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WO2024259275A1 - Near real time sialic acid quantitation of glycoproteins - Google Patents

Near real time sialic acid quantitation of glycoproteins Download PDF

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Publication number
WO2024259275A1
WO2024259275A1 PCT/US2024/034067 US2024034067W WO2024259275A1 WO 2024259275 A1 WO2024259275 A1 WO 2024259275A1 US 2024034067 W US2024034067 W US 2024034067W WO 2024259275 A1 WO2024259275 A1 WO 2024259275A1
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hours
polypeptides
column
acid
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Letha CHEMMALIL
Mathura RAMAN
Christopher Michael CHUMSAE
Hongqing DING
Anthony Michael LEONE
Tanmay Abhay KULKARNI
Priya Rajkumar SINGH
Rong Li
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Bristol Myers Squibb Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis

Definitions

  • Glycosylation has been shown to have an effect on protein activity, antigenicity, stability and pharmacokinetic/pharmacodynamic properties.
  • sialylation needs to be optimized to ensure the prolonged circulatory half-life of glycoproteins in serum.
  • natural and recombinant forms of erythropoietin carry three sialylated N-glycans and one sialylated O-glycan.
  • Engineering of erythropoietin to a hypersialylated form has shown to exhibit better pharmacokinetic properties, especially a longer half-life in the bloodstream (Varki A, et al. Essentials of Glycobiology.2nd edition.
  • Certain aspects of the disclosure are directed to methods for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA
  • the released sialic acids are mixed with a sialic acid labeling agent, thereby labeling the sialic acids, prior to d).
  • Certain aspects of the disclosure are directed to methods for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid
  • two, three, four, or five or more of the steps are automated. In some aspects, all of the steps are automated. [0010] In some aspects, one, two, three, four, or five or more of the steps are performed in a closed system. In some aspects, all of the steps are performed in a closed system. [0011] In some aspects, the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected automatically. [0012] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column.
  • the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column.
  • the desialylation agent is an acid or an exoglycosidase.
  • the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof.
  • the exoglycosidase is a sialidase.
  • the sialic acid labeling agent is 1,2-diamino-4,5- methylenedioxybenzene (DMB) or o-phenylenediamine.
  • the analytical device is a UPLC, uHPLC or HPLC.
  • FIG.1 are overlaid chromatograms showing the amount of sialic acids released from glycoproteins after either enzymatic digestion with sialidase (black line) or acid hydrolysis (blue line).
  • FIG.2 is a chromatogram showing the amount of labeled sialic acids after 2 hours (black line), 3 hours (blue line), or 4 hours (green line) of labeling with DMB.
  • FIGs.3A-3B are chromatograms showing UPLC separation of DMB labeled NANA and NGNA from either a mixture of NANA and NGNA (blue line) (FIG.3A) or isolated NANA (blue line) and NGNA (red line) (FIG.3B). The reagent peak is shown in red (FIG.3A) or green (FIG.3B).
  • FIGs.4A-4B are chromatograms showing UPLC separation of NANA and NGNA.
  • FIG.4A is a chromatogram showing UPLC separation of 1.9 pmol (blue line), 3.9 pmol (orange line), 7.85 pmol (dark green line), 15.6 pmol (dark blue line), 31.25 pmol (brown line), 62.5 pmol (pink line), 125 pmol (light blue line) 250 pmol (bright green line), 500 pmol (blue line), or 1000 pmol (black line) of NANA and NGNA.
  • FIG. 4B is a chromatogram showing UPLC separation of NANA and NGNA (blue line). Acid was run as a control (black line).
  • FIG.5 is a workflow schematic for online sialic acid quantitation.
  • sialic acid is designated as critical quality attributes (CQA) for such biotherapeutic glycoproteins and is an essential regulatory requirement to monitor and control the sialic acid to a specified level.
  • CQA critical quality attributes
  • the harvest decision is often based on the sialic acid content.
  • the current paradigm of prolonged offline analysis for sialic acid quantitation conflicts with the requirement of getting rapid sialic acid results for the timely harvest decision.
  • the online method described in this manuscript is intended to acquire near-real-time sialic acid data. With this approach the harvest decision can be made with better precision and accuracy. With the deployment of such a novel integrated platform, no human intervention is required.
  • the present disclosure provides a method for measuring an amount of sialic acid in polypeptides in near real time, the method comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated.
