Determination of degree of modification of therapeutic proteins by 1h-nmr spectroscopy

Abstract

To provide a method for determining the average number of first polymers covalently bound to a protein-first polymer conjugate in solution.SOLUTION: The present invention includes: a) performing measurement of 1H nuclear magnetic resonance on a solution containing a protein-first polymer conjugate; b) processing measurement values of the 1H nuclear magnetic resonance; c) integrating the measurement values of the 1H nuclear magnetic resonance at a first type of proton peak; d) calibrating the result of c); e) integrating the measurement values of the 1H nuclear magnetic resonance at a second type of proton peak, the second type of proton peak including a signal generated by at least one proton in the first polymer; f) dividing a result of e) by the number of second type protons per monomer of the first polymer; and g) dividing the result of f) by the average number of monomers in the first polymer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 62 / 980,757, filed February 24, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Protein modification has been used for many years to enhance the pharmacokinetic properties of biologically active molecules. Water-soluble polymers such as polyethylene glycol (PEG), polysialic acid (PSA), dextran, hydroxyethyl starch (HES), and their derivatives conjugated to proteins can dramatically improve the water solubility of the modified molecules, reduce immunogenicity through a shielding effect, and protect biomolecules from proteolytic enzymes, all of which can effectively extend their in vivo half-life. Numerous long-acting therapeutic proteins are on the market, most of which are based on PEGylation technology (Turecek et al., J. Pharm. Sci. 2016, 105, 460-475).

[0003] One of the key parameters for analytical characterization of these conjugates is the modification degree (the number of water-soluble polymers attached to the protein). Obtaining the modification degree of these conjugates is important for monitoring industrial production processes and for consistent product quality.

[0004] Determination of the number of polymers bound to proteins (degree of modification) in polymer-protein conjugates Traditionally, determining the degree of modification has involved at least two or three experimental measurements.

[0005] First, the protein concentration of the conjugate in the sample was confirmed. Assays that can determine protein concentration include fluorescent / spectrophotometric assays, the Lowry assay, the bicinchoninic acid (BCA) assay, the Bradford assay, copper ion-based assays, and several other UV and colorimetric protein assays.

[0006] Second, the concentration of the water-soluble polymer in the conjugate in the sample was determined. The test depends on the type of polymer attached, and each polymer has its own assay. In some assays, the concentration of the water-soluble polymer is determined by experimentally measuring the total water-soluble polymer in the sample and also experimentally measuring the free water-soluble polymer in the sample.

[0007] For example, the concentration of PSA in a protein sample is measured by the resorcinol assay. In this assay, the polymer is completely hydrolyzed under strongly acidic conditions, and the formation of colored complexes between each monomer and resorcinol is monitored photometrically. The final PSA concentration can be determined using a calibration curve. A drawback of this assay is that the sample is destroyed during the analysis process.

[0008] As another example, the concentration of PEG is determined by an HPLC-based method. A drawback of this assay is that the sample is destroyed during the analysis.

[0009] Each experimental measurement introduces some degree of error, reducing the precision of the obtained values. These errors can have serious consequences when high-purity pharmaceutical products are desired. Lack of precision in the obtained values may require multiple tests to determine the correct values of release parameters and / or stricter release specifications to ensure a compliant product is released. Tighter release specifications may result in desirable batches of product being unnecessarily discarded. Lack of precision in determining the target level of protein modification may make it more difficult to determine the optimal conditions for polymer-protein coupling and may require multiple tests for confirmation. Therefore, lack of precision may also increase the time it takes to develop an optimal manufacturing process for polymer-protein conjugates. Lack of precision may also affect the product being developed.

[0010] Providing high accuracy modification analysis with a reduced number of experimental measurements improves quality control analysis in industrial manufacturing processes. This and other advantages are provided by the invention described herein. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Turecek et al.,J.Pharm.Sci.2016,105,460-475 Summary of the Invention

[0012] The present invention relates, inter alia, to a method for determining the average number of first polymers covalently attached to a protein-first polymer conjugate in solution, comprising: a) For a solution containing a protein-first polymer conjugate 1 performing H nuclear magnetic resonance measurements; b) the relevant 1 processing the H nuclear magnetic resonance measurements; c) the corresponding peak of the first proton 1integrating H nuclear magnetic resonance measurements, wherein the first type proton peak comprises a signal generated by at least one proton on the side chain of at least one amino acid in the protein-first polymer conjugate; d) calibrating the results of c); e) The corresponding second proton peak 1 integrating H nuclear magnetic resonance measurements, wherein the second type proton peak comprises a signal generated by at least one proton in the first polymer; f) dividing the result of e) by the number of second type protons per monomer of the first polymer; and g) dividing the result of f) by the average number of monomers in the first polymer. thereby determining the average number of first polymers covalently attached to protein-first polymer conjugates in solution. [Brief explanation of the drawings]

[0013] [Figure 1] NMR integral of H3 versus PSA degree of rFIX-PSA mixture. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention I. Definitions and Abbreviations As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to an "active agent" includes a single active agent as well as a combination of two or more different active agents. It is understood that the present teachings are not limited to the particular dosage forms, carriers, or the like disclosed herein, as these may vary.

[0015] The abbreviations used herein generally have their conventional meaning within the chemical and biological arts.

[0016] As used herein, the term "poly" means at least 2. For example, a polyvalent metal ion is a metal ion with a valency of at least two.

[0017] "Moiety" refers to a radical of a molecule that is attached to the rest of the molecule.

[0018] symbol TIFF2025118907000002.tif4128, whether used as a bond or displayed perpendicular to a bond, indicates the point at which the displayed moiety is attached to the rest of the molecule.

[0019] The phrase "pharmaceutically acceptable" means, within the scope of medical judgment, moieties or compounds suitable for use in humans without causing undesired biological effects such as, for example, excessive toxicity, irritation, allergic response, and the like.

[0020] As used herein, the term "protein" refers to a series of 10 or more covalently linked amino acids that perform a biological function. Protein molecules contemplated include full-length proteins, precursors of full-length proteins, biologically active subunits or fragments of full-length proteins, and biologically active derivatives and variants of any of these forms of proteins. Thus, the term protein includes (1) proteins having greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more amino acid sequence identity to a polypeptide encoded by a reference nucleic acid described herein or to an amino acid sequence described herein over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids, and / or (2) proteins that specifically bind to antibodies (e.g., polyclonal or monoclonal antibodies) generated against an immunogen comprising a reference amino acid sequence described herein, an immunogenic fragment thereof, and / or a conservatively modified variant thereof.

[0021] As used herein, a "biologically active derivative" or "biologically active variant" includes any derivative or variant of a molecule that has substantially the same functional properties (e.g., binding properties) and / or biological properties and / or the same structural basis (e.g., peptide backbone or basic polymer unit) of the molecule.

[0022] An "analog," such as a "variant" or "derivative," is a compound, such as a protein or a first polymer, that is substantially similar in structure and possesses the same biological activity, although in some cases the biological activity may be to a different extent. For example, a polypeptide variant refers to a polypeptide that shares a substantially similar structure and possesses the same biological activity as a reference polypeptide. Variants or analogs differ in their amino acid sequence composition compared to the naturally occurring polypeptide from which the analog is derived based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and / or at one or more internal regions (e.g., fragments) of the naturally occurring polypeptide sequence, (ii) insertion or addition of one or more amino acids at one or more termini of the polypeptide (typically "additions" or "fusions") and / or at one or more internal regions (typically "insertions") of the naturally occurring polypeptide sequence, or (iii) substitution of one or more amino acids for other amino acids in the naturally occurring polypeptide sequence. By way of example, a "derivative" is a type of analog and refers to a polypeptide that shares the same or substantially similar structure as, for example, a chemically modified reference polypeptide.

[0023] Variant polypeptides are a type of analog polypeptide, including insertional variants, in which one or more amino acid residues are added to the amino acid sequence of a protein of the present invention. The insertions can be located at either or both termini of the protein and / or within internal regions of the protein amino acid sequence. Insertional variants with additional residues at either or both termini include, for example, fusion proteins and proteins containing amino acid tags or other amino acid labels. In one embodiment, the blood coagulation protein molecule optionally contains an N-terminal Met when the molecule is recombinantly expressed in bacterial cells such as E. coli.

[0024] Deletion variants have one or more amino acid residues removed from the protein polypeptides described herein. Deletions can be made at one or both ends of the protein polypeptide and / or by removing one or more residues within the protein amino acid sequence. Thus, deletion variants include fragments of the protein polypeptide sequence.

[0025] In substitution variants, one or more amino acid residues of a protein polypeptide are removed and replaced with alternative residues. In one embodiment, the substitutions are conservative in nature, and conservative substitutions of this type are well known in the art. Alternatively, the present invention encompasses substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp. 71-77] and are listed below. conservative substitution TIFF2025118907000003.tif100170

[0026] Alternatively, exemplary conservative substitutions are listed here. Conservative substitution II TIFF2025118907000004.tif153170

[0027] The present invention may suitably comprise, consist of, or consist essentially of the elements of the claims set out herein.

[0028] II. Introduction The present invention provides a method for determining the degree of modification of water-soluble polymer-protein conjugates by quantitative nuclear magnetic resonance (Q-NMR). Q-NMR measurement of the degree of modification offers numerous advantages over conventional methods, including at least the following: First, Q-NMR data is acquired in a single measurement, whereas conventional methods require two or more measurements. Fewer measurements reduces overall error. Second, Q-NMR requires less sample than conventional methods, which may reduce this requirement by at least 50%. Third, Q-NMR sample preparation is simpler than conventional sample preparation. In an exemplary embodiment, a Q-NMR sample is simply prepared by adding 10% DO to the material and transferring it to an NMR tube. Fourth, Q-NMR analysis is non-destructive to the sample, whereas conventional methods, such as resorcinol testing, are. Sample recovery is a major advantage of Q-NMR analysis. Fifth, Q-NMR measurements are collected on a timescale of seconds / minutes, whereas conventional measurements often require several hours to collect.

[0029] Proton NMR spectroscopy Proton nuclear magnetic resonance ( 1 H-NMR (also known as NMR) is a spectroscopic technique that detects energy absorbed by changes in nuclear spin states. NMR uses large magnets to probe the intrinsic spin properties of atomic nuclei. Like all spectroscopic techniques, NMR uses components of electromagnetic radiation (radio frequency) to drive transitions (resonances) between nuclear energy levels. NMR is the only spectroscopic technique with inherently quantitative resonance intensities. Indeed, NMR resonance decay coefficients are identical for all nuclei of a particular type, eliminating the need for calibration with reference materials for each detected resonance. In an exemplary embodiment, the NMR spectroscopy is not 2D-NMR spectroscopy.

[0030] Quantitative proton NMR (Q-NMR): Q-NMR has been in use for decades, and spectrometers have now reached a level of sensitivity where good quantitative data can be routinely obtained.

[0031] III. method In an exemplary embodiment, the invention provides a method for determining the average number of first polymers covalently attached to a protein-first polymer conjugate in a solution. The method comprises: a) For a solution containing a protein-first polymer conjugate 1 performing H nuclear magnetic resonance measurements; b) the relevant 1 processing the H nuclear magnetic resonance measurements; c) the corresponding peak of the first proton 1 integrating H nuclear magnetic resonance measurements, wherein the first type proton peak comprises a signal generated by at least one proton on the side chain of at least one amino acid in the protein-first polymer conjugate; d) calibrating the results of c); e) The corresponding second proton peak 1 integrating H nuclear magnetic resonance measurements, wherein the second type proton peak comprises a signal generated by at least one proton in the first polymer; f) dividing the result of e) by the number of second type protons per monomer of the first polymer; and g) dividing the result of f) by the average number of monomers in the first polymer. thereby determining the average number of first polymers covalently attached to the protein-first polymer conjugates in the solution.

[0032] Protein-first polymer conjugate In exemplary embodiments, the protein in the protein-first polymer conjugate is described herein. In exemplary embodiments, the protein in the protein-first polymer conjugate is Factor VIII. In exemplary embodiments, the protein in the protein-first polymer conjugate is full-length Factor VIII. In exemplary embodiments, the protein in the protein-first polymer conjugate is B-domain deleted Factor VIII. In exemplary embodiments, the protein in the protein-first polymer conjugate is Factor IX. In exemplary embodiments, the protein in the protein-first polymer conjugate is Factor VIIa. In exemplary embodiments, the protein in the protein-first polymer conjugate is von Willebrand factor. In exemplary embodiments, the protein in the protein-first polymer conjugate is bovine pancreatic trypsin inhibitor. In exemplary embodiments, the protein in the protein-first polymer conjugate is albumin. In exemplary embodiments, the protein in the protein-first polymer conjugate is bovine serum albumin. In an exemplary embodiment, the protein in the protein-first polymer conjugate is human serum albumin. In an exemplary embodiment, the protein in the protein-first polymer conjugate is granulocyte colony-stimulating factor (G-CSF). In an exemplary embodiment, the protein in the protein-first polymer conjugate is phenylalanine ammonia lyase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is adenosine deaminase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is asparaginase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is L-asparaginase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is erythropoietin. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon.In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2a. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2b. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon beta-1a. In an exemplary embodiment, the protein in the protein-first polymer conjugate is tumor necrosis factor. In an exemplary embodiment, the protein in the protein-first polymer conjugate is tumor necrosis factor alpha. In an exemplary embodiment, the protein in the protein-first polymer conjugate is human growth hormone. In an exemplary embodiment, the protein in the protein-first polymer conjugate is uricase.

[0033] In exemplary embodiments, the first polymer in the protein-first polymer conjugate is described herein. In exemplary embodiments, the first polymer in the protein-first polymer conjugate is polyethylene glycol (PEG). In exemplary embodiments, the first polymer in the protein-first polymer conjugate is polysialic acid (PSA).

