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

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 under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 200,576, filed March 16, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates to methods for detecting post-translational modifications of polypeptides, in particular online methods for detecting post-translational modifications of recombinant proteins produced in cell culture in real time. [Background technology]

[0003] Biopharmaceuticals, including therapeutic proteins such as fusion proteins, growth factors, cytokines, enzymes, hormones, and monoclonal antibodies (mAbs), represent the fastest growing segment 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 host cells such as Chinese hamster ovary (CHO), NS0, or Sp2 / 0. Mammalian systems are often preferred because many biopharmaceuticals are too large and complex to be produced by microorganisms or because they require post-translational modifications (PTMs) that are only possible in mammalian host cells. However, PTMs can affect the physicochemical characteristics of biomolecules, which can affect activity, protein-protein interactions, biological function, efficacy, and stability, resulting in various levels of protein heterogeneity. Therefore, PTMs are critical quality attributes (CQAs) that are characterized, controlled, and monitored during process development and manufacturing 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. Among these modifications, the most common and naturally occurring are phosphorylation, acetylation, glycosylation (O-linked and N-linked), deamidation, oxidation, hydroxylation, methylation, ubiquitination, pyroglutamate, sulfation, and cleavage. Non-Patent Document 1.

[0005] Glycosylation is a common PTM that involves the attachment of glycans at specific sites on proteins, e.g., at asparagine residues (N-linked glycosylation) or serine (Ser) or threonine (Thr) residues (O-linked glycosylation). N-linked glycosylation occurs at consensus sequences (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 added 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 glycans undergo further trimming and modification resulting in various N-glycan structures. O-linked glycans are usually attached to serine (Ser) or threonine (Thr) residues via an α-O-glycosidic bond formed between N-acetylgalactosamine (GalNAc) and the hydroxyl group (-OH) of the 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. The O-GalNAc precursor is processed to generate various O-GalNAc glycans. Non-Patent Document 2.

[0006] The traditional process for analyzing PTMs involves manually collecting samples at the end of cell culture, shipping the samples to a laboratory where the samples are concentrated, purified, and prepared for analysis. The traditional process can take 3-5 days, resulting in development and manufacturing delays and increased costs. Because the 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 with cell culture. Therefore, analytical methods that can be performed in real time to characterize the product during production so that production parameters can be adjusted to keep the product within the desired specifications are desirable.

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

[0008] There remains a need for improved processes for monitoring the post-translational modifications (PTMs) of recombinantly produced 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 Summary of the Invention [Means for solving the problem]

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

[0011] In one aspect, a method for detecting post-translational modifications of a protein is provided, the method being performed by a flow injection analyzer (FIA) controlled by at least one processor. In one aspect, the FIA ​​is a sequential injection analyzer (SIA). In one aspect, the FIA ​​is a direct injection analyzer (DIA). In one aspect, the method comprises: (a) containing a first sample containing a protein in a retention reservoir; (b) delivering, via the FIA, a first volume of binding buffer to an affinity column within the FIA; (c) transferring the first sample from the holding reservoir to the affinity column via the FIA; (d) delivering, via the FIA, a first volume of elution buffer to the affinity column, thereby eluting proteins in the first sample from the affinity column to form a first eluate; (e) transferring the first eluate to a protein concentration detection unit via the FIA; (f) determining by at least one processor a concentration of protein in the first eluate; (g) containing a second sample containing the protein in the retention reservoir; (h) delivering a second volume of 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 via the FIA ​​to the affinity column, thereby eluting proteins in the second sample from the affinity column to form a second eluate; (k) transferring the second eluate to a digestion chamber via the FIA; (l) delivering a digestion reagent via the FIA ​​to digest the proteins in the second eluate to form a peptide mixture; and (m) transferring the peptide mixture to a peptide mapping unit via FIA and detecting post-translational modifications of proteins.

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

[0013] In one embodiment, the affinity column comprises a Protein A column.

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

[0015] In one embodiment, the elution time of the proteins in the first sample from the affinity column to form the first eluate is controlled by at least one processor based on a predetermined elution time. In one embodiment, the elution time of the proteins in the second sample from the affinity column to form the second eluate is controlled by at least one processor based on a predetermined elution time. In one embodiment, the predetermined elution times of the proteins in the first sample and the second sample are the same. In one embodiment, the predetermined elution times of the proteins in the first sample and the second sample are determined experimentally. In one embodiment, the predetermined elution times of the proteins in the first sample and the second sample are determined experimentally prior to 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 the 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 prior to performing the online method described herein. In one embodiment, the spectrophotometer measures the UV absorbance of the first eluate at 280 nm and calculates the protein concentration based on the calibration curve by at least one processor. In one embodiment, the spectrophotometer measures the UV absorbance of the first eluate at 205 nm and calculates the protein concentration based on the calibration curve by at least one processor.

[0017] In one aspect, the proteins in the second eluate are enzymatically digested with (l). In one aspect, the enzymatic digestion of the proteins 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 FIA. In one aspect, the proteolytic enzyme comprises trypsin.

[0018] In one aspect, the proteolytic enzyme comprises a thermostable trypsin. (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 embodiment, the digestion reagent comprises a digestion buffer, a reducing agent, and a protease. In one embodiment, the digestion buffer has a pH of about 6.0 to about 7.5. In one embodiment, the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP). In one embodiment, the second eluate is contacted with an amount of protease determined by at least one processor based on the concentration of the first eluate. In one embodiment, the protease comprises trypsin. In one embodiment, the protease comprises thermostable trypsin. In one embodiment, the second eluate is contacted with the protease at an enzyme:protein ratio of about 1:10 to about 1:100. In one embodiment, the second eluate is contacted with the protease at an enzyme:protein ratio of about 1:20 to about 1:30. In one embodiment, 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 embodiment, incubating the digestion mixture comprises incubating for about 5 minutes to about 30 minutes at a temperature of about 36° C. to about 85° C. In one embodiment, incubating the digestion mixture comprises incubating for about 15 minutes to about 30 minutes at a temperature of about 50° C. to about 75° C. In one embodiment, incubating the digestion mixture comprises incubating for about 15 minutes to about 30 minutes at a temperature of about 70° C. to about 72° C.

[0021] In one aspect, the proteolytic enzyme comprises a thermostable trypsin, and the method further comprises: (i) introducing via FIA a first amount of a digestion buffer comprising a digestion buffer, a reducing agent, and a thermostable trypsin into the second eluate 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 incubation mixture; (iii) introducing a second amount of thermostable trypsin via FIA into the incubation 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.

[0022] In one embodiment, a second amount of digestion buffer is added to the first incubation mixture along with a second amount of (iii) heat stable trypsin via FIA.

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

[0024] In one aspect, the method is performed online.

[0025] In one aspect, the post-translational modification includes glycosylation, oxidation, deamidation, isomerization, glycosylation, N-terminal leader sequence, N-terminal cyclization, C-terminal lysine retention, amide formation, or a combination thereof, In one aspect, the glycosylation includes N-linked glycosylation, O-linked glycosylation, or a combination thereof.

[0026] In one aspect, a method for producing a recombinant protein is provided. In one aspect, the method comprises: (a) culturing host cells under conditions in which the recombinant protein is expressed; (b) detecting post-translational modifications of a protein according to the methods described herein; (c) modifying one or more cell culture parameters based on the post-translational modifications detected in (b).

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

[0028] In one aspect, 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 methods described herein.

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

[0030] In one aspect, a recombinant protein produced by the methods described herein is provided. In one aspect, a pharmaceutical composition is provided comprising a recombinant protein produced by the methods described herein and a pharma- ceutically acceptable carrier.

[0031] In one aspect, an online method for monitoring post-translational modifications of proteins is provided. In one aspect, the method is performed by a flow injection analyzer (FIA). In one aspect, the FIA ​​is a sequential injection analyzer (SIA). In one aspect, the FIA ​​is a direct injection analyzer (DIA). In one aspect, the FIA ​​comprises a bi-directional pump controlled by at least one processor. In one aspect, the FIA ​​comprises a syringe pump and a multi-port valve controlled by at least one processor. In one aspect, the FIA ​​comprises two or more syringe pumps and two or more multi-port valves. In one aspect, the FIA ​​comprises two syringe pumps and two multi-port valves.

[0032] In one aspect, the method comprises: (a) containing a first sample containing a protein in a retention reservoir of an FIA; (b) introducing a first volume of binding buffer into an affinity column within the FIA ​​by operation of a multiport valve; (c) advancing the first sample from the holding reservoir to the affinity column by operation of the syringe pump, and binding proteins in the first sample to the affinity column; (d) introducing a first volume of elution buffer into the affinity column by operation of the multiport valve to elute the bound protein to form a first eluate; (e) advancing the first eluate to a spectrophotometer within the FIA ​​by operation of a syringe pump; (f) determining, by at least one processor, a concentration of protein in the first eluate according to the measured UV absorbance; (g) containing a second sample containing the protein in the retention reservoir; (h) introducing a second volume of binding buffer into the affinity column by operation of the multiport valve; (i) advancing a second sample from the holding reservoir to the affinity column by operation of the syringe pump, thereby binding proteins in the second sample to the affinity column; (j) introducing a second volume of elution buffer into the affinity column by operation of the multiport valve to elute the bound protein to form a second eluate; (k) advancing the second eluate to a digestion chamber within the FIA ​​by operation of a syringe pump; and (l) introducing a digestion reagent into the second eluate in the digestion chamber by operation of the multiport valve to digest the proteins to form a peptide mixture; and (m) advancing the peptide mixture by the action of a syringe pump to a peptide mapping unit to detect post-translational modifications of the proteins.

[0033] In one aspect, digesting the protein comprises: (i) introducing a digestion buffer, a reducing agent, and a proteolytic enzyme into the second eluate in the digestion chamber by operation of the multiport valve and mixing to form a first digestion mixture; (ii) incubating the first digestion mixture in a digestion chamber to form a first incubation mixture; (iii) introducing a second volume of digestion buffer and proteolytic enzyme into the incubation mixture by operation of the multiport valve to form a second digestion mixture; and (iv) incubating the second digestion mixture in a digestion chamber to form a peptide mixture.

[0034] In one embodiment, determining the concentration of the protein in the first eluate in (f) is performed by at least one processor according to a predetermined calibration curve. In one embodiment, the protein in the first sample is eluted from the affinity column based on a predetermined elution time to form a first eluate. In one embodiment, the protein in the second sample is eluted from the affinity column based on a predetermined elution time to form a 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 proteolytic enzyme introduced into the digestion chamber is determined by at least one processor based on the concentration of the first eluate (previously determined). In one embodiment, the proteolytic enzyme comprises trypsin. In one embodiment, the trypsin comprises a thermostable trypsin. In one embodiment, the second eluate is contacted with the trypsin at a trypsin:protein ratio of about 1:10 to about 1:100. In one embodiment, the second eluate is contacted with the trypsin at a trypsin:protein ratio of about 1:20 to about 1:30.