  • NANA N- acetylneuraminic acid
  • NGNA N-glycolyln
  • the method of a) to e) can occur near time, i.e., within or less than about five hours. In some aspects, the method of a) to e) can occur within or less than about four hours. In some aspects, the method of a) to e) can occur within or less than about three hours. In some aspects, the method of a) to e) can occur within or less than about two hours.
  • a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
  • the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower), unless indicated otherwise.
  • the term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • “at least 18 nucleotides of a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
  • polypeptide refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, and can refer to a minimal chain comprising two amino acids linked together via a peptide bond.
  • a "protein,” “peptide,” “peptide fragment,” “amino acid chain,” “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids are generically included in the definition of a "polypeptide”.
  • the term further includes proteins, which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, or amidation.
  • the terms “neuraminic acid” or “5-amino-3,5-dideoxy-D-glycero-D-galacto-non- 2-ulosonic acid” refer to an acidic amino sugar with a backbone formed by nine carbon atoms.
  • Sialic acid or “sialic acid residue” refer to any member of a family of nine-carbon carboxylated sugars. Sialic acids include a group of ⁇ 50 chemical variants of the nine carbon sugar neuraminic acid that are strategically placed at the tip of glycans. The most common member of the family is N-acetyl-neuraminic acid (also known as NANA, Neu5Ac, Neu5Ac, and 2-keto-5-acetamido-3,5-dideoxy-D-glycero-D- galactononulopyranos-l-onic acid).
  • NANA NANA
  • Neu5Ac Neu5Ac
  • Neu5Ac 2-keto-5-acetamido-3,5-dideoxy-D-glycero-D- galactononulopyranos-l-onic acid
  • N-glycolyl- neuraminic acid also known as NGNA, Neu5Gc, NeuGc.
  • Near real time means events in real time or nearly in real-time, without significant or meaningful delay that would affect results.
  • “Real-time” data is data that's collected, processed, and analyzed on a continual basis. It's information that's available for use immediately after being generated. Near real time pertains “to the timeliness of data or information which has been delayed by the time required for analysis, data processing and communication.
  • real time means as an event occurs, i.e., the actual time and event or process occurs.
  • near real time includes two events occurring in time within or less than about five hours, about four hours, about three hours, or about two hours. In some aspects, “near real time” means two or more events in time occurring within or less than five hours. In some aspects, “near real time” means two or more events occurring in time within or less than four hours. In some aspects, “near real time” means two or more events occurring in time within or less than three hours. In some aspects, “near real time” means two or more events occurring in time within or less than two hours. In some aspects, “near real time” means two or more events occurring in time between five hours and two hours.
  • the term "elution buffer” refers to the buffer, which is typically used to remove (elute) the polypeptide from the purification device (e.g. a chromatographic resin, polypeptide binding column) to which it was applied earlier. Typically, the elution buffer is selected so that separation of the polypeptide of interest from unwanted aggregates/impurities can be accomplished.
  • the term "desialylation agent” refers to an agent that removes sialic acids from a polypeptide. Desialylation agent allows a silyl group on a polypeptide to be exchanged for a proton.
  • Various fluoride salts e.g.
  • binding complex refers to the complex formed when a polypeptide binds to a polypeptide-binding column.
  • the terms “automated” or “automatic” refer to a system that operates, i.e., proceeds step-by-step through various mechanical movements and actions, with little or no intervention or control from a human operator. In general, the online system described herein is controlled via computer or other suitable control module.
  • the term “closed system” refers to a system that is closed to the outside environment.
  • the term “cell culture” refers to the cultivation of cells in a liquid medium, in the present context, for the purpose of producing polypeptides.
  • cell culture media refers to any liquid, semi-solid or solid media that can be used to support the growth of a cell or microorganism.
  • biologicalreactor refers to a vessel within which cells are grown under controlled conditions.
  • the vessel may comprise a stainless steel tank, or tank of other material, or a lined tank, or a polymer bag of dimensions suitable to grow the number of cells required to produce a target amount of the desired polypeptide; equipped with sensors and inputs to permit monitoring of critical process parameters and adjustments as necessary to maintain ideal conditions for cell growth and polypeptide production.
  • exoglycosidase refers to glycoside hydrolase enzymes that cleave the glycosidic linkage of a terminal monosaccharide in an oligosaccharide or polysaccharide.
  • sialidase or “neuraminidase” refer to a class of enzymes that are glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids and sialic acids on glycosylated molecules.
  • Near Real Time Quantitation Methods of Sialic Acids [0048] In some aspects, the present disclosure provides methods for measuring an amount of sialic acid in polypeptides in near real time.
