CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63/409,456, filed on September 23, 2022, which is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under the National Science Foundation (CBET- 1911478) and the Office of Naval Research (N00014-20- 1-2731). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention generally relates to methods for producing lower immunogenic biomolecule-polymer conjugates from an initial source of biomolecule- polymer conjugate, wherein the lower immunogenic biomolecule-polymer conjugates are more hydrophilic conjugate species than the higher immunogenic biomolecule-polymer conjugates.

BACKGROUND

[0004] Polyethylene glycol (PEG) is generally considered inert and non-immunogenic. For this reason, PEG is commonly conjugated to various drugs to result in PEGylated drug conjugates. However, mounting evidence indicates that PEG can elicit an immunogenic response partly due to the amphiphilic nature of PEG. Indeed, antibodies against PEG are known to be elicited in subjects who have been administered PEGylated drugs. The evidence indicates that PEG, although substantially inert, can still trigger a certain level of biological interactions through its intrinsic hydrophobicity.

[0005] Moreover, a sample of biomolecule-polymer conjugate is generally not uniform in structure, and for this reason can contain a range of lower immunogenic and higher immunogenic forms. A method for isolating the lower immunogenic forms of biomolecule- polymer conjugates would be a significant advance but has thus far remained elusive.

SUMMARY

[0006] The present disclosure is foremost directed to a method for selectively isolating lower immunogenic biomolecule-polymer conjugates from an initial source of biomolecule- polymer conjugate. The method includes eluting the initial source of biomolecule-polymer conjugate through a hydrophobic interaction chromatography (HIC) medium at an eluent salt concentration greater than 0.15 M, and se1ectively isolating more hydrophilic conjugate species that pass through the HIC column at the salt concentration greater than 0.15 M while leaving more hydrophobic conjugate species bound to the HIC medium, wherein the more hydrophilic conjugate species being isolated have a lower immunogenicity than the hydrophobic conjugate species bound to the HIC column. In some embodiments, the polymer is or includes PEG. In other embodiments, the polymer is or includes a zwitterionic material, such as a poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), poly(trimethylamine N-oxide) (pTMAO), or poly(phosphorylcholine) (pPC).

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGS, la-lj. FIG. la shows structures of hydrophilic polymers for protein conjugation studied in this application. FIGS, lb to Ih are eluent profiles of the polymers (10k PeOX, 10k mPEG, 10k HO-PEG, 5k mPEG, 5k HO-PEG, 10k PmOX, and 10k PCB, respectively) on a Capto butyl HIC column. Polymers were first loaded and isocratically eluted with a 4M NaCl solution, followed by gradient elution by changing NaCl concentration from 4 M to 0 M. Solution conductivity (dashed line, left y-axis) and UV signal at 214 nm (solid line, right y-axis) were monitored and recorded. FIGS, li and Ij show HIC (eluent) profiles of PCB and PmOX, respectively, using the same eluent method while changing 4 M NaCl to 2 M AS buffer.

[0008] FIG. 2 is a graph showing polymer immunogenicity indexes versus hydrophobicity ranking. The immunogenicity index was defined as the arithmetic sum of log values of IgG and IgM titers. The theoretical minimum of immunogenicity index (horizontal dash line) was 4 as all serum dilution started from 100-fold. Vertical dash lines divide the polymers into three regions based on immunogenicity levels (left to right: low to high); P values were calculated using one-way ANOVA followed by Tukey post hoc test. Significance, P<0.05. #: Data for each group was significantly lower than other groups.

[0009] FIGS. 3a-3f. Eluent profiles are shown for native Asp (FIG. 3a), PEG-Asp (FIG. 3b), and PCB-Asp (FIG. 3c) on Capto butyl HIC column. Proteins or protein conjugates were loaded and isocratic eluted with 4 M NaCl solution, followed by a gradient elution when changing NaCl concentration from 4 M to 0 M. FIG. 3d is a graph comparing anti- whole conjugate immunogenicity indexes of PCB-Asp and PEG-Asp. FIG. 3e is a graph comparing Anti- Asp immunogenicity indexes of Asp, PEG-Asp and PCB-Asp. FIG. 3f is a graph comparing Anti-Asp immunogenicity index before and after the HIC treatment of PCB-Asp conjugates. PCB-Asp w/ HIC represented the non-binding portion from HIC column. P values were calculated using two-tailed student t test. Multiple comparisons were performed using one-way ANOVA followed by Tukey post hoc test. Significance: *, P<0.05, *** P<0.001.

[0010] FIGS. 4a-4g. Eluent profiles are shown for native Uricase casp (FIG. 4a), PCB-20k- Uricase casp (FIG. 4b), Uricase BF (FIG. 4c), PCB-20k-Uricase BF single conjugation (FIG. 4d), and PCB-20k-Uricase BF double conjugation (FIG. 4e) on a Capto butyl HIC Column. Proteins or conjugates were loaded, isocratically eluted with a 4 M NaCl solution, and then followed by gradient elution by changing the NaCl concentration from 4 M to 0 M. Solution conductivities (dashed line, left y-axis) and UV signal at 280nm (solid line, right y- axis) were monitored and recorded (FIG. 4f) for GPC curves of uricase proteins and conjugates. FIG. 4g shows an SDS-PAGE of ladders (Lane 1), Uricase BF (Lane 2), PCB- 20k-Uricase BF double conjugation before HIC treatment (Lane 3) and PCB-20k-Uricase BF double conjugation after HIC treatment (Lane 4).

[0011] FIGS. 5a-5b. Uricase BF and related PCB conjugates were used to control kidney maldevelopment in a uricase-deficient mice model. Uricase-deficient mice were injected with 5 mg/kg of uricase or uricase conjugates IV for three weekly doses. Mice were sacrificed two weeks after the last dose. Serum and kidneys were harvested for analysis. FIG. 5a is a graph showing that HIC purified PCB double conjugated Uricase BF showed lowest immunogenicity. FIG. 5b is a graph showing that HIC purified PCB double conjugated Uricase BF also maintained the kidney growth in uricase-deficient mice comparable to the heterozygote (healthy control) mice. DETAILED DESCRIPTION

[0012] The present invention is foremost directed to a novel method for selectively isolating lower immunogenic biomolecule-polymer conjugates from an initial source of biomolecule- polymer conjugate. The lower immunogenic biomolecule-polymer conjugates are more hydrophilic conjugate species than the higher immunogenic biomolecule-polymer conjugates. The method can advantageously be applied to any polymer-biomolecule (e.g., polymer-drug, polymer-small molecule, or polymer-protein) conjugate wherein the polymer is necessarily non-toxic and safe for administration to a living subject. Although not wishing to be bound by theory, it is believed that the more hydrophilic (lower immunogenic) conjugate species possess a higher polymer coverage than the more hydrophobic (higher immunogenic) conjugate species. The term “polymer coverage” is used herein to mean a surface area coverage of polymer molecules on the biomolecule- polymer conjugate. The polymer coverage of the lower immunogenic conjugate species may be, for example, at least or greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and the higher immunogenic conjugate species have a lower coverage than any of the foregoing. In some embodiments, the coverage of the lower immunogenic conjugate species is 100% while the higher immunogenic conjugate species is lower than 100% or lower than or no more than any of the values given above.

[0013] More specifically, the method includes eluting the initial source of biomolecule- polymer conjugate through a hydrophobic interaction chromatography (HIC) medium at an eluent salt concentration greater than 0.15 M. In different embodiments, the salt concentration of the eluent is at least or greater than, for example, 0.2 M, 0.25 M, 0.3 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, or 4 M. At the eluent salt concentration greater than 0.15 M, more hydrophilic conjugate species remain in the eluent while more hydrophobic (less hydrophilic) conjugated species become bound to the HIC medium.

Thus, the process selectively isolates more hydrophilic conjugate species that pass through the HIC column at the salt concentration greater than 0.15 M while leaving more hydrophobic conjugate species bound to the HIC medium. As indicated earlier above, the more hydrophilic conjugate species being isolated have a lower immunogenicity than the hydrophobic conjugate species bound to the HIC column. In some embodiments, the more hydrophilic conjugate species being isolated have a reduced amount of at least one impurity relative to the initial source of biomolecule-polymer conjugate. The impurity may include, for example, at least one of unconjugated biomolecule and aggregates of biomolecule- polymer conjugates.

[0014] In some embodiments, the hydrophobic interaction chromatography (HIC) medium comprises at least one hydrophobic ligand. The at least one hydrophobic ligand may be selected from, for example, alkyl (e.g., butyl, hexyl, and octyl) groups, aryl (e.g., phenyl) groups, and polypropylene glycol. In specific embodiments, the HIC media contains an agarose medium functionalized with octyl groups.

[0015] In some embodiments, the eluent comprises a salt selected from chloride salts, sulfate salts, citrate salts, ammonium sulfate, sodium sulfate, sodium citrate, sodium chloride, and combinations thereof. The eluent may include a cation selected from Ba ²⁺ ; Ca ²⁺ ; Mg ²⁺ ; Li ⁺ ; Cs ⁺ ; Na ⁺ ; K ⁺ ; Rb ⁺ ; NH4 ⁺ ; tetramethylammonium; tetraethylammonium and combinations thereof. The eluent may include an anion selected from Cl; Br ; NO3’; SO4 ² '; PO4 ³ '; CH iCOO'; SCN- and combinations thereof. The eluent typically has a salt concentration in the range of 10 M-0.5 M.