[0034] In exemplary embodiments, a protein-first polymer conjugate is described herein. In exemplary embodiments, the protein-first polymer conjugate is Factor VIII-PEG. In exemplary embodiments, the protein-first polymer conjugate is full-length Factor VIII-PEG. In exemplary embodiments, the protein-first polymer conjugate is B-domain deleted Factor VIII-PEG. In exemplary embodiments, the protein-first polymer conjugate is Factor IX-PEG. In exemplary embodiments, the protein-first polymer conjugate is Factor VIIa-PEG. In exemplary embodiments, the protein-first polymer conjugate is von Willebrand factor-PEG. In exemplary embodiments, the protein-first polymer conjugate is bovine pancreatic trypsin inhibitor-PEG. In exemplary embodiments, the protein-first polymer conjugate is albumin-PEG. In exemplary embodiments, the protein-first polymer conjugate is bovine serum albumin-PEG. In an exemplary embodiment, the protein-first polymer conjugate is human serum albumin-PEG. In an exemplary embodiment, the protein-first polymer conjugate is granulocyte colony-stimulating factor-PEG (G-CSF-PEG). In an exemplary embodiment, the protein-first polymer conjugate is phenylalanine ammonia-lyase-PEG. In an exemplary embodiment, the protein-first polymer conjugate is adenosine deaminase-PEG. In an exemplary embodiment, the protein-first polymer conjugate is asparaginase-PEG. In an exemplary embodiment, the protein-first polymer conjugate is L-asparaginase-PEG. In an exemplary embodiment, the protein-first polymer conjugate is erythropoietin-PEG. In an exemplary embodiment, the protein-first polymer conjugate is interferon-PEG. In an exemplary embodiment, the protein-first polymer conjugate is interferon α-2-PEG.In an exemplary embodiment, the protein-first polymer conjugate is interferon alpha-2a-PEG. In an exemplary embodiment, the protein-first polymer conjugate is interferon alpha-2b-PEG. In an exemplary embodiment, the protein-first polymer conjugate is interferon beta-1a-PEG. In an exemplary embodiment, the protein-first polymer conjugate is tumor necrosis factor-PEG. In an exemplary embodiment, the protein-first polymer conjugate is tumor necrosis factor alpha-PEG. In an exemplary embodiment, the protein-first polymer conjugate is human growth hormone-PEG. In an exemplary embodiment, the protein-first polymer conjugate is uricase-PEG.

[0035] In an exemplary embodiment, the protein-first polymer conjugate is Factor VIII-PEG. In an exemplary embodiment, the protein-first polymer conjugate is full-length Factor VIII-PSA. In an exemplary embodiment, the protein-first polymer conjugate is B-domain deleted Factor VIII-PSA. In an exemplary embodiment, the protein-first polymer conjugate is Factor IX-PSA. In an exemplary embodiment, the protein-first polymer conjugate is Factor VIIa-PSA. In an exemplary embodiment, the protein-first polymer conjugate is von Willebrand factor-PSA. In an exemplary embodiment, the protein-first polymer conjugate is bovine pancreatic trypsin inhibitor-PSA. In an exemplary embodiment, the protein-first polymer conjugate is albumin-PSA. In an exemplary embodiment, the protein-first polymer conjugate is bovine serum albumin-PSA. In an exemplary embodiment, the protein-first polymer conjugate is human serum albumin-PSA. In an exemplary embodiment, the protein-first polymer conjugate is granulocyte colony-stimulating factor-PSA (G-CSF-PSA). In an exemplary embodiment, the protein-first polymer conjugate is phenylalanine ammonia-lyase-PSA. In an exemplary embodiment, the protein-first polymer conjugate is adenosine deaminase-PSA. In an exemplary embodiment, the protein-first polymer conjugate is asparaginase-PSA. In an exemplary embodiment, the protein-first polymer conjugate is L-asparaginase-PSA. In an exemplary embodiment, the protein-first polymer conjugate is erythropoietin-PSA. In an exemplary embodiment, the protein-first polymer conjugate is interferon-PSA. In an exemplary embodiment, the protein-first polymer conjugate is interferon alpha-2-PSA. In an exemplary embodiment, the protein-first polymer conjugate is interferon alpha-2a-PSA.In an exemplary embodiment, the protein-first polymer conjugate is interferon alpha-2b-PSA. In an exemplary embodiment, the protein-first polymer conjugate is interferon beta-1a-PSA. In an exemplary embodiment, the protein-first polymer conjugate is tumor necrosis factor-PSA. In an exemplary embodiment, the protein-first polymer conjugate is tumor necrosis factor alpha-PSA. In an exemplary embodiment, the protein-first polymer conjugate is human growth hormone-PSA. In an exemplary embodiment, the protein-first polymer conjugate is uricase-PSA.

[0036] a) 1 H nuclear magnetic resonance measurements In an exemplary embodiment, a solution is provided in which the protein-first polymer conjugate is dissolved. 1 The time required to perform H nuclear magnetic resonance measurements is approximately proportional to the concentration of the protein-first polymer conjugate in solution. In other words, the total measurement time for a diluted protein-first polymer conjugate solution may be longer than for a more concentrated protein-first polymer conjugate solution. If the solution is too dilute, an acceptable signal-to-noise ratio may not be obtained within a reasonable total measurement time. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 1.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 1.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 2.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 2.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 2.5. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 2.5 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 2.8. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 2.8 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 3.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 3.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 3.2. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 3.2 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 3.4. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 3.4 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 3.8. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 3.8 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 4.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 4.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 4.5. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 4.5 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 5.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 5.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 6.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 6.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 7.0. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 7.0 and 20. In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 8.0. In an exemplary embodiment, the signal to noise ratio of the solution is between 8.0 and 20. In an exemplary embodiment, the signal to noise ratio of the solution is greater than 9.0. In an exemplary embodiment, the signal to noise ratio of the solution is between 9.0 and 20.In an exemplary embodiment, the signal-to-noise ratio of the solution is greater than 10. In an exemplary embodiment, the signal-to-noise ratio of the solution is between 10 and 20. The signal-to-noise ratio can be calculated based on the ratio of the peak heights of the spectra.

[0037] In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.01 mg / mL to about 10 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.01 mg / mL to about 0.1 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.1 mg / mL to about 1 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 1 mg / mL to about 10 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.01 mg / mL to about 1 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.1 mg / mL to about 10 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.08 mg / mL to about 0.3 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.15 mg / mL to about 0.5 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.25 mg / mL to about 0.75 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.33 mg / mL to about 1.0 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.2 mg / mL to about 0.6 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.3 mg / mL to about 0.65 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.5 mg / mL to about 1.0 mg / mL. In exemplary embodiments, the concentration of the protein-first polymer conjugate in the solution is from about 0.65 mg / mL to about 1.2 mg / mL. In an exemplary embodiment, the concentration of the protein-first polymer conjugate in the solution is from about 0.45 mg / mL to about 0.9 mg / mL.

[0038] In an exemplary embodiment, the protein-first polymer conjugate is purified from the reaction mixture in which it was synthesized to remove materials such as unconjugated first polymer. In an exemplary embodiment, the protein-first polymer conjugate solution is purified. In an exemplary embodiment, the protein-first polymer conjugate is 1 Prior to performing H nuclear magnetic resonance measurements, the compound is purified from the reaction mixture in which it was synthesized. In an exemplary embodiment, the purification is by a member selected from the group consisting of ion exchange chromatography, size exclusion chromatography, affinity chromatography, ultrafiltration, diafiltration, hydrophilic interaction chromatography, normal phase chromatography, reverse phase chromatography, and simulated moving bed chromatography.

[0039] The solution in which the protein-first polymer conjugate is dissolved is a liquid capable of dissolving a provided amount of the protein-first polymer conjugate. Examples of solvents suitable for dissolving the protein-first polymer conjugate include deuterated solvents. Examples of deuterated solvents include DO, MeOD, and deuterated DMSO. In an exemplary embodiment, the solvent is about 1% DO to about 99.9% DO. In an exemplary embodiment, the solvent is about 1% to about 99.9% DO selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 1% DO to about 10% DO. In an exemplary embodiment, the solvent is about 1% to about 10% DO selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 9% DO to about 20% DO. In an exemplary embodiment, the solvent is about 9% to about 20% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 19% to about 30% DO. In an exemplary embodiment, the solvent is about 19% to about 30% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 29% to about 40% DO. In an exemplary embodiment, the solvent is about 29% to about 40% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 39% to about 50% DO. In an exemplary embodiment, the solvent is about 39% to about 50% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 49% DO to about 60% DO. In an exemplary embodiment, the solvent is about 49% to about 60% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 59% DO to about 70% DO.In an exemplary embodiment, the solvent is about 59% to about 70% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 69% DO to about 80% DO. In an exemplary embodiment, the solvent is about 69% to about 80% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 79% DO to about 90% DO. In an exemplary embodiment, the solvent is about 79% to about 90% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. In an exemplary embodiment, the solvent is about 89% DO to about 99% DO. In an exemplary embodiment, the solvent is about 89% to about 99% deuterated solvent selected from DO, MeOD, deuterated DMSO, and combinations thereof. For any of the embodiments in this paragraph, the remaining solvent is HO. In an exemplary embodiment, the solvent is about 7% to about 13% DO, with the remaining solvent contributed by HO. For any of the embodiments in this paragraph, the amount of deuterated solvent also includes MeOD or deuterated DMSO. For example, the solvent is HO. In an exemplary embodiment, an additional component is present in the solution. In an exemplary embodiment, an additional component such as tetramethylsilane, 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS), 3-(trimethylsilyl)propion-2,2,3,3-d4 acid sodium salt (TMSP-d4), or octamethylcyclotetrasiloxane (D4) is used for calibration purposes. In an exemplary embodiment, the additional component can help maintain the protein-first polymer conjugate in solution, for example, in MeOD or deuterated DMSO. These additional components may have protons that do not interfere with the collection of data for the first or second proton peaks. In an exemplary embodiment, the protein-first polymer conjugate concentration in solution is about 1 mg / mL.

[0040] In an exemplary embodiment, the solution is provided in an NMR tube.1 The quality of a H nuclear magnetic resonance measurement is proportional to the diameter of the NMR tube. In an exemplary embodiment, a solution in an NMR tube with a smaller diameter is measured for a longer period of time than the same sample in an NMR tube with a larger diameter. A standard NMR tube has a diameter of approximately 5 mm. Larger diameter NMR tubes require more sample volume.

[0041] In an exemplary embodiment, the NMR tube has a diameter of about 1 mm to about 20 mm. In an exemplary embodiment, the NMR tube has a diameter of about 1 mm to about 10 mm. In an exemplary embodiment, the NMR tube has a diameter of about 3 mm to about 7 mm. In an exemplary embodiment, the NMR tube has a diameter of about 5 mm.

[0042] In an exemplary embodiment, the NMR tube is standard size. In an exemplary embodiment, the NMR tube has a dimension of 5 mm x 4.2 mm (outer diameter x inner diameter). In an exemplary embodiment, the NMR tube has a length of about 5 inches to about 9 inches. In an exemplary embodiment, the NMR tube has a length of about 7 inches.

[0043] In an exemplary embodiment, the solution is introduced into an apparatus capable of performing nuclear magnetic resonance measurements. In an exemplary embodiment, the manufacturer of the NMR apparatus is Bruker. NMR apparatus that may be utilized in the present invention are described on the Bruker website: https: / / www.bruker.com / en / products-and-solutions / magnetic-resonance.html. Additional NMR instrument manufacturers include Agilent Technologies (Varian NMR spectrometers), Anasazi Instruments (https: / / www.aiinmr.com), Jeol (https: / / www.jeol.co.jp / en / products / category_nmr.html), Magritek (https: / / magritek.com), Nanalysis (https: / / www.nanalysis.com), Oxford Instruments: (https: / / nmr.oxinst.com), Qonetec (http: / / www.qonetec.com / spectrometers), and ThermoFisher Scientific (https: / / www.thermofisher.com / at / en / home / industrial / spectroscopy-elemental-isotope-analysis / molecular-spectroscopy / nuclear-magnetic-resonance-nmr.html). The NMR instrument used in the present invention has a magnetic field strength of about 60 MHz to about 1.5 GHz, or about 200 MHz to about 700 MHz, or about 300 MHz to about 600 MHz, or about 300 MHz, or about 600 MHz. In an exemplary embodiment, the measurement time of the NMR instrument is about 1 second to about 10 hours. In an exemplary embodiment, the measurement time is about 1 second to 1 minute, or about 1 minute to 10 minutes, or about 10 minutes to 30 minutes, or about 30 minutes to about 1 hour, or about 1 hour to 2 hours, or about 2 hours to about 5 hours, or about 5 hours to about 10 hours. Generally, the longer the measurement time, the higher the signal-to-noise ratio, enabling more accurate measurements.Those skilled in the art can modify the measurement parameters to fit the specifications of available spectrometers, however, using a high-field spectrometer may shorten measurement times and increase signal-to-noise ratios (S / N).

[0044] In an exemplary embodiment, 1 H nuclear magnetic resonance measurements are performed on the solution in an NMR tube. 1 Some acquisition parameters to consider when performing H nuclear magnetic resonance measurements include recycle delay, pulse sequence, pulse width, number of scans, and temperature. Processing parameters such as baseline correction, integration, digitization, or deconvolution are also parameters that may be optimized.

[0045] Examples of acquisition parameters include: - Repeat Delay Time: The repeat delay is the time until the atomic spins are reset; it is a waiting period. The repeat delay, generally referred to as d1, is displayed at the beginning of the pulse sequence. In practice, this delay occurs after the acquisition time. Nuclear spins do not immediately return to equilibrium. Not allowing sufficient time for relaxation between pulses results in variable signal decay and inaccurate integration. In an exemplary embodiment, measurements are performed with a repeat delay of about 0.1 seconds to about 1 minute. In an exemplary embodiment, measurements are performed with a repeat delay of about 1 second to about 10 minutes. In an exemplary embodiment, measurements are performed with a repeat delay of about 0.5 seconds to about 1.5 seconds. In an exemplary embodiment, measurements are performed with a repeat delay of about 1 second to about 5 seconds. In an exemplary embodiment, measurements are performed with a repeat delay of about 4 seconds to about 30 seconds. In an exemplary embodiment, measurements are performed with a repeat delay of about 10 seconds to about 20 seconds. In an exemplary embodiment, measurements are performed with a repeat delay of about 13 seconds to about 17 seconds. In an exemplary embodiment, measurements are performed with a repeat delay time of about 1 second to about 20 seconds.

