Methods of analyzing amino acid content of a therapeutic protein

Abstract

Provided herein are methods for analyzing the amino acid content of a therapeutic protein. In exemplary embodiments, the method comprises exchanging by diafiltration the therapeutic protein into a second solution from a first solution to obtain an exchanged protein solution; incubating the exchanged protein solution at an acidic pH at a temperature greater than 110 °C to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein; combining the hydrolyzed sample with activated charcoal; and separating the hydrolyzed sample from the activated charcoal by filtration, centrifugation, or both filtration and centrifugation; and analyzing the amino acid content of the hydrolyzed sample.

Description

METHODS OF ANALYZING AMINO ACID CONTENT OF A THERAPEUTIC PROTEINCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 596,666, filed on November 7, 2023, is hereby claimed, and the entire disclosure thereof is incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: 104,682 byte XML file named "10285-W001-SEC_Sequence_Listing"; created on October 30, 2024.BACKGROUND

[0003] Equipped with the cellular machinery for carrying out human-like post-translational modifications, mammalian cells are the preferred hosts for the production of many protein biopharmaceuticals. The manufacture of these post-translationally modified proteins by mammalian cells can be subject to high variability, however, due to batch-to-batch variability, process drifts, and manufacturing changes. Thus, there is a need for monitoring and controlling such modifications during the manufacturing process, especially those modifications which are critical to the protein's in vivo therapeutic function, e.g., critical quality attributes (CQAs). Though product quality analyses carried out during the production of the protein have been limited by the lengthy time requirements for such analyses, recent efforts have moved the industry toward automated and high-throughput techniques, combined with multivariate data analysis, to provide relevant process knowledge in near real-time. See, e.g., Pais et al., Current Opinion in Biotechnology 2014, 30:161-167.

[0004] Conventionally, protein quality analyses include isolation of the protein from the sample obtained from the manufacturing process (e.g., a cell culture sample taken from a bioreactor), enzymatic digestion of the protein into peptide fragments, and chromatographic separation of the peptide fragments followed by mass spectrometry (MS) analytics. Depending on the complexity of the three-dimensional structure of the protein analyte, pre-digestion processing steps, including, for instance, denaturation, reduction, and alkylation, may be needed to open up the protein for digestion. One or more post-digestion steps are generally included to stop the digestion and prepare the peptide fragment yield for separation and MS analysis. In some instances, a particular modification may very low in abundance. There exists a need for more sensitive, in-depth analyses of the protein for detection of the modification.

[0005] Thus, there is a need in the art for improved methods of detecting low abundance modifications of a therapeutic protein.SUMMARY

[0006] Presented herein for the first time are data demonstrating the feasibility and advantages of a method of analyzing the amino acid content of a therapeutic protein. The method is highly sensitive in that it can detect low abundance or low frequency amino acids and distinguish between structurally similar amino acids which differ by only small structural variations. For instance, the method can detect hydroxyproline present at low abundance (e.g., 3 residues) within a therapeutic protein comprising over 1100 amino acids and can successfully distinguish its presence over proline residues which is structurally similar but for the hydroxyl group present in hydroxyproline. The methods of the present disclosure are thus useful for detecting post translational modifications (PTM) in a therapeutic protein, e.g., a modified or isomerized amino acid, which forms, for example, during manufacture or upon storage. As the method can detect structural changes of a therapeutic protein in a drug product, the methods are advantageous for evaluating product quality, manufacturing process steps, and overall stability and safety of a drug product.

[0007] Accordingly, provided herein are methods of analyzing the amino acid content of a therapeutic protein. In exemplary embodiments, the method comprises (a) exchanging by diafiltration the therapeutic protein into a second solution from a first solution to obtain an exchanged protein solution; (b) incubating the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein; (c) combining the hydrolyzed sample with activated charcoal; (d) separating the hydrolyzed sample from the activated charcoal; and (e) analyzing the amino acid content of the hydrolyzed sample. In various aspects, the first solution comprises less than 5 different proteins. In various instances, the therapeutic protein is the only protein present in the first solution. In exemplary aspects, the first solution has a bioburden less than 100 CFU / mL, such as less than 10 CFU / mL. In exemplary aspects, the first solution does not comprise or consist of a tissue sample, such as a homogenized tissue sample or a blood sample. In exemplary instances, the first solution is a liquid drug product or a liquid drug substance. The first solution, in various aspects, comprises one or more excipients selected from the group of: a salt, a surfactant, sugar and / or a polyol. In various aspects, the first solution comprises a buffer and a sugar. The buffer in various aspects is acetate, succinate, citrate, histidine, glutamate, phosphate, tris, or the like. The sugar in exemplary instances is sucrose, glucose, lactose, arabinose, glucosamine, galactosamine, N-acetyl galactosamine, galactose, or fructose. Surfactants are surface active agents that are amphipathic (having a polar head and hydrophobic tail). Surfactants preferentially accumulate at interfaces,resulting in reduced interfacial tension. In some aspects, the surfactant present in the first solution is an amphipathic and / or nonionic surfactant. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (e.g. polysorbate 20, polysorbate 80), alkylaryl polyethers, e.g. oxyethylated alkyl phenol (e.g. Triton™ X-100), and poloxamers (e.g. Pluronics®, e.g. Pluronic® F68), and combinations of any of the foregoing, either within a class of surfactants or among classes of surfactants. Polysorbate 20 and polysorbate 80 (and optionally mixtures thereof) are particularly contemplated. Exemplary polyols include but are not limited to glycerol, mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, and isomalt.

[0008] The second solution, in exemplary aspects, has the same pH as the first solution. In exemplary instances, the second solution lacks sugar or comprises only a small amount, e.g., less than about 1% (w / v), less than about 0.5% (w / v), less than about 0.1% (w / v), or less than about 0.01% (w / v), of sugar. In exemplary instances, the diafiltration is performed with at least 2 or more diavolumes of the second solution. In exemplary instances, the diafiltration is performed with at least 3 or 4 diavolumes of the second solution. In exemplary instances, the diafiltration is performed with at least 4 diavolumes of the second solution. In various aspects, the method comprises combining the exchanged protein solution with an acid pH and a temperature greater than about 110 °C to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein. In exemplary instances, the acid is hydrochloric acid. The hydrochloric acid is greater than 8N, for instance, or greater than 10 N or about 12 N hydrochloric acid, in various aspects. In exemplary instances, the method comprises incubating the exchanged protein solution at an acidic pH, e.g., less than about 4.2, and a temperature greater than about 110 °C, e.g., about 115 °C to about 130 °C, to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein. The acidic pH in various instances is less than about 3.5. In exemplary aspects, the exchanged protein solution is incubated at about 120 °C. In various instances, the exchanged protein solution is incubated at the acidic pH and the temperature greater than about 110°C for at least 1 or 2 hours, optionally, about 3 hours. In exemplary aspects, the method comprises combining the hydrolyzed sample with activated charcoal wherein about 2.5 mg to about 5 mg activated charcoal is combined with the hydrolyzed sample for every 1 mg therapeutic protein in the first solution. In exemplary embodiments, the hydrolyzed sample is separated from the activated charcoal by (i) centrifugation, (ii) filtration, or (iii) both centrifugation and filtration. In various aspects, the centrifugation is performed at about 13,000 x g to about 14,000 x g for about 3 min to about 5 min and / or the filtration is performed with a 0.22 pm filter. In exemplary aspects, the method comprises separating the hydrolyzed sample from the activated charcoal by (i) centrifuging to obtain an activated charcoal pellet and a supernatant and (ii) passing the supernatant through a filter. In various aspects, themethod comprises analyzing the amino acid content of the hydrolyzed sample through an analytical technique, and in various instances, the analytical technique has a limit of detection (LOD) in the nanomolar or picomolar range. In various aspects, the method comprises analyzing the amino acid content of the hydrolyzed sample through a technique which does not comprise a colorimetric assay. The method in exemplary instances is capable of detecting low abundance amino acids which represent less than 0.5% of the amino acids of the therapeutic protein. In various aspects, the method comprises chromatographically separating the amino acids of the therapeutic protein after the hydrolyzed sample is separated from the activated charcoal. Optionally, the amino acids of the therapeutic protein are chromatographically separated by a high-performance liquid chromatography (HPLC), optionally, a reversed phase-HPLC (RP-HPLC). The method of the present disclosure in various instances comprises chromatographically separating the amino acids of the therapeutic protein by hydrophilic interaction liquid chromatography (HILIC). In exemplary aspects, the method further comprises labeling the amino acids of the therapeutic protein with a detectable label, and this labeling occurs after the hydrolyzed sample is separated from the activated charcoal and before chromatographically separating the amino acids of the therapeutic protein. In exemplary aspects, the detectable label absorbs ultraviolet light at a specific wavelength, e.g., 260 nm. In exemplary instances, the detectable label is 6-Aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC). In exemplary instances, a transmitted UV (TUV) or a photo diode array (PDA) detector is used to detect the detectable label-amino acid complex, and, in various aspects, the TUV or PDA detects during the chromatographic separation, e.g., the HPLC. In alternative or additional aspects, the method comprises analyzing the amino acid content through mass spectrometry. In various instances, the mass of the chromatographically separated amino acids is determined through mass spectrometry. Optionally, when mass spectrometry is carried out, labeling of the amino acids of the therapeutic protein is not necessary. In various aspects, the method further comprises concentrating the hydrolyzed sample after the hydrolyzed sample is separated from the activated charcoal but before the amino acid content of the hydrolyzed sample is analyzed. In exemplary aspects, the method comprises concentrating the hydrolyzed sample, labeling the amino acids of the therapeutic protein with a detectable label, and chromatographically separating the amino acids of the therapeutic protein. In exemplary instances, the method comprises concentrating the hydrolyzed sample, chromatographically separating the amino acids of the therapeutic protein, and performing mass spectrometry on the separated amino acids. In exemplary instances, the therapeutic protein comprises one or more antibody domains. In exemplary aspects, the therapeutic protein comprises one or more antibody heavy chains. Optionally, the therapeuticprotein comprises an Fc domain and / or a Fab fragment. In various instances, the therapeutic protein is an antibody or comprises a single chain variable (scFv) domain or is a bispecific antibody.