  • Sialic acids are negatively charged monosaccharides present as terminal oligosaccharide residues on many glycans.
  • NANA N-acetylneuraminic acid
  • NGNA N-glycolylneuraminic acid
  • NANA is found in both human and non-human cells, NGNA is synthesized by all mammalian cells except human cells.
  • the presence of terminal NANA has been shown to impact various key properties of glycosylated proteins, including protein stability, circulatory half-life, solubility, and thermal stability (Lewis, A. M., et al. PloS one, 11(6), e0157111).
  • NGNA has been shown to be highly immunogenic in humans. Hence, maintaining a desired level NANA and low levels of NGNA in protein therapeutics is important to achieve better pharmacokinetic properties and longer half-life in the bloodstream.
  • the present disclosure provides measuring or assessing the sialic acid content during the upstream process as well as during downstream purification processes.
  • the conductivity of the wash buffer is adjusted to achieve a desired sialic acid to protein ratio. To decide on the strength of wash buffer, the sialic acid content at the termination of the preceding unit operation can be a pre-requisite.
  • the timely synchronization of data generation and harvest execution and downstream processing steps are inefficient and imperfect tasks to carryout.
  • Monitoring and controlling of sialylation, especially maintaining the levels of NANA and NGNA in biotherapeutics during all stages of the product life cycle are critical to ensure pharmacokinetics/pharmacodynamics, safety and efficacy of these drugs.
  • the pharmacokinetics or serum half-life of protein therapeutics is influenced by the terminal sialylation, which masks the galactose moiety from the hepatocyte asialoglycoprotein receptor, reducing glomeruli clearance in the kidneys (Buettner, M., et al. Front. Immunol., 02 November 2018 Sec. T Cell Biology. Vol.
  • Sialic acid for instance, increases the serum half- life of numerous recombinant glycoproteins including erythropoietin (EPO), interferon ⁇ , interferon ⁇ , and IgG antibodies by masking the terminal galactose and GlcNAc residues from the hepatocyte ASGP receptor, preventing endocytosis to extend prolong circulatory lifetime (See id.). Therefore, the present disclosure provides obtaining sialic acid results with quick turn-around-time. [0050] Continuous bioprocesses can increase production flexibility, overall reduction of facility footprints and increased productivity, securing reduced overall production cost.
  • EPO erythropoietin
  • interferon ⁇ interferon ⁇
  • IgG antibodies by masking the terminal galactose and GlcNAc residues from the hepatocyte ASGP receptor, preventing endocytosis to extend prolong circulatory lifetime (See id.). Therefore, the present disclosure provides obtaining sialic acid results with quick turn-around-time
  • the present disclosure provides an online analysis of complex techniques such a glycan analysis and peptide mapping. The prsent disclosure thus provides methods to maintain sialic acid content by making harvest decision based on the levels of sialic acid.
  • the present disclosure provides use of ⁇ SIA system as a sample preparation platform coupled with SegFlow sampling device and an online ultra-performance liquid chromatography (UPLC) system.
  • UPLC ultra-performance liquid chromatography
  • online sampling using segFlow and automatic sample preparations using ⁇ SIA system along with chromatographic separation using UPLC facilitates near-real-time monitoring of sialic acid contents in the bioreactors.
  • the approach presented in the present disclosure provides the opportunity to make the harvest decision purely based on the sialic acid content of cell culture harvest to maintain consistent mole/mole ratio of sialic acid to protein from batch-to-batch.
  • the method of the present disclosure comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated.
  • NANA N-acetylneuraminic acid
  • NGNA N- glycolylneuraminic acid
  • two, three, four, or more of a) to e) are automated. In some aspects, two of a) to e) are automated. In some aspects, three of a) to e) are automated. In some aspects, four of a) to e) are automated. In some aspects, all of a) to e) are automated. [0056] In some aspects, one, two, three, four, or more of a) to e) are performed in a closed system. In some aspects, one of a) to e) are performed in a closed system. In some aspects, two of a) to e) are performed in a closed system. In some aspects, three of a) to e) are performed in a closed system.
  • the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected from a cell lysate. In some aspects, the sample is collected automatically. In some aspects, the sample is collected after protein purification. In some aspects, the sample is collected from downstream after protein-A purification.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, a hydrophilic interaction column, a reverse phase column, or an ion exchange column.
