as a function of increasing ligand concentration.
  • FIG.14I Percentage change in secondary structural motifs of Hb, ⁇ -Gal and IgG between the native protein and 1mM PFNA treated samples. ⁇ -sheet structures were further differentiated into parallel and antiparallel conformations. Hb, ⁇ -Gal and IgG were prioritized for analysis based on their significant spectral shift in the presence of 1mM PFNA.
  • FIGs.14J-14L Sensograms for BSA (FIG.
  • FIG.15 depicts the melting temperature (Tm) of 5 test proteins dissolved in either PBS (Aq.) or Perfluorooctane (PFOc).
  • FIGs.16A-16C depict that the thermal stability of ⁇ -gal, GFP, and Trypsin is enhanced when the proteins are dissolved in a PFNA/PFOC mixture, compared to aqueous solutions of the proteins.
  • FIGs.17A-17B depict the uptake of GFP in human lung A549 cells after 2 and 24 hours of incubation in the absence/presence of PFNA.
  • FIGs.18A-18B depict the uptake of Cy5-labeled BSA in human lung A549 cells after 2 and 24 hours of incubation in the absence/presence of PFNA. Results are shown in relative fluorescence units (RFU).
  • FIGs.19A-19B depicts the uptake of Texas Red-labeled Transferrin in human lung A549 cells after 2 and 24 hours of incubation in the absence/presence of PFNA. Results are shown in relative fluorescence units (RFU).
  • FIGs.20A-20D depicts annexin-V/PI co-staining of A549 cells treated without or with PFNA at 2 and 24 hours.
  • FIGs.20A-20C Flow cytometry scatter plots.
  • FIG.20D quadrant data comparing the population of unaffected cells (native), to cells displaying lipid translocation (Lip. Tran.) membrane permeabilization (Mem. Perm.), or both.
  • FIG.21 depicts confocal micrographs of eGFP-PC9 cells in the absence (top) and presence (bottom) of PFNA.
  • FIGs.24A-24B show that PFOc samples remain sterile after contamination with E. coli.
  • FIG.24A Diagram of contamination experiment.
  • FIG.24B Samples in PBS (top) and PFOc (bottom) after contamination with E. coli.
  • FIG.25 depicts selected fluorochemical compounds 6 (6), 7 (7), 8 (8), 9 (9), and 10 (10), which were based off Formula II with 6, 7, 8, 9, and 10 repeats (indicated as ‘x’ in Formula II), respectively.
  • FIG.26 depicts fluorochemical compounds 21, 22, 23, and 24, which are based on Formula VIII.
  • FIGs.27A-27D depict the fluorochemical-mediated dispersion of test proteins bovine serum albumin (BSA, FIG.27A), ⁇ -Galactosidase ( ⁇ -Gal, FIG.27B), trypsin (FIG.27C), and rabbit serum immunoglobulin G (IgG, FIG.27D) into solutions containing fluorochemical compounds.
  • BSA bovine serum albumin
  • ⁇ -Galactosidase ⁇ -Gal, FIG.27B
  • trypsin FIG.27C
  • IgG rabbit serum immunoglobulin G
  • Results are displayed as percent soluble protein relative to initial loading.
  • the compound ‘0’ indicates a control sample absent of an additive.
  • FIG.28 depicts a viability assay for HepG2 cells treated with two first generation compounds and two second generation compounds.
  • FIG.29 depicts additional fluorochemical compounds 25, 26, 27, 28, and 29, based on Formula VIII.
  • FIGs.30A-30B depict additional fluorochemical compounds, based on Formula IX.
  • FIGs.31A-31B depict additional fluorochemical compounds based on Formula X.
  • FIG.32 depicts time-dependent enzymatic activity curves of ⁇ -Gal in plasma delivered systemically in either saline ( ⁇ -GalSaline) or extracted PFOc ( ⁇ -GalExt. PFOc) vehicle.
  • FIGs.34A-34B depict representative histopathologic images of lung, kidney, liver, and spleen tissue section from C57BL/6J mice 24 hours after administration of saline, ⁇ - GalSaline or ⁇ -GalExt. PFOc.
  • FIG.34B Same data as 34A, collected at 40X magnification.
  • FIG.35 depicts a structural library of protein dispersants (PD).
  • PD protein dispersants
  • FIG.36A-36D depict dispersion efficiency of (FIG.36A) bovine serum albumin (BSA), (FIG.36B) ⁇ -Galactosidase ( ⁇ -Gal), (FIG.36C) rabbit serum immunoglobulin G (IgG) and (FIG.36D) trypsin in PFOc using the indicated dispersant (PD:protein molar ratio of 1000:1). Results displayed as percent soluble protein relative to initial loading. PD-7 is highlighted via cross-hatching to aid indexing results to dispersant structure.
  • FIG.38B Indicates the concentration at which 50% inhibition of cell (IC50) occurs.
  • FIG.41A-41B represent 2 ⁇ m and 500 nm, respectively.
  • FIG.42 depicts superimposed 1 H NMR region demonstrating the downfield shift of PD-7’s -COOH protein in the absence and presence of BSA.
  • FIG.43 depicts a superimposed 1 H NMR spectra of PD-7 (60 mM) and PD-7:BSA (1000:1 molar ratio) complex. Inset shows magnified region of PD-7 -COOH proton chemical shift ( ⁇ ).
  • FIG.47B PD-2 (60 mM) and PD-2:BSA (1000:1 molar ratio) complex. Inset shows magnified region of PD-2 -CF x proton chemical shift ( ⁇ ).
  • FIG.47 depicts stacked FTIR spectra of PD-7, BSA and the PD-7:BSA complex after elution into PBS.
  • FIGs.48A-48D depict residue-specific hydrogen-bonding interactions of PD-2 (grey) and PD-7 (black) with (FIG.48A) Hb, (FIG.48B) ⁇ -Gal, (FIG.48C) GFP and (FIG.48D) trypsin.
  • FIGs.50A-50B depict the molecular model of (FIG.50A) PD-2 and (FIG.50B) PD-7 docked to the surface of BSA, with the indicated distance of representative hydrogen bonds.
  • FIG.51 depicts residue-specific hydrogen-bonding interactions of PD-2 (grey) and PD-7 (black) with BSA. Data reported as the percentage of total hydrogen bonds divided by the frequency normalized residue count in the protein.
  • FIGs.52A-52D depict the change in number of hydrogen bonds and free energy after interaction with the protein surface, as well as free energy of dispersant oligomerization, for PD-2 (grey) and PD-7 (black) over the 10 ns simulation time.
  • FIGs.52C-52D Free energy plots for dispersant interactions with Hb (a), ⁇ -Gal (b), GFP (c) and trypsin (d).
  • FIG.53 depicts representative photographs of agar plates streaked with BSA formulations in PBS (top panel) or PFOc (lower panel) contaminated with the indicated pathogen.
  • MRSA methicillin resistant Staphylococcus aureus.
  • perfluorinated compound refers to an organofluorine compound that contains only carbon-fluorine and carbon-carbon bonds and may or may not also contain heteroatoms.
  • perfluorinated compounds include, but are not limited to, perfluoroalkul substances.
  • perfluoroalkyl substance abbreviated as “PFAS,” refers to organofluorine compounds that possess C-F bonds and other heteroatom functional groups (e.g. -OH, -CO 2 H).
  • the dispersant compound is perfluorinated.
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; and the protein of interest is dissolved in the fluorinated solvent.
  • the dispersant compound interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone and amino acid side chains of the protein of interest.
  • the protein of interest comprises a non-native secondary structure.
  • the dispersant compound is perfluorinated.
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; the protein of interest is dissolved in the fluorinated solvent; the fluorinated solvent is chosen from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, or perfluorodecalin; and the perfluorinated dispersant compound is chosen from a compound that comprises: Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII.
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; the protein of interest is dissolved in the fluorinated solvent; the fluorinated solvent is chosen from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, or perfluorodecalin; and the perfluorinated dispersant compound is chosen from: perfluorohexane (Compound 0), perfluorooctane (Compound 1), perfluorotetradecane (Compound 2), trifluoroacetic acid (Compound 3), perfluoropropionic acid (Compound 4), perfluoropentanoic acid (Compound 5
  • the dispersant compound is a perfluorinated compound chosen from the group that includes: perfluorohexane (Compound 0), perfluorooctane (Compound 1), perfluorotetradecane (Compound 2), trifluoroacetic acid (Compound 3), perfluoropropionic acid (Compound 4), perfluoropentanoic acid (Compound 5), perfluorooctanoic acid (Compound 6), perfluoronanonic acid (Compound 7), perfluorodecanoic acid (Compound 8), perfluoroundecanoic acid (Compound 9), perfluorododecanoic acid (Compound 10), perfluorotetradecanoic acid (Compound 11), perfluorooctadecanoic acid (Compound 12), perfluorocyclohexanecarboxylic acid (Compound 13), (2S) 2-FMOC-amino-3-pentafluorophenyl propanoic acid (
  • the dispersant compound is perfluorinated.