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

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

[0038] In one aspect, the proteolytic enzyme comprises a thermostable trypsin, and the method further comprises: (i) introducing a first amount of a digestion buffer, a reducing agent, and a thermostable trypsin into the second eluate by operation of the multiport valve 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 an incubation mixture; (iii) introducing a second amount of digestion buffer and thermostable trypsin into the incubation mixture by operation of the multiport valve 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.

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

[0040] In one embodiment, the peptide mixture comprises peptides containing less than about 100 amino acids.

[0041] In one aspect, an online system for monitoring post-translational modifications of a protein of interest is provided. In one aspect, the system comprises: (a) a holding reservoir configured to accommodate a first sample and a second sample, each of which comprises a protein of interest; (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 in fluid communication with the first multiport valve and configured to receive the first sample and the second sample from the retention reservoir; (e) a spectrophotometer within the FIA ​​configured to receive a first eluate from the affinity column, where the first eluate comprises proteins from a first sample, the spectrophotometer configured to determine UV absorbance and calculate a protein concentration of the first sample based on a pre-defined calibration curve; (f) a second syringe pump configured to advance a second eluate from the affinity column to a digestion chamber, where the second eluate comprises proteins from the second sample, and where 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 the second syringe pump. [Brief description of the drawings]

[0042] [Figure 1] 1 is a schematic diagram of a system configured to provide a process implementation of an automated method consistent with the methods described herein. [Diagram 2] FIG. 1 is a schematic diagram of a computer system configured to provide automated method process control consistent with methods described herein. [Diagram 3] FIG. 2 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] FIG. 2 is a schematic diagram of a second syringe pump and a second valve system for carrying out the online automated methods described herein. [Diagram 5] 1 is a schematic illustration of the online automated method described herein. [Figure 6] 1 is a schematic illustration of an alternative method described herein. [Figure 7] 1 is a further schematic illustration of the online automated method described herein. [Figure 8] 1 is a flow chart of the peptide mapping process described herein. [Figure 9A]Graph showing calibration curve and UV determination of polypeptides using a Protein A capture step. [Figure 9B] UV profile used to perform concentration determination of the first eluate, showing that the same amount of protein was captured for the second eluate, which was later used for protein digestion. [Figure 10A] FIG. 1 is a graph showing the correlation between the % glycosylation of a sample IgG1 monoclonal antibody determined using the online peptide mapping method described herein and that determined using the glycan release (Oligo 2-AB) method. [Figure 10B] 1 is a graph showing the % quantification of mannose monitored over time (days 7, 10, and 14) during cell culture using the online peptide mapping method described herein. Replicate experiments at each time point show a consistent level of % quantification. [Figure 11A] 1 is a graph showing a direct linear relationship between G0F detection using the online peptide mapping method and G0F detection using the glycan release (Oligo 2-AB) method. [Figure 11B] 1 is a graph showing a direct linear relationship between G1F detection using the online peptide mapping method and the glycan release (Oligo 2-AB) method. [Figure 11C] 1 is a graph showing the direct linear relationship between Man5 detection using the online peptide mapping method and the glycan release (Oligo 2-AB) method. [Figure 11D] 1 is a graph showing a direct linear relationship between G0F-GN detection using the online peptide mapping method and G0F-GN detection using the glycan release (Oligo 2-AB) method. [Figure 12A] 1 is a graph showing the oxidation levels at specific methionine residues over time in cell culture, as determined using the online peptide mapping method described herein. [Figure 12B]1 is a graph showing the level of N-terminal cyclization over time in cell culture, as determined using the online peptide mapping method described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0044] A.Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise specified by context, singular terms shall include the plural and plural terms shall include the singular. For example, unless otherwise specified, "a" or "an" shall include the plural, 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 particular range described and the values ​​related to the endpoints of the particular 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 of components in a composition used in the description of the present invention. The term "about" refers to variations in numerical quantities that may occur, for example, due to typical measuring and handling procedures used in the manufacture of a compound, composition, concentrate, or formulation; due to accidental errors in these procedures; due to differences in the manufacture, source, or purity of starting materials or components used to carry out the method, and other similar requirements. The term "about" also encompasses amounts that differ due to changes over time in a formulation having a particular initial concentration or initial mixture, and amounts that differ due to mixing or processing a formulation having a particular initial concentration or initial mixture. When modified by the term "about", the claims appended hereto include such equivalents.

[0046] Generally, the nomenclatures and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes.

[0047] As used herein, the term "polypeptide" refers to a molecule of any length that contains two or more amino acid residues joined together by peptide bonds. As used herein, the term "peptide" refers to a polypeptide that contains about 2 to about 50 amino acids joined together by peptide bonds. The term "protein" refers to a polypeptide that contains more than about 100 amino acid residues, or more than about 1000 amino acid residues joined together by peptide bonds. The sequence of amino acids in a polypeptide is referred to as the primary structure. In one aspect, a protein has a secondary structure, e.g., by the formation of a hydrogen bond between an oxygen atom of a first amino acid and a nitrogen atom of a second amino acid resulting in a two-dimensional alpha helix or beta pleated sheet. In one aspect, a protein has a three-dimensional tertiary structure, e.g., by 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 aspect, a protein has a quaternary structure, e.g., by the presence of two or more polypeptide chains, e.g., an antibody. In one aspect, the peptide has no 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 are not limited to, therapeutic proteins. The term "therapeutic protein" refers to a protein having one or more biological activities useful for treating, preventing, or ameliorating a disorder or disease.

[0049] As used herein, the terms "antibody" and "immunoglobulin" may be used interchangeably and refer to a group of proteins or polypeptides that contain at least one binding domain formed by folding of the polypeptide chains to have a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant features 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 regions of each light / heavy chain pair form the antibody binding site. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. 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 its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Light chains are classified as either lambda or kappa chains based on the amino acid sequence of the light chain constant region. The antibodies can 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)), including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two different epitope-binding fragments, CDR-grafted, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, anti-idiotypic (anti-Id) antibodies, intrabodies, and desired antigen-binding fragments thereof, which can include recombinantly produced antibody fragments.

[0050] The term "antigen-binding fragment" or "immunologically active fragment" refers 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 may be recombinantly produced include, but are not limited to, antibody fragments that contain variable heavy and light chain domains, such as single chain Fv (scFv), single chain antibodies, Fab fragments, Fab' fragments, 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 a biological material, such as a nucleic acid or protein, that has been altered or produced artificially or synthetically (i.e., non-naturally), by human intervention (e.g., using recombinant DNA processes), including, for example, a protein expressed using a recombinant expression vector transfected into a host cell.

[0052] The term "cell culture" refers to methods and techniques used to create and maintain a population of host cells capable of producing a biomolecule of interest, such as a protein of interest, as well as methods for producing and harvesting the biomolecule of interest. In "cell culture", the protein may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In one aspect, the cell culture may include mammalian host cells. Culture conditions suitable 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 may be cultured, for example, in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, in suspension, or attached to a solid substrate.

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

[0054] "Conditioned medium" refers to culture medium that contains a biomolecule of interest that is released or secreted by cells into the cell culture medium.

[0055] "Clarified cell culture medium" refers to 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 aspect, clarified cell culture medium includes a cell culture supernatant.

[0056] "Bioreactor" refers to a vessel designed to control the growth of cell cultures. The term bioreactor can refer to any scale of cell culture, from individual flasks and shake flasks or wave bags, to 1 liter bioreactors, and up to large-scale industrial bioreactors. In one aspect, the bioreactor is a small-scale bioreactor, for example, having a volume of about 125 ml to about 500 ml. In one aspect, the bioreactor is a large-scale bioreactor, for example, having a volume of about 100 liters to about 20,000 liters or more. Various bioreactors are known, including, but not limited to, stirred-stank, fixed-bed, fluidized-bed, bubble column, and airlift bioreactors. Large-scale or commercial-scale cell cultures (e.g., for the production of therapeutic proteins) can be maintained for several days or even weeks. During this period, the culture can be supplemented with a concentrated feed medium containing components such as nutrients and amino acids that are consumed during the course of the cell culture. In one aspect, during the cell culture process in a batch process, the medium in the bioreactor is not replenished. In one aspect, during the cell culture process in a fed-batch bioreactor, the medium in the bioreactor is replenished with additional components, such as nutrients or cell culture medium. In one aspect, medium is continuously added to the bioreactor and spent medium and product 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 the particular cell but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain modifications may occur in the progeny, either due to mutation or environmental influences, but are still included within the scope of the term "host cell." Host cells can include prokaryotic and eukaryotic cells. In one aspect, the host cell is a mammalian host cell, such as a Chinese hamster ovary cell (CHO), a human fetal kidney cell (e.g., HEK293), a monkey kidney cell (e.g., COS), a mouse myeloma cell (e.g., NS0 or SP2), or a human primary fetal retina cell (PER.C6).

[0058] "Critical Quality Attributes" (CQAs) are chemical, physical, or biological characteristics of a therapeutic protein that may affect the safety and / or efficacy of the product. CQAs can include product-specific variants, including, for example, 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, leachables, and other raw materials.

[0059] "Post-translational modification" (PTM) refers to an alteration of a polypeptide as a result of the covalent addition of a functional group to or cleavage of a peptide bond within the polypeptide chain, including, but not limited to, glycosylation, oxidation, deamidation, isomerization, glycosylation, N-terminal leader sequences, N-terminal cyclization, C-terminal lysine retention, amide formation, or a combination thereof. In one aspect, glycosylation includes N-linked glycosylation, O-linked glycosylation, or a combination thereof.

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

[0061] "Glycoform" refers to isoforms of a protein that differ in their glycosylation profile (i.e., the number and / or type of glycans attached).

[0062] "Glycan" refers to an oligosaccharide attached to a polypeptide chain, including N-linked and O-linked glycans. N-linked glycans (also called N-glycans) are attached to the polypeptide chain at the amide nitrogen of an asparagine (Asp) or arginine (Arg) residue via an N-glycosidic bond. N-linked glycosylation occurs at the consensus sequence Asn-X-Ser / Thr, where X is any amino acid except proline. O-linked glycans (or O-glycans) are attached to the polypeptide chain at a serine (Ser) or threonine (Thr) residue via an α-O-glycosidic bond. 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 that involve the stepwise addition and / or removal of individual monosaccharides. There are many types of glycans that can be classified by the presence or absence of various terminal sugars, including agalactosylglycans with zero terminal galactose moieties (e.g., G0, G0-GlcNAc, G0F, G0F-GlcNAc), glycans with one terminal galactose moiety (e.g., G1, G1F, G1F-GlcNAc), and glycans with two terminal galactose moieties (e.g., G2F, G2F+sialic acid). Some glycosylation patterns may be beneficial to protein function, while others may be detrimental.

[0063] "Process Analytical Technology" (PAT) refers to a system for designing, analyzing, and controlling manufacturing through the timely (i.e., during processing) measurement of critical quality and performance attributes of raw and in-process materials and processes.

