Determination of the free N-terminus of pegfilgrastim using acid protease

Abstract

The present disclosure provides materials and methods for determining the presence of an N-terminal modification on a therapeutic protein and / or the efficiency of an N-terminal modification, such as PEGylation, at the N-terminus of a therapeutic protein, such as filgrastim (the PEGylated version is therefore pegfilgrastim).

Description

Technical Field

[0001] Incorporation by reference of electronically submitted materials A sequence listing that is part of this disclosure has been submitted as a text file simultaneously with this specification. The name of the text file containing this sequence listing is "55200_Seqlisting.txt", created on March 18, 2021, and its size is 2,0,64 bytes. The subject matter of this sequence listing is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] Pegfilgrastim (Neulasta®) is produced by attaching a polyethylene glycol (PEG) polymer to filgrastim (granulocyte colony-stimulating factor; G-CSF, GCSF) using conditions that result in linkage at the N-terminal amine of filgrastim upon reaction with a PEG-aldehyde. However, even under special conditions, some proportion of PEG-aldehyde may react with other primary amine groups in filgrastim. The filgrastim molecule contains five primary amine groups, the first and most desirable being located at the N-terminus, while the other four are located on the side chains of lysine residues at positions 17, 24, 35, and 41, respectively. To determine that PEGylation is indeed occurring at the N-terminus and not on the side chains of lysine residues, the analytical method must be able to distinguish the N-terminus from all lysine residues, and among these lysine residues, Lys-17 is the most difficult to distinguish due to its proximity to the N-terminus. When PEG is present at various sites, chromatographic separation of PEGylated filgrastim is difficult. Therefore, fragmentation techniques must be applied to cleave PEGylated filgrastim into smaller fragments. Due to the large size of PEG (approximately 20 kDa) and its heterogeneous nature, separation of filgrastim fragments with PEG located at various sites remains challenging. For example, to distinguish whether the PEG molecule is attached to the N-terminus (e.g., the N-terminal methionine, if present) or to Lys-17, these two residues must be separated by either chemical or enzymatic methods. Historically, Edman degradation has been used to evaluate N-terminal PEGylation of modified polypeptides, but further methods are needed in this field to evaluate the efficiency of PEGylation or other conjugations at the N-terminus of filgrastim. [Overview of the project] [Means for solving the problem]

[0003] As described herein, the disclosure provides materials and methods for determining the presence of N-terminal modifications on therapeutic proteins and / or the efficiency of N-terminal modifications (e.g., PEGylation; thus the PEGylated version is filgrastim) at the N-terminus of therapeutic proteins such as filgrastim.

[0004] In one embodiment, the present disclosure provides a method for determining the amount of the unmodified (e.g., “free”) N-terminus of a polypeptide, comprising: (a) incubating a sample containing the polypeptide and a nonspecific protease under conditions that allow cleavage at one or more sites within the polypeptide and a single cleavage between the N-terminal amino acid at position 1 and the first lysine amino acid; (b) separating the cleavage product produced in step (a); and determining the amount of the unmodified free N-terminus of the polypeptide by comparing it with a control standard. In one embodiment, the polypeptide is recombinant.

[0005] In one embodiment, the present disclosure provides a method for measuring the amount of the unmodified (e.g., “free”) N-terminus of a human granulocyte colony-stimulating factor (G-CSF) polypeptide, comprising: (a) incubating a sample containing the G-CSF polypeptide and a nonspecific protease under conditions that allow cleavage at one or more sites within the G-CSF polypeptide and a single cleavage between the N-terminal methionine at position 1 and the lysine at position 16; (b) separating the cleavage product produced in step (a); and (c) measuring the amount of the unmodified free N-terminus of the G-CSF polypeptide by comparison with a control standard. In one embodiment, the G-CSF polypeptide is recombinant.

[0006] In other embodiments, a method is provided which is the method described above, wherein the sample comprises a mixture of a modified G-CSF polypeptide and an unmodified G-CSF polypeptide, the modified G-CSF polypeptide comprising at least one polyethylene glycol (PEG) modification.

[0007] In yet another embodiment, a method is provided in which the G-CSF polypeptide is selected from the group consisting of pegfilgrastim (Neulasta®), pegfilgrastim-jmdb (Fulphila®), INN-pegfilgrastim (Pelgraz®), Lapelga®, Pelmeg®, pegfilgrastim-cbqv (Udenyca®), pegfilgrastim-bmez (Ziextenzo®), and Grasustek®. In one embodiment, the G-CSF polypeptide is pegfilgrastim (Neulasta®).

[0008] The disclosure also provides, in various embodiments, the aforementioned method, wherein a nonspecific protease cleaves between leucine at position 15 and leucine at position 16, generating peptides (peptides M1-L15) with a length of 15 amino acids.

[0009] In another embodiment, the method described above is provided, wherein the nonspecific protease is pepsin.

[0010] This disclosure provides, in various embodiments, the method described above, wherein the conditions in step (a) include incubation for a period of time of (a) about 5 minutes to about 60 minutes, at a pH of about 1.5 to about 4.0, at a temperature of about 25°C to about 60°C. In one embodiment, the conditions include incubation for a period of time of (a) about 2.2, at a pH of about 37°C, and at a temperature of (c) about 15 minutes.

[0011] In yet another embodiment, a method is provided in which the separation of step (b) is carried out under conditions that allow for the separation of peptide M1-L15 from other cleavage products. In one embodiment, the separation of step (b) is carried out by a method selected from chromatography and electrophoresis. In another embodiment, the chromatography is selected from the group consisting of high-performance liquid chromatography (HPLC) and ultrahigh-performance liquid chromatography (UHPLC). In yet another embodiment, the HPLC is reverse-phase HPLC (RP-HPLC). In yet another embodiment, the chromatography comprises a column and trifluoroacetic acid (TFA) at a concentration of about 0.01 v / v% to about 0.2 v / v%. In a related embodiment, the concentration of TFA is about 0.02 v / v% to about 0.03 v / v%. In yet another embodiment, the concentration of TFA is about 0.025 v / v%.