  • the dispersant compound is a perfluorinated compound chosen from the group that includes: perfluorooctanoic acid (Compound 6), perfluoronanonic acid (Compound 7), perfluorodecanoic acid (Compound 8) perfluoroundecanoic acid (Compound 9), perfluorododecanoic acid (Compound 10), 2,2-(1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane)- 2-cyanoacetic acid (Compound 21), 2,2-(1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane)-2- acetic acid (Compound 22), 2,2-(1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluorodecane)-2- cyanoacetic acid (Compound 23), 2,2-(1,1,1,2,2,3,3,4,4,5,5,6,6,7,7
  • the dispersant compound is a perfluorinated compound chosen from the group that includes any compound described in any one of Tables 2-6. In some embodiments, the dispersant compound is a perfluorinated compound with a chemical formula chosen from a group that is described in Table 2. In some embodiments, the chemical formula of the perfluorinated dispersant compound is based off of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, or Formula VII. In some embodiments, the perfluorinated dispersant compound comprises the SMILES ID chosen from the group as described in Table 6. In some embodiments, the perfluorinated dispersant compound comprises a chemical name as chosen from the group that is described in Table 5.
  • the fluorinated solvent comprises Compound 0 (perfluorohexane) and the dispersant compound also comprises Compound 0.
  • Attorney Docket No.148411.002502 PATENT the protein of interest is dispersed in perfluorohexane in the presence of Compound 0.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest dose not phase separate when dispersed in perfluorohexane.
  • the protein of interest phase separates when dispersed in perfluorohexane, wherein the dispersion efficiency is about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or about 10%.
  • the dispersion efficiency is between about 1% and about 10%.
  • the dispersion efficiency is about 5%. In some embodiments, the dispersion efficiency is less than 5%.
  • the fluorinated solvent comprises Compound 0 (perfluorohexane) and the dispersant compound is chosen from the group that includes perfluorooctane (Compound 1), perfluorotetradecane (Compound 2), trifluoroacetic acid (Compound 3), perfluoropropionic acid (Compound 4), perfluoropentanoic acid (Compound 5), perfluorooctanoic acid (Compound 6/Compound 6), perfluoronanonic acid (Compound 7/Compound 7), perfluorodecanoic acid (Compound 8/Compound 8), perfluoroundecanoic acid (Compound 9/Compound 9), perfluorododecanoic acid (Compound 10/Compound 10), perfluorotetradecanoic acid (Compound 11), perfluorooctadecanoic acid (Compound 12), perfluorocyclohexanecarboxylic acid (Compound 13), (2S) 2-FMOC-a
  • the dispersant compound comprises Formula I: (Formula I), wherein the formula is (CF 2 ) V (CF 3 ) 12. In some embodiments, v is 6 (perfluorooctane). In some embodiments, v is 12 (perfluorotetradecane). In some embodiments, the dispersant compound comprises Compound 1. In some embodiments, Compound 1 comprises perfluorooctane (PFO). In some embodiments, Compound 1 comprises the formula C 8 F 18 . In some embodiments, the dispersant compound comprises Formula I ((CF2)V(CF3)2), wherein v is 6 ((CF2)6(CF3)2), and the compound is Compound 1 comprising perfluorooctane.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 1 that also comprises Attorney Docket No.148411.002502 PATENT perfluorooctane.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluoro-1-3- dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 1.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 1.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 1.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo-transferrin
  • URE urease
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 1, wherein the molar ratio of Compound 1 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 1, wherein the molar ratio of Compound 1 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 1, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 1.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 1, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, Attorney Docket No.148411.002502 PATENT 20%, or 25%.
  • the dispersion efficiency is between about 1% and about 25%. In some embodiments, the dispersion efficiency is about 15%.
  • the dispersant compound comprises Compound 2. In some embodiments, Compound 2 comprises perfluorotetradecane. In some embodiments, Compound 2 comprises the formula C14F30. In some embodiments, the dispersant compound comprises Formula I ((CF 2 ) V (CF 3 ) 2 ), wherein v is 12 ((CF 2 ) 12 (CF 3 ) 2 ), and the compound is Compound 2 comprising perfluorotetradecane.
  • perfluorotetradecane is 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14- triacontafluorotetradecane.
  • the molecular weight of Compound 2 is about 738 g/mol, or about the molecular weight of perfluorotetradecane.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 2.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 2.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 2.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 2. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 2. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 2. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 2. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 2.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 2.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight Attorney Docket No.148411.002502 PATENT of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa.
  • the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa. In some embodiments, the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 2, wherein the molar ratio of Compound 2 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 2, wherein the molar ratio of Compound 2 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 2, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 2. In some embodiments, the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 2, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%. In some embodiments, the dispersion efficiency is between about 1% and about 25%. In some embodiments, the dispersion efficiency is about 15%.
  • the dispersant compound comprises Formula II: (Formula II), wherein the formula is (CF2)X x is 0, 1, 3, 6, 7, 8, 9, 10, 12, or 16.
  • x is 0 (trifluoroacetic acid).
  • x is 1 (perfluoropropionic acid).
  • x is 3 (perfluoropentanoic acid).
  • x is 6 (perfluorooctanoic acid).
  • x is 7 (perfluoronanonic acid).
  • x is 8 (perfluorodecanoic acid).
  • x is 9 (perfluoroundecanoic acid).
  • x is 10 (perfluorododecanoic acid). In some embodiments, x is 12 (perfluorotetradecanoic acid). In some embodiments, x is 16 (perfluorooctadecanoic acid).
  • the dispersant compound comprises Compound 3. In some embodiments, Compound 3 comprises trifluoroacetic acid (TFA). In some embodiments, Compound 3 comprises the formula C 2 HF 3 O 2 .
  • the dispersant Attorney Docket No.148411.002502 PATENT compound comprises Formula II ((CF 2 ) X CF 3 COOH), wherein x is 0 ((CF 2 ) 0 CF 3 COOH), and the compound is Compound 3 comprising trifluoroacetic acid.
  • trifluoroacetic acid is 2,2,2-trifluoroacetic acid.
  • the molecular weight of Compound 3 is about 114 g/mol, or about the molecular weight of TFA.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 3.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 3.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 3.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 3. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 3. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 3. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 3. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 3.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 3.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is Attorney Docket No.148411.002502 PATENT dispersed in perfluorohexane in the presence of Compound 3, wherein the molar ratio of Compound 3 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 3, wherein the molar ratio of Compound 3 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 3, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 3.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 3, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%. In some embodiments, the dispersion efficiency is between about 1% and about 25%.
  • the dispersant compound comprises Compound 4.
  • Compound 4 comprises perfluoropropionic acid (PFPrA).
  • Compound 4 comprises the formula C3HF5O2.
  • the dispersant compound comprises Formula II ((CF2)XCF3COOH), wherein x is 1 (CF 2 CF 3 COOH), and the compound is Compound 4 comprising perfluoropropionic acid.
  • perfluoropropionic acid is 2,2,3,3,3-pentafluoropropanoic acid .
  • the molecular weight of Compound 4 is about 164 g/mol, or about the molecular weight of PFPrA.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 4.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 4.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 4. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 4. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 4. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 4. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 4.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 4.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 4.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 4, wherein the molar ratio of Compound 4 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 4, wherein the molar ratio of Compound 4 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 4, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 4.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 4, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%. In some embodiments, the dispersion efficiency is between about 1% and about 25%.
  • the dispersion efficiency is about 15%.
  • the dispersant compound comprises Compound 5.
  • Compound 5 comprises perfluoropentanoic acid (PFPeA).
  • Compound 5 comprises the formula C 5 HF 9 O 2 .
  • the dispersant compound comprises Formula II ((CF2)XCF3COOH), wherein x is 3 ((CF 2 ) 3 CF 3 COOH), and the compound is Compound 5 comprising perfluoropentanoic acid.
  • perfluoropentanoic acid is 2,2,3,3,4,4,5,5,5-nonafluoropentanoic acid.
  • the molecular weight of Compound 5 is about 264 g/mol, or about the Attorney Docket No.148411.002502 PATENT molecular weight of PFPeA.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 5.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 5. In some embodiments, the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 5. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 5. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 5. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 5.