  • the polypeptide binding column is a size exclusion column. In some aspects, the polypeptide binding column is a hydrophobic interaction column. In some aspects, the polypeptide binding column is an ion exchange column. In some aspects, the polypeptide binding column is a hydrophilic interaction column. In some aspects, the polypeptide binding column is a reverse phase column. [0059] In some aspects, the polypeptide binding column is an affinity column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column.
  • the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a FLAG column.
  • the desialylation agent is an acid or a proteolytic enzyme. [0061] In some aspects, the desialylation agent is a proteolytic enzyme. In some aspects, the proteolytic enzyme is a exoglycosidase. In some aspects, the exoglycosidase is a sialidase. [0062] In some aspects, the desialylation agent is an acid. In some aspects, the acid is phosphoric acid, acetic acid, sulfuric acid, or any combination thereof.
  • the acid is phosphoric acid. In some aspects, the acid is acetic acid. In some aspects, the acid is sulfuric acid. [0063] For quantification, free sialic acid is often derivatized with sialic acid labeling agents (e.g., chromophores and/or fluorophores) to achieve stabilization and significant enhancement in detection sensitivity. In some aspects, the sialic acid is labeled with a sialic acid labeling agent. In some aspects, the sialic acid labeling agent is 1,2-diamino- 4,5-methylenedioxybenzene (DMB) or o-phenylenediamine.
  • DMB 1,2-diamino- 4,5-methylenedioxybenzene
  • the sialic acid labeling agent is 1,2-diamino-4,5-methylenedioxybenzene (DMB). In some aspects, the sialic acid labeling agent is o-phenylenediamine. [0064] In some aspects, the labeling of the sialic acids is automated. In some aspects, the labeling of sialic acid takes about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour, or about 0.5 hours. In some aspects, the labeling of sialic acid takes about 3.5 hours, 3 hours, or 2.5 hours. In some aspects, the labeling of sialic acid takes about 3 hours.
  • sialic acid-binding proteins include lectins, antibodies, and recombinant sialic acid-binding proteins (Zhou X, et al. Cells.2020; 9(2):273).
  • Lectins are sugar-binding proteins that can specifically recognize glycans on glycoconjugates.
  • Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL) are commonly used to preferentially bind to sialic acid (Zhou X, et al. Cells.2020; 9(2):273).
  • the sialic acid is not labeled.
  • the analytical device is a UPLC, HPLC, uHPLC, or LC/MS. In some aspects, the analytical device is a UPLC or a HPLC. In some aspects, the analytical device is a UPLC. In some aspects, the analytical device is a HPLC. In some aspects, the analytical device is a uHPLC. In some aspects, the analytical device is LC/MS. [0067] In some aspects, the amount of total sialic acid per polypeptide is calculated by Equation 1.
  • X.XX ⁇ g is the amount of NANA injected and 309.27 ⁇ g is the molecular weight of NANA. Equation 2. 2 0 ⁇ ⁇ ( ⁇ ⁇ ⁇ . ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) where X.XX ⁇ g is the amount of NANA injected, and 309.27 ⁇ g is the molecular weight of NANA. [0069] In some aspects, the amount of NGNA per polypeptide in the sample is calculated by Equation 3. X.XX ⁇ g is the amount of NGNA injected and 325.27 ⁇ g is the molecular weight of NGNA. Equation 3.
  • the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture.
  • the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture.
  • DANA N-acetylmannosamine (ManNAc), fetuin, CuCl 2 , dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture.
  • a) to e) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours.
  • a) to f) take about 5 hours.
  • a) to e) take about 2 hours.
  • the method further comprises quantifying the titer of the polypeptides in the sample prior to a).
  • the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide- binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer.
  • the analytical device is a UV spectrophotometer, a fluorescence spectrometer, UPLC, HPLC, uHPLC, or LC/MS. In some aspects, the analytical device is a UV spectrophotometer.
  • the analytical device is a fluorescence spectrometer. In some aspects, the analytical device is a UPLC or a HPLC. In some aspects, the analytical device is a UPLC. In some aspects, the analytical device is a HPLC. In some aspects, the analytical device is a uHPLC. In some aspects, the analytical device is a LC/MS. [0075] In some aspects, after determining the titer of the polypeptides, a specific amount of polypeptides in the sample is collected in a second sample. In some aspects, the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to f).
  • one of i) to iii) are automated. In some aspects, two or three of i) to iii) are automated. In some aspects, two of i) to iii) are automated. In some aspects, all of i) to iii) are automated.