[0012] In other embodiments, the method described above is provided, wherein step (c) of measurement is performed by mass spectrometry. In one embodiment, the mass spectrometry is selected from electrospray MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

[0013] In yet another embodiment, a method is provided which is the method described above, wherein the control standard comprises a known amount of modified G-CSF polypeptide and a known amount of unmodified G-CSF polypeptide.

[0014] In one embodiment, the present disclosure provides a method for measuring the amount of non-PEGylated free N-terminus of pegfilgrastim (Neulasta®), comprising: (a) incubating a sample containing pegfilgrastim (Neulasta®) and a non-specific protease under conditions that allow cleavage at one or more sites within the pegfilgrastim (Neulasta®) and only one cleavage between the N-terminal methionine at position 1 and the lysine at position 16, the conditions comprising incubating at (a) a pH of about 2.2, (b) a temperature of about 37°C, and (c) for a time period of about 15 minutes; (b) separating the cleavage products generated in step (a) by reverse-phase HPLC (RP-HPLC); and (c) measuring the amount of non-PEGylated free N-terminus of pegfilgrastim (Neulasta®) by comparing to a control standard. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for measuring the amount of the unmodified N-terminus of human granulocyte colony-stimulating factor (G-CSF) polypeptide, (a) Incubating a sample containing the G-CSF polypeptide and a nonspecific protease under conditions that allow for cleavage at one or more sites within the G-CSF polypeptide, and for a single cleavage between the N-terminal methionine at position 1 and the lysine at position 16; (b) A step of separating the cleavage product generated in step (a); and (c) A step of measuring the amount of the unmodified free N-terminus of the G-CSF polypeptide by comparing it with a control standard. A method that includes this. (Item 2) The method according to item 1, wherein the G-CSF polypeptide is a recombinant. (Item 3) The method according to item 1 or 2, wherein the sample comprises a mixture of a modified G-CSF polypeptide and an unmodified G-CSF polypeptide, the modified G-CSF polypeptide comprising at least one polyethylene glycol (PEG) modification. (Item 4) The G-CSF polypeptide is selected from the group consisting of pegfilgrastim (Neulasta®), pegfilgrastim-jmdb (Fulphila®), INN-pegfilgrastim (Pelgraz®), Lapelga®, Pelmeg®, pegfilgrastim-cbqv (Udenyca®), pegfilgrastim-bmez (Ziextenzo®), and Grasustek®, according to any one of items 1 to 3. (Item 5) The method described in item 4, wherein the G-CSF polypeptide is pegfilgrastim (Neulasta®). (Item 6) The method according to any one of items 1 to 5, wherein the nonspecific protease cleaves between the leucine at position 15 and the leucine at position 16, producing peptides (peptides M1-L15) with a length of 15 amino acids. (Item 7) The method according to any one of items 1 to 6, wherein the nonspecific protease is pepsin. (Item 8) The method according to any one of items 1 to 7, wherein the conditions in step (a) include (a) a pH of about 1.5 to about 4.0, (b) a temperature of about 25°C to about 60°C, and (c) incubation for a time of about 5 minutes to about 60 minutes. (Item 9) The method according to item 8, wherein the conditions include (a) incubation at a pH of about 2.2, (b) incubation at a temperature of about 37°C, and (c) incubation for a time of about 15 minutes. (Item 10) The method according to any one of items 1 to 9, wherein the separation in (b) is carried out under conditions that allow for the separation of peptide M1-L15 from other cleavage products. (Item 11) The method according to item 10, wherein the separation in step (b) is carried out by a method selected from chromatography and electrophoresis. (Item 12) The chromatography method according to item 11, wherein the chromatography is selected from the group consisting of high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC). (Item 13) The method according to item 10, wherein the HPLC is reverse-phase HPLC (RP-HPLC). (Item 14) The chromatography is the method according to item 12 or 13, comprising a column and trifluoroacetic acid (TFA) at a concentration of about 0.01 v / v% to about 0.2 v / v%. (Item 15) The method according to item 14, wherein the concentration of the TFA is approximately 0.02 v / v% to approximately 0.03 v / v%. (Item 16) The method according to item 15, wherein the concentration of the TFA is approximately 0.025 v / v%. (Item 17) The method according to any one of items 1 to 16, wherein step (c) of the measurement procedure is performed by mass spectrometry. (Item 18) The mass spectrometry is selected from electrospray MS and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) as described in item 17. (Item 19) The control standard is the method according to any one of items 1 to 18, comprising a known amount of modified G-CSF polypeptide and a known amount of unmodified G-CSF polypeptide. <000014​​​​​​​​​​​​​​​​​​​​​​​​​​The peptide mapping profile of filgrastim shows pepsin digestion to produce the major N-terminal peptides M1-L15. Chromatograms were obtained on an Agilent 1260 system with an acetonitrile gradient of 2%–35% acetonitrile over 30 minutes. The mobile phase contained 0.02% (v / v) TFA. [Figure 4] This shows a comparison of the peptide map profiles of filgrastim and pegfilgrastim digested with pepsin. [Figure 5A] The overlapping profiles of pegfilgrastim and pegfilgrastim supplemented with 5% filgrastim are shown using 0.02% (v / v) TFA eluent in six different UPLC column lots. [Figure 5B] The overlapping profiles of pegfilgrastim and pegfilgrastim supplemented with 5% filgrastim are shown using 0.025% (v / v) TFA eluent in six different UPLC column lots. [Figure 5C] The overlapping profiles of pegfilgrastim (black trace) and pegfilgrastim with 5% filgrastim added (blue trace) using 0.03% (v / v) TFA eluent from four different UPLC column lots are shown. [Figure 6] The profile of a pegfilgrastim reference standard with 5% filgrastim added, using 0.025% (v / v) TFA in the eluent, is shown. [Figure 7] A comparison of chromatograms of pepsin digests of pegfilgrastim, neupogen, epogen, and romiplostim is shown. [Figure 8] The plots show the determined percentage of free N-terminal methionine for each additive level. [Figure 9] The residual plots for the percentage of free N-terminal methionine determined at each level are shown. [Figure 10] This shows a comparison of the stability of chromatograms from Level 3 (approximately 2% free N-terminal methionine) samples. [Figure 11-1](Figure 11A) Figure 11A shows robustness with respect to total area. (Figure 11B) Shows robustness with respect to noise (between peaks). (Figure 11C) Shows robustness with respect to P2 RT. [Figure 11-2] (Figure 11D) Shows robustness with respect to P2 area. (Figure 11E) Shows robustness with respect to P3 RT. [Figure 11-3] (Figure 11F) Shows robustness with respect to P3 area. (Figure 11G) Shows robustness with respect to P1 RT. [Figure 12] This demonstrates the robustness of determining the percentage of free N-terminal methionine. [Modes for carrying out the invention]