  • the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 5.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 5.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 5.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo-transferrin
  • URE urease
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest is dispersed in perfluorohexane in the Attorney Docket No.148411.002502 PATENT presence of Compound 5, wherein the molar ratio of Compound 5 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 5, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 5.
  • the dispersant compound comprises Formula II ((CF2)XCF3COOH), wherein x is 6 ((CF2)6CF3COOH), and the compound is Compound 6 comprising perfluorooctanoic acid.
  • perfluorooctanoic acid is 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8- pentadecafluorooctanoic acid.
  • the molecular weight of Compound 6 is about 414 g/mol, or about the molecular weight of PFOA.
  • the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 6.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 6.
  • Attorney Docket No.148411.002502 PATENT In some embodiments, the protein of interest is dispersed in perfluorohexane in the presence of Compound 6.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 6, wherein the molar ratio of Compound 6 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 6, wherein the molar ratio of Compound 6 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 6, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 6.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 6, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%.
  • the dispersion efficiency is between about 1% and about 40%. In some embodiments, the dispersion efficiency is about 35%. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the protein of interest has a molecular weight of about 5 kDa to about 600 kDa. In some embodiments, the dispersion efficiency of the protein of interest is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the protein of interest has a molecular weight of about 5 kDa to about 100 kDa.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at Attorney Docket No.148411.002502 PATENT least about 35% when the protein of interest has a molecular weight of about 5 kDa to about 600 kDa. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the solvent accessible surface area (SASA) is between about 1 x10 4 ⁇ and about 15 x10 4 ⁇ .
  • SASA solvent accessible surface area
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the hydrodynamic radii of the protein of interest is between about 2 and to 8 nm. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the isoelectric point (pI) of the protein of interest is between about 4 to about 8.
  • pI isoelectric point
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the ⁇ helicity content of the protein of interest is between 0% and about 35%. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the ⁇ sheet content of the protein of interest is between 0% and about 60%.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is between about 5% to about 100% when the protein of interest contains between about 0% to about 30% polar residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is between about 35% to about 100% when the protein of interest contains between about 0% to about 30% polar residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is between about 5% to about 100% when the protein of interest contains between about 0% to about 45% nonpolar residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOA is between about 35% to about 100% when the protein of interest contains between about 0% to about 45% nonpolar residues.
  • the protein of interest dissolved in PFH in the presence of PFOA wherein the dispersant compound PFOA interacts with the protein of interest via hydrogen bonding with solvent shell H 2 O molecules interacting with the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the dispersant compound PFOA interacts with the protein of interest via hydrogen Attorney Docket No.148411.002502 PATENT bonding with nitrogen donors present in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the dispersant compound PFOA interacts with the protein of interest via hydrogen bonding with oxygen donors present in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the dispersant compound PFOA interacts with the protein of interest via hydrogen bonding with sulfur donors present in the amino acid side chains of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFOA wherein the carboxylic acid group of the dispersant compound PFOA interacts with the protein of interest via hydrogen bonding to the nitrogen, oxygen, or sulfur donors in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFOA, wherein the one or more fluorine group of the dispersant compound PFOA interacts with the protein of interest via hydrogen bonding to the nitrogen, oxygen, or sulfur donors in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the dispersant compound PFOA makes hydrophobic contacts with protein solvent accessible amino acids.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the Gibbs free energy between the PFOA and the protein of interest is between about -20 to about -50 kcal/mol.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the Gibbs free energy between the PFOA and the docked rotamers from the protein of interest is between about -20 to about -50 kcal/mol.
  • the structure of the protein of interest changes when dissolved in PFH in the presence of PFOA.
  • the protein of interest comprises a non-native secondary structure.
  • the change in molar ellipticity of the protein of interest is between about 2 deg cm -2 dmol -1 to about -6 deg cm -2 dmol -1 .
  • the change in ⁇ -sheet content is between about -20% to about 20%. In some embodiments, the change in parallel ⁇ -sheet content is between about -20% to about 20%. In some embodiments, the change in antiparallel ⁇ -sheet content is between about -20% to about 20%. In some embodiments, the change in ⁇ -helix content is between about -20% to about 20%. In some embodiments, the change in turns content is between about -20% to about 20%.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the binding of the protein to the PFOA is 1:1.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the binding of the protein to the PFOA is not 1:1. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFOA, wherein the thermal stability of the protein is increased compared to the thermal stability of the protein of interest dissolved in an aqueous solvent. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFOA, wherein the melting temperature of the protein is increased by about 5 °C, 10 °C, 20 °C, 30 °C, or 40 °C compared to the melting temperature of the protein of interest in an aqueous solvent.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the protein of interest is active at 25 °C and is also active at 75 °C, 80 °C, 85 °C, or 90 °C. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFOA, wherein the activity is reduced by about 1% to about 25% when the temperature is about 75 °C, 80 °C, 85 °C, or 90 °C, compared to when the temperature is about 25 °C.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the activity is increased by about 1% to about 25% when the temperature is about 75 °C, 80 °C, 85 °C, or 90 °C, compared to when the temperature is about 25 °C.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the protein comprises a reduced risk of contamination compared to when the protein is dissolved in an aqueous solvent.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein the protein comprises a reduced risk of bacterial or viral contamination compared to when the protein is dissolved in PBS.
  • the protein of interest is dissolved in PFH in the presence of PFOA, wherein Attorney Docket No.148411.002502 PATENT the protein comprises a reduced risk of E. coli contamination compared to when the protein is dissolved in PBS.
  • the dispersant compound comprises Compound 7.
  • Compound 7 comprises perfluoronanonic acid (PFNA).
  • PFNA perfluoronanonic acid
  • Compound 7 comprises the formula C9HF17O2.
  • the dispersant compound comprises Formula II ((CF 2 ) X CF 3 COOH), wherein x is 7 ((CF 2 ) 7 CF 3 COOH), and the compound is Compound 7 comprising perfluoronanonic acid.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 7.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 7.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 7.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 7. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 7. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 7. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 7. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 7.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 7.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa.
  • the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa. In some embodiments, the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa.
  • the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa. In some embodiments, the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 7, wherein the molar ratio of Compound 7 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 7, wherein the molar ratio of Compound 7 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 7, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 7. In some embodiments, the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 7, wherein the dispersion efficiency is about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the dispersion efficiency is about 50%. In some embodiments, the dispersion efficiency is about 55%. In some embodiments, the dispersion efficiency is about 60%.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the protein of interest has a molecular weight of about 5 kDa to about 600 kDa. In some embodiments, the dispersion efficiency of the protein of interest is at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the protein of interest has a molecular weight of about 5 Attorney Docket No.148411.002502 PATENT kDa to about 100 kDa.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 55% to about 100 when the protein of interest has a molecular weight of about 5 kDa to about 500 kDa. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the solvent accessible surface area (SASA) is between about 1 x10 4 ⁇ and about 15 x10 4 ⁇ .
  • SASA solvent accessible surface area
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is at least about 70%, 80%, or 90% when the hydrodynamic radii of the protein of interest is between about 7 to about 8 nm. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 30% to about 100% when the hydrodynamic radii of the protein of interest is between about 2 and to 8 nm. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 50% to about 100% when the hydrodynamic radii of the protein of interest is between about 2 to about 4 nm.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to about 15% nonpolar residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% nonpolar residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to about 15% tyrosine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to about 15% tryptophan residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to about 15% proline residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 2% to about 10% glycine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 2% to about 15% alanine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 4% to about 12% valine residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 4% to about 14% leucine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to Attorney Docket No.148411.002502 PATENT about 6% isoleucine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0.25% to about 3% methionine residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 1.5% to about 6% phenylalanine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 1% to about 5% tyrosine residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 0% to about 5% tryptophan residues.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFNA is between about 40% to about 100% when the protein of interest contains between about 3% to about 8% proline residues.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with solvent shell H 2 O molecules interacting with the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone of the protein of interest, wherein the amino acid comprises serine, threonine, cysteine, methionine, tyrosine, tryptophan, aspartate, glutamate, asparagine, glutamine, histidine, leucine, or arginine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with oxygen donors present in the protein backbone of the protein of Attorney Docket No.148411.002502 PATENT interest, wherein the amino acid comprises arginine, histidine, lysine, aspartate, glutamate, asparagine, glutamine, serine, threonine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.