[0046] - Number of scans: In exemplary embodiments, the measurements are performed from about 10 scans to about 10,000 scans. In exemplary embodiments, the measurements are performed from about 10 scans to about 30 scans. In exemplary embodiments, the measurements are performed from about 90 scans to about 400 scans. In exemplary embodiments, the measurements are performed from about 200 scans to about 400 scans. In exemplary embodiments, the measurements are performed from about 2750 scans to about 325 scans. In exemplary embodiments, the measurements are performed from about 390 scans to about 1000 scans. In exemplary embodiments, the measurements are performed from about 990 scans to about 2000 scans. In exemplary embodiments, the measurements are performed from about 1300 scans to about 1700 scans. In exemplary embodiments, the measurements are performed from about 1990 scans to about 4000 scans. In an exemplary embodiment, measurements are performed at about 2700 scans to about 3300 scans. In an exemplary embodiment, measurements are performed at about 3990 scans to about 6000 scans. In an exemplary embodiment, measurements are performed at about 5990 scans to about 10,000 scans.

[0047] - Total measurement time: The total measurement time depends on the number of scans and the duration of the pulse sequence. In an exemplary embodiment, the measurement is performed for about 0.1 seconds to about 12 hours. In an exemplary embodiment, the measurement is performed for about 0.1 seconds to about 3 hours. In an exemplary embodiment, the measurement is performed for about 0.25 hours to about 4 hours. In an exemplary embodiment, the measurement is performed for about 0.25 hours to about 1.5 hours. In an exemplary embodiment, the measurement is performed for about 1.5 hours to about 4 hours. In an exemplary embodiment, the measurement is performed for about 1 hour to about 3 hours. In an exemplary embodiment, the measurement is performed for about 1 hour to about 2 hours. In an exemplary embodiment, the measurement is performed for about 1.5 hours to about 3.5 hours. In an exemplary embodiment, the measurement is performed for about 1 second to about 2 hours. In an exemplary embodiment, the measurement is performed for about 10 seconds to about 1 hour. In an exemplary embodiment, the measurement is performed for about 1 minute to about 50 minutes. In an exemplary embodiment, the measurement is performed for about 10 minutes to about 45 minutes.

[0048] - Temperature: Temperature affects the relaxation time and chemical shift of a sample. In an exemplary embodiment, measurements are performed at about -10°C to about 100°C. In an exemplary embodiment, measurements are performed at about 1°C to about 90°C. In an exemplary embodiment, measurements are performed at about 1°C to about 40°C. In an exemplary embodiment, measurements are performed at about 5°C to about 35°C. In an exemplary embodiment, measurements are performed at about 10°C to about 30°C. In an exemplary embodiment, measurements are performed at about 15°C to about 25°C. In an exemplary embodiment, measurements are performed at about 20°C to about 30°C. In an exemplary embodiment, measurements are performed at about 20°C to about 25°C.

[0049] - Pulse sequence: A pulse sequence is a series of pulse signals, wait times, and acquisition times, and can be repeated multiple times to improve the signal-to-noise ratio. In an exemplary embodiment, the utilized pulse sequence appropriately suppresses the signal contribution from HO. In an exemplary embodiment, the pulse sequence is the Bruker pulse sequence "zgesgp." In an exemplary embodiment, the pulse sequence uses excitation sculpting. In an exemplary embodiment, the pulse sequence uses z-gradients only, with a gradient ratio of gp1:gp2=31:11. In an exemplary embodiment, the pulse sequence uses a relaxation delay time of about 1 second to about 15 seconds. In an exemplary embodiment, the pulse sequence is found in T.-L. Hwang, AJ Shaka, J. Magn. Reson., Ser. A, 112, 275-279 (1995), which is incorporated herein by reference in its entirety for all purposes.

[0050] - Pulse width: Applying a pulse sequence at an appropriate frequency rotates the bulk magnetization of the nuclear spins by a specific angle. A pulse is generally expressed in terms of this rotation angle. The rotation angle depends on the pulse width. The pulse width is entered in microseconds. In an exemplary embodiment, the pulse width is about 0.10 ms to about 200 ms. In an exemplary embodiment, the pulse width is about 100 ms to about 200 ms. In an exemplary embodiment, the pulse width is about 0.9 ms to about 100 ms. In an exemplary embodiment, the pulse width is about 1 ms to about 110 ms. In an exemplary embodiment, the pulse width is about 0.9 ms to about 10 ms. In an exemplary embodiment, the pulse width is about 2 ms to about 50 ms. In an exemplary embodiment, the pulse width is about 5 ms to about 40 ms. In an exemplary embodiment, the pulse width is about 25 ms to about 75 ms. In an exemplary embodiment, the pulse width is about 25 ms to about 150 ms. In an exemplary embodiment, the pulse width is approximately 40 ms to 180 ms.

[0051] - Spectral width: In exemplary embodiments, measurements are performed over a spectral width of about -4 ppm to about +10 ppm. In exemplary embodiments, measurements are performed over a spectral width of about +1 ppm to about +20 ppm. In exemplary embodiments, measurements are performed over a spectral width of about -1 ppm to about +8 ppm. In exemplary embodiments, measurements are performed over a spectral width of about +1 ppm to about +10 ppm.

[0052] - Receiver Gain: NMR receiver gain is a parameter often selected to maximize the signal-to-noise ratio. For optimal sensitivity, diluted analytes must be observed with high NMR receiver gain, and interfering strong solvent signals must be suppressed. In an exemplary embodiment, measurements are performed without optimized receiver gain. In an exemplary embodiment, measurements are performed with optimized receiver gain.

[0053] b) Processing In an exemplary embodiment, data acquired in an NMR measurement is processed, which includes one or more of Fourier transformation, phasing, baseline correction, zero-filling, peak picking, multiplet analysis, integration, digitization, and deconvolution.

[0054] - Fourier transform: In NMR, a Fourier transform is performed to extract a frequency domain spectrum from the raw time domain FID. Spectra from a single FID may have a low signal-to-noise ratio, but this can be easily improved by averaging repeated acquisitions. In an exemplary embodiment, the measurements are processed with a Fourier transform.

[0055] - Phase alignment: In high-resolution NMR, peaks need to be as narrow and symmetric as possible. This is equivalent to saying that the FID signal must be in phase with the instrument's receiver. Phasing involves both a zero-order and, often, a first-order adjustment. Zero-order (PH0) phase is the same throughout the spectrum for all peaks. First-order (PH1) phase is applied to the peaks in linearly varying amounts starting from a "pivot" point where the first-order phase adjustment is zero.

[0056] - Baseline correction: 1 In H NMR spectra, baseline artifacts can be present that can adversely affect the identification and quantification of NMR resonances. Baseline correction algorithms range from manual to fully automated methods. 1 The present invention is applicable to H nuclear magnetic resonance measurements. In an exemplary embodiment, baseline correction is manual. In an exemplary embodiment, baseline correction is automatic.

[0057] - Zero filling: Zero filling involves adding zeros (possibly many) to the FID to improve the digital resolution of the FT spectrum.

[0058] - Peak Picking: In peak picking, a peak is selected and its chemical shift is displayed.

[0059] - Integral operations: 1 The integral intensities of signals in a H NMR spectrum give the ratio of the number of hydrogens giving rise to the signal, thus facilitating the calculation of the total number of hydrogens present in the sample. Single signals can be calibrated with values given manually by the operator, while other integral values are calculated proportionally and automatically.

[0060] - Digitalization: In an exemplary embodiment, the data acquired in the NMR measurement is digitized.In an exemplary embodiment, the data acquired in the NMR measurement is not digitized.

[0061] - Deconvolution: In an exemplary embodiment, the data acquired in the NMR measurement is deconvoluted.In an exemplary embodiment, the data acquired in the NMR measurement is not deconvoluted.

[0062] In an exemplary embodiment, the measurements are processed with automatic phase correction. In an exemplary embodiment, the measurements are processed with automatic baseline correction. In an exemplary embodiment, the measurements are processed with a line broadening window function of approximately 0.01 to 10 Hz. In an exemplary embodiment, the measurements are processed with an exponential window function. In an exemplary embodiment, the measurements are processed with DSS axis calibration.

[0063] c) Protein peak integration In an exemplary embodiment, the first proton peak is integrated. Any proton peak generated by a proton on an amino acid in a protein can be selected. In an exemplary embodiment, the proton peak of the amino acid does not overlap with the proton peak generated by a proton on a solvent molecule. In an exemplary embodiment, the proton peak of the amino acid does not overlap with the proton peak generated by a proton on the first polymer. As used herein, "non-overlapping" means that the signal between the two peaks returns to baseline, or within 2%, or within 5%, or within 7%, or within 10%, or returns to baseline.

[0064] In an exemplary embodiment, the first type proton peak is a peak of a first type proton on at least one amino acid on the protein. In an exemplary embodiment, the protein has a molecular weight of about 1 kDa to about 10 kDa. In an exemplary embodiment, the protein has a molecular weight of about 5 kDa to about 15 kDa. In an exemplary embodiment, the protein has a molecular weight of about 10 kDa to about 35 kDa. In an exemplary embodiment, the protein has a molecular weight of about 20 kDa to about 60 kDa. In an exemplary embodiment, the protein has a molecular weight of about 30 kDa to about 70 kDa. In an exemplary embodiment, the protein has a molecular weight of about 50 kDa to about 250 kDa. In an exemplary embodiment, the protein has a molecular weight of about 200 kDa to about 500 kDa. In an exemplary embodiment, the protein has a molecular weight of about 450 kDa to about 1,000 kDa. In an exemplary embodiment, the protein has a molecular weight of about 900 kDa to about 4,000 kDa. In an exemplary embodiment, the protein has a molecular weight of about 3,000 kDa to about 10,000 kDa. In an exemplary embodiment, the protein has a molecular weight of about 9,000 kDa to about 20,000 kDa. In an exemplary embodiment, the protein has a molecular weight of about 500 kDa to about 20,000 kDa. In an exemplary embodiment, the first type proton is on at least one amino acid of the blood coagulation protein. In exemplary embodiments, the blood coagulation protein has a biological activity selected from the group consisting of Factor II, Factor III, Factor V, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand factor, protein C, antithrombin III, thrombin (FII), protein S, tPA, PAI-1, tissue factor (TF), and ADAMTS13 protease. In exemplary embodiments, the protein has the biological activity of Factor VIIa. In exemplary embodiments, the protein has the biological activity of Factor VIII. In exemplary embodiments, the protein has the biological activity of Factor IX. In exemplary embodiments, the protein has the biological activity of human serum albumin.

[0065] In an exemplary embodiment, the first type proton peak is generated by a first type proton on one amino acid of the protein in the protein-first polymer conjugate. Any proton peak contributed by a proton on at least one amino acid of the protein in the protein-first polymer conjugate can be selected. In an exemplary embodiment, the first type proton peak includes peaks generated by first type protons on two or more amino acids of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak is generated by first type protons on two, three, four, or five amino acids of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak is generated by at least one proton on the side chain of an alanine of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak is generated by at least one proton on the side chain of a valine of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on a leucine side chain of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on an isoleucine side chain of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on a tryptophan side chain of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on a histidine side chain of the protein in the protein-first polymer conjugate.

[0066] In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on the side chain of valine of the protein in the protein-first polymer conjugate and at least one proton on the side chain of leucine of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on the side chain of valine of the protein in the protein-first polymer conjugate and at least one proton on the side chain of isoleucine of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peak comprises a peak generated by at least one proton on the side chain of leucine of the protein in the protein-first polymer conjugate and at least one proton on the side chain of isoleucine of the protein in the protein-first polymer conjugate. In an exemplary embodiment, the first type proton peaks include peaks generated by at least one proton on the side chain of valine of the protein in the protein-first polymer conjugate, at least one proton on the side chain of leucine of the protein in the protein-first polymer conjugate, and at least one proton on the side chain of isoleucine of the protein in the protein-first polymer conjugate.

[0067] In an exemplary embodiment, the integral is fixed. In an exemplary embodiment, the integral is centered.

[0068] d) Calibration In an exemplary embodiment, the calibration comprises equating the result of c) to the number of first type protons in the protein-first polymer conjugate.

[0069] In an exemplary embodiment, the protein in the protein-first polymer conjugate is Factor VIII. In an exemplary embodiment, the protein in the protein-first polymer conjugate is full-length Factor VIII. In an exemplary embodiment, the protein in the protein-first polymer conjugate is B-domain deleted Factor VIII. In an exemplary embodiment, the protein in the protein-first polymer conjugate is Factor IX. In an exemplary embodiment, the protein in the protein-first polymer conjugate is Factor VIIa. In an exemplary embodiment, the protein in the protein-first polymer conjugate is von Willebrand factor. In an exemplary embodiment, the protein in the protein-first polymer conjugate is granulocyte colony-stimulating factor (G-CSF). In an exemplary embodiment, the protein in the protein-first polymer conjugate is phenylalanine ammonia lyase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is adenosine deaminase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is asparaginase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is L-asparaginase. In an exemplary embodiment, the protein in the protein-first polymer conjugate is erythropoietin. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2a. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon alpha-2b. In an exemplary embodiment, the protein in the protein-first polymer conjugate is interferon beta-1a. In an exemplary embodiment, the protein in the protein-first polymer conjugate is tumor necrosis factor.In an exemplary embodiment, the protein in the protein-first polymer conjugate is tumor necrosis factor alpha. In an exemplary embodiment, the protein in the protein-first polymer conjugate is human growth hormone. In an exemplary embodiment, the protein in the protein-first polymer conjugate is uricase. For any of the proteins described in this paragraph, the first type proton peak comprises a peak generated by at least one proton on the side chain of valine. For any of the proteins described in this paragraph, the first type proton peak comprises a peak generated by at least one proton on the side chain of leucine. For any of the proteins described in this paragraph, the first type proton peak comprises a peak generated by at least one proton on the side chain of isoleucine. For any of the proteins described in this paragraph, the first type proton peak comprises a peak generated by at least one proton on the side chain of tryptophan. For any of the proteins described in this paragraph, the first type proton peaks include peaks generated by at least one proton on the side chain of valine, at least one proton on the side chain of leucine, and at least one proton on the side chain of isoleucine.