[0064] "On-line" refers to an analytical process where automated sampling of process solutions occurs without human intervention. "In-line" refers to an analytical process where a sensor is placed in the flowing material stream to perform the analysis. "At-line" refers to a process where a sample is transferred from the process to an analytical device in physical proximity to the process and the analytical results are returned within a relatively short period of time (i.e., hours). "Off-line" refers to a process where a sample is removed from the process and analyzed at another location and the analytical results are not returned within a relatively short period of time (e.g., days). Both offline and at-line analysis require human intervention to manually remove a 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] By "real-time" it is meant that the analytical process is performed without significant time delay, e.g., within a time short enough that information obtained from the analytical process can be applied to decisions regarding optimization of process conditions, e.g., cell culture conditions in a bioreactor can be optimized while a sampled cell culture is still in progress. In one embodiment, the results of the analytical process, including the Protein A capture step and protein concentration determination, are available within less than about 5 hours, less than about 4 hours, or less than about 3 hours. In one embodiment, the results of the analytical process are available within less than about 2.5 hours. In contrast, in offline processes, the results of the analytical process typically require at least one day and up to two days.

[0066] "Automated" refers to a process that is performed without human invention. In one embodiment, an automated process is performed by one or more devices. In one embodiment, an automated process includes a process in which the sample preparation is online and automatic. In one embodiment, the online automated sample preparation process is performed on 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, the 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 a liquid handling system, 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" refers to something that is determined prior to the initiation of a process, e.g., prior to the initiation of a method described herein. For example, a "predetermined calibration curve" used in determining protein concentration refers to a calibration curve that is created using a representative sample with a known protein concentration. For example, a calibration curve can be created by obtaining a sample with a known concentration and performing a serial dilution, e.g., a 10-fold serial dilution, to obtain about 3 to about 10 samples of known concentration. In one embodiment, the UV absorbance of the serially diluted samples is then determined along with the UV absorbance of a blank that contains all reagents except the protein. The calibration curve is created by plotting the UV absorbance against the known protein concentration of the sample. In one embodiment, the "predetermined elution time" is determined by loading a known amount of a representative sample onto a column and determining the amount of time required to elute the sample from the column. In one embodiment, the elution time refers to the amount of time that elapses between the time the sample is loaded onto the column and the time the sample is no longer eluted from the column when an elution reagent is applied at a known rate. In one aspect, elution time refers to the amount of time that elapses between the time that a sample is loaded onto a column and the time that the maximum concentration of the sample is eluted from the column when elution reagent is applied at a known rate. In one aspect, elution time can refer to the volume of elution reagent at a known flow rate that is required to elute all of the sample from the column. A "predetermined" post-translational modification refers to a known post-translational modification of a protein of interest that is identified prior to the methods described herein that are used to identify and / or quantify the predetermined post-translational modification.

[0068] "Concurrently", when used in connection with method steps as described herein, refers to methods in which the steps are performed at substantially the same time. Operations performed "substantially at the same time" can mean that the operations are performed in parallel, but can also include situations in which two operations are performed within a short time of each other, for example, within about 30 minutes of each other, or within about 15, about 10, about 5, about 4, about 3, about 2, or about 1 minute of each other. In one embodiment, method steps are performed "concurrently" if the performance of at least a portion of one method step overlaps in time with the performance of a portion of another method step. "Concurrently" does not require strictly parallel action. For example, not all method steps need to start or finish at the same time. In one embodiment, "concurrently" can mean that all reagents required for a method step are combined in the same reaction mixture, such that the reactions occur in the same reaction volume and / or during the same incubation period.

[0069] "Substantially the same" or "substantially identical," when used in reference to two process parameters, including but not limited to amount, volume, time, temperature, pressure, concentration, and flow rate, means that the amounts or quantifications of the two process parameters are functionally equivalent, e.g., 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 microliter volumes of sample are injected into and carried by a carrier stream for automated sample processing. Flow injection analysis can be performed by a flow injection analyzer (FIA), which as used herein may include or encompass a sequential injection analyzer (SIA) or a direct injection analyzer (DIA). In one aspect, a flow injection analyzer uses a constant flow rate of a carrier stream to move a sample through the analyzer. In one aspect, a flow injection analyzer includes a bidirectional pump, e.g., a syringe pump, that can reverse or stop the flow of the carrier stream to mix the sample with reagents or to assay sample components.

[0071] As used herein, "in fluid communication" means that a fluid, e.g., a liquid, can flow from one component, instrument, or device to another component, instrument, or device in a system. The flow may be achieved by one or more intermediate components, instruments, or devices, and may or may not be selectively shut off (e.g., by a valve).

[0072] As used herein, "coupled" means that two or more components, instruments, or devices are movably or fixedly joined or otherwise attached by one or more intermediate components, instruments, or devices.

[0073] As used herein, "upstream" and "downstream" refer to relative locations with respect to the flow direction of the carrier stream within a system. The term "upstream" refers to a system component or location that is closer to the input of the system compared to another component or location. The term "downstream" refers to a system component or location that is closer to the outlet of the system compared to another component or location.

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

[0075] "Affinity chromatography" refers to a chromatographic process in which biomolecules of interest, e.g., proteins, in a mobile phase are retained by the stationary phase of a chromatographic column based on specific, reversible binding interactions between the components of interest and compounds immobilized on the chromatographic matrix, including but not limited to interactions between receptors and ligands, enzymes and substrates, or antigens and antibodies, that result in chromatographic separation, rather than on 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 a stationary phase.

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

[0078] "Mass spectrometry" (MS) refers to an analytical technique in which compounds are detected, identified, and / or measured based on their mass-to-charge ratio (m / z). MS typically involves ionizing compounds, separating the ionized molecules by mass-to-charge ratio, detecting the ionized molecules, and producing a mass spectrum. A known method of protein ionization is electrospray ionization (ESI). "Mass spectrometer" refers to an analytical instrument that can detect, identify, and / or quantify compounds based on their mass-to-charge ratio. A mass spectrometer typically includes three parts: an ion source, a mass analyzer, and a detector.

[0079] As used herein, "liquid chromatography / mass spectrometry" (LC / MS) refers to a method in which components in a sample, such as peptides in a peptide mixture, are separated by liquid chromatography and then ionized and characterized by their mass-to-charge ratio.

[0080] "Tandem mass spectrometry" (MS / MS) refers to a technique for detecting, identifying, and / or measuring compounds using a multi-step process involving two or more steps of mass spectrometry, where 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 aspect, peptide mapping can be used to verify the sequence of a biomolecule. In one aspect, peptide mapping can be used to identify and quantify post-translational modifications (PTMs) of peptides produced in cell culture. In one aspect, peptide mapping can be used for multi-attribute monitoring (MAM) of critical quality attributes (CQAs).

[0082] Many methods have been developed to analyze PTMs. One of the most widely used approaches involves peptide mapping, which involves liquid chromatography (LC) / mass spectrometry (MS). In one method, glycans are chemically labeled, for example with 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. Because the glycans are removed from the protein, no site-specific information can be obtained by the release of the glycans. In another method, glycoproteins are digested into peptides using enzymes such as trypsin prior to LC / MS analysis. Because the glycans remain on the peptides, site-specific information can be obtained regarding the PTMs of the peptides, for example glycosylation. In one aspect, peptides identified by mass spectrometry are used as surrogate representatives of intact proteins obtained from cell culture media.

[0083] Provided herein are automated online systems and methods 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 require more than two days, e.g., up to about three or about four days, to complete, the online peptide mapping methods described herein provide a fully automated process that can provide information on glycosylation propensity, post-translational modifications, and other site-specific information in less than one day, or even as little as 2.5 hours. In one aspect, the systems described herein provide fully automated methods for sample preparation and peptide mapping analysis. In one aspect, the methods provide fully automated systems for protein capture, protein concentration determination, and protein digestion. In one aspect, the online peptide mapping methods described herein are used to detect one or more known post-translational modifications (PTMs) associated with a protein of interest. In one aspect, the online peptide mapping methods described herein are used to detect one or more predetermined post-translational modifications associated with a protein of interest. Advantageously, site-specific information on PTMs can be obtained using the methods described herein.

[0084] 1-4 provide an overview of a system configured to execute or perform the automated methods described herein.

[0085] Figure 1 is a schematic diagram of a system configured to provide an automated method process implementation consistent with the automated methods and systems described herein. Figure 1 illustrates an automated process control system 100 configured to perform the automated processing methods described herein. In Figure 1, one embodiment of a network environment is shown. The network environment can include one or more process control devices 200 in communication with one or more of 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 analytical equipment 160 via one or more networks 202.

[0086] As shown in Figures 3 and 4, the FIA ​​150 may include at least a first syringe pump 302, a second syringe pump 402, a first valve 301, and a second valve 401. Further, the FIA ​​150 may include at least one affinity column 313, at least one spectrophotometer 414, and at least one digestion chamber 415. In addition, the FIA ​​150 may include further components such as tubing, t-connections, valves, and other components necessary to route fluids to appropriate destinations. Furthermore, the FIA ​​150 may contain, include, or be otherwise connected to sources of samples, reagents, reactants, water, reducing agents, enzymes, buffers, etc., and a waste sink for disposal of waste products. The FIA ​​150 may be configured as a flow injection analyzer and / or with any or all of the functionality 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 may be obtained from the one or more bioreactors 140 either manually or through an automated feed process to the FIA ​​150. Further description of the bioreactors 140 consistent with the methods described herein is provided below.

[0088] The one or more analytical devices of the automated process control system 100 may include a peptide mapping system equipped with one or more mass analyzers.

[0089] Data and information stored by the automated process control system 100 may include the following information: As used herein, "automated process control system data" refers to any and all data that may be recorded and stored on or in a memory associated with the automated process control system 100. The automated process control system data may be stored in any suitable data format and may be sorted by production batch, production date, or any other suitable parameter. As used herein, "process information" refers to information regarding variables and parameters of sample processing methods and techniques, including, but not limited to, timing, temperature, dosage amounts, calibration factors, flow rates, and models. As used herein, "production information" refers to information regarding processed samples, past sample runs, and the like. Each of the above data and / or information may be accessed in near real-time by the process control device 200 discussed herein. Data and process information associated with the automated process control system 100 may be stored, for example, in a data retention system 201, described below, 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 smart phone, a tablet computing device, a computing system integrated with the FIA ​​150, and / or other device that may be programmed to interface with the bioreactor 140, the FIA ​​150, and / or the mass analyzer 160. In an aspect, any or all of the functionality of the process control device 200 may be implemented as part of a cloud computing platform. The process control device 200 is further described below with reference to FIG. 2.

[0091] 1 represents an example of an automated process control system 100 and process control device 200 configured to provide the automated methods described herein. Although shown connected via a network 202, any suitable series of individual or network connections can be used to enable 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] The 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 standard for communicating between mobile devices, including standards designated as 2G, 3G, 4G, or 5G. Wireless standards may use various channel access methods, such as FDMA, TDMA, CDMA, or SDMA. In an aspect, different types of data may be transmitted over different links and standards. Alternatively, the same type of data may be transmitted over different links and standards. Network communication may occur over any suitable protocol, including, for example, http, tcp / ip, udp, ethernet, ATM, etc.