  • the amino acid comprises arginine, histidine, lysine, aspartate, glutamate, asparagine, glutamine, serine, threonine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalan
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with oxygen donors present in the protein backbone of the protein of interest, wherein the amino acid comprises serine, threonine, cysteine, methionine, tyrosine, tryptophan, aspartate, glutamate, asparagine, glutamine, histidine, leucine, or arginine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the amino acid side chains of the protein of interest wherein the amino acid comprises tryptophan, asparagine, glutamine, histidine, or arginine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone and amino acid side chains of the protein of interest, wherein the amino acid comprises tryptophan, asparagine, glutamine, histidine, or arginine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with oxygen donors present in the amino acid side chains of the protein of interest, wherein the amino acid comprises serine, threonine, tyrosine, aspartate, glutamate, asparagine, or glutamine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with oxygen donors present in the protein backbone and amino acid side chains of the protein of interest, wherein the amino acid comprises serine, threonine, tyrosine, aspartate, glutamate, asparagine, or glutamine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding with sulfur donors present in the amino acid side chains of the protein of interest, wherein the amino acid comprises cysteine or methionine.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein the carboxylic acid group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the H2O molecules interacting with the protein of interest.
  • the protein of interest dissolved in PFH in the presence of Attorney Docket No.148411.002502 PATENT PFNA wherein the carboxylic acid group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the nitrogen donors present in the protein backbone of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein the carboxylic acid group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the oxygen donors present in the amino acid side chains of the protein of interest.
  • the amino acid is serine, threonine, tyrosine, aspartate, or glutamate.
  • the protein of interest dissolved in PFH in the presence of PFNA, wherein the carboxylic acid group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the nitrogen donors present in the amino acid side chains of the protein of interest.
  • the amino acid is histidine.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein the carboxylic acid group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the sulfur donors present in the amino acid side chains of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein one or more fluorine group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the H 2 O molecules interacting with the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein one or more fluorine group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the nitrogen donors present in the protein backbone of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFNA wherein one or more fluorine group of the dispersant compound PFNA interacts with the protein of interest via hydrogen bonding to the oxygen donors present in the amino acid side chains of the protein of interest.
  • the amino acid is serine, threonine, tyrosine, asparagine, or glutamine.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA makes hydrophobic contacts with protein solvent accessible amino acids.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the dispersant compound PFNA makes hydrophobic contacts with protein solvent accessible amino acids, wherein the solvent accessible amino acid is G, A, S, T, C, V, L, I, M, P, F, Y, W, D, E, N, Q, H, L, or R.
  • the solvent accessible amino acid is G, A, S, T, V, L, I, P, F, Y, W, D, E, N, Q, H, L, or R. In some embodiments, the solvent accessible amino acid is S, T, Y, D, H, L, or R.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the Gibbs free energy between the PFNA and the protein of interest is between about -20 to about -50 kcal/mol.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the Gibbs free energy between the PFNA and the docked rotamers from the protein of interest is between about -20 to about -50 kcal/mol.
  • the structure of the protein of interest changes when dissolved in PFH in the presence of PFNA.
  • the protein of interest comprises a non-native secondary structure.
  • the change in molar ellipticity of the protein of interest is between about 2 deg cm -2 dmol -1 to about -6 deg cm -2 dmol -1 .
  • the change in molar ellipticity of the protein of interest is between about 1 deg cm -2 dmol -1 to about -1 deg cm -2 dmol -1 . In some embodiments, the molar ellipticity of the protein of interest decreases by about -1 deg cm -2 dmol -1 to about -6 deg cm -2 dmol -1 . In some embodiments, the molar ellipticity of the protein of interest increases by about 1 deg cm -2 dmol -1 to about 2 deg cm -2 dmol -1 . In some embodiments, the change in ⁇ -sheet content is between about -20% to about 20%.
  • the protein of interest is active in an aqueous solvent at 25 °C, but the activity is reduced when the protein of interest is dissolved in an aqueous solvent at 75 °C, 80 °C, 85 °C, or 90 °C. In some embodiments, the activity is reduced by about 80% or by about 90%. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFNA, wherein the protein of interest is active at 25 °C and is also active at 75 °C, 80 °C, 85 °C, or 90 °C.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the activity is reduced by about 1% to about 25% when the temperature is about 75 °C, 80 °C, 85 °C, or 90 °C, compared to when the temperature is about 25 °C. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFNA, wherein the activity is reduced by about 1% to about 10% when the temperature is about 75 °C, 80 °C, 85 °C, or 90 °C, compared to when the temperature is about 25 °C.
  • the protein of interest is dissolved in PFH in the presence of PFNA, wherein the protein comprises a reduced risk of contamination compared to when the protein is dissolved in an aqueous solvent. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFNA, wherein the protein comprises a reduced risk of bacterial or viral contamination compared to when the protein is dissolved in PBS. In some Attorney Docket No.148411.002502 PATENT embodiments, the protein of interest is dissolved in PFH in the presence of PFNA, wherein the protein comprises a reduced risk of E. coli contamination compared to when the protein is dissolved in PBS. In some embodiments, the dispersant compound comprises Compound 8.
  • Compound 8 comprises Formula II ((CF2)XCF3COOH), wherein x is 8 ((CF2)8CF3COOH), and the compound is Compound 8 comprising perfluorodecanoic acid.
  • Compound 8 is Compound 8.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 8.
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, Attorney Docket No.148411.002502 PATENT 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa.
  • the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa. In some embodiments, the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa.
  • the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa. In some embodiments, the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 8, wherein the molar ratio of Compound 8 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 8, wherein the molar ratio of Compound 8 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 8, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 8. In some embodiments, the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 8, wherein the dispersion efficiency is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or about 80%. In some embodiments, the dispersion efficiency is between about 10% and about 80%. In some embodiments, the dispersion efficiency is about 80%. In some embodiments, the dispersion efficiency is about 50%.
  • the molecular weight of Compound 9 is about 564 g/mol, or about the molecular weight of PFUnDA.
  • Compound 9 comprises Formula II ((CF2)XCF3COOH), wherein x is 9 ((CF2)9CF3COOH), and the compound is Compound 9 comprising perfluoroundecanoic acid.
  • Compound 9 is Compound 9.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 9. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 9. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 9. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 9. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 9.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 9.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 9.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 9, wherein the molar ratio of Compound 9 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the Attorney Docket No.148411.002502 PATENT presence of Compound 9, wherein the molar ratio of Compound 9 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 9, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 9.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 9, wherein the dispersion efficiency is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or about 80%. In some embodiments, the dispersion efficiency is between about 5% and about 80%. In some embodiments, the dispersion efficiency is about 80%. In some embodiments, the dispersion efficiency is about 50%. In some embodiments, the dispersion efficiency is about 25%. In some embodiments, the dispersant compound comprises Compound 10. In some embodiments, Compound 10 comprises perfluorododecanoic acid.
  • Compound 10 comprises the formula C12HF23O2.
  • the dispersant compound comprises Formula II ((CF2)X(CF3)2COOH), wherein x is 10 ((CF2)10CF3COOH), and the compound is Compound 10 comprising perfluorododecanoic acid.
  • perfluorododecanoic acid is 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,12-tricosafluorododecanoic acid.
  • the molecular weight of Compound 10 is about 614 g/mol, or about the molecular weight of perfluorododecanoic acid.
  • Compound 10 comprises Formula II ((CF2)X(CF3)2COOH), wherein x is 10 ((CF2)10CF3COOH), and the compound is Compound 10 comprising perfluorododecanoic acid.
  • Compound 10 is Compound 10.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 10.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 10. In some embodiments, the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 10. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 10. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 10. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 10.
  • the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant Attorney Docket No.148411.002502 PATENT compound comprises Compound 10.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 10.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 10.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo- transferrin
  • URE urease
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 11. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 11. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 11. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 11. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 11.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 11.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 11, wherein the molar ratio of Compound 11 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 11, wherein the molar ratio of Compound 11 is about 1000:1 with the protein of interest.
  • perfluorooctadecanoic acid is 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18- pentatriacontafluorooctadecanoic acid.
  • the molecular weight of Compound 12 is about 914 g/mol, or about the molecular weight of perfluorooctadecanoic acid.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 12.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 12.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 12.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 12.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 12.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the dispersant compound comprises Formula III: (Formula III). In some the formula C 6 F 11 COOH. In some embodiments, Formula III is acid. In some embodiments, Compound 13 comprises perfluorocyclohexanecarboxylic acid. In some embodiments, Compound 13 comprises the formula C7HF11O2. In some embodiments, the dispersant compound comprises Formula III (C 6 F 11 COOH), and the compound is Compound 13 comprising perfluorocyclohexanecarboxylic acid.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 13.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 13.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 13.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 13. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 13. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 13. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 13. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 13.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 13.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- Attorney Docket No.148411.002502 PATENT transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 13, wherein the molar ratio of Compound 13 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 13, wherein the molar ratio of Compound 13 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 13, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 13.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 13, wherein the dispersion efficiency is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or about 80%.