[0009] As the presently disclosed methods of analyzing the amino acid content of a therapeutic protein are useful for detecting the presence or absence of a post translational modification (PTM) in a therapeutic protein, the present disclosure additionally provides a method of detecting the presence or absence of a post translational modification (PTM) in a therapeutic protein. In exemplary embodiments, the method comprises analyzing the amino acid content of a therapeutic protein in accordance with the methods of analyzing the amino acid content of a therapeutic protein of the present disclosure. In various aspects, the PTM is a modified or isomerized amino acid, such as hydroxyproline. Since the presently disclosed methods of analyzing the amino acid content of a therapeutic protein are also useful for detecting structural changes of a therapeutic protein in a drug product, the present disclosure further provides methods of detecting structural changes of a therapeutic protein in a drug product. In exemplary embodiments, the method comprises analyzing the amino acid content of a therapeutic protein in accordance with the methods of analyzing the amino acid content of a therapeutic protein of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is an illustration of an exemplary configuration for exchanging the protein into a second solution from a first solution.

[0011] Figure 2 is a photograph of dark-brown test samples obtained in Example 2.

[0012] Figure 3 is a photograph of samples obtained in Example 3.

[0013] Figure 4A is a photograph of standard (STD) curve samples (either 0, 0.5, 1, 2.5, 5.0, or 10 standard) and a test sample (~1 mg). Figure 4B is a photograph of standard (STD) curve samples, test samples comprising either 0.75 mg or 1.5 mg protein, negative (Neg.) control samples, and blank samples.

[0014] Figure 5 is an overlay of chromatograms depicting UV absorbance at 260 nm of hydroxyproline standard (black line), proline standard (blue line), and a mixture of hydroxyproline standard and proline standard (green line).

[0015] Figure 6A is an overlay of chromatograms depicting UV absorbance at 260 nm of a test sample comprising hydrolyzed TP1 (black line) and a test sample comprising hydrolyzed TP1 with a hydroxyproline standard (blue line). Figure 6B is a zoomed view of the boxed overlay of Figure 6A. Hydroxyproline peaks are labeled as "HYP".

[0016] Figure 6C is an overlay of chromatograms depicting UV absorbance at 260 nm of a test sample comprising hydrolyzed TP1 ("Test Sample") and negative control sample ("Negative Control"). Hydroxyproline peaks are labeled. Figure 6D is a zoomed view of the boxed overlay of Figure 6C. Hydroxyproline peaks are labeled as "HYP".

[0017] Figure 6E is a pair of the zoomed views of Figures 6B (left) and 6D (right). Hydroxyproline peaks are labeled as "HYP".

[0018] Figure 7 is a chromatogram of UV absorbance at 260 nm of test sample comprising hydrolyzed TP1 with hydroxyproline and proline elution peaks integrated and % Peak Area reported. Hydroxyproline peak is labeled as "HYP". Proline peak is labeled as "PRO".

[0019] Figure 8A is an overlay of chromatograms depicting UV absorbance at 260 nm of a negative control sample ("Negative Control Sample" line) and a negative control sample spiked with a hydroxyproline standard ("Negative Control Sample w / hydroxyproline standard spike" line). Figure 8B is an overlay of chromatograms of Test Sample comprising hydrolyzed TP2 with hydroxyproline standard ("Test Sample of TP2 w / hydroxyproline standard spike" line) and Test Sample comprising hydrolyzed TP2 without hydroxyproline standard; "Test Sample of TP2" line).DETAILED DESCRIPTION

[0020] Provided herein are sensitive methods of analyzing the amino acid content of a protein, e.g., a therapeutic protein. For instance, the method, in exemplary instances, detects low-abundant amino acids which represent, e.g., less than 0.5% of the amino acids of the therapeutic protein. In various instances, the method detects an amino acid that represents less than 0.4% of the amino acids of the therapeutic protein. In various aspects, the method detects an amino acid that represents less than 0.3% of the amino acids of the therapeutic protein. The method, in exemplary aspects, detects low-abundance PTMs that represent less than 1% of the total amount of the PTM and unmodified amino acid. The method, in exemplary aspects, detects low abundance PTMs that represent less than 0.5% of the total amount of the PTM and unmodified amino acid. In exemplary embodiments, the method comprises (a) exchanging by diafiltration the protein, e.g., therapeutic protein, into a second solution from a first solution to obtain an exchanged protein solution; (b) incubating the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C to obtain a hydrolyzed sample comprising amino acids of the protein, e.g., therapeutic protein; (c) combining the hydrolyzed sample with activated charcoal; (d) separating the hydrolyzed sample from the activated charcoal; and (e) analyzing the amino acid content of the hydrolyzed sample.

[0021] The methods of the present disclosure advantageously are not limited to any one type of protein, though the methods are suitable for analyzing the amino acid content of a therapeuticprotein for, e.g., detecting a PTM in the therapeutic protein which PTM has potential for posing a safety issue. As the method can detect structural changes of a therapeutic protein in a drug product, the methods are advantageous for evaluating product quality, identifying problematic manufacturing process steps, and analyzing the overall stability and safety of a drug product.

[0022] In exemplary embodiments, the protein of which the amino acid content is analyzed comprises one or more polypeptide chains. In various aspects, the protein comprises only one polypeptide chain. In various instances, the protein comprises more than one polypeptide chain, e.g., two, three, or four polypeptide chains. In various aspects, the polypeptide chain(s) of the protein each comprise at least 25, at least 50, at least 75, or at least 100 amino acids.

[0023] In exemplary instances, the protein, e.g., therapeutic protein, comprises one or more domains of an antibody, e.g., antibody domains, immunoglobulin domains. As used herein, the term "antibody" which is synonymous with "immunoglobulin", refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions, as described in, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 4thed., Elsevier Science Ltd. / Garland Publishing, 1999. For example, an antibody may be an IgG which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and varies among other antibodies that bind to different antigens. The constant region of the antibody may function to recruit cells and molecules of the immune system. The variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4thed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0024] The general structure and properties of CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0025] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, lgG2, lgG3, and lgG4. IgM has subclasses, including, but not limited to, IgMl and lgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgGl, lgG2, lgG3 or lgG4. In exemplary aspects, the therapeutic protein comprises one or more antibody heavy chains.

[0026] An antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab')? fragment and a pFc' fragment. In exemplary aspects of the present disclosure, the fusion protein of the present disclosure comprises an antigen binding antibody fragment. As used herein, the term "antigen binding antibody fragment" or "antigen-binding fragment" or "antigen-binding portion" refers to a portion of an antibody that is capable of binding to the antigen of the antibody. In exemplary instances, the antigen binding antibody fragment is a Fab fragment or a F(ab' fragment. Optionally, the protein of which the amino acid content is analyzed comprises an Fc domain and / or a Fab fragment.

[0027] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as "antibody protein products". Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH / VH (discussed below). The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimericantibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH / VH of camelid heavy chain Abs as well as single domain Abs (sdAb). The building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ~15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012). Other antibody protein products include a single chain antibody (SCA); a diabody; a triabody; a tetrabody; bispecific or trispecific antibodies, and the like. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97- 106 (2015). In various instances, the protein, e.g., therapeutic protein, of which the amino acid content is analyzed is an antibody or comprises a single chain variable (scFv) domain or is a bispecific antibody.

[0028] In various aspects, the amino acid content of the entire protein is determined by the presently disclosed method. In alternative aspects, the amino acid content for only a portion of the protein is determined by the presently disclosed method. In exemplary instances, the presently disclosed method is used to detect a PTM. The PTM may be a modified or isomerized amino acid, including but not limited to a lipidated amino acid (e.g., palmitoylated, myristoylated), a phosphorylated amino acid, a glycosylated amino acid, a ubiquitinated amino acid, a nitrosylated amino acid, a methylated amino acid, an acetylated amino acid, an oxidated amino acid, a reduced amino acid, a deamidated amino acid, a deaminated amino acid, an aggregated amino acid, a denatured amino acid, a precipitated amino acid, a hydrolysed amino acid, an isomerized amino acid, or a N-terminal and C-terminal modified amino acid (e.g., an esterified, acylated, acetylated, or cyclized N- or C-terminal amino acid). The PTM in various aspects is a deamidated Asn or Gin, a deaminated Glu, Ser, or Gly, a glycated Lys, a glycosylated Asn, an oxidated Met, Trp, or His, an isomerized Asp, a fragmented or clipped Asp or Pro, a hydroxylated Lys or Pro. In various aspects, the method determines the presence or absence of PTM. In exemplary instances, the PTM is a hydroxyproline.

[0029] In exemplary aspects, the protein of which the amino acid content is analyzed is a therapeutic protein. As used herein, the term "therapeutic protein" refers to any molecule, which may be naturally-occurring or engineered or synthetic, comprising at least one polypeptide chain, which, when administered to a subject, is intended for achieving a therapeutic effect for treatmentof a disease or medical condition. Therapeutic proteins can include antigen binding proteins such as antibodies and proteins comprising antibody fragments, as well as growth factors and enzymes. In various aspects, the therapeutic protein comprises more than one proline. In various instances, the therapeutic protein comprises two or more, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, proline residues. Optionally, the therapeutic protein comprises more than one lysine. In various instances, the therapeutic protein comprises two or more, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, lysine residues. In various aspects, the therapeutic protein comprises one or more lysine residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a proline.