[0093] Network 202 may be any type and / or form of network. The geographical scope of the network may vary widely, and network 202 may be a body area network (BAN), a personal area network (PAN), a local area network (LAN), such as an intranet, a metropolitan area network (MAN), a wide area network (WAN), or the Internet. The topology of network 202 may be of any form, including, for example, any of the following: point-to-point, bus, star, ring, mesh, or tree. Network 202 may be of any such network topology known to those skilled in the art that can support the operations described herein. Network 202 may utilize layers or stacks of different technologies and protocols, including, for example, Ethernet protocols, Internet Protocol Suite (TCP / IP), ATM (Asynchronous Transfer Mode) techniques, SONET (Synchronous Optical Networking) protocols, or SDH (Synchronous Digital Hierarchy) protocols. The TCP / IP Internet Protocol Suite may include an application layer, a transport layer, an Internet layer (including, for example, IPv4 and IPv4), or a link layer. Network 202 may be some type of broadcast network, telecommunications network, data communications network, or computer network.

[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 media or devices may 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 memories (RAM), read only memories (ROM), erasable programmable read only memories (EPROM or flash memory), static random access memories (SRAM), portable compact disk read only memories (CD-ROM), digital versatile disks (DVD), memory sticks, and the like.

[0095] 2 is a schematic diagram of a computer system or process control device configured to provide automated method process control consistent with the methods described herein. The process control device 200 includes one or more processors 110 (also referred to herein interchangeably as processors 110, processor(s) 110, or processor 110 for convenience), one or more storage devices 120, and / or other components. In alternative aspects, the functions of the processors may be implemented by hardware (e.g., through the use of application specific integrated circuits ("ASICs"), programmable gate arrays ("PGAs"), field programmable gate arrays ("FPGAs"), or the like), or by any combination of hardware and software. The storage device 120 includes any type of non-transitory computer-readable storage medium and / or non-transitory computer-readable storage device. Such computer-readable storage medium or device may store computer-readable program instructions for causing the processor to perform one or more of the methods described herein. Examples of computer readable storage media or devices may 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, but not limited to, 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 disc read only memory (CD-ROM), digital versatile disc (DVD), memory sticks.

[0096] The processor 110 is programmed by one or more computer program instructions stored on the storage device 120, which represent software protocols. For example, the processor 110 is programmed by a process control manager 252, a data collection manager 254, a calibration manager 256, an enzyme determination manager 258, and a user interface manager 260. It will be understood that the functions of the various managers discussed herein are representative and not limiting. Additionally, the storage device 120 may function as a data retention system 201 that provides data storage. As used herein, for convenience, the various "managers" are described as performing operations when in fact the managers program the processor 110 (and thus the process control device 200) to perform operations.

[0097] The various components of the process control device 200 cooperate to provide control of one or more FIAS 150 and one or more analytical instruments 160, as well as to provide interfaces through which users or other systems can interface with these systems.

[0098] The process control manager 252 is a software protocol that runs 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 adjustment of one or more process parameters of the FIA ​​150 and the analyzer 160. As used herein, the term "process parameter" as previously mentioned refers to any parameter or variable of a production process that can be adjusted by a user through the process control device 200. Process parameters include, but are not limited to, paths, flow rates, temperatures, incubation periods, addition of reagents, timing of various process steps, etc. The determination of the control signals may be based on process or production information received by the data collection 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 of the processes that may be performed by the FIA ​​150 and analyzer 160 described herein. For example, the process control manager 252 may be configured to control the flow, routing, and timing of all samples, fluids, reagents, temperatures, agitation, incubation periods, etc. throughout the FIA ​​150 during an automated process, via automated control of the various drives of the FIA ​​150.

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

[0101] In another aspect, the process control manager 252 may be configured to communicate and / or otherwise interact with a calibration manager 256 and an enzyme determination manager 258. The calibration manager 256 and the enzyme determination manager 258 are described in more detail below.

[0102] In one aspect, the process control manager 252 is configured to control the operation of the FIA ​​150 and the analyzer 160, particularly as described further below with respect to the automated methods 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 described in more detail below. In another embodiment, the process control manager 252 controls the flow of the sample through the affinity column 313 to achieve a predetermined volume of eluate as an output. The process control manager 252 may control the flow of the sample to achieve a predetermined volume of eluate within a predetermined elution time. The process control manager 252 is further configured to control the flow of the eluate from an output of the affinity column 313 to a spectrophotometer 414 so that the protein concentration of the eluate can be determined and / or measured by the data collection manager 254, as described below. In one embodiment, the process control manager 252 is configured to control the flow of the eluate from an output of the affinity column 313 to a digestion chamber so that the proteins can be digested by adding an amount of digestion reagent determined by the process control manager 252, as described below. 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 collection manager 254 is a software protocol that runs on the process control device 200. The data collection manager 254 is configured to access one or more of the FIA ​​150, the analyzer 160, the affinity column 155, the spectrophotometer 156, and the digester chamber 415 to collect and / or gather data and information associated with the automated process described herein. The automated system data may include, for example, production information, archived data, and / or extracted data that may be obtained 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 the analyzer 160. The data collection 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 collection manager 254 is configured to interface with the spectrophotometer 156 to perform protein concentration measurements / determinations on the eluate produced or output by the affinity column 313 .

[0106] The calibration manager 256 is a software protocol that runs 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 made by the spectrophotometer 414. The calibration manager 256 is configured to communicate and cooperate with the process control manager 252 and the data collection 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 collection manager 254 to determine the protein concentration in the sample eluate.

[0107] The enzyme determination manager 258 is a software protocol that runs 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 an automated processing procedure. As 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 collection manager 254 operates to obtain protein concentration measurements that, in combination with calibration information, can be used by the process control manager 252 to determine the protein concentration level in the eluted sample. The enzyme determination manager 258 can use the determined protein concentration level to determine the appropriate amount of digestive enzyme to be added to the sample digestion step.

[0108] In operation, the enzyme determination manager 258 can communicate and cooperate with the process control manager 252 to provide information of the determined digestive enzymes for the amount of enzyme added to the second sample, determined by the protein concentration determination of the first sample. As mentioned 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 predefined timing and volume processing parameters to the first sample and the second sample processed by the affinity column 313, the process control manager 252 may be able to achieve a first sample eluate and a second sample eluate having substantially similar concentration values. Thus, as mentioned above, the protein concentration measurements performed on the first sample eluate may be applied by the enzyme determination manager 258 to determine the appropriate amount of digestive enzymes to be added to the second sample eluate.

[0109] The user interface manager 260 is a software protocol that runs on the process control device 200. The user interface manager 260 is configured to provide a user interface that enables a user to interact 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, a touch screen, a keyboard, a mouse, a controller, a joystick, or a 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 suitable 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 full set of interface options, while a smartphone or tablet may be provided with a user interface that is limited to status updates.

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

[0111] 3 is a schematic diagram of a first syringe pump and a first valve system associated with the FIA ​​150 for carrying out the on-line automated methods described herein. In operation, the process control manager 252 of the process control device controls the first valve 301 and the first syringe pump 302 of the FIA ​​150 to properly deliver and mix fluids during the automated process methods described herein. Table 1 below provides an exemplary listing of various connections to the first valve 301 and the first syringe pump 302.

[0112] The first valve 301 includes a number of input ports 304 and an output port 308. The terms "input port" and "output port" are used herein to distinguish between ports at either end of the first valve 301 and do not necessarily refer to the flow direction associated therewith. The first valve 301 may be, for example, a multi-port rotary valve. The process control manager 252 controls a valve automatic selector means (e.g., a stepper motor, servo motor, or other actuator device, not shown) configured to rotate the valve 301 to provide fluid communication between selected input ports 304 and output ports 308. The output port 308 is connected to a 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 stepper motor, servo motor, or other actuator device, not shown) configured to operate the port selection device 311 to align one of the selected plurality of 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 and place a particular syringe pump port 305 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 stepper motor, linear drive, servo motor, or other actuator device, not shown) to provide bidirectional pressure to aspirate and expel 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 depressing and retracting the syringe plunger when driven by the syringe pump drive means. The actuator syringe 306 can be any type of syringe configured to apply pressure bidirectionally to the FIA ​​150 system to aspirate and expel fluid.

[0114] FIG. 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 actuate the first valve 301 to establish a fluid connection between a selected input port 304 and an output port 308. The process control manager 252 may actuate the port selection device 311 to create a fluid connection between a selected syringe pump port 305 and an actuator syringe 306. The actuator syringe 306 may then be actuated to aspirate a particular amount of fluid into the actuator syringe 306 via the selected syringe pump port 305. The port selection device 311 may then be actuated again by the process control manager 252 to create fluid communication between the communication line 309 and the syringe pump actuator 306. The actuator syringe 306 may then be actuated again to aspirate fluid from the first valve 301 into the actuator syringe 306. The actuator syringe 306 and port selection device 311 can then be further actuated to draw additional fluid into the actuator syringe 306 and / or to move fluid in the actuator syringe out through one of the syringe pump ports 305.

[0116] The process control manager 252 can provide accurate and precise control of the actuator syringe 306 to transfer appropriate amounts of fluid through the FIA ​​150 system.

[0117] 4 is a schematic diagram of a second syringe pump and a second valve system associated with the FIA ​​150 for carrying out the on-line automated methods described herein. In operation, the process control manager 252 of the process control device controls the second valve 401 and the second syringe pump 402 of the FIA ​​150 to properly deliver and mix fluids during the automated process methods described herein. Table 1 below provides an exemplary listing 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 between ports at either end of the first valve 301 and do not necessarily refer to the flow direction associated therewith. The process control manager 252 controls a valve automatic selector means (e.g., a stepper motor, 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 the second syringe pump 402 via a communication line 409. The second syringe pump 402 includes a port selection device 411 and a driving 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 stepper motor, servo motor, or other actuator device, not shown) configured to operate the port selection device 411 to align one of the selected plurality of syringe pump ports 405 with the actuator syringe 406. The syringe pump automatic selector means is controlled by the process control manager 252 to select and place a particular syringe pump port 305 in fluid communication with the valve 301 via the communication line 309.

[0119] The actuator syringe 406 is a syringe that is connected to a syringe pump drive means (e.g., a stepper motor, linear drive, servo motor, or other actuator device, not shown) and provides bidirectional pressure to aspirate and expel 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 depressing and retracting the syringe plunger when driven by the syringe pump drive means. The actuator syringe 406 can be any type of syringe configured to apply pressure bidirectionally to the FIA ​​150 system to aspirate and expel fluid.

[0120] Figure 4 also shows an affinity column 313 connected at its input to the first valve 301 and at its output to a multi-way junction 412. The multi-way junction 412 is further connected to a spectrophotometer 414. Figure 4 also shows an S105 digestion chamber 415 connected to a second syringe pump 402 via a syringe pump port 405.

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

[0122] Table 1 below provides examples of various fluid connections, fluid reservoirs / sources, fluid sinks / outlets / drains, etc. within the FIA ​​150 consistent with the system described herein.