[0016] This disclosure addresses the aforementioned needs in the art by providing methods and materials useful for determining the presence of N-terminal modifications on therapeutic proteins and / or for determining the efficiency of N-terminal modifications at the N-terminus of therapeutic proteins such as filgrastim (e.g., PEGylation in one embodiment; thus the PEGylated version is pegfilgrastim).

[0017] In various embodiments, pegfilgrastim is digested under acidic conditions with pepsin, a nonspecific protease. The digested peptides are separated by reverse-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection. The proteolytic peptide containing 15 residues at the N-terminus is used to quantify free N-terminal methionine by standard detection methods by adding a known amount of filgrastim to the pegfilgrastim sample. The pepsin peptide map profile of the sample is compared to a pegfilgrastim reference standard to confirm its identity.

[0018] definition As used herein, “filgrastim” refers to filgrastim (Neupogen®) and may be used interchangeably with “G-CSF.” Biosimilar biosimilars similarly intended by this disclosure include, but are not limited to, filgrastim-aafi (Nivestym®), tbo-filgrastim (Granix®), and filgrastim-sndz (Zarxio®).

[0019] As used herein, “Pegfilgrastim” refers to Pegfilgrastim (Neulasta®), which is a PEGylated version of filgrastim. Biosimilar products similarly intended by this disclosure include, but are not limited to, Pegfilgrastim-jmdb (Fulphila®), INN-Pegfilgrastim (Pelgraz®), Lapelga®, Pelmeg®, Pegfilgrastim-cbqv (Udenyca®), Pegfilgrastim-bmez (Ziextenzo®), and Grasustek®.

[0020] As used herein, the term "G-CSF" means "granulocyte colony-stimulating factor." As used herein, G-CSF may be chemically or genetically modified and may be produced by recombinant methods known in the art. G-CSF may be modified, for example, by PEG (filgrastim) or other molecules. In one embodiment, G-CSF is modified at the N-terminus. In another embodiment, G-CSF is modified with N-terminal methionine. Unless otherwise stated, the term G-CSF refers to filgrastim, and the terms PEGylated G-CSF or G-CSF conjugate refer to pegfilgrastim.

[0021] The phrase "at least one" as used herein may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0022] Polyethylene glycol, or PEG, is a polyether compound used in a wide range of applications, from industrial production to pharmaceuticals. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is generally represented as H-(O-CH2-CH2)n-OH.

[0023] As used herein, the term “non-specific protease” means an enzyme that catalyzes proteolysis, which is the breakdown of a protein into smaller polypeptides or single amino acids, without strictly requiring an amino acid sequence substrate. Representative non-specific proteases without strict substrate requirements, as intended herein, include pepsin (and its precursor pepsinogen), chymotrypsin, elastase, papain, protease type XIII, and thermolysin.

[0024] As used herein, the terms “protein” and “polypeptide” are interchangeable and mean any chain of at least five naturally occurring or non-naturally occurring amino acids linked by peptide bonds. As used herein, the terms “isolated” and “purified” are interchangeable and mean reducing the amount of heterogeneous elements (e.g., biomacromolecules such as proteins or DNA) that may be present in a sample containing the protein of interest by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or more. The presence of heterogeneous proteins may be assayed by any suitable method, such as high-performance liquid chromatography (HPLC), gel electrophoresis and staining, and / or ELISA assays.

[0025] Method for measuring unmodified polypeptides As described herein, the Disclosure provides a method for determining the amount of the unmodified free N-terminus of a human granulocyte colony-stimulating factor (G-CSF) polypeptide in one embodiment, comprising: (a) incubating a sample containing the G-CSF polypeptide and a nonspecific protease under conditions that allow cleavage at one or more sites within the G-CSF polypeptide and a single cleavage between the N-terminal methionine at position 1 and the lysine at position 17; (b) separating the cleavage product produced in step (a); and (c) determining the amount of the unmodified free N-terminus of the G-CSF polypeptide by comparison with a control standard.

[0026] In various other embodiments, the methods described herein are useful for measuring the amount of the free N-terminus of any polypeptide (e.g., a recombinant polypeptide for therapeutic use, such as an antibody).

[0027] PEGylation is a universal therapeutic technique used to provide diverse conjugations with aptamers, enzymes, proteins, low molecular weight drugs, and antibodies, and this PEGylation is expanding clinical applications in the biopharmaceutical industry. PEGylation is the process by which polyethylene glycol (PEG) chains are conjugated to proteins (therapeutic proteins), peptides, or any other molecule. The PEGylation process increases the molecular weight of the therapeutic protein, and (therefore) this process can protect the therapeutic protein from proteolytic enzymes and degradation, potentially improving its pharmacokinetics.

[0028] In one embodiment of this disclosure, the efficiency of N-terminal PEGylation is determined with respect to filgrastim. In other embodiments, various other N-terminal modifications (other than PEGylation) are considered, including, but not limited to, polysaccharides such as dextran and heparosan. In addition to PEG, other polymer moieties are also useful conjugation partners with G-CSF. For example, in particular in International Publication No. 02 / 09766, biocompatible protein-polymer compounds are disclosed produced by the conjugation of a biologically active protein with a biocompatible polymer derivative. This biocompatible polymer is a highly reactive branched polymer, and the resulting conjugate contains a long linker between the polymer and the polypeptide. Examples of biocompatible polymers relating to International Publication No. 02 / 09766 are: PEG, PPG, polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and its derivatives, polyamino acids, polyurethanes, polyphosphazenes, poly(L-lysine), polyalkylene oxides (PAO), water-soluble polymers such as polysaccharides and dextran, and non-immunogenic polymers such as polyvinyl alcohol and polyacrylamide.

[0029] International Publication No. 96 / 11953 describes protein compounds with chemically modified N-terminuses and methods for producing them. Specifically, it describes G-CSF compositions obtained by conjugating a water-soluble polymer to the N-terminus of G-CSF. Examples of water-soluble polymers listed in International Publication No. 96 / 11953 are: ethylene glycol-propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymer or random copolymer), poly(n-vinylpyrrolidone) polyethylene glycol, PPG homopolymer, polypropylene oxide / ethylene oxide copolymer, or polyoxyethylated polyol. Other modifications are described in U.S. Patent No. 8,207,112, which is incorporated herein by reference in its entirety.

[0030] As described in U.S. Patent No. 5,824,784 (which is incorporated herein by reference in its entirety), substantially homogeneous mixtures of monopolymer / protein conjugates can be obtained by both N-terminally modified G-CSF (e.g., filgrastim) and reductive alkylation methods (utilizing the different reactivity of various types of primary amino groups available for derivatization (lysine vs. N-terminus)). “Substantially homogeneous,” as used herein, means that the observed polymer / protein conjugate molecules have only one polymer moiety. The preparation may contain unreacted (i.e., lacking polymer moiety) protein. An example of a preparation is provided which, as confirmed by peptide mapping and N-terminal sequencing and as described in U.S. Patent No. 5,824,784, consists of at least 90% monopolymer / protein conjugate and up to 10% unreacted protein. Preferably, the N-terminally monoPEGylated material constitutes at least 95% of the preparation (as in the examples below), and most preferably, the N-terminally monoPEGylated material constitutes at least 99% or more of the preparation. Monopolymer / protein conjugates possess biological activity. The “substantially homogeneous” N-terminally PEGylated G-CSF preparations provided herein are sufficiently homogeneous to demonstrate the advantages of homogeneous preparations (e.g., ease of clinical application in terms of lot-to-lot pharmacokinetic predictability).

[0031] Chemotherapy-induced neutropenia (CIN) is a common and serious complication of myelosuppressive chemotherapy. This complication is associated with significant morbidity and mortality and can increase the cost of cancer treatment. In this case, colony-stimulating factors are needed to restore cells crucial for immune function. For more than 20 years, granulocyte colony-stimulating factor (G-CSF; filgrastim) has been the cornerstone of CIN treatment and prevention, and has been shown to reduce the risk of neutropenia, lower the incidence of febrile neutropenia, reduce the incidence of infection, decrease the need for antibiotic treatment, and promote neutrophil recovery in a variety of patient settings.

[0032] Filgrastim is a recombinant non-pegylated human granulocyte colony-stimulating factor (G-CSF) analog. Filgrastim is marketed by Amgen under the trade name Neupogen® (first approved in 1998) and by Pfizer as the biosimilar Nivestym®. Neupogen® / filgrastim is approved for a variety of indications. tbo-filgrastim, marketed by Sicor Biotech and approved by the FDA on August 29, 2012, contains the same active ingredient as Neupogen® and is biosimilar, but formulated for shorter action. The FDA has also approved the biosimilar Zarxio® (filgrastim-sndz), indicated for use under the same conditions as Neupogen. Zarxio® is marketed by Sandoz.

[0033] Pegfilgrastim is a PEGylated form of filgrastim, a recombinant human granulocyte colony-stimulating factor (G-CSF) analog. Pegfilgrastim is used, among several reasons, to reduce the incidence of infections, such as those seen in febrile neutropenia, in non-myeloid cancer patients undergoing myelosuppressive anticancer treatment. Due to filgrastim's relatively short circulating half-life, a longer-acting form of the drug has been developed by covalently conjugating a 20 kDa PEG portion to the N-terminus of filgrastim (via a methionine residue). Due to its longer half-life and slower elimination rate compared to filgrastim, pegfilgrastim requires less frequent administration. However, pegfilgrastim retains the same biological activity as filgrastim, binding to the same G-CSF receptor and stimulating neutrophil proliferation, differentiation, and activation.

[0034] Pegfilgrastim, originally developed by Amgen, was first approved by the FDA in 2002 and marketed as Neulasta®. Several biosimilars of pegfilgrastim (Fulphila®, Pelgraz® or Lapelga®, Pelmeg®, Udenyca®, Ziextenzo®, and Grasustek®) have been approved by Health Canada, the European Union (EU), and the FDA for the same therapeutic indications.

[0035] The amino acid sequences of filgrastim and pegfilgrastim are as follows: [ka]

[0036] The 17 N-terminal residues of filgrastim are MTPLGPASSLPQSFLLK (SEQ ID NO: 2). None of these residues are readily cleaved by commonly used proteases such as trypsin (cleaved after K and R), Lys-C (cleaved after K), Glu-C (cleaved after E), Asp-N (cleaved before D), and Arg-C (cleaved after R). Consequently, Edman degradation (performed by an automated N-terminal sequencer) has historically been used as an assay to release pegfilgrastim by cleaving the residues one by one from the N-terminus. The unPEGylated free N-terminus was determined by the methionine residues recovered in the first Edman degradation cycle.