  • the dispersion efficiency is between about 5% and about 80%. In some embodiments, the dispersion efficiency is about 80%. In some embodiments, the dispersion efficiency is about 25%. In some embodiments, the dispersion efficiency is about 10%.
  • the dispersant compound comprises Formula IV: (Formula IV). In some embodiments, Formula IV comprises (2S) 2-FMOC-amino-3- pentafluorophenyl propanoic acid. In some embodiments, Formula IV comprises an 9- fluorenylmethyloxycarbonyl (FMOC) protecting group. In some embodiments, Formula IV Attorney Docket No.148411.002502 PATENT comprises Fmoc-L-pentafluorophenylalanine.
  • Formula IV comprises (2S)-2-( ⁇ [(9H-fluoren-9-yl)methoxy]carbonyl ⁇ amino)-3-(2,3,4,5,6- pentafluorophenyl)propanoic acid.
  • the dispersant compound comprises Compound 14.
  • Compound 14 comprises (2S)-2-( ⁇ [(9H- fluoren-9-yl)methoxy]carbonyl ⁇ amino)-3-(2,3,4,5,6-pentafluorophenyl)propanoic acid.
  • Compound 14 comprises the formula C 24 H 16 F 5 NO 4 .
  • the dispersant compound comprises Formula 14 (C24H16F5NO4), wherein and the compound is Compound 14 comprising (2S)-2-( ⁇ [(9H-fluoren-9- yl)methoxy]carbonyl ⁇ amino)-3-(2,3,4,5,6-pentafluorophenyl)propanoic acid.
  • the molecular weight of Compound 14 is about 477 g/mol, or about the molecular weight of (2S)-2-( ⁇ [(9H-fluoren-9-yl)methoxy]carbonyl ⁇ amino)-3-(2,3,4,5,6- pentafluorophenyl)propanoic acid.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 14.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 14.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 14.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 14. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 14. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 14. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 14. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 14.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 14.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of about 27 Attorney Docket No.148411.002502 PATENT kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa. In some embodiments, the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 14, wherein the molar ratio of Compound 14 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 14, wherein the molar ratio of Compound 14 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 14, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 14. In some embodiments, the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 14, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, the dispersion efficiency is between about 1% and about 10%. In some embodiments, the dispersion efficiency is about 5%.
  • the dispersant compound comprises Formula V: (Formula V), wherein the formula is C 6 F 5 R COH. In some embodiments, R is OH and the compound is pentafluorophenol. In some embodiments, R is COH and the compound is pentafluorobenzaldehyde. In some embodiments, the dispersant compound comprises Compound 15. In some embodiments, Compound 15 comprises pentafluorophenol. In some embodiments, Compound 15 comprises the formula C6HF5O. In some embodiments, the dispersant compound comprises Formula V (C6F5R), where R is OH (C6F5OH), and the compound is Compound 15 comprising pentafluorophenol.
  • pentafluorophenol is 2,3,4,5,6-pentafluorophenol.
  • the molecular weight of Compound 15 is Attorney Docket No.148411.002502 PATENT about 184 g/mol, or about the molecular weight of pentafluorophenol.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro- 1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 15.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 15. In some embodiments, the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 15. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 15. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 15. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 15.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo- transferrin
  • URE urease
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 15, wherein the molar ratio of Compound 15 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the Attorney Docket No.148411.002502 PATENT protein of interest.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 16.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 16.
  • the protein of interest can include, but is Attorney Docket No.148411.002502 PATENT not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 16, wherein the molar ratio of Compound 16 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 17.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the dispersion efficiency is between about 5% and about 60%. In some embodiments, the dispersion efficiency is about 60%. In some embodiments, the dispersion efficiency is about 25%. In some embodiments, the dispersion efficiency is about 10%. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the protein of interest has a molecular weight of about 5 kDa to about 600 kDa.
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the solvent accessible surface area (SASA) is between about 1 x10 4 ⁇ and about 15 x10 4 ⁇ . In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the hydrodynamic radii of the protein of interest is between about 2 and to 8 nm.
  • SASA solvent accessible surface area
  • the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when the ⁇ sheet content of the protein of interest is between 0% and about 60%. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is between about 5% to about 100% when the protein of interest contains between about 0% to about 30% polar residues. In some embodiments, the dispersion efficiency of the protein of interest dissolved in PFH in the presence of PFOS is between about 35% to about 100% when the protein of interest contains between about 0% to about 30% polar residues.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the dispersant compound PFOS interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the dispersant compound PFOS interacts with the protein of interest via hydrogen bonding with oxygen donors present in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFOS wherein the carboxylic acid group of the Attorney Docket No.148411.002502 PATENT dispersant compound PFOS interacts with the protein of interest via hydrogen bonding to the nitrogen, oxygen, or sulfur donors in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFOS wherein the one or more fluorine group of the dispersant compound PFOS interacts with the protein of interest via hydrogen bonding to the H2O molecules interacting with the protein of interest.
  • the protein of interest dissolved in PFH in the presence of PFOS, wherein the one or more fluorine group of the dispersant compound PFOS interacts with the protein of interest via hydrogen bonding to the nitrogen, oxygen, or sulfur donors in the protein backbone or the amino acid side chains of the protein of interest.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the dispersant compound PFOS makes hydrophobic contacts with protein solvent accessible amino acids.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the Gibbs free energy between the PFOS and the protein of interest is between about -20 to about -50 kcal/mol.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the Gibbs free energy between the PFOS and the docked rotamers from the protein of interest is between about -20 to about -50 kcal/mol.
  • the structure of the protein of interest changes when dissolved in PFH in the presence of PFOS.
  • the protein of interest comprises a non-native secondary structure.
  • the change in molar ellipticity of the protein of interest is between about 2 deg cm -2 dmol -1 to about -6 deg cm -2 dmol -1 .
  • the change in ⁇ -sheet content is between about -20% to about 20%.
  • the change in parallel ⁇ -sheet content is between about -20% to about 20%. In some embodiments, the change in antiparallel ⁇ -sheet content is between about -20% to about 20%. In some embodiments, the change in ⁇ -helix content is between about -20% to about 20%. In some embodiments, the change in turns content is between about -20% to about 20%.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the binding of the protein to the PFOS is 1:1. In some embodiments, the protein of interest is dissolved in PFH in the presence of PFOS, wherein the binding of the protein to the PFOS is not 1:1.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the thermal stability of the protein is increased compared to the thermal stability of the protein of interest dissolved in an aqueous solvent.
  • the protein of interest is dissolved in PFH in the presence of PFOS, wherein the melting Attorney Docket No.148411.002502 PATENT temperature of the protein is increased by about 5 °C, 10 °C, 20 °C, 30 °C, or 40 °C compared to the melting temperature of the protein of interest in an aqueous solvent.
  • the protein of interest is active in an aqueous solvent at 25 °C, but is not active in an aqueous solvent at 75 °C, 80 °C, 85 °C, or 90 °C. In some embodiments, the protein of interest is active in an aqueous solvent at 25 °C, but the activity is reduced when the protein of interest is dissolved in an aqueous solvent at 75 °C, 80 °C, 85 °C, or 90 °C. In some embodiments, the activity is reduced by about 80% or by about 90%.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 23, wherein the dispersion efficiency is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
  • the dispersion efficiency is between about 5% and about 100%.
  • the dispersion efficiency is about 100%.
  • the dispersion efficiency is at least about 80%.
  • the Attorney Docket No.148411.002502 PATENT dispersion efficiency is at least about 50%.
  • the dispersion efficiency is at least about 25%.
  • Compound 24 comprises Formula VIII: (Formula VIII), wherein the formula is C 5 H 8 wherein R 1 is C 8 F 17 , and wherein R 2 is H.
  • the Compound 24 comprises a chemical group R1_Extended, wherein R1_Extended is C 10 H 5 F 17 .
  • the Compound 24 comprises two chemical groups R1_Extended, wherein R1_Extended is C10H5F17.
  • the Compound 24 comprises two chemical groups R1_Extended, wherein R1_Extended is C10H5F17, and additionally comprises the group R2, wherein R2 is H.
  • Compound 24 comprises Formula VIII_B: (Formula VIII_B), wherein the formula is 2(R2)COOH, wherein R1_Extended is C10H5F17 and wherein R 2 is H.