[0123] [Table 1]

[0124] With reference to the automated process system 100 described above, a schematic overview of the online automated method is provided in FIG. 5. Briefly, the clarified cell culture medium is delivered to a sample preparation unit including 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 the barrel of a syringe pump. In one embodiment, the temperature of the digestion chamber can be controlled. In one embodiment, the prepared sample is transferred to an analysis device. In one embodiment, the analysis device 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] 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, and the protein digestion, data acquisition, and data processing steps are performed online. In one embodiment, the sample preparation steps, including, for example, protein capture, concentration determination, and protein digestion steps, are performed offline, and the data acquisition and data processing steps are performed online.

[0126] In one aspect, a method for detecting post-translational modifications (PTMs) of proteins is provided. In one aspect, the method is performed by a flow injection analyzer (FIA) controlled by at least one processor. In one aspect, the flow injection analyzer (FIA) is a sequential injection analyzer (SIA). In one aspect, the flow injection analyzer (FIA) is a direct injection analyzer (DIA). As mentioned 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 receiving a first sample comprising a protein of interest in a holding reservoir of an FIA, such as FIA 150 described above. In one embodiment, the first sample is obtained from a bioreactor, such as bioreactor 140 described above. In one embodiment, bioreactor 140 is coupled to the FIA. In one embodiment, the FIA ​​includes a sampling device coupled to bioreactor 140 and configured to obtain cell culture medium or clarified cell culture medium from bioreactor 140. In one embodiment, an automated system interface is used to interface bioreactor 140 with the FIA, such as a Modular Automated Sampling Technology (MAST™) sampling system (Lonza, Bend, OR, USA) or a Seg-Flow automated sampling system (BioProcess International Europe, Vienna, Austria). In one embodiment, a 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 vessel 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 the FIA ​​via the FIA. In one embodiment, the sample loading step includes transferring a first sample from a retention reservoir to the affinity column via the FIA. In one embodiment, the sample elution step includes delivering a first volume of elution buffer to the affinity chromatography column via the FIA, and eluting proteins in the first sample from the affinity chromatography column to form a first eluate. In one embodiment, the proteins in the first sample are eluted from the affinity chromatography column based on a predetermined elution time 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 the automatic control system. In one embodiment, proteins in the first sample are eluted from the affinity column based on a predetermined elution volume to form a first eluate. In one embodiment, the elution volume is controlled by a process control manager of a process control device associated with the 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 at-line. 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 at-line.

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

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

[0132] In one embodiment, the protein concentration determination is performed manually, as shown in Figure 6. In one embodiment, the protein concentration determination is performed offline. In one embodiment, the protein concentration determination is performed at-line.

[0133] After the protein concentration of the first eluate is determined, the method includes receiving a second sample comprising the protein in the holding reservoir. In one embodiment, the second sample is obtained from the bioreactor. In one embodiment, the second sample comprises cell culture medium. In one embodiment, the second sample comprises 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 sample and the second sample are obtained from the cell culture at substantially the same time. In one embodiment, the first sample and the second sample 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 of 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 the affinity column via the FIA. In one embodiment, the sample loading step includes transferring a second sample from a retention reservoir to the affinity column via the FIA. In one embodiment, the sample elution step includes delivering a second volume of elution buffer to the affinity column via the FIA, and eluting the proteins in the second sample from the affinity column to form a second eluate. In one embodiment, the proteins in the second sample are eluted from the affinity column based on a predetermined elution time to form a second eluate. In one aspect, the elution time (and optionally other process parameters such as elution buffer volume) is controlled by at least one processor, e.g., via a process control manager, to substantially match a pre-defined elution time of the first elution, thereby resulting in a second elution. In one aspect, the second elution has a protein concentration substantially similar to that of the first elution. In one aspect, based on the pre-defined elution volume, proteins in the second sample are eluted from the affinity column to form a second elution. In one aspect, the elution time of the second sample is controlled by the process control manager to substantially match the elution time of the first sample. In one aspect, the elution volume of the second sample is controlled by the process control manager 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 at-line. 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 at-line.

[0136] In one aspect, the method includes digesting the proteins in the second eluate to form a peptide mixture. In one aspect, the proteins in the second eluate are enzymatically digested. In one aspect, digesting the proteins in the second eluate includes introducing a digestion reagent to the second eluate via the FIA ​​to form a digestion mixture. In one aspect, digesting the proteins in the second eluate includes introducing a digestion reagent to the second eluate via the FIA ​​to form a digestion mixture, the digestion reagent including a digestion buffer, a reducing agent, and a proteolytic enzyme. In one aspect, the second eluate is contacted with an amount of proteolytic enzyme determined by at least one processor, e.g., an enzyme determination manager, based on the concentration of the first eluate. In one aspect, the method includes mixing the digestion mixture, e.g., via a syringe pump of the FIA ​​controlled by a process control manager. In one aspect, the method includes incubating the digestion mixture, e.g., under control of a process control manager.

[0137] In one embodiment, the proteolytic enzyme comprises thermostable trypsin. In one embodiment, the method includes introducing a first amount of digestion buffer, reducing agent, and thermostable trypsin to the second eluate via the FIA ​​and mixing 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 above incubation process aspects may be controlled via a process control manager.

[0138] In one embodiment, the method includes introducing a second amount of thermostable trypsin to the incubation mixture via the FIA ​​and mixing 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 above incubation process aspects may be controlled via a process control manager.

[0139] In one embodiment, the protein digestion step is performed manually, as shown in Figure 6. In one embodiment, the protein digestion step is performed offline. In one embodiment, the protein digestion step is performed at-line.

[0140] In one embodiment, the method comprises transferring the peptide mixture via FIA to an analytical device, such as a peptide mapping unit (which may include, for example, a mass spectrometer), and detecting certain post-translational modifications (PTMs) of proteins.

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

[0142] In one embodiment, the method includes receiving a first sample comprising a protein in a holding reservoir of an FIA. In one embodiment, the sample is advanced through the FIA ​​by 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 about 10 μL / sec to about 200 μL / sec.

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

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

[0145] In one embodiment, the method includes transferring the first eluate to a protein concentration detection unit by operation of a syringe pump. In one embodiment, the method includes advancing the first eluate to a spectrophotometer by operation of a syringe pump. In one embodiment, the method includes determining, by at least one processor, a concentration of protein in the first eluate according to the measured UV absorbance. In one embodiment, the first eluate is transferred to a waste container after the protein concentration is determined. In one 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 receiving a second sample in a retention reservoir of the FIA. In one embodiment, the method includes advancing the sample through the FIA ​​by operation of a syringe pump. In one embodiment, the method includes delivering a second volume of binding buffer to the affinity column via a multiport valve. In one embodiment, the method includes transferring the second sample from the retention reservoir to the affinity column by operation of a syringe pump. In one embodiment, proteins in the second sample bind to the affinity column. In one embodiment, the method includes introducing a second volume of elution buffer to the affinity column by operation of the multiport valve to elute the bound proteins to form a second eluate. In one embodiment, proteins in the second sample are eluted from the affinity column based on a predetermined elution time to form a second eluate. In one embodiment, proteins in the second sample are eluted from the affinity column based on a predetermined elution volume to form a second eluate. In one aspect, the first and second eluents have substantially the same elution volume.In some aspects, the operation of the syringe pump and the multiport valve may be controlled by a process control manager.

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

[0148] In one embodiment, the proteolytic enzyme comprises a thermostable trypsin. In one embodiment, the method comprises introducing a first amount of a digestion buffer, a reducing agent, and a thermostable trypsin into the second eluate by operation of a multiport valve and mixing to form a first digestion mixture. In one embodiment, the method comprises incubating the first digestion mixture at a temperature of about 70° C. to about 75° C. for about 10 minutes to about 20 minutes. In one embodiment, the method comprises introducing a second amount of a digestion buffer and a thermostable trypsin into the first digestion mixture by operation of a multiport valve and mixing to form a second digestion mixture. In one embodiment, the method comprises 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.

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

[0150] C.Cell culture In one embodiment, the method is used to detect post-translational modifications (PTMs) of a protein 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 culture are known. In general, an expression vector is prepared that contains a coding sequence that encodes the protein. In one aspect, the coding sequence is operably linked to one or more regulatory sequences. Regulatory sequences are known and include, but are not limited to, promoters, enhancers, and other expression control elements that control the transcription or translation of the coding sequence. "Operatively linked" or "operably linked" means that the regulatory sequence is placed in an appropriate position relative to the coding sequence to effect expression of the coding sequence. In one aspect, the regulatory sequence is adjacent to the coding sequence. In one aspect, the regulatory sequence acts in trans or remotely to control the expression of the coding sequence. In one aspect, the recombinant expression vector includes one or more additional sequences, such as an origin of replication and / or a selection marker.

[0152] In one embodiment, the recombinant protein comprises two or more polypeptide chains. In one embodiment, the recombinant protein is an antibody. For proteins comprising two or more polypeptide chains, the coding sequences for 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 secretion of the protein from the host cell.

[0153] For recombinant expression of a protein, a recombinant expression vector encoding the protein is transfected into a host cell. Transfection methods are well known.

[0154] Host cells may include prokaryotic, yeast, or higher eukaryotic cells. In one embodiment, the host cell is a mammalian host cell. Suitable mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO cells), NS0 myeloma cells, COS cells, SP2 cells, COS-7 cells, human embryonic 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 carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL), human lung cells (W138), human hepatocytes (Hep G2), human hepatoma cells (Hep G2), mouse mammary carcinoma 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 grown in a suitable cell culture medium under suitable culture conditions (e.g., temperature, pH, agitation, and dissolved oxygen levels) for expression of recombinant proteins. 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), Iscove's Modified Dulbecco's Medium, and Minimal Essential Medium-alpha (MEM-alpha). In one aspect, 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 aspect, the host cells are cultured in a small scale bioreactor. In one aspect, the host cells are cultured in individual flasks, shake flasks, or wave bags. In one aspect, the host cells are cultured in a bioreactor having a volume of about 125 ml to about 500 ml. In one aspect, the host cells are cultured in a large scale bioreactor. In one aspect, the host cells are cultured in a bioreactor having a volume of about 100 liters to about 20,000 liters or more. In one aspect, the host cells are cultured in a stirred tank, fixed bed, fluidized bed, bubble column, or airlift bioreactor.

[0157] In one aspect, the cell culture is supplemented with a concentrated feed medium containing components such as nutrients and amino acids during the course of the cell culture. In one aspect, during the cell culture process in a batch process, the medium in the bioreactor is not supplemented. In one aspect, 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 aspect, medium is continuously added to the bioreactor and spent medium and products are removed throughout the cell culture process in a perfusion bioreactor.