[0030] Exchanging the Protein into a Second Solution From a First Solution

[0031] The presently disclosed method of analyzing amino acid content of a protein, e.g., therapeutic protein, comprises exchanging the protein into a second solution from a first solution to obtain an exchanged protein solution. The exchanging in various aspects occurs by diafiltration (DF), a technique that utilizes a membrane to remove, substitute, or reduce the concentration of salts and / or solvents from a first solution containing the protein, while increasing the concentration of the salts and / or solvents of a new or second solution or diafiltration buffer. In various aspects of the presently disclosed methods, the exchanging by diafiltration (DF) removes, substitutes, or reduces the concentration of the salts and / or solvents of the first solution while increasing the salts and / or solvents of the second solution to effect a replacement or exchange of the first solution for the second solution. The DF may be carried out according to, e.g., Schwartz, Larry. "Diafiltration: A Fast, Efficient Method for Desalting, or Buffer Exchange of Biological Samples". Literature Library; "Protein Concentration and Diafiltration by Tangential Flow Filtration" (PDF). Millipore; Sweeney, Scott F.; Woehrle, Gerd H.; Hutchison, James E. (2006). "Rapid Purification and Size Separation of Gold Nanoparticles via Diafiltration". Journal of the American Chemical Society. 128 (10): 3190-3197. doi:10.1021 / ja0558241. PMID 16522099; Schwartz, Larry. "Diafiltration for Desalting or Buffer Exchange" (PDF). BioProcess International; "Diafiltration (Buffer Exchange) Using Hollow Fiber Membranes instead of Dialysis Tubing - Automated Diafiltration"; Bowen, W. Richard; Mohammad, A. Wahab (Aug 1998). "Diafiltration by nanofiltration: Prediction and optimization". AIChE Journal.44 (8): 1799-1812. doi:10.1002 / aic.690440811; Limayem, Imene; Charcosset, Catherine; Fessi, Hatem (2004). "Purification of nanoparticle suspensions by a concentration / diafiltration process". Separation and Purification Technology. 38 (1): 1-9. doi:10.1016 / j.seppur.2003.10.002; Sheth, Jignesh P.; et al. (2003). "Nanofiltration-based diafiltration process for solvent exchange in pharmaceutical manufacturing". Journal of Membrane Science. 211 (2): 251-261. doi:10.1016 / s0376-7388(02)00423-4; Millipore Mechanical Brief: Protein Concentration andDiafiltration by Tangential Flow Filtration, available at chrome- extension: / / efaidnbmnnnibpcajpcglclefindmkaj / viewer.html?pdfurl=http%3A%2F%2Fwolfson.huji.ac .il%2Fpurification%2FPDF%2Fdialysis%2FMILLIPORE_TFF.pdf&clen=2842762&chunk=true; or Koros et al., Pure & Appl Chem 68: 1479-1489; Ng et al., Separation Science 2: 499-502 (1976); Tkacik and Michaels, Bio / Technol 9: 941-946 (1991); van Reis et al., Biotech Bioeng 56: 71-82 (1997); van Reis et al., J Membrane Sci 130: 123-140 (1997); Encyclopedia of Bioprocess Technology, John Wiley and Sons, Inc., New York (1999); Zeman and Zydney, U.S. Patent Application Publication Nos. 2006 / 0051347 and 2014 / 0370003; Maruthamuthu et al., Trends in Biotechnology, 38(10): 1169- 1186 (2020); Kovacs, Z. (2016). Continuous Diafiltration: Cocurrent and Countercurrent Modes. In: Drioli, E., Giorno, L. (eds) Encyclopedia of Membranes. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-662-44324-8_638; and Schwartz, L., Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications; the contents of each are incorporated herein by reference. In exemplary instances, the diafiltration is carried out using an Amicon® filter device, e.g., an Amicon® ultra filter or ultra centrifugal filter device, or a Centricon® centrifugal filter unit, e.g., a Centricon® Plus-70. In various aspects, the diafiltration results in an exchanged protein solution, which is the second solution comprising the protein, e.g., therapeutic protein.

[0032] Figure 1 illustrates an exemplary configuration for exchanging the protein into a second solution from a first solution. The direction of movement of solutions is shown with solid black arrows. In exemplary instances, as second solution is added to a first solution in a retentate vessel to create a mixture, a pump causes movement of the solution in the retentate vessel to move through the membrane to obtain a retentate, which comprises the protein, and a permeate, which comprises one or more components of the first solution. The pump causes movement of the retentate to the retentate vessel and the permeate to the permeate vessel. The movement of the solutions continues in this manner until the volume of the second solution added to the retentate vessel is essentially the same as the volume of permeate collected in the permeate vessel. The volume of the second solution added to the retentate vessel in various instances is about 3 times the volume of the first solution. Optionally, the volume of the second solution is about 4 times or 5 times the volume of the first solution. In various aspects, the volume of the first solution is referred to as a "diavolume" and optionally, the volume of the second solution added to the retentate vessel is 3, 4, or 5 diavolumes. In various instances, the volume of the second solution added to the retentate vessel is 6, 7, 8, 9, or 10 diavolumes. Generally, the higher the number of diavolumes the greater percent exchange is accomplished via diafiltration.

[0033] In exemplary instances, the diafiltration is carried out using an Amicon® filter device, e.g., an Amicon® ultra filter or ultra centrifugal filter device, or a Centricon® centrifugal filter unit, e.g., a Centricon® Plus-70. In exemplary instances, the method comprises continuous diafiltration by tangential flow. In various aspects, the protein is exchanged into a volume of about 50 pL to about 500 pL of the second solution. In exemplary instances, the volume is about 50 pL to about 400 pL, about 50 pL to about 300 pL, about 50 pL to about 200 pL, about 50 pL to about 100 pL, about 100 pL to about 500 pL, about 200 pL to about 500 pL, about 300 pL to about 500 pL or about 400 pL to about 500 pL. In some aspects, the volume is about 100 pL to about 300 pL, about 150 pL to about 250 pL, or about 200 pL. In various aspects, the protein is exchanged into about 200 pL of the second solution. In various aspects, the diafiltration results in an exchanged protein solution, which is the second solution comprising the protein, e.g., therapeutic protein.

[0034] In various aspects, the first solution comprises the protein, e.g., therapeutic protein, and at least one other protein. For example, the first solution comprises in certain aspects, less than 5 different proteins, one of which is the therapeutic protein. The first solution in various aspects comprises in certain aspects, less than 4 different proteins, less than 3 different proteins, or two different proteins, one of which is the therapeutic protein. In exemplary aspects, the first solution has a bioburden less than 100 CFU / mL, such as less than 50 CFU / mL, less than 25 CFU / mL, or less than 10 CFU / mL. In various aspects, the first solution does not comprise, or does not consist of blood, a cell culture supernatant, tissue culture supernatant, urine, plasma, or serum. The first solution in various aspects is not a biological sample obtained from a subject, e.g., a mammal, a human.

[0035] In various aspects, the first solution is a sample of in-process material. In exemplary aspects, the therapeutic protein is recombinantly produced by cells in a cell culture and the cell culture supernatant is the first solution. In various instances, the amino acid content of the therapeutic protein is analyzed after a chromatography, e.g., a chromatography used to purify the therapeutic protein from a cell culture supernatant. In exemplary instances the chromatography is an ion exchange chromatography (e.g., a cation exchange chromatography or an anion exchange chromatography). The chromatography may be a capture or affinity chromatography which is used, for example, to separate the recombinant therapeutic protein from other proteins, aggregates, DNA, viruses and other such impurities. In exemplary instances, the chromatography comprises or consists of a chromatography carried out with Protein A (e.g., Protein A attached to a resin). In alternative aspects, the chromatography comprises or consists of an intermediate or polish chromatography, which in various aspects further purifies the recombinant therapeutic protein, removing bulk contaminants, adventitious viruses, trace impurities, aggregates, isoforms, etc. Thechromatography in various aspects, is performed in bind and elute mode, where the therapeutic protein of interest is bound to the chromatography medium and the impurities flow through, or in flow-through mode, where the impurities are bound and the therapeutic protein flows through. Examples of such chromatography methods include, e.g., ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reverse phase chromatography and gel filtration, or methods comprising two or more of the listed items. In various aspects, the chromatography comprises an affinity chromatography (e.g., a Protein A or Protein G affinity chromatography), a capture chromatography, intermediate chromatography, and / or polish chromatography. The chromatography may be one described in International Patent Application Publication No. WO 2022 / 061092, which is incorporated herein by reference. In some aspects, the amino acid content of the therapeutic protein is analyzed after a virus inactivation and neutralization, virus filtration, or a buffer exchange. The method in various instances is implemented as part of a lot release assay. The first solution in some aspects is a sample of a manufacturing lot. In various instances, the presently disclosed method is purposed to assure that batches of drug products meet each appropriate specification and appropriate statistical quality control criteria as a condition for their approval and release, pursuant to 21 CFR 211.165. Terminology, including, but not limited to "lot" and "in-process" accord with their meaning as defined in 21 Code of Federal Regulations (CFR) Section 210.3.