[0070] Valine has two equivalent methyl groups attached to the beta carbon, which corresponds to six protons that are equivalent (i.e., have the same electronic environment) in NMR. Valine also has one proton attached to the beta carbon and one proton attached to the alpha carbon.

[0071] Leucine has two equivalent methyl groups attached to the gamma carbon, which corresponds to six protons that are NMR equivalent (i.e., have the same electronic environment). Leucine has one proton attached to the gamma carbon, two protons attached to the beta carbon, and one proton attached to the alpha carbon.

[0072] Isoleucine has three protons attached to the delta carbon, two protons attached to the gamma carbon, two protons attached to the beta carbon, and one proton attached to the alpha carbon.

[0073] Tryptophan has a characteristic unique proton (NH) at approximately 11 ppm, which can also be used for internal calibration.

[0074] Examples of determining the number of first type protons in the protein of the first polymer conjugate are found in Example A6, Example B7 (part b), Example C7, Example C8, Example D3, and Example E.

[0075] To calibrate the results using a reference sample with a known structure or a calibration curve, the integral value of the first proton can be arbitrarily selected as long as the value is constant between the measurement of the reference sample and the measurement of the protein-first polymer conjugate solution. However, the integral value of the NMR spectrum does not reflect the number of corresponding protons in the NMR sample molecule. Whether the number of first protons of the amino acid isoleucine is 6 or 7 does not affect the results obtained. This is because the K value used to correspond to the shift between the integral value of the modification degree of the reference sample and the actual value. NMR The coefficient is affected by the number of primary protons (approximately 1 ppm) of the amino acid. 1 The H-NMR integrals can be calibrated. If the correct number of these protons is used, other integrals of other peaks will also correspond to the correct number of corresponding protons in the molecules of the NMR sample (K NMR will be close to 1).

[0076] e) Integration of the first polymer peak In an exemplary embodiment, the second proton peak is integrated. Any proton peak contributed by a proton on the first polymer can be selected. In an exemplary embodiment, the proton peak of the first polymer does not overlap with the proton peak generated by a proton on a solvent molecule. In an exemplary embodiment, the proton peak of the first polymer does not overlap with the proton peak generated by a proton on a protein.

[0077] In an exemplary embodiment, the first polymer is selected from the group consisting of polyalkylene glycols, polysaccharides, polyalkylene glycols (PAGs), polyoxazolines, polyacryloylmorpholines, polyvinyl alcohols (PVAs), polycarboxylates, polyvinylpyrrolidones, polyphosphazenes, polyoxazolines, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, and poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF). In an exemplary embodiment, the first polymer is selected from the group consisting of polyethylene glycols (PEGs), polypropylene glycols (PPGs), polyoxazolines, polyacryloylmorpholines, polyvinyl alcohols (PVAs), polycarboxylates, polyvinylpyrrolidones, polyphosphazenes, polyoxazolines, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, and poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF). In exemplary embodiments, the first polymer is selected from the group consisting of polysialic acid (PSA), pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, and carboxymethyldextran. In exemplary embodiments, the first polymer is selected from the group consisting of HES (hydroxyethyl starch), poly(ethylene oxide) (PEO), polyoxyethylene (POE), polyvinyl alcohol, hydroxyethyl cellulose, and dextran.

[0078] In an exemplary embodiment, the first polymer comprises polysialic acid. In another exemplary embodiment, the first polymer is polysialic acid. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid. In another exemplary embodiment, the first polymer is (α-2,8) polysialic acid. In an exemplary embodiment, the first polymer comprises (α-2,9) polysialic acid. In another exemplary embodiment, the first polymer is (α-2,9) polysialic acid. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and (α-2,9) polysialic acid. In another exemplary embodiment, the first polymer is (α-2,8) polysialic acid and (α-2,9) polysialic acid. TIFF2025118907000005.tif118170

[0079] In an exemplary embodiment, the first polymer comprises polysialic acid and polyethylene glycol. In another exemplary embodiment, the first polymer is polysialic acid and polyethylene glycol. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and polyethylene glycol. In another exemplary embodiment, the first polymer is (α-2,8) polysialic acid and polyethylene glycol. In an exemplary embodiment, the first polymer comprises (α-2,9) polysialic acid and polyethylene glycol. In another exemplary embodiment, the first polymer is (α-2,9) polysialic acid and polyethylene glycol. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and (α-2,9) polysialic acid and polyethylene glycol. In another exemplary embodiment, the first polymer is (α-2,8) polysialic acid and (α-2,9) polysialic acid and polyethylene glycol.

[0080] In an exemplary embodiment, the first polymer comprises a polysialic acid as described herein, and the second species proton peak is generated from a proton on the sialic acid portion of the polysialic acid. In an exemplary embodiment, the first polymer comprises a polysialic acid as described herein, and the second species proton peak is generated from a proton on the sialic acid portion of the polysialic acid. 3a, H 3b In an exemplary embodiment, the first polymer comprises a polysialic acid as described herein, and the second proton peak is generated from a proton selected from the group consisting of H, H, H, and H. 3a or H 3b In an exemplary embodiment, the first polymer comprises a polysialic acid as described herein, and the second proton peak is generated from a proton selected from H 3a is generated from

[0081] In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid, and the second species proton peak is generated from protons on sialic acid moieties on the (α-2,8) polysialic acid. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid, and the second species proton peak is generated from H on the (α-2,8) polysialic acid. 3a , H 3b In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid, and the second proton peak is generated from a proton selected from the group consisting of H on the (α-2,8) polysialic acid. 3a or H 3b In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid, and the second proton peak is generated from a proton selected from H on the (α-2,8) polysialic acid. 3a is generated from

[0082] In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and polyethylene glycol, and the second species proton peak is generated from the protons on the (α-2,8) polysialic acid. In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and polyethylene glycol, and the second species proton peak is generated from the H on the (α-2,8) polysialic acid. 3a , H 3bIn an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and polyethylene glycol, and the second proton peak is generated from a proton selected from the group consisting of H on the (α-2,8) polysialic acid. 3a or H 3b In an exemplary embodiment, the first polymer comprises (α-2,8) polysialic acid and polyethylene glycol, and the second proton peak is generated from a proton selected from H on the (α-2,8) polysialic acid. 3a is generated from

[0083] In an exemplary embodiment, the first polymer comprises polyethylene glycol. In another exemplary embodiment, the first polymer is polyethylene glycol.

[0084] In an exemplary embodiment, the first polymer comprises polyethylene glycol and the second type proton peak is on the ethylene moiety. TIFF2025118907000006.tif18170

[0085] In an exemplary embodiment, the first polymer has a molecular weight of about 10 kDa to about 110 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 1 kDa to about 11 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 9 kDa to about 21 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 19 kDa to about 31 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 29 kDa to about 41 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 39 kDa to about 51 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 49 kDa to about 61 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 59 kDa to about 71 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 69 kDa to about 81 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 79 kDa to about 91 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 89 kDa to about 101 kDa. In an exemplary embodiment, the first polymer has a molecular weight of about 99 kDa to about 111 kDa.

[0086] f) Division In an exemplary embodiment, the result of step e) is divided by the number of second-type protons per monomer of the first polymer. In an exemplary embodiment, the second-type proton peak is divided by the number of second-type protons per monomer of the first polymer. In an exemplary embodiment, the number of monomers in the first polymer is determined by this calculation.

[0087] In an exemplary embodiment, the first polymer comprises a polysialic acid as described herein, and the second type proton peak is generated by a sialic acid proton as described herein. In an exemplary embodiment, the first polymer is an (α-2,8) polysialic acid and polyethylene glycol as described herein, and the second type proton peak is generated by an H on the (α-2,8) polysialic acid. 3a It is produced by protons.

[0088] In an exemplary embodiment, the first polymer is a polyethylene glycol as described herein, and the second proton peak is generated by an ethylene proton as described herein.

[0089] g) Division In an exemplary embodiment, the result of step f) is divided by the average number of monomers in the first polymer. In an exemplary embodiment, the number of monomers in the first polymer is divided by the number of second-type protons per monomer of the first polymer. In an exemplary embodiment, the number of first polymers covalently attached to the protein-first polymer conjugate is determined.

[0090] IIIa. Additional Embodiments In absolute quantification (measurement of the absolute purity of a material), a known weight of a reference substance is generally added to a known weight of an analyte in an appropriate solvent. Relative quantification is another very common method for obtaining the ratio between two analytes in the same sample. The integral of one peak assigned to one of the components is compared with the integral of another peak assigned to the other component, and after taking into account the number of hydrogen atoms in each peak, the molar ratio of the two compounds is obtained.

[0091] IIIb. K NMR In industrial-scale production of protein-first polymer conjugates, there is variability in the molecular weight of the protein as well as the molecular weight of the first polymer. Furthermore, when using NMR techniques, there may be trace signals from other solution components (proteins, solvents, impurities) in the first or second proton peaks. In an exemplary embodiment, a correction factor can be applied to the calculations described herein to account for this variability. The correction factor is K NMR In an exemplary embodiment, the result of e) is expressed as K NMR In an exemplary embodiment, the result of f) is divided by K NMR In an exemplary embodiment, the result of step g) is divided by K NMR Divide by.

[0092] K NMR can be calculated according to any of several methods.

[0093] IIIb.i) K NMR standard material Example "C" In an exemplary embodiment, the number of valine, leucine, and isoleucine residues present in the protein is multiplied by the number of first-type protons (signal: 1 ppm or less) each contains to obtain the total number of first-type protons (signal: 1 ppm or less) in the protein. The protein multiplet around 1 ppm is integrated using automatic baseline correction, and the integral value is calibrated to the value of the total number of first-type protons (signal: 1 ppm or less). In an exemplary embodiment, the integral of the second-type proton is set, and the peak is integrated using the integration command with automatic baseline correction. In an exemplary embodiment, this integral value is multiplied by the molecular weight of one polymer unit and divided by the molecular weight of each polymer chain (which is equivalent to dividing by the average number of repeating units in each polymer chain). In an exemplary embodiment, the result is divided by the degree of polymerization of the sample obtained by a conventional assay.

[0094] IIIb.ii) K NMR Calibration curve of Example "A" In an exemplary embodiment, a calibration curve is generated. In an exemplary embodiment, the calibration curve is obtained by generating NMR measurements for a series of solutions, each solution containing the same but known amount of protein and a different but known amount of the first polymer. The slope of the curve generated from the series of solutions, taking into account the number of protons per first polymer chain, is defined as K NMR For example, a polymer chain with an average polymer chain length of 69 is PSA, and the second proton peak is H 3a If generated by NMR = slope / 69(69 H per PSA chain) 3a proton).

[0095] In an exemplary embodiment, K NMR is measured by preparing a calibration curve of five samples containing the same amount of rFIX and different but known amounts of PSA (not bound to protein). The fact that the PSA used (native PSA, oxo-PSA, or PSA oxime) is not bound to protein does not interfere with the measurement, as the electronic environment of each proton changes only to the extent that no change in the proton shift is expected.

[0096] IIIb.iii) K NMR Alternative assays for Example "B" When using conventional methods to determine the degree of modification (MD) of some samples without utilizing standards, the K NMR Calculate the average and K NMR Example B shows good agreement between the conventional and NMR methods (RSD less than 5%).

[0097] IV. Comparison of conventional and NMR methods Quantifying the degree of modification is crucial for monitoring industrial production processes and matching product specifications. However, tests such as the resorcinol assay combined with a fluorescence test (used to determine the PSA degree of PSA-rFVIII) suffer from a large imprecision (approximately ±15%), which leads to great difficulties during product development, production, and lifecycle management in trying to avoid out-of-specification batches. 1Using H NMR spectrometry, the degree of protein modification can be determined in a very reliable manner, significantly improving the accuracy of the method (±5%). By integrating protein regions of the NMR spectrum where the identity and number of protons are known, and cointegrating signals specific to water-soluble polymers, the protein / polymer ratio and therefore the degree of modification can be estimated. For example, the first-order signals from valine, leucine, and isoleucine can be used as internal calibration references for integration, knowing the numbers of these amino acids from the protein's primary structure. The integrals of the internal references can be set to the corresponding values.

[0098] The water-soluble polymer specific proton signal can then be integrated. The degree of modification can be calculated by dividing the value of this second integral by the number of protons per chain that generate this specific signal (= the number of repeat units where only one proton per repeat unit generates a specific polymer signal). A correction factor (K NMR ) is advantageous for matching the actual degree of modification and balancing peak overlap.

[0099] IVa. Adynovate An example of a PEGylated FVIII product is Adynovate / Adynovi, a long-acting rFVIII product manufactured by Takeda. Adynovate is produced by covalently attaching Nektar Therapeutics' branched 20 kDa PEG reagent using N-hydroxysuccinimide (NHS) chemistry to Advate, a full-length rFVIII product produced by Takeda.

[0100] IVb. PSA-rFVIII Another example of a long-acting FVIII is polysialylated rFVIII. PSA is a linear homopolymer composed of N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulo-pyranos-1-one) monomers. Sialic acid is typically found as an α-glycoside occupying the terminus of hetero-oligosaccharides in glycoconjugates such as glycoproteins and glycolipids, making it a ubiquitous compound in most biological tissues. Furthermore, this polysaccharide is susceptible to selective oxidation of the vicinal diol at the non-reducing end. This feature enables the functionalization of PSA, which can then be used for the chemical modification of therapeutic proteins such as coagulation factors. In PSA-rFVIII, PSA concentration is determined by a combination of a fluorescent assay to assess protein concentration and a resorcinol test to quantify total PSA. In particular, this product suffers from a large imprecision regarding the degree of modification (approximately ±15%), leading to great difficulties in avoiding out-of-specification batches during product development, production, and lifecycle management.

[0101] The present invention is further illustrated by the following examples, which are not intended to define or limit the scope of the invention. [Example]

[0102] The following examples illustrate the synthesis of representative compounds used in the present invention, and the following reference examples illustrate the synthesis of intermediates in their preparation. These examples are not intended to, and should not be construed to, limit the scope of the invention. It will be apparent that the invention may be practiced otherwise than as specifically described herein. Many modifications and variations of the present invention are possible in light of the teachings herein and, therefore, are within the scope of the invention.