[0016] The biomolecule is any molecule found in, derived from, or intended for administration into a living organism. Biomolecules intended for administration into a living organism typically have a positive effect on the living organism, such as treatment of a disease or condition. The biomolecule may have any size, such as up to, above, or below 10,000 g/mol (10 kD), 20 kD, 30 kD, 40 kD, 50 kD, or 100 kD. In some embodiments, the biomolecule in the biomolecule-polymer conjugate is a small molecule, such as a drug. The small molecule typically has a molecular weight of up to or less than 6,500 g/mol, or more typically, up to or less than 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1,500, 1,250, or 1000 g/mol. Some examples of small molecules include Amberstatin269, Calicheamicin, Camptothecin, huB4-DGN462, Ravtansine (DM4 ), Duocarmycin, DX-8951 (DXd), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Pyrrolobenzodiazepine (PBD), SN-38, Tubulysin, dolastatin 10, dolastatin 15, mertansine (DM1) and auristatin PE. In some embodiments, the biomolecule is a protein or oligopeptide, such as an antibody, antibody fragment, enzyme, enzyme active site, or metalloprotein. In some embodiments, the biomolecule is a nucleic acid or oligonucleotide, such as an RNA (e.g., mRNA) or DNA (e.g., plasmid or vector). [0017] In some embodiments, the biomolecule may be, for example, a drug, protein, nucleic acid, glycoprotein, lipid, or virus. In particular embodiments, the biomolecule is a protein, such as a recombinant protein. Some examples of recombinant proteins include Adenosine deaminase, Asparaginase, Interferon-a-2b, Interferon-a-2a, Granulocyte colony-stimulating factor (G-CSF), Human growth hormone (hGH), Erythropoietin, Anti-tumor necrosis factor (TNF) a Fab’, Phenylalanine Ammonia Lyase, interleukin-2, interleukin- 10, interleukin- 12, and urate oxidase (uricase).

[0018] As noted above, the polymer in the conjugate should be non-toxic and safe for administration to a living subject. In some embodiments, the polymer is a PEG polymer or is a copolymer that includes a PEG segment (block).

[0019] In some embodiments, the polymer is or includes a polypeptide containing: a. a plurality of negatively charged amino acids; b. a plurality of positively charged amino acids; and c. a plurality of additional amino acids independently selected from the group consisting of proline, serine, threonine, asparagine, glutamine, glycine, and derivatives thereof; and wherein the ratio of the number of positively charged amino acids to the number of positively charged amino acids is from about 1:0.5 to about 1:2.

[0020] In some embodiments, the plurality of negatively charged amino acids may be independently selected from the group consisting of aspartic acid, glutamic acid, and derivatives thereof. In some embodiments, the plurality of positively charged amino acids is independently selected from the group consisting of lysine, histidine, arginine, and derivatives thereof. In some embodiments, the positively charged amino acids and negatively charged amino acids constitute from about 20% to about 95% (or 20-80%) of the total number of amino acids present in the charged domain. The polypeptide may comprise from about 6 to about 1000 (or, e.g., 6-500 or 6-250) amino acids. In some embodiments, the ratio of positively charged amino acids to negatively charged amino acids is from about 1:0.7 to about 1:1.4 (or e.g., 1:0.7 to 1:1.2 or 1:0.7 to 1:1). In some embodiments, the polypeptide comprises at least two (or at least three or four) pairs comprising a positively charged amino acid adjacent to a negatively charged amino acid. For example, the polypeptide may contain a glutamic acid (E) residue and a lysine (K) residue, wherein the polymer may thus be referred to as an EK polymer. [0021] In other embodiments, the polymer is zwitterionic, which may or may not include one or more PEG segments. In some embodiments, the polymer is a zwitterionic polymer and the biomolecule is a protein or oligopeptide. The zwitterionic polymer may include an ammonium group and an anionic group, wherein the anionic group may be a carboxylate, sulfate, sulfonate, phosphate, phosphonate, or nitrate group. In some embodiments, the zwitterionic polymer is a betaine polymer. The average number of strands of the zwitterionic polymer in the protein-poly mer conjugate is typically between about 1 and 40 per protein subunit. In different embodiments, the average number of strands in the zwitterionic polymer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, or 40, or a range bounded by any two of these values, e.g., 1-30, 1-20, 2-20, 4-20, 6-20, or 6-10.

[0022] In some embodiments, the polymer is a zwitterionic polymer and the biomolecule is a protein, and the average number of strands of the zwitterionic polymer in the protein- polymer conjugate is between about 1 and 40 per protein subunit. In sub-embodiments, the average number of strands of the zwitterionic polymer in the protein-polymer conjugate is between about 1 and 30, or a between about 1 and 20, or between about 2 and 20, or between about 3 and 20, or between about 4 and 20, or between about 5 and 20, or between about 6 and 20, or between about 6 and 10, per protein subunit

[0023] In some embodiments, the zwitterionic polymer has the following formula: wherein: Mi and M2 are independently selected from a functional group suitable for polymerization by addition, condensation or free radical polymerization; Ki and K2 are independently selected from -(CH ₂ ) _X -, -(CH(CN)) _X -, -C(=O)NH(CH ₂ ) _X -, - C(=O)(CH ₂ ) _X -, - C(=O)O(CH ₂ ) _X -, -C(=O)OC(=O)O(CH ₂ )X-, -(CH ₂ ) _X -O-(CH ₂ ) _X -, C(=S)(CH ₂ ) _X , -NH(CH ₂ )X and -(CH ₂ ) _X -S-S-(CH ₂ )X-, wherein x at each occurrence is an integer independently selected from 0 to 20; m and n are independently integers from 5 to about 10,000; and Z is independently selected from zwitterionic group consisting of:

and

[0024] In the above structures, R1 and R8 are selected from the group consisting of hydrogen, fluorine, trifluoromethyl, C1-C ₂ 0 alkyl, and C6-C12 aryl groups, and CN; R4, R5, R6, and R7 are independently selected from the group consisting of hydrogen, C1-C ₂ 0 alkyl, C6-C12 aryl, cyclic alkyl, fluoroalkyl, oxide (O ) or void (when substituent is absent); L is C or Si or void (when substituent is absent); Li, L3, L ₄ , and L5 are independently selected from -(CH ₂ )x-, -(CH(CN))X-, -C(=0)NH(CH ₂ )X-, -C(=0)0(CH ₂ )X-, - C(=0)0C(=0)0(CH ₂ ) -, -(CH ₂ )X-O-(CH ₂ )X-, and -(CH ₂ )X-S-S-(CH ₂ )X-, where x at each occurrence is an integer independently selected from 0 to 20, combinations thereof, or void (when substituent is absent); L ₂ is independently selected from -(CH2)x-, or -(CH(CN))x-, where x is an integer from 1 to 20; Ai is N or P; Bi is C, S, SO, PO, or PO ₂ "; B ₂ is C or SO; C1 is PO ₄ ; and M is selected from C1, Br, I, SO ₄ , NO ₃ , C1O ₄ , BF ₄ , PF ₆ , N(SO ₂ CF ₃ ) ₂ , SO3CF3, RCOO (R is C1-C ₂ 0 alkyl), lactate, benzoate, salicylate, or void (when substituent is absent).

[0025] In some embodiments, the zwitterionic polymer is derived from a zwitterionic monomer which may be fluorinated or non-fluorinated. The zwitterionic monomer compositions are capable of being polymerized into zwitterionic polymers. As used herein, the term “zwitterionic monomer” refers to a polymerizable molecule containing at least one zwitterionic moiety, wherein a zwitterionic moiety contains both negative and positively charged groups. In typical embodiments, the zwitterionic monomer contains a vinyl (olefin) group attached to a zwitterionic portion, wherein the vinyl group is polymerizable by vinyl addition as well known in the art to produce a polymer containing zwitterionic pendant groups and a polyvinyl backbone. In the zwitterionic monomer, the zwitterionic moiety may alternatively be attached to polymerizable groups other than vinyl or methacryl groups, such as those polymerizable groups that can react to form backbones containing siloxane bonds, ester bonds, urethane bonds, urea bonds, and carbonate bonds (the resulting polymer may thus be, respectively, e.g., a zwitterionic polysiloxane, polyester, polyurethane, polyurea, or polycarbonate). Moreover, the zwitterionic monomer may, in some embodiments, contain a zwitterionic moiety that functions as a polymerizable group, which results in a zwitterionic polymer containing a backbone that is zwitterionic.

[0026] In some embodiments, the zwitterionic monomer has the following general structure:

[0027] In Formula (1), A is a polymerizable group (e.g., olefin, siloxane, diacid, diester, diol, acid-hydroxy, isocyanate, or diisocyanate); L is a bond or linking portion; and Z is a zwitterionic moiety or a zwitterionic precursor moiety containing a protecting group capable of deprotection to form a charged group. In some embodiments, one or more hydrogen atom in A, L, and/or Z in Formula (1) is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. The fluorinated hydrocarbon group may or may not include one or more oxygen or nitrogen atoms. The fluorinated hydrocarbon group may be partially fluorinated or fully fluorinated (perfluorinated). Some examples of fluorinated hydrocarbon groups include CF3, CH2CF3, (CF ₂ ) _r CF ₃ , CH(CF ₃ ) ₂ , and CF(CF ₃ ) ₂ , wherein r is, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, for any of the formulas in this disclosure, the fluorinated hydrocarbon group has the formula -(CH ₂ ) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group.

[0028] The zwitterionic monomer may more particularly have the following structure: [0029] In Formula (la), A and Z are as defined under Formula (1); and m is an integer of at least 1. In different embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, or 12, or m is within a range therein, e.g., 1-12 or 3-12, wherein the methylene groups may or may not be interrupted by a heteroatom-containing group, such as -O-, -NR’-, -C(O)O-, or -C(O)NR-). The group R’ may be a hydrogen atom or hydrocarbon group, wherein the hydrocarbon groups typically contains 1-12 carbon atoms, as described above.

[0030] In some embodiments, the zwitterionic monomer more particularly has the following structure:

[0031] In Formula (lb), R ^a is H or an alkyl group containing 1-3 carbon atoms (e.g., methyl, ethyl, n-propyl, or isopropyl, wherein the alkyl group optionally contains one, two, three, or more fluorine atoms); X is O or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms; Z is a zwitterionic moiety or a zwitterionic precursor moiety containing a protecting group capable of deprotection to form a charged group; and m is an integer of at least 1 or as provided above. In some embodiments, the monomer of Formula (lb) is non- fluorinated. In some embodiments, one or more hydrogen atoms in Formula (lb) (e.g., in olefin group, R ^a , X, methylene group under m, or Z) is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1- 12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above.