[0037] This disclosure provides the first use of a nonspecific protease for cleaving an N-terminal residue in various embodiments, the first use in which a single clean cut is generated between the N-terminal methionine and Lys-17. Since pepsin is a nonspecific protease, it can potentially cleave at various sites between the two residues. As described herein, conditions for generating a clean cut between residues Leu-15 and Leu-16 of SEQ ID NO: 2 have been identified. In addition, since pepsin acts under acidic conditions that denature the protein, reduction / alkylation is unnecessary, making sample preparation much simpler. The resulting proteolytic peptide is separated by reverse-phase HPLC in certain embodiments and monitored by UV absorbance. The N-terminal peptide, referred herein as "M1-L15," is well separated from other peaks, and the accurate and reproducible free N-terminus is used for more quantitative purposes. Slowly eluting PEGylated N-terminal peptides may be used for identification purposes.

[0038] As is known in the art, pepsin is an endopeptidase that breaks down proteins or polypeptides into smaller peptides or amino acids. Pepsin is produced in the chief cells of the stomach lining and is one of the major digestive enzymes in the digestive systems of humans and many other animals, aiding in the digestion of proteins in food. Pepsin is an aspartate protease, using the catalytic aspartate at its active site. Pepsin is one of two major proteases in the human digestive system, the other two being chymotrypsin and trypsin. During the digestive process, these enzymes (each specialized in cleaving the bonds between specific types of amino acids) work together to break down dietary proteins into their components (i.e., peptides and amino acids that can be readily absorbed by the small intestine). Pepsin is most efficient at cleaving peptide bonds between hydrophobic amino acids such as phenylalanine, tryptophan, tyrosine, and leucine. Pepsinogen, the enzyme precursor of pepsin, is released from chief cells in the stomach wall. When mixed with hydrochloric acid in gastric juice, pepsinogen is activated and becomes pepsin.

[0039] The separation of digestion products can be achieved in many ways according to this disclosure. For example, according to some embodiments, chromatography and electrophoresis are considered. For example, high-performance liquid chromatography (HPLC), ultrahigh-performance liquid chromatography (UHPLC), reverse-phase HPLC (RP-HPLC), hydrophilic interaction chromatography (HILIC), and ion exchange chromatography.

[0040] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described and can therefore be diverse. Similarly, it should be understood that the terminology used herein is intended only to describe specific embodiments and not to limit them, since the scope of the present invention is limited only by the appended claims.

[0041] Where a range of values ​​is specified, the intermediate values ​​between the upper and lower limits of this range (up to one-tenth of the lower limit unit, unless otherwise clearly indicated in the context) and any other specified or intermediate values ​​within this specified range are included in the present invention. The upper and lower limits of these smaller ranges may independently be included in this smaller range and are also included in the present invention, subject to any limitations specifically excluded in this specified range. If a specified range includes one or both limits, the range excluding one or both of these included limits is also included in the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and illustrate methods and / or materials relating to citations in this publication.

[0043] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” should be noted to include multiple referents unless otherwise explicitly indicated by the context. For example, a reference to “conformation switching probe” includes multiple such conformation switching probes, a reference to “microfluidic device” includes one or more microfluidic devices and their equivalents known to those skilled in the art, and so on. Furthermore, it should be noted that the claims may be written to exclude any element, such as any optional element. Accordingly, this statement is intended to serve as an antecedent for the use of exclusive terminology such as “only,” “only,” etc., in relation to the enumeration of elements of the claims or the use of “negative” limitations.

[0044] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or readily combined with any of the features of several other embodiments without departing from the scope and spirit of the invention. Any enumerated method may be performed in the order of the enumerated events or in any other logically possible order. This is intended to support all such combinations. [Examples]

[0045] Example 1 Pepsin digestion and separation of products A. Protease selection Figure 1 shows the sequence of pegfilgrastim, with potential PEGylation sites highlighted in red. Cleavage must occur between Met-1 and Lysine-17 to distinguish the N-terminus from lysine-17. Since no cleavage site exists for typical specific proteases, nonspecific proteases such as pepsin or chymotrypsin were used.

[0046] Evaluations of pepsin and chymotrypsin showed that pepsin is more promising than chymotrypsin. As seen in Figure 2, the chymotrypsin digests of filgrastim and pegfilgrastim showed insufficient separation between the free N-terminal methionine peak and the peaks of other peptides. In addition, since pepsin acts at an acidic pH when proteins are denatured, reduction / alkylation is unnecessary, making sample preparation much easier and simpler.

[0047] Pepsin digestion of B. filgrastim To perform pepsin digestion of filgrastim or pegfilgrastim, 120 μL of a 1 mg / mL protein sample was used, followed by the addition of 60 μL of 0.3 M phosphate buffer (pH 2.2), and 10 μL of 0.48 mg / mL pepsin solution. The mixture was then incubated at 50°C for 5 minutes or at 37°C for 15 minutes. After incubation, the digestion was quenched by adding 10 μL of 1N NaOH.

[0048] Digestion conditions were tested using the Filgrastim Reference Standard (RS) to maximize free N-terminal peptide. Similar results were obtained by optimizing digestion conditions at 50°C for 5 minutes or 37°C for 15 minutes. The final condition of 37°C for 15 minutes was selected because it was likely to result in a condition with less relative error and greater robustness in relation to the longer digestion time.

[0049] To determine how the N-terminal region is digested by pepsin, filgrastim (RS) was digested and analyzed on an Agilent 1290 HPLC system at 50°C using a Waters CSH 100×2.1 mm column, eluting at 0.2 mL / min with an acetonitrile gradient containing 0.02% (v / v) TFA in each mobile phase. This HPLC was directly connected to a Thermo Scientific LTQ-Orbitrap system, and mass and MS / MS data were collected to identify each eluted peptide.

[0050] Figure 3 shows the peptide map profile of filgrastim RS digested with pepsin. Pepsin digestion of filgrastim produced the major N-terminal peptide M1-L15. This peptide was sufficiently isolated in the chromatogram without any interference from other peptides, suggesting its potential use for quantifying free N-terminal methionine. Other N-terminal peptides (e.g., M1-L10, M1-S13, M1-F14 (co-eluting with peptides F114-E124), and M1-L16 (co-eluting with peptides L51-L76)) were also observed, but as seen in the mass spectrometry intensities of each peptide, they were all present in lower amounts compared to the M1-L15 peptide. Since these four low-abundance peptides should not significantly affect the quantification accuracy, they were not considered in the quantification of free N-terminal methionine.

[0051] Pepsin digestion of C. pegyfilgrastim Pegfilgrastim samples were digested with pepsin at 37°C for 15 minutes, and the peptide map profiles were compared with those of filgrastim (Figure 4). Peptides were identified by online mass spectrometry detection. The profiles were remarkably similar, except for the disappearance of the N-terminal free peptide (M1-L15) and the appearance of the PEGylated N-terminal peptide (PEG-M1-L15), indicating that the presence of PEGylation does not affect pepsin digestion. Due to the heterogeneous nature of PEGylation, a clear mass could not be determined for the very slow elution peak in pegfilgrastim. This peak was isolated and analyzed by MALDI-TOF MS and N-terminal sequencing. MALDI-TOF yielded a broad peak with a mass of approximately 23000 Da, and N-terminal sequencing confirmed that this sequence was the N-terminal M1-L15, with threonine (the actual second residue) observed instead of methionine in the first cycle.

[0052] D. Robustness of pepsin digestion Due to the nonspecific nature of pepsin, pepsin substances obtained from different sources or vendor lots may exhibit different activities, and therefore, there is concern that different peptide map profiles may be created. To test the robustness of pepsin digestion, a filgrastim sample (lot 1039502) was digested using pepsin substances from six different sources, and the resulting chromatograms were compared. A description of the six pepsin substances is shown in Table 1.

[0053] [Table 1]

[0054] Chromatograms showed that pepsin-based digestion is robust and reproducible regardless of the pepsin source, as long as the pepsin activity is 2500 units / mg or higher. The MS intensities of various N-terminal peptides created from this dataset demonstrate the reproducibility of digestion and the superiority of M1-L15 when various pepsin sources are used.

[0055] E. Separation by chromatography To quantify free N-terminal methionine in pegfilgrastim using the N-terminal peptide M1-L15, this peptide must be separated from all other nearby eluting peptides. A sample containing 95% pegfilgrastim reference standard (RS) and 5% filgrastim RS was prepared, and chromatographic separation was optimized using this sample. A Waters CSH C18 column (2.1 × 100 mm, 1.7 μm) was used. After some gradient optimization, the following chromatographic conditions were selected. Mobile phase A: 0.025% TFA in water Mobile phase B: 0.025% TFA in acetonitrile Column temperature: 50℃ Detection wavelength: 214nm Flow rate: 0.2mL / min gradient:

[0056] [Table 2]

[0057] Figures 5A–5C show peptide map profiles near the target peptide, and six different UPLC column lots were tested at three different TFA concentrations (0.02, 0.025, and 0.03 v / v%). The peptide identification of the labeled peaks is shown in Table 2.

[0058] [Table 3]

[0059] Figures 5A-5C clearly show that the separation of peptides M1-L15 from neighboring peptides (P1-pre and P1-post) depends on the column lot being used. Fortunately, the separation of these three peaks can be optimized by changing the TFA concentration in the mobile phase. If co-elution of two peaks occurs with a particular column lot, separation can be achieved by changing the TFA concentration from 0.02% (v / v) to 0.03% (v / v), as illustrated in Figures 5A-5C. A TFA concentration of 0.025% (v / v) was selected because, in all six column lots, sufficient separation of the P1 peak was achieved with 0.025% (v / v) TFA compared to the other two concentrations.

[0060] Example 2 Eligibility of the method A. Chromosomes and Carryovers To establish specificity, the main reference peaks (P2, P3, and P4), as well as several neighboring peaks of interest, were initially identified by online mass spectrometry (MS) detection (Figure 6). Due to the heterogeneous nature of the mPEG-aldehyde, a clear mass could not be determined from the mass spectrum of peak P4. As described in Section 5.1.3, N-terminal sequencing confirmed that peak P4 is PEG-M1-L15.

[0061] To establish the specificity of this assay method to product-related components, a chromatographic profile specific to the pegfilgrastim drug substance (DS) after pepsin digestion must be achieved. Products of similar size or manufactured at the same site (e.g., Neupogen® (filgrastim), Epogen® [epoetin alfa, (EPO)], and Nplate® (romiplostim)) were analyzed in parallel with pegfilgrastim, and the resulting chromatograms are shown in Figure 7. These chromatograms are clearly different, establishing the specificity of this assay for distinguishing these products. Among these products, Neupogen differs from pegfilgrastim only in the PEGylation of its N-terminus, which can be clearly distinguished by the absence of reference peak P4 (PEGylated N-terminal peptide) and the appearance of peak P1 (Free N-terminal peptide) in the Neupogen pepsin digest.

[0062] To evaluate the carryover of this method, an enzyme blank was injected after injecting digested pegfilgrastim RS supplemented with 5% filgrastim RS. Carryover was calculated based on the relative percentage of peak area determined for each reference peak in the blank run compared to the supplemented pegfilgrastim RS sample. No peaks were detected in the retention time region both before and after the blank run, and the carryover for all of these peaks is 0%.

[0063] B. Linearity Linearity experiments were designed to determine the assay's ability (within a given range) to obtain test results directly proportional to the percentage level of free N-terminal impurities. To establish the linearity of this method, pegfilgrastim RS with filgrastim RS added at levels of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% (resulting in final free N-terminal methionine levels of approximately 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%, respectively) were triple digested and analyzed (Table 4). In addition to the analysis of unadded pegfilgrastim RS (two values ​​per linearity run), a total of six levels are available for linearity evaluation.

[0064] [Table 4]

[0065] [Table 5]

[0066] In Figure 8, the determined levels of free N-terminal methionine (%) were plotted against the additive level. Linear regression was performed to determine the slope, y-intercept, and R. 2 The values ​​are obtained. Similarly, Figure 9 shows the residual plot, from which the sum of squared residuals and the standard deviation of residuals are calculated. Table 5 shows these determined values ​​regarding the linearity of the measurements within the investigated range.