[0103] In the following examples, all temperatures are given in degrees Celsius and all parts and percentages are by weight unless otherwise indicated. Reagents may be purchased from commercial suppliers such as Sigma-Aldrich Chemical Company and may be used without further purification unless otherwise indicated. Reagents may also be prepared according to standard literature procedures known to those skilled in the art. Solvents may be purchased from Sigma-Aldrich in Sure-Seal bottles and used as received. All solvents may be purified using standard methods known to those skilled in the art unless otherwise indicated. The reactions shown below were generally carried out at ambient temperature unless otherwise indicated. Proton magnetic resonance ( 1 H NMR spectra were recorded using an NMR spectrometer operating at a field strength of 600 MHz. Chemical shifts are reported in the form of delta (δ) values given in parts per million (ppm) relative to an internal standard such as tetramethylsilane (TMS). Alternatively, for samples in DO / HO, the spectral reference (SR) frequency value of the DSS sample was used to calibrate its methyl signal at 0.00 ppm. 1 The H NMR spectrum was referenced. Peak multiplicities are designated as follows: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; dt, doublet of triplet; q, quartet; br, broadband; m, multiplet. Coupling constants are given in Hertz (Hz).

[0104] Starting materials used were either available from commercial sources or prepared according to literature procedures and had experimental data in accordance with those reported.

[0105] Example A demonstrates the use of NMR methods on protein-first polymer conjugate samples when enough unconjugated protein is available to establish a calibration curve. PSA-FIX is the protein-first polymer conjugate in this set of examples.

[0106] Example B demonstrates the use of the NMR method when sufficient unconjugated protein is not available to establish a calibration curve. Here, researchers have several conjugated protein samples. The degree of modification of these protein-first polymer conjugate samples has been assessed by conventional methods, and degree of polymerization values are available. After measuring the NMR spectra of these samples, researchers can find convenient K values that match the results of both methods within the range of degrees of polymerization of the sample set. NMR The coefficients were selected. This method allows researchers to "translate" results from conventional methods to NMR methods. PSA-rFVIII is the protein-first polymer conjugate in this set of examples.

[0107] Example C demonstrates the use of NMR methods in yet another situation where sufficient unconjugated protein is not available to establish a calibration curve. Here, researchers have one protein-first polymer conjugate sample, and the degree of modification of this protein-first polymer conjugate is being assessed by NMR. PEG-HSA is the conjugate in this set of examples.

[0108] Example D describes the use of NMR methods to determine the degree of PEGylation in samples of PEG-rFVIII conjugates.

[0109] Example E describes the use of NMR methods to determine the degree of PEGylation in samples of PEG-bovine pancreatic trypsin inhibitor (BPTI) conjugates.

[0110] Example F describes the use of NMR methods to determine the degree of PEGylation in samples of PEG-rFIX conjugates.

[0111] Example A1: Generation of PSA-rFIX conjugates PSA-rFIX was prepared as described in US Pat. No. 8,809,501B2 (Example 11).

[0112] 25 mg of FXI (V = 4.63 mL) was placed in a 50 mL vial. Coupling buffer (V = 13.73 mL, 20 mM histidine, 5 mM CaCl2, 50 mM NaCl, pH 6) was added, and the mixture was stirred at ambient temperature. Aminooxy-PSA reagent (55.7 mg, V = 1.39 mL, MW 20.5 kDa) was added, followed by m-toluidine solution (5 mL, 50 mM) and NaIO4 solution (250 μL, 10 mM). The pH of the reaction was adjusted to 6.0. After completion of the reaction, the reaction was quenched using L-cysteine solution (256 μL, 1 M). The conjugate was purified using strong anion exchange chromatography and hydrophobic interaction chromatography (HIC).

[0113] Example A2: Determination of total rFIX concentration using conventional methods - Bradford assay The Coomassie (Bradford) protein assay is a quantitative assay used to determine the total rFIX concentration (conjugated and unconjugated) in samples from the production and purification process. This type of assay was first published by Bradford in 1976 (Anal. Biochem 72, 248-254).

[0114] The assay was performed over a range of standard concentrations from 5 to 50 μg / mL. Standard concentrations of 5, 7, 10, 20, 39, 40, and 50 μg / mL were prepared by diluting the PSA-rFIX standard with HO. The assay was performed by mixing 800 μL of sample with 200 μL of Bradford reagent (BIORAD). The mixture was incubated at 20–25°C for 5–60 minutes and then vortexed again immediately before measurement. The extinction coefficients of the solutions (sample and standard) were measured spectrophotometrically at a wavelength of 596 nm against a blank solution (800 μL HO + 200 μL reagent). The absorbance data at 595 nm (y-axis) were plotted as a function of concentration (μg / mL). The resulting curves were fitted using a four-parameter curve fit to calculate the total rFIX concentration in samples containing PSA-rFIX conjugates.

[0115] The total rFIX concentration was determined to be 2.38 mg / mL by Bradford assay.

[0116] Example A3: Determination of total PSA concentration using conventional methods - resorcinol assay The resorcinol assay was performed as described by Svennerholm in 1957 (Biochimica et Biophysica Acta, 24, 604-611). The assay is based on the following principle: TIFF2025118907000007.tif50170

[0117] N-acetylneuraminic acid (pyranose form) (NANA) is converted to the furanose form in the presence of concentrated hydrochloric acid and rapidly dehydrated to furfural. This furfural forms a blue complex with resorcinol (1,3-benzenediol) in the presence of Cu(II) ions. This is shaken in an organic solvent (butyl acetate / butanol) and measured photometrically at 585 nm. The absorbance of the blue complex is proportional to the concentration of N-acetylneuraminic acid (NANA).

[0118] To determine the total PSA content in preparations of polysialylated rFIX (PSA-rFIX), a reagent solution was prepared by mixing 20 mL of aqueous resorcinol solution with 500 μL of an aqueous solution of CuSO4 × 5HO (concentration 0.1 M). 37% hydrochloric acid was then added to bring the final volume to 200 mL.

[0119] The assay range was 10 μg / mL to 100 μg / mL. For the reference curve, five different concentrations of NANA in HO were prepared (10, 25, 50, 75, and 100 μg / mL). 400 μL of each sample (PSA-rFIX sample and reference sample) was vigorously mixed with 400 μL of resorcinol reagent. The samples were then incubated at 99°C for 40 minutes in a thermomixer at 450 rpm and cooled to -15°C or below for 10 minutes. To extract the colored complex, 800 μL of organic extraction solution was added and mixed vigorously. This solution was centrifuged at 13,000 rpm for 5 minutes. The upper organic phase was separated and transferred to a 1 mL cuvette. The absorbance was then measured at 585 nm in a spectrophotometer, and the total PSA concentration was calculated from the reference curve. All measurements were performed in duplicate.

[0120] Total PSA concentration was determined to be 1.43 mg / mL by resorcinol assay.

[0121] Example A4: Conventional Method—Calculation of the PSA Modification Degree of PSA-rFIX Conjugate Samples The degree of PSA modification of the PSA-rFIX conjugate is described using the following formula: TIFF2025118907000008.tif9170

[0122] Based on the data obtained in the above assay, assuming that the molecular weight of rFIX is 55 kDa and that of PSA is 20.5 kDa, the degree of PSA modification of the PSA-rFIX conjugate is as follows: TIFF2025118907000009.tif9170

[0123] By conventional methods, the degree of PSA modification of the unknown sample PSA-rFIX conjugate was determined to be 1.61 mol PSA / mol rFIX.

[0124] Example A5: Q-NMR Method: Acquisition and Processing An aqueous sample of PSA-FIX (0.5 mL, 1 mg / mL) in a 2 mL vial was thawed by standing at room temperature for 30 minutes. The sample was transferred to a 5 mm NMR tube with a Pasteur pipette. 56 μL of DO (99.9 atomic % D) was added to the NMR tube, and the tube was sealed with a polyethylene cap. The contents were then homogenized by shaking. Finally, air bubbles were removed by gentle tapping or a brief immersion in an ultrasonic bath. The NMR tube was wiped and placed in an NMR spinner according to the spectrometer supplier's specifications.

[0125] Acquisition parameters Proton NMR spectra were obtained on a 600 MHz AVANCE BRUKER spectrometer as follows. Spectrometer frequency: 600MHz pulse program:zgesgp; Temperature (℃):25±5; Number of scans: 3000 SW(ppm):>14(approx.+10~-4); Receiver Gain: Optimized D1 (seconds):1

[0126] Processing parameters Proton NMR spectra were processed using BRUKER Topspin software V3.5 as follows. Window function: EM (line broadening: 0.3 Hz); The following command was executed. ft: Fourier transform apk:Auto phase correction abs: Automatic baseline correction Axis calibration: After calibrating the methyl signal at 0.00 ppm, the spectral reference frequencies were matched to the DSS SR values.

[0127] Example A6: K by calibration curve NMR Acquisition of Five samples of rFIX (approximately 0.5 mL, each containing approximately 0.5 mg of protein) were mixed with different amounts of oxoPSA 20 kDa (alternatively, NatPSA or PSA oxime 20 kDa could be used). In sample 1, 0.5 equivalents (= 0.5 × n rFIX) of PSA chain were added to the protein; in sample 2, 1 equivalent (= 1 × n rFIX) of PSA chain was added to the protein; in sample 3, 1.5 equivalents (= 1.5 × n rFIX) of PSA chain were added to the protein; in sample 4, 2.25 equivalents (= 2.25 × n rFIX) of PSA chain were added to the protein; and in sample 5, 3 equivalents (= 3 × n rFIX) of PSA chain were added to the protein. The results are shown in Table 1.

[0128] [Table 1]

[0129] The proton NMR spectrum of each sample was acquired according to the parameters listed in Example A5.

[0130] The number of valine, leucine, and isoleucine residues present in the protein was multiplied by the number of first-order protons they each contain (signal: 1 ppm or less, amino acids V, L, and I). For rFIX: Valine: 35 residues x 6 first-order protons = 210H Leucine: 21 residues x 6 first-order protons = 126H Isoleucine: 22 residues x 7 first-order protons = 154H

[0131] The protein has a total of 490 first-order protons.

[0132] The internal calibration of the protein multiplet, ranging from approximately 1.05 to 0.6 ppm, was set to a value of 490 using the integrate command with automatic baseline correction.

[0133] Integration of PSA H3a (equatorial) was performed as follows.

[0134] The peak center is determined (approximately 2.65 ppm) (appears as a doublet) and the peak is integrated for ±0.04 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0135] For each spectrum, the integral of the H3a peak (approximately 2.65 ppm) was measured after internal calibration of the protein multiplets in the range of approximately 1.05 to 0.6 ppm. Table 2 shows the results.

[0136] [Table 2]

[0137] A calibration curve can be drawn and a correlation between the theoretical PSA level and the integral value of the H3a signal can be established (Figure 1). K NMR can be determined by dividing the slope of the calibration curve by the number of units in the polymer chain. The average molecular weight of the PSA polymer utilized in the conjugate was 20 kDa. There are approximately 69 sialic acid units in a 20 kDa PSA chain. K NMR =144.18 / 69=2.09

[0138] Example A7: Q-NMR measurement of PSA modification degree of PSA-rFIX conjugate samples The degree of PSA modification of a full-length FIX preparation based on the recombinant product Rixubis (Windyga et al., Ther. Adv. Hematol. 2014, 5, 168-180) was determined as follows: 1 Determined using H-NMR techniques.

[0139] An unknown sample of PSA-rFIX conjugate was measured by NMR. Integration was performed using the integrate command and automatic baseline correction. The proton NMR spectrum of the sample was acquired according to Example A5, and the protein multiplets were internally calibrated at a value of 490 using the integrate command with automatic baseline correction in the range of approximately 1.05 to 0.6 ppm. 3a The integral of the peak (approximately 2.65 ppm) was measured.

[0140] PSA H 3a An equatorial integration was set, which appeared as a doublet. The peak center was determined (approximately 2.65 ppm), and the peak was integrated ±0.04 ppm on either side of the peak center using the integrate command with automatic baseline correction. H 3a Peak integral = 196.12

[0141] The calibration curve for Example A6 was determined as follows. y=144.18x-22.683 where y is the integral of the second proton and x is the degree of PSA modification of the PSA-rFIX conjugate sample. 3a The peak integral (196.12) was selected as the second proton. The degree of PSA modification (x) of the unknown rFIX sample was determined to be 1.52 mol PSA / mol rFIX by Q-NMR.

[0142] Example A8: Determination of the relative accuracy of the Q-NMR method in determining the degree of PSA modification of PSA-FIX conjugates By conventional methods, the degree of PSA modification to rFIX of the unknown sample was determined to be 1.61 mol PSA / mol rFIX.

[0143] The degree of PSA modification of the unknown sample with rFIX was determined to be 1.52 mol PSA / mol rFIX by Q-NMR.

[0144] The relative accuracy of the Q-NMR method in determining the degree of PSA modification of PSA-FIX conjugates is as follows: 1.52 / 1.6=0.95

[0145] These results are in agreement within ±5%. The Q-NMR method has the advantage that only one measurement of the sample is required, and the protein-first polymer conjugate is not destroyed. Conventional methods require two or more measurements, during which the protein-first polymer conjugate is also destroyed.

[0146] Example B1: Generation of PSA-rFVIII conjugate 1706.30 mg of FVIII (V = 407.31 mL) was placed in a 2 L reactor. Coupling buffer (V = 1143.95 mL, 20 mM histidine, 5 mM CaCl2, 50 mM NaCl, pH 6) was added, and the mixture was stirred at ambient temperature. Aminooxy-PSA reagent (6459.32 mg, V = 165.2 mL, MW 20 kDa) was added, followed by m-toluidine solution (308.11 mL, 50 mM) and NaIO4 solution (15.40 mL, 40 mM). The pH of the reaction was adjusted to 6.0. After the reaction was complete (120 min), the reaction was quenched using L-cysteine solution (30.3 mL, 1 M) and incubated for 60 min. The conjugate was purified using HIC and SEC chromatography followed by an ultrafiltration (UF) step to give 793.9 g of bulk drug substance (BDS) (1.3 mg FVIII / mL).