[0032] In other embodiments, the zwitterionic monomer more particularly has the following structure: [0033] In Formula (1c), R ^a , X, and m are as defined under Formula (lb), and C ₁ and C ₂ are independently selected as positively charged and negatively charged groups to form a zwitterionic moiety C ₁ -C ₂ . In some embodiments, the monomer of Formula (1c) is non- fluorinated. In some embodiments, one or more hydrogen atoms on the olefin group, R ^a , X, methylene group under m, C ₁ , and/or C ₂ is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above.

[0034] The zwitterionic monomer of Formula (1c) may more particularly have the following structure:

(1c-1)

In Formula (1c-1), R ^a , X, and m are as defined under Formula (lb), and R ¹ and R ² are independently selected from hydrocarbon groups containing 1-12 carbon atoms, wherein at least one of R ¹ and R ² contains at least one fluorine atom. C ₁ ⁺ and C ₂ " are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ - C ₂ -. The dashed line represents an optional bond. The C ₁ ⁺ atom or group may be or include, for example, a positively charged nitrogen atom, phosphorus atom, or sulfur atom. In some embodiments, the monomer of Formula (1c-1) is non-fluorinated. In some embodiments, any one or more of R ^a , X, and methylene group under m is optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above.

[0035] The zwitterionic monomer of Formula (1c) may more particularly have the following structure:

(1c-2)

[0036] In Formula (1c-2), R ^a , X, and m are as defined under Formula (lb), and R ¹ and R ² are independently selected from R ^a or hydrocarbon groups containing 1-12 carbon atoms, wherein R ¹ and/or R ² (or at least one) optionally contains at least one fluorine atom, as defined earlier above. In some embodiments, the monomer of Formula (1c-2) is non- fluorinated. In some embodiments, at least one hydrogen atom on R ^a , X, methylene group under m, R ¹ , and/or R ² is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above.

[0037] The zwitterionic monomer of Formula (1c) may more particularly have the following structure:

[0038] In Formula (1c-3), R ^a , X, and m are as defined under Formula (1b), and R ² is selected from R ^a or hydrocarbon groups containing 1-12 carbon atoms and optionally containing one ore more fluorine atoms, as defined earlier above. C ₁ ⁺ and C ₂ - are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ - C ₂ -. The dashed line represents an optional bond. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non- fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and optionally, precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. In some embodiments, any one or more of R ^a , X, and methylene group under m is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom.

[0039] The zwitterionic monomer of Formula (1c-3) may more particularly have the following structure: (1c-4)

[0040] In Formula (1c-4), R ^a , X, and m are as defined under Formula (lb), and R ² is selected from R ^a or hydrocarbon groups containing 1-12 carbon atoms and optionally containing one ore more fluorine atoms, as defined earlier above. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non- fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and optionally, precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. In some embodiments, the monomer of Formula (1c-4) is non-fluorinated. In some embodiments, any one or more of R ^a , X, methylene group under m, and/or R ² is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom.

[0041] Alternatively, Z may contain a positively charged group indirectly bound to a negatively charged group via a linker, such as in the following structure:

[0042] In Formula (Id), R ^a , X, and m are as defined under Formula (lb). The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c , R ^d , R ^e , and R ^f are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon group containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Notably, R ^c and R ^d are each independently present twice for each methylene linkage. One or both of R ^e and R ^f are alternatively selected from positive and negative charges. The variables C ₁ and C ₂ are independently selected as positively charged and negatively charged groups (moieties) to form a zwitterionic moiety. Some examples of positively charged moieties include ammonium (-NR ^a 2 ⁺ -), phosphonium (-PR -), and sulfonium moieties. Some examples of negatively charged moieties include terminal oxide (-O'), carboxylate (-C(O)O-), phosphate (-OPO3 ), phosphonate (-PO3'), sulfate (-OSO3'), and sulfonate (-SC3'). In some embodiments, C ₁ is neutral and R ^e is a protecting group capable of deprotection to form a charged group, or C ₂ is neutral and R ^f is a protecting group capable of deprotection to form a charged group. The protecting group may be, for example, a bulky alkyl group (e.g., t-butyl) forming an ester with a carboxylate. Protecting groups may be removed by standard chemical means to produce a charged group to form a zwitterion. Thus, a monomer in which C ₁ or C ₂ contains a protecting group can be considered a zwitterionic monomer precursor. In some embodiments, C ₁ or C ₂ is a neutral amine group, which can be reacted with an amine- reactive compound, such as an alkyl halide, to result in a quaternary ammonium group. Thus, a monomer in which C ₁ or C ₂ is a neutral amine can also be considered a zwitterionic monomer precursor. In some embodiments, the monomer of Formula (Id) is non- fluorinated. In some embodiments, any of R ^c , R ^d , R ^e , and R ^f has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0043] In more particular embodiments, the zwitterionic monomer of Formula (Id) has the following structure:

[0044] In Formula (1e), R ^a , X, m, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c , R ^d , R ^e , R ^f , and R ^g are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, R ^f is alternatively a negative charge. The variable C ₂ is a negatively charged group. Optionally, C ₂ is neutral and R ^f is a protecting group capable of deprotection to form a charged group. In some embodiments, the monomer of Formula (le) is non-fluorinated. In some embodiments, R ^g and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2)x-(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1- 12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0045] In some embodiments, the zwitterionic monomer of Formula (le) may more particularly have the following structure:

[0046] In Formula (le-1), R ^a , X, m, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c , R ^d , R ^e , and R ^g are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. The variable C ₂ ’ is a negatively charged group, such as a carboxylate, sulfonate, sulfate, phosphonate, or phosphate group. In some embodiments, R ^g and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1- 12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the monomer of Formula (le-1) is non-fluorinated.

[0047] In some embodiments, the zwitterionic monomer of Formula (Id) may more particularly have the following structure: [0048] In Formula (le-2), R ^a , X, m, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Each R ^e is independently selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a , wherein one or both R ^e may contain at least one fluorine atom. C ₁ ⁺ and C ₂ " are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ - C ₂ -. The dashed line represents an optional bond. In some embodiments, the monomer of Formula (le-2) is non- fluorinated. In some embodiments, one or more of R ^a , X, methylene group under m, R ^c , and R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0049] In some embodiments, the zwitterionic monomer of Formula (le-2) may more particularly have the following structure:

[0050] In Formula (le-3), R ^a , X, and m are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Each R ^e is independently selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a , wherein at least one R ^e contains at least one fluorine atom. C ₁ ⁺ and C ₂ ’ are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ -C ₂ ‘. The dashed line represents an optional bond. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non-fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and optionally, precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. In some embodiments, the monomer of Formula (le-3) is non-fluorinated. In some embodiments, any one or more of R ^a , X, methylene group under m, R ^c , and R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom.

[0051] In some embodiments, the zwitterionic monomer of Formula (le-3) may more particularly have the following structure:

[0052] In Formula (le-4), R ^a , X, and m are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. R ^e is selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a . The variable C ₂ - is a negatively charged group, such as a carboxylate, sulfonate, sulfate, phosphonate, or phosphate group. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non-fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and optionally, precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. In some embodiments, the monomer of Formula (le-4) is non-fluorinated. In some embodiments, any one or more of R ^a , X, methylene group under m, R ^c , and/or R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the monomer of Formula (le-4) is non- fluorinated.

[0053] In some embodiments, the zwitterionic monomer of Formula (le-4) may more particularly have the following structure:

[0054] In Formulas (le-5) and (le-6), R ^a , X, and m are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. R ^e is selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a . The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non-fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and optionally, precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. In some embodiments, any one or more of R ^a , X, methylene group under m, R ^c , and/or R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the monomer of Formula (le-5) and/or (le-6) is non- fluorinated.

[0055] The zwitterionic monomer of Formula (le-1) may more particularly have any of the following structures: [0056] In any of the above formulas, R ^a , X, and m are as defined above. The variable p is an integer of at least 1. The variables R ^c , R ^d , R ^e , R ^f , R ^g , and R ^h are selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1- 12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, the variable R ^f is alternatively a protecting group capable of deprotection to form a carboxylate group. In some embodiments, R ^s and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms. In some embodiments, only or at least R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the monomer of Formula (le-1-1), (le-1-2), (le- 1-3), and/or (le-1- 4) is non-fluorinated.

[0057] In other embodiments of Formula (le), the zwitterionic monomer has the following structure:

[0058] In Formula (If), R ^a , X, and m are as defined earlier above. The variable C ₂ ⁺ is a positively charged group. The variable p is an integer of at least 1. The variables R ^c , R ^d , and R ^f are selected from hydrogen atom, fluorine atom, hydrocarbon group, and fluorinated hydrocarbon group containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, R ^f is alternatively absent. In some embodiments, the monomer of Formula (11 is non- fluorinated.

[0059] In particular embodiments of Formula (If), the zwitterionic monomer has the following structure:

(1f-1)

[0060] In Formula (If- 1), R ^a , X, and m are as defined above, and p is an integer of at least 1. The variables R ^c , R ^d , R ³ , R ⁴ , and R ⁵ are selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, the monomer of Formula (lf-1) is non-fluorinated.