[0036] In various instances, the protein of which the amino acid content is analyzed, e.g., therapeutic protein, is the only protein present in the first solution. In exemplary aspects, the first solution comprises only one protein, e.g., therapeutic protein, and the one protein is analyzed for amino acid content by the presently disclosed method. In exemplary instances, the first solution is a liquid drug product or a liquid drug substance. For instance, the first solution is a formulated therapeutic protein, or the first solution comprises the therapeutic protein in a formulation buffer. The first solution, in various aspects, comprises one or more excipients selected from the group of: a salt, a surfactant, sugar and / or a polyol. In various aspects, the first solution comprises a buffer and sugar. In exemplary aspects, the first solution is a formulation buffer comprising a buffer, e.g., acetate or glutamic acid, and a sugar, e.g., sucrose, and optionally a surfactant, e.g., polysorbate 20, polysorbate 80. In exemplary aspects, the first solution has the same pH as the second solution. In various aspects, the first solution comprises or consists of a pharmaceutical formulation. In various aspects, the first solution comprises a buffer and a sugar. The buffer in various aspects is acetate, succinate, citrate, histidine, glutamate, phosphate, tris, or the like. The sugar in exemplary instancesis sucrose, glucose, lactose, arabinose, glucosamine, galactosamine, N-acetyl galactosamine, galactose, or fructose. Surfactants are surface active agents that are amphipathic (having a polar head and hydrophobic tail). Surfactants preferentially accumulate at interfaces, resulting in reduced interfacial tension. In some aspects, the surfactant present in the first solution is an amphipathic and / or nonionic surfactant. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (e.g. polysorbate 20, polysorbate 80), alkylaryl polyethers, e.g. oxyethylated alkyl phenol (e.g. Triton™ X-100), and poloxamers (e.g. Pluronics®, e.g. Pluronic® F68), and combinations of any of the foregoing, either within a class of surfactants or among classes of surfactants. Polysorbate 20 and polysorbate 80 (and optionally mixtures thereof) are particularly contemplated. Exemplary polyols include but are not limited to glycerol, mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, and isomalt.

[0037] Without being bound by theory, it is contemplated herein that the presence of sugars during temperature-induced hydrolysis of the therapeutic protein can lead to caramelization of the sugars, which can interfere with colorimetric determination of amino acids following temperature- induced hydrolysis, especially for low abundance amino acids (See Example 2 and Figure 2). In exemplary instances, the second solution lacks sugar or comprises only a small amount, e.g., less than about 1% (w / v), less than about 0.5% (w / v), less than about 0.1% (w / v), less than about 0.01% (w / v), of sugar. In exemplary instances, the second solution is identical to the first solution but lacks sugar. In exemplary instances, the diafiltration is performed with at least 2 or more diavolumes of the second solution. In exemplary instances, the diafiltration is performed with at least 3 or 4 diavolumes of the second solution. In exemplary instances, the diafiltration is performed with at least 4 diavolumes of the second solution.

[0038] An exchanged protein solution is obtained following diafiltration. The exchanged protein solution comprises the protein, e.g., therapeutic protein, in the second solution.

[0039] Hydrolysis

[0040] In exemplary aspects, the method comprises combining the exchanged protein solution (e.g., the protein in the second solution) at an acidic pH and a temperature greater than about 110 °C to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein. Without being bound by theory, it is believed that combining the exchanged protein solution at the acidic pH and high temperature leads to hydrolysis of the peptide bonds linking the individual amino acids of the polypeptide chain(s) of the protein. Thus, the hydrolyzed sample so obtained comprises a mixture of the individual amino acids of the therapeutic protein. In various instances, combining the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C leads to hydrolysis ofgreater than 90% of the peptide bonds of the protein. In various instances, combining the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C leads to hydrolysis of greater than 95% of the peptide bonds of the protein. In various instances, combining the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C leads to hydrolysis of greater than 98% of the peptide bonds of the protein. In various instances, combining the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C leads to hydrolysis of nearly 100% of the peptide bonds of the protein. In exemplary instances, the method comprises incubating the exchanged protein solution at an acidic pH, e.g., less than about 4.2, and a temperature greater than about 110 °C, e.g., about 115 °C to about 130 °C, to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein. The acidic pH in various instances is less than about 4.0 (e.g., less than 3.9, less than 3.8, less than 3.7, less than 3.6, or less than about 3.5). In exemplary aspects, the acidic pH is less than 3.5, optionally, about 3.0 to about 3.5. In various aspects, the temperature greater than about 110 °C is less than about 125 °C. In various instances, the temperature greater than about 110 °C is greater than 115 °C and less than about 125 °C. In exemplary aspects, the exchanged protein solution is incubated at about 120 °C. In various instances, the exchanged protein solution is incubated at the acidic pH and the temperature greater than about 110°C for at least 1 or 2 hours, optionally, about 3 hours. In various instances, the exchanged protein solution is incubated at the acidic pH and the temperature greater than about 110°C for at least 1.25 hours, at least 1.50 hours, at least 1.75 hours, at least 2.0 hours, at least 2.25 hours, at least 2.50 hours, at least 2.75 hours, or at least 3.0 hours. In exemplary aspects, combining the exchanged protein solution at an acidic pH comprises combining the exchanged protein solution with an acid. The acid in various aspects is acetic acid or hydrochloric acid. In exemplary instances, the acid is hydrochloric acid. The acid, e.g., hydrochloric acid, is greater than 8N. For instance, the acid is greater than 10 N or about 12 N hydrochloric acid, in various aspects. In exemplary instances, 12N hydrochloric acid is added to the exchanged protein solution and heated at 120 °C for about 3 hours. Optionally, about 200 pL of 12 N hydrochloric acid is added to the exchanged protein solution and heated at 120 °C for about 3 hours. In various aspects, the volume of 12 N hydrochloric acid added to the exchanged protein solution is about 50 pL to about 500 pL. In exemplary instances, the volume of 12 N hydrochloric acid is about 50 pL to about 400 pL, about 50 pL to about 300 pL, about 50 pL to about 200 pL, about 50 pL to about 100 pL, about 100 pL to about 500 pL, about 200 pL to about 500 pL, about 300 pL to about 500 pL or about 400 pL to about 500 pL. In some aspects, the volume of 12 N hydrochloric acid is about 100 pL to about 300 pL, about 150 pL to about 250 pL, or about 200 pL. In various aspects, the volume of 12 N hydrochloric acid added to the exchanged protein solution is equivalent to the volume of the exchanged protein solution.

[0041] Activated Charcoal

[0042] In exemplary aspects of the presently disclosed methods, activated charcoal is combined with the hydrolyzed sample. In various aspects, the activated charcoal is mixed with the hydrolyzed sample. Without being bound by theory, the activated charcoal acts to bind to impurities found in the hydrolyzed sample that may interfere with analysis of the amino acid content. In exemplary instances, about 2.5 mg to about 5.0 mg activated charcoal is combined with the hydrolyzed sample for every 1 mg therapeutic protein in the first solution. In exemplary instances, about 3.0 mg to about 5.0 mg or about 3.5 mg to about 5.0 mg or about 4.0 mg to about 5.0 mg or about 4.5 mg to about 5.0 mg activated charcoal is combined with the hydrolyzed sample for every 1 mg therapeutic protein in the first solution. In exemplary instances, about 2.5 mg to about 4.5 mg or about 2.5 mg to about 4.0 mg or about 2.5 mg to about 3.5 mg or about 2.5 mg to about 3.0 mg activated charcoal is combined with the hydrolyzed sample for every 1 mg therapeutic protein in the first solution.

[0043] After activated charcoal is combined (e.g., mixed) with the hydrolyzed sample, the activated charcoal is removed or separated from the hydrolyzed sample. In exemplary embodiments, the hydrolyzed sample is separated from the activated charcoal by (i) centrifugation, (ii) filtration, or (iii) both centrifugation and filtration. In various aspects, the centrifugation is performed at about 13,000 x g to about 14,000 x g for, e.g., about 3 min to about 5 min. In various instances, the filtration is performed with a filter having a pore size of about 0.1 pm to about 0.4 pm, for example a 0.22 pm filter. In exemplary aspects, the method comprises separating the hydrolyzed sample from the activated charcoal by (i) centrifuging to obtain an activated charcoal pellet and a supernatant and (ii) passing the supernatant through a filter, e.g., a 0.22 pm filter.

[0044] Concentrating the Sample

[0045] In various instances, the method further comprises concentrating the hydrolyzed sample following the centrifugation and / or filtration. In various aspects, the method further comprises removing solvent from the hydrolyzed sample following the centrifugation and / or filtration. In exemplary aspects, the method further comprises evaporating to dryness the supernatant or the filtered supernatant comprising the hydrolyzed sample by, for example heating at 40 °C for more than 8 hours under vacuum. In various aspects, the supernatant or the filtered supernatant comprising the hydrolyzed sample is dried using a rotary evaporator, e.g., a rotovap. In exemplary instances, the dried sample is resuspended with a small volume of a solution. In various aspects, the small volume of solution comprising the concentrated hydrolyzed sample comprising the amino acids of the therapeutic protein is a working volume for analysis of the amino acid content. In exemplary aspects, the dried pellet is resuspended in less than 100 pL, less than 50 pL or less than 25pL solution. In various aspects, the dried pellet is resuspended in 20 pL solution. In various instances, the dried pellet is resuspended in water. In various aspects, the method comprises separating the hydrolyzed sample from the activated charcoal by (i) centrifuging to obtain an activated charcoal pellet and a supernatant and (ii) passing the supernatant through a filter, e.g., a 0.22 pm filter, and subsequently evaporating to dryness the filtered supernatant using a rotary evaporator to obtain a dried pellet, and then resuspending the dried pellet with less than 50 pL water.

[0046] Separation of the Amino Acids of Hydrolyzed Sample & Detection Thereof

[0047] In exemplary aspects, the method comprises analyzing the amino acid content of the hydrolyzed sample, e.g., amino acid analysis. In various aspects, the method comprises analyzing the amino acid content of the hydrolyzed sample through an analytical technique, and in various instances, the analytical technique has a limit of detection (LOD) in the nanomolar or picomolar range. Without being bound by theory, it is contemplated herein that conventional colorimetric assays, such as chemical-based assays, such as that described in Examples 2 and 3 and an ELISA, e.g., described in Example 4, do not achieve LODs in the nanomolar or picomolar range, and thus may not be suitable for detecting low-abundance amino acids (See, e.g., Example 4 and Figures 3A-3B). In various aspects, the method comprises analyzing the amino acid content of the hydrolyzed sample through a technique which does not comprise a colorimetric assay. The method in exemplary instances is capable of detecting a single amino acid which represents less than 0.1% of the amino acids of the therapeutic protein.

[0048] In various aspects, analyzing the amino acid content of the hydrolyzed sample comprises chromatographically separating the amino acids of the therapeutic protein. Desirably, the chromatographic separation occurs after the hydrolyzed sample is combined with and then separated from the activated charcoal. In various instances, the chromatographic separation occurs after the hydrolyzed sample is combined with and then separated from the activated charcoal and after the hydrolyzed sample is evaporated to dryness and resuspended in a small volume of a solution. Optionally, the amino acids of the therapeutic protein are chromatographically separated by a high-performance liquid chromatography (HPLC), optionally, a reversed phase-HPLC (RP-HPLC). Reversed phased chromatography, e.g., RP-HPLC, is described in great detail in the prior art. See, for instance, Reversed Phase Chromatography: Principles and Methods, ed. AA, Amersham Biosciences, Buckinghamshire, England (1999). In various instances, the amino acids of the hydrolyzed sample are separated by RP-HPLC. In exemplary instances, the amino acids are chromatographically separated, and the separation is characterized as having high resolution. In various aspects, the resolution of the separation of the peaks of each amino acid is at least or about 1.0, optionally, atleast or about 1.1, at least or about 1.2, at least or about 1.3, or at least or about 1.4. In various aspects, the resolution of the separation of the peaks of each molecular species is at least or about 1.5, optionally, at least or about 1.6, at least or about 1.7, at least or about 1.8, or at least or about 1.9. Optionally, the resolution of the separation of the peaks corresponding to each molecular species (e.g., the resolution of the separation between the peak of the duplex and the peak of the sense single strand) is at least or about 2.0 (e.g., at least or about 2.1, at least or about 2.2, at least or about 2.3, at least or about 2.4. In various aspects, the resolution of the separation is at least or about 2.4. In exemplary instances, the resolution is at least or about 2.5, at least or about 3.0, or at least or about 4.0. In various aspects, the resolution of the separation of the peaks of each amino acid is a range bound by any two of the listed values. In various aspects, the resolution is a United States Pharmacopeia (USP) resolution and may be calculated using the USP Resolution equation (Equation 1) which uses the baseline peak width calculated using lines tangent to the peak at 50% height:> 2.0 R ~ 1.7wherein R = resolution, Rt = retention time, and W1+W2 = sum of peak widths at 50% peak height[Equation 1](Taken from "Empower System Suitability: Quick Reference Guide" Waters Corp. (2002))

[0049] In exemplary aspects, the hydrolyzed sample is chromatographically separated such that the amino acids of the therapeutic protein elute from the column at different elution times. For example, the prolines of the therapeutic protein elute from the column at an elution time that is different from the elution time of lysine. Additionally, in various instances, the amino acids of the therapeutic protein elute from the column at elution times different from the post translationally modified form of the unmodified amino acid. For instance, the elution time of proline is different from the elution time of hydroxyproline.

[0050] The method of the present disclosure in various instances comprises chromatographically separating the amino acids of the therapeutic protein by a hydrophobic interaction liquid chromatography (HILIC), wherein the hydrophobic amino acids (or labeled forms thereof) elute later than less hydrophobic amino acids (or labeled forms thereof).

[0051] In various instances, the method comprises chromatographically separating the amino acids of the hydrolyzed sample and then detecting at least some of the separated amino acids. In various instances, the method comprises labeling the amino acids of the therapeutic protein with a detectable label and then chromatographically separating the labeled amino acids of the therapeuticprotein via, e.g., RP-HPLC. In various aspects, the method comprises labeling the amino acids of the therapeutic protein with a detectable label before chromatographically separating the amino acids. In exemplary aspects, the detectable label absorbs ultraviolet light at a specific wavelength, e.g., 260 nm. In exemplary instances, the detectable label is 6-Aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC). In exemplary instances, a transmitted UV (TUV) or a photo diode array (PDA) detector is used to detect the detectable label-amino acid complex, and, in various aspects, the TUV or PDA detects during the chromatographic separation, e.g., the HPLC. In various instances, the detectable label is a fluorescent label, and the method comprises detecting the fluorescence during the chromatographic separation. See, e.g., Torres et al., Anal Chem 94: 16538-16542 (2022). In various aspects, the method comprises combining the hydrolyzed sample with activated charcoal, separating the hydrolyzed sample from the activated charcoal, evaporating to dryness, then resuspending the sample in a small volume of a solution, then labeling the amino acids with a detectable label followed by chromatographically separating the labeled amino acids.

[0052] In alternative or additional aspects, the method comprises analyzing the amino acid content through mass spectrometry. In various instances, the mass of the chromatographically separated amino acids is determined through mass spectrometry. Optionally, when mass spectrometry is carried out, labeling of the amino acids of the therapeutic protein is not necessary. In various aspects, the method comprises concentrating the hydrolyzed sample after the hydrolyzed sample is separated from the activated charcoal but before the amino acid content of the hydrolyzed sample is analyzed. In exemplary aspects, the method comprises combining the hydrolyzed sample with activated charcoal, separating the hydrolyzed sample from the activated charcoal, evaporating to dryness, then resuspending the sample in a small volume of a solution, then performing mass spectrometry on the separated amino acids.

[0053] Use

[0054] As the presently disclosed methods of analyzing the amino acid content of a therapeutic protein are useful for detecting the presence or absence of a post translational modification (PTM) in a therapeutic protein, the present disclosure additionally provides a method of detecting the presence or absence of a post translational modification (PTM) in a therapeutic protein. In exemplary embodiments, the method comprises analyzing the amino acid content of a therapeutic protein in accordance with the methods of analyzing the amino acid content of a therapeutic protein of the present disclosure. In various aspects, the PTM is a modified or isomerized amino acid, such as hydroxyproline.

[0055] Since the presently disclosed methods of analyzing the amino acid content of a therapeutic protein are also useful for detecting structural changes of a therapeutic protein in a drug product, the present disclosure further provides methods of detecting structural changes of a therapeutic protein in a drug product. In exemplary embodiments, the method comprises analyzing the amino acid content of a therapeutic protein in accordance with the methods of analyzing the amino acid content of a therapeutic protein of the present disclosure.

[0056] Because the presently disclosed methods of analyzing the amino acid content of a therapeutic protein are further useful for evaluating product quality, manufacturing process steps, and overall stability and safety of a drug product, the present disclosure furthermore provides methods of evaluating product quality, manufacturing process steps, and overall stability and safety of a drug product. In exemplary embodiments, the method comprises analyzing the amino acid content of a therapeutic protein in accordance with the methods of analyzing the amino acid content of a therapeutic protein of the present disclosure. In exemplary aspects, the product quality of a drug product of a particular lot is evaluated. Accordingly, a sample of a manufacturing lot is used in the presently disclosed method of analyzing the amino acid content as the first solution. In exemplary instances, a manufacture process step or a combination of manufacture process steps is evaluated in terms of whether a protein is structurally altered during the manufacture process step(s). Accordingly, a sample of an in-process material that has undergone the step(s) is used in the presently disclosed method of analyzing the amino acid content as the first solution.

[0057] Additional Steps

[0058] The methods disclosed herein, in various aspects, comprise additional steps. For example, in some aspects, the methods of the present disclosure are part of a manufacture process step and the method thus comprises manufacture process step(s) which can be, for instance, one or more upstream steps or downstream steps involved in producing, purifying, and formulating a recombinant protein, e.g., a therapeutic protein. Optionally, the downstream steps are any one of those downstream processing steps described herein or known in the art. In exemplary embodiments, the method comprises steps for generating host cells that express the therapeutic protein. The host cells, in some aspects, are prokaryotic host cells, e.g., E. coli or Bacillus subtilis, or the host cells, in some aspects, are eukaryotic host cells, e.g., yeast cells, filamentous fungi cells, protozoa cells, insect cells, or mammalian cells (e.g., CHO cells). Such host cells are described in the art. See, e.g., See, e.g., Kunert et al., Appl. Microbiol Biotechnol. 100: 3451-61 (2016). For example, the methods comprise, in some instances, introducing into host cells a vector comprising a nucleic acid comprising a nucleotide sequence encoding the therapeutic protein, or a polypeptide chain thereof.

[0059] In exemplary embodiments, the methods disclosed herein comprise steps for isolating and / or purifying the recombinant therapeutic protein from the culture. In exemplary aspects, the method comprises one or more chromatography steps including, but not limited to, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography.

[0060] In various aspects, the first solution of the method is a drug substance. In various instances, the methods of the present disclosure comprise one or more steps for preparing the drug substance into a drug product. For example, the method comprises in various aspects finishing or polishing the drug substance and / or filling it into a container.