  • Compound 24 comprises the chemical formula 2,2- (R1_Extended)-2-acetic acid, wherein R1_Extended is the chemical group 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluorodecane.
  • Compound 24 comprises 2,2-(1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluorodecane)-acetic acid.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo- transferrin
  • URE urease
  • Compound 25 comprises Formula VIII: (Formula VIII), wherein the formula is C 5 H 8 wherein R 1 is C 4 F 9 , and wherein R 2 is CN.
  • the Compound 25 comprises a chemical group R1_Extended, wherein R1_Extended is C 6 H 5 F 9 .
  • the Compound 25 comprises two chemical groups R1_Extended, wherein R1_Extended is C6H5F9.
  • the Compound 25 comprises two chemical groups R1_Extended, wherein R1_Extended is C6H5F9, and additionally comprises the group R2, wherein R2 is CN.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 25, wherein the dispersion efficiency is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
  • the dispersion efficiency is between about 5% and about 100%.
  • the dispersion efficiency is about 100%.
  • the dispersion efficiency is at least about 80%.
  • the dispersion efficiency is at least about 50%.
  • the dispersion efficiency is at least about 25%.
  • the dispersion efficiency is at least about 15%.
  • the dispersant compound comprises Compound 26.
  • the Compound 26 comprises a chemical group R1, wherein R1 is C 4 F 9 .
  • the Compound 26 comprises two chemical groups R1, wherein R1 is C4F9.
  • the Compound 26 comprises the group R2, wherein R2 is CN.
  • the Compound 26 comprises two chemical groups R1, wherein R1 is C4F9, and additionally comprises the group R2, wherein R2 is H.
  • Compound 26 comprises Formula VIII: (Formula VIII), wherein the formula is C 5 H 8 wherein R 1 is C 4 F 9 , and wherein R 2 is H.
  • the Compound 26 comprises a chemical group R1_Extended, wherein R1_Extended is C6H5F9. In some embodiments, the Compound 26 comprises two chemical groups R1_Extended, wherein R1_Extended is C 6 H 5 F 9 . In some embodiments, the Compound 26 comprises two chemical groups R1_Extended, wherein R1_Extended is C 6 H 5 F 9 , and additionally comprises the group R 2 , wherein R 2 is H. In some embodiments, Compound 26 comprises Formula VIII_B: (Formula VIII_B), wherein the formula is 2(R2)COOH, wherein R1_Extended is C6H5F9 and wherein R2 is H.
  • the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 26, wherein the molar ratio of Compound 26 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, Attorney Docket No.148411.002502 PATENT 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 26, wherein the molar ratio of Compound 26 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 26, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 26.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 26, wherein the dispersion efficiency is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
  • the dispersion efficiency is between about 5% and about 100%.
  • the dispersion efficiency is about 100%.
  • the dispersion efficiency is at least about 80%.
  • the dispersion efficiency is at least about 50%.
  • the dispersion efficiency is at least about 25%.
  • the dispersion efficiency is at least about 15%.
  • the dispersant compound comprises Compound 27.
  • the Compound 27 comprises a chemical group R1, wherein R1 is C4F9. In some embodiments, the Compound 27 comprises two chemical groups R1, wherein R1 is C4F9. In some embodiments, the Compound 27 comprises the group R2, wherein R2 is CN. In some embodiments, the Compound 27 comprises two chemical groups R1, wherein R1 is C4F9, and additionally comprises the group R2, wherein R2 is COOH. In some embodiments, Compound 27 comprises Formula VIII: (Formula VIII), wherein the formula is C5H8 wherein R1 is C4F9, and wherein R2 is COOH.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Compound 27.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 27.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 27.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 27. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 27. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 27. In some embodiments, the fluorinated solvent is perfluoro-1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Compound 27. In some embodiments, the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Compound 27.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 27.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo- transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa. In some embodiments, the protein of interest has a molecular weight of about 464 kDa, 483 kDa, or 520 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 27, wherein the molar ratio of Compound 27 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 27, wherein the molar ratio of Compound 27 is about 1000:1 with the protein of interest.
  • the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa. In some embodiments, the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 28, wherein the molar ratio of Compound 28 is about 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1250:1, 1500:1, 1750:1, 2000:1, 2500:1, 3000:1, 4000:1, or 5000:1 with the protein of interest.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 28, wherein the molar ratio of Compound 28 is about 1000:1 with the protein of interest.
  • the dispersion efficiency is at least about 80%. In some embodiments, the dispersion efficiency is at least about 50%. In some embodiments, the dispersion efficiency is at least about 25%. In some embodiments, the dispersion efficiency is at least about 15%.
  • the dispersant compound comprises Compound 29. In some embodiments, the Compound 29 comprises a chemical group R1, wherein R1 is C8F17. In some embodiments, the Compound 29 comprises two chemical groups R1, wherein R1 is C 8 F 17 . In some embodiments, the Compound 29 comprises the group R 2 , wherein R 2 is CN.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Compound 29. In some embodiments, the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Compound 29. In some embodiments, the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Compound 29. In some embodiments, the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Compound 29. In some embodiments, the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Compound 29.
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 29, wherein the molar ratio of Compound 29 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 29, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 29.
  • the dispersant compound comprises Formula IX, wherein R 1 is COOH, wherein R 2 is the lysosomal targeting group C7H15N2O, wherein R3 is C8F17, and wherein R4 is H.
  • the dispersant compound comprises Compound 30: (Compound 30).
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; the protein of interest is dissolved in the fluorinated solvent; the fluorinated solvent is chosen from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, or perfluorodecalin; and the perfluorinated dispersant compound comprises Formula IX, wherein the compound is Attorney Docket No.148411.002502 PATENT Compound 30 comprising the chemical formula that can be derived from the SMILES ID from Table 6.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3- dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Formula IX, wherein the compound is Compound 30.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluoro- 1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Formula IX.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Formula IX.
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; the protein of interest is dissolved in the fluorinated solvent; the fluorinated solvent is chosen from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, or perfluorodecalin; and the perfluorinated dispersant comprises Formula X: Attorney Docket No.148411.002502 PATENT (Formula X), wherein R1 is CN, H, or targeting group C7H15N2O, wherein R3 is C4F9, C6F13, C8F17, and wherein R4 is CN, H, or COOH.
  • Formula X Attorney Docket
  • a composition comprising a fluorinated solvent, a perfluorinated dispersant compound, and a protein of interest, wherein: the perfluorinated dispersant compound interacts with the protein of interest; the protein of interest is dissolved in the fluorinated solvent; the fluorinated solvent is chosen from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, or perfluorodecalin; and the perfluorinated dispersant compound comprises Formula X, wherein the compound is Compound 31 comprising the chemical formula that can be derived from the SMILES ID from Table 6.
  • the fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3-dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Formula X.
  • the Attorney Docket No.148411.002502 PATENT fluorinated solvent is chosen from the group that includes perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoromethylcyclohexane, perfluoro-1-3- dimethylcyclohexane, or perfluorodecalin, and the perfluorinated dispersant compound comprises Formula X, wherein the compound is Compound 31.
  • the fluorinated solvent is perfluorohexane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluorooctane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluoropentane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluoroheptane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluoromethylcyclohexane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluoro- 1-3-dimethylcyclohexane and the perfluorinated dispersant compound comprises Formula X.
  • the fluorinated solvent is perfluorodecalin and the perfluorinated dispersant compound comprises Formula X.
  • the protein of interest is dispersed in perfluorohexane in the presence of a compound comprising Formula IX.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, or 200 kDa. In some embodiments, the protein of interest has a molecular weight of about 150 kDa. In some embodiments, the protein of interest has a molecular weight of at least 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, or 600 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of a compound comprising Formula IX, wherein the molar ratio of a compound comprising Formula IX is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of a compound comprising Formula IX, but does not phase separate when dispersed in perfluorohexane in the absence of a compound comprising Formula IX.
  • the protein of interest can include, but is not limited to, human hemoglobin (Hb), green fluorescent protein (GFP), bovine serum albumin (BSA), ⁇ -Galactosidase ( ⁇ -Gal), rabbit serum immunoglobulin (IgG), bovine apo-transferrin (TFN), or urease (URE).
  • Hb human hemoglobin
  • GFP green fluorescent protein
  • BSA bovine serum albumin
  • IgG rabbit serum immunoglobulin
  • TNF bovine apo-transferrin
  • URE urease
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest has a molecular weight of at least 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, or 100 kDa. In some embodiments, the protein of interest has a molecular weight of about 27 kDa, 64.5 kDa, 66.5 kDa, 76 kDa, or 81 kDa.