  • the polypeptides are fusion proteins. In some aspects, the polypeptides are therapeutic proteins. In some aspects, the polypeptides are antibodies. In some aspects, the polypeptides are bispecific or multi-specific polypeptides. [0078] In some aspects, the method further comprises determining when to harvest polypeptides from a cell culture based on the sialic acid content of the polypeptides.
  • the method further comprises determining when to harvest polypeptides from a bioreactor based on the sialic acid content of the polypeptides. In some aspects, the method further comprises harvesting the polypeptides from the bioreactor when the polypeptides contain the desired sialic acid content. In some aspects, the polypeptides are harvested when the polypeptides have a desired sialic acid to protein ratio.
  • harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column; and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, a reverse phase column, a hydrophilic interaction column, or an ion exchange column.
  • the polypeptide binding column is a size exclusion column.
  • the polypeptide binding column is a hydrophobic interaction column. In some aspects, the polypeptide binding column is a reverse phase column. In some aspects, the polypeptide binding column is a hydrophilic interaction column. In some aspects, the polypeptide binding column is an ion exchange column. [0082] In some aspects, the polypeptide binding column is an affinity column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column.
  • the affinity column is a Myc column. In some aspects, the affinity column is a FLAG column. [0083] In some aspects, one, two, or three of aa) to cc) are automated. In some aspects, all of aa) to cc) are automated. [0084] In some aspects, wash buffer is moved through the polypeptide binding column. In some aspects, the conductivity of the wash buffer is adjusted to achieve a desired sialic acid to protein ratio. [0085] In some aspects, the wash buffer is prepared based on the sialic acid content of the polypeptides. In some aspects, the wash buffer is modified based on the sialic acid content of the polypeptides.
  • the wash buffer comprises NaCl, KCl, Na2SO4, K2SO4, Na3PO4 NaOAc, or any combination thereof.
  • the wash buffer comprises NaCl, KCl, Na 2 SO 4 , K 2 SO 4 , or any combination thereof.
  • the wash buffer comprises Na 3 PO 4 , NaOAc, or any combination thereof.
  • the amount of Na3PO4, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified based on the sialic acid content of the polypeptides.
  • the amount of Na3PO4, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified to increase the sialic acid content of the polypeptides.
  • the amount of Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified to increase the amount of NANA on the polypeptides.
  • 20 mM to 70 mM Na3PO4 e.g., 40 mM, 50 mM, or 60 mM
  • 200 mM to 400 mM NaOAc e.g., 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM
  • the pH of the wash buffer is pH 7.0.
  • a conductivity range of the wash buffer is between about 7 and 13 mS/cm.
  • 20 mM to 70 mM Na 3 PO 4 e.g., 40 mM, 50 mM, or 60 mM
  • 200 mM to 400 mM NaOAc e.g., 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM
  • the pH of the wash buffer is pH 7.0.
  • a conductivity range of the wash buffer is between about 7 and 13 mS/cm.
  • the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated.
  • NANA N-acetylneuraminic acid
  • NGNA N-gly
  • the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein two, three, four, or five or more of a) to f) are automated.
  • NANA N-acetylneuraminic acid
  • two or more of a) to f) are automated. In some aspects, three or more of a) to f) are automated. In some aspects, four or more of a) to f) are automated. In some aspects, five or more of a) to f) are automated. In some aspects, all of a) to f) are automated.
  • the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one, two, three, four, or five or more of a) to f) are performed in a closed system.
  • NANA N-acetyl
  • one or more of a) to f) are performed in a closed system. In some aspects, two or more of a) to f) are performed in a closed system. In some aspects, three or more of a) to f) are performed in a closed system. In some aspects, four or more of a) to f) are performed in a closed system. In some aspects, five or more of a) to f) are performed in a closed system. In some aspects, all of a) to f) are performed in a closed system. [0094] In some aspects, the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected automatically.
  • the sample is collected after protein purification. In some aspects, the sample is collected after protein-A purification.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column.
  • the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column.
  • X.XX ⁇ g is the amount of NANA injected and 309.27 ⁇ g is the molecular weight of NANA. Equation 2.
  • X.XX ⁇ g is the amount of NANA injected, and 309.27 ⁇ g is the molecular weight of NANA.
  • the amount of NGNA per polypeptide in the sample is calculated by Equation 3.
  • X.XX ⁇ g is the amount of NGNA injected and 325.27 ⁇ g is the molecular weight of NGNA. Equation 3.
  • the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture media.
  • DANA N-acetylmannosamine (ManNAc), fetuin, CuCl 2 , dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture media.
  • ManNAc N-acetylmannosamine
  • