System and method for online detection of post-translational modifications of polypeptides

Abstract

Provided herein are methods and systems for monitoring post-translational modifications and glycosylation of polypeptides, in particular online (i.e., automated) methods for monitoring the post-translational modifications of recombinant proteins produced in cell culture in real time. Methods and systems for detecting and quantifying post-translational modifications of proteins include flow injection analysis (FIA), affinity columns, spectrometry, digestion, and peptide mapping.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 200,576, filed on Mar. 16, 2021, under 35 U.S.C. § 119(e), the entire disclosure of which is hereby incorporated by reference for all purposes.

[0002] This disclosure relates to methods for detecting post - translational modifications of polypeptides, particularly to an online method for real - time detection of post - translational modifications of recombinant proteins produced in cell culture.

Background Art

[0003] Biopharmaceuticals, including therapeutic proteins such as fusion proteins, growth factors, cytokines, enzymes, hormones, and monoclonal antibodies (mAbs), represent one of the fastest - growing areas in the pharmaceutical industry. Typically, biopharmaceuticals are recombinantly produced in cell culture using genetically engineered host cells, including microbial systems such as Escherichia coli or Saccharomyces cerevisiae, fungal systems such as Aspergillus, and mammalian systems such as Chinese hamster ovary (CHO), NS0, or Sp2 / 0 host cells. Mammalian systems are often preferred because many biopharmaceuticals are too large and complex to be produced by microorganisms or require post - translational modifications (PTMs) that are only possible in mammalian host cells. However, PTMs can affect the physicochemical characteristics of biomolecules, thereby potentially affecting activity, protein - protein interactions, biological functions, efficacy, and stability, resulting in various levels of protein heterogeneity. Therefore, PTMs are important quality attributes (CQAs) that are characterized, controlled, and monitored during the process development and manufacture of biopharmaceuticals.

[0004] Common PTMs include glycosylation, deamidation, isomerization, and oxidation, incomplete disulfide bond formation, glycation, N-terminal glutamine cyclization, and C-terminal lysine processing. Of these modifications, the most common and spontaneously occurring are phosphorylation, acetylation, glycosylation (O- and N-linked), deamidation, oxidation, hydroxylation, methylation, ubiquitination, pyroglutamate, sulfation, and cleavage. Non-patent document 1.

[0005] Glycosylation is a common PTM (Protein Therapeutic Modulation) involving the attachment of glycans to specific sites on proteins, such as asparagine residues (N-linked glycosylation) or serine (Ser) or threonine (Thr) residues (O-linked glycosylation). N-linked glycosylation occurs at the consensus sequence (Asn-X-Ser / Thr, where X is any amino acid except proline) and is initiated in the endoplasmic reticulum (ER), where a glycan chain (Glc3Man9GlcNAc2) is attached to the Asn residue of the consensus sequence. After the glycan chain is trimmed by various glycoside hydroxylase enzymes, the glycoprotein is transported to the Golgi apparatus, where the glycan undergoes further trimming and modification, resulting in various N-glycan structures. O-linked glycans typically attach to serine (Ser) or threonine (Thr) residues via an α-O-glycosidic bond formed between N-acetylgalactosamine (GalNAc) and the hydroxyl group (-OH) of a Ser or Thr residue. O-glycan biosynthesis is initiated in the Golgi apparatus by the transfer of GalNAc from UDP-GalNAc to a Ser or Thr residue. O-GalNAc precursors are then processed to produce various O-GalNAc glycans. (Non-patent document 2.)

[0006] Conventional processes for analyzing PTMs (Post-Tissue Manufacturing Models) involve manually collecting samples at the end of cell culture, sending them to a laboratory for concentration, purification, and preparation for analysis. These conventional processes can take 3-5 days, potentially resulting in development and manufacturing delays and increased costs. Since analysis is typically performed at the end of the process, the results reflect the overall outcome of the process. Furthermore, if the product does not meet the required specifications, the entire process must be repeated, starting from cell culture. Therefore, there is a need for analytical methods that can be performed in real time to characterize the product during the manufacturing process, allowing for adjustments to manufacturing parameters to maintain the product within the desired specifications.

[0007] Non-patent document 3 describes a method for real-time monitoring of glycosylation using a microsequential injection system coupled with an ultra-high-performance liquid chromatography (UPLC) system as a sample preparation platform for real-time monitoring of antibody-glycan profiles. For glycan analysis, glycans were enzymatically released from the antibody using PNGase F (P0704, New England BioLabs, Ipswich, MA) and separated using a UPLC column.

[0008] Improved processes are still needed to monitor post-translational modifications (PTMs) of recombinant proteins in real time. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Virag et al. (2020) “Current Trends in the Analysis of Post-translational Modifications,” Chromatographia.83:1-10(doi.org / 10.1007 / s10337-019-03796-9) [Non-Patent Document 2] Zhang et al. (2016) “Glycan analysis of therapeutic glycoproteins,” MAbs.8(2):205-215 [Non-Patent Document 3] Tharmalingham et al. (2015) “A Framework for Real-Time Glycosylation Monitoring (RT-GM) in Mammalian Cell Culture,” Biotech.Bioeng.112(6):1146-1154 [Overview of the project] [Means for solving the problem]

[0010] The present invention relates to methods and systems for detecting, identifying, or monitoring post-translational modifications (PTMs) of recombinant proteins, and more particularly to online methods or systems for real-time monitoring of PTMs of recombinant proteins in cell culture.

[0011] In one embodiment, a method is provided for detecting post-translational modifications of proteins, the method being performed by a flow injection analyzer (FIA) controlled by at least one processor. In one embodiment, the FIA ​​is a sequential injection analyzer (SIA). In one embodiment, the FIA ​​is a direct injection analyzer (DIA). In one embodiment, the method is (a) Contain the first sample containing protein within the holding reservoir; (b) Delivering a first volume of binding buffer to the affinity column in the FIA ​​via the FIA; (c) Transferring the first sample from the holding reservoir to the affinity column via FIA; (d) Delivering a first volume of elution buffer to the affinity column via FIA, thereby eluting the protein in the first sample from the affinity column to form a first eluate; (e) Transferring the first eluate to the protein concentration detection unit via FIA; (f) Determining the concentration of the protein in the first eluate by at least one processor; (g) Contain the second sample containing protein within the holding reservoir; (h) Delivering a second volume of binding buffer to the affinity column via FIA; (i) Transferring the second sample from the holding reservoir to the affinity column via FIA; (j) Delivering a second volume of elution buffer to the affinity column via FIA, thereby eluting proteins from the second sample from the affinity column to form a second eluate; (k) Transferring the second eluate to the digestion chamber via FIA; (l) Delivering digestion reagents via FIA and digesting proteins in the second eluate to form a peptide mixture; and (m) The process includes transferring a peptide mixture to a peptide mapping unit via FIA and detecting post-translational modifications of the protein.

[0012] In one embodiment, the first and second samples are obtained from a cell culture. In one embodiment, the cell culture is located in a bioreactor. In one embodiment, the bioreactor is a batch bioreactor, a fed-boil bioreactor, or a perfusion bioreactor.

[0013] In one embodiment, the affinity column includes a protein A column.

[0014] In one embodiment, the concentration of the protein in the first eluate of (f) is determined by at least one processor according to a predetermined calibration curve.

[0015] In one embodiment, the elution time of proteins in a first sample from an affinity column for forming a first eluate is controlled by at least one processor based on a predetermined elution time. In one embodiment, the elution time of proteins in a second sample from an affinity column for forming a second eluate is controlled by at least one processor based on a predetermined elution time. In one embodiment, the predetermined elution times of proteins in the first and second samples are the same. In one embodiment, the predetermined elution times of proteins in the first and second samples are determined experimentally. In one embodiment, the predetermined elution times of proteins in the first and second samples are determined experimentally before performing the online method described herein.

[0016] In one embodiment, the protein concentration detection unit includes a spectrophotometer. In one embodiment, the spectrophotometer measures the UV absorbance of a first eluate and calculates the protein concentration based on a predetermined calibration curve by at least one processor. In one embodiment, the calibration curve is determined experimentally. In one embodiment, the calibration curve is determined experimentally before performing the online method described herein. In one embodiment, the spectrophotometer measures the UV absorbance of a first eluate at 280 nm and calculates the protein concentration based on a calibration curve by at least one processor. In one embodiment, the spectrophotometer measures the UV absorbance of a first eluate at 205 nm and calculates the protein concentration based on a calibration curve by at least one processor.

[0017] In one aspect, the protein in the second eluate is enzymatically digested in (l). In one aspect, the enzymatic digestion of the protein involves contacting the second eluate with a proteolytic enzyme selected from trypsin, chymotrypsin, pepsin, thermolysin, papain, pronase, endopeptidase Arg-C, peptidyl-Asp metalloendopeptidase (endopeptidase Asp-N), glutamyl endopeptidase (Glu-C endopeptidase), and lysyl endopeptidase (Lys-C endopeptidase) via FIA. In one aspect, the proteolytic enzyme includes trypsin.

[0018] In one aspect, the proteolytic enzyme includes thermostable trypsin. In one aspect, the method (i) introducing a digestion reagent into the second eluate via FIA to form a digestion mixture; (ii) mixing the digestion mixture; and (iii) incubating the digestion mixture.

[0019] In one aspect, the digestion reagent includes a digestion buffer, a reducing agent, and a proteolytic enzyme. In one aspect, the digestion buffer has a pH of about 6.0 to about 7.5. In one aspect, the reducing agent includes tris(2-carboxyethyl)phosphine (TCEP). In one aspect, the second eluate is contacted with an amount of proteolytic enzyme determined by at least one processor based on the concentration of the first eluate. In one aspect, the proteolytic enzyme includes trypsin. In one aspect, the proteolytic enzyme includes thermostable trypsin. In one aspect, the second eluate is contacted with the proteolytic enzyme at an enzyme:protein ratio of about 1:10 to about 1:100. In one aspect, the second eluate is contacted with the proteolytic enzyme at an enzyme:protein ratio of about 1:20 to about 1:30. In one aspect, the second eluate is contacted with trypsin at a trypsin:protein ratio of about 1:10 to about 1:100. In one aspect, the second eluate is contacted with trypsin at a trypsin:protein ratio of about 1:20 to about 1:30.

[0020] In one aspect, incubating the digestion mixture includes incubating at a temperature of about 36°C to about 85°C for about 5 minutes to about 30 minutes. In one aspect, incubating the digestion mixture includes incubating at a temperature of about 50°C to about 75°C for about 15 minutes to about 30 minutes. In one aspect, incubating the digestion mixture includes incubating at a temperature of about 70°C to about 72°C for about 15 minutes to about 30 minutes.

[0021] In one aspect, the proteolytic enzyme includes thermostable trypsin, and the method (i) introducing a first amount of digestion buffer containing a digestion buffer, a reducing agent, and thermostable trypsin into a second eluate via FIA and mixing to form a first digestion mixture; (ii) incubating the first digestion mixture at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a first incubated mixture; (iii) introducing a second amount of thermostable trypsin into the incubated mixture via FIA and mixing to form a second digestion mixture; and (iv) incubating the second digestion mixture at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a peptide mixture.

[0022] In one aspect, a second amount of digestion buffer is added to the first incubated mixture via FIA together with the second amount of thermostable trypsin in (iii).

[0023] In one aspect, the peptide mapping unit includes a mass spectrometer (MS). In one aspect, detecting post-translational modifications includes peptide mapping analysis performed using mass spectrometry (MS). In one aspect, detecting post-translational modifications includes peptide mapping analysis performed using liquid chromatography / mass spectrometry (LC / MS). In one aspect, the liquid chromatography includes ultra-high performance liquid chromatography (UPLC).

[0024] In one embodiment, this method is carried out online.

[0025] In one embodiment, post-translational modifications include glycosylation, oxidation, deamidation, isomerization, saccharification, N-terminal leader sequence, N-terminal cyclization, C-terminal lysine retention, amide formation, or combinations thereof. In one embodiment, glycosylation includes N-linked glycosylation, O-linked glycosylation, or combinations thereof.

[0026] In one embodiment, a method for producing recombinant protein is provided. In one embodiment, this method is (a) Culturing host cells under conditions in which recombinant proteins are expressed; (b) detecting post-translational modifications of proteins in accordance with the methods described herein; (c)(b) comprising modifying one or more cell culture parameters based on the post-translational modifications detected in (c)(b).

[0027] In one embodiment, the host cells are cultured in a bioreactor which is a batch bioreactor, a fed-boil bioreactor, or a perfusion bioreactor. In one embodiment, the host cells are mammalian host cells. In one embodiment, the host cells are CHO, HEK 293, COS, NS0, SP2, or PER.C6 host cells.

[0028] In one embodiment, one or more of the following cell culture parameters: pH; CO2 level; dissolved oxygen (dO2); temperature; amount or type of nutrients; presence and type of glycan precursors; or combinations thereof are modified based on post-translational modifications detected using the method described herein.

[0029] In one embodiment, the protein contains at least 1000 amino acid residues. In one embodiment, the protein is recombinantly produced. In one embodiment, the protein contains a glycoprotein. In one embodiment, the protein contains a therapeutic protein. In one embodiment, the protein contains a therapeutic antibody or its antigen-binding fragment. In one embodiment, the protein contains a fusion protein.

[0030] In one embodiment, a recombinant protein produced by the method described herein is provided. In another embodiment, a pharmaceutical composition is provided comprising the recombinant protein produced by the method described herein and a pharmaceutically acceptable carrier.

[0031] In one embodiment, an online method for monitoring post-translational modifications of a protein is provided. In one embodiment, the method is performed using a flow injection analyzer (FIA). In one embodiment, the FIA ​​is a sequential injection analyzer (SIA). In one embodiment, the FIA ​​is a direct injection analyzer (DIA). In one embodiment, the FIA ​​includes a bidirectional pump controlled by at least one processor. In one embodiment, the FIA ​​includes a syringe pump and a multiport valve controlled by at least one processor. In one embodiment, the FIA ​​includes two or more syringe pumps and two or more multiport valves. In one embodiment, the FIA ​​includes two syringe pumps and two multiport valves.

[0032] In one aspect, this method is (a) Contain the first sample containing the protein in the FIA's retention reservoir; (b) The operation of a multiport valve to introduce a first volume of binding buffer into the affinity column in the FIA; (c) The syringe pump operates to advance the first sample from the holding reservoir to the affinity column, thereby binding the proteins in the first sample to the affinity column; (d) The operation of the multiport valve introduces a first volume of elution buffer into the affinity column, eluting the bound protein to form a first eluate; (e) The syringe pump is used to advance the first eluate into the spectrophotometer in the FIA; (f) Determining the concentration of the protein in the first eluate according to the measured UV absorbance by at least one processor; (g) Contain the second sample containing protein within the holding reservoir; (h) The operation of the multiport valve introduces a second volume of binding buffer into the affinity column; (i) The syringe pump operates to advance the second sample from the holding reservoir to the affinity column, thereby binding the proteins in the second sample to the affinity column; (j) The operation of the multiport valve introduces a second volume of elution buffer into the affinity column, thereby eluting the bound protein and forming a second eluate; (k) The syringe pump is used to advance the second eluate into the digestion chamber within the FIA; and (l) The operation of the multiport valve introduces the digestive reagent into the second eluate in the digestion chamber, digesting the protein to form a peptide mixture; and (m) The operation of a syringe pump to advance the peptide mixture to a peptide mapping unit and to detect post-translational modifications of the protein.

[0033] In one aspect, digesting protein is (i) By the operation of the multiport valve, the digestion buffer, reducing agent, and proteolytic enzyme are introduced into the second eluate in the digestion chamber and mixed to form the first digestion mixture; (ii) Incubating the first digestion mixture in the digestion chamber to form the first incubation mixture; (iii) By the operation of a multiport valve, a second volume of digestion buffer and proteolytic enzyme is introduced into the incubation mixture to form a second digestion mixture; and (iv) Incubating a second digestion mixture in a digestion chamber to form a peptide mixture.

[0034] In one embodiment, the concentration of the protein in the first eluate of (f) is determined by at least one processor according to a predetermined calibration curve. In one embodiment, based on a predetermined elution time, the protein in the first sample is eluted from the affinity column to form the first eluate. In one embodiment, based on a predetermined elution time, the protein in the second sample is eluted from the affinity column to form the second eluate.

[0035] In one embodiment, the digestion buffer has a pH of about 6.0 to about 7.5. In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). In one embodiment, the amount of protease introduced into the digestion chamber is determined by at least one processor based on the concentration of the first eluate (already determined). In one embodiment, the protease includes trypsin. In one embodiment, the trypsin includes heat-stable trypsin. In one embodiment, the second eluate is brought into contact with trypsin at a trypsin:protein ratio of about 1:10 to about 1:100. In one embodiment, the second eluate is brought into contact with trypsin at a trypsin:protein ratio of about 1:20 to about 1:30.

[0036] In one embodiment, the first digestion mixture is incubated at a temperature of approximately 36°C to approximately 85°C for approximately 5 minutes to approximately 30 minutes. In one embodiment, the first digestion mixture is incubated at a temperature of approximately 50°C to approximately 75°C for approximately 15 minutes to approximately 30 minutes. In one embodiment, the first digestion mixture is incubated at a temperature of approximately 70°C to approximately 72°C for approximately 15 minutes to approximately 30 minutes.

[0037] In one embodiment, the second digestion mixture is incubated at a temperature of approximately 36°C to approximately 85°C for approximately 5 minutes to approximately 30 minutes. In one embodiment, the second digestion mixture is incubated at a temperature of approximately 50°C to approximately 75°C for approximately 15 minutes to approximately 30 minutes. In one embodiment, the second digestion mixture is incubated at a temperature of approximately 70°C to approximately 72°C for approximately 15 minutes to approximately 30 minutes.

[0038] In one embodiment, the protease includes heat-stable trypsin, and this method (i) By operating a multiport valve, a first volume of digestion buffer, reducing agent, and heat-stable trypsin is introduced into a second eluate and mixed to form a first digestion mixture; (ii) Incubate the first digestion mixture at a temperature of approximately 70°C to 75°C for approximately 10 to 20 minutes to form an incubated mixture; (iii) By the operation of a multiport valve, a second amount of digestion buffer and thermostable trypsin is introduced into the incubation mixture and mixed to form a second digestion mixture; and (iv) Incubating the second digestion mixture at a temperature of approximately 70°C to approximately 75°C for approximately 10 to 20 minutes to form a peptide mixture.

[0039] In one embodiment, the protein contains at least about 1000 amino acid residues.

[0040] In one embodiment, the peptide mixture includes peptides containing fewer than approximately 100 amino acids.

[0041] In one embodiment, an online system is provided for monitoring post-translational modifications of a target protein. In one embodiment, the system is: (a) A holding reservoir configured to contain a first sample and a second sample, each containing the target protein; (b) A first syringe pump configured to advance the first and second samples from a holding reservoir through a flow injection analyzer (FIA); (c) A first multi-port valve in fluid communication with a first syringe pump; (d) An affinity column configured to be in fluid communication with a first multiport valve and to contain the first and second samples from a holding reservoir; (e) A spectrophotometer in a FIA configured to contain a first eluate from an affinity column, wherein the first eluate contains protein from a first sample, and the spectrophotometer is configured to determine UV absorbance and calculate the protein concentration of the first sample based on a predetermined calibration curve; (f) A second syringe pump configured to advance a second eluate from an affinity column into a digestion chamber, wherein the second eluate contains proteins from a second sample, and the proteins in the second eluate are digested based on the protein concentration of the first sample determined in (e); and (g) A second injection multiport valve in fluid communication with a second syringe pump, comprising: [Brief explanation of the drawing]

[0042] [Figure 1] This is a schematic diagram of a system configured to provide an automated process implementation consistent with the method described herein. [Figure 2] This is a schematic diagram of a computer system configured to provide process control of an automated method consistent with the method described herein. [Figure 3] This is a schematic diagram of a first syringe pump and a first valve system for carrying out the online automated method described herein. [Figure 4] This is a schematic diagram of a second syringe pump and a second valve system for carrying out the online automated method described herein. [Figure 5] This is a schematic diagram of the online automated method described herein. [Figure 6] This is a schematic diagram of the alternative method described herein. [Figure 7] This is a further schematic illustration of the online automated method described herein. [Figure 8] This is a flowchart of the peptide mapping process described herein. [Figure 9A]This graph shows the calibration curve and UV determination of polypeptides using the protein A capture process. [Figure 9B] This is the UV profile used to determine the concentration of the first eluate, and it shows that the same amount of protein was captured in the second eluate, which was later used for protein digestion. [Figure 10A] This graph shows the correlation between the glycosylation percentage of sample IgG1 monoclonal antibodies determined using the online peptide mapping method described herein and that determined using the glycan release (Oligo 2-AB) method. [Figure 10B] This graph shows the quantified percentage of mannose monitored over time (days 7, 10, and 14) during cell culture using the online peptide mapping method described herein. Repeated experiments at each time point yield a consistent level of quantification. [Figure 11A] This graph shows a direct linear relationship between GF detection using online peptide mapping and GF detection using glycan release (oligo2-AB) method. [Figure 11B] This graph shows a direct linear relationship between G1F detection using online peptide mapping and G1F detection using glycan release (oligo2-AB) method. [Figure 11C] This graph shows a direct linear relationship between Man5 detection using online peptide mapping and Man5 detection using glycan release (oligo2-AB) method. [Figure 11D] This graph shows a direct linear relationship between G0F-GN detection using online peptide mapping and G0F-GN detection using glycan release (oligo2-AB) method. [Figure 12A] This graph shows the oxidation levels of specific methionine residues in cell culture over time, as determined using the online peptide mapping method described herein. [Figure 12B]This graph shows the level of N-terminal cyclization over time in cell culture, determined using the online peptide mapping method described herein. [Modes for carrying out the invention]

[0043] The present invention relates to methods and systems for detecting, identifying, or monitoring post-translational modifications (PTMs) of recombinant polypeptides, and more particularly to online methods or systems for real-time monitoring of PTMs of recombinant proteins in cell culture.

[0044] A.Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. For example, unless otherwise specified, “a” or “an” shall include plural forms, e.g., “one or more” or “at least one,” and the term “or” may mean “and / or.” The terms “including,” “includes,” and “included” are not limiting. Any type of range provided herein includes all values ​​within the specified range and values ​​at the endpoints of the specified range.

[0045] As used herein, the term “about” is used to modify, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, and ranges thereof of components in a composition, as used in the description of the present invention. The term “about” refers to the variation in numerical quantities that may occur, for example, due to typical measurement and handling procedures used in the manufacture of a compound, composition, concentrate, or formulation; due to accidental errors in these procedures; or due to differences in the manufacture, source, or purity and other similar requirements of the starting materials or components used to carry out the method. The term “about” also encompasses different quantities resulting from changes over time in a formulation having a particular initial concentration or initial mixture, and different quantities resulting from mixing or processing of a formulation having a particular initial concentration or initial mixture. Where modified by the term “about,” the claims appended herein include such equivalents.

[0046] Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry, and hybridization described herein are well known and commonly used in the art. In this specification, amino acids may be referred to by either the commonly known three-letter code or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by the commonly accepted one-letter code.

[0047] As used herein, the term “polypeptide” refers to a molecule of any length containing two or more amino acid residues joined to one another by peptide bonds. As used herein, the term “peptide” refers to a polypeptide containing about 2 to about 50 amino acids joined to one another by peptide bonds. The term “protein” refers to a polypeptide containing more than about 100 amino acid residues, or more than about 1000 amino acid residues, joined to one another by peptide bonds. The sequence of amino acids within a polypeptide is referred to as the primary structure. In one embodiment, a protein has a secondary structure, resulting from the formation of a hydrogen bond between, for example, the oxygen atom of a first amino acid and the nitrogen atom of a second amino acid, which results in a two-dimensional alpha-helix or beta-pleated sheet. In one embodiment, a protein has a three-dimensional tertiary structure, for example, from the formation of one or more interchain disulfide (SS) bonds or other interactions between amino acid R groups (including, but not limited to, hydrogen bonds, ionic bonds, covalent bonds, and hydrophobic interactions). In one embodiment, a protein has a quaternary structure, for example, from the presence of two or more polypeptide chains, such as an antibody. In one embodiment, the peptide does not have a secondary, tertiary, or quaternary structure.

[0048] The term "protein" may refer to antibodies and other non-antibody proteins (including, but not limited to, enzymes, receptors, ligands, secreted proteins, fusion proteins, or fragments thereof). Proteins may be of scientific or commercial interest, and may include, but not limited to, therapeutic proteins. The term "therapeutic protein" refers to a protein that has one or more biological activities useful for treating, preventing, or improving a disorder or disease.

[0049] As used herein, the terms “antibody” and “immunoglobulin” are interchangeable and refer to a group of proteins or polypeptides that include at least one binding domain formed by the folding of a polypeptide chain having a three-dimensional binding space with an inner surface shape and charge distribution complementary to the characteristic features of the antigenic determinant of the antigen. Conventional or naturally occurring antibodies typically have a tetrameric form of two pairs of polypeptide chains, each pair having one “light” chain and one “heavy” chain. The variable region of each light / heavy chain pair forms the antibody binding site. Each light chain is linked to the heavy chain by one covalent disulfide bond, although the number of disulfide bonds differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intrachain disulfide crosslinks. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Light chains are classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. Antibodies may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, e.g., G1m(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)), and may include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments (e.g., bispecific antibodies), CDR-grafted antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, anti-idiotype (anti-Id) antibodies, intracellular antibodies, and their desired antigen-binding fragments, and may also include recombinantly produced antibody fragments.

[0050] The terms “antigen-binding fragment” or “immunologically active fragment” refer to a fragment of an antibody that contains at least one antigen-binding site and retains the ability to specifically bind to an antigen. Examples of antibody fragments that can be recombinantly produced include, but are not limited to, antibody fragments containing variable heavy and light chain domains, such as single-chain Fv(scFv), single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments. Antibody fragments may also include epitope-binding fragments or derivatives of any of the antibodies listed above.

[0051] The term "recombinant" refers to biomolecules, such as nucleic acids or proteins, that have been artificially or synthetically (i.e., unnaturally) modified or produced by human intervention (e.g., using recombinant DNA processes), including, for example, proteins expressed using recombinant expression vectors transfected into host cells.

[0052] The term "cell culture" refers to methods and techniques used to create and maintain a population of host cells capable of producing a target biomolecule, such as a target protein, as well as methods for producing and collecting the target biomolecule. During "cell culture," proteins may be produced intracellularly, in the perimembrane space, or directly secreted into the culture medium. In one embodiment, the cell culture may include mammalian host cells. Suitable culture conditions for mammalian cells are known in the art. See, for example, *Animal cell culture: A Practical Approach*, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian host cells can be cultured in suspension or attached to a solid substrate, with or without microcarriers, in, for example, a fluidized bed bioreactor, a hollow fiber bioreactor, a roller bottle, a shaking flask, or a stirred tank bioreactor.

[0053] "Cell culture medium" or "culture medium" refers to a liquid or gel containing appropriate energy sources and nutrients necessary for the survival and growth of cells during cell culture. Typical cell culture media include amino acids; energy sources, usually in the form of carbohydrates such as glucose; vitamins; free fatty acids; buffers; salts; and trace essential elements. Depending on the requirements of the cells being cultured and / or the desired cell culture parameters, cell culture media may also be supplemented with additional components or components at increased concentrations, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, or trace elements.

[0054] "Acclimatized medium" refers to a culture medium that contains the target biomolecules released or secreted into the cell culture medium by the cells.

[0055] "Clarified cell culture medium" refers to a culture medium from which host cells and / or cell debris have been removed, for example, by centrifugation, microfiltration, tangential flow filtration, alternating tangential flow filtration, and / or depth filtration. In one embodiment, the clarified cell culture medium includes the cell culture supernatant.

[0056] A "bioreactor" refers to a vessel designed to control the growth of cell cultures. The term bioreactor can refer to cell cultures of any scale, from individual flasks and shaking flasks or wave bags to 1-liter bioreactors and up to large-scale industrial bioreactors. In one embodiment, a bioreactor is a small-scale bioreactor, for example, having a volume of about 125 ml to about 500 ml. In another embodiment, a bioreactor is a large-scale bioreactor, for example, having a volume of about 100 liters to about 20,000 liters or more. Various types of bioreactors are known, and they include, but are not limited to, stirred-stank, fixed-bed, fluidized-bed, bubble tower, and air-lift bioreactors. Large-scale or commercial-scale cell cultures (e.g., for the production of therapeutic proteins) can be maintained for several days or even several weeks. During this period, the culture may be supplemented with concentrated feed medium containing nutrients and components such as amino acids that are consumed during the process of cell culture. In one embodiment, the culture medium in the bioreactor is not replenished during the cell culture process in a batch process. In another embodiment, during the cell culture process in a fed-batch bioreactor, the culture medium in the bioreactor is replenished with additional components, such as nutrients or cell culture medium. In another embodiment, the culture medium is continuously added to the bioreactor, and used medium and products are removed throughout the cell culture process in a perfusion bioreactor.

[0057] A “host cell” or “recombinant host cell” is a cell into which an expression vector has been introduced. The term “host cell” refers not only to a specific cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell, as certain modifications may occur in the progeny due to either mutation or environmental influences, but they are still included in the scope of the term “host cell.” Host cells can include prokaryotic and eukaryotic cells. In one embodiment, the host cell is a mammalian host cell, e.g., Chinese hamster ovary cell (CHO), human fetal kidney cell (e.g., HEK293), monkey kidney cell (e.g., COS), mouse myeloma cell (e.g., NS0 or SP2), or human primary fetal retinal cell (PER.C6).

[0058] "Critical Quality Characteristics" (CQAs) are chemical, physical, or biological characteristics of a therapeutic protein that may affect the safety and / or efficacy of the product. CQAs may include product-specific variants, such as size and sequence variants, variants due to aggregation or fragmentation, and charge variants. Other variants include post-translational modifications (PTMs) such as glycosylation, oxidation, deamidation, isomerization, N-terminal processing, and C-terminal processing. CQAs also include process-related impurities, including but not limited to host cell proteins, DNA, leachates, and other raw materials.

[0059] Post-translational modification (PTM) refers to changes in a polypeptide resulting from the covalent addition of a functional group to a polypeptide chain or the cleavage of a peptide bond within a polypeptide chain, including but not limited to glycosylation, oxidation, deamidation, isomerization, glycation, N-terminal leader sequencing, N-terminal cyclization, C-terminal lysine retention, amide formation, or combinations thereof. In one embodiment, glycosylation includes N-linked glycosylation, O-linked glycosylation, or combinations thereof.

[0060] A "glycoprotein" refers to a protein that contains one or more carbohydrate or sugar moieties attached along the entire length of its polypeptide chain. A "glycopeptide" refers to a peptide that contains one or more carbohydrate or sugar moieties attached along the entire length of its polypeptide chain.

[0061] A "glycoform" refers to an isoform of a protein that has a different glycosylation profile (i.e., the number and / or type of attached glycans).

[0062] "Glycan" refers to oligosaccharides attached to polypeptide chains, including N-linked and O-linked glycans. N-linked glycans (also called N-glycans) attach to polypeptide chains via N-glycosidic bonds at the amide nitrogen of asparagine (Asp) or arginine (Arg) residues. N-linked glycosylation occurs in a consensus sequence (Asn-X-Ser / Thr, where X is any amino acid except proline). O-linked glycans (or O-glycans) attach to polypeptide chains via α-O-glycosidic bonds at serine (Ser) or threonine (Thr) residues. There is no general consensus sequence for O-linked glycosylation. Both N-linked and O-linked glycosylation occur in the endoplasmic reticulum (ER) and Golgi apparatus in reaction pathways involving the stepwise addition and / or removal of individual monosaccharides. There are many types of glycans that can be classified according to the presence or absence of various terminal sugars. These include, for example, agalactosylglycans with zero terminal galactoses (e.g., G0, G0-GlcNAc, G0F, G0F-GlcNAc), glycans with one terminal galactose (e.g., G1, G1F, G1F-GlcNAc), and glycans with two terminal galactoses (e.g., G2F, G2F+sialic acid). Some glycosylation patterns can be beneficial to protein function, while others can be detrimental.

[0063] Process Analysis Technology (PAT) refers to systems for designing, analyzing, and controlling manufacturing by measuring critical quality and performance characteristics of raw materials, in-process materials, and processes in a timely manner (e.g., during processing).

[0064] "Online" refers to an analytical process where automated sampling of process solutions is performed without human intervention. "In-line" refers to an analytical process where sensors are placed in the flow of a moving material for analysis. "At-line" refers to a process where the sample is transferred from the process to an analytical instrument physically close to the process, and the analysis results are returned relatively quickly (i.e., within a few hours). "Offline" refers to a process where the sample is removed from the process and analyzed elsewhere, and the analysis results are not returned within a relatively short time (e.g., within a few days). Both offline and at-line analysis require human intervention, such as manually taking the sample from the system, followed by sample preparation and analysis. Online and in-line analysis can be fully automated and performed without human intervention.

[0065] "Real-time" means that the analytical process is carried out without significant time delays, for example, within a sufficiently short time so that the information obtained from the analytical process can be applied to decisions regarding the optimization of process conditions, such as optimizing cell culture conditions in a bioreactor while the cell culture from which the sample was taken is still in progress. In one embodiment, the results of an analytical process including the protein A capture step and protein concentration determination are available within approximately 5 hours, approximately 4 hours, or approximately 3 hours. In another embodiment, the results of the analytical process are available within approximately 2.5 hours. In contrast, with offline processes, the results of the analytical process generally take at least 1 day and up to 2 days.

[0066] "Automated" refers to a process that is carried out without human invention. In one embodiment, an automated process is carried out by one or more devices. In one embodiment, an automated process includes a process in which sample preparation is online and automated. In one embodiment, the online automated sample preparation process is carried out by a fluid injection analyzer (FIA). In one embodiment, the fluid injection analyzer is a sequential injection analyzer (SIA). In one embodiment, the fluid injection analyzer is a direct injection analyzer (DIA). In one embodiment, an automated process includes an automated analyzer, such as an automated peptide mapping unit. The term "automated" does not include offline automated processes, such as processes using liquid handling systems, such as the Hamilton Liquid Handling System (Hamilton Company, Reno, NV, USA) or the TECAN Liquid Handling System (TECAN Group Ltd., Maennedorf, Switzerland).

[0067] "Predetermined" means determined before the start of the process, for example, before the start of the method described herein. For example, "predetermined calibration curve" used in determining protein concentrations refers to a calibration curve created using representative samples with known protein concentrations. For example, a calibration curve can be created by obtaining a sample of known concentration and performing serial dilutions, for example, 10-fold serial dilutions, to obtain about 3 to about 10 samples of known concentrations. In one embodiment, the UV absorbance of the serially diluted samples is then determined together with the UV absorbance of a blank containing all reagents except protein. The calibration curve is created by plotting the UV absorbance against the known protein concentrations of the samples. In one embodiment, "predetermined elution time" is determined by loading a known amount of representative sample onto a column and determining the amount of time required to elute the sample from the column. In one embodiment, elution time refers to the amount of time elapsed between the time the sample is loaded onto the column and the time at which no further sample is eluted from the column, when the elution reagent is applied at a known rate. In one embodiment, elution time refers to the amount of time elapsed between the time the sample is loaded onto the column and the time the maximum concentration of the sample is eluted from the column, when the elution reagent is applied at a known rate. In one embodiment, elution time may refer to the volume of elution reagent at a known flow rate required to elute all of the sample from the column. A “predetermined” post-translational modification refers to a known post-translational modification of the target protein identified prior to the method described herein, which is used to identify and / or quantify a predetermined post-translational modification.

[0068] When used in reference to the steps of the method herein, “simultaneously” means a method in which the steps are carried out substantially at the same time. Actions carried out “substantially at the same time” may mean that the actions are carried out in parallel, but may also include situations in which the two actions are carried out within a short time interval from each other, for example, within about 30 minutes from each other, or within about 15, 10, 5, 4, 3, 2, or 1 minute from each other. In one embodiment, if the execution of at least part of one method step overlaps in time with the execution of part of another method step, the method steps are carried out “simultaneously.” “Simultaneously” does not require strictly parallel actions. For example, not all method steps need to start or finish at the same time. In one embodiment, “simultaneously” may mean that all the reagents required for the method step are combined in the same reaction mixture so that the reaction occurs in the same reaction volume and / or during the same incubation period.

[0069] When used with respect to two process parameters, including but not limited to quantity, volume, time, temperature, pressure, concentration, and flow rate, “substantially the same” or “substantially identical” means that the quantities or quantifications of the two process parameters are functionally equivalent, for example, the difference between the two process parameters is less than about 10%, less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%.

[0070] Flow injection analysis (FIA) refers to an analytical technique in which a microliter volume of sample is injected into a carrier flow for automated sample processing and moved by the carrier flow. Flow injection analysis can be performed by a flow injection analyzer (FIA), which may or may include a sequential injection analyzer (SIA) or a direct injection analyzer (DIA) as used herein. In one embodiment, the flow injection analyzer uses a carrier flow of a constant flow rate to move the sample through the analyzer. In one embodiment, the flow injection analyzer includes a bidirectional pump, such as a syringe pump, which can reverse or stop the flow of the carrier flow for mixing the sample with a reagent or for assaying sample components.

[0071] As used herein, “fluidally connected” means that a fluid, such as a liquid, can flow from one component, apparatus, or device within a system to another. The flow may be realized by one or more intermediate components, apparatus, or devices, which may or may not be selectively blocked (e.g., by valves).

[0072] As used herein, “connected” means that two or more components, equipment, or devices are movably, fixedly connected, or otherwise attached by one or more intermediate components, equipment, or devices.

[0073] As used herein, “upstream” and “downstream” refer to positions relative to the direction of carrier flow within a system. The term “upstream” refers to a system component or position closer to the system’s input compared to another component or position. The term “downstream” refers to a system component or position closer to the system’s outlet compared to another component or position.

[0074] "Column chromatography" refers to a process in which components in a liquid sample are separated based on the differential adsorption of components in the sample as the mobile phase of the sample flows over the stationary phase.

[0075] "Affinity chromatography" refers to a chromatographic process in which a target biomolecule in the mobile phase, such as a protein, is retained by the stationary phase of a chromatography column, based on specific and reversible binding interactions between the target component and a compound immobilized on the chromatography matrix (including, but not limited to, receptor-ligand, enzyme-substrate, or antigen-antibody interactions) that result in chromatographic separation, rather than general protein characteristics such as isoelectric point, hydrophobicity, or size.

[0076] "Protein A chromatography" refers to an affinity chromatography process in which antibodies are purified from a sample based on the affinity between the Fc portion of the antibody and the IgG-binding domain of protein A immobilized on the stationary phase.

[0077] Liquid chromatography (LC) is a process in which one or more components of a liquid sample are selectively retained as the sample passes through a column. The retention of one or more components in the sample arises from the distribution of components between the stationary phase and the mobile phase. Liquid chromatography includes, but is not limited to, reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and ultrahigh-performance liquid chromatography (UHPLC).

[0078] Mass spectrometry (MS) is an analytical technique in which compounds are detected, identified, and / or measured based on their mass-to-charge ratio (m / z). MS typically involves ionization of the compound, separation of the ionized molecules by mass-to-charge ratio, detection of the ionized molecules, and creation of a mass spectrum. A known method of protein ionization is electrospray ionization (ESI). A mass spectrometer is an analytical instrument capable of detecting, identifying, and / or quantifying compounds based on their mass-to-charge ratio. A mass spectrometer typically consists of three parts: an ion source, a mass spectrometer, and a detector.

[0079] As used herein, "liquid chromatography / mass spectrometry" (LC / MS) refers to a method of separating components in a sample, such as peptides in a peptide mixture, by liquid chromatography, then ionizing them, and characterizing them by their mass-to-charge ratio.

[0080] Tandem mass spectrometry (MS / MS) is a technique for detecting, identifying, and / or measuring compounds using a multi-step process that includes two or more steps of mass spectrometry, wherein fragmentation is performed during the mass spectrometry process.

[0081] B. Overview Peptide mapping is an important analytical tool used during the development of cell culture processes and during the production of biomolecules using cell culture processes. In one embodiment, peptide mapping may be used to confirm the sequence of a biomolecule. In one embodiment, peptide mapping may be used to identify and quantify post-translational modifications (PTMs) of peptides produced in cell culture. In one embodiment, peptide mapping may be used for multi-characteristic monitoring (MAM) of critical quality characteristics (CQAs).

[0082] Numerous methods have been developed to analyze PTMs. One of the most widely used approaches involves peptide mapping, which includes liquid chromatography (LC) / mass spectrometry (MS). In one method, glycans are chemically labeled using, for example, 2-aminobenzamide (2-AB) or 2-aminobenzoic acid (2-AA), enzymatically or chemically cleaved from glycoproteins, purified, and analyzed by liquid chromatography (LC) using a fluorescence detector. This process is called the "fluorescently labeled glycan release" method. Since the glycans are removed from the protein, site-specific information cannot be obtained by glycan release. In another method, the glycoprotein is digested into peptides using an enzyme such as trypsin before LC / MS analysis. Since the glycans remain on the peptides, site-specific information regarding the peptide's PTM, such as glycosylation, can be obtained. In one embodiment, peptides identified by mass spectrometry are used as surrogate representatives of intact proteins obtained from cell culture media.

[0083] This specification provides an automated online system and method for analyzing in-process samples for real-time monitoring of product quality and process control during cell culture. In contrast to conventional peptide mapping approaches that may take more than two days to complete, for example, up to approximately three or four days, the online peptide mapping method described herein provides a fully automated process that can provide information on glycosylation tendency, post-translational modifications, and other site-specific information in less than one day, or even less than 2.5 hours. In one embodiment, the system described herein provides a fully automated method for sample preparation and peptide mapping analysis. In one embodiment, the method provides a fully automated system for protein capture, protein concentration determination, and protein digestion. In one embodiment, the online peptide mapping method described herein is used to detect one or more known post-translational modifications (PTMs) associated with a target protein. In one embodiment, the online peptide mapping method described herein is used to detect one or more predetermined post-translational modifications associated with a target protein. Advantageously, site-specific information regarding PTMs can be obtained using the method described herein.

[0084] Figures 1-4 provide an overview of a system configured to perform or carry out the automated method described herein.

[0085] Figure 1 is a schematic diagram of a system configured to provide a process implementation of an automated method consistent with the automated methods and systems described herein. Figure 1 illustrates an automated process control system 100 configured to carry out the automated processing method described herein. Figure 1 shows one embodiment of a network environment. The network environment may include one or more process control devices 200 which communicate with one or more of the following via one or more networks 202: a bioreactor 140, a flow injection analyzer (FIA) 150, one or more data retention systems 201, one or more other process control devices 200, and one or more analyzers 160.

[0086] As shown in Figures 3 and 4, the FIA150 may include at least a first syringe pump 302, a second syringe pump 402, a first valve 301, and a second valve 401. Furthermore, the FIA150 may include at least one affinity column 313, at least one spectrophotometer 414, and at least one digestion chamber 415. In addition, the FIA150 may include further components such as tubing materials, T-connectors, valves, and other components necessary to deliver fluids to appropriate destinations. Furthermore, the FIA150 may include, or may otherwise include, sources of samples, reagents, reactants, water, reducing agents, enzymes, buffers, etc., and a drainage sink for waste disposal. The FIA150 may be configured as a flow injection analyzer and / or having either or all of the functions of a sequential injection analyzer (SIA) or a direct injection analyzer (DIA).

[0087] The automated process control system 100 may further include one or more bioreactors 140. Samples for processing via the automated process control system 100 can be obtained from one or more bioreactors 140 either manually or through an automated supply process to the FIA ​​150. A further description of bioreactors 140 consistent with the methods described herein will be provided later.

[0088] One or more analytical instruments of the automated process control system 100 may include a peptide mapping system equipped with one or more mass spectrometers.

[0089] The data and information stored by the automated process control system 100 may include the following: As used herein, “automated process control system data” means all data that can be recorded and stored on or within the memory associated with the automated process control system 100. The automated process control system data can be stored in any preferred data format and can be sorted by production batch, production date, or any other preferred parameter. As used herein, “process information” means information about variables and parameters of sample handling methods and techniques, including but not limited to timing, temperature, additive amount, calibration coefficient, flow rate, and model. As used herein, “production information” means information about processed samples, past sample runs, etc. Each of the above data and / or information can be accessed in near real-time by the process control devices 200 discussed herein. The data and process information associated with the automated process control system 100 may be stored, for example, in the data retention system 201 described later and / or in the memory of one or more process control devices 200.

[0090] The process control device 200 may be configured as a server (e.g., having one or more server blades, processors, etc.), a personal computer (e.g., a desktop computer, a laptop computer, etc.), a smartphone, a tablet computing device, a computing system integrated with the FIA ​​150, and / or other devices that can be programmed to interface with the bioreactor 140, the FIA ​​150, and / or the mass spectrometer 160. In one embodiment, some or all of the functions of the process control device 200 may be implemented as part of a cloud computing platform. The process control device 200 will be described further later with reference to Figure 2.

[0091] The network environment shown in Figure 1 represents an example of an automated process control system 100 and process control device 200 configured to provide the automated method described herein. Although shown connected via network 202, any suitable set of individual or network connections can be used to allow the process control device 200 to control the bioreactor 140, FIA 150, and analyzer 160, and to access necessary resources such as various data retention systems 201.

[0092] Network 202 may be connected via wired or wireless links. Wired links may include digital subscriber lines (DSL), coaxial cable lines, Ethernet, or fiber optic lines. Wireless links may include Bluetooth®, Bluetooth Low Energy (BLE), ANT / ANT+, ZigBee, Z-Wave, Thread, Wi-Fi®, Worldwide Interoperability for Microwave Access (WiMAX®), Mobile WiMAX®, WiMAX®-Advanced, NFC, SigFox, LoRa, Random Phase Multiple Access (RPMA), Weightless-N / P / W, infrared channels, or satellite bands. Wireless links may also include any cellular network standards for communication between mobile devices, including standards certified as 2G, 3G, 4G, or 5G. Various channel access methods may be used in wireless standards, such as FDMA, TDMA, CDMA, or SDMA. In one embodiment, different types of data may be transmitted over different links and standards. In another embodiment, the same type of data may be transmitted over different links and standards. Network communication may be conducted over any preferred protocol, including, for example, http, TCP / IP, UDP, Ethernet, ATM, etc.

[0093] Network 202 can be any type and / or form of network. The geographical range of the network can vary widely, and Network 202 can be a Body Area Network (BAN), Personal Area Network (PAN), Local Area Network (LAN), e.g., an intranet, Metropolitan Area Network (MAN), Wide Area Network (WAN), or the Internet. The topology of Network 202 can be any form, e.g., including any of the following: point-to-point, bus, star, ring, mesh, or tree. Network 202 can be any such network topology known to those skilled in the art that can assist in the operation described herein. Network 202 can utilize different layers or stacks of techniques and protocols, e.g., Ethernet protocol, Internet Protocol Suite (TCP / IP), ATM (Asynchronous Transfer Mode) technique, SONET (Synchronous Optical Networking) protocol, or SDH (Synchronous Digital Hierarchy) protocol. The TCP / IP Internet Protocol Suite may include an application layer, transport layer, internet layer (e.g., including IPv4 and IPv4), or link layer. Network 202 may be a broadcast network, telecommunications network, data communication network, or computer network of some kind.

[0094] The data retention system 201 may include any type of computer-readable storage medium and / or computer-readable storage device. Such computer-readable storage medium or device may be configured to store data and provide access to data. Examples of computer-readable storage mediums or devices include, but are not limited to, electronic storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination thereof, such as computer diskettes, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disk read-only memory (CD-ROM), digital multipurpose disks (DVDs), memory sticks, and the like.

[0095] Figure 2 is a schematic diagram of a computer system or process control device configured to provide process control of an automated method consistent with the method described herein. The process control device 200 includes one or more processors 110 (hereinafter, for convenience, also interchangeably referred to as multiple processors 110, processor(s)) 110, or processor 110), one or more storage devices 120, and / or other components. In another embodiment, the functions of the processors may be performed by hardware (e.g., through the use of application-specific integrated circuits ("ASICs"), programmable gate arrays ("PGAs"), field-programmable gate arrays ("FPGAs"), etc.), or by any combination of hardware and software. The storage devices 120 include any type of non-temporary computer-readable storage medium and / or non-temporary computer-readable storage device. Such computer-readable storage mediums or devices can store computer-readable program instructions for causing the processors to perform one or more of the methods described herein. Examples of computer-readable storage media or devices include, but are not limited to, electronic storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination thereof, such as computer diskettes, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disk read-only memory (CD-ROM), digital multipurpose disks (DVDs), and memory sticks (but are not limited to these examples).

[0096] The processor 110 is programmed by one or more computer program instructions stored in the storage device 120, which represent a software protocol. For example, the processor 110 is programmed by a process control manager 252, a data acquisition manager 254, a calibration manager 256, an enzyme determination manager 258, and a user interface manager 260. It should be understood that the functions of the various managers discussed herein are representative and not limited. Furthermore, the storage device 120 may function as a data retention system 201 that provides data storage. For convenience as used herein, various “managers” are described as performing operations, but this actually means that the managers are programmed to perform operations on the processor 110 (and therefore the process control device 200).

[0097] The various components of the process control device 200 work together to provide control for one or more FIAS 150s and one or more analyzers 160, and also provide interfaces for a user or other system to interface with these systems.

[0098] The process control manager 252 is a software protocol that operates on the automated process control system 100. The process control manager 252 is configured to provide one or more control signals to the FIA ​​150 and the analyzer 160. The control signals provided by the process control manager 254 are configured to cause adjustments to one or more process parameters of the FIA ​​150 and the analyzer 160. As used herein, the aforementioned “process parameter” refers to any parameter or variable of the production process that can be adjusted by the user through the process control device 200. Process parameters include, but are not limited to, routes, flow rates, temperatures, incubation periods, reagent additions, and the timing of various process steps. The determination of the control signals may be based on process information or production information received by the data acquisition manager 254, as discussed herein.

[0099] The control signals provided by the process control manager 252 may be used to initiate, control, and / or update any processes that the FIA ​​150 and analyzer 160 described herein can perform. For example, the process control manager 252 is configured to control the flow, path, and timing of all samples, fluids, reagents, temperature, stirring, incubation periods, etc., throughout the FIA ​​150 during an automated process, via automatic control of the various drive means of the FIA ​​150.

[0100] In one embodiment, the process control manager 252 provides process monitoring functions. The process control manager 252 may be configured to access all information measured, generated, and / or stored by the process control device 200. The process control manager 254 may further be configured to provide any such information to the user via the user interface manager 260.

[0101] In another embodiment, the process control manager 252 may be configured to communicate with and / or interact with the calibration manager 256 and the enzyme determination manager 258. The calibration manager 256 and the enzyme determination manager 258 will be described in more detail later.

[0102] In one embodiment, the process control manager 252 is configured to control the operation of the FIA ​​150 and the analyzer 160, as will be further described later, particularly with respect to the automated method described herein.

[0103] In one embodiment, the process control manager 252 controls the flow of the sample through the affinity column 313 for a predetermined elution time, as will be described in more detail later. In another embodiment, the process control manager 252 controls the flow of the sample through the affinity column 313 to produce a predetermined volume of eluate as an output. The process control manager 252 can control the flow of the sample to produce a predetermined volume of eluate within a predetermined elution time. The process control manager 252 is further configured to control the flow of eluate from the output section of the affinity column 313 to the spectrophotometer 414, as will be described later, so that the protein concentration of the eluate can be determined and / or measured by the data acquisition manager 254. In one embodiment, the process control manager 252 is configured to control the flow of eluate from the output section of the affinity column 313 to the digestion chamber, as will be described later, so that the protein can be digested by adding an amount of digestion reagent determined by the process control manager 252. In one embodiment, the process control manager 252 is configured to control the incubation time and temperature of the sample in the digestion chamber 415.

[0104] The data acquisition manager 254 is a software protocol that operates on the process control device 200. The data acquisition manager 254 is configured to access one or more of the FIA ​​150, analyzer 160, affinity column 155, spectrophotometer 156, and digestion chamber 415 to collect and / or gather data and information associated with the automated process described herein. Automated system data may include, for example, production information, archive data, and / or extracted data that can be acquired in near real time, as well as process information and process parameter information, and any other information or data created or measured by the FIA ​​150 and analyzer 160. The data acquisition manager 254 is further configured to access one or more data retention systems 201 to store and / or receive automated processing system data stored in the data retention systems 201.

[0105] The data acquisition manager 254 is configured to interface with the spectrophotometer 156 and perform protein concentration measurement / determination on the eluate generated or output by the affinity column 313.

[0106] The calibration manager 256 is a software protocol that operates on the process control device 200. The calibration manager 256 is configured to receive and manage calibration information related to the determination of protein concentration based on UV absorbance measurements performed by the spectrophotometer 414. The calibration manager 256 is configured to communicate and cooperate with the process control manager 252 and the data acquisition manager 254 to collect raw data from the spectrophotometer 414. The calibration information stored and managed by the calibration manager 256 can be applied by the process control manager 252 to the raw data (e.g., UV absorbance data) collected by the data acquisition manager 254 to determine the protein concentration in the sample eluate.

[0107] The Enzyme Determination Manager 258 is a software protocol that operates on the process control device 200. The Enzyme Determination Manager 258 is configured to determine the amount of enzyme to be added to the sample during the automated processing procedure. As will be described in more detail below, during the automated processing procedure, the first sample can be eluted through the affinity column 313 for a predetermined elution time controlled by the process control manager 252 to obtain a sample eluate. The data acquisition manager 254 operates to obtain protein concentration measurements, which, combined with calibration information, can be used by the process control manager 252 to determine the protein concentration level in the eluted sample. Using the determined protein concentration level, the Enzyme Determination Manager 258 can determine the appropriate amount of digestive enzyme to be added to the sample digestion step.

[0108] During operation, the enzyme determination manager 258 can communicate and cooperate with the process control manager 252 to provide information on the determined digestive enzymes related to the amount of enzyme to be added to the second sample, which is determined by the protein concentration determination of the first sample. As described above, the process control manager 252 is configured to precisely control the timing and volume of the sample flow through the affinity column 313. By applying the same predetermined timing and volume processing parameters to the first and second samples processed by the affinity column 313, the process control manager 252 may be able to achieve first and second sample elutes with substantially similar concentration values. Therefore, as described above, the protein concentration measurements performed on the first sample elute can be applied by the enzyme determination manager 258 to determine the appropriate amount of digestive enzyme to be added to the second sample elute.

[0109] The User Interface Manager 260 is a software protocol that operates on the process control device 200. The User Interface Manager 260 is configured to provide a user interface that enables user interaction with the process control device 200. The User Interface Manager 260 is configured to receive input from any user input source, including but not limited to touchscreens, keyboards, mice, controllers, joysticks, and voice control. The User Interface Manager 260 is configured to provide a user interface such as a text-based user interface, a graphical user interface, or any other preferred user interface. The User Interface Manager 260 may be configured to provide different user interface services depending on the type of client device. For example, a laptop or desktop computer may be provided with a user interface that includes a complete set of interface options, while a smartphone or tablet may be provided with a user interface limited to status updates.

[0110] In one embodiment, the user interface manager 260 may provide one or more users with access to any or all process and / or production information relating to the automated process control system 100 via a user interface. The user interface manager 260 may enable users to perform various tasks on the bioreactor 140, FIA 150, and analyzer 160. For example, the user interface manager 260 may enable users to directly adjust or control one or more process parameters.

[0111] Figure 3 is a schematic diagram of the first syringe pump and first valve system accompanying the FIA150 for carrying out the online automated method described herein. During operation, the process control manager 252 of the process control device controls the first valve 301 and the first syringe pump 302 of the FIA150 to properly deliver and mix the fluid during the automated process method described herein. Table 1 below provides an illustrative list of various connections to the first valve 301 and the first syringe pump 302.

[0112] The first valve 301 includes a plurality of input ports 304 and an output port 308. The terms “input port” and “output port” are used herein to distinguish the ports at both ends of the first valve 301 and do not necessarily refer to the direction of flow associated with them. The first valve 301 may be, for example, a multi-port rotary valve. The process control manager 252 controls an automatic valve selector means (e.g., a stepping motor, a servo motor, or other actuator device; not shown) configured to rotate the valve 301 to provide fluid communication between the selected input port 304 and the output port 308. The output port 308 is connected to the first syringe pump 302 via a communication line 309. The first syringe pump 302 includes a port selection device 311 and a drive syringe 306. The port selection device 311 includes a plurality of syringe pump ports 305 and a syringe pump automatic selector means (e.g., a stepping motor, a servo motor, or other actuator device; not shown) configured to be operated to align one of the selected syringe pump ports 305 with the actuator syringe 306. The syringe pump automatic selector means is controlled by the process control manager 252 to select a specific syringe pump port 305 and position it in fluid communication with the actuator syringe 306.

[0113] The actuator syringe 306 is a syringe driven by a syringe pump drive means (e.g., a stepping motor, linear drive, servo motor, or other actuator device; not shown) that provides bidirectional pressure for aspirating and pushing fluid through one of the selected syringe pump ports 305. The actuator syringe 306 is further configured to mix fluid in the syringe chamber via the pushing and retracting of the syringe plunger when driven by the syringe pump drive means. The actuator syringe 306 may be any type of syringe configured to apply bidirectional pressure to the FIA150 system to aspirate and push fluid.

[0114] Figure 3 further illustrates an affinity column 313 connected to the first valve 301 by a V208 input port 304.

[0115] During operation, the process control manager 252 may activate the first valve 301 to establish a fluid connection between the selected input port 304 and the output port 308. The process control manager 252 may also activate the port selection device 311 to create a fluid connection between the selected syringe pump port 305 and the actuator syringe 306. The actuator syringe 306 is then driven, and a specific amount of fluid can be drawn into the actuator syringe 306 through the selected syringe pump port 305. The port selection device 311 can then be activated again by the process control manager 252 to create a fluid connection between the communication line 309 and the syringe pump actuator 306. The actuator syringe 306 is then driven again, and fluid can be drawn into the actuator syringe 306 from the first valve 301. The actuator syringe 306 and port selection device 311 may then be further actuated to draw additional fluid into the actuator syringe 306 and / or to move the fluid in the actuator syringe out through one of the syringe pump ports 305.

[0116] The process control manager 252 can provide precise and accurate control of the actuator syringe 306 to transfer the appropriate amount of fluid through the FIA150 system.

[0117] Figure 4 is a schematic diagram of a second syringe pump and second valve system accompanying the FIA150 for carrying out the online automated method described herein. During operation, the process control manager 252 of the process control device controls the second valve 401 and the second syringe pump 402 of the FIA150 to properly deliver and mix the fluid during the automated process method described herein. Table 1 below provides an illustrative list of various connections to the second valve 401 and the second syringe pump 402.

[0118] The second valve 401 includes a plurality of input ports 404 and an output port 408. The second valve 401 may be, for example, a multi-port rotary valve. The terms “input port” and “output port” are used herein to distinguish the ports at both ends of the first valve 301 and do not necessarily refer to the direction of flow associated with them. The process control manager 252 controls an automatic valve selector means (e.g., a stepping motor, a servo motor, or other actuator device; not shown) configured to rotate the valve 401 to provide fluid communication between the selected input port 404 and the output port 408. The output port 408 is connected to a second syringe pump 402 via a communication line 409. The second syringe pump 402 includes a port selection device 411 and a drive syringe 406. The port selection device 411 includes a plurality of syringe pump ports 405 and a syringe pump automatic selector means (e.g., a stepping motor, a servo motor, or other actuator device; not shown) configured to be operated to align one of the selected syringe pump ports 405 with an actuator syringe 406. The syringe pump automatic selector means is controlled by the process control manager 252 to select a specific syringe pump port 305 and position it in fluid communication with a valve 301 via a communication line 309.

[0119] The actuator syringe 406 is a syringe connected to a syringe pump drive means (e.g., a stepping motor, linear drive, servo motor, or other actuator device; not shown) and provides bidirectional pressure for aspirating and pushing fluid through one of the selected syringe pump ports 405. The actuator syringe 406 is further configured to mix fluid in the syringe chamber via the pushing and retracting of a syringe plunger when driven by the syringe pump drive means. The actuator syringe 406 may be any type of syringe configured to apply bidirectional pressure to the FIA150 system to aspirate and push fluid.

[0120] Figure 4 further illustrates an affinity column 313, which is connected to a first valve 301 at its input end and to a multi-directional connector 412 at its output end. The multi-directional connector 412 is further connected to a spectrophotometer 414. Figure 4 further illustrates an S105 digestion chamber 415, which is connected to a second syringe pump 402 via a syringe pump port 405.

[0121] During operation, the second valve 401 and the second syringe pump 402 operate in the same manner as the first valve 301 and the first syringe pump 302. The process control manager 252 provides control signals to the valve automatic selector means, the syringe pump automatic selector means, and the syringe pump drive means to control and adjust the connections of the FIA ​​150 and generate / provide fluid flow throughout the FIA ​​150 according to process and production parameters.

[0122] Table 1 below provides examples of various fluid connections, fluid reservoirs / sources, fluid sinks / outlets / drains, etc., within the FIA150, consistent with the systems described herein.

[0123] [Table 1]

[0124] Referring to the automated process system 100 described above, Figure 5 provides a schematic overview of the online automated method. Briefly, the clarified cell culture medium is delivered to a sample preparation unit which includes a protein capture unit, a protein concentration determination unit, and a protein digestion unit. In one embodiment, the protein capture unit includes a chromatography column. In one embodiment, the protein capture unit includes an affinity chromatography column (also referred to herein as an affinity column). In one embodiment, the protein concentration determination unit includes a spectrophotometer. In one embodiment, the protein digestion unit includes a digestion chamber. In one embodiment, the protein digestion chamber is a syringe pump barrel. In one embodiment, the temperature of the digestion chamber can be controlled. In one embodiment, the prepared sample is transferred to an analyzer. In one embodiment, the analyzer includes a peptide mapping unit. In one embodiment, the peptide mapping unit includes a data acquisition unit and a data processing unit. In one embodiment, the data acquisition unit includes a liquid chromatography column (LC) and a mass spectrometer (MS).

[0125] Figure 6 is a schematic diagram of the method described herein, in which some steps are performed manually or offline. In one embodiment, the protein capture and concentration determination steps for sample preparation are performed offline, while the protein digestion, data acquisition, and data processing steps are performed online. In another embodiment, for example, the sample preparation steps, including the protein capture, concentration determination, and protein digestion steps, are performed offline, while the data acquisition and data processing steps are performed online.

[0126] In one embodiment, a method for detecting post-translational modifications (PTMs) of proteins is provided. In one embodiment, the method is performed by a flow injection analyzer (FIA) controlled by at least one processor. In one embodiment, the flow injection analyzer (FIA) is a sequential injection analyzer (SIA). In one embodiment, the flow injection analyzer (FIA) is a direct injection analyzer (DIA). As described above, examples of methods or systems for online automated flow injection analysis are shown in Figures 1-4.

[0127] In one embodiment, the method includes housing a first sample containing the target protein in a holding reservoir of a FIA, for example, the FIA ​​150 described above. In one embodiment, the first sample is obtained from a bioreactor, for example, the bioreactor 140 described above. In one embodiment, the bioreactor 140 is connected to the FIA. In one embodiment, the FIA ​​includes a sample collection device connected to the bioreactor 140 and configured to obtain cell culture medium or clarified cell culture medium from the bioreactor 140. In one embodiment, an automated system interface used to connect the bioreactor 140 and the FIA ​​is used, for example, the Modular Automated Sampling Technology (MAST) sample collection system (Lonza, Bend, OR, USA) or the Seg-Flow automated sample collection system (BioProcess International Europe, Vienna, Austria). In one embodiment, the sample is obtained manually from the bioreactor and introduced into the FIA ​​retention reservoir, for example, by introducing the FIA ​​retention reservoir into a container containing the cell culture sample. In one embodiment, the first sample comprises cell culture medium. In one embodiment, the first sample comprises clarified cell culture medium. In one embodiment, the first sample has a volume of about 20 μL to about 1000 μL.

[0128] In one embodiment, the method includes a first protein capture step. In one embodiment, the protein capture step includes an affinity chromatography step. In one embodiment, the affinity chromatography step includes column equilibration, sample loading, and sample elution. In one embodiment, the column equilibration step includes delivering a first volume of binding buffer to an affinity chromatography column in an FIA via a FIA. In one embodiment, the sample loading step includes transferring a first sample from a holding reservoir to an affinity column via an FIA. In one embodiment, the sample elution step includes delivering a first volume of elution buffer to an affinity chromatography column via an FIA and eluting proteins in the first sample from the affinity chromatography column to form a first eluate. In one embodiment, based on a predetermined elution time, proteins in the first sample are eluted from the affinity chromatography column to form a first eluate. In one embodiment, the predetermined elution time is controlled by a process control manager of a process control device associated with an automated control system. In one embodiment, proteins in a first sample are eluted from an affinity column based on a predetermined elution volume, forming a first eluate. In one embodiment, the elution volume is controlled by a process control manager of a process control device associated with an automated control system.

[0129] In one embodiment, the protein capture step is performed manually, as shown in Figure 6. In one embodiment, the protein capture step is performed offline. In one embodiment, the protein capture step is performed atline. In one embodiment, the affinity chromatography step is performed manually. In one embodiment, the affinity chromatography step is performed offline. In one embodiment, the affinity chromatography step is performed atline.

[0130] In one embodiment, the first eluate is transferred to a protein concentration detection unit via FIA under the control of a process control manager. In one embodiment, the protein capture process from column equilibration to elution takes approximately 15 minutes to approximately 1 hour. In another embodiment, the protein capture process takes less than approximately 30 minutes.

[0131] In one embodiment, the method includes determining the concentration of protein in a first eluate by at least one processor. In one embodiment, determining the concentration of protein in the first eluate is performed by at least one processor according to a predetermined calibration curve. In one embodiment, the concentration of protein in the first eluate is determined by measuring the absorbance of the sample and calculating the protein concentration based on a predetermined calibration curve. For example, a process control manager can work with a data acquisition manager to acquire UV absorbance data from a spectrophotometer and work with a calibration manager to acquire a predetermined calibration curve and apply it to the acquired UV absorbance data. In one embodiment, the concentration of protein in the first eluate is determined by measuring the UV absorbance of the sample at approximately 190 nm to approximately 280 nm, or approximately 205 nm to approximately 280 nm, and calculating the protein concentration based on a predetermined calibration curve. In one embodiment, after determining the protein concentration, the method includes transferring the first eluate to a waste container T106 via the operation of the FIA. In one embodiment, the concentration determination process takes approximately 5 to 30 minutes, or less than 10 minutes.

[0132] In one embodiment, protein concentration determination is performed manually, as shown in Figure 6. In another embodiment, protein concentration determination is performed offline. In yet another embodiment, protein concentration determination is performed atline.

[0133] After the protein concentration of the first eluate is determined, the method includes containing a second sample containing the protein in a holding reservoir. In one embodiment, the second sample is obtained from a bioreactor. In one embodiment, the second sample includes cell culture medium. In one embodiment, the second sample includes clarified cell culture medium. In one embodiment, the second sample is obtained from the same cell culture from which the first sample was obtained. In one embodiment, the first and second samples are obtained from the cell culture at substantially the same time. In one embodiment, the first and second samples are obtained from the cell culture within at least about 30 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute from each other. In one embodiment, the second sample has a volume of about 20 μL to about 1000 μL.

[0134] In one embodiment, the method includes a second protein capture step. In one embodiment, the second protein capture step includes an affinity chromatography step. In one embodiment, the second affinity chromatography step includes column equilibration, sample loading, and sample elution. In one embodiment, the column equilibration step includes delivering a second volume of binding buffer to an affinity column via a FIA. In one embodiment, the sample loading step includes transferring a second sample from a holding reservoir to an affinity column via a FIA. In one embodiment, the sample elution step includes delivering a second volume of elution buffer to an affinity column via a FIA and eluting proteins from the second sample from the affinity column to form a second eluate. In one embodiment, based on a predetermined elution time, proteins from the second sample are eluted from the affinity column to form a second eluate. In one embodiment, the elution time (and optionally other process parameters such as the volume of elution buffer) is controlled by at least one processor, for example via a process control manager, so as to substantially match a predetermined elution time for the first eluate, thereby obtaining a second eluate. In one embodiment, the second eluate has a protein concentration substantially similar to that of the first eluate. In one embodiment, based on a predetermined elution volume, proteins in the second sample are eluted from the affinity column to form a second eluate. In one embodiment, the elution time of the second sample is controlled by a process control manager so as to substantially match the elution time of the first sample. In one embodiment, the elution volume of the second sample is controlled by a process control manager so as to substantially match the elution volume of the first sample.

[0135] In one embodiment, the protein capture step is performed manually, as shown in Figure 6. In one embodiment, the protein capture step is performed offline. In one embodiment, the protein capture step is performed atline. In one embodiment, the affinity chromatography step is performed manually. In one embodiment, the affinity chromatography step is performed offline. In one embodiment, the affinity chromatography step is performed atline.

[0136] In one embodiment, the method includes digesting the protein in a second eluate to form a peptide mixture. In one embodiment, the protein in the second eluate is enzymatically digested. In one embodiment, digesting the protein in the second eluate includes introducing a digestion reagent into the second eluate via an FIA to form a digestion mixture. In one embodiment, digesting the protein in the second eluate includes introducing a digestion reagent, comprising a digestion buffer, a reducing agent, and a protease, into the second eluate via an FIA to form a digestion mixture. In one embodiment, the second eluate is brought into contact with a protease in an amount determined by at least one processor, for example, an enzyme determination manager, based on the concentration of the first eluate. In one embodiment, the method includes mixing the digestion mixture via a syringe pump of an FIA controlled, for example, by a process control manager. In one embodiment, the method includes incubating the digestion mixture, for example, under the control of a process control manager.

[0137] In one embodiment, the protease comprises heat-stable trypsin. In one embodiment, the method includes introducing a first volume of digestion buffer, a reducing agent, and heat-stable trypsin into a second eluate via FIA and mixing them to form a first digestion mixture. In one embodiment, the first digestion mixture is incubated at a temperature of about 65°C to about 85°C for about 5 minutes to about 30 minutes to form an incubation mixture. In one embodiment, the digestion mixture is incubated at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form an incubation mixture. In one embodiment, the digestion mixture is incubated at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form an incubation mixture. The embodiments of the incubation process described above may be controlled via a process control manager.

[0138] In one embodiment, the method includes introducing a second amount of thermostable trypsin into an incubation mixture via a FIA and mixing it to form a second digestion mixture. In one embodiment, the second digestion mixture is incubated at a temperature of about 65°C to about 85°C for about 5 minutes to about 30 minutes to form a peptide mixture. In one embodiment, the second digestion mixture is incubated at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a peptide mixture. In one embodiment, the second digestion mixture is incubated at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a peptide mixture. The embodiments of the incubation process described above may be controlled via a process control manager.

[0139] In one embodiment, the protein digestion process is performed manually, as shown in Figure 6. In one embodiment, the protein digestion process is performed offline. In another embodiment, the protein digestion process is performed atline.

[0140] In one embodiment, the method includes transferring a peptide mixture via FIA to an analytical instrument, such as a peptide mapping unit (which may include, for example, a mass spectrometer), and detecting a predetermined post-translational modification (PTM) of the protein.

[0141] In one embodiment, an online method for monitoring post-translational modifications of a protein is provided. In one embodiment, the method is carried out by a flow injection analyzer (FIA) controlled by a process control device including at least one processor. In one embodiment, the flow injection analyzer includes at least one bidirectional pump. In one embodiment, the FIA ​​includes at least one syringe pump controlled by at least one processor. In one embodiment, the FIA ​​includes at least one multiport valve controlled by at least one processor. In one embodiment, the FIA ​​includes two syringe pumps. In one embodiment, the FIA ​​includes two multiport valves. In one embodiment, the multiport valve is a 10-port valve.

[0142] In one embodiment, the method includes containing a first sample containing a protein in a holding reservoir of a FIA. In one embodiment, the sample is advanced through the FIA ​​by the operation of one or more syringe pumps controlled by at least one processor. In one embodiment, the sample is advanced through the FIA ​​at a flow rate of approximately 10 μL / sec to approximately 200 μL / sec.

[0143] In one embodiment, a first volume of binding buffer is introduced into the affinity column in the FIA ​​via a multiport valve by the operation of the multiport valve.

[0144] In one embodiment, the sample is advanced through the FIA ​​system to the affinity column by the operation of a syringe pump. In one embodiment, the method includes transferring a first sample from a holding reservoir to the affinity column by the operation of a syringe pump. In one embodiment, the protein in the first sample binds to the affinity column. In one embodiment, the method includes introducing a first volume of elution buffer into the affinity column by the operation of a multiport valve to elute the bound protein and form a first eluate. In one embodiment, based on a predetermined elution time, the protein in the first sample is eluted from the affinity column to form a first eluate. In one embodiment, based on a predetermined elution volume, the protein in the first sample is eluted from the affinity column to form a first eluate. In some embodiments, the operation of the syringe pump may be controlled by a process control manager.

[0145] In one embodiment, the method includes transferring a first eluate to a protein concentration detection unit by the operation of a syringe pump. In another embodiment, the method includes advancing the first eluate to a spectrophotometer by the operation of a syringe pump. In another embodiment, the method includes determining the concentration of protein in the first eluate according to the measured UV absorbance by at least one processor. In another embodiment, the first eluate is transferred to a waste container after the protein concentration has been determined. In another embodiment, the concentration of protein in the first eluate is determined by at least one processor according to a predetermined calibration curve.