[0158] In one aspect, the method includes obtaining a first sample and a second sample from a cell culture. In one aspect, the first sample and the second sample are obtained from the same cell culture. In one aspect, the first sample and the second sample are obtained from the cell culture at substantially the same time. As used herein, two operations performed "substantially at the same time" can mean that the two operations are performed in parallel, but also includes situations where the two operations are performed within a short time of each other. In one aspect, the first and the sample are obtained within about 30 minutes of each other. In one aspect, the first and the sample 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 protein of interest in a sample. In one embodiment, the protein is recombinantly produced. In one embodiment, the recombinantly produced protein comprises 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 aspect, the protein is a glycoprotein. In one aspect, the protein is a therapeutic antibody or an antigen-binding fragment thereof. In one aspect, the protein is a receptor or a receptor ligand. In one aspect, the protein is a fusion protein.

[0161] Recombinant proteins can be produced intracellularly, in the periplasmic space, or directly secreted into the cell culture medium. If the polypeptide protein is produced intracellularly, the first step of the purification process usually involves lysis of the cells, for example by mechanical shearing, osmotic shock, or enzymatic treatment, which releases the entire contents of the cell into a homogenate and generates cell debris that can be removed by differential centrifugation or filtration.

[0162] In one aspect, the protein is a therapeutic protein. As used herein, a "therapeutic protein" is a protein that has one or more biological activities useful for treating, preventing, or ameliorating a disease or at least one symptom of a disease.

[0163] E. Post-translational Modifications "Post-translational modification" (PTM) refers to one or more covalent modifications made to a polypeptide during or after protein synthesis. In one aspect, PTMs are introduced by an enzyme or enzymatic pathway. PTMs can result in varying levels of protein heterogeneity, which can have significant effects on protein structure and function, including but not limited to biological activity, binding activity, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of recombinantly produced proteins. Some PTMs can be beneficial to the physicochemical characteristics of therapeutic proteins, while others can be detrimental. The amount and type of PTMs can be affected by cell culture conditions and are considered critical quality attributes (CQAs) that need to be monitored during the manufacture of recombinantly produced therapeutic proteins.

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

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

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

[0167] In one embodiment, the PTM comprises a high mannose glycan. As used herein, "high mannose glycan" refers to an N-glycan structure that contains 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 Provided herein is an automated online system or method for detecting post-translational modifications (PTMs) in a polypeptide. In one aspect, a method or system is provided for detecting PTMs in a recombinantly produced polypeptide. In one aspect, a method or system is provided for detecting PTMs in a protein. In one aspect, a method or system is provided for detecting PTMs in a recombinantly produced protein. In one aspect, a method or system is provided for detecting PTMs in a therapeutic protein. In one aspect, a method or system is provided for detecting PTMs in a recombinantly produced therapeutic protein.

[0169] In one aspect, an automated online sample preparation system or method is provided. In one aspect, the automated online sample preparation system or method includes one or more of a protein capture step, a concentration determination step, and a protein digestion step. In one aspect, 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 aspect, an automated online sample preparation system or method is provided, in which proteins in a sample are prepared for an analytical process (e.g., for detecting one or more PTMs). In one aspect, the automated online sample preparation system or method prepares proteins in a sample for mass spectrometry (MS). In one aspect, the automated online sample preparation system or method prepares proteins in a sample for liquid chromatography / mass spectrometry (LC / MS). In one aspect, the automated online sample preparation system or method prepares proteins in a sample for an automated LC / MS system. In one aspect, the automated online sample preparation system or method prepares proteins in a sample for an integrated LC / MS system.

[0170] A schematic overview of the online automated method is provided in Figure 5. 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 device, 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 of the method steps may be performed manually or offline. In one embodiment, the protein capture and concentration determination steps for sample preparation are performed offline, and the protein digestion, data acquisition, and data processing steps are performed online. In one embodiment, the sample preparation steps, including, for example, protein capture, concentration determination, and protein digestion steps, are performed offline, and the data acquisition and data processing steps are performed online.

[0172] 1. Flow Injection Analysis In one embodiment, the automated sample preparation is performed using a flow injection analyzer (FIA) optionally included in an automated process control system including 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 a valve. 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 discussion of an automated process implemented by an automated process control system 100, including at least one process control device 200, at least one bioreactor 140, at least one FIA ​​150, and at least one analyzer 160, may be understood with reference to FIGS. 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 the second valve 401 of the FIA ​​150 is merely exemplary. Other arrangements and configurations may 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, the first syringe pump 302 and the second syringe pump 402 may be swapped. In another example, alternative or additional ports and / or connections may be used. In the following discussion, 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 automated aspects of the system are controlled by the process control device 200, as previously described with reference to FIG.

[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 coupled to the FIA. In one embodiment, the FIA ​​includes a sampling device coupled 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 interface the bioreactor 140 and the FIA, such as a Modular Automated Sampling Technology (MAST™) sampling system (Lonza, Bend, OR, USA) or a 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, such as by introducing the FIA's holding reservoir into a vessel containing the cell culture sample. In one embodiment, a sample having a volume of about 20 μL to about 1000 μL is introduced into a holding reservoir of the FIA. 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, the filtration comprises microfiltration or depth filtration. In one embodiment, the sample comprises a polypeptide. In one embodiment, the host cells in the cell culture are removed from a bioreactor. In one embodiment, the host cells in the cell culture are removed during cell culture (e.g., in a perfusion bioreactor). In one embodiment, the sample comprises a recombinantly produced polypeptide. In one embodiment, the sample comprises a protein. In one embodiment, the sample comprises a recombinantly produced protein. In one embodiment, the sample comprises a recombinantly produced therapeutic protein.

[0175] 2. Protein Capture In one aspect, the method includes a protein capture step in which a protein of interest in a sample is purified to separate it from impurities, such as host cell proteins or other product-related impurities, present in the sample. In one aspect, the sample includes clarified cell culture medium from which cells and other cellular debris have been removed. In one aspect, the protein capture step includes affinity chromatography.

[0176] "Affinity chromatography" refers to a chromatographic process in which a target biomolecule, e.g., a protein, in a mobile phase is retained by a stationary phase of a chromatographic column based on a specific and reversible binding interaction between the target component and a compound immobilized on a chromatographic matrix. In one embodiment, affinity chromatography includes a column equilibration step, a sample loading step, and a sample elution step. In one embodiment, the chromatographic 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 / sec to about 100 μL / sec.

[0177] In one embodiment, the method comprises 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 a buffer used as an equilibration buffer, a loading buffer, and / or a washing buffer.

[0178] In one embodiment, the protein capture step takes about 15 minutes to about 2 hours. In one embodiment, the protein capture step takes about 15 minutes to about 1 hour. In one embodiment, the protein capture step takes less than about 30 minutes.

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

[0180] In one embodiment, the sample is loaded onto the 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, about 20 μL to about 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 about 10 μL / sec to about 200 μL / sec. The sample can be accessed by the FIA ​​105 at the V209 input port 304 and pumped by the first syringe pump 302 to the affinity column 313 via the V208 input port 304.

[0181] In one embodiment, the method includes a column washing step, for example to remove proteins that interact weakly or non-specifically with the affinity chromatography resin. In one embodiment, the wash buffer has the same composition as the equilibration buffer and / or the loading buffer. In one embodiment, the term "binding buffer" refers to the buffer used as the equilibration buffer, the loading buffer, and the wash buffer. In one embodiment, the wash buffer has a different composition than the equilibration buffer and / or the loading buffer. In one embodiment, the loaded column is washed with about 5 to about 10 column volumes (CV) of wash buffer. In one embodiment, the loaded column is washed with wash buffer at a flow rate of about 10 μL / sec to about 200 μL / sec. In one embodiment, the wash buffer is accessed by the FIA ​​105 at the S202 syringe pump port 305 and delivered by the second syringe pump 402 to the affinity column 313 via the S108 syringe pump port 405.

[0182] In one embodiment, the protein of interest is eluted from the column in an elution step. In one embodiment, the protein of interest is eluted from the column by passing an elution buffer over the column on which the sample is immobilized. In one embodiment, the protein of interest is eluted from the column using an elution buffer having a different pH or salt concentration than the loading buffer. In one embodiment, the elution is a gradient elution, e.g., where the composition of the elution buffer changes linearly over time. In one embodiment, the elution is a step elution, e.g., where the composition of the elution buffer is 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 eluent. In one embodiment, the elution buffer is applied to the column at a flow rate of about 10 μL / sec to about 200 μL / sec to elute the protein of interest from the column. In one embodiment, the solution eluted from the column containing the protein of interest is referred to as the eluate. The elution buffer may be accessed by the FIA ​​105 at the V203 input port 305 and delivered by a first syringe pump 302 to the affinity column 313 via the V208 input port 304.

[0183] In one embodiment, the protein is an antibody, antibody fragment, or other protein that comprises an Fc region, e.g., an Fc fusion protein. In one embodiment, the affinity column comprises an immunoglobulin-binding protein. In one embodiment, the affinity column comprises an immunoglobulin-binding bacterial protein. In one embodiment, the affinity column comprises 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 involves a column with a protein obtained from Staphylococcus aureas. In one embodiment, Protein A chromatography involves a column with a protein produced by recombinant or synthetic methods. In one embodiment, Protein A chromatography involves a column with a Protein A variant that retains the ability to bind to the Fc region of an immunoglobulin.

[0185] In one embodiment, the protein of interest comprises an N- or C-terminal tag to facilitate protein purification, including, but not limited to, tags comprising streptavidin, biotin, poly-histidine (His), or glutathione-S-transferase (GST).

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

[0187] As shown in FIG. 7, the eluate obtained from the protein capture step can be transferred to a protein concentration detection unit or a protein digestion step through the operation of the FIA ​​150. 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 pumped from the affinity column 313 by the second syringe pump 402 and transferred to a protein concentration determination unit (e.g., a spectrophotometer 414) through a multi-way connection 412. In one embodiment, the second sample is purified by affinity chromatography using the affinity column 313, and a second eluate is obtained. The second eluate is transferred to a protein digestion step. The second eluate can be pumped from the affinity column 313 by the second syringe pump 402 and transferred to the S105 digestion chamber 415 through 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 can be enabled through the operation of a second syringe pump 402, which draws the eluate from the affinity column 313 through a multi-way valve 412 via an S108 syringe pump port 405 and pushes the eluate back through the multi-way valve 412 to the spectrophotometer 414. 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 about 190 nm to about 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 UV absorbance at about 175 nm to about 280 nm. In one embodiment, the protein concentration in the sample is determined by measuring UV absorbance at about 280 nm. In one embodiment, the protein concentration in the sample is determined by measuring UV absorbance due to the peptide backbone. In one embodiment, the protein concentration in the sample is determined by measuring UV absorbance at about 190 nm to about 220 nm. In one embodiment, the protein concentration in the sample is determined by measuring UV absorbance at about 205 nm. In one 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 spectrophotometrically and the protein concentration is calculated based on a predetermined calibration curve. In one embodiment, the concentration determination step takes about 5 minutes to about 30 minutes, or less than about 10 minutes. The UV absorbance data can be measured by the data collection manager 254, which in conjunction with the process control manager 252 and the calibration manager 256 can determine the protein concentration as described above.