[0061] Therapeutic Proteins

[0062] Therapeutic proteins, including those that bind to one or more of the following, can be processed and analyzed in the disclosed methods. These include CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding. HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor. Cell adhesion molecules, for example, LFA-1, Mol, pl50, 95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin. Growth factors, such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mui lerian-inhibiting substance, human macrophage inflammatory protein (MIP-I -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF- a and TGF-P, including TGF-pi, TGF-P2, TGF-P3, TGF- P4, or TGF- P 5, insulin-like growth factors-l and -II (IGF-I and IGF-II), des(l-3)-IGF-l (brain IGF-I), and osteoinductive factors. Insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins. Coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-l-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens. Colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM- CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor (c-fms). Receptors and receptor- associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors ("TPO-R," "c-mpl"), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors. Receptor ligands, including, for example, OX40L, the ligand for the 0X40receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A-chain, relaxin B-chain, and prorelaxin; interferons and interferon receptors, including for example, interferon-a, -p, and -y, and their receptors. Interleukins and interleukin receptors, including IL-I to IL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, among others. Viral antigens, including an AIDS envelope viral antigen. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies.Myostatins, TALL proteins, including TALL-1, amyloid proteins, including but not limited to amyloidbeta proteins, thymic stromal lymphopoietins ("TSLP"), RANK ligand ("RANKL" or "OPGL"), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.

[0063] Exemplary therapeutic proteins, include Activase® (Alteplase); alirocumab, Aranesp® (Darbepoetin-alfa), Epogen® (Epoetin alfa, or erythropoietin); Avonex® (Interferon P-la); Bexxar® (Tositumomab); Betaseron® (Interferon-P); bococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see US8080243); Campath® (Alemtuzumab); Dynepo® (Epoetin delta); Velcade® (bortezomib); MLN0002 (anti-a4P7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (Epoetin alfa); Erbitux® (Cetuximab); evolocumab; Genotropin® (Somatropin); Herceptin® (Trastuzumab); Humatrope® (somatropin [rDNA origin] for injection); Humira® (Adalimumab); Infergen® (Interferon Alfacon-1); Natrecor® (nesiritide); Kineret® (Anakinra), Leukine® (Sargamostim); LymphoCide® (Epratuzumab); BenlystaTM (Belimumab); Metalyse® (Tenecteplase); Mircera® (methoxy polyethylene glycol-epoetin beta); Mylotarg® (Gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); SolirisTM (Eculizumab);Pexelizumab (Anti-C5 Complement); MEDI-524 (Numax®); Lucentis® (Ranibizumab); Edrecolomab (,Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (Nimotuzumab); Omnitarg (Pertuzumab, 2C4); Osidem® (IDM-I); OvaRex® (B43.13); Nuvion® (visilizumab); Cantuzumab mertansine (huC242-DMI); NeoRecormon® (Epoetin beta); Neumega® (Oprelvekin); Neulasta® (Pegylated filgastrim, pegylated G-CSF, pegylated hu-Met-G-CSF); Neupogen® (Filgrastim); Orthoclone OKT3® (Muromonab-CD3), Procrit® (Epoetin alfa); Remicade® (Infliximab), Reopro® (Abciximab), Actemra® (anti-l L6 Receptor mAb), Avastin® (Bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (Rituximab); Tarceva® (Erlotinib); Roferon-A®-(lnterferon alfa-2a); Simulect® (Basiliximab); StelaraTM (Ustekinumab);Prexige® (lumiracoxib); Synagis® (Palivizumab); 146B7-CHO (anti-l L15 antibody, see US7153507), Tysabri® (Natalizumab); Valortim® (MDX-1303, anti-B. anthracis Protective Antigen mAb);ABthraxTM; Vectibix® (Panitumumab); Xolair® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGI and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRI fused to IgGI Fc), Zenapax® (Daclizumab); Zenapax® (Daclizumab), Zevalin® (Ibritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-lg), anti-a4P7 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti- CD23 mAb (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); SimponiTM (Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab, anti-a5pi integrin mAb); MDX-010 (Ipilimumab, anti-CTLA-4 mAb and VEGFR-I (IMC-18F1); anti-BR3 mAb; anti-C. difficile Toxin A and Toxin B C mAbs MDX-066 (CDA-I) and MDX-1388); anti-CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibodies; anti-TSLP receptor antibody (US8101182); anti-TSLP antibody designated as A5 (US7982016); (anti-CD3 mAb (NI-0401);Adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAbs); MDX-1333 (anti- IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti- CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxinl mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibodies (see, US8715663 or US7592429) anti-sclerostin antibody designated as Ab-5 (US8715663 or US7592429); anti- ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF Receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNa mAb); anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-l L12 / IL23p40 mAb (Briakinumab); anti-IL-23pl9 mAb (LY2525623); anti- 1113 mAb (CAT-354); anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti-IL5 Receptor mAb; anti-integrin receptors mAb (MDX-018, CNTO 95); anti-l PIO Ulcerative Colitis mAb (MDX- 1100); anti- LLY antibody; BMS-66513; anti-Mannose Receptor / hCGP mAb (MDX-1307); anti-mesothelin dsFv- PE38 conjugate (CAT-5001); anti-PdlmAb (MDX-1 106 (ONO- 4538)); anti-PDGFRa antibody (IMC- 3G3); anti-TGFP mAb (GC-1008); anti-TRAIL Receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti- ZP3 mAb (HuMax-ZP3); NVS Antibody #1; NVS Antibody #2; and an amyloid-beta monoclonal antibody.

[0064] Examples of therapeutic proteins suitable for the methods and pharmaceutical formulations include those shown in Table G. Other examples of suitable therapeutic proteins include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab,apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, bemarituzumab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab,simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox.

[0065] Antibodies also include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab, and antibodies selected from Table A.Table AExamples of therapeutic antibodiesTarget (informal HC* Type (including LC* T LC* SEQ ID HC* SEQ ID name) allotypes)ype PNO: NO: anti-amyloid IgGl (f) (R;EM) Kappa 9.0 2 3GMCSF (247) lgG2 Kappa 8.7 4 5CGRPR lgG2 Lambda 8.6 6 7RANKL lgG2 Kappa 8.6 8 9Sclerostin (27H6) lgG2 Kappa 6.6 10 11IL-1R1 lgG2 Kappa 7.4 12 13Myostatin IgGl (z) (K;EM) Kappa 8.7 14 15B7RP1 lgG2 Kappa 7.7 16 17Amyloid IgGl (za) (K;DL) Kappa 8.7 18 19GMCSF (3.112) lgG2 Kappa 8.8 20 21CGRP (32H7) lgG2 Kappa 8.7 22 23CGRP (3B6.2) lgG2 Lambda 8.6 24 25PCSK9 (8A3.1) lgG2 Kappa 6.7 26 27PCSK9 (492) lgG2 Kappa 6.9 28 29CGRP lgG2 Lambda 8.8 30 31Hepcidin lgG2 Lambda 7.3 32 33TNFR p55) lgG2 Kappa 8.2 34 35OX40L lgG2 Kappa 8.7 36 37HGF lgG2 Kappa 8.1 38 39GMCSF lgG2 Kappa 8.1 40 41Glucagon R lgG2 Kappa 8.4 42 43GMCSF (4.381) lgG2 Kappa 8.4 44 45Sclerostin (13F3) lgG2 Kappa 7.8 46 47CD-22 IgGl (f) (R;EM) Kappa 8.8 48 49INFgR IgGl (za) (K;DL) Kappa 8.8 50 51Ang2 lgG2 Kappa 7.4 52 53Target (informal HC* Type (including LC* T LC* SEQ ID HC* SEQ ID name) allotypes)ype PNO: NO:TRAILR2 IgGl (f) (R;EM) Kappa 8.7 54 55EGFR lgG2 Kappa 6.8 56 57IL-4R lgG2 Kappa 8.6 58 59IL-15 IgGl (f) (R;EM) Kappa 8.8 60 61IGF1R IgGl (za) (K;DL) Kappa 8.6 62 63IL-17R lgG2 Kappa 8.6 64 65Dkkl (6.37.5) lgG2 Kappa 8.2 66 67Sclerostin lgG2 Kappa 7.4 68 69TSLP lgG2 Lambda 7.2 70 71Dkkl (llHlO) lgG2 Kappa 8.2 72 73PCSK9 lgG2 Lambda 8.1 74 75GIPR (2G10.006) IgGl (z) (K;EM) Kappa 8.1 76 77Activin lgG2 Lambda 7.0 78 79Sclerostin (2B8) lgG2 Lambda 6.7 80 81Sclerostin lgG2 Kappa 6.8 82 83 c-fms lgG2 Kappa 6.6 84 85 a4P7 lgG2 Kappa 6.5 86 87*HC - antibody heavy chain; LC - antibody light chain. Each sequence of referenced SEQ. ID NO: includes a signal peptide. Most HC signal peptides are the first 19 amino acids and most LC signal peptide are 22 amino acids (Haryadi et al., PloS One 10(2): e0116878).

[0066] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLESEXAMPLE 1

[0067] This example describes the discovery of a hydroxylated amino acid post-translational modification (PTM) of a therapeutic protein.

[0068] Peptide mapping analysis of a novel therapeutic protein (TP1) was carried out to determine the presence of PTMs and assess its overall post-manufacture quality. Briefly, the peptide mapping analysis comprised a two-step enzymatic digestion of TP1 to create peptide fragments, chromatographic separation of the peptide fragments, followed by mass spectrometry (MS) analysis. Additional processing of the sample occurring before the enzymatic digestion included a denaturation, alkylation, and reduction, and post-digestion processing included a quench to halt enzymatic digestion and / or a buffer exchange. The results of the peptide mapping suggested the presence of three hydroxyproline residues, each within a peptide fragment that is present in triplicate in TP1. The peptide fragment also included a lysine residue, and, because hydroxyproline occurs less frequently, relative to hydroxylysine, and since hydroxylysine could potentially impact TP1, an orthogonal technique confirming the identity of the hydroxylated amino acid PTM presenton TP1 was needed. Since, this PTM represented three amino acids out of the 1144 amino acids ofTP1, it was determined that a high level of sensitivity was required of the orthogonal technique.

[0069] This example demonstrated the need for a highly sensitive method for analyzing the amino acid content of a therapeutic protein to confirm the identity of the hydroxylated amino acid PTM present on the therapeutic protein.EXAMPLE 2

[0070] This example describes a first attempt at determining amino acid content of a therapeutic protein to identify the hydroxylated amino acid PTM.

[0071] As a first approach, a colorimetric-based assay was selected for identifying the hydroxylated amino acids of TP1. The assay determines hydroxyproline concentration of a sample by reacting oxidized hydroxyproline with 4-(Dimethylamino)benzaldehyde (DMAB), which results in the formation of a colorimetric product detectable at 560 nm in a manner which is proportional to the hydroxyproline content. To prepare TP1 for this colorimetric-based assay, a first solution comprising about 1 mg to about 2 mg TP1 in 10 mM glutamate buffer with 9% (w / v) sucrose (pH 4.2) was exchanged via diafiltration into a second solution comprising 10 mM glutamate buffer (pH 4.2). The exchanged solution was diluted with water to achieve a diluted TP1 concentration less than 10 mg / mL and the volume was about 100 pL to about 400 pL. The diluted TP1 solution was then subjected to a heated acid hydrolysis which was initiated by adding an equal volume of 12 N hydrochloric acid and then incubating at 120 °C for 3 hours. The heated acid hydrolysis led to hydrolysis of the peptide bonds of TP1 yielding a hydrolyzed solution comprising individual amino acids of TP1. This solution was evaporated to dryness in a vacuum centrifuge heated to 60°C, then resuspended in 100 pL water. The resuspended solution was used in the colorimetric-based assay of the Hydroxyproline Assay Kit (Catalog No. MAK008, Sigma, St. Louis, Missouri) according to the supplied instructions. Hydroxyproline concentration of test samples was determined using a standard curve made with samples comprising varying concentrations of a hydroxyproline standard. The standard curve correlated hydroxyproline concentration to absorbance at 560 nm. A summary of the procedure carried out is provided in Table 1.TABLE 1T1

[0072] Figure 2 is a representative photo of the test samples (at different dilutions) obtained upon carrying out the procedure of Table 1. As shown in this figure, the samples were notably dark brown and outside the color spectrum of the standard curve samples (which appeared similar to Wells 1A- 1H and 2A-2H of Figure 3). Based on the visual appearance of the test samples, it was deemed highly unlikely that meaningful results would be obtained upon reading the absorbance of these samples. Thus, the concentration of hydroxyproline of the test samples was not determined, and the identity of the hydroxylated PTM of TPl remained unconfirmed.

[0073] This example demonstrated that the procedure outlined in Table 1 was unsuccessful at determining the amino acid content of the therapeutic protein.EXAMPLE 3

[0074] This example describes a second attempt at determining amino acid content of a therapeutic protein to identify the hydroxylated amino acid PTM.

[0075] A second attempt at identifying the hydroxylated amino acid PTM of TP1 was made by carrying out a modified version of the procedure described in Example 2. The modified version additionally included incubating the hydrolyzed solution with activated charcoal and then centrifuging to create a supernatant and an activated charcoal pellet. A summary of the modified version is provided in Table 2, and the differences relative to Table 1 are underlined.TABLE 2

[0076] Figure 3 is a representative photo of the samples obtained from the procedure of Table 2. Standard curve samples ranging from 0 to 1.0 pg / mL standard hydroxyproline were run in duplicate in Columns 1 and 1, and the color spectrum of the standard curve samples varied in orange colors. The wells in Columns 3 and 4 comprised test samples and blank samples, and as shown in Figure 3, the colors of the blank samples and the test samples (Columns 3 and 4) were outside the color spectrum of the standard curve samples. It was notable however that none of the samples appeared dark brown like those pictured in Figure 2. Based on the visual appearance of the test samples, it was deemed highly unlikely that meaningful results would be obtained upon reading the absorbance of these samples. Thus, the concentration of hydroxyproline of the test samples was not determined, and the identity of the hydroxylated PTM of TPl remained unconfirmed.

[0077] This example demonstrated that the procedure outlined in Table 2 was unsuccessful at determining the amino acid content of the therapeutic protein.EXAMPLE 4

[0078] This example describes a third attempt at determining amino acid content of a therapeutic protein to identify the hydroxylated amino acid PTM.

[0079] In a third attempt, the same procedure outlined in Table 2 was carried out, except that a hydroxyproline-targeting ELISA was performed in place of the chemical-based colorimetric assay performed in Examples 2 and 3. The Hydroxyproline (HYP) ELISA Kit (Catalog No. 194648, US Biological, Salem, Massachusetts) was used according to manufacturer's instructions. Briefly, samples and HYP-horseradish peroxidase (HRP) conjugates were loaded to a microtiter plate coated with a monoclonal anti-HYP antibody. Following incubation and subsequent washing, a substrate for the HRP enzyme was added to the sample. A quench solution was added to stop the HRP reaction, resulting in a yellow solution that absorbs at 450 nm. A standard curve correlating absorbance at 450 nm and HYP concentration was created using the standard curve samples (comprising 0, 0.5, 1, 2.5., 5.0, and 10 pg / mL HYP) included in the assay kit.

[0080] Figure 4A is a representative photo of the standard curve samples (Wells A-F and H) and a test sample (Well G). As shown in Figure 4A, the standard curve samples ranged in the intensity ofyellow color, while the color of the test sample appeared outside the standard curve color spectrum. The absorbance at 450 nm of the test sample was a negative value, and thus the ELISA was repeated.

[0081] The ELISA was repeated using an increased amount of TP1 (1.5 mg) and a decreased amount of TP1 (0.75 mg), relative to the amount of test sample of the first ELISA (1.0 mg). Figure 4B is a representative photo of the standard curve samples run in duplicate (Wells 1A-1F and 2A-2F), test samples (Wells 3A, 3B, 3C, and 3D), blank samples (Wells 1G, 1H, 2G, and 2H) and negative control samples (3E, 3F, 3G, 3H) wherein a protein (0.75 mg or 1.5 mg) known to not have any hydroxyproline was processed through the procedure of Table 2. As shown in Figure 4B, the visual appearance of the test samples was similar to that of the blank samples and the negative controls and all of the test samples were outside the standard curve color spectrum. As expected, the absorbance values of the test samples, blank samples, and negative control samples were outside the range of absorbance of the standard curve samples.

[0082] This example demonstrated that the procedure outlined in Table 2 using an ELISA was unsuccessful at determining the amino acid content of the therapeutic protein.EXAMPLE 5

[0083] This example describes a fourth approach for determining amino acid content of a therapeutic protein to identify the hydroxylated amino acid PTM of TP1.

[0084] In previous attempts to identify the hydroxylated amino acid PTM of TP1, a colorimetricbased assay was used but failed to identify the hydroxylated amino acid PTM of TP1. In this fourth approach, a more sensitive analytic technique was carried out in place of the colorimetric-based assay. A summary of this procedure is provided in TABLE 3, wherein the italicized aspects are unique to this procedure relative to the procedure of Table 2. Following resuspension of the dried sample comprising amino acids of TP1, a label, 6-aminoquibolyl-N-hydroxysuccinimidyl carbamate (AQC), which reacts with the primary and secondary amines of the amino acids of TP1, was added to form stable urea-type derivatives. These AQC-amino acid derivatized samples were subsequently separated using reversed-phase liquid chromatography (RP-LC) in which the least hydrophobic derivatized amino acids elute at an earlier time compared to when more hydrophobic derivatized amino acids elute. The separation was monitored using UV absorbance at 260 nm, the wavelength at which the AQC-amino acid derivatized samples absorbs UV radiation, allowing for pico-mole (pmol) sensitivity.TABLE 3

[0085] Table 4 lists the samples and controls used in this experiment. Each sample was injected on an HPLC to achieve separation of the individual amino acids of the sample. The amino acids were then identified by retention time using a control amino acid profile provided by the kit manufacturer.TABLE 4

[0086] Figure 5 is an overlay of chromatograms obtained upon injecting the samples: Pro (blue, middle line), HYP (black, lower line), and Pro + HYP (green, upper line). As shown in this figure, HYP (retention time of about 1.50) is separated from Pro (retention time of around 5.4). Figure 5 demonstrates that this analytic technique is sensitive enough to resolve hydroxyproline vs proline species. Figure 6A is an overlay of spectra of Test Sample (black, lower line) and Test Sample + HYP (blue, upper line) and Figure 6B is a zoomed view of the boxed chromatograms of Figure 6A. As shown in Figure 6B, the hydroxyproline present in the Test Sample + HYP (blue, upper line) has a retention time of about 1.5 min. A small peak at this retention time is observed in the Test Sample chromatogram (black). Figure 6C is an overlay of spectra of Test Sample (black, lower line) and Negative Control (blue, upper line) and Figure 6D is a zoomed view of the boxed chromatograms of Figure 6C. As shown in Figure 6D, a small peak is observed in the Test Sample spectrum (black) at about 1.50 min (the retention time of HYP (Figure 6B) and no peak is shown in the Negative Control (blue line) at this retention time. Figure 6E provides a side-by-side comparison of zoomed views of Figure 6B (HYP (blue, upper line) and Test Sample (black, lower line) and Figure 6D (Negative Control (blue, upper line) and Test Sample (black, lower line). As shown in Figure 6E, the black peak at about 1.50 min is small but is present when compared to the Negative Control at this retention time.Taken together, these results confirm the presence of hydroxyproline on TP1.

[0087] Figure 7 shows the low abundance of hydroxyproline in the Test Samples. The trace of UV absorbance at 260 nm was integrated on two peaks identified as hydroxyproline and proline by elution time comparison to a control profile provided by the kit manufacturer. Hydroxyproline and proline percent peak areas were compared to assess the relative level of modified vs unmodified proline, which showed the level was 0.44% hydroxyproline to 99.56% proline.

[0088] This example demonstrated that the method of Table 3 was sufficient for determining amino acid content of a therapeutic protein to identify a hydroxylated amino acid PTM.EXAMPLE 6

[0089] This example describes a method of determining amino acid content to identify a hydroxylated amino acid PTM for a different therapeutic protein.

[0090] In Examples 1-5, TP1, a molecule comprising an antibody domain fused to an scFV, was used. In this example, a different therapeutic protein referred to herein as "TP2" was used. Similar to TP1, TP2 comprised half an antibody fused to an scFv, but differed from TP1 in that the binding site of the antibody half had sequence differences. The procedure summarized in Table 3 were carried out with TP2. In Table 3, (1), a solution comprising 30 mg / mL TP2 in formulation buffer was exchanged into the second solution comprising 25 mM sodium acetate, pH 5.2. The same samples and controls described in Table 4 were used in this experiment, except that the Test Samples comprised TP2 instead of TP1. As in Example 5, each sample was injected on an HPLC to achieve separation of the individual amino acids of the sample. The amino acids were then identified by retention time using a control amino acid profile provided by the kit manufacturer.

[0091] Exemplary chromatograms are provided in Figures 8A and 8B. Figure 8A is an overlay of chromatograms for Negative Control (blue line) and Negative Control + HYP. As shown in this figure, hydroxyproline has a retention time of about 1.75 min. Figure 8B is an overlay of chromatograms of Test Sample + HYP (black line) and Test Sample (without HYP; blue line). As shown in Figure 8B, the small peak of the blue spectra at around 1.75 represents hydroxyproline confirming that the Test Sample of TP2 comprise hydroxyproline.

[0092] This example demonstrated that the method of Table 3 was sufficient for determining amino acid content of another therapeutic protein to identify a hydroxylated amino acid PTM.EXAMPLE 7

[0093] This example describes another exemplary method of determining amino acid content of a therapeutic protein to identify a hydroxylated amino acid PTM.

[0094] In Examples 5 and 6, detection of the amino acids of the therapeutic protein was carried out by labeling the amino acids and detecting them with a UV detector attached to the HPLC. In an alternative method, a different mode of detection can be used. Table 5 describes a method in which detection of the amino acids is carried out via mass spectrometry. Differences between the methods of Examples 5 and 6 are italicized in Table 5. Note that in Table 5, amino acids are not labeled before chromatographic separation.TABLE 5

[0095] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0096] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms including the indicated component(s) but not excluding other elements (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0097] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0098] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, orexemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0099] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein.Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A method for analyzing the amino acid content of a therapeutic protein, comprising: a. exchanging by diafiltration the therapeutic protein into a second solution from a first solution to obtain an exchanged protein solution; b. incubating the exchanged protein solution at an acidic pH and a temperature greater than about 110 °C to obtain a hydrolyzed sample comprising amino acids of the therapeutic protein; c. combining the hydrolyzed sample with activated charcoal; d. separating the hydrolyzed sample from the activated charcoal by (i) centrifugation, (ii) filtration, or (iii) both centrifugation and filtration; and e. analyzing the amino acid content of the hydrolyzed sample.

2. The method of claim 1, wherein the first solution comprises less than 5 different proteins.

3. The method of claim 2, wherein the therapeutic protein is the only protein present in the first solution.

4. The method of any one of the preceding claims, wherein the first solution has a bioburden less than 100 CFU / mL, such as less than 10 CFU / mL.

5. The method of any one of the preceding claims, wherein the first solution is a liquid drug product or a liquid drug substance.

6. The method of any one of claims 1 to 5, wherein the first solution comprises one or more excipients selected from the group of: a salt, a surfactant, sugar and / or a polyol.

7. The method of any one of the preceding claims, wherein the first solution comprises a buffer and a sugar.

8. The method of any one of claims 1 to 7, wherein the second solution has the same pH as the first solution.

9. The method of any one of claims 1 to 8, wherein the second solution lacks sugar.

10. The method of any one of claims 1 to 9, wherein the diafiltration is performed with at least 4 diavolumes of the second solution.

11. The method of any one of the preceding claims, wherein (b) comprises combining the exchanged protein solution with an acid.

12. The method of claim 11, wherein the acid is hydrochloric acid.

13. The method of claim 12, wherein the hydrochloric acid is 12 N hydrochloric acid.

14. The method of any one of the preceding claims, wherein the acidic pH is less than about 4.2.

15. The method of claim 14, wherein the acidic pH is less than about 3.5.

16. The method of any one of the preceding claims, wherein the exchanged protein solution is incubated at about 115 °C to about 130 °C.

17. The method of any one of the preceding claims, wherein the exchanged protein solution is incubated at about 120 °C.

18. The method of any one of the preceding claims, wherein the exchanged protein solution is incubated at the acidic pH and the temperature greater than about 110°C for at least 1 or 2 hours.

19. The method of claim 18, wherein the exchanged protein solution is incubated at the acidic pH and the temperature greater than about 110°C for about 3 hours.

20. The method of any one of the preceding claims, wherein about 2.5 mg to about 5 mg activated charcoal is combined with the hydrolyzed sample for every 1 mg therapeutic protein in the first solution.

21. The method of any one of the preceding claims, wherein the centrifugation is performed at about 13,000 x g to about 14,000 x g for about 3 min to about 5 min.

22. The method of any one of the preceding claims, wherein the filtration is performed with a 0.22 pm filter.

23. The method of any one of the preceding claims, comprising separating the hydrolyzed sample from the activated charcoal by (i) centrifuging to obtain an activated charcoal pellet and a supernatant and (ii) passing the supernatant through a filter.

24. The method of any one of the preceding claims, wherein the amino acid content is analyzed by an analytical technique having a limit of detection (LOD) in the nanomolar or picomolar range.

25. The method of any one of the preceding claims, wherein said analyzing comprises detecting an amino acid having a concentration in the nanomolar or picomolar range.

26. The method of any one of the preceding claims, wherein the method can detect an amino acid which constitutes less than 0.1% of the amino acids of the therapeutic protein.

27. The method of any one of the preceding claims, wherein the analysis of the amino acid content does not comprise a colorimetric assay.

28. The method of any one of the preceding claims, comprising chromatographically separating the amino acids of the therapeutic protein after the hydrolyzed sample is separated from the activated charcoal.

29. The method of claim 28, wherein the amino acids of the therapeutic protein are chromatographically separating by a high-performance liquid chromatography (HPLC), optionally, a reversed phase-HPLC (RP-HPLC).

30. The method of claim 28 or 29, comprising a hydrophilic interaction liquid chromatography (HILIC).

31. The method of any one of claims 28-30, further comprising labeling the amino acids of the therapeutic protein with a detectable label after the hydrolyzed sample is separated from the activated charcoal and before chromatographically separating the amino acids of the therapeutic protein.

32. The method of claim 31, wherein the detectable label is an ultraviolet (UV) absorbing label.

33. The method of claim 32, wherein the label is 6-Aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC).

34. The method of any one of claims 29-33, wherein the HPLC is run with a transmitted UV (TUV) detector or photo diode array (PDA) detector.

35. The method of any one of claims 28-34, comprising analyzing the amino acid content through mass spectrometry.

36. The method of any one of the preceding claims, further comprising, after (d) and before (e), concentrating the hydrolyzed sample.

37. The method of claim 36, comprising concentrating the hydrolyzed sample, labeling the amino acids of the therapeutic protein with a detectable label, and chromatographically separating the amino acids of the therapeutic protein.

38. The method of claim 36, comprising concentrating the hydrolyzed sample, chromatographically separating the amino acids of the therapeutic protein, and performing mass spectrometry on the separated amino acids.

39. The method of any one of the preceding claims, wherein (e) comprises determining the presence or absence of a modified or isomerized amino acid, such as hydroxyproline.

40. The method of any one of the preceding claims, wherein the therapeutic protein comprises one or more antibody domains.

41. The method of any one of the preceding claims, wherein the therapeutic protein comprises one or more antibody heavy chains.

42. The method of any one of the preceding claims, wherein the therapeutic protein comprises an Fc domain.

43. The method of any one of the preceding claims, wherein the therapeutic protein comprises a Fab fragment.

44. The method of any one of the preceding claims, wherein the therapeutic protein is an antibody.

45. The method of any one of claims 1 to 44, wherein the therapeutic protein comprises a single chain variable (scFv) domain.

46. The method of any one of claims 1 to 45, wherein the therapeutic protein is a bispecific antibody.

47. A method of detecting the presence or absence of a post translational modification (PTM) in a therapeutic protein, comprising analyzing the amino acid content of a therapeutic protein in accordance with any one of the preceding claims.

48. A method of detecting structural changes of a therapeutic protein in a drug product, comprising analyzing the amino acid content of a therapeutic protein in accordance with any one of the preceding claims.

Images