[0061] The zwitterionic polymer, as attached to the biomolecule, may be derived from or include any of the zwitterionic monomers described above, such as those in Formulas (1), (1a), (1b), (1c), (1c-1), (1d), (1e), (1e-1), (1e-1-1), (1e-1-2), (1e- 1-3), (1e-1-4), (1f), and (1f- 1). As used herein, the term “zwitterionic polymer” refers to a polymer containing zwitterionic moieties, wherein a zwitterionic moiety contains both negative and positively charged groups covalently attached to the polymer. In one set of embodiments, the zwitterionic polymer is produced by polymerization of a polymerizable zwitterionic monomer or a monomer containing a precursor for a zwitterionic group. Zwitterionic monomers are electrically neutral monomers that include equal numbers of positive and negative charges per monomer. In another set of embodiments, the zwitterionic polymer is produced by polymerization of equal numbers of monomers containing negatively charged groups and monomers containing positively charged groups. In some cases, the polymer is produced solely from zwitterionic monomers or equal numbers of positively and negatively charged monomers, in which case the zwitterionic polymer has 100 mole percent (100 mol%) zwitterionic moieties. In other cases, the polymerization process may include uncharged monomers, which provides a zwitterionic copolymer having less than 100 mole percent zwitterionic moieties. For example, when a polymerizable zwitterionic monomer and polymerizable comonomer are present in equal proportions in the polymerization mixture, the product is a zwitterionic polymer having 50 mol% zwitterionic moieties. The uncharged monomer may be, for example, ethylene, propylene, styrene, methyl acrylate, methyl methacrylate, 2-hydroxyethyl acrylate, 2-ethoxyethyl acrylate, 2-(2- ethoxyethoxy)ethyl acrylate, acrylamide, vinyl alcohol, or acrylonitrile. Any of the zwitterionic polymers described in this application may function as the polymer in the biomolecule-polymer conjugate which is processed according to the method described here for isolating lower immunogenic forms of the biomolecule-polymer conjugate.

[0062] In some embodiments, the zwitterionic polymer is a homopolymer of any of the foregoing zwitterionic monomers. In other embodiments, the polymer is a copolymer (e.g., block, random, or alternating) containing any two or more of the above zwitterionic monomers, in the absence or presence of a non- zwitterionic monomer. In other embodiments, the zwitterionic polymer is a copolymer containing one or two of any of the above zwitterionic monomers and one or more non- zwitterionic monomers (e.g., styrene, butadiene, or a fluorinated vinyl molecule).

[0063] The zwitterionic polymers may be, for example, any of the polymers of the Formulas (I), (la), (lb), (Ic), (Ic-1), (Id), (le), (Ie-1), (Ie-1-1), (Ie-1-2), (Ie-1-3), (Ie-1-4), (If), and (If- 1). The polymers may be homopolymers of any of the zwitterionic monomers described above, or the polymers may be copolymers containing any of the zwitterionic monomers described above copolymerized with a non-zwitterionic monomer (e.g., vinyl alcohol, acrylate, methacrylate, trifluoroethylene, tetrafluoroethylene, styrene, or butadiene, or fluorinated versions of any one of these).

[0064] The zwitterionic polymer may have the following formula:

[0065] In Formula (I), A’ is a backbone of the polymer formed by polymerization of A. The backbone (A) can be any polymeric backbones known in the art, as described above and which may include linear, branched, and cyclic backbone structures. The backbone may be, for example, olefinic (e.g., polyvinyl or poly methacryl), poly siloxane, polyester, polyurethane, polyurea, polycarbonate, polypeptide, polyimide, polyphosphazene, poly epoxy, phenolic polymer, poly sulfone, or a poly sulfide. In the case of vinyl monomers, as largely described above, the backbone has a polyethylene (polyvinyl) structure, which may or may not be substituted with one or more fluorine atoms. The variable L is a bond or a linking portion, as described above, which may or may not be substituted with one or more fluorine atoms. The linker (L) can be any of the linkers commonly included in pendant groups of polymers, e.g., linear or branched alkyl linkers or cyclic linkers, any of which may contain precisely or at least 1, 2, 3, 4, 5, or 6 carbon atoms and optionally containing one or more heteroatoms (typically selected from oxygen and nitrogen atoms). Z is a zwitterionic moiety or a zwitterionic precursor moiety containing a protecting group capable of deprotection to form a charged group, as described above. The subscript n is typically an integer of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 50,000, or 100,000 units, or a number of units within a range bounded by any two of the foregoing values. In some embodiments, at least one hydrogen atom in A’, L, and/or Z in Formula (I) is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above. As indicated earlier, Formula (I) includes copolymers of Formula (I), unless otherwise specified. In particular embodiments, Z is selected from the group consisting of carboxybetaine, sulfobetaine, phosphobetaine, and trialky lamine-N-oxide zwitterionic moieties. In some embodiments, the zwitterionic polymer of Formula (I) is non-fluorinated.

[0066] In some embodiments of Formula (I), the zwitterionic polymer has the following formula: wherein A’ is a backbone, as described above; Z is a zwitterionic moiety or a zwitterionic precursor moiety, as described above; n is an integer of at least 2, and m is an integer of at least 1, as described earlier above. In particular embodiments, Z is selected from the group consisting of carboxybetaine, sulfobetaine, phosphobetaine, and trialkylamine-N-oxide zwitterionic moieties. In some embodiments, the zwitterionic polymer of Formula (la) is non-fluorinated. [0067] In other embodiments of Formula (I), the zwitterionic polymer has the following formula:

[0068] In Formula (lb), R ^a is H or an alkyl group containing 1-3 carbon atoms, as described earlier above. Some examples of alkyl groups containing 1-3 carbon atoms include methyl, ethyl, n-propyl, and isopropyl. In some embodiments, R ^a is H or methyl. The variable X is 0 or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, as described earlier above. In some embodiments, R ^b is H or methyl. The variable Z is a zwitterionic moiety or a zwitterionic precursor moiety containing a protecting group capable of deprotection to form a charged group, as described earlier above. In particular embodiments, Z is selected from the group consisting of carboxybetaine, sulfobetaine, phosphobetaine, and trialky lamine-N-oxide zwitterionic moieties. The subscript n is typically an integer of at least 2, 5, or 10 (e.g., at least or greater than 10, 50, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 50,000, or 100,000 units, or a number of units within a range bounded by any two of the foregoing values). The subscript m is an integer of at least 1, such as a value of precisely or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a value within a range bounded by any two of the foregoing values (e.g., 1-12, 1-10, 1-6, 1-4, 1-3, 2-4, or 2-3). In some embodiments, at least one hydrogen atom in Formula (lb) is substituted by a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, the zwitterionic polymer of Formula (lb) is non-fluorinated.

[0069] In some embodiments, the group Z represents a single zwitterionic group containing a positive and negative charge in the same Z group. The group Z may be selected from, for example, carboxybetaine, sulfobetaine, phosphobetaine, and trialkylamine-N'-oxide zwitterionic moieties. The resulting polymer may be, for example, a poly(carboxybetaine), poly(sulfobetaine), poly(phosphobetaine), and poly(trialkylamine-N-oxide), as well known in the art. In other embodiments, a certain number of the Z groups are positively charged (i.e. , Z ⁺ ) and an equal number of Z groups are negatively charged (i.e., Z ) to result in zwitterionic pairs (i.e., Z ⁺ Z' zwitterionic pairs).

[0070] Notably, although the formulas and sub-formulas provided throughout this disclosure may appear to depict zwitterionic homopolymers (100 mol% zwitterionic moieties), the formulas and sub-formulas thereof include the possibility that one or more non- zwitterionic or uncharged monomer units is situated (inserted) between zwitterionic monomeric units depicted in any of these formulas, thereby resulting in a copolymer. If zwitterionic monomeric units (such as any of those described above, i.e., where n = 1) are labeled as A units, and non-zwitterionic or uncharged monomeric units are labeled as B units, the copolymer can have any of the known copolymer arrangements, including alternating (e.g., A-B-A-B), block (e.g., A-A-A-A-B-B-B-B), or random (e.g., A-B-B-A-B- A-A-B-A-B-B). The zwitterionic polymer may also include more than one type of non- zwitterionic or uncharged monomer unit, such as in the structures A-B-C-A-B-C (repeating), A-A-A-B-B-B-C-C-C (block), or A-C-B-B-C-A-B-C-A-C-A-B-C-A-B-C-B (random), wherein B and C represent non-zwitterionic or uncharged monomeric units. The copolymers may also include more than one type of zwitterionic units, in which case A can represent two or more types of zwitterionic monomer units.

[0071] In some embodiments, Z contains a positively charged group directly bound to a negatively charged group to result in the following polymer structure:

[0072] In Formula (Ic), R ^a , X, n, and m are as defined earlier above, and C ₁ and C ₂ are independently selected as positively charged and negatively charged groups to form a zwitterionic moiety C1-C ₂ . Some examples of positively charged moieties include ammonium (-NRV-) and phosphonium moieties. Some examples of negatively charged moieties include terminal oxide (-O ), carboxylate (-C(O)O-), phosphate (-OPO3 ), phosphonate (-PO3 ), sulfate (-OSO3 ), and sulfonate (-SO3 ). In some embodiments, C ₁ is positively charged and C ₂ is negatively charged. For example, C ₁ may be an ammonium moiety and C ₂ may be oxide, which together results in an ammonium A-oxide zwitterionic group. As the ammonium moiety is also attached to a carbon atom of the polymer, the ammonium A-oxide zwitterionic group is also herein referred to as a trialkylamine- A-oxide group. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In separate or further specific embodiments, X is 0 or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In some embodiments, at least one hydrogen atom on R ^a , X, methylene group under m, C ₁ , and/or C ₂ is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In other embodiments, C ₁ is negatively charged and C ₂ is positively charged.

[0073] In some embodiments, the zwitterionic polymer of Formula (Ic) more particularly has the following structure:

[0074] In Formula (Ic- 1), R ^a , X, and m are as defined under Formula (lb), and R ¹ and R ² are independently selected from hydrocarbon groups containing 1-12 carbon atoms, wherein R ¹ and R ² are optionally fluorinated. C ₁ ⁺ and C ₂ - are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ - C ₂ -. The dashed line represents an optional bond. The C ₁ ⁺ atom or group may be or include, for example, a positively charged nitrogen atom, phosphorus atom, or sulfur atom. Any one or more of R ^a , X, and methylene group under m is optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, as described above. In some embodiments, the zwitterionic polymer of Formula (Ic-1) is non-fluorinated.