[0147] One lot was split into two and used for Examples B2-B5. Separate lots were prepared for Examples B6-B7. Separate lots were prepared for Examples B8-B10. Separate lots were prepared for Examples B11-B12. Separate lots were prepared for Example B13. Separate lots were prepared for Example B14.

[0148] In the conventional method described in Examples B2 and B3, PSA-rFVIII conjugate was produced in six separate reactions. Each reaction product was labeled A to F. Each of Samples A to F was further divided into two. One of the two aliquots was subjected to a fluorescence test according to Example B2. The other aliquot was subjected to a resorcinol test according to Example B3. The MD was calculated in Example B4. The (relative) accuracy of this conventional method was then investigated in Example B5.

[0149] In the Q-NMR method described in Examples B6 to B8, PSA-rFVIII conjugates were produced in six separate reactions. Each reaction product was labeled 1 to 6. MD was calculated in Example B4. The (relative) accuracy of this conventional method was then investigated in Example B5.

[0150] Example B2: Determination of total rFVIII concentration using conventional methods - fluorometric assay Due to the high purity of FVIII, the total protein measurement is equivalent to the FVIII protein concentration. The determination of the total FVIII protein content in PSA-rFVIII was performed by spectroscopy. The analysis was based on the intrinsic fluorescence emitted by aromatic amino acids such as tryptophan, tyrosine, and phenylalanine, measured at 350 nm. The intensity of FVIII fluorescence was proportional to its concentration. To ensure uniform exposure of aromatic amino acids, FVIII was incubated with guanidine hydrochloride, which acts as a chaotropic agent, prior to fluorescence measurement.

[0151] Data evaluation was performed using a calibration curve of several dilutions of a home-made standard lot with known protein concentration.

[0152] To measure the protein content, PSA-rFVIII bulk drug substance containing 1000 IU of rFVIII, as determined by a chromogenic assay, was dissolved in 2.5 mL of water for injection (WFI). Then, 1 mL of 8 M guanidine HCl aqueous solution was added. This solution was then diluted with dilution buffer to obtain FVIII concentrations of 10-50 μg / mL.

[0153] This buffer was prepared as follows.

[0154] In a 1000 mL beaker, 0.24 g of Tris base, 1.31 g of sodium chloride, 0.06 g of calcium chloride dihydrate, and 0.02 g of glutathione were dissolved in 900 mL of WFI. 0.24 g of HEPES was added, and 0.03 g of Tween 80 was dissolved in 10 mL of WFI. The pH was adjusted to 7.0 with 1 N hydrochloric acid and transferred to a 1000 mL volumetric flask containing WFI water. Finally, 8.00 g of mannitol and 2.00 g of trehalose were dissolved in 100 mL of this solution.

[0155] The standard used was rFVIII based on Takeda's recombinant full-length rFVIII. Reference curve standard points of 10, 20, 30, 40, and 50 μg / mL were prepared.

[0156] The fluorescence of each of samples A to F was measured at 350 nm using a spectrophotometer with an excitation wavelength of 285 nm. All measurements were performed in duplicate.

[0157] Total rFVIII protein results: Table 3 below provides the total rFVIII concentrations of samples A-F by fluorescent assay.

[0158] [Table 3]

[0159] The average total rFVIII protein concentration for samples A to F was 1689.83 mg / mL.

[0160] Example B3: Determination of total PSA concentration using conventional methods - resorcinol assay Assay Principle The polysialylation degree of PSA-rFVIII can be calculated by conventional methods by measuring the concentration of bound PSA and the concentration of FVIII protein. The amount of bound PSA is determined by calculating the difference between total PSA and free PSA. Total PSA is measured by using resorcinol assay, and free PSA is measured by using size exclusion chromatography HPLC method. Protein concentration is measured by fluorescence method.

[0161] Determination of total PSA Resorcinol assays were performed as described by Svennerholm in 1957 (Biochimica et Biophysica Acta, 24, 604-611). To determine the total PSA content in preparations of polysialylated rFVIII (PSA-rFVIII), a reagent solution was prepared by mixing 20 mL of aqueous resorcinol solution with 500 μL of an aqueous solution of CuSO4 × 5H2O (concentration 0.1 M). 37% hydrochloric acid was then added to bring the final volume to 200 mL.

[0162] The assay range was 10 μg / mL to 100 μg / mL. For the reference curve, five different concentrations of NANA were prepared in HO (10, 25, 50, 75, and 100 μg / mL). 400 μL of each sample (PSA-rFVIII sample and reference sample) was vigorously mixed with 400 μL of resorcinol reagent. The samples were then incubated in a thermomixer at 450 rpm at 99°C for 40 minutes and cooled to a temperature below -15°C for 10 minutes. To extract the colored complex, 800 μL of organic extraction solution was added and mixed vigorously. This solution was centrifuged at 13,000 rpm for 5 minutes. The upper organic phase was separated and transferred to a 1-mL cuvette. The absorbance was then measured at 585 nm in a spectrophotometer, and the total PSA concentration was calculated from the reference curve. Measurements for each of samples A to F were performed in duplicate.

[0163] Total PSA results: Table 4 below provides the total PSA concentrations of Samples A-F by fluorescent assay.

[0164] [Table 4]

[0165] The mean total PSA concentration in samples A to F was 1.09 mg / mL.

[0166] Example B4: Conventional Method - Calculation of PSA Modification Degree of PSA-rFVIII Conjugate Samples The degree of PSA modification of the PSA-rFVIII conjugate is described using the following formula: TIFF2025118907000014.tif9170

[0167] Based on the data obtained in the above assay, assuming that the molecular weight of rFVIII is 280 kDa and the molecular weight of PSA is 20 kDa, the PSA modification degree of each PSA-rFVIII conjugate of samples A to F is as follows: TIFF2025118907000015.tif9170

[0168] [Table 5]

[0169] The average PSA modification degree of the PSA-rFVIII conjugates of Samples A to F was 9.05.

[0170] Example B5: Determination of the (relative) accuracy of conventional methods in determining the degree of PSA modification of PSA-rFVIII The robustness of the conventional method was evaluated by measuring the fluorescence values of resorcinol and six samples (A–F) from the same batch. Samples A and B were measured on day 1, C and D on day 2, and E and F on day 3.

[0171] SD / RSD results for total PSA concentration: The standard deviation (SD) and relative standard deviation (RSD) were calculated for the total PSA concentrations of samples A to F. The standard deviation (SD) was 0.02 mg / mL, and the relative standard deviation (RSD) was 1.68%.

[0172] SD / RSD results for total rFVIII protein concentration: The standard deviation (SD) and relative standard deviation (RSD) were calculated for the total rFVIII protein concentrations of samples A to F. The standard deviation (SD) was 49.08 mg / mL, and the relative standard deviation (RSD) was 2.90%.

[0173] SD / RSD results of PSA modification degree of PSA-rFVIII conjugate: The standard deviation (SD) and relative standard deviation (RSD) were calculated for the PSA modification degree of the PSA-rFVIII conjugates of samples A to F. The standard deviation (SD) was 0.25 mol PSA / mol FVIII, and the relative standard deviation (RSD) was 2.78%.

[0174] Example B6: Q-NMR Method: Acquisition and Processing Aqueous samples of PSA-rFVIII (0.5 mL, 0.8 mg / mL) in 2 mL vials were thawed by standing at room temperature for 30 minutes. The samples were transferred to 5 mm NMR tubes with a Pasteur pipette. 56 μL of DO (99.9 atomic % D) was added to each NMR tube, and the tubes were sealed with polyethylene caps. The contents were then homogenized by shaking. Finally, air bubbles were removed by gentle tapping or brief immersion in an ultrasonic bath. The NMR tubes were wiped and placed in an NMR spinner according to the spectrometer supplier's specifications.

[0175] Acquisition parameters Proton NMR spectra were obtained on a 600 MHz AVANCE BRUKER spectrometer as follows. Spectrometer frequency: 600MHz pulse program:zgesgp; Temperature (℃):25±5; Number of scans: 3000 SW(ppm):>14(approx.+10~-4); Receiver Gain: Optimized D1 (seconds):1

[0176] Processing parameters Proton NMR spectra were processed using BRUKER Topspin software V3.5 as follows. Window function: EM (line broadening: 0.3 Hz); The following command was executed. ft: Fourier transform apk:Auto phase correction abs: Automatic baseline correction Axis calibration: After calibrating the methyl signal at 0.00 ppm, the spectral reference frequencies were matched to the DSS SR values.

[0177] Example B7: K by using an alternative assay NMR Decision a) Obtaining PSA MD of Samples 1 to 6 by conventional methods Six samples of PSA-rFVIII conjugates were analyzed. All PSA materials described in the document refer to 20 kDa PSA chain materials (approximately 69 polysialic acid units per chain). According to Examples B2-B4, a fluorescence assay in combination with a resorcinol assay was applied to each of the samples, revealing the following PSA modification degrees:

[0178] [Table 6]

[0179] b) Acquisition of "crude" PSA MD of samples 1 to 6 by Q-NMR method The number of valine, leucine, and isoleucine residues present in the protein rFVIII is multiplied by the number of first-order protons (signal: ≦1 ppm) that each of them contains. For full-length rFVIII: Valine: 125 residues x 6 first-order protons = 750 H Leucine: 222 residues x 6 first-order protons = 1332H Isoleucine: 110 residues x 7 first-order protons = 770 H

[0180] This protein has a total of 2852 first-order protons. The protein multiplet is integrated between 1.045 and 0.63 ppm using the integrate command with automatic baseline correction, and the integral is calibrated at 2852.

[0181] PSA H 3a Set the integration to (equatorial). This peak appears as a doublet. Determine the peak center (approximately 2.65 ppm) and integrate this peak for ±0.04 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0182] The value of this integral was multiplied by the molecular weight of one polymer unit and divided by the molecular weight of each PSA chain (which is equivalent to dividing by the average number of repeat units in each polymer chain). The crude PSA MD was determined as follows:

[0183] [Table 7]

[0184] c) K using data from conventional methods and Q-NMR data NMR Calculation of Next, the ratio of the value in Table 7 / the value in Table 6 was calculated.

[0185] [Table 8]

[0186] The values in Table 8 are correction factors for each batch, introduced to make the PSA MD measured by NMR consistent with the PSA MD measured by conventional methods (fluorescence assay / resorcinol assay).

[0187] Averaging these correction factors across six samples (MD = 5.5–15.6) yielded a K of 1.31. NMR is generated. K NMR is specific for PSA-rFVIII conjugates with MDs of 5.5 to 15.6.

[0188] K NMR accounts for protein heterogeneity as well as overlapping peaks within the integration range.

[0189] d)K NMR SD / RSD results for: Conjugate MD ranges from 5.5 to 15.6, with K NMR The relative standard deviation was estimated: RSD = 4.1% (SD = 0.05).

[0190] Example B8: Determination of corrected MD of PSA-rFVIII conjugate samples by Q-NMR method In subsequent analytical experiments, PSA-rFVIII conjugate samples were prepared according to Example B1.

[0191] Q-NMR analysis of this PSA-rFVIII conjugate sample was performed as described in Examples B6 and B7b. The crude PSA modification degree was determined to be 15.52.

[0192] The corrected PSA modification degree was calculated by multiplying the crude PSA modification degree by the K obtained in Example B7c. NMR was obtained by dividing by Modified PSA qualification level: 15.52 / 1.31=11.85

[0193] Example B9: Comparison of Q-NMR results of Example B8 with analysis of samples by conventional methods The PSA-rFVIII conjugate sample of Example B8 was subjected to the conventional method (fluorescence / resorcinol assay) described in Examples B2 to B5. The PSA MD was as follows: PSA modification degree by fluorescence and resorcinol assay = 10.8

[0194] Example B10: Determination of the (relative) accuracy of the Q-NMR method in determining the degree of PSA modification of PSA-rFVIII conjugates By conventional methods, the PSA MD of the PSA-rFVIII conjugate of Example B8 was determined to be 10.8 mol PSA / mol rFVIII.

[0195] By Q-NMR methods, the corrected PSA MD of the PSA-rFVIII conjugate of Example B8 was determined to be 11.85 mol PSA / mol rFVIII.

[0196] The relative accuracy of the Q-NMR method in determining the degree of PSA modification of PSA-FIX conjugates is as follows: 11.85 / 10.8=1.10 These results are in agreement within ±10%.

[0197] Example B11: To illustrate the (relative) accuracy of the NMR method, six samples were prepared from the same lot containing PSA-FVIII conjugate and subjected to the acquisition and processing parameters described in Example B6 and the integration procedure described in Example B7.

[0198] This experiment revealed that the relative standard deviation (RSD) of the PSA modification degree for these six samples was less than 2%. The results are shown in the following table.

[0199] [Table 9]

[0200] The degree of modification of PSA by Q-NMR assay is 11.1±0.2.

[0201] Example B12: Relative accuracy of Example B11 Traditional Method This experiment (see Example B11) revealed that the PSA potency of the six samples, when measured by conventional methods, had a relative standard deviation (RSD) of 2.78%.

[0202] Q-NMR method This experiment (see Example B11) revealed that the PSA potency of the six samples, as measured by Q-NMR method, had a relative standard deviation (RSD) of 1.48%.

[0203] Example B13: Influence of the spectrometer on Q-NMR measurements Two identical NMR samples of the PSA-rFVIII conjugate were prepared according to Example B1. One sample was sent to an NMR facility in Austria equipped with a Bruker 600 MHz spectrometer, and the second sample was sent to an NMR facility in Germany equipped with a Bruker 600 MHz spectrometer, using the acquisition and processing parameters described in Example B6.

[0204] The degree of PSA modification was calculated. *Calculated PSA modification of sample measured in Austria: 9.56 *Calculated PSA modification of samples measured in Germany: 9.25

[0205] The relative precision of PSA modification for samples measured in Austria and samples measured in Germany is as follows: 9.25 / 9.56=0.968

[0206] These results are in agreement with ±5% < 5%. The precision of the results obtained was independent of the spectrometer.

[0207] Example B14: Effect of scan number on Q-NMR measurements A PSA-rFVIII conjugate sample was prepared according to Example B1. The NMR spectrum of this sample (PSA-rFVIII concentration = 0.8 mg / mL) was measured three times in succession using the acquisition and processing parameters described in Example B6: the first using 3000 scans, the second using 1500 scans, and the third using 16 scans.

[0208] The spectra were processed to determine the degree of PSA modification. *PSA modification degree of sample calculated from 3000 scans: 12.52 *PSA modification degree of sample calculated from 1500 scans: 12.42 *PSA modification degree of sample calculated from 16 scans: 11.80

[0209] The relative precision of the PSA modification degree for the sample with 16 scans and the sample measured with 3000 scans is as follows: 11.80 / 12.52=0.942

[0210] Even at low resolution (16 scans), reasonable correlation was obtained with higher resolution spectra (3000 scans).

[0211] Example C1: Generation of PEG-HSA conjugates used as reference samples HSA contains 60 lysine residues, and the more exposed residues can be chemically modified with branched NHS PEG with a molecular weight of 20 kD to form amide bonds.

[0212] Human albumin (MW 66.5 kD, 0.4813 μmol, 0.291 mL, protein concentration 110 mg / mL) in 20 mM phosphate buffer (pH 7) was diluted with 50 mM phosphate buffer (pH 7) (7.709 mL) and reacted with PEG-NHS solution (MW 20 kD, 4.813 μmol, 1 mL, PEG concentration 96 mg / mL) in 2 mM hydrogen chloride for 2 h at ambient temperature. The coupling reaction was quenched by reacting the mixture with 1 M glycine solution (80 μL) for 1 h at ambient temperature. To remove glycine, excess PEG-NHS, and free NHS, the mixture (9 mL) was dialyzed against 20 mM phosphate buffer (pH 7) (2 L, buffer exchanged after 3 and 4 days) at ambient temperature for 5 days using a Float-A-Lyzer G2 dialysis device (Spectrum Laboratories; MWCO 50 kD, 10 mL), followed by dialysis against 20 mM phosphate buffer (pH 7) (2 L, buffer exchanged after 1, 2, and 4 days) at ambient temperature for 8 days using a Float-A-Lyzer G2 dialysis device (Spectrum Laboratories; MWCO 100 kD, 10 mL) to obtain a clear, colorless product solution. The clear, colorless product solution was then used directly for conventional or NMR testing.

[0213] Example C2: Determination of Total HSA Concentration Using Conventional Methods - Nephelometry Nephelometry is a method used for the immunochemical measurement of proteins in complex biological mixtures, including plasma, serum, urine, and other body fluids. When an antigen solution is mixed with a corresponding antibody solution, immune complexes form, causing the solution to become turbid. Nephelometry measures the turbidity of a solution by measuring the decrease in the intensity of light scattered by these immune complexes. Using defined conditions, the intensity of scattered light is proportional to the amount of immune complexes in the solution. The following procedure was performed on a BN ProSpec nephelometer according to the BN ProSpec instruction manual [BN ProSpec Instruction Manual, Siemens. Version 1.2, Ausgabedatum 2005 / 05]. The total HSA concentration was determined to be 1.16 mg / mL.

[0214] Example C3: Determination of total PEG concentration using conventional methods—HPLC The PEG concentration in HSA samples is measured using an Agilent HPLC1200 system equipped with an evaporative light scattering detector (ELSD) and an Onyx Monolith C18 column (4.6 x 100 mm). Prior to analyzing the samples by HPLC, the PEG-rFVIII protein in Adynovate is enzymatically digested with pronase K and reduced with dithiothreitol (DTT). Then, 100 μL of sample (PEG concentration 3–45 μg / mL) is injected and analyzed using the following separation conditions: Eluent A: 0.1% TFA in H2O, Eluent B: 0.1% TFA in CH3CN. Gradient: 0 min 25% B; 6 min 65% B; 6,1 min 25 B; 9 min 25% B. Flow rate: 2mL / min.

[0215] For the reference curve, use PEG standards of different concentrations (PEG concentrations: 3 to 45 μg / mL). The ELSD signal is integrated. The concentration of total PEG is calculated from the reference curve between the common logarithm of the concentration and the common logarithm of the peak area of the calibration standard.

[0216] The total PEG concentration was determined to be 0.630 mg / mL.

[0217] Example C4: Conventional Method—Calculation of the PEG Modification Degree of PEG-HSA Conjugates Based on the data obtained in the above assay, assuming that the molecular weight of albumin is 66.5 kDa and the molecular weight of PEG is 20 kDa, the degree of PEG modification of HSA is as follows: TIFF2025118907000021.tif25170

[0218] By conventional methods, the degree of PEG modification for albumin in the reference sample was determined to be 1.81 mol PEG / mol HSA.

[0219] Example C5: HSA contains 60 lysine residues, and the more exposed residues can be chemically modified with branched NHS PEG with a molecular weight of 20 kD to form amide bonds.

[0220] Human albumin (MW 66.5 kD, 0.4813 μmol, 0.291 mL, protein concentration 110 mg / mL) in 20 mM phosphate buffer (pH 7) was diluted with 50 mM phosphate buffer (pH 7) (7.709 mL) and reacted with PEG-NHS solution (MW 20 kD, 9.626 μmol, 1 mL, PEG concentration 192 mg / mL) in 2 mM hydrogen chloride at ambient temperature for 2 h. The coupling reaction was quenched by reacting the mixture with 1 M glycine solution (80 μL) at ambient temperature for 1 h. To remove glycine, excess PEG-NHS, and free NHS, the mixture (9 mL) was dialyzed against 20 mM phosphate buffer (pH 7) (2 L, buffer exchanged after 3 and 4 days) at ambient temperature for 5 days using a Float-A-Lyzer G2 dialysis device (Spectrum Laboratories; MWCO 50 kD, 10 mL), followed by dialysis against 20 mM phosphate buffer (pH 7) (2 L, buffer exchanged after 1, 2, and 4 days) at ambient temperature for 8 days using a Float-A-Lyzer G2 dialysis device (Spectrum Laboratories; MWCO 100 kD, 10 mL) to obtain a clear, colorless product solution with a protein concentration of 1.83 mg / mL. The protein concentration was determined by nephelometry (details available from Siemens Healthcare Diagnostics GmbH).

[0221] Example C6: Q-NMR Method: Acquisition and Processing Preparation of samples containing conjugates and Q-NMR acquisition / processing analysis An aqueous sample of PEG-HSA (0.5 mL, 0.5–5 mg / mL) in a 2 mL vial was thawed by leaving it at room temperature for 30 minutes. The sample was transferred to a 5 mm NMR tube with a Pasteur pipette. 56 μL of DO (99.9 atomic % DO) was added to the NMR tube, and the tube was sealed with a polyethylene cap. The contents were then homogenized by shaking the tube. Finally, air bubbles were removed by gentle tapping or by briefly immersing the tube in an ultrasonic bath. The NMR tube was wiped and placed in an NMR spinner according to the spectrometer supplier's specifications.

[0222] Acquisition parameters Proton NMR spectra were obtained on a 600 MHz AVANCE BRUKER spectrometer as follows. Spectrometer frequency: 600MHz pulse program:zgesgp; Temperature (℃):25±5; Number of scans: 3000 SW(ppm):>14(approx.+10~-4); Receiver Gain: Optimized D1 (seconds):1

[0223] Processing parameters Proton NMR spectra were processed using BRUKER Topspin software V3.5 as follows. Window function: EM (line broadening: 0.3 Hz); The following command was executed. ft: Fourier transform apk:Auto phase correction abs: Automatic baseline correction Axis calibration: After calibrating the methyl signal at 0.00 ppm, the spectral reference frequencies were matched to the DSS SR values.

[0224] Example C7:K NMR Measurement of K of PEG-HSA conjugates NMR Measurement method This method is useful when one sample with a known value is available by conventional methods. Using a PEG-HSA conjugate sample with a known degree of PEG modification, the K NMR In Example C4, the degree of PEG modification was determined by conventional methods to be 1.81 mol PEG / mol HSA.

[0225] The proton NMR spectrum of the reference sample was obtained and processed according to Example C6.

[0226] The number of valine, leucine, and isoleucine residues present in HSA is multiplied by the number of first-order protons (signal: 1 ppm or less) they each contain. For HSAs: Valine: 43 residues x 6 first-order protons = 258H Leucine: 64 residues x 6 first-order protons = 384H Isoleucine: 9 residues x 7 first-order protons = 63H

[0227] This protein has a total of 705 primary protons. The protein multiplet is integrated over the range of approximately 0.89-0.5 ppm using the integrate command with automatic baseline correction, and the integral is calibrated to the value 705.

[0228] Set the integration of the PEG signal. This peak appears as a singlet. Determine the peak center (3.555 ppm) and integrate this peak ±0.01 ppm on either side of the peak center using the integrate command with automatic baseline correction. An integration value of 100114 protons was obtained.

[0229] The value of this integral is divided by the average number of CH2PEG protons present in each ethylene glycol unit (4 protons). 20000 g / mol / 44 g / mol = 455 PEG units (per 20 kDa PEG chain) 455 PEG units (per PEG reagent) x 4 protons (per ethylene glycol unit) = 1820 H

[0230] On average, there are 1820 CH2 protons per 20 kDa PEG chain. 100114 / 1820=55.0

[0231] This result is then divided by the known degree of PEG modification of the reference sample. Thus, the K NMR is obtained. 55.03 / 1.81=30.4

[0232] K of PEG-HSA conjugates NMR is 30.4.

[0233] Example C8: Q-NMR Modification Degree Q-NMR measurement of the degree of PEG modification of PEG-HSA conjugates The PEG MD of an unknown PEG-HSA sample was determined using Q-NMR techniques. The sample was prepared according to Example C5.

[0234] The proton NMR spectrum was acquired and processed according to Example C6, with the PEG peak centered at 3.555 ppm (similar to the reference spectrum).

[0235] The number of valine, leucine, and isoleucine residues present in rFVIII is multiplied by the number of first type protons (signal: ≦1 ppm) they each contain.

[0236] For HSAs: Valine: 43 residues x 6 first-order protons = 258H Leucine: 64 residues x 6 first-order protons = 384H Isoleucine: 9 residues x 7 first-order protons = 63H

[0237] This protein has a total of 705 first-order protons. The protein multiplet is integrated in the range of approximately 0.89–0.5 ppm (similar to the reference spectrum) using the integrate command with automatic baseline correction, and the integral is calibrated to the value 705.

[0238] Set the integration of the PEG signal. This peak appears as a singlet. Set the peak center at 3.555 ppm and integrate this peak for ±0.01 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0239] The integral value of the PEG peak (117876 H) was obtained.

[0240] The value of this integral was divided by the average number of CH2PEG protons present in each PEG chain unit. This result was then used to calculate the NMR coefficient (K NMR ) (see Example C7). This accounts for protein heterogeneity as well as overlapping peaks in the integration range. The result obtained is the degree of PEG modification of the PEG-HSA conjugate in the unknown sample. 117876 / (1820x30.4)=2.13

[0241] According to NMR, the degree of PEG modification of the PEG-HSA conjugate in the unknown sample is 2.13.

[0242] Using NMR to determine the MD of a sample, it can be confirmed that the results are the same as those obtained by conventional methods.

[0243] Calculation of the degree of PEGylation of unknown sample PEG-albumin using conventional methods The PEG concentration of the unknown sample was determined to be 1.22 mg PEG / mL using RP-HPLC / ELSD.

[0244] Based on the data obtained in the assay, the degree of PEGylation of HSA is calculated using the same calculation scheme as that enumerated for PSA-rFVIII in Example B2. TIFF2025118907000022.tif9170Molecular weight of PEG reagent: 20kDa Albumin molecular weight = 66.5 kDa Protein concentration = 1.83 mg / mL TIFF2025118907000023.tif8170PEG MD=2.22

[0245] The PEG degree of the unknown sample (PEG-albumin) by the conventional method is 2.22 PEG chains per protein molecule, which is consistent with the value obtained according to the NMR method.

[0246] Example C9: Determination of the (relative) accuracy of the Q-NMR method in determining the degree of PEG modification of PEG-HSA conjugates By conventional methods, the degree of PEG modification of the unknown sample PEG-HSA conjugate was determined to be 2.22 mol PEG / mol HSA.

[0247] The degree of PEG modification of the unknown sample of PEG-HSA conjugate was determined to be 2.13 mol PEG / mol HSA by Q-NMR.

[0248] The relative accuracy of the Q-NMR method in determining the PEG modification degree of PEG-HSA conjugates is as follows: 2.13 / 2.22=0.96 These results are in agreement within ±5%.

[0249] Example D1: Determination of the PEG modification degree of PEG-rFVIII conjugate samples Generation of PEG-rFVIII conjugates The aqueous sample of PEG-rFVIII in the vial can be thawed by leaving it at room temperature for 30 minutes. The sample can be transferred to a 5 mm NMR tube with a Pasteur pipette. After adding DO (99.9 atomic % D) to the NMR tube and sealing the tube with a polyethylene cap, the contents can be homogenized by shaking the tube. Finally, air bubbles can be removed by gentle tapping or briefly immersing the tube in an ultrasonic bath. The NMR tube can be wiped and placed in an NMR spinner according to the spectrometer supplier's specifications.

[0250] Q-NMR acquisition parameters Proton NMR spectra can be obtained on a 600 MHz AVANCE BRUKER spectrometer as follows. Spectrometer frequency: 600MHz pulse program:zgesgp; Temperature (℃):25±5; Number of scans: 3000 SW(ppm):>14(approx.+10~-4); Receiver Gain: Optimized D1 (seconds):1

[0251] Q-NMR processing parameters Proton NMR spectra can be processed using BRUKER Topspin software V3.5 as follows. Window function: EM (line broadening: 0.3 Hz); The following commands can be executed: ft: Fourier transform apk:Auto phase correction abs: Automatic baseline correction Axis calibration: After calibrating the methyl signal at 0.00 ppm, the spectral reference frequencies can be matched with the DSS SR values.

[0252] Example D2: Determination of total rFVIII concentration using conventional method - Fluorescence method The degree of PEG modification of the PEG-rFVIII conjugate can be calculated by conventional methods by measuring the concentration of bound PEG and the concentration of FVIII protein, which is measured by a fluorescence method.

[0253] Example D3: Conventional Method—Measurement of Bound PEG Concentration Using HPLC Assay Principle The PEG modification degree of the PEG-rFVIII conjugate can be calculated by conventional methods by measuring the concentration of PEG and the concentration of FVIII protein. The procedure is similar to the determination of PEG concentration in Example C and the determination of FVIII in Example B. Both parameters are determined using RP-HPLC method.

[0254] Determination of total PEG Total PEG in PEG-rFVIII conjugate samples is measured using an Agilent HPLC1200 system equipped with an evaporative light scattering detector (ELSD) and an Onyx Monolith C18 column (4.6 x 100 mm). Before analyzing the samples by HPLC, the rFVIII in the PEG-rFVIII conjugates is enzymatically digested with pronase K and reduced with dithiothreitol (DTT). Then, 100 μL of sample (PEG concentration 3–45 μg / mL) is injected and analyzed using the following separation conditions: Eluent A: 0.1% TFA in H2O, Eluent B: 0.1% TFA in CH3CN. Gradient: 0 min 25% B; 6 min 65% B; 6,1 min 25 B; 9 min 25% B. Flow rate: 2mL / min.

[0255] For the reference curve, use PEG standards of different concentrations (PEG concentrations: 3 to 45 μg / mL). The ELSD signal is integrated. The concentration of total PEG is calculated from the reference curve between the common logarithm of the concentration and the common logarithm of the peak area of the calibration standard.

[0256] Calculation of the degree of PEGylation of Adynovate® Based on the data obtained in the above assays, the degree of PEGylation of Adynovate® is calculated using the same calculation scheme as that enumerated for PSA-rFVIII in Example C.

[0257] K NMRTo determine this, Adynovate® samples with known PEG degrees are set as standards against which other PEG-rFVIII conjugates are measured.

[0258] Proton NMR spectra of reference samples were acquired and processed as above.

[0259] The number of valine, leucine, and isoleucine residues present in rFVIII is multiplied by the number of first-order protons (signal: 1 ppm or less) they each contain. For full-length rFVIII: Valine: 125 residues x 6 first-order protons = 750 H Leucine: 222 residues x 6 first-order protons = 1332H Isoleucine: 110 residues x 7 first-order protons = 770 H

[0260] This protein has a total of 2852 first-order protons. The protein multiplet is integrated in the range of approximately 1.05–0.6 ppm (depending on the peak resolution) using the integration command with automatic baseline correction, and the integral is calibrated to the value 2852.

[0261] Set the integration for the PEG signal. This peak appears as a singlet. Determine the center of the peak (approximately 3.67 ppm) and integrate this peak for ±0.01 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0262] The value of this integral is divided by the average number of CH2PEG protons present in each PEG chain unit, and then this result is divided by the known PEG degree of the reference sample. Thus, the K NMR is obtained.

[0263] Q-NMR measurement of the degree of PEG modification of Adynovate® samples The number of valine, leucine, and isoleucine residues present in rFVIII is multiplied by the number of first-order protons (signal: 1 ppm or less) they each contain. For full-length rFVIII: Valine: 125 residues x 6 first-order protons = 750 H Leucine: 222 residues x 6 first-order protons = 1332H Isoleucine: 110 residues x 7 first-order protons = 770 H

[0264] This protein has a total of 2852 first-order protons. The protein multiplet is integrated in the range of approximately 1.05–0.6 ppm (depending on the peak resolution) using the integration command with automatic baseline correction, and the integral is calibrated to the value 2852.

[0265] Set the integration for the PEG signal. This peak appears as a singlet. Determine the center of the peak (approximately 3.67 ppm) and integrate this peak for ±0.01 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0266] This integral was divided by the average number of CH2PEG protons present in each PEG chain unit, and the result was calculated as the NMR coefficient (K NMR ) which accounts for protein heterogeneity as well as overlapping peaks within the integration range. The result obtained is the degree of PEG modification on full-length rFVIII.

[0267] Example E: MD measurement of PEGylated BPTI Preparation of PEGylated BPTI For PEGylation of bovine pancreatic trypsin inhibitor (BPTI or aprotinin; Ascenzi et al., Curr. Protein Pept. Sci. 2003, 4, 231-251), the MS(PEG)4 methyl-PEG-NHS-ester reagent from Thermofisher Scientific was used. This PEG reagent has a short, four-unit PEG chain and contains a terminal NHS ester to modify lysine residues to form stable amide bonds. It also contains a terminal methoxy end-cap. PEGylation of BPTI using NHS technology is described in US7550427B2 and CN101412995B.

[0268] Recombinant BPTI was purchased from Sigma-Aldrich (Saint Louis, MO, USA). A solution of BPTI (1 mg / mL) was prepared in 20 mM Hepes buffer (pH 7.4) containing 150 mM NaCl. The PEGylation reaction was carried out at room temperature for 1 hour with a 40 mM excess relative to the number of lysine residues (5 Lys). The reaction was then stopped by adding glycine (final concentration: 100 mM). Finally, the conjugate was purified by cation exchange chromatography on SP-Sepharose FF (GE-Healthcare) in 50 mM phosphate buffer (pH 7.2). The reaction mixture was applied to the column, followed by a five-column flush with phosphate buffer (pH 7.2). The conjugate was eluted from the column using a linear gradient of 0 to 0.8 M NaCl. Fractions containing the conjugate are concentrated by UF / DF using a Vivaspin2 device made of polyethersulfone with a molecular weight cut-off of 3 kDa (Sigma-Aldrich, Saint Louis, MO, USA).

[0269] Measurement of the degree of PEG modification of PEG-BPTI conjugates by Q-NMR BPTI 1 The 1 H NMR spectrum has been published by Masson and Wuthrich (FEBS Letters 1973, 31, 114-118).

[0270] Multiply the number of valine, leucine, and isoleucine residues present in the protein by the number of first-order protons (signal: 1 ppm or less) they each contain. For BPTI: Valine: 1 residue x 6 first-order protons = 6H Leucine: 2 residues x 6 first-order protons = 12H Isoleucine: 2 residues x 7 first-order protons = 14H

[0271] This protein has a total of 32 primary protons. The protein multiplet is integrated in the range of approximately 1.05-0.6 ppm (depending on the peak resolution) using the integration command with automatic baseline correction, and the integral is calibrated at a value of 32. If another peak interferes with the integration, a closet baseline point is used.

[0272] Set the integration for PEG. This peak appears as a singlet. Determine the peak center (approximately 3.67 ppm) and integrate this peak for ±0.01 ppm on either side of the peak center using the integrate command with automatic baseline correction.

[0273] The value of this integral is divided by the average number of CH2PEG protons present in each PEG chain. The quotient is then calculated as the NMR coefficient (K NMR ) which accounts for protein heterogeneity as well as overlapping peaks within the integration range. This factor can be calculated from modification data measured by MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry. This is outlined in US7550427B2.

[0274] The results obtained are: 1 The degree of PEG modification on BPTI determined by 1 H NMR spectroscopy.

[0275] Example F: MD measurement of PEGylated FIX Human coagulation factor IX is a 461-amino acid serine protease with a molecular weight of 57 kDa. Its structure consists of a Gla domain, an EGF domain, an activation peptide, and a catalytic domain. The activation peptide contains an N-glycan (Makino et al., J. Biochem. 2000, 128(2):175-180), and can be selectively PEGylated using the same approach as described for PSA-rFVIII in Example B (reaction of FVIII with aminooxy PSA). To this end, recombinant factor IX is PEGylated via its N-glycan using a 20 kDa aminooxy reagent (Sunbright GL2-200CA) from NOF (NOF Corporation, Tokyo, Japan) as described in US8642737B2.

[0276] Factor IX 1 The 1 H NMR spectrum has been published by Freedman et al. (J. Biol. Chem 1995, 270(14), 7980-7987).

[0277] Multiply the number of valine, leucine, and isoleucine residues present in the protein by the number of primary protons (signals below 1 ppm) each contains. The protein multiplet is integrated in the range of approximately 1.05 to 0.6 ppm (depending on the peak resolution) using the integration command with automatic baseline correction, and the integral value is calibrated. If another peak interferes with the integration, a closet baseline point is used. Set the integral for PEG. This peak appears as a singlet. Determine the peak center (approximately 3.67 ppm) and integrate this peak using the integration command with automatic baseline correction for ±0.01 ppm on either side of the peak center.

[0278] The value of this integral is divided by the average number of CH2PEG protons present in each PEG chain. The quotient is then calculated as the NMR coefficient (K NMR) which accounts for protein heterogeneity as well as overlapping peaks within the integration range. This factor can be calculated from data on the degree of modification measured by conventional methods (e.g., HPLC methods such as those described for PSA-rFVIII in Example B).

[0279] The results obtained are: 1 1 is the degree of PEG modification on coagulation factor IX determined by 1 H NMR spectroscopy.

[0280] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations therein will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for determining the average number of first polymers covalently attached to a protein-first polymer conjugate in a solution, comprising: a) adding to the solution containing the protein-first polymer conjugate １ performing H nuclear magnetic resonance measurements; b) the above １ processing the H nuclear magnetic resonance measurements; c) the first proton peak １ integrating H nuclear magnetic resonance measurements, wherein the first type proton peak comprises a signal generated by at least one proton on the side chain of at least one amino acid in the protein-first polymer conjugate; d) calibrating the results of c); e) the second proton peak １ integrating H nuclear magnetic resonance measurements, wherein the second type proton peak comprises a signal generated by at least one proton in the first polymer; f) dividing the result of e) by the number of second type protons per monomer of the first polymer; and g) dividing the result of f) by the average number of monomers in the first polymer. thereby determining the average number of said first polymers covalently attached to said protein-first polymer conjugates in said solution.

2. The method further comprises: ＮＭＲ further comprising the step of dividing by K ＮＭＲ The method of claim 1 , wherein is obtained from a standard or a calibration curve or a surrogate assay.

3. 2. The method of claim 1, wherein the calibrating step comprises equating the result of step c) with the number of first type protons in the protein-first polymer conjugate.

4. the integrating step in step c) is fixed; and The integrating step in step e) is fixed; The method of claim 1.

5. The integration step of c) is centered on the first proton peak, and The integration step of e) is centered on the second proton peak. The method of claim 1.

6. b) is １ The H nuclear magnetic resonance measurement was In Fourier transform, Automatic phase correction Automatic baseline correction With a linewidth broadening window function of approximately 0.01 to 10 Hz, With an exponential window function, and DSS axis calibration The method of claim 1 , further comprising processing.

7. a) is １ H nuclear magnetic resonance measurement At a temperature of about 15°C to 35°C, Approximately 16 to 10,000 scans A spectral width of about +10 ppm to about -4 ppm, with a repeat delay time of about 0.1 seconds to about 120 seconds, and With optimized receiver gain The method of claim 1 , comprising:

8. 2. The method of claim 1, wherein the first type proton peak consists essentially of a signal generated by the at least one proton on the side chain of the at least one amino acid in the protein-first polymer conjugate.

9. 2. The method of claim 1, wherein the first type proton peak comprises signals generated by six protons on the methyl group of valine, six protons on the methyl group of leucine, six protons on the methyl group of isoleucine, and a proton on the methine group of isoleucine.

10. 2. The method of claim 1, wherein the first type proton peaks consist essentially of signals generated by six protons on the methyl group of valine, six protons on the methyl group of leucine, six protons on the methyl group of isoleucine, and a proton on the methine group of isoleucine.

11. The method of claim 1, wherein the first proton peak is located on an amino acid of a protein having blood coagulation biological activity.

12. 12. The method of claim 11, wherein the protein is selected from the group consisting of Factor VII, Factor VIIa, Factor VIII, Factor IX, and von Willebrand factor (VWF).

13. 13. The method of claim 12, wherein the protein is Factor VIII.

14. 2. The method of claim 1, wherein the first proton peak is located on an amino acid of a protein having human serum albumin biological activity or bovine pancreatic trypsin inhibitor biological activity.

15. 2. The method of claim 1, wherein the second type proton peak consists essentially of a signal produced by at least one proton in the first polymer.

16. The first polymer in the protein-first polymer conjugate is polysialic acid, and the second proton peak is H on a sialic acid monomer in the polysialic acid. ３ａ The method of claim 1 , wherein the protons are generated from protons.

17. The method of claim 1 , wherein the first polymer is a water-soluble polymer.

18. The method of claim 1 , wherein the first polymer is a polyalkylene glycol or a polysaccharide.

19. 20. The method of claim 18, wherein the first polymer is a polyalkylene glycol and the polyalkylene oxide is polyethylene glycol.

20. 20. The method of claim 18, wherein the first polymer is a polysaccharide, and the polysaccharide is polysialic acid.

21. 1. A method for determining the average number of polysialic acid polymers covalently attached to a Factor VIII-polysialic acid conjugate in a solution, comprising: a) adding to a solution containing the Factor VIII-polysialic acid conjugate, At a temperature of about 15°C to 35°C, Approximately 16 to 10,000 scans A spectral width of about +10 ppm to about -4 ppm, with a repeat delay time of about 0.1 seconds to about 120 seconds, and With optimized receiver gain １ performing H nuclear magnetic resonance measurements; b) the above １ The H nuclear magnetic resonance measurement was In Fourier transform, Automatic phase correction Automatic baseline correction With a 0.3Hz line broadening window function, With an exponential window function, and DSS axis calibration treating, c) the first proton peak consisting essentially of signals generated by six protons on the methyl group of valine, six protons on the methyl group of leucine, six protons on the methyl group of isoleucine, and protons on the methine group of isoleucine. １ performing center integrating of H nuclear magnetic resonance measurements; d) calibrating the results of c); e) H on the sialic acid monomer in the polysialic acid polymer ３ａ The second proton peak essentially consists of signals generated from protons. １ performing a centered integration of the H nuclear magnetic resonance measurements; f) dividing the result of e) by 1; g) dividing the result of f) by the average number of monomers in the polysialic acid polymer; and The result of step g) is K ＮＭＲ Dividing by K ＮＭＲ is obtained from a standard or calibration curve or surrogate assay, A method comprising:

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