  • the protein of interest is dispersed in perfluorohexane in the presence of Compound 30, wherein the molar ratio of Compound 30 is about 1000:1 with the protein of interest.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 30, but does not phase separate when dispersed in perfluorohexane in the absence of Compound 30.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of Compound 30, wherein the dispersion efficiency is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, Attorney Docket No.148411.002502 PATENT 85%, 90%, 95%, or about 100%. In some embodiments, the dispersion efficiency is between about 5% and about 100%. In some embodiments, the dispersion efficiency is at least about 10%. In some embodiments, the protein of interest is dispersed in perfluorohexane in the presence of Compound 31.
  • the protein of interest phase separates when dispersed in perfluorohexane in the presence of the dispersion compound comprising the formula of Formula: I, II, III, IV, V, VI, VII, VIII, IX, or X, wherein the dispersion efficiency is about 100%. In some embodiments, the protein of interest phase separates when dispersed in perfluorohexane in the presence of the dispersion compound comprising the formula of Formula: I, II, III, IV, V, VI, VII, VIII, IX, or X, wherein the dispersion efficiency is about at least 80%.
  • composition of any proceeding embodiment, wherein the dispersant compound interacts with the protein of interest via hydrogen bonding with nitrogen donors present in the protein backbone and amino acid side chains of the protein of interest.
  • Embodiment 14 The composition of any proceeding embodiments, wherein the protein of interest comprises a non-native secondary structure.
  • Embodiment 15 The composition of embodiment 1, wherein the fluorinated solvent is perfluorohexane and the dispersant compound is perfluoronanonic acid.
  • Embodiment 16 The composition of embodiment 1, wherein the fluorinated solvent is perfluorooctane and the dispersant compound comprises Formula VIII: Attorney Docket No.148411.002502 PATENT (Formula VIII), wherein R 1 is C 6 F 13 Embodiment 16.
  • the second is a distinct optimum of the tail length at eight perfluorocarbons (FIG.9A).
  • PFNA perfluorocarbons
  • ⁇ -Gal which showed a stepwise increase in dispersion efficiency as the perfluorocarbon tail was increased from -(CF)7CF3 (compound 7) to -(CF) 10 CF 3 (compound 10).
  • a sharp drop off in dispersion efficiency was observed at - (CF)12CF3 (compound 11).
  • PFNA hydrogen bonding frequency of PFNA’s carboxyl group (FIG.12B) and aliphatic fluorines (FIG.12C) with the solvent shell, backbone, and surface amino acid side chains of each test protein.
  • results in FIG.12B demonstrated that PFNA’s carboxyl group (hydrogen bond donor/acceptor) markedly favored hydrogen bonding with protein backbone oxygens (acceptor), that ranged in relative frequencies from 0.30 for BSA to 0.86 for Hb. Apart from tyrosine in the case of BSA, amino acid side chains infrequently formed hydrogen bonds with PFNA’s carboxyl group ( ⁇ 0.20).
  • Results in FIG.12C showed a complementary analysis for hydrogen bonding of fluorine atoms in PFNA’s perfluorinated tail. Compared to oxygen, fluorine is a relatively poor hydrogen bond acceptor due to its high electronegativity and low polarizability.
  • Additional perfluorinated small molecule compounds containing FTags were selected (FIG.26) and tested against select first generation perfluorinated small molecule compounds Attorney Docket No.148411.002502 PATENT (FIG.25).
  • the second-generation compounds each contained an FTag that contained two R 1 groups and one R 2 group.
  • the R 1 group was C 6 F 13 for compound 21 and compound 22 and was C8F17 for compound 23 and compound 24.
  • the R2 group was CN for compound 21 and compound 23 and was H for compound 22 and compound 24.
  • the FTag compounds were dissolved in PFOc (perfluorooctane) and added to dried protein to achieve a final protein:FTag molar ratio of 1:1000 (1uM:1mM).
  • Dispersion efficiency was about the same between the first and second generation perfluorinated small molecule compounds when mixed with ⁇ -gal (FIG.27B).
  • the second generation perfluorinated small molecule compounds had similar or greater dispersion efficiency when mixed with trypsin compared to PFNA (FIG.27C).
  • Dispersion efficiency was about the same between the first and second generation perfluorinated small molecule compounds when mixed with rabbit IgG (FIG. 27D).
  • viability assays were performed by seeding HepG2 (liver cancer cell line) onto 96 well plates and allowing the cells to adhere overnight. DMEM cell culture media containing various concentrations of each FTag compound were then added, and the plates were incubated at 37 °C overnight.
  • Kidneys were examined for signs of necrosis, cellular infiltration, and hemorrhage. Livers were examined for hepatic cell necrosis, inflammation, and hemorrhage. Spleens were examined for changes in white and red pulp structure, as well as signs of abnormal cellular infiltration. Serological hematologic, renal, and hepatic toxicology screens showed statistically significant changes in blood urea nitrogen (BUN), red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) between ⁇ -Gal delivered from PFOc versus saline (FIG.33).
  • BUN blood urea nitrogen
  • RBC red blood cell count
  • HGB hemoglobin
  • HCT hematocrit
  • Example 4 Development and Validation of Optimized Perfluorchemical Dispersion Reagents for Extremophilic Protein Formulations Introduction
  • perfluorochemical additives allow proteins to disperse in non- aqueous perfluorocarbon (PFC) liquids, maintaining their structure and function even at high temperatures, and providing resistance to bacterial, fungal, and proteolytic contamination.
  • PFC perfluorocarbon
  • Disclosed herein is an optimized family of perfluorochemical dispersion reagents with enhanced PFOc solubilization efficiencies and biocompatibility. Using a multi-faceted approach, compound PD-7 was demonstrated to show superior performance, sterility, and safety compared to PFNA.
  • PD-7 separates from the protein surface in physiological solutions, reducing the risk of tissue bioaccumulation of fluorinated reagents, addressing concerns over the toxicity of perfluoroalkyl substances.
  • the single perfluorinated tail containing protein dispersants (PD-1 to PD-5) were procured commercially, whereas bivalent perfluorinated derivatives (PD-6 to PD-11) were synthesized through modification of reported protocols.
  • This cell line was chosen as liver is the primary site of metabolism and toxicity for many perfluorinated amphiphiles.
  • PD-7 was the least toxic, with an IC50 ⁇ 1.0 mM. Thus, PD-7 was prioritized as the lead candidate for further development of thermally stabile protein dispersions, discussed below.
  • Evaluation of Thermally Induced Structural Changes of PD-7 To determine structural integrity of proteins in aqueous and fluorinated environments at elevated temperatures, circular dichroism (CD) was used to probe secondary structure. Proteins were either diluted in PBS (aqueous) or dispersed in PFOc at the same designated concentrations according to protocol described above.
  • samples were subjected to elevated temperature condition (90 °C) well beyond their native denaturation temperature for 30 minutes. After that, samples were Attorney Docket No.148411.002502 PATENT allowed to cool down to room temperature before retrieval.
  • proteins were extracted into PBS before performing the ensuing assays.
  • an equal volume of protein samples was added to their substrates dissolved in PBS (4 mg/mL ONPG for ⁇ -Gal or 1 mg/mL BAEE for Trypsin). After incubation, ONPG and BAEE substrate conversions were measured by taking absorbance using a microplate reader at 420 nm or 400 nm, respectively.
  • FTIR Fourier transform infrared
  • FTIR spectrometry was then performed on powdered samples using LN-MCT detector (4000 – 800 cm-1, backward output). Data is represented by average +- SD of wavenumber among replicates depicting corresponding characteristic bonds.
  • An equal volume of PBS was added to the BSA:PD-7 sample in PFOc, and vortexing the phase separated mixture for ⁇ 2 seconds.
  • PD-2 in contrast, favors hydrogen bonding with the protein surface, rather than self-assembly, leading to the formation of dispersant islands that leave large areas exposed to the fluorous solvent.
  • the weak van der Waals interactions between PD-7 and protein surfaces explains the additional benefit of rapid decoupling in ionic solutions observed during FTIR experiments (FIG.47).
  • Evaluating PFOc Protein Formulation Resilience Against Contaminants Aqueous protein formulations can be compromised by contaminating pathogens, or the biologic inactivated by exposure to environmental disinfectants and acids. In the context of bacterial and fungal contamination, it was hypothesized that removal of the water solvent required for microorganismal survival should make the PFOc protein formulations intrinsically sterile.