[0075] In particular embodiments of Formula (Ic), C ₁ is an ammonium moiety and C ₂ is oxide, which together results in an ammonium A-oxide (-NRa2+ ) zwitterionic group. The resulting polymer is a poly(trialkylammonium oxide), i.e., pTMAO, and may have the following structure:

(Ic-2)

[0076] In Formula (Ic-2), R ^a , X, n, and m are as defined earlier above, and R ¹ and R ² are independently selected from R ^a , as defined earlier above. In some embodiments, R ¹ and R ² are both alkyl, or more particularly, both methyl. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In separate or further specific embodiments, X is 0 or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In some embodiments, at least one hydrogen atom on R ^a , X, methylene group under m, R ¹ , and/or R ² is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1- 12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, the zwitterionic polymer of Formula (Ic-2) is non-fluorinated.

[0077] In some embodiments, the polymer of Formula (Ic) more particularly has the following structure:

(Ic-3)

[0078] In Formula (Ic-3), R ^a , X, and m are as defined under Formula (lb), and R ² is selected from R ^a or hydrocarbon groups containing 1-12 carbon atoms and optionally containing one ore more fluorine atoms, as defined earlier above. C ₁ ⁺ and C ₂ " are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ - C ₂ -. The dashed line represents an optional bond. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non- fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. Any one or more of R ^a , X, and methylene group under m is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the zwitterionic polymer of Formula (Ic-3) is non-fluorinated

[0079] In some embodiments, the polymer of Formula (Ic-3) more particularly has the following structure:

[0080] In Formula (Ic-4), R ^a , X, and m are as defined under Formula (lb), and R ² is selected from R ^a or hydrocarbon groups containing 1-12 carbon atoms and optionally containing one ore more fluorine atoms, as defined earlier above. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non-fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. Any one or more of R ^a , X, methylene group under m, and/or R ² is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the zwitterionic polymer of Formula (Ic-4) is non-fluorinated

[0081] In some embodiments of Formula (I), Z contains a positively charged group indirectly bound to a negatively charged group via a linker. The resulting polymer may then have the following structure:

[0082] In Formula (Id), R ^a , X, n, m, R ^c , R ^d , R ^e , and R ^f are as defined earlier above. The subscript p is an integer of at least 1, such as a value of precisely or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a value within a range bounded by any two of the foregoing values (e.g., 1-12, 1-10, 1-6, 1-4, 1-3, 2-4, or 2-3). The variables R ^c , R ^d , R ^e , and R ^f are selected from hydrogen atom, hydrocarbon group, and fluorinated hydrocarbon group containing 1- 12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Notably, R ^c and R ^d are each independently present twice for each methylene linkage. One or both of R ^e and R ^f may alternatively be selected from positive and negative charges. The variables C ₁ and C ₂ are independently selected as positively charged and negatively charged groups to form a zwitterionic moiety. Some examples of positively charged moieties include ammonium (-NRV-), phosphonium (-PRV-), and sulfonium moieties. Some examples of negatively charged moieties include terminal oxide (-0 ), carboxylate (-C(O)O- ), phosphate (-OPO3 ), phosphonate (-PO3 ), sulfate (-OSO3 ), and sulfonate (-SO3 ). In some embodiments, C ₁ is positively charged and C ₂ is negatively charged. For example, C ₁ may be an ammonium or phosphonium moiety and C ₂ may be carboxylate, sulfate, sulfonate, phosphate, or phosphonate moiety. In other embodiments, C ₁ is negatively charged and C ₂ is positively charged. For example, C ₁ may be a phosphate, phosphonate, sulfate, or sulfonate moiety and C ₂ may be an ammonium or phosphonium moiety. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In other separate or further specific embodiments, X is O or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In some embodiments, C ₁ is optionally neutral and R ^e is a protecting group capable of deprotection to form a charged group, or C ₂ is optionally neutral and R ^f is a protecting group capable of deprotection to form a charged group. In some embodiments, any of R ^c , R ^d , R ^e , and R ^f has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the zwitterionic polymer of Formula (Id) is non-fluorinated.

[0083] In particular embodiments of Formula (Id), the polymer has the following structure:

[0084] In Formula (le), R ^a , X, n, m, p, R ^c , R ^d , R ^e , R ^f , and R ^g are as defined earlier above. The variables R ^c , R ^d , R ^e , R ^f , and R ^g are independently selected from hydrogen atom, hydrocarbon group, and fluorinated hydrocarbon group containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, except that a fluorine atom is not directly attached to nitrogen. In some embodiments, R ^f is alternatively a negative charge. The variable C ₂ is a negatively charged group. Optionally, C ₂ is neutral and R ^f is a protecting group capable of deprotection to form a charged group. The variable p is an integer of at least 1, as described earlier above. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. In some embodiments, R ^g and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the zwitterionic polymer of Formula (le) is non-fluorinated. [0085] In particular embodiments of Formula (le), C ₁ is an ammonium moiety and C ₂ is a negatively charged moiety, such as a carboxylate, sulfate, sulfonate, phosphate, or phosphonate moiety, which together results in a spaced zwitterionic group. The resulting polymer may have the following structure:

[0086] In Formula (Ie-1), R ^a , X, n, m, p, R ^c , R ^d , R ^e , and R ^g are as defined earlier above.

The variables R ^c , R ^d , R ^e , and R ⁸ are selected from hydrogen atom and fluorinated hydrocarbon group containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms, except that a fluorine atom is not directly attached to nitrogen. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variable C ₂ ’ is a negatively charged group (e.g., carboxylate, sulfonate, phosphonate, phosphinate, sulfate, or phosphate). In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In separate or further specific embodiments, X is 0 or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In embodiments where C ₂ is a carboxylate moiety, the polymer of Formula (le-1) can generally be referred to as a poly(carboxybetaine). In embodiments where C ₂ " is a sulfonate moiety, the polymer of Formula (le-1) can generally be referred to as a poly(sulfobetaine). In embodiments where C ₂ is a phosphate moiety, the polymer of Formula (le-1) can generally be referred to as a poly (phosphobetaine). In some embodiments, R ^g and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fully fluorinated hydrocarbon group containing 1- 12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the zwitterionic polymer of Formula (Ie-1) is non-fluorinated.

[0087] In some embodiments, the polymers of Formula (Ie-1) have the following structure: (Ie-2)

[0088] In Formula (Ie-2), R ^a , X, m, p, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Each R ^e is independently selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a , wherein one or both R ^e may contain at least one fluorine atom. C1 ⁺ and C ₂ - are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C1-C2. The dashed line represents an optional bond. Any one or more of R ^a X, methylene group under m, R ^c , and R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, only or at least R ^c or R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2) _X -(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the zwitterionic polymer of Formula (Ie-2) is non-fluorinated.

[0089] In some embodiments, the polymers of Formula (Ie-2) have the following structure:

(Ie-3).

[0090] In Formula (Ie-3), R ^a , X, m, p, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. Each R ^e is independently selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a , wherein at least one R ^e contains at least one fluorine atom. C ₁ ⁺ and C ₂ " are positively charged and negatively charged atoms or groups, respectively, to form a zwitterionic moiety C ₁ ⁺ -C ₂ ". The dashed line represents an optional bond. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non-fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH ₂ ) _q contains at least one F atom. Any one or more of R ^a , X, methylene group under m, R ^c , and R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the zwitterionic polymer of Formula (Ie-3) is non-fluorinated.

[0091] In some embodiments, the polymers of Formula (Ie-3) have the following structure:

(Ie-4) [0092] In Formula (Ie-4), R ^a , X, m, p, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. R ^e is selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a . The variable C ₂ " is a negatively charged group, such as a carboxylate, sulfonate, sulfate, phosphonate, or phosphate group. The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non- fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2) _q contains at least one F atom. Any one or more of R ^a , X, methylene group under m, R ^c , and/or R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the zwitterionic polymer of Formula (Ie-4) is non- fluorinated.

[0093] In some embodiments, the polymer of Formula (Ie-4) may more particularly have any of the following structures: [0094] In Formulas (le-5) and (le-6), R ^a , X, m, p, R ^c , R ^d , and R ^e are as defined earlier above. The variable p is an integer of at least 1. The variable p may be, for example, 1, 2, 3, 4, 5, or 6, or an integer within a range bounded by any two of the foregoing values. The variables R ^c and R ^d are independently selected from hydrogen atom, hydrocarbon group, fluorine atom, and fluorinated hydrocarbon groups containing 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. R ^e is selected from hydrocarbon groups containing 1-12 carbon atoms and R ^a . The subscript q is an integer of precisely or at least 1. In different embodiments, q is precisely or at least 1, 2, 3, or 4, or q is within a range bounded by any two of the foregoing values. R ¹ is a fluorinated or non- fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms and precisely or at least one, two, three, or more fluorine atoms, as described above. In some embodiments, at least the carbon in R ¹ attaching to (CH2)q contains at least one F atom. Any one or more of R ^a , X, methylene group under m, R ^c , and/or R ^d is/are optionally substituted by fluorine or a fluorinated hydrocarbon group, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and at least one fluorine atom. In some embodiments, the zwitterionic polymer of Formula (Ie-4), (Ie-5), or (Ie-6) is non- fluorinated.

[0095] In particular embodiments of Formula (Ie-1), the zwitterionic polymers may have any of the following structures:

(Ie-1-2)

[0096] In the above formulas, R ^a , X, n, m, p, R ^c , R ^d , R ^e , R ^f , R ^g , and R ^h are as defined earlier above. The variables R ^c , R ^d , R ^e , R ^f , R ^g , and R ^h are independently selected from hydrogen atom, hydrocarbon groups containing 1-12 carbon atoms, and partially or fully fluorinated hydrocarbon groups containing 1-12 carbon atoms, which may contain precisely or at least one, two, three, four, five, or six fluorine atoms. The variable p is an integer of at least 1, as described earlier above. In some embodiments, R ^g and/or R ^e are selected from fluorinated hydrocarbon groups that do not contain an ether linkage or that do not contain oxygen and/or nitrogen atoms. In some embodiments, R ^g and/or R ^e has the formula -(CH2)x-(RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, 2-6, 2-4, or 3-6 carbon atoms. In some embodiments, only or at least R ^d is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms, or more particularly, the formula -(CH2)x (RF), wherein x is precisely or at least 1, 2, 3, 4, 5, or 6, and RF is a partially or fluorinated hydrocarbon group containing 1-12, 1-10, 1-8, or 1-6 carbon atoms. In some embodiments, the zwitterionic polymer of Formula (Ie-1-1), (Ie-1-2), (Ie-1-3), or (Ie-1-4) is non- fluorinated.