[0146] In one embodiment, the method includes containing a second sample in a holding reservoir of an FIA. In one embodiment, the method includes advancing the sample through the FIA ​​by the operation of a syringe pump. In one embodiment, the method includes delivering a second volume of binding buffer to an affinity column via a multiport valve. In one embodiment, the method includes transferring the second sample from the holding reservoir to the affinity column by the operation of a syringe pump. In one embodiment, the protein in the second sample binds to the affinity column. In one embodiment, the method includes introducing a second volume of elution buffer into the affinity column by the operation of a multiport valve to elute the bound protein and form a second eluate. In one embodiment, based on a predetermined elution time, the protein in the second sample is eluted from the affinity column and a second eluate is formed. In one embodiment, based on a predetermined elution volume, the protein in the second sample is eluted from the affinity column and a second eluate is formed. In one embodiment, the first and second eluates have substantially the same elution volume. In some embodiments, the operation of the syringe pump and multiport valve can be controlled by a process control manager.

[0147] In one embodiment, the method includes advancing a second eluate into a digestion chamber within a FIA by the operation of a syringe pump. In one embodiment, the method includes introducing a digestion reagent into the second eluate by the operation of a multiport valve to digest the proteins in the second eluate and form a peptide mixture. In one embodiment, digesting the proteins includes introducing a digestion buffer, a reducing agent, a protease, or a combination thereof into the second eluate in the digestion chamber by the operation of a multiport valve to form a first digestion mixture. In one embodiment, the method includes mixing the first digestion mixture. In one embodiment, the digestion chamber is an actuator syringe, for example, the internal barrel of an actuator syringe, and the first digestion mixture is mixed by pushing down and retracting the plunger of the syringe. In one embodiment, the first digestion mixture is incubated in the digestion chamber to form an incubation mixture. In one embodiment, the method includes introducing a second volume of protease into the first incubation mixture by the operation of a multiport valve to form a second digestion mixture. In one embodiment, a second volume of digestion buffer is introduced into a first incubation mixture. In one embodiment, the second digestion mixture is incubated to form a peptide mixture. In one embodiment, the method includes mixing the second digestion mixture. In one embodiment, the second digestion mixture is mixed by pushing down and retracting the plunger of a syringe. In one embodiment, the amount of protease introduced into the second digestion mixture is determined by at least one processor based on the concentration of the first eluate. In one embodiment, the operation of the syringe pump and multiport valve may be controlled by a process control manager.

[0148] In one embodiment, the protease comprises heat-stable trypsin. In one embodiment, the method includes introducing a first volume of digestion buffer, a reducing agent, and heat-stable trypsin into a second eluate by the operation of a multiport valve and mixing them to form a first digestion mixture. In one embodiment, the method includes incubating the first digestion mixture at a temperature of about 70°C to about 75°C for about 10 to about 20 minutes. In one embodiment, the method includes introducing a second volume of digestion buffer and heat-stable trypsin into the first digestion mixture by the operation of a multiport valve and mixing them to form a second digestion mixture. In one embodiment, the method includes incubating the second digestion mixture at a temperature of about 70°C to about 75°C for about 10 to about 20 minutes to form a peptide mixture.

[0149] In one embodiment, the method includes advancing a peptide mixture to a peptide mapping unit by the operation of a syringe pump and detecting post-translational modifications (PTMs) of a protein. In another embodiment, the method includes advancing a peptide mixture to a peptide mapping unit by the operation of a syringe pump and detecting a predetermined PTM of a protein.

[0150] C.Cell culture In one embodiment, the method is used to detect post-translational modifications (PTMs) of proteins in a sample. In one embodiment, the protein is a recombinant protein. In one embodiment, the protein is a therapeutic protein. In one embodiment, the protein is a recombinantly produced therapeutic protein. In one embodiment, the sample is obtained from a cell culture.

[0151] Methods for expressing recombinant proteins in cell cultures are known. Generally, expression vectors containing a coding sequence that encodes a protein are prepared. In one embodiment, the coding sequence is operatively ligated with one or more regulatory sequences. Regulatory sequences are known and include, but are not limited to, promoters, enhancers, and other expression regulatory elements that control the transcription or translation of the coding sequence. "Operatally ligated" or "operably ligated" means that the regulatory sequence is positioned appropriately relative to the coding sequence to result in the expression of the coding sequence. In one embodiment, the regulatory sequence is adjacent to the coding sequence. In one embodiment, the regulatory sequence controls the expression of the coding sequence by acting trans or remotely. In one embodiment, the recombinant expression vector includes one or more additional sequences, such as origins of replication and / or selection markers.

[0152] In one embodiment, the recombinant protein comprises two or more polypeptide chains. In another embodiment, the recombinant protein is an antibody. In the case of a protein comprising two or more polypeptide chains, the coding sequences of the individual polypeptide chains can be inserted into the same or separate expression vectors. Methods for inserting genes into expression vectors are known. In one embodiment, the recombinant expression vector encodes a signal peptide that facilitates the secretion of a protein from a host cell.

[0153] To induce recombinant expression of a protein, a recombinant expression vector encoding that protein is transfected into host cells. Transfection methods are well known.

[0154] The host cell may include cells of prokaryotes, yeasts, or higher eukaryotes. In one embodiment, the host cell is a mammalian host cell. Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary cells (CHO cells), NS0 myeloma cells, COS cells, SP2 cells, COS-7 cells, human fetal kidney cells (HEK 293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL), human lung cells (W138), human hepatocytes (Hep G2), human hepatocarcinoma cells (Hep G2), mouse mammary cancer cells (MMT), TRI cells, MRC 5 cells, and FS4 cells. In one embodiment, the mammalian host cell is a CHO, HEK 293, COS, NS0, SP2, or PER.C6 host cell.

[0155] During cell culture, transformed host cells are cultured in a suitable cell culture medium under suitable culture conditions (e.g., temperature, pH, agitation, and dissolved oxygen level) for recombinant protein expression. Commercially available cell culture media include, but are not limited to, Ham's F10® (Sigma), Minimal Essential Medium® (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium® (DMEM) (Sigma), Iskov Modified Dulbecco's Medium, and Minimal Essential Medium-Alpha (MEM-Alpha). In one embodiment, the cell culture medium is supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or other energy sources.

[0156] In one embodiment, the host cells are cultured in a small-scale bioreactor. In another embodiment, the host cells are cultured in individual flasks, shaking flasks, or wave bags. In another embodiment, the host cells are cultured in a bioreactor having a volume of approximately 125 ml to approximately 500 ml. In another embodiment, the host cells are cultured in a large-scale bioreactor. In another embodiment, the host cells are cultured in a bioreactor having a volume of approximately 100 liters to approximately 20,000 liters or more. In another embodiment, the host cells are cultured in a stirred tank, fixed bed, fluidized bed, bubble tower, or airlift bioreactor.

[0157] In one embodiment, the cell culture is supplemented with a concentrated feed medium containing nutrients and amino acids during the cell culture process. In one embodiment, the medium in the bioreactor is not supplemented during the cell culture process in a batch process. In one embodiment, during the cell culture process in a fed-batch bioreactor, the medium in the bioreactor is supplemented with additional components, such as nutrients or cell culture medium. In one embodiment, the medium is continuously added to the bioreactor, and used medium and products are removed throughout the cell culture process in a perfusion bioreactor.

[0158] In one embodiment, the method includes obtaining a first sample and a second sample from a cell culture. In one embodiment, the first sample and the second sample are obtained from the same cell culture. In one embodiment, the first sample and the second sample are obtained from the cell culture at substantially the same time. As used herein, two operations performed at substantially the same time may mean that the two operations are performed in parallel, but also include situations in which the two operations are performed within a short time of each other. In one embodiment, the first and second samples are obtained within about 30 minutes of each other. In one embodiment, the first and second samples are obtained within about 15, about 10, about 5, about 4, about 3, about 2, or about 1 minute of each other.

[0159] D. Recombinant Proteins In one embodiment, the method is used to detect post-translational modifications (PTMs) of a target protein in a sample. In one embodiment, the protein is recombinantly produced. In one embodiment, the recombinantly produced protein contains at least about 1000 amino acid residues. In one embodiment, the recombinantly produced protein has a molecular weight of at least about 10 kDa, or at least about 20 kDa, or at least about 30 kDa.

[0160] In one embodiment, the protein is a glycoprotein. In one embodiment, the protein is a therapeutic antibody or its antigen-binding fragment. In one embodiment, the protein is a receptor or receptor ligand. In one embodiment, the protein is a fusion protein.

[0161] Recombinant proteins can be produced intracellularly, in the perimembrane space, or directly secreted into the cell culture medium. When polypeptide proteins are produced intracellularly, the first step of the purification process typically involves lysis of the cells, such as by mechanical shearing, osmotic shock, or enzymatic treatment, which releases the entire contents of the cells into a homogenate, resulting in cell debris that can be removed by fractional centrifugation or filtration.

[0162] In one embodiment, the protein is a therapeutic protein. As used herein, “therapeutic protein” is a protein having one or more biological activities useful for treating, preventing or improving a disease or at least one symptom of a disease.

[0163] E. Post-translation modification Post-translational modifications (PTMs) refer to one or more covalent modifications performed on a polypeptide during or after protein synthesis. In one embodiment, PTMs are introduced by enzymes or enzymatic pathways. PTMs can result in varying levels of protein heterogeneity, which can significantly affect the structure and function of recombinantly produced proteins, including but not limited to their biological activity, binding activity, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity. Some PTMs may be beneficial to the physicochemical characteristics of therapeutic proteins, while others may be detrimental. The amount and type of PTMs may be affected by cell culture conditions and are considered critical quality characteristics (CQAs) that need to be monitored during the production of recombinantly produced therapeutic proteins.

[0164] In one embodiment, a method for detecting PTMs of a protein is provided. In another embodiment, a method for detecting PTMs of a recombinantly produced protein is provided. In yet another embodiment, a method for detecting PTMs of a therapeutic protein is provided.

[0165] In one embodiment, PTM includes glycosylation. In one embodiment, glycosylation includes N-linked glycosylation, O-linked glycosylation, or a combination thereof. In one embodiment, PTM includes oxidation, deamidation, isomerization, glycation, or a combination thereof. In one embodiment, PTM includes, but is not limited to, C-terminal modifications such as lysine retention, glycine loss, amide formation, or a combination thereof. In one embodiment, PTM includes, but is not limited to, N-terminal modifications such as leader sequence retention, N-terminal cyclization, or a combination thereof.

[0166] In one embodiment, the PTM includes N-linked glycans. In one embodiment, the PTM includes O-linked glycans. In one embodiment, the PTM includes agalactosylglycans with zero terminal galactose moieties, e.g., G0, G0-GlcNAc, G0F, G0F-GlcNAc, or combinations thereof. In one embodiment, the PTM includes glycans with one terminal galactose moiety, e.g., G1, G1F, G1F-GlcNAc, or combinations thereof. In one embodiment, the PTM includes glycans with two terminal galactose moieties, e.g., G2F, G2F+sialic acid, or combinations thereof.

[0167] In one embodiment, PTM contains a high-mannose glycan. As used herein, “high-mannose glycan” refers to an N-glycan structure containing about 3, about 4, about 5, about 6, about 7, about 8, or about 9 mannose residues, which may also be referred to as Man4, Man5, Man6, Man7, Man8, or Man9 glycan, respectively.

[0168] F. Sample Preparation This specification provides an automated online system or method for detecting post-translational modifications (PTMs) within a polypeptide. In one embodiment, a method or system for detecting PTMs in recombinantly produced polypeptides is provided. In one embodiment, a method or system for detecting PTMs in proteins is provided. In one embodiment, a method or system for detecting PTMs in recombinantly produced proteins is provided. In one embodiment, a method or system for detecting PTMs in therapeutic proteins is provided. In one embodiment, a method or system for detecting PTMs in recombinantly produced therapeutic proteins is provided.

[0169] In one embodiment, an automated online sample preparation system or method is provided. In one embodiment, the automated online sample preparation system or method includes one or more of the following steps: a protein capture step, a concentration determination step, and a protein digestion step. In one embodiment, an automated online sample preparation system or method is provided in which proteins in a sample are prepared for an analytical process, such as mass spectrometry. In one embodiment, an automated online sample preparation system or method is provided in which proteins in a sample are prepared for an analytical process (for example, to detect one or more PTMs). In one embodiment, the automated online sample preparation system or method prepares proteins in a sample for mass spectrometry (MS). In one embodiment, the automated online sample preparation system or method prepares proteins in a sample for liquid chromatography / mass spectrometry (LC / MS). In one embodiment, the automated online sample preparation system or method prepares proteins in a sample for an automated LC / MS system. In one embodiment, the automated online sample preparation system or method prepares proteins in a sample for an integrated LC / MS system.

[0170] Figure 5 provides a schematic overview of the online automated method. Briefly, the clarified cell culture medium is delivered to a sample preparation unit which includes a protein capture unit, a protein concentration determination unit, and a protein digestion unit. The prepared sample is then transferred to an analytical instrument, such as a peptide mapping unit which includes a data acquisition unit and a data processing unit. In one embodiment, the data acquisition unit includes liquid chromatography (LC) and mass spectrometry (MS).

[0171] As shown in Figure 6, some steps of the method may be performed manually or offline. In one embodiment, the protein capture and concentration determination steps for sample preparation are performed offline, while the protein digestion, data acquisition, and data processing steps are performed online. In another embodiment, for example, the sample preparation steps, including the protein capture, concentration determination, and protein digestion steps, are performed offline, while the data acquisition and data processing steps are performed online.

[0172] 1. Flow injection analysis In one embodiment, automated sample preparation is performed using a flow injection analyzer (FIA) optionally included in an automated process control system that includes one or more process control devices, as discussed with respect to Figures 1 and 2. Flow injection analyzers are commercially available, for example, from FIAlab Instruments, Inc. (Seattle, WA, USA). In one embodiment, the FIA ​​is a sequential injection analyzer (SIA). In one embodiment, the FIA ​​is a direct injection analyzer (DIA). In one embodiment, the FIA ​​includes a bidirectional pump and valves. In one embodiment, the FIA ​​includes two or more pumps and two or more valves. In one embodiment, the FIA ​​includes two pumps and two valves. In one embodiment, the bidirectional pump is a syringe pump. In one embodiment, the valve is a rotary valve. In one embodiment, the valve is a multi-port valve. In one embodiment, the valve is a 10-port rotary valve. In one embodiment, the flow injection analyzer is a ProSIA dual-pump injection analyzer (FIAlab Instruments, Inc., Seattle, WA, USA).

[0173] The following considerations relating to an automated process implemented by an automated process control system 100, which includes at least one process control device 200, at least one bioreactor 140, at least one FIA ​​150, and at least one analyzer 160, can be understood by referring to Figures 1-4 and Table 1. The arrangement of reservoirs, inputs, outputs, sinks, drains, and equipment with respect to the various input and output ports of the first valve 301 and second valve 401 of the FIA ​​150 are merely examples. Other arrangements and configurations can be used without departing from the methods and systems described herein. For example, the roles of the first valve 301 and the second valve 401, and the first syringe pump 302 and the second syringe pump 402 can be swapped. In another example, alternative or additional ports and / or connections can be used. In the following discussion, as previously mentioned with reference to Figure 2, it will be understood that the first valve 301, the second valve 401, the first syringe pump 302, the second syringe pump 402, and any other automatic aspects of the system are controlled by the process control device 200.

[0174] In one embodiment, the sample is introduced into the holding reservoir of the FIA. The sample may be introduced by any suitable means, including manual and automated methods. In one embodiment, the bioreactor 140 is connected to the FIA. In one embodiment, the FIA ​​includes a sampling device connected to the bioreactor 140 and configured to obtain cell culture medium or clarified cell culture medium from the bioreactor 140. In one embodiment, an automated system interface is used to connect the bioreactor 140 and the FIA, for example, the Modular Automated Sampling Technology (MAST) sampling system (Lonza, Bend, OR, USA) or the Seg-Flow automated sampling system (BioProcess International Europe, Vienna, Austria). In one embodiment, the sample is obtained manually from the bioreactor and introduced into the holding reservoir of the FIA, for example, by introducing the holding reservoir of the FIA ​​into a container containing the cell culture sample. In one embodiment, a sample having a volume of approximately 20 μL to approximately 1000 μL is introduced into the FIA's retention reservoir. In one embodiment, the sample is obtained from a cell culture. In one embodiment, the sample comprises cell culture medium. In one embodiment, the sample comprises clarified cell culture medium. In one embodiment, the cell culture medium is clarified by centrifugation or filtration. In one embodiment, filtration includes microfiltration or depth filtration. In one embodiment, the sample comprises polypeptides. In one embodiment, host cells in the cell culture are removed from the bioreactor. In one embodiment, host cells in the cell culture are removed during cell culture (e.g., in a perfusion bioreactor). In one embodiment, the sample comprises recombinantly produced polypeptides. In one embodiment, the sample comprises proteins. In one embodiment, the sample comprises recombinantly produced proteins. In one embodiment, the sample comprises recombinantly produced therapeutic proteins.

[0175] 2. Protein capture In one embodiment, the method includes a protein capture step to purify the target protein in a sample in order to separate the target protein from impurities such as host cell proteins or other product-related impurities present in the sample. In one embodiment, the sample includes a clarified cell culture medium from which cells and other cell debris have been removed. In one embodiment, the protein capture step includes affinity chromatography.

[0176] Affinity chromatography refers to a chromatographic process in which a target biomolecule in the mobile phase, such as a protein, is retained by the stationary phase of a chromatography column based on specific and reversible binding interactions between the target component and a compound immobilized on the chromatography matrix. In one embodiment, affinity chromatography includes a column equilibration step, a sample loading step, and a sample elution step. In one embodiment, the chromatography column has a volume of about 50 μL to about 300 μL, or about 50 μL to about 200 μL. In one embodiment, affinity chromatography includes low-pressure affinity chromatography. In one embodiment, affinity chromatography is performed at a flow rate of about 10 μL / second to about 100 μL / second.

[0177] In one embodiment, the method includes a column equilibration step, a loading step, a washing step, and an elution step. In one embodiment, the same buffer is used in one or more of the column equilibration step, the loading step, and the washing step. In one embodiment, different buffers are used in one or more of the equilibration step, the loading step, and the washing step. In one embodiment, the term “binding buffer” is used to refer to the buffer used as the equilibration buffer, the loading buffer, and / or the washing buffer.

[0178] In one embodiment, the protein capture process takes approximately 15 minutes to 2 hours. In another embodiment, the protein capture process takes approximately 15 minutes to 1 hour. In yet another embodiment, the protein capture process takes less than 30 minutes.

[0179] In one embodiment, the column is equilibrated by passing an equilibration buffer over the column before loading the sample. In one embodiment, the equilibration buffer may be referred to as the binding buffer. In one embodiment, the column is equilibrated with approximately 5 to approximately 10 column volumes (CV) of the equilibration buffer. In one embodiment, the column is equilibrated with the equilibration buffer at a flow rate of approximately 10 μL / sec to approximately 200 μL / sec. The binding buffer (or equilibration buffer) may be accessed by the FIA150 via the S201 input port and delivered to the affinity column T105 through the FIA150.

[0180] In one embodiment, the sample is loaded onto an equilibrated column. In one embodiment, the sample is loaded into a loading buffer having the same composition as the equilibration buffer. In one embodiment, the term “binding buffer” refers to a buffer used as both the equilibration buffer and the loading buffer. In one embodiment, approximately 20 μL to approximately 1000 μL of sample is loaded onto the column in the loading buffer. In one embodiment, the sample is loaded onto the affinity column at a flow rate of approximately 10 μL / sec to approximately 200 μL / sec. The sample can be accessed by the FIA ​​105 at the V209 input port 304 and delivered to the affinity column 313 via the V208 input port 304 by the first syringe pump 302.

[0181] In one embodiment, the method includes a column washing step for removing proteins that interact weakly or nonspecifically with the affinity chromatography resin, for example. In one embodiment, the washing buffer has the same composition as the equilibration buffer and / or loading buffer. In one embodiment, the term “binding buffer” refers to the buffer used as the equilibration buffer, loading buffer, and washing buffer. In one embodiment, the washing buffer has a different composition from the equilibration buffer and / or loading buffer. In one embodiment, the loaded column is washed with about 5 to about 10 column volumes (CV) of washing buffer. In one embodiment, the loaded column is washed with washing buffer at a flow rate of about 10 μL / sec to about 200 μL / sec. In one embodiment, the washing buffer is accessed by FIA105 at the S202 syringe pump port 305 and delivered to the affinity column 313 via the S108 syringe pump port 405 by a second syringe pump 402.

[0182] In one embodiment, the target protein is eluted from the column during the elution process. In one embodiment, the target protein is eluted from the column by passing an elution buffer over a column on which the sample is immobilized. In one embodiment, the target protein is eluted from the column using an elution buffer having a different pH or salt concentration than the loading buffer. In one embodiment, elution is a gradient elution, for example, in which the composition of the elution buffer changes linearly over time. In one embodiment, elution is a step elution, for example, in which the composition of the elution buffer remains constant at each step. In one embodiment, the elution time of the sample is predetermined. As used herein, "elution time" refers to the amount of time elapsed between the time the sample is loaded onto the column and the time the sample is eluted from the column. In one embodiment, the elution time is the amount of time elapsed between the time the sample is loaded onto the column and the maximum concentration of the eluate. In one embodiment, the target protein is eluted from the column by applying the elution buffer to the column at a flow rate of approximately 10 μL / sec to approximately 200 μL / sec. In one embodiment, the solution eluted from the column containing the target protein is referred to as the eluate. The elution buffer can be accessed by the FIA105 at the V203 input port 305 and delivered to the affinity column 313 via the V208 input port 304 by the first syringe pump 302.

[0183] In one embodiment, the protein is an antibody, an antibody fragment, or another protein containing an Fc region, such as an Fc fusion protein. In one embodiment, the affinity column contains an immunoglobulin-binding protein. In one embodiment, the affinity column contains an immunoglobulin-binding bacterial protein. In one embodiment, the affinity column contains an immunoglobulin-binding bacterial protein such as protein A, protein G, protein A / G, or protein L. In one embodiment, the affinity column is a protein A column. In one embodiment, the affinity column is a protein G column. In one embodiment, the affinity column is a protein A / G column. In one embodiment, the affinity column is a protein L column. In one embodiment, the column is a protein M column.

[0184] "Protein A chromatography" refers to chromatography involving a specific and reversible interaction between the Fc portion of an immunoglobulin molecule and the IgG-binding domain of protein A. In one embodiment, protein A chromatography includes a column using a protein obtained from Staphylococcus aureas. In another embodiment, protein A chromatography includes a column using a protein produced by recombinant or synthetic methods. In yet another embodiment, protein A chromatography includes a column using a protein A variant that retains the ability to bind to the Fc region of immunoglobulins.

[0185] In one embodiment, the target protein includes an N-terminal or C-terminal tag for facilitating protein purification, which includes, but is not limited to, tags containing streptavidin, biotin, polyhistidine (His), or glutathione-S-transferase (GST).

[0186] In one embodiment, the affinity column matrix includes a monoclonal antibody that specifically binds to recombinantly produced proteins.

[0187] As shown in Figure 7, the eluate obtained from the protein capture step can be transferred to a protein concentration detection unit or a protein digestion step via the operation of the FIA150. In one embodiment, the first sample is purified by affinity chromatography, and the first eluate is transferred to a protein concentration determination unit. The first eluate can be drawn out of the affinity column 313 by a second syringe pump 402 and transferred to a protein concentration determination unit (e.g., a spectrophotometer 414) via a multidirectional connector 412. In another embodiment, the second sample is purified by affinity chromatography using the affinity column 313 to obtain a second eluate. The second eluate is transferred to a protein digestion step. The second eluate can be drawn out of the affinity column 313 by a second syringe pump 402 and transferred to the S105 digestion chamber 415 via the V104 input port 404 of the second valve 401.

[0188] 3.Concentration determination In one embodiment, the eluate from the affinity chromatography step is transferred to a protein detection unit. In one embodiment, the protein detection unit measures the concentration of the sample protein using a spectrophotometer 414. In one embodiment, the transfer of the eluate from the affinity column 313 to the spectrophotometer 414 may be made possible by the operation of a second syringe pump 402, which aspirates the eluate from the affinity column 313 through a multidirectional valve 412 via the S108 syringe pump port 405 and pushes the eluate back to the spectrophotometer 414 through the multidirectional valve 412. In one embodiment, the concentration of the protein in the sample is determined by measuring the UV absorbance. In one embodiment, the concentration of the protein in the sample is determined by measuring the UV absorbance from approximately 190 nm to approximately 280 nm. In one embodiment, the concentration of the protein in the sample is determined by measuring the UV absorbance due to the presence of the amino acids tyrosine and tryptophan. In one embodiment, the protein concentration in the sample is determined by measuring the UV absorbance from approximately 175 nm to approximately 280 nm. In another embodiment, the protein concentration in the sample is determined by measuring the UV absorbance at approximately 280 nm. In yet another embodiment, the protein concentration in the sample is determined by measuring the UV absorbance attributable to the peptide backbone. In yet another embodiment, the protein concentration in the sample is determined by measuring the UV absorbance from approximately 190 nm to approximately 220 nm. In yet another embodiment, the protein concentration in the sample is determined by measuring the UV absorbance at approximately 205 nm. In yet another embodiment, the protein concentration in the sample is determined by a colorimetric assay, for example, using the Bradford method or the Lowry method.

[0189] In one embodiment, the UV absorbance of the first eluate is determined by spectrophotometric method, and the protein concentration is calculated based on a predetermined calibration curve. In one embodiment, the concentration determination process takes approximately 5 to 30 minutes, or less than 10 minutes. UV absorbance data can be measured by the data acquisition manager 254, and in conjunction with the process control manager 252 and the calibration manager 256, the protein concentration can be determined as described above.

[0190] 4. Automated protein digestion in solution In one embodiment, the eluate obtained from the protein capture step is transferred to a digestion chamber. In another embodiment, the second eluate obtained from the protein capture step is transferred to a digestion chamber. In yet another embodiment, the second eluate obtained from the affinity chromatography step is transferred to a digestion chamber. The second eluate can be drawn out from the affinity column 313 by a second syringe pump 402 and transferred to the digestion chamber 415 via the T101 multidirectional valve 412 and the S108 syringe pump port.

[0191] In one embodiment, the proteins in the eluate obtained from the affinity chromatography column are digested to form a peptide mixture for use in an analytical process, such as peptide mapping. In one embodiment, the proteins in the eluate obtained from the affinity chromatography column are digested into peptides with an average size of less than approximately 3000 Da, less than approximately 2500 Da, less than approximately 2000 Da, or less than approximately 1500 Da. In one embodiment, the proteins in the eluate from the affinity chromatography column are digested into peptides with an average size of approximately 500 Da to approximately 5000 Da. In one embodiment, the proteins in the eluate from the affinity column are digested into peptides with an average size of approximately 1000 Da to approximately 5000 Da. In one embodiment, the protein in the eluate from the affinity chromatography column is digested into peptides ranging in average size from about 500 Da, about 750 Da, or about 1000 Da to a maximum of about 1500 Da, 2000 Da, 2500 Da, or about 3000 Da. In one embodiment, the protein in the eluate obtained from the affinity chromatography column is digested to form a peptide mixture containing peptides with lengths of about 2 to about 100 amino acids, at least about 5 or about 10 amino acids, and a maximum of about 15, about 25, about 50, or about 100 amino acids, or about 5 to about 50, or about 10 to about 25, or about 10 to about 15 amino acids.

[0192] Conventional enzymatic protein digestion methods typically include denaturation, reduction, alkylation, buffer exchange, and digestion steps. Denaturation can be achieved by increasing the temperature or by using chemical denaturants, such as chaotropic agents, salts, or surfactants, including but not limited to guanidine hydrochloride and urea. Once the protein is denatured, the disulfide bonds are reduced by adding a reducing agent, such as dithiothreitol (DTT), glutathione, β-mercaptoethanol (β-ME), or tris(2-carboxyethyl)phosphine (TCEP). To prevent the reformation of disulfide bonds, alkylating agents, such as iodoacetic acid, iodoacetamide, acrylamide, or chloroacetamide, are added.

[0193] Reagents used in conventional protein digestion methods, such as chemical denaturants or alkylating agents, can degrade proteases; therefore, these reagents are typically removed before the addition of proteases, for example, using buffer exchange. Once the chemical denaturants and alkylating agents have been removed, proteases can be added to digest the protein. Conventional protein digestion processes are typically carried out at 37°C at a neutral pH and take approximately 12 to 24 hours, generally around 18 hours.

[0194] Traditionally, protein digestion has been stopped by adding acids or other chemicals, such as formic acid (FA), hydrochloric acid (HCl), or acetic acid (HAc); or L-cyanide bromide (CNBr), 2-nitro-5-thiocyanobenzoic acid (NTCB), or hydroxylamine.

[0195] This specification provides a time-efficient and streamlined method for protein digestion. In one embodiment, the protein digestion method is automated and online. While conventional protein digestion processes can take up to 4 hours, in one embodiment, the automated online protein digestion method takes approximately 5 minutes to a maximum of approximately 1 hour. In one embodiment, protein digestion takes approximately 5 minutes to approximately 30 minutes. In one embodiment, protein digestion takes approximately 15 minutes to approximately 30 minutes. In one embodiment, protein digestion takes less than approximately 30 minutes. In one embodiment, the online protein digestion method does not include alkylation or buffer exchange steps.

[0196] In one embodiment, protein denaturation, reduction, and digestion are carried out in the same reaction mixture. In one embodiment, protein denaturation, reduction, and digestion are carried out in the same digestion chamber. In one embodiment, protein denaturation, reduction, and digestion are carried out simultaneously. In one embodiment, the reducing agent is introduced into the digestion chamber 415 via the S103 syringe pump port. In one embodiment, approximately 5 μL to approximately 20 μL of the reducing agent is introduced into the digestion chamber 415 via the S103 syringe pump port. In one embodiment, a protease, such as trypsin, is introduced into the digestion chamber 415 via the S102 syringe pump port. In one embodiment, approximately 5 μL to approximately 20 μL of the protease is introduced into the digestion chamber 415 via the S102 syringe pump port. In one embodiment, approximately 5 μL to approximately 20 μL of trypsin is introduced into the digestion chamber 415 via the S102 syringe pump port. In one embodiment, the digestion buffer is introduced into the digestion chamber 415 via the V104 input port 404 of the second valve 401. In another embodiment, the digestion buffer is introduced into the digestion chamber 415 via the V111 valve output port 408, the S109 syringe pump port, and the S105 syringe pump port. In another embodiment, approximately 100 μL to 400 μL of digestion buffer is introduced into the digestion chamber 415. In another embodiment, the reagent is combined with the second eluate in the digestion chamber using syringe 402 406. In another embodiment, the protein in the second eluate is denatured in the digestion chamber 415.

[0197] In one embodiment, protein digestion is carried out at a high temperature. In one embodiment, the protein is denatured using high-temperature denaturation. In one embodiment, protein digestion is carried out using a heat-stable protease. In one embodiment, protein digestion is carried out at a high temperature, for example, at a temperature of about 35°C to about 85°C. In one embodiment, protein digestion is carried out at a temperature of at least about 35°C, about 40°C, about 45°C, about 50°C, about 60°C, or about 70°C, and up to a maximum of about 75°C, about 80°C, or about 85°C. In one embodiment, protein digestion is carried out at a temperature of at least about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C, and up to a maximum of about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In one embodiment, protein digestion is carried out at a temperature of about 50°C to about 85°C. In one embodiment, protein digestion is carried out at a temperature of approximately 65°C to approximately 80°C. In another embodiment, protein digestion is carried out at a temperature of approximately 70°C to approximately 75°C. In yet another embodiment, protein digestion is carried out at a temperature of approximately 70°C to approximately 72°C.

[0198] In one embodiment, protein digestion includes contacting a protein solution with a digestion reagent to form a digestion mixture. In one embodiment, the digestion reagent includes a digestion buffer, a reducing agent, and a proteolytic enzyme. In one embodiment, a digestion mixture is formed by contacting an eluate from an affinity column with a digestion reagent. In one embodiment, a digestion mixture is formed by contacting an eluate from an affinity column with a digestion reagent containing a digestion buffer, a reducing agent, and a proteolytic enzyme. In one embodiment, a digestion mixture is formed by contacting a second eluate from an affinity column with a digestion buffer, a reducing agent, and a proteolytic enzyme.

[0199] In one embodiment, the digestion buffer has a pH of approximately 6.0 to approximately 8.0, or approximately 6.5 to approximately 7.5. In one embodiment, the digestion buffer has a pH starting from approximately 6.5. In one embodiment, the digestion buffer is from the Thermo Fisher SMART digestion kit (Thermo Fisher, Waltham, MA, USA).

[0200] In one embodiment, the reducing agent includes tris(2-carboxyethyl)phosphine (TCEP).

[0201] In one embodiment, the protease includes, for example, trypsin, chymotrypsin, pepsin, thermolysin, papain, pronase, endopeptidase Arg-C, peptidyl-Asp metalloendopeptidase (endopeptidase Asp-N), glutamyl endopeptidase (Glu-C endopeptidase), and lysyl endopeptidase (Lys-C endopeptidase). In one embodiment, the protease includes trypsin. In one embodiment, the protease includes heat-stable trypsin. In one embodiment, the protease includes SMART trypsin (Thermo Fisher Scientific, Waltham, MA, USA).

[0202] In one embodiment, the reagents used for protein digestion do not interfere with mass spectrometry (MS). For example, ionization in MS may be hindered by the presence of contaminants in the sample, including but not limited to salts and / or surfactants, particularly high concentrations of salts and / or surfactants. In one embodiment, the reagents used for protein digestion do not interfere with the ionization process in MS. In one embodiment, the protein digestion method does not include a buffer exchange step.

[0203] Alternatively, the protein digestion method includes a buffer exchange step. In one embodiment, the protein digestion method includes one or more reagents that may interfere with ionization in MS, including, but not limited to, chemically denaturing reagents removed using the buffer exchange step, such as chaotropic agents such as guanidine hydrochloride and urea; reducing agents such as dithiothreitol (DTT) glutathione or β-mercaptoethanol (β-ME); or alkylating agents such as iodoacetic acid, iodoacetamide, acrylamide, or chloroacetamide. In one embodiment, the buffer exchange reagent is delivered to the digestion chamber via a multiport valve. In one embodiment, the digestion method is carried out at room temperature, although the process will take longer at room temperature.

[0204] In one embodiment, the amount of proteolytic enzyme used in the digestion mixture of the second eluate is determined based on the protein concentration of the first eluate. In one embodiment, the second eluate is brought into contact with trypsin at a trypsin:protein ratio of approximately 1:10 to approximately 1:100. In another embodiment, the second eluate is brought into contact with trypsin at a trypsin:protein ratio of approximately 1:20 to approximately 1:30. In one embodiment, the trypsin:protein ratio is at least about 1:10 and at most about 1:100, or at least about 1:10 or 1:20 and at most about 1:30, about 1:40, about 1:50, or about 1:100, or about 1:10 to about 1:100, about 1:10 to about 1:50, about 1:10 to about 1:40, about 1:10 to about 1:30, about 1:10 to about 1:20, about 1:20 to about 1:100, about 1:20 to about 1:50, about 1:20 to about 1:40, or about 1:20 to about 1:30.

[0205] In one embodiment, the second eluate is brought into contact with the digestion buffer, reducing agent, and protease simultaneously. As used herein, “simultaneously,” when used in connection with the steps of the method herein, means that the steps are carried out substantially concurrently (i.e., the execution of at least part of one method step overlaps in time with the execution of part of another method step). “Simultaneously” does not require strictly parallel actions; that is, all method steps do not need to start or finish at the same time. In one embodiment, “simultaneously” may mean that all reagents required for the method step are combined in the same reaction mixture so that the reaction occurs in the same reaction volume and during the same incubation period. In one embodiment, the digestion mixture comprises the digestion buffer, reducing agent, protease, and the second eluate. In one embodiment, the digestion mixture is prepared by introducing the second eluate and other reagents into the chamber of a syringe, and the digestion mixture is mixed by pushing down and retracting the plunger of the syringe. In one embodiment, the digestion chamber is used as an incubation chamber. For example, the second eluate can be transferred from the affinity column 313 to the chamber of the drive syringe 406, as described above. The drive syringe 406 can further access the protease via the S102 syringe pump port 405. The second eluate and protease, along with any other suitable reagents, can then be mixed in the chamber of the drive syringe 406.

[0206] In one embodiment, the second eluate is sequentially contacted with a digestion buffer, a reducing agent, and a proteolytic enzyme. As used herein, “sequentially” means, when used in relation to the steps of the method herein, a method in which the steps are carried out at different points in time, for example, a method in which separate events occur in the practice of the method. In one embodiment, the sequential steps are carried out during separate incubation periods. In one embodiment, the sequential steps are carried out in different reaction mixtures. In one embodiment, the sequential method steps are carried out at different times, but in the same reaction chamber.