[0190] 4. Automated Protein Digestion in Solution In one embodiment, the eluate from the protein capture step is transferred to the digestion chamber. In one embodiment, the second eluate from the protein capture step is transferred to the digestion chamber. In one embodiment, the second eluate from the affinity chromatography step is transferred to the digestion chamber. The second eluate can be pumped from the affinity column 313 by the second syringe pump 402 and transferred to the digestion chamber 415 through the T101 multi-way valve 412, the S108 syringe pump port, and the S105 digestion chamber 415.

[0191] In one embodiment, the proteins in the eluate 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 from the affinity chromatography column are digested to peptides having an average size of less than about 3000 Da, less than about 2500 Da, less than about 2000 Da, or less than about 1500 Da. In one embodiment, the proteins in the eluate from the affinity chromatography column are digested to peptides having an average size of about 500 Da to about 5000 Da. In one embodiment, the proteins in the eluate from the affinity column are digested to peptides having an average size of about 1000 Da to about 5000 Da. In one aspect, proteins in the eluate from the affinity chromatography column are digested into peptides having an average size of about 500 Da, about 750 Da, or about 1000 Da, up to about 1500 Da, 2000 Da, 2500 Da, or about 3000 Da. In one aspect, proteins in the eluate obtained from the affinity chromatography column are digested to form a peptide mixture comprising peptides about 2 to about 100 amino acids in length, at least about 5 or about 10 amino acids in length, and up to about 15, about 25, about 50, or about 100 amino acids in length, or about 5 to about 50, or about 10 to about 25, or about 10 to about 15 amino acids in length.

[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 detergents, including but not limited to guanidine hydrochloride and urea. Once the protein is denatured, disulfide bonds are reduced by adding a reducing agent, such as dithiothreitol (DTT), glutathione, b-mercaptoethanol (b-ME), or tris(2-carboxyethyl)phosphine (TCEP). To prevent the reformation of disulfide bonds, an alkylating agent, such as iodoacetic acid, iodoacetamide, acrylamide, or chloroacetamide, is added.

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

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

[0195] Provided herein is a time-efficient, streamlined protein digestion method. In one aspect, the protein digestion method is automated and online. In one aspect, the automated online protein digestion method takes from about 5 minutes up to about 1 hour, whereas conventional protein digestion processes can take up to 4 hours. In one aspect, the protein digestion takes from about 5 minutes to about 30 minutes. In one aspect, the protein digestion takes from about 15 minutes to about 30 minutes. In one aspect, the protein digestion takes less than about 30 minutes. In one aspect, the online protein digestion method does not include an alkylation or buffer exchange step.

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

[0197] In one embodiment, protein digestion is carried out at high temperature. In one embodiment, proteins are denatured using high temperature denaturation. In one embodiment, protein digestion is carried out using a thermostable protease. In one embodiment, protein digestion is carried out at 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 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 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, the protein digestion is carried out at a temperature of about 65° C. to about 80° C. In one embodiment, the protein digestion is carried out at a temperature of about 70° C. to about 75° C. In one embodiment, the protein digestion is carried out at a temperature of about 70° C. to about 72° C.

[0198] In one aspect, the protein digestion comprises contacting the protein solution with a digestion reagent to form a digestion mixture. In one aspect, the digestion reagent comprises a digestion buffer, a reducing agent, and a proteolytic enzyme. In one aspect, the eluate from the affinity column is contacted with the digestion reagent to form a digestion mixture. In one aspect, the eluate from the affinity column is contacted with a digestion reagent comprising a digestion buffer, a reducing agent, and a proteolytic enzyme to form a digestion mixture. In one aspect, a second eluate from the affinity column is contacted with a digestion buffer, a reducing agent, and a proteolytic enzyme to form a digestion mixture.

[0199] In one aspect, the digestion buffer has a pH of about 6.0 to about 8.0, or about 6.5 to about 7.5. In one aspect, the digestion buffer has a pH from about 6.5. In one aspect, the digestion buffer is from Thermo Fisher SMART Digestion Kit (Thermo Fisher, Waltham, MA, USA).

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

[0201] In one aspect, the proteolytic enzyme 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 aspect, the proteolytic enzyme includes trypsin. In one aspect, the proteolytic enzyme includes thermostable trypsin. In one aspect, the proteolytic enzyme 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 detergents, especially high concentrations of salts and / or detergents. 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] Instead, 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 chemical denaturing reagents, such as chaotropic agents such as guanidine hydrochloride and urea; reducing agents such as dithiothreitol (DTT), glutathione, or b-mercaptoethanol (b-ME); or alkylating agents such as iodoacetic acid, iodoacetamide, acrylamide, or chloroacetamide, which are removed using a buffer exchange step. In one embodiment, the buffer exchange reagent is delivered to the digestion chamber through a multi-port valve. In one embodiment, the digestion method is performed at room temperature, which would result in the process taking longer.

[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 contacted with trypsin at a trypsin:protein ratio of about 1:10 to about 1:100. In one embodiment, the second eluate is contacted with trypsin at a trypsin:protein ratio of about 1:20 to about 1:30. In one aspect, the trypsin:protein ratio is at least about 1:10 and up to about 1:100, or at least about 1:10 or 1:20 and up to about 1:30, about 1:40, about 1:50, or about 1:100, or from about 1:10 to about 1:100, from about 1:10 to about 1:50, from about 1:10 to about 1:40, from about 1:10 to about 1:30, from about 1:10 to about 1:20, from about 1:20 to about 1:100, from about 1:20 to about 1:50, from about 1:20 to about 1:40, or from about 1:20 to about 1:30.

[0205] In one aspect, the second eluate is contacted with the digestion buffer, the reducing agent, and the proteolytic enzyme simultaneously. As used herein, "simultaneously," when used in connection with the method steps described herein, refers to a method in which the steps are performed at substantially the same time (i.e., the performance of at least a portion of one method step overlaps in time with the performance of a portion of another method step). "Simultaneously" does not require strictly parallel action; that is, not all method steps need to start or end at the same time. In one aspect, "simultaneously" can mean that all reagents required for a method step are combined in the same reaction mixture such that the reactions occur in the same reaction volume and during the same incubation period. In one aspect, the digestion mixture comprises the digestion buffer, the reducing agent, the proteolytic enzyme, and the second eluate. In one aspect, the digestion mixture is prepared by introducing the second eluate and other reagents into a chamber of a syringe, and the digestion mixture is mixed by depressing and retracting the plunger of the syringe. In one aspect, the digestion chamber is used as an incubation chamber. For example, the second eluate may be transferred from the affinity column 313 as previously described to the chamber of the drive syringe 406. The drive syringe 406 may further have access to the protease via the S102 syringe pump port 405. The second eluate and the protease, as well as any other suitable reagents, may then be mixed in the chamber of the drive syringe 406.

[0206] In one embodiment, the second eluate is contacted with a digestion buffer, a reducing agent, and a proteolytic enzyme sequentially. As used herein, "sequentially," when used in connection with the method steps described herein, refers to a method in which the steps are performed at different times, e.g., separate events occur in the practice of the method. In one embodiment, the sequential steps are performed during separate incubation periods. In one embodiment, the sequential steps are performed in different reaction mixtures. In one embodiment, the sequential method steps are performed at different times, but in the same reaction chamber.

[0207] In one embodiment, the temperature of the incubation chamber can be increased, for example, to incubate the digestion mixture at a temperature of about 35°C to about 85°C. In one embodiment, the digestion 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 about 75°C, about 80°C, or about 85°C. In one embodiment, the digestion 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 about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In one embodiment, the digestion mixture is incubated at a temperature of about 50°C to about 85°C. In one embodiment, the digestion mixture is incubated at a temperature of about 65°C to about 80°C. In one embodiment, the digestion mixture is incubated at a temperature of about 70°C to about 55°C. In one embodiment, the digestion mixture is incubated at a temperature of about 70° C. to about 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, incubating the digestion mixture comprises incubating for about 5 minutes to about 30 minutes at a temperature of about 36° C. to about 85° C. In one embodiment, incubating the digestion mixture comprises incubating for about 15 minutes to about 30 minutes at a temperature of about 50° C. to about 75° C. In one embodiment, incubating the digestion mixture comprises incubating for about 15 minutes to about 30 minutes at a temperature of about 70° C. to about 72° C.

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

[0210] G. Peptide Mapping Analysis In one embodiment, the peptide mixture is transferred to an analytical device. In one embodiment, the analytical device includes a peptide mapping unit. In one embodiment, as shown in FIG. 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 analyzer. For example, the peptide mixture can be drawn from the digestion chamber 415 through the V108 input valve 404 by the operation of the second syringe pump 402 into an analytical device, such as a peptide mapping unit.

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

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

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

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

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

[0216] In one embodiment, the mass analyzer separates gas phase ions by mass to charge ratio (m / z). In one embodiment, the mass analyzer comprises tandem mass spectrometry (MS / MS), which comprises two or more steps of mass analysis in which fragmentation is performed during the mass analysis 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 the data acquisition unit to identify and / or quantify post-translational modifications (PTMs) of proteins. In one embodiment, peptides identified by the mass spectrometer are used as surrogates of intact proteins obtained from the cell culture medium. In one embodiment, the data processing provides information regarding the amino acid sequence of the protein and / or the presence, location, and / or amount of one or more PTMs. In one embodiment, the data processing unit provides information regarding a given PTM. In one embodiment, the data processing takes about 5 minutes to about 30 minutes, or less than about 10 minutes. In an embodiment, the data processing unit of the peptide mapping unit can interface with and / or otherwise communicate with the process control device 200. In some embodiments, the functions of the data processing unit of the peptide mapping unit can be performed by software running on the process control device 200 without the need for a data processing unit.

[0218] H. Modulation of post-translational modifications / modifications to cell culture conditions In one aspect, a method of producing a recombinant protein is provided. In one aspect, the method includes culturing a host cell under conditions in which the recombinant protein is expressed; detecting a post-translational modification (PTM) of the protein according to the methods described herein; and modifying one or more cell culture parameters based on the detected post-translational modification. In one aspect, a recombinant protein produced by the methods described herein is provided. In one aspect, a pharmaceutical composition is provided comprising a recombinant protein produced by the methods described herein and a pharma- ceutically acceptable carrier.

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

[0220] I. INCORPORATION BY REFERENCE All references cited herein (including patents, patent applications, articles, textbooks, etc.), and the references cited therein, to the extent they have not already been cited, are hereby incorporated by reference in their entirety. 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 to obtain clarified cell culture medium (CCM), which 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 to control the system components and to collect and analyze data. 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] A first sample of 0.3 mL of CCM was obtained from the holding reservoir and injected through valve 1 of the FIA ​​onto the Protein A affinity column pretreated with equilibration buffer. The first sample IgG1 mAb was eluted from the Protein A column as follows: 500 μL of binding buffer was injected onto the affinity column at 50 μL / s–100 μL / s; 300 μL of CM was injected at 50 μL / s–100 μL / s; 1 mL of binding buffer was injected for washing at 50 μL / s–100 μL / s; and 100–300 μL of elution buffer was injected at 20 μL / s.