  • a hypodermic needle was streaked across a lawn of the human bacterial pathogens E. coli, P. aeruginosa, K. pneumoniae or Methicillin-resistant S. aureus (MRSA), as well as the human fungal pathogen C. albicans prepared on agar.
  • the contaminated needle was then submerged into BSA protein formulations prepared in either PBS or PFOc solvents. Contaminated liquid formulations were incubated at 37 °C overnight and replated onto agar plates to assess growth. Results in FIG.53 show, as expected, BSA in PBS was readily contaminated by all five pathogens, as demonstrated by the emergence of viable colonies on the plate.
  • PFOc samples remained sterile, despite the high pathogen concentrations inoculated into the samples.
  • a possible explanation for this is that contaminating pathogens dehydrate when submerged in the non-aqueous PFOc formulation, leading to their elimination.
  • protein formulations can be compromised by incidental contact with environmental proteases, oxidizing cleaners, and acidic disinfectants.
  • aliquots of either proteinase K, a chlorine and sodium hydroxide mixture (bleach), or hydrochloric acid were added to PFOc and PBS protein samples (FIG.54A-54C).
  • ⁇ -Gal bioactivity was determined after exposure to potent proteolytic enzyme, proteinase K. Lyophilized ⁇ -Gal (1 ⁇ M) and proteinase K (10 ⁇ M) was either diluted in PBS Attorney Docket No.148411.002502 PATENT or dispersed in PFOc. An equal volumetric amount of ⁇ -Gal and proteinase K stocks were mixed and incubated together for 24 hours. The remaining active ⁇ -Gal was evaluated by adding ONPG substrate diluted in PBS at 4 mg/mL directly to the samples. This action eluted ⁇ -Gal from PFOc solvent and immediately starting ONPG conversion without further ⁇ -Gal disruption from residue proteinase K that was also eluted into aqueous buffer.
  • the enzymatic activity was calculated from measuring absorbance (420 nm) after 15-minute conversion. Data was normalized to ⁇ -Gal activity that was carried by the same solvent, without proteinase K. In the presence of proteinase K, ⁇ -Gal dissolved in PBS was completely inactivated, while there was no statistically significant change in activity for PFOc formulations under similar conditions (FIG.54A). A likely explanation for this is that the proteinase K protein is also coated by the PD-7 additive after addition to the PFOc solvent, leading to its segregation from the co-dispersed ⁇ -Gal protein. Next, resistance against oxidizer contaminant was demonstrated in ⁇ -Gal exposed to a mixture of sodium hypochlorite (bleach).
  • stock of lyophilized ⁇ -Gal was prepared then dispersed in PBS or PFOc.
  • a small volumetric quantity (1% v/v) of 10% bleach was added to the protein samples.
  • an equal volume of PBS containing 4 mg/mL ONPG substrate was added to the sample and incubated for 5 minutes to allow for complete substrate conversion.
  • absorbance was taken at 420 nm on the eluted samples, and relative activity was calculated by normalizing absorbance of converted ONPG in oxidizer- treated samples against their corresponding untreated group. Resistance against acid contaminant was demonstrated in ⁇ -Gal exposed to a strong acid, hydrochloric acid (HCl).
  • stock of lyophilized B-Gal was prepared then dispersed in PBS or PFOc.
  • ⁇ -Gal formulations were intravaneously administered to C57BL/6 mice and monitored time dependent serum bioavailability (FIG.55). This experiment utilized a fluorescent conversion substrate to monitor the amount of functional protein in serum, thereby investigating changes to both bioavailability and bioactivity. Due to the large solution volumes administered (150 ⁇ L, ⁇ 15% of mouse blood volume), PFOc dispersed proteins were extracted into sterile saline before injection to avoid hyponatremia.
  • the bivalent perfluorinated carboxylic acid PD-7 was validated as a lead second-generation candidate due to its high dispersion efficiencies (>95%) and low cytotoxicity (IC50 ⁇ 1.0 mM). Subsequent thermostability experiments showed that proteins dispersed into PFOc using PD-7 had near complete retention of bioactivity at temperatures up to 90°C. A series of biophysical and in silico assays elucidated the mechanistic basis for this thermal stabilization.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Dermatology (AREA)
  • Dispersion Chemistry (AREA)
  • Emulsifying, Dispersing, Foam-Producing Or Wetting Agents (AREA)
  • Paints Or Removers (AREA)
EP23895200.6A 2022-10-07 2023-10-06 Zusammensetzungen mit in fluorierten lösungsmitteln gelösten proteinen sowie verfahren zur herstellung und verwendung davon Pending EP4598563A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263414209P 2022-10-07 2022-10-07
PCT/US2023/034700 WO2024112388A2 (en) 2022-10-07 2023-10-06 Compositions comprising proteins dissolved in fluorinated solvents, and methods of making and using the same

Publications (1)

Publication Number Publication Date
EP4598563A2 true EP4598563A2 (de) 2025-08-13

Family

ID=91196403

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23895200.6A Pending EP4598563A2 (de) 2022-10-07 2023-10-06 Zusammensetzungen mit in fluorierten lösungsmitteln gelösten proteinen sowie verfahren zur herstellung und verwendung davon

Country Status (2)

Country Link
EP (1) EP4598563A2 (de)
WO (1) WO2024112388A2 (de)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9156938B2 (en) * 2010-08-19 2015-10-13 Massachusetts Institute Of Technology Compositions, methods, and systems comprising fluorous-soluble polymers
WO2019023706A1 (en) * 2017-07-28 2019-01-31 The Penn State Research Foundation ULTRASOUND-SENSITIVE PEPTIDE PARTICLES FOR ADMINISTRATION OF SPACE-RESOLVED MOLECULES

Also Published As

Publication number Publication date
WO2024112388A9 (en) 2024-08-02
WO2024112388A2 (en) 2024-05-30
WO2024112388A3 (en) 2024-07-11

Similar Documents

Publication Publication Date Title
Miller et al. Perfluorocarbons in chemical biology
Gopalakrishnan et al. Therapeutic potential of foldamers: from chemical biology tools to drug candidates?
Townsend et al. Imidazole derivatives improve charge reduction and stabilization for native mass spectrometry
Jha et al. Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammonium-based ionic liquids
Lee et al. Modulating protein structure with fluorous amino acids: increased stability and native-like structure conferred on a 4-helix bundle protein by hexafluoroleucine
Hilinski et al. Stitched α-helical peptides via bis ring-closing metathesis
Lee et al. Fluorous effect in proteins: de novo design and characterization of a four-α-helix bundle protein containing hexafluoroleucine
Violette et al. N, N ‘-Linked oligoureas as foldamers: chain length requirements for helix formation in protic solvent investigated by circular dichroism, NMR spectroscopy, and molecular dynamics
Garcia et al. Self‐assembly of an amino acid derivative into an antimicrobial hydrogel biomaterial
Fanghänel et al. Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state
Müntener et al. New lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins
Tressler et al. (2 S, 4 R)-and (2 S, 4 S)-Perfluoro-tert-butyl 4-Hydroxyproline: Two Conformationally Distinct Proline Amino Acids for Sensitive Application in 19F NMR
Burade et al. Self-assembly of fluorinated sugar amino acid derived α, γ-cyclic peptides into transmembrane anion transport
US20100021344A1 (en) Beta-peptide lyotropic liquid crystals and methods of manufacture and use thereof
Chen et al. The interplay of disulfide bonds, α-helicity, and hydrophobic interactions leads to ultrahigh proteolytic stability of peptides
Mason et al. Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream
Xiao et al. Clickable tryptophan modification for late-stage diversification of native peptides
Kitamura et al. Rational design of potent and selective inhibitors of an epoxide hydrolase virulence factor from Pseudomonas aeruginosa
Balasco et al. Self-assembled materials based on fully aromatic peptides: the impact of tryptophan, tyrosine, and dopa residues
Castelletto et al. Conformation and aggregation of selectively PEGylated and lipidated gastric peptide hormone human PYY3–36
Mouhib et al. Influence of detergent and lipid composition on reconstituted membrane proteins for structural studies
Hohmann et al. Introducing aliphatic fluoropeptides: Perspectives on folding properties, membrane partition and proteolytic stability
Chae et al. Tripod amphiphiles for membraneprotein manipulation
Judy et al. Prevention of insulin fibrillation by biocompatible choline-amino acid based ionic liquids: biophysical insights
Pariary et al. Targeted inhibition of amyloidogenesis using a non-toxic, serum stable strategically designed cyclic peptide with therapeutic implications

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250407

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)