[0097] Notably, any of the above zwitterionic polymers containing an ammonium group may be modified by replacing N ⁺ in the structure with P ⁺ to generate an equivalent number of phosphonium zwitterionic polymers. Such polymers may have the general structure: or the following more particular structures: and

[0098] In other embodiments of Formula (Id), C ₁ is a phosphate moiety and C ₂ is a positively charged moiety, such as an ammonium or phosphonium moiety, which together results in a spaced zwitterionic group. The resulting polymers may have the following structure:

[0099] In Formula (If), R ^a , X, n, and m are as defined earlier above. The variable C ₂ ⁺ is a positively charged moiety that forms a zwitterionic spaced pair with the phosphate moiety in Formula (If). Some examples of positively charged moieties include ammonium (- and phosphonium moieties. The variable p is an integer of at least 1, as described earlier above. The variables R ^c , R ^d , and R ^f are selected from hydrogen atom, hydrocarbon group containing 1-12 carbon atoms, and fluorinated hydrocarbon group containing 1-12 carbon atoms containing precisely or at least one, two, three, four, five, or six fluorine atoms. In some embodiments, R ^f is alternatively absent. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In other separate or further specific embodiments, X is 0 or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In some embodiments, the zwitterionic polymer of Formula (If) is non-fluorinated.

[00100] In particular embodiments of Formula (If), C ₂ ⁺ is an ammonium moiety. The resulting polymers may have the following structure:

[00101] In Formula (If- 1), R ^a , X, n, and m are as defined earlier above. The variable p is an integer of at least 1, as described earlier above. The variables R ^c , R ^d , R ³ , R ⁴ , and R ⁵ are selected from hydrogen atom, hydrocarbon group containing 1-12 carbon atoms, and fluorinated hydrocarbon group containing 1-12 carbon atoms, which may contain precisely or at least one, two, three, four, five, or six fluorine atoms, wherein at least one of R ^c , R ^d , R ³ , R ⁴ , and R ⁵ is said fluorinated hydrocarbon group. In separate or further specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, R ^a is H or methyl. In separate or further specific embodiments, X is O or NR ^b , wherein R ^b is H or an alkyl group containing 1-3 carbon atoms, or R ^b is H or methyl. In some embodiments, the zwitterionic polymer of Formula (If- 1) is non-fluorinated.

[00102] Some specific examples of zwitterionic polymers within the above formulas include:

(xvi). In some embodiments, at least one hydrogen atom in each structure above is substituted with a fluorine atom or fluorinated hydrocarbon group. In some embodiments, any of the zwitterionic polymers above is non-fluorinated.

[00103] Other types of zwitterionic polymers within the scope of Formula (I) include:

(xxii), wherein q can have any of the values given above for m (e.g., at least 1). In some embodiments, at least one hydrogen atom in each structure is substituted with a fluorine atom or fluorinated hydrocarbon group. In some embodiments, any of the above zwitterionic polymers is non- fluorinated.

[00104] In other embodiments, the zwitterionic polymer is a mixed-charged zwitterionic polymer containing the following components: a. a plurality of first repeating units selected from negatively charged repeating units polymerized from monomers selected from 2-carboxyethyl acrylate, 2-carboxyethyl acrylate, 3-sulfopropyl methacrylate, lauryl methacrylate, and D-glucuronic acid, and repeating units having latent negatively charged groups polymerized from monomers selected from isobutyl methacrylate, 2,2,2-trifluoroethyl methacrylate, and ethyl glycolate methacrylate, reactive to provide negatively charged groups; and b. a plurality of second repeating units selected from positively charged repeating units polymerized from monomers selected from 2-(dimethylamino)ethyl methacrylate, 2- (diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl]trimethylammonium chloride, 2-aminoethyl methacrylate hydrochloride, and N-acetylglucosamine; and repeating units having latent positively charged groups polymerized from monomers selected from a monomer comprising an imide and a monomer comprising an oxyimino group, reactive to provide positively charged groups.

[00105] The zwitterionic polymers may alternatively be mixed-charged zwitterionic copolymers. Such polymers may be conveniently represented by the following formula:

[00106] In Formula (II), A’ and A” are backbones of different copolymer segments. L and L’ are same or different linking portions. The variable C ₁ ⁺ is a positively charged group, and C ₂ is a negatively charged group, wherein C ₁ ⁺ and C ₂ " together form a zwitterionic system. The variables n and n’ are independently selected from integers of at least 2. In some embodiments, at least one hydrogen atom in A’, A”, L, L’, C ₁ ⁺ , and/or C ₂ " in Formula (I) is substituted by a fluorinated hydrocarbon group or fluorine atom, wherein the fluorinated hydrocarbon group contains 1-12 carbon atoms and precisely or at least one, two, three, four, five, or six fluorine atoms. In other embodiments, the zwitterionic polymer of Formula (II) is non- fluorinated. In some embodiments, A’ and A” are olefinic. Notably, the polymer of Formula (II) may have any of the known copolymer arrangements, including block, alternating, periodic, and random arrangements. Moreover, although Formula (II) depicts a binary copolymer, the copolymer of Formula (II), may or may not contain one or more additional units or segments to result in a ternary or quaternary copolymer.

[00107] Any of the zwitterionic and non-zwitterionic polymers described in this application may function as a polymer in the biomolecule-polymer conjugate which is processed according to the method described here for isolating lower immunogenic forms of the biomolecule-polymer conjugate. In some embodiments, the polymer in the biomolecule- polymer conjugate is non-zwitterionic, such as a PEG-containing or polypeptide-containing polymer. In other embodiments, the polymer in the biomolecule-polymer conjugate is zwitterionic, such as any of the polymers of Formulas (I), (la), (lb), (Ic), (Ic-1), (Id), (le), (Ie-1), (Ie-1-1), (Ie-1-2), (le- 1-3), (Ie-1-4), (If), (If- 1), or (II), or a mixed-charged copolymer. The polymer may also be a copolymer containing at least two different units selected from PEG, polypeptide, and zwitterionic units, wherein the copolymer may be block, random, alternating, star, or brush. The copolymer may be, for example, a PEG- polypeptide, PEG-zwitterionic, polypeptide-zwitterionic, or PEG-polypeptide-zwitterionic copolymer. In some embodiments, the polymer may exclude PEG. Notably, any biomolecule disclosed in this application may be combined with any of the types of polymers described in this application to form a biomolecule-polymer conjugate.

[00108] Examples have been set forth below for the purpose of illustration and to describe the best mode of the invention at the present time. However, the scope of this invention is not to be in any way limited by the examples set forth herein.

EXAMPLES

[00109] Example 1

[00110] Overview

[00111] In this study, the following four synthetic polymers were studied: PEG, PCB, poly(2-methyl-2-oxazoline) (PmOX), and poly(2-ethyl-2-oxazoline) (PeOX), as typical examples to establish the ranking of the intrinsic hydrophobicity of the polymers. The structures of these polymers are shown in FIG. la. Before on-column tests, the polymer solubilities were first examined in two types of salted buffers, 0-4 M sodium chloride (NaCl) and 0-2 M ammonium sulfate (AS), which are also typical ‘salting out’ substances used in HIC. As a high salt buffer leads to polymer aggregation or liquid-liquid phase separation, polymer solubility can be monitored by the change of O.D. values at 350nm. Generally, all polymers steadily dissolved in the buffer containing up to 4 M of NaCl while only PeOX had a slight OD increase at the highest salt buffer. The difference in solubility was revealed in AS solutions, a stronger ‘salting out’ substance according to the Hofmeister series (A. M. Hyde et al., Org. Process Res. Dev., 21, 1355-1370, 2017). PeOX showed the lowest solubility limit, followed by MPEG and PmOX, which formed phase separation at 1.2M AS. Only PCB solution kept clear even in 2 M AS buffer. Thus, a solubility -based polymer hydrophobicity ranking may be roughly given as PCB<mPEG~PmOX<PeOX.

[00112] Column based HIC tests were then run to establish detailed hydrophobicity rankings. Typical HIC columns are functionalized by butyl, octyl, or phenyl groups. Based on the ‘like dissolves like’ rule, a butyl group functionalized HIC medium was selected to reveal potential hydrophobicity coming from polymer backbone or pendant groups. A column HIC run was performed as follows: polymers were first loaded and isocratically eluted at 4 M NaCl until all non-binding or weak binding portions passed through, followed by gradient elution from 4 M to 0 M NaCl to free trapped sample portions. The eluent profiles of polymers were monitored by UV signals at 214 nm. As the PEG backbone lacks spectral signals in the UV-Vis range, mono-maleimide modified derivatives were used to evaluate PEG hydrophobicity in all HIC tests. The conductivity of the eluent buffer was monitored simultaneously and the value corresponding to the washout peak was recorded to describe the strength of hydrophobic interactions between the polymer and the column.

[00113] Polymer solubility test

[00114] The solubility of polymers was measured by a plate-based method (Application note 28-9964-9949 AA, High-throughput screening of HIC media in PreDictor™ plates for capturing recombinant Green Fluorescent Protein from E. coli (2011). GE Healthcare Bio- Sciences AB). Generally, light scattering of polymer solutions (0.5mg/ml in 50mM phosphate buffer, pH=7.2) at 350 nm were recorded in the presence of two salt types (sodium chloride and ammonium sulfate) at different concentrations in 96- well UV- transparent microplates. O.D. at 350 nm was plotted with respect to salt concentration to determine the change in polymer solubility.