[0207] In one embodiment, the temperature of the incubation chamber can be raised, for example, to incubate the digested mixture at a temperature of about 35°C to about 85°C. In one embodiment, the digested mixture is incubated at a temperature of at least about 35°C, about 40°C, about 45°C, about 50°C, about 60°C, or about 70°C, and up to a maximum of about 75°C, about 80°C, or about 85°C. In one embodiment, the digested mixture is incubated at a temperature of at least about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C, and up to a maximum of about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In one embodiment, the digested mixture is incubated at a temperature of about 50°C to about 85°C. In one embodiment, the digested mixture is incubated at a temperature of about 65°C to about 80°C. In one embodiment, the digested mixture is incubated at a temperature of about 70°C to about 55°C. In one embodiment, the digested mixture is incubated at a temperature of approximately 70°C to 72°C. The temperature of the digestion chamber can be controlled as a process parameter, for example, via a process control device.

[0208] In one embodiment, incubation of the digestion mixture includes incubation at a temperature of about 36°C to about 85°C for about 5 minutes to about 30 minutes. In one embodiment, incubation of the digestion mixture includes incubation at a temperature of about 50°C to about 75°C for about 15 minutes to about 30 minutes. In one embodiment, incubation of the digestion mixture includes incubation at a temperature of about 70°C to about 72°C for about 15 minutes to about 30 minutes.

[0209] In one embodiment, the digestion method comprises two steps: In one embodiment, the first digestion mixture is incubated at a temperature of approximately 36°C to approximately 85°C for approximately 5 minutes to approximately 30 minutes. In one embodiment, the first digestion mixture is incubated at a temperature of approximately 50°C to approximately 75°C for approximately 15 minutes to approximately 30 minutes. In one embodiment, the first digestion mixture is incubated at a temperature of approximately 70°C to approximately 72°C for approximately 15 minutes to approximately 30 minutes. In one embodiment, additional proteolytic enzymes and / or digestion buffers are added to the first digestion mixture to form a second digestion mixture. In one embodiment, the second digestion mixture is incubated at a temperature of approximately 36°C to approximately 85°C for approximately 5 minutes to approximately 30 minutes. In one embodiment, the second digestion mixture is incubated at a temperature of approximately 50°C to approximately 75°C for approximately 15 minutes to approximately 30 minutes. In one embodiment, the second digestion mixture is incubated at a temperature of approximately 70°C to approximately 72°C for approximately 15 minutes to approximately 30 minutes.

[0210] G peptide mapping analysis In one embodiment, the peptide mixture is transferred to an analyzer. In one embodiment, the analyzer includes a peptide mapping unit. In one embodiment, as schematically shown in Figure 8, the peptide mapping unit includes three components: a peptide separation component, a detection or data acquisition unit, and a data processing unit. In one embodiment, the data acquisition unit includes an ion source and a mass spectrometer. For example, the peptide mixture can be aspirated from the digestion chamber 415 to the analyzer, such as the peptide mapping unit, via the V108 input valve 404 by the operation of a second syringe pump 402.

[0211] In one embodiment, an automated peptide mapping system is used. Automated peptide mapping systems are commercially available and include, for example, the BioAccord Peptide Mapping System (Waters Corporation, Milford, MA, USA).

[0212] In one embodiment, peptide mapping takes approximately 30 minutes to approximately 2 hours, or less than 1 hour.

[0213] In one embodiment, the peptide mapping unit includes a peptide separation component. In one embodiment, the peptide separation component includes a chromatography column. In one embodiment, peptides in a peptide mixture are separated by column chromatography. In one embodiment, column chromatography includes liquid chromatography (LC). Liquid chromatography includes, but is not limited to, ultra-high-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC). In one embodiment, peptides in a peptide mixture are separated by UPLC. In one embodiment, peptides in a peptide mixture are separated by HPLC.

[0214] In one embodiment, the peptide mapping unit includes a data acquisition unit. In one embodiment, the data acquisition unit includes a mass spectrometer (MS). In one embodiment, the data acquisition unit includes an ion source and a mass spectrometer. In one embodiment, the data acquisition unit includes a peptide separator, an ion source, and a mass spectrometer. See Figure 8.

[0215] In one embodiment, an ion source ionizes peptides in a peptide mixture. Methods for ionizing peptides are known and include, but are not limited to, electrospray ionization (ESI).

[0216] In one embodiment, the mass spectrometer separates gas-phase ions by mass-to-charge ratio (m / z). In one embodiment, the mass spectrometer includes tandem mass spectrometry (MS / MS), which includes two or more steps of mass spectrometry in which fragmentation is performed during the mass spectrometry process.

[0217] In one embodiment, the peptide mapping unit includes a data processing unit. In one embodiment, the data processing unit includes a processor that processes data from a data acquisition unit to identify and / or quantify post-translational modifications (PTMs) of proteins. In one embodiment, peptides identified by a mass spectrometer are used as surrogate samples of intact proteins obtained from cell culture media. In one embodiment, the data processing provides information about the amino acid sequence of the protein, and / or the presence, location, and / or quantity of one or more PTMs. In one embodiment, the data processing unit provides information about a given PTM. In one embodiment, the data processing takes about 5 to 30 minutes, or less than 10 minutes. In one embodiment, the data processing unit of the peptide mapping unit can interface with and / or communicate with a process control device 200. In some embodiments, the functions of the data processing unit of the peptide mapping unit may be performed by software operating on the process control device 200 without requiring the data processing unit.

[0218] H. Modification of post-translational modifications / Modification of cell culture conditions In one embodiment, a method for producing a recombinant protein is provided. In one embodiment, the method comprises: culturing host cells under conditions in which the recombinant protein is expressed; detecting post-translational modifications (PTMs) of the protein according to the method described herein; and modifying one or more cell culture parameters based on the detected PTMs. In one embodiment, a recombinant protein produced by the method described herein is provided. In one embodiment, a pharmaceutical composition is provided comprising a recombinant protein produced by the method described herein and a pharmaceutically acceptable carrier.

[0219] In one embodiment, one or more of the following cell culture parameters: pH; CO2 level; dissolved oxygen (dO2); temperature; amount or type of nutrients; presence and type of glycan precursors; or combinations thereof, are modified based on the PTM detected by the method described herein. In one embodiment, one or more cell culture parameters are adjusted in "real time" based on the PTM detected by the method described herein. In one embodiment, one or more cell culture parameters are adjusted within the cell culture or bioreactor from which the sample was taken. In one embodiment, one or more cell culture parameters are adjusted within the cell culture or bioreactor within approximately less than 1 day, less than 12 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2.5 hours after the sample was taken from the cell culture or bioreactor.

[0220] I. Incorporation by reference All references cited herein (including patents, patent applications, articles, textbooks, etc.), and references cited therein, are incorporated herein by reference in their entirety, to the extent that they have not already been cited. For example, this disclosure provides the following embodiments. [1] A method for detecting post-translational modifications of proteins, performed by a flow injection analyzer (FIA) controlled by at least one processor, (a) Containing the first sample containing the protein in a holding reservoir; (b) Delivering a first volume of binding buffer to the affinity column in the FIA ​​via the FIA; (c) Transferring the first sample from the holding reservoir to the affinity column via the FIA; (d) Delivering a first volume of elution buffer to the affinity column via the FIA, thereby eluting the protein in the first sample from the affinity column to form a first eluate; (e) Transferring the first eluate to the protein concentration detection unit via the FIA; (f) Determining the concentration of the protein in the first eluate using at least one processor; (g) Containing the second sample containing the protein in the retention reservoir; (h) Delivering a second volume of the binding buffer to the affinity column via the FIA; (i) Transferring the second sample from the holding reservoir to the affinity column via the FIA; (j) Delivering a second volume of elution buffer to the affinity column via the FIA, thereby eluting the protein from the second sample from the affinity column to form a second eluate; (k) Transferring the second eluate to the digestion chamber via the FIA; (l) Delivering a digestion reagent via the FIA ​​to digest the protein in the second eluate and form a peptide mixture; and (m) Transferring the peptide mixture to a peptide mapping unit via the FIA ​​and detecting the post-translational modification of the protein, Methods that include... [2] The method according to claim 1, wherein the FIA ​​is a sequential injection analyzer (SIA) or a direct injection analyzer (DIA). [3] The method according to 1, wherein the first sample and the second sample are obtained from a cell culture. [4] The method according to 3, wherein the cell culture is located in a bioreactor. [5] The method according to 4, wherein the bioreactor includes a batch bioreactor, a fed-boil bioreactor, or a perfusion bioreactor. [6] The method according to claim 1, wherein the affinity column includes a protein A column. [7] The method according to (f), wherein determining the concentration of the protein in the first eluate is performed by the at least one processor according to a predetermined calibration curve. [8] The method according to (d), further comprising controlling the elution time of the protein in the first sample eluted from the affinity column for forming the first elution of (d) by the at least one processor based on a predetermined elution time. [9] The method according to 8, further comprising controlling by the at least one processor the elution time of the protein in the second sample eluted from the affinity column for forming the second elution of (j) based on the same predetermined elution time as the first sample of (d).

[10] The method according to any one of claims 1 to 9, wherein the protein concentration detection unit includes a spectrophotometer.

[11] The method according to 10, wherein the spectrophotometer measures the UV absorbance of the first eluate and the at least one processor calculates the protein concentration based on a calibration curve.

[12] The method according to any one of claims 1 to 11, wherein the protein in the second eluate is enzymatically digested with (l).

[13] The method according to 12, wherein the enzymatic digestion of the protein comprises contacting the second eluate with a proteolytic enzyme selected from trypsin, chymotrypsin, pepsin, thermolysin, papain, pronase, endopeptidase Arg-C, peptidyl-Asp metalloendopeptidase (endopeptidase Asp-N), glutamyl endopeptidase (Glu-C endopeptidase), and lysyl endopeptidase (Lys-C endopeptidase) via the FIA.

[14] The method according to 13, wherein the proteolytic enzyme comprises trypsin.

[15] The method according to 14, wherein the proteolytic enzyme comprises heat-stable trypsin.

[16] (l) (i) Introducing a digestive reagent via the FIA ​​to form a digestive mixture; (ii) Mixing the digested mixture; and (iii) Incubate the digested mixture, The method described in any one of the above 12 to 15, including the method described in the above 12 to 15.

[17] The method according to 16, wherein the digestion buffer comprises a digestion buffer with a pH of approximately 6.0 to approximately 7.5.

[18] The method according to 16 or 17, wherein the digestion reagent comprises a reducing agent containing tris(2-carboxyethyl)phosphine (TCEP).

[19] The method according to any one of claims 16 to 18, wherein the digestive reagent comprises a proteolytic enzyme.

[20] The method according to any one of claims 16 to 19, wherein the second eluate is brought into contact with a protease in an amount determined by the at least one processor based on the concentration of the first eluate determined by (g). [twenty one] The method according to 20, wherein the proteolytic enzyme includes trypsin. [twenty two] The method according to 20 or 21, wherein the second eluate is brought into contact with trypsin in a trypsin:protein ratio of approximately 1:10 to approximately 1:100. [twenty three] The method according to 20 or 21, wherein the second eluate is brought into contact with trypsin in a trypsin:protein ratio of approximately 1:20 to approximately 1:30. [twenty four] The method according to any one of claims 16 to 23, wherein incubation of the digested mixture includes incubation at a temperature of about 36°C to about 85°C for about 5 minutes to about 30 minutes. [twenty five] The method according to any one of claims 16 to 24, wherein incubation of the digested mixture comprises incubation at a temperature of about 50°C to about 75°C for about 15 minutes to about 30 minutes.

[26] The method according to any one of claims 16 to 25, wherein incubation of the digested mixture comprises incubation at a temperature of about 70°C to about 72°C for about 15 minutes to about 30 minutes.

[27] The proteolytic enzyme includes heat-stable trypsin, and the method is (i) Introducing a first amount of digestion buffer, reducing agent, and thermostable trypsin into the second eluate via the FIA ​​and mixing them to form a first digestion mixture; (ii) Incubate the first reaction mixture at a temperature of approximately 70°C to approximately 75°C for approximately 10 to 20 minutes to form the first incubation mixture; (iii) introducing a second amount of thermostable trypsin into the incubation mixture via the FIA ​​and mixing it to form a second digestion mixture; and (iv) Incubate the second digestion mixture at a temperature of approximately 70°C to approximately 75°C for approximately 10 to 20 minutes to form a peptide mixture. The method according to 16, including the method described above.

[28] The method according to 27, wherein a second amount of digestion buffer is added to the first incubation mixture together with the second amount of thermostable trypsin of (iii) via the FIA.

[29] The method according to any one of claims 1 to 28, wherein the peptide mapping unit includes a mass spectrometer (MS).

[30] The method according to any one of claims 1 to 29, wherein the detection of post-translational modifications includes peptide mapping analysis performed using mass spectrometry (MS).

[31] The method according to 30, wherein detection of post-translational modifications includes peptide mapping analysis performed using liquid chromatography / mass spectrometry (LC / MS).

[32] The method according to 31, wherein the liquid chromatography includes ultra-high performance liquid chromatography (UPLC).

[33] The method described in any one of items 1 to 32 above, which is carried out online.

[34] The method according to any one of claims 1 to 33, wherein the post-translational modification includes glycosylation, oxidation, deamidation, isomerization, saccharification, N-terminal leader sequence, N-terminal cyclization, C-terminal lysine retention, amide formation, or a combination thereof.

[35] The method according to 34, wherein the glycosylation includes N-linked glycosylation, O-linked glycosylation, or a combination thereof.

[36] A method for producing recombinant proteins, (a) Culturing host cells under conditions in which the recombinant protein is expressed; (b) detecting the post-translational modification of the protein in accordance with the method described in any one of items 1 to 35 above; (c) Modify one or more cell culture parameters based on the post-translational modifications detected in (b), Methods that include...

[37] The method according to 36, wherein the host cells are cultured in a bioreactor including a batch bioreactor, a fed-boil bioreactor, or a perfusion bioreactor.

[38] The method according to 36 or 37, wherein the host cell is a mammalian host cell.

[39] The method according to 38, wherein the host cell is a CHO, HEK 293, COS, NS0, SP2, or PER.C6 host cell.

[40] One or more of the following cell culture parameters: pH; CO2 level; dissolved oxygen amount (dO2); temperature; amount or type of nutrient; presence and type of glycan precursor; or a combination thereof are modified based on the post-translation modification detected in (b), the method according to any one of items 36 to 39 above.

[41] The method according to any one of items 1 to 40 above, wherein the protein contains at least about 1000 amino acid residues.

[42] The method according to any one of items 1 to 41 above, wherein the protein is recombinantly produced.

[43] The method according to any one of items 1 to 42 above, wherein the protein contains a glycoprotein.

[44] The method according to any one of items 1 to 43 above, wherein the protein contains a therapeutic protein.

[45] The method according to any one of items 1 to 44 above, wherein the protein contains a therapeutic antibody or an antigen-binding fragment thereof.

[46] The method according to any one of items 1 to 44 above, wherein the protein contains a fusion protein.

[47] A recombinant protein produced by the method according to item 35 above.

[48] A pharmaceutical composition comprising the recombinant protein according to item 46 above and a pharmaceutically acceptable carrier.

[49] An online method for monitoring post-translational modification of a protein, performed by a flow injection analyzer (FIA) including a syringe pump and a multiport valve controlled by at least one processor, (a) accommodating a first sample containing the protein in the holding reservoir of the FIA; (b) introducing a first volume of binding buffer into an affinity column in the FIA by operation of the multiport valve; (c) The syringe pump operates to advance the first sample from the holding reservoir to the affinity column, thereby binding the protein in the first sample to the affinity column; (d) The operation of the multiport valve introduces a first volume of elution buffer into the affinity column, thereby eluting the bound protein and forming a first eluate; (e) The syringe pump is used to advance the first eluate to the spectrophotometer in the FIA; (f) The concentration of the protein in the first eluate is determined by at least one processor according to the measured UV absorbance; (g) Containing the second sample containing the protein in the retention reservoir; (h) The operation of the multiport valve introduces a second volume of the binding buffer into the affinity column; (i) The syringe pump operates to advance the second sample from the holding reservoir to the affinity column, thereby binding the protein in the second sample to the affinity column; (j) The operation of the multiport valve introduces a second volume of elution buffer into the affinity column, thereby eluting the bound protein and forming a second eluate; (k) The operation of the syringe pump to advance the second eluate into the digestion chamber within the FIA; and (l) The operation of the multiport valve introduces a digestion reagent into the second eluate in the digestion chamber, digests the protein, and forms a peptide mixture; and (m) The operation of the syringe pump advances the peptide mixture to the peptide mapping unit and detects the post-translational modification of the protein. Online methods, including those mentioned above.

[50] (m) digests the aforementioned protein, (i) The operation of the multiport valve introduces the digestion buffer, reducing agent, and proteolytic enzyme into the second eluate in the digestion chamber, and mixes them to form the first digestion mixture; (ii) Incubating the first digestion mixture in the digestion chamber to form a first incubation mixture; (iii) By operating the multiport valve, a second volume of digestion buffer and proteolytic enzyme is introduced into the incubation mixture to form a second digestion mixture; and (iv) Incubating the second digestion mixture in the digestion chamber to form a peptide mixture, The method described in 49, including the method described above.

[51] The method according to 50, wherein the digestion buffer has a pH of approximately 6.0 to approximately 7.5.

[52] The method according to 50 or 51, wherein the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

[53] The method according to any one of claims 49 to 52, wherein the concentration of the protein in the first eluate of (f) is performed by the at least one processor according to a predetermined calibration curve.

[54] The method according to any one of claims 49 to 53, further comprising controlling the elution time of the protein in the first sample eluted from the affinity column for forming the first elution in (d) by the at least one processor based on a predetermined elution time.

[55] The method according to 41, further comprising controlling by at least one processor the elution time of the protein in the second sample eluted from the affinity column for forming the second elution of (j) based on the same predetermined elution time as the first sample of (d).

[56] The method according to any one of claims 49 to 55, wherein the amount of proteolytic enzyme introduced in (i) is determined by the at least one processor based on the concentration of the first eluate determined in (g).

[57] The method according to any one of claims 49 to 52, wherein the proteolytic enzyme comprises trypsin.

[58] The method according to 57, wherein the trypsin includes heat-stable trypsin.

[59] The method according to 57 or 58, wherein the second eluate is brought into contact with trypsin in a trypsin:protein ratio of approximately 1:10 to approximately 1:100.

[60] The method according to 57 or 58, wherein the second eluate is brought into contact with trypsin in a trypsin:protein ratio of approximately 1:20 to approximately 1:30.

[61] The method according to any one of the 50 to 60, wherein the first digested mixture of (ii) is incubated at a temperature of about 36°C to about 85°C for about 5 minutes to about 30 minutes.

[62] The method according to any one of claims 50 to 61, wherein the first digested mixture of (ii) is incubated at a temperature of about 50°C to about 75°C for about 15 minutes to about 30 minutes.

[63] The method according to any one of claims 50 to 62, wherein the first digested mixture of (ii) is incubated at a temperature of about 70°C to about 72°C for about 15 minutes to about 30 minutes.