[0067] [Table 6]

[0068] C. Accuracy (reproducibility / intermediate accuracy) Reproducibility and intermediate precision were evaluated for both the peptide map profile and the quantification of free N-terminal methionine. To assess the reproducibility and intermediate precision of the peptide map profile, a total of six runs were performed, each consisting of four sample injections and four blank injections, by two different analysts in two different laboratories and using three different columns across four different HPLC systems. The total area under the curve (tAUC), peak area (pAUC), and reference peak retention time (RT) ratios (P2 / P4 and P3 / P4) for each sample injection, as well as the inter-peak noise for each enzyme-only blank injection and the difference in retention time of the reference standard with bracketing filgrastim added, were recorded and are shown in Table 6. The maximum CV% for retention time and peak area parameters per run were less than 0.3% and 7%, respectively. These results demonstrate good reproducibility of this peptide map profile.

[0069] [Table 7]

[0070] To evaluate the reproducibility and intermediate precision of free N-terminal methionine quantification, a total of four runs were performed using pegfilgrastim samples with 1.5% filgrastim added, with triple analysis in each run, using four different HPLC systems, two different analysts in two different laboratories, and three different columns. Table 7 shows the results of these analyses regarding the reproducibility standard deviation and intermediate precision standard deviation.

[0071] [Table 8]

[0072] D. Accuracy The accuracy of this method in measuring the amount of free N-terminal methionine was evaluated using data collected from linearity experiments. To obtain the accurate theoretical amount of free N-terminal methionine in each sample, the small amount of N-terminal methionine in unadded pegfilgrastim RS was first determined from six system fit runs during the linearity evaluation (determined to be an average of 0.471%, as shown in Table 8).

[0073] [Table 9]

[0074] Due to the low levels of free N-terminal methionine in unadded pegfilgrastim RS, measurement errors can be ignored when calculating the theoretical concentration of the added sample. The theoretical level of free N-terminal methionine is expressed by the following formula: (0.471% VPF + 100% VF) / (VPF + VF) Therefore, it can be calculated based on the amount of added filgrastim (VF) and the amount of unadded pegfilgrastim RS (VPF).

[0075] Table 9 compares the theoretical percentage of free N-terminal methionine in samples with the experimentally determined percentage at various levels of free N-terminal methionine. Precision was calculated from the average determined value of triple analysis and is expressed as a percentage in Table 9. Measurement precision was found to be acceptable at the 1.5% additive level and all other measured concentrations.

[0076] [Table 10]

[0077] E. Range Within the range of free N-terminal methionine levels from 1.0% to 3.0%, the data presented in this report exhibited acceptable linearity, precision, and accuracy, demonstrating that this method can determine the percentage of free N-terminal methionine within this range.

[0078] F. Limit of Detection (LOD) and Limit of Quantification (LOQ) In one embodiment described herein, the protocol of the method requires performing two injections of pegfilgrastim RS and two injections of pegfilgrastim RS supplemented with 5% filgrastim RS in each sequence, thereby facilitating the determination of the percentage of N-terminal methionine in pegfilgrastim RS. This linearity evaluation consists of three sequences, yielding six measurements of the percentage of free N-terminal methionine in pegfilgrastim RS. In addition, three further runs for intermediate precision evaluation yielded six further measurements. The standard deviation of the twelve measurements was calculated, and these standard deviations were used to determine the LOD and LOQ according to the following equations. The results are shown in Table 10. LOD=3.3σ LOQ = 10σ

[0079] [Table 11]

[0080] G. Stability of the sample after preparation To demonstrate the stability of digested samples in a cooled autoinjector before analysis, two digested samples were injected before and after a long sequence (at 16-hour intervals) and on day 2 (at 42-hour intervals). No significant differences were observed in the resulting chromatograms (Figure 10). The determined difference in the percentage of free N-terminal methionine in the samples at 16 hours and 42 hours at 4°C was less than 0.4% (Table 11), suggesting that the samples are stable for up to 42 hours at 4°C.

[0081] [Table 12]

[0082] Example 3 Robustness The robustness of this method was evaluated by performing a seven-factor designed experiment (DOE) (Table 12). The lot factors for the HPLC column and enzyme were categorical, and the matrix was prepared using Minitab® 15.1.30.0 statistical software. One analysis was performed per run on the control sample (pegfilgrastim RS with 1.5% filgrastim RS added).

[0083] [Table 13]

[0084] To evaluate the robustness of this method for peptide map profiles, the total peak area, inter-peak noise (p-interval), P2 / P4 and P3 / P4 ratios related to retention time, and the average peak area from the center point experiment are calculated. Acceptable ranges for each parameter were established using the following equations. Acceptable range = mean of center point experiments ± 3 * (StdDev) * (Mn) Here, Mn=1.403 is the multiplier for intermediate-precision measurements (n=24) (Hahn and Meeker, 2011), and StdDev is the standard deviation obtained from the intermediate-precision experiments. As can be seen in Figures 11A-11G, the total peak area, noise (between p), peak area, and retention time of the four reference peaks are all within acceptable limits.

[0085] To evaluate the robustness of this method for determining free N-terminal methionine, the average value of free N-terminal methionine determined from center point experiments is calculated. The acceptable range for other experiments is calculated using the following equation. Acceptable range = mean of center point experiment ± 3 * (intermediate precision standard deviation) * (Mn) Here, Mn=1.698 is the multiplier for intermediate precision measurements (n=12) (Hahn, Gerald J., and William Q. Meeker. Statistical intervals: a guide for practitioners. Vol.92. John Wiley & Sons, 2011). As can be seen in Figure 12, the determined percentage of free N-terminal methionine was within an acceptable range in all experiments, demonstrating the robustness of this method in determining the level of free N-terminal methionine.

[0086] Example 4 Representative protocols - materials and methods The following materials and methods, as further disclosed in the above examples, provide representative methods for verifying the identity of pegfilgrastim and for determining the percentage level of free N-terminal methionine with excellent specificity, precision, accuracy, linearity, LOD, LOQ, range, and robustness.