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

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

[0225] Tris(2-carboxyehtyl)phosphine (TCEP), 0.01 mL (concentration 500 mM), 0.3 mL of digestion buffer, and trypsin were injected into the barrel of syringe pump 2 through 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 depressing and retracting the plunger of the syringe, and the mixture was incubated at 70° C. for 15 min to obtain a sample containing tryptic digest peptides ("tryptic peptides").

[0226] Example 2. Online peptide mapping Peptide mapping of tryptic peptides from the FIA ​​sample preparations of Example 1 was analyzed by liquid chromatography coupled with UV detection and mass spectrometry using a Waters BioAccord™ Integrated Peptide Mapping System (comprising an ACQUITY™ UPLC™ I-Class PLUS System equipped with a ultraviolet (TUV) detector coupled in-line to an ACQUITY RDa™ mass spectrometer).

[0227] The chromatographic conditions were as follows: temperature 65°C; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile; gradient elution (100%-40%); and flow rate: 0.2 ml / min. The analysis time for mobile phase A was 40 min. UV detection 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 of the Protein A eluate was evaluated.

[0230] A calibration curve was generated by injecting known concentrations of reference standards (RS) in known volumes onto the affinity column. 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 onto the affinity column. UV response readings were recorded in the built-in calibrator table. A calibration curve was generated using the calculated slope and coefficients. The calibration data was saved as a calibration file.

[0231] Before running the digestion process, a calibration file was opened in the FIA ​​software. A cell culture media sample of unknown concentration was then injected into the affinity column. The UV response was obtained and entered into the calibration file, and the protein concentration was calculated. During the digestion method run, live feedback of titer information was taken and fitted to the enzyme calculations.

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

[0233] FIG. 9B shows the UV profile used to perform concentration determination of the first eluate, showing that the same amount of protein was captured for the second eluate, which was subsequently used for protein digestion.

[0234] Example 4. Glycan profiling: online methods compared to glycan release The glycan profile determined using the online peptide mapping method was compared to the results of the glycan profile using a conventional glycan release method (2-aminobenzamide (Oligo 2-AB) glycan detection). Briefly, the glycosylation profile of the 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] Briefly, 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 the 2AB labeling reagents, they were combined with the glycans and incubated at 65°C for 3 hours. The labeled glycans were then washed to remove the access reagent and dried.

[0236] Glycosylation of ten glycans (G0, G0F, G0F-GlcNAc, G0-GlcNAc, G1, G1F, G2F, G2f+SA, Man5, and Man6) was determined using the online peptide mapping and Oligo2-AB glycan release methods described in Example 1. As shown in FIG. 10A, the glycosylation profile determined using the online method showed a similar trend to the glycosylation profile determined using the Oligo-2AB procedure. FIG. 10B shows the quantification percentage of one of the glycan species (mannose) monitored during the cell culture period. Replicate experiments at each time point show a consistent level of quantification percentage.

[0237] Example 5. Glycan profiling: online methods compared to glycan release Glycan trends were determined for sample IgG1 mAb from four different bioreactors at three time points (12 time points in total) using the online peptide mapping and Oligo 2-AB glycan release methods according to the procedures described in Example 4. There was a direct linear relationship between the two methods, indicating that the results were comparable for the types of glycosylation tested.

[0238] Figures 11A-E show the correlation between the two methods for five 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)).

[0239] Example 6. Detection of post-translational modifications Oxidation is a common post-translational modification found at various methionine sites within monoclonal antibodies. Methionine oxidation is an important product quality attribute monitored during the cell culture process. Figure 12A shows that oxidation levels at specific methionine residues remained 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 more than 95% when measured on days 7, 10, and 14 of cell culture. Repeated experiments at each time point show 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] If a molecule has more than one glycosylation site, peptide mapping analysis can provide site-specific glycosylation information. For example, the amino acids at which glycosylation occurs can be determined at the peptide level, providing information about the site-specific glycosylation of the intact protein. In contrast, in glycan release methods, all site-specific information about glycosylation is lost when the glycans are cleaved from the protein for analysis.

Claims

1. 1. A method for detecting post-translational modifications of proteins, the method being carried out by a flow injection analyzer (FIA) controlled by at least one processor, comprising: (a) containing a first sample containing the protein in a retention reservoir; (b) delivering a first volume of binding buffer via the FIA ​​to an affinity column within the FIA; (c) transferring the first sample from the retention reservoir to the affinity column via the FIA; (d) delivering a first volume of an elution buffer to the affinity column via the FIA, thereby eluting the proteins in the first sample from the affinity column to form a first eluate; (e) transferring the first eluate via the FIA ​​to a protein concentration detection unit; (f) determining by said at least one processor a concentration of said protein in said first eluate; (g) containing a 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 retention 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 proteins in the second sample from the affinity column to form a second eluate; (k) transferring the second eluate via the FIA ​​to a digestion chamber; (l) delivering a digestion reagent via the FIA ​​to digest the proteins in the second eluate to form a peptide mixture; and (m) transferring said peptide mixture to a peptide mapping unit via said FIA to detect said post-translational modifications of said proteins; A method comprising:

2. 2. The method of claim 1, wherein the FIA ​​is a sequential injection analyzer (SIA) or a direct injection analyzer (DIA), and the protein concentration detection unit optionally includes a spectrophotometer, which optionally measures the UV absorbance of the first eluate and calculates the protein concentration based on a calibration curve by the at least one processor.

3. 2. The method of claim 1, wherein the first sample and the second sample are obtained from a cell culture, optionally wherein the cell culture is in a bioreactor, optionally wherein the bioreactor is a batch bioreactor, a fed-batch bioreactor, or a perfusion bioreactor.

4. 2. The method of claim 1, further comprising controlling, by the at least one processor, an elution time of the proteins in the first sample eluted from the affinity column to form the first eluate in (d) based on a predetermined elution time.

5. 5. The method of claim 4, further comprising controlling by the at least one processor an elution time of the protein in the second sample eluted from the affinity column to form the second eluate in (j) based on the same predetermined elution time as the first sample in (d).

6. 6. The method of any one of claims 1 to 5, wherein the proteins in the second eluate are enzymatically digested with (l), and optionally the enzymatic digestion of the proteins comprises contacting the second eluate via the FIA ​​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).

7. (l) is (i) introducing a digestion reagent via the FIA ​​to form a digestion mixture; (ii) mixing the digestion mixture; and (iii) incubating the digestion mixture; The method of claim 6, comprising:

8. 8. The method of claim 7, wherein the digestion buffer comprises a digestion buffer at a pH of 6.0 to 7.5, and optionally the digestion reagent comprises a reducing agent comprising tris(2-carboxyethyl)phosphine (TCEP) and / or a proteolytic enzyme.

9. 9. The method of claim 7 or 8, wherein incubating the digestion mixture comprises incubating at a temperature between 36°C and 85°C for between 5 minutes and 30 minutes.

10. The proteolytic enzyme comprises a thermostable trypsin, and the method further comprises: (i) introducing a first amount of a digestion buffer, a reducing agent, and a thermostable trypsin into the second eluate via the FIA ​​and mixing to form a first digestion mixture; (ii) incubating the first reaction mixture at a temperature of 70° C. to 75° C. for 10 minutes to 20 minutes to form a first incubation mixture; (iii) introducing a second amount of thermostable trypsin into the incubation mixture via the FIA ​​and mixing to form a second digestion mixture; and (iv) incubating the second digestion mixture at a temperature of 70° C. to 75° C. for 10 minutes to 20 minutes to form a peptide mixture; The method of claim 7, comprising:

11. 1. An automated method for monitoring post-translational modifications of proteins, the method being carried out by a flow injection analyzer (FIA) comprising a syringe pump and a multiport valve controlled by at least one processor, (a) containing a first sample containing the protein in a holding reservoir of the FIA; (b) introducing a first volume of binding buffer into an affinity column within the FIA ​​by operation of the multiport valve; (c) propelling the first sample from the retention reservoir to the affinity column by operation of a syringe pump, thereby binding the proteins in the first sample to the affinity column; (d) introducing a first volume of elution buffer into the affinity column by operation of the multiport valve to elute the bound protein to form a first eluate; (e) advancing the first eluate to a spectrophotometer within the FIA ​​by operation of the syringe pump; (f) determining, by the at least one processor, a concentration of protein in the first eluate according to the measured UV absorbance; (g) containing a second sample containing the protein in the retention reservoir; (h) introducing a second volume of the binding buffer into the affinity column by operation of the multiport valve; (i) propelling the second sample from the retention reservoir to the affinity column by operation of the syringe pump, thereby binding the proteins in the second sample to the affinity column; (j) introducing a second volume of elution buffer into the affinity column by operation of the multiport valve to elute the bound protein to form a second eluate; (k) advancing the second eluate to a digestion chamber within the FIA ​​by operation of the syringe pump; and (l) introducing a digestion reagent into the second eluate in the digestion chamber by operation of the multiport valve to digest the proteins to form a peptide mixture; and (m) advancing the peptide mixture to a peptide mapping unit by operation of the syringe pump to detect post-translational modifications of the protein; The automated method includes:

12. (l) digesting the protein, (i) introducing a digestion buffer, a reducing agent, and a proteolytic enzyme into the second eluate in the digestion chamber by operation of the multiport valve and mixing to form a first digestion mixture; (ii) incubating the first digestion mixture in the digestion chamber to form a first incubation mixture; (iii) introducing a second volume of digestion buffer and proteolytic enzyme into the incubation mixture by operation of the multiport valve to form a second digestion mixture; and (iv) incubating the second digestion mixture in the digestion chamber to form a peptide mixture; The method of claim 11 , comprising:

13. 13. The method of claim 12, wherein the digestion buffer has a pH of 6.0 to 7.5, and optionally the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

14. The proteolytic enzyme comprises a thermostable trypsin, and the method further comprises: (i) introducing a first amount of a digestion buffer, a reducing agent, and a thermostable trypsin into the second eluate by operation of the multiport valve and mixing to form a first digestion mixture; (ii) incubating the digestion mixture at a temperature of 70° C. to 75° C. for 10 minutes to 20 minutes to form a first incubation mixture; (iii) introducing a second amount of digestion buffer and thermostable trypsin into the incubation mixture by operation of the multiport valve and mixing to form a second digestion mixture; and (iv) incubating the second digestion mixture at a temperature of 70° C. to 75° C. for 10 minutes to 20 minutes to form a peptide mixture; The method of claim 12, comprising:

15. 1. An automated system for monitoring post-translational modifications of proteins, comprising: (a) a retention reservoir configured to accommodate a first sample and a second sample comprising 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 in fluid communication with the first multiport valve and configured to receive the first sample and the second sample from the retention reservoir; (e) a spectrophotometer configured to receive a first eluate from the affinity column, the first eluate comprising proteins from the first sample, the spectrophotometer configured to determine UV absorbance and calculate a 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 to a digestion chamber, the second eluate comprising proteins from the second sample, the proteins in the second eluate being 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; , an automated system.

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