[64] The method according to any one of claims 50 to 63, wherein the second digestion mixture of (iv) is incubated at a temperature of about 36°C to about 85°C for about 5 minutes to about 30 minutes.

[65] The method according to any one of claims 50 to 64, wherein the second digested mixture of (iv) is incubated at a temperature of about 50°C to about 75°C for about 15 minutes to about 30 minutes.

[66] The method according to any one of items 50 to 65, wherein the second digestion mixture in (iv) is incubated at a temperature of about 70°C to about 72°C for about 15 minutes to about 30 minutes.

[67] The proteolytic enzyme includes thermostable trypsin, and the method includes (i) introducing, by operation of the multiport valve, a first amount of digestion buffer, reducing agent, and thermostable trypsin into the second eluate and mixing to form a first digestion mixture; (ii) incubating the digestion mixture at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a first incubated mixture; (iii) introducing, by operation of the multiport valve, a second amount of digestion buffer and thermostable trypsin into the incubated mixture and mixing to form a second digestion mixture; and (iv) incubating the second digestion mixture at a temperature of about 70°C to about 75°C for about 10 minutes to about 20 minutes to form a peptide mixture, The method according to item 50, comprising

[68] The method according to any one of items 49 to 67, wherein the protein contains at least about 1000 amino acid residues.

[69] The method according to any one of items 49 to 68, wherein the peptide mixture contains peptides containing less than about 100 amino acids.

[70] An online system for monitoring post-translational modification of a protein, comprising (a) a holding reservoir configured to accommodate a first sample and a second sample containing the protein; (b) a first syringe pump configured to advance the first sample and the second sample from the holding reservoir through a flow injection analyzer (FIA); (c) a first multiport valve in fluid communication with the first syringe pump; (d) an affinity column configured to be in fluid communication with the first multiport valve and to contain the first and second samples from the holding reservoir; (e) A spectrophotometer configured to contain a first eluate from the affinity column, wherein the first eluate contains protein from the first sample, and the spectrophotometer is configured to determine UV absorbance and calculate the protein concentration of the first sample based on a predetermined calibration curve; (f) A second syringe pump configured to advance a second eluate from the affinity column into a digestion chamber, wherein the second eluate contains protein from the second sample, and the protein in the second eluate is digested based on the protein concentration of the first sample determined in (e); and (g) A second injection multiport valve in fluid communication with the second syringe pump, Online systems, including those mentioned above. [Examples]

[0221] Example 1. Automated sample preparation Recombinant cells expressing the sample IgG1 monoclonal antibody (IgG1 mAb) were cultured in a perfusion bioreactor. Cell culture medium (CM) was processed in the perfusion bioreactor to remove cells and cell debris, obtaining clarified cell culture medium (CCM). This was introduced into the holding reservoir of a ProSIA dual-pump injection analyzer (FIAlab® Instruments, Inc., Seattle, WA, USA). Proprietary SIAsoft® software (FIAlab® Instruments, Inc., Seattle, WA, USA) was used for system component control and data acquisition and analysis. Samples and reagents were introduced via two syringe pumps (syringe pump 1 and syringe pump 2) and their associated 10-port valves (valve 1 and valve 2, respectively).

[0222] 0.3 mL of the first sample of CCM was obtained from the holding reservoir and injected via valve 1 of the FIA ​​into a Protein A affinity column pre-treated with equilibration buffer. The IgG1 mAb of the first sample was eluted from the Protein A column as follows: 500 μL of binding buffer was injected into the affinity column at 50 μL / sec to 100 μL / sec. 300 μL of CM was injected at 50 μL / sec to 100 μL / sec. 1 mL of binding buffer was injected for washing at 50 μL / sec to 100 μL / sec. 100 to 300 μL of elution buffer was injected at 20 μL / sec.

[0223] The eluate was collected and transferred to a UV spectrometer via FIA, where the absorbance at 280 nm was measured to determine the concentration of IgG1 mAb in the first sample.

[0224] 300 μL of the second CCM sample (or the same volume as the first CCM sample) was obtained from the holding reservoir and injected via valve 1 of the FIA ​​into a protein A affinity column pre-treated with equilibration buffer. IgG1 mAb from the second sample was eluted from the protein A column using the gradient elution method described above. The eluate was collected and transferred via the FIA ​​to the barrel of syringe pump 2.

[0225] 0.01 mL of tris(2-carboxyethyl(carboxyehtyl))phosphine (TCEP) (concentration 500 mM), 0.3 mL of digestion buffer, and trypsin were injected into the barrel of syringe pump 2 via valve 2 of the FIA. The amount of trypsin added was calculated based on the protein concentration determined for the first sample. The digestion reagents were then mixed by pushing down and retracting the syringe plunger, and the mixture was incubated at 70°C for 15 minutes to obtain a sample containing trypsin digestion peptide ("trypsin peptide").

[0226] Example 2. Online peptide mapping The peptide mapping of the trypsin peptide from the FIA ​​sample preparation of Example 1 was performed by liquid chromatography coupled with UV detection and mass spectrometry using the Waters BioAccord® integrated peptide mapping system (including the ACQUITY® ULC® I-Class PLUS system, which features an in-line ultraviolet (TUV) detector connected to an ACQUITY® RDa® mass spectrometer).

[0227] The chromatographic conditions were as follows: temperature 65°C; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution; gradient elution (100%~40%); and flow rate: 0.2 ml / min. The analysis time for mobile phase A was 40 minutes. UV detection was performed at 220 nm.

[0228] For mass spectrometry, peptides were detected in the mass range of 50–2000 m / z using MS and tandem MS (MS / MS) detection.

[0229] Example 3. Determination of protein concentration The feasibility of determining the protein concentration in the protein A eluate was evaluated.

[0230] Calibration curves were created by injecting reference standards (RS) of known concentrations into affinity columns in known volumes. A blank, calibrator 1 (RS 0.1 mg / mL), calibrator 2 (RS 0.5 mg / mL), calibrator 3 (RS 1.0 mg / mL), and calibrator 4 (RS 2.0 mg / mL) were injected separately into the affinity column. UV response readings were recorded in the built-in calibrator table. Calibration curves were created using the calculated slopes and coefficients. Calibration data were saved as a calibration file.

[0231] Before performing the digestion process, the calibration file was opened within the FIA ​​software. Next, a cell culture medium sample of unknown concentration was injected into the affinity column. The UV response was obtained and entered into the calibration file to calculate the protein concentration. During the execution of the digestion method, live feedback of titer information was captured and fitted to the enzyme calculation values.

[0232] The sample calibration curve is shown in Figure 9A, with concentration on the x-axis and the corresponding UV response on the y-axis. Each calibration point on the left-hand calibration curve represents a single UV measurement of a reference standard (RS) at a known concentration of the IgG sample. The UV profile of the affinity column is shown in the right panel. The calibration curve was constructed using four different concentrations of reference standard (RS). The slope of the calibration curve was calculated using a first-order polynomial fitted model.

[0233] Figure 9B shows the UV profile used to determine the concentration of the first eluate, which indicates that the same amount of protein was captured in the second eluate, which was later used for protein digestion.

[0234] Example 4. Glycan profiling: Online method compared to glycan release. The glycan trends determined using online peptide mapping were compared with the results of glycan trends using a conventional glycan release method (2-aminobenzamide (oligo2-AB) glycan detection). Briefly, the glycosylation profile of sample monoclonal IgG1 was determined using the online peptide mapping method described in Example 1 and the ProZyme 2AB labeling kit (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions.

[0235] In short, IgG1-containing samples were treated with PNGase F enzyme to cleave glycans from the protein backbone. The released glycans were purified and collected using a solid-phase extraction preparation kit. After preparing 2AB labeling reagents, they were combined with the glycans and incubated at 65°C for 3 hours. The labeled glycans were then washed, the access reagents were removed, and the mixture was dried.

[0236] The glycosylation of 10 glycans (G0, G0F, G0F-GlcNAc, G0-GlcNAc, G1, G1F, G2F, G2f+SA, Man5, and Man6) was determined using the online peptide mapping method and the Oligo2-AB glycan release method described in Example 1. As shown in Figure 10A, the glycosylation profiles determined using the online method showed a similar trend to the glycosylation profiles determined using the Oligo2AB procedure. Figure 10B shows the quantification percentage of one of the glycan species (mannose) monitored during the cell culture period. Repeated experiments at each time point yield a consistent level of quantification percentage.

[0237] Example 5. Glycan profiling: Online method compared to glycan release. Following the procedure described in Example 4, the glycan trend was determined for sample IgG1 mAb obtained from four different bioreactors (a total of 12 time points) at three time points using online peptide mapping and oligo-2-AB glycan release methods. A direct linear relationship was observed between the two methods, indicating that the results were equivalent for the type of glycosylation tested.

[0238] Figures 11A-E show the correlation between two methods for five types of glycans: G0F (CC=0.9322); G1F (CC=0.9229); Man 5 (CC=0.9979); C0F-GN (CC=0.9938); and G2F+SA (CC=0.8488, within the assay variability and limit of quantification (LOQ) range).

[0239] Example 6. Detection of post-translational modifications Oxidation is a common post-translational modification observed at various methionine sites within monoclonal antibodies. Methionine oxidation is an important product quality characteristic to monitor during the cell culture process. Figure 12A shows that oxidation levels at specific methionine residues were maintained below 2% when measured on days 7, 9, and 11 of cell culture, compared to a reference standard (RS).

[0240] N-terminal cyclization is a common post-translational modification found in the N-terminal sequences of antibody heavy and light chains. Figure 12B shows that the level of N-terminal cyclization was maintained at over 95% when measured at days 7, 10, and 14 of cell culture. Repeated experiments at each time point demonstrate a consistent level of modification in the heavy and light chains.

[0241] Example 7. Site-specific glycosylation Unlike the conventional oligo-2-AB glycan release method described in Example 2, the online peptide mapping method described in Example 1 can be used to obtain site-specific glycosylation information.

[0242] When a molecule has two or more glycosylation sites, peptide mapping analysis can provide information on site-specific glycosylation. For example, the amino acid that undergoes glycosylation can be determined at the peptide level, providing information on site-specific glycosylation of an intact protein. In contrast, in the glycan release method, all site-specific information regarding glycosylation is lost when the glycan is cleaved from the protein for analysis.

Claims

1. A method for detecting post-translational modifications of a protein, which is performed by a flow injection analyzer (FIA) controlled by at least one processor, (a) Containing the sample containing the protein in a holding reservoir; (b) Delivering a first volume of binding buffer to the affinity column in the FIA ​​via the FIA; (c) Transferring a first volume of the sample from the holding reservoir to the affinity column via the FIA; (d) Delivering a first volume of elution buffer to the affinity column via the FIA, thereby eluting the protein from the first volume of sample from the affinity column to form a first eluate; (e) Transferring the first eluate to the protein concentration detection unit via the FIA; (f) Determining the concentration of the protein in the first eluate using at least one processor; (g) Containing the sample containing the protein in the holding reservoir; (h) Delivering a second volume of the binding buffer to the affinity column via the FIA; (i) Transferring a second volume of the sample from the holding reservoir to the affinity column via the FIA, wherein the first volume and the second volume of the sample are the same; (j) Delivering a second volume of elution buffer to the affinity column via the FIA, thereby eluting the protein from the second volume of sample from the affinity column to form a second eluate; (k) Transferring the second eluate to the digestion chamber via the FIA; (l) Delivering a digestion reagent via the FIA ​​to digest the protein in the second eluate to form a peptide mixture, wherein the protein in the second eluate is digested based on the protein concentration of the first volume of sample determined in (f); and (m) Transferring the peptide mixture to a peptide mapping unit via the FIA ​​and detecting the post-translational modifications of the protein, Methods that include...

2. The method according to claim 1, wherein the FIA ​​is a sequential injection analyzer (SIA) or a direct injection analyzer (DIA).

3. The method according to claim 2, wherein the protein concentration detection unit includes a spectrophotometer.

4. The method according to claim 3, wherein the spectrophotometer measures the UV absorbance of the first eluate and the at least one processor calculates the protein concentration based on a calibration curve.

5. The method according to claim 1, wherein the first volume of sample and the second volume of sample are obtained from a cell culture.

6. The method according to claim 5, wherein the cell culture is located in a bioreactor.

7. The method according to claim 6, wherein the bioreactor is a batch bioreactor, a fed-boil bioreactor, or a perfusion bioreactor.

8. The method according to claim 1, further comprising controlling the elution time of the protein in the first volume of sample eluted from the affinity column for forming the first eluate of (d) by the at least one processor based on a predetermined elution time.

9. The method according to claim 8, further comprising controlling by at least one processor the elution time of the protein in the second volume of sample eluted from the affinity column for forming the second elution of (j) based on the same predetermined elution time as the first volume of sample of (d).

10. The method according to any one of claims 1 to 9, wherein the protein in the second eluate is enzymatically digested with (l).

11. The method according to claim 10, wherein the enzymatic digestion of the protein comprises contacting the second eluate with a proteolytic enzyme selected from trypsin, chymotrypsin, pepsin, thermolysin, papain, pronase, endopeptidase Arg-C, peptidyl-Asp metalloendopeptidase (endopeptidase Asp-N), glutamyl endopeptidase (Glu-C endopeptidase), and lysyl endopeptidase (Lys-C endopeptidase) via the FIA.

12. (l) is, (i) Introducing a digestion reagent through the FIA ​​to form a digestion mixture; (ii) Mixing the digested mixture; and (iii) Incubate the digestion mixture. The method according to claim 10, including the method described in claim 10.

13. The method according to claim 12, wherein the digestion reagent comprises a digestion buffer with a pH of 6.0 to 7.

5.

14. The method according to claim 13, wherein the digestion reagent comprises a reducing agent containing tris(2-carboxyethyl)phosphine (TCEP) and / or a proteolytic enzyme.

15. The method according to claim 12 or 13, wherein incubation of the digested mixture comprises incubation at a temperature of 36°C to 85°C for 5 to 30 minutes.

16. The proteolytic enzyme includes heat-stable trypsin, and the method is (i) Introducing a first amount of digestion buffer, reducing agent, and heat-stable trypsin into the second eluate via the FIA ​​and mixing them to form a first digestion mixture; (ii) Incubate the first digestion mixture at a temperature of 70°C to 75°C for 10 to 20 minutes to form the first incubation mixture; (iii) introducing a second amount of heat-stable trypsin into the incubation mixture via the FIA ​​and mixing it to form a second digestion mixture; and (iv) Incubate the second digestion mixture at a temperature of 70°C to 75°C for 10 to 20 minutes to form a peptide mixture. The method according to claim 14, including the method described in claim 14.

17. An automated method for monitoring post-translational modifications of proteins, performed by a flow injection analyzer (FIA) including a syringe pump and a multi-port valve controlled by at least one processor, (a) Containing a first volume of sample containing the protein in the holding reservoir of the FIA; (b) By operating the multiport valve, a first volume of binding buffer is introduced into the affinity column in the FIA; (c) The syringe pump is used to advance the first volume of sample from the holding reservoir to the affinity column, thereby binding the protein in the first volume of sample to the affinity column; (d) The operation of the multiport valve introduces a first volume of elution buffer into the affinity column and elutes the bound protein to form a first eluate; (e) The syringe pump is used to advance the first eluate to the spectrophotometer in the FIA; (f) The concentration of the protein in the first eluate is determined by the at least one processor according to the measured UV absorbance; (g) Containing a second volume of the sample containing the protein in the holding reservoir; (h) Introducing a second volume of the binding buffer into the affinity column by operating the multiport valve; (i) The syringe pump is used to advance the second volume of sample from the holding reservoir to the affinity column, thereby binding the protein in the second volume of sample to the affinity column; (j) By operating the multiport valve, a second volume of elution buffer is introduced into the affinity column, and the bound protein is eluted to form a second eluate; (k) The operation of the syringe pump to advance the second eluate into the digestion chamber within the FIA; and (l) The operation of the multiport valve introduces a digestion reagent into the second eluate in the digestion chamber, digesting the protein to form a peptide mixture, where the protein in the second eluate is digested based on the protein concentration of the first volume of sample determined in (f); and (m) The operation of the syringe pump advances the peptide mixture to the peptide mapping unit and detects the post-translational modification of the protein. An automated method, including

18. (l) Digestion of the aforementioned protein (i) By operating the multiport valve, the digestion buffer, reducing agent, and proteolytic enzyme are introduced into the second eluate in the digestion chamber and mixed to form the first digestion mixture; (ii) Incubating the first digestion mixture in the digestion chamber to form a first incubation mixture; (iii) By operating the multiport valve, a second volume of digestion buffer and proteolytic enzyme is introduced into the incubation mixture to form a second digestion mixture; and (iv) Incubating the second digestion mixture in the digestion chamber to form a peptide mixture. The method according to claim 17, including the method described in claim 17.

19. The method according to claim 18, wherein the digestion buffer has a pH of 6.0 to 7.

5.

20. The method according to claim 19, wherein the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

21. The proteolytic enzyme includes heat-stable trypsin, and the method is (i) By operating the multiport valve, a first amount of digestion buffer, reducing agent, and heat-stable trypsin is introduced into the second eluate and mixed to form a first digestion mixture; (ii) Incubate the digested mixture at a temperature of 70°C to 75°C for 10 to 20 minutes to form a first incubation mixture; (iii) By operating the multiport valve, a second amount of digestion buffer and heat-stable trypsin is introduced into the incubation mixture and mixed to form a second digestion mixture; and (iv) Incubate the second digestion mixture at a temperature of 70°C to 75°C for 10 to 20 minutes to form a peptide mixture. The method according to claim 18, including the method described in claim 18.

22. An automated system for monitoring post-translational modifications of proteins, (a) A retaining reservoir configured to contain a first volume of sample and a second volume of sample containing the protein; (b) A first syringe pump configured to advance the first volume of the sample and the second volume of the sample from the holding reservoir through a flow injection analyzer (FIA); (c) A first multi-port valve in fluid communication with a first syringe pump; (d) an affinity column configured to be in fluid communication with the first multiport valve and to contain the first volume of sample and the second volume of sample from the holding reservoir; (e) A spectrophotometer configured to contain a first eluate from the affinity column, wherein the first eluate contains protein from a first volume of sample, and the spectrophotometer is configured to determine UV absorbance and calculate the protein concentration of the first volume of sample based on a predetermined calibration curve; (f) A second syringe pump configured to advance a second eluate from the affinity column to a digestion chamber, wherein the second eluate contains protein from the second volume of sample, and the protein in the second eluate is digested based on the protein concentration of the first volume of sample determined in (e); and (g) A second injection multiport valve in fluid communication with the second syringe pump, An automated system, including

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