[00115] Hydrophobic interaction chromatography (HIC)

[00116] HIC experiments were performed on a pure protein purification system equipped with a butyl group functionalized HIC column (HiTrap Capto Butyl, 4.7 ml, GE health). 4M NaCl or 2M AS solution buffered with 50 mM phosphates (pH 7.2) were used as high salt mobile phases and for column equilibration. lOOpl of polymer solution (lOmg/ml) in the corresponding mobile phase was loaded onto the columns. Isocratic eluent was run at 1.0 ml/min for lOmin or until the complete elution of weak bounded peaks. All bounded peaks were washed out using a linear gradient eluent (lOmin at 1.0 ml/min, from 100% to 0% salt solution), followed by another lOmin of eluent with 50mM phosphate buffer. Sample elution was monitored by UV (214nm for polymers, 254nm for protein and conjugates) and conductivity detectors. [00117] Preparation of polymer KLH conjugates

[00118] Polymer-KLH conjugates were prepared according to a vendor provided protocol. Typically, amine-terminated polymers were first activated by Traut’s reagent (amine/Traut’s reagent: 1/2). The thiol activated polymers were purified by desalting column (Bio-rad) and then mixed with maleimide modified KLH in PBS (protein cone. 4mg/ml) and kept at 4°C overnight. Unreacted polymers and small molecular impurities were removed by extensive centrifugal filtration (MW cutoff, lOOkDa). 660 nm protein assay was used to determine KLH amount in every conjugate formulation. The corresponding unconjugated KLH was used as standard sample in the 660 nm protein assay. To determine polymer density of each KLH conjugate formulation, a PBS solution containing 5 mg of KLH was buffer exchanged with DI water and re-confirmed protein content by 660nm protein assay. Then the solution was lyophilized and weighed. Weight difference and related polymer molecular weight were used to calculate the number of attached polymer chains.

[00119] Preparation of PCB-Asp and PEG- Asp conjugates

[00120] Preparation of PCB-ASP and PEG- ASP conjugates were performed according to an earlier report (B. Li, et al., Nano Lett., 20(6), 4693-4699, 2011). 10 mg Asp was dissolved in 5 ml pH7.4 PBS buffer and then AMAS crosslinker (2.16mg, 40mg/mL in DMSO) was added into the solution. After Ih of incubation at room temperature, activated ASP (AMAS-ASP) was concentrated 3 times by centrifugal filtration (MW cutoff, 30k). PCB- SH (200mg, lOkDa) or mPEG-SH (lOOmg, 5kDa), prepared by the same method mentioned above, were mixed with 5 mg of AMAS-Asp, respectively. The mixtures were kept shaking at 4°C overnight. Unreacted polymers and small molecular impurities were removed by extensive centrifugal filtration (MW cutoff, lOOkDa). Protein contents were determined by 660 nm protein assay. Asp and Asp conjugates were further characterized by HIC mentioned above (4M NaCl to 0M NaCl, Capto butyl column). SDS-PAGE was used to characterize molecular weight and distribution of Asp and Asp conjugate formulations. Samples in reducing SDS loading buffer were loaded on a 4-12% polyacrylamide gel. An electrophoresis process was run in bis-tri buffer under 140V for 40 min. [00122] SEC for protein conjugates

[00123] Analytical SEC was performed on a quaternary high-performance liquid chromatography system equipped with an SEC column. Sample profiles were monitored by a UV detector, a multi-angle light scattering (MALS) detector, and a differential refractive index (dRI) detector. The flow rate was set at 0.5 mL/min with the mobile phase PBS (pH 7.4) containing 0.02% sodium azide as a preservative. Lab scale high-throughput SEC was performed on an FPLC system equipped with a SEC column (Superdex 200 increase 10/300 gl). PBS was used as mobile phase. Isocratic eluent was run at 0.7ml/min and samples were monitored by UV signal at 254nm.

[00124] Animal study

[00125] C57bl/6 mice, a widely used animal model in pharmaceutical evaluation for drugs, were chosen here to study the immunogenicity of polymer and polymer-protein conjugates. C57LB/6 mice (male, body weight 20-25 g) from Jackson Laboratories were randomized into treatment groups with a sample size of five animals per group. For polymer immunogenicity studies, mice were S.C. injected with polymer-KLH conjugates on day 1 at a dose of 2.5 mg/kg (protein amount) and boosted with another dose on day 15. All the mice were sacrificed on day 22 and serum were collected and stored at -20°C for further test. For immunogenicity studies of Asp conjugates, mice were S.C. injected with Asp or Asp conjugates at a dose of 2.5 mg/kg (protein amount) on the 1 ^st day of every week. After 3 doses of injection, all the mice were euthanized on day 28 and blood serum were collected and stored at -20°C for further test.

[00126] Antibody detection by indirect ELISA

[00127] Indirect ELISA is the main or the only widely accepted method to evaluate antibody (Ab) generation against a new antigen such as zwitterionic pCB polymer when the antigen- specific monoclonal antibody against this antigen is not available. Polymer-BSA conjugates, used in ELISA plate coatings for detecting anti-polymer Abs, were prepared with similar polymer density to reduce plate coating variations. Antigens used in anti- Asp Abs and anti- whole conjugate Abs tests were un-modified asparaginase and corresponding polymer- Asp conjugates that were injected into mice. To avoid interference from detergents, PEG based surfactants like Tween or Triton-X were excluded from solutions in the ELISA experiment. Typically, 100 μL of polymer-BSA conjugates or Asp (10 μg/mL of protein concentration) solution in 0.1 M sodium carbonate buffer (pH 9.4), was added into each well of the 96-well plates. Plates were incubated at 4 °C overnight to achieve antigen coating. After discarding antigen solutions, wells were washed five times using Tris buffered saline (TBS IX, pH 7.4) and then filled with 200ul of blocking buffer (1% BSA in IX TBS buffer). After incubation at room temperature for 1 h, blocking buffer was aspirated and all wells were washed by TBS buffer five times. Serial dilutions of serum samples in blocking buffer were added to the plates (100 pL/well) and the plates were incubated for 1 h at 37 °C. The plates were then washed five times with TBS buffer. Subsequently, goat anti-mouse secondary antibody HRP conjugate solution was added to the plates (100 pL/well, dilution 1:20000). After Ih of incubation at 37°C, the solution was discarded, and the plates were washed five times using TBS buffer. Then, 100 pL/well of HRP substrate was added. The plates were shaken for 30 min, and 50 pL stop solution (0.2 M H2SO4) was added to each well. Absorbance at 450 nm (signal) and 570 nm (background) was recorded by a microplate reader.

[00128] Statistics

[00129] All measurements are represented as mean ± standard deviation. The two-tailed student t-test was used to assess statistical differences between a pair of groups. Multiple comparisons were performed using one-way ANOVA followed by Tukey post hoc test. Significant difference was assumed at P < 0.05.

[00130] Discussion

[00131] HIC curves are shown in FIGS. Ib-lj. As shown, PeOX (10k) and mPEG (10k) showed the highest hydrophobicity as they completely bind to the column at 4 M NaCl. Eluent of binding PeOX(lOk) required a lower salt concentration (peak, 115.7 mS/cm), indicating its stronger column interactions than mPEG(lOk). Most of mPEG(5k) passed through the butyl column without decreasing salt concentration, but a broadened and delayed eluent peak profile indicated the existence of weak interactions. Changing the terminal group from methyl to hydroxyl did not significantly alter curve patterns, but both peaks of HO-PEG (5k) and HO-PEG (10k) left-shifted compared to methyl terminated derivatives, suggesting reduced hydrophobicity. No column binding occurred in HIC tests for PCB (10k) and PmOX (10k) as they eluted along with 4M NaCl immediately leaving sharp peaks. A huge difference was revealed after 4M NaCl was changed to 2M AS as an eluent buffer. PmOX (Figure li) completely bound to the column in 2M AS, and washing out the trapped PmOX molecules required a very low AS concentration (85.6 mS/cm, equals 0.82M). The peak of PCB (10k) slightly right-shifted compared to the one in 4M NaCl test, but no broadened peak occurred. This phenomenon was likely due to the shrinkage of the hydrodynamic size of PCB induced by a stronger 'salting out' effect rather than the consequence of hydrophobic interactions. The size exclusion effect of the porous column medium may be playing a main role here. To summarize, a more detailed hydrophobicity ranking was revealed as follows: PCB(10k)<<PmOX(10k)<HO- PEG(5k)~mPEG(5k) < HO-PEG(lOk)- mPEG (10k)< PeOX(lOk).

[00132] Next, each polymer was conjugated to a high immunogenic carrier protein, keyhole limpet hemocyanin (KLH), to induce anti-polymer Abs (B. Li et al., Angew. Chem. Int. Ed., 57, 13873-13876, 2018). Similar polymer density was achieved to make a fair comparison. Each group of C57bl/6 mice (n=5) was then treated with two doses of polymer- KLH conjugate via biweekly subcutaneous (s.c.) injections. Serum samples for the Ab test were collected one week later after the 2 ^nd injection and Ab titers were measured by indirect ELISA. For the quantitative comparison of polymer immunogenicity, a parameter called ‘immunogenicity index’ was employed (B. Li et al., supra), which was defined as an arithmetic sum of the log values of IgG and IgM titers. It should be noted that the theoretical minimum of immunogenicity index in the current study is 4 because serum dilution for IgG and IgM ELISA tests started from 100-fold.

[00133] The relationship between polymer immunogenicity and their intrinsic hydrophobicity is summarized in FIG. 2. Seven polymers can be divided into three groups based on the level of immunogenicity indexes. PCB (10k), with the most hydrophilicity, possessed the lowest immunogenicity. PEGs (5k) and PmOX (10k), which all passed through the butyl column at 4M NaCl, showed similar and modest immunogenicity index values. PEGs (10k) and PeOX (10k), that completely bound to the HIC column, triggered the highest level of anti-polymer Abs. The order of polymer immunogenicity indexes is estimated as: PCB (10k) < PmOX (10k) ~ HO-PEG (5k) ~ mPEG (5k) < HO-PEG (10k) ~ mPEG (10k) ~PeOX (10k), which matches with their hydrophobicity ranking. To better understand structural effects, polymers sharing similar structures were compared in their immunogenicity indexes. Additional methylene moiety in every repeating unit makes PeOX more hydrophobic than PmOX. As a result, PeOX induced higher antibody levels than PmOX did (P=0.0048). PEGs (10k) showed significantly higher immunogenicity indexes than PEGs (5k) as increased molecular weight enhanced overall hydrophobicity interactions. However, turning the methoxy terminal group into a more hydrophilic hydroxyl group did not provide a significant improvement over PEG immunogenicity. Previous reports showed that the terminal hydroxyl group of PEG induced stronger complement activation than the methoxy group terminated PEG (Y. Arima et al., Biomaterials, 29, 551-560, 2008). While OH is more hydrophilic, its complement activation should be also taking into account when it comes to polymer immunogenicity.

[00134] The correlation between polymer hydrophobicity and immunogenicity can provide a new way to mitigate anti-polymer Abs by reducing the hydrophobic feature of a polymer. In clinical practice, anti-drug Ab (ADA) for the whole polymer-protein conjugate is more meaningful because it is a combined result contributed by the immunogenicity of both protein and polymer. The measurement of ADA can also be performed in both preclinical and clinical trials. Thus, further efforts were aimed at determining whether the ‘hydrophobicity-immunogenicity’ correlation can be applied to the relationship between the hydrophobicity of a polymer-protein conjugate and its ADA. MPEG (5k) modified Asparaginase (Asp) with a similar structure to Oncaspar® (FDA approved Pegylated therapeutic protein), was prepared and studied here. 10k PCB-Asp conjugate was also prepared with similar polymer density and particle size for a fair comparison. SDS-PAGE and SEC data confirmed their compositions. Asp and Asp conjugates were then loaded on the butyl HIC column and eluted with 4M NaCl by the same method used in polymer tests.

[00135] Native Asp (FIG. 3a) showed a similar curve pattern as 10k PEGs and PeOX did but needed a lower NaCl concentration to be released from the column, which indicates its high level of hydrophobicity. After modification with 5k mPEG, the entire PEG- Asp conjugates (FIG. 3b) still tightly bind to the HIC column at 4 M NaCl ,but the main eluent peak advanced from 43.6 to 51.9 mS/cm compared to Asp protein. Although 5k mPEG showed very weak column binding at 4 M NaCl, it only slightly reduced the apparent hydrophobicity of the Asp conjugate. Modification of Asp with multiple PEG chains made the protein conjugate more like a large star-shaped mPEG nanoparticle; thus, the HIC profile of mPEG- Asp conjugate was predicted to behave like high molecular weight PEGs.

[00136] A large contrast was observed in PCB-Asp conjugate (FIG. 3c). Most of PCB-Asp freely passed through the butyl column as same as free PCB polymer did although the apparent molecular weight of PCB increased tens of times. Antibodies were induced in C57BL/6 mice by three weekly s.c. injections of Asp or Asp conjugates. The calculated immunogenicity index of the whole PEG- Asp conjugate reached 5.99 while PCB-Asp did not induce any positive signals (FIG. 3d). This result confirmed that the apparent hydrophobicity of polymer-protein conjugates may also be used to predict their potential immunogenicity.

[00137] With a similar purpose, Anti- Asp immunogenicity indexes for Asp, PEG- Asp and PCB-Asp were further compared (FIG. 3e). As shown, these correlate well with their hydrophobicity ranking as shown in FIGS. 3a, 3b and 3c. PEG reduced Asp immunogenicity by shielding surface epitopes, which was reflected as improved hydrophilicity. It is noted that the anti-protein index was lower than the anti-conjugate index detected in those mice treated with PEG- Asp, indicating the existence of PEG- specific Abs.

[00138] PCB significantly mitigated Asp immunogenicity, but anti-Asp Abs were still detected in PCB-Asp treated mice although small. Thus, the non-binding portion of PCB- Asp from HIC purification (or further purified PCB-ASP conjugate) was further tested in mice by the same three-injection route. No anti-Asp Abs could be detected in this experiment (FIG. 3f). Interestingly, SEC curves and SDS-PAGE did not reveal any significant difference caused by HIC treatment. A slight increase in average PCB density from 9.1 to 9.3 chains per Asp monomer was found by SEC-MALS measurement. There are many factors that can affect the surface chemistry and hydrophilicity of polymer-protein conjugates such as the hydrophilicity, molecular weight, chain number, and distribution of a polymer to shield the hydrophobic regions of a protein. The HIC method in this work picks up the overall most hydrophilic polymer-protein conjugates. The conventional purification of polymer-protein conjugates highly relies on liquid chromatography technologies like SEC, ion exchange, hydrophobic interaction, or affinity chromatography. These techniques are effective to separate mono-, di-, or even tri-polymer conjugated proteins from the reaction mixture but the process becomes tedious, and inefficient, particularly when completely shielding polymers over the polymer-protein conjugate is required. By combining the super-hydrophilic PCB modification and the HIC purification technique, the best PCB-protein conjugates can be selectively collected in a scalable process, which benefits both drug production and quality control. [00139] Example 2

[00140] Uricase proteins from two species, Candida sp (casp). and bacillus fastidiosus (BF) were conjugated with PCB polymer (MW 20,000 Da) according to a similar procedure that was used to prepare PCB-Asp as described above. Generally, 10 mg Uricase was dissolved in 4 ml pH 7.4 PBS buffer and then AMAS crosslinker (2.16 mg prepared at 40mg/mL in DMSO) was added into the solution. After Ih of incubation at room temperature, activated Uricase (AMAS-Uri) was concentrated 3 times by centrifugal filtration (MW cutoff, 30kDa). PCB-SH (200 mg, 20kDa), prepared by the same method mentioned above, was mixed with 5 mg of AMAS-Uri, respectively. The mixture was shaken at 4°C overnight. Unreacted polymers and small molecular impurities were removed by extensive centrifugal filtration (MW cutoff, lOOkDa). Protein contents were determined by 660 nm protein assay. Uricase and PCB conjugates were further characterized by hydrophobic interaction chromatography (HIC) mentioned above (4M NaCl to 0M NaCl, Capto butyl column). SDS-PAGE and size exclusion chromatography (SEC) were used to characterize molecular weight and distribution of Uricases and PCB-Uricase conjugates.

[00141] HIC profiles of native Uricase casp and PCB-20k-Uricase casp conjugate are similar to Asp and PCB-Asp, respectively. As shown in FIG. 4a and 4b, Uricase casp tightly binds to the HIC column at 4 M NaCl while a part of PCB 20k modified Uricase casp could freely pass through the HIC column. It is reported that Uricase from Candida sp. has almost 12 water- accessible lysines per uricase monomer. This supports the observation that a single step of PCB conjugation successfully yields high PCB-density uricase conjugates. Conjugates with the highest hydrophilicity could be isolated by the illustrated HIC process (FIG 4a and 4b). The same procedure was also performed to purify PCB-20k- uricase BF. As shown in FIGS. 4c and 4d, one-step PCB-20k modification did not introduce enough polymer onto the protein to pass through the HIC column at 4M NaCl. A structure simulation was performed to check if Uricase BF has similar lysine numbers available for conjugation as the Uricase casp. Based on a published crystal structure of a Uricase BF (PDB file 4R8X), it was herein found that there are only 2-3 water-accessible lysines per uricase BF monomer, which explains the observation that the single step PCB- 20k conjugation could not shield the hydrophobic portions on Uricase BF tetramer. To further increase PCB density, a second conjugation step was performed by reacting PCB- 20k-NH2 with carboxy groups on the protein surface. 100 mg PCB-20k-NH2, 1 mg EDC and 0.5 mg NHS were added into a MOPS solution (pH 6.5) containing 2.5 mg of PCB- 20k-Uricase BF. The whole mixture was stirred at 4°C overnight. Unreacted polymers and small molecules were removed by dialysis. Double conjugated PCB -20k- Uricase BF was also tested on the HIC column and a sharp unbound peak was observed (FIG. 4e). SEC (FIG. 4f) and SDS-PAGE (FIG. 4g) assay confirmed the formation of a new conjugate with higher PCB density.

[00142] Example 3

[00143] Uricase BF and related PCB conjugates were used to control kidney maldevelopment in a uricase-deficient mouse model. Uricase-deficient mice were injected with 5 mg/kg of uricase or uricase conjugates intravenously for three weekly doses. Mice were sacrificed two weeks after the last dose. Serum and kidneys were harvested for analysis. The HIC purified PCB double conjugated Uricase BF showed a significant reduction in immunogenicity compared to the PCB double conjugated Uricase BF that was not processed through the HIC (FIG 5a). FIG 5b shows HIC purified PCB double conjugated Uricase BF also maintained kidney growth in uricase-deficient mice comparable to the heterozygote (healthy control) mice. This demonstrates that even with highly polymer conjugated Uricase, the use of the HIC purification method plays a key role in improving drug safety and efficacy.

[00144] The above experimental results established rankings in hydrophobicity for hydrophilic polymers and their polymer-protein conjugates by the HIC technique. Immunogenicity reflected by the generation of Abs was also evaluated for these polymers and their polymer-protein conjugates. The results demonstrated a relationship between polymer hydrophobicity and immunogenicity. Higher polymer hydrophobicity induced a higher level of anti-polymer Abs when the polymer is conjugated to an immunogenic protein. The correlation became more apparent when those polymers sharing a similar structure were compared. Although the reduction in polymer hydrophobicity by altering polymer structures or reducing molecular weights may mitigate polymer immunogenicity issues to a certain extent, using a super-hydrophilic alternative and further combining with HIC purification has herein been shown to be a useful alternative means for mitigating polymer immunogenicity in principle and practice.

[00145] While there have been shown and described what are at present considered the preferred embodiments of the invention, those skilled in the art may make various changes and modifications which remain within the scope of the invention defined by the appended claims.