[0087] Pegfilgrastim is digested with pepsin under acidic conditions. The digested peptides are separated by reverse-phase chromatography and detected by UV at 214 nm. The percentage of free N-terminal methionine (without PEG) is determined by adding a known amount (5%) of filgrastim to the pegfilgrastim sample. The pepsin peptide map profile of the sample is compared to the pegfilgrastim reference standard to confirm its identity.

[0088] To perform pepsin digestion of filgrastim or pegfilgrastim (additional sample and normal sample), 120 μL of a 1 mg / mL protein sample and 10 μL of a 0.48 mg / mL pepsin solution were thoroughly mixed in 60 μL of 0.3 M phosphate buffer (pH 2.2) and incubated at 37°C for 15 minutes. After incubation, this digestion was quenched by adding 10 μL of 1N NaOH.

[0089] 15 μg of digested sample peptide was separated on a Waters CSH reverse-phase column (2.1 × 100 mm) at 50°C by a 40-minute acetonitrile gradient with 0.025% TFA (2–25% for 19 minutes, followed by 25–30% for 10 minutes, then 30–99% for 10 minutes, followed by column washing and re-equilibrium). The flow rate was maintained at 0.2 mL / min, and UV detection was achieved at 214 nm.

[0090] The following formula

number

[0091] Further embodiments may be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in this application datasheet are incorporated herein by reference in their entirety. Aspects of this embodiment may be modified as necessary to adopt the concepts of various patents, applications, and publications to provide further embodiments.

[0092] These and other modifications may be made to the embodiments in consideration of the detailed description above. In general, the terms used in the following claims should not be construed to limit these claims to the specific embodiments disclosed herein and in the claims, but rather to include all possible embodiments along with the entire scope of equivalents to which such claims are entitled. Accordingly, these claims are not limited by this disclosure.

Claims

1. A method for measuring the amount of the unmodified N-terminus of human granulocyte colony-stimulating factor (G-CSF) polypeptide, (a) Incubating the sample containing the G-CSF polypeptide and pepsin under conditions that allow for cleavage at one or more sites within the G-CSF polypeptide and a single cleavage between the N-terminal methionine at position 1 and the lysine at position 16; (b) A step of separating the cleavage product generated in step (a); and (c) A step of measuring the amount of the unmodified free N-terminus of the G-CSF polypeptide by comparing it with a control standard containing a mixture of the N-terminally modified G-CSF polypeptide and the unmodified G-CSF polypeptide. A method that includes this.

2. The method according to claim 1, wherein the G-CSF polypeptide is a recombinant.

3. The method according to claim 1 or 2, wherein the sample comprises a mixture of a modified G-CSF polypeptide and an unmodified G-CSF polypeptide, and the modified G-CSF polypeptide comprises at least one polyethylene glycol (PEG) modification.

4. The method according to any one of claims 1 to 3, wherein the G-CSF polypeptide is selected from the group consisting of pegfilgrastim (Neulasta®), pegfilgrastim-jmdb (Fulphila®), INN-pegfilgrastim (Pelgraz®), Lapelga®, Pelmeg®, pegfilgrastim-cbqv (Udenyca®), pegfilgrastim-bmez (Ziextenzo®), and Grasustek®.

5. The method according to claim 4, wherein the G-CSF polypeptide is pegfilgrastim (Neulasta®).

6. The method according to any one of claims 1 to 5, wherein pepsin cleaves between leucine at position 15 and leucine at position 16 to produce peptides (peptides M1-L15) with a length of 15 amino acids.

7. The method according to any one of claims 1 to 6, wherein the conditions in step (a) include (i) a pH of 1.5 to 4.0, (ii) a temperature of 25°C to 60°C, and (iii) incubation for a time of 5 minutes to 60 minutes.

8. The method according to claim 7, wherein the conditions include (i) incubation at a pH of 2.2, (ii) incubation at a temperature of 37°C, and (iii) incubation for a period of 15 minutes.

9. The method according to any one of claims 1 to 8, wherein the separation in step (b) is carried out under conditions that allow for separation of peptide M1-L15 from other cleavage products.

10. The method according to claim 9, wherein the separation in step (b) is carried out by a method selected from chromatography and electrophoresis.

11. The method according to claim 10, wherein the chromatography is selected from the group consisting of high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC).

12. The method according to claim 11, wherein the HPLC is a reverse-phase HPLC (RP-HPLC).

13. The method according to claim 11 or 12, wherein the chromatography comprises a column and trifluoroacetic acid (TFA) at a concentration of 0.01 v / v% to 0.2 v / v%.

14. The method according to claim 13, wherein the concentration of the TFA is 0.02 v / v% to 0.03 v / v%.

15. The method according to claim 14, wherein the concentration of the TFA is 0.025 v / v%.

16. The method according to any one of claims 1 to 15, wherein the measurement step (c) is performed by mass spectrometry.

17. The method according to claim 16, wherein the mass spectrometry is selected from electrospray MS and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

18. The method according to any one of claims 1 to 17, wherein the control standard comprises a known amount of modified G-CSF polypeptide and a known amount of unmodified G-CSF polypeptide.

19. A method for measuring the amount of non-PEG-treated free N-terminus of pegfilgrastim (Neulasta®), (a) Incubating a sample containing pegfilgrastim (Neulasta®) and pepsin under conditions that allow cleavage at one or more sites within the pegfilgrastim (Neulasta®) and a single cleavage between the N-terminal methionine at position 1 and the lysine at position 16, wherein the conditions include (i) incubation at a pH of 2.2, (ii) incubation at a temperature of 37°C, and (iii) incubation for a time of 15 minutes; (b) A step of separating the cleavage product generated in step (a) by reverse-phase HPLC (RP-HPLC); and (c) A step of measuring the amount of the un-PEGylated free N-terminus of the pegfilgrastim (Neulasta®) by comparing it with a control standard containing a mixture of the N-terminally modified G-CSF polypeptide and the unmodified G-CSF polypeptide. A method that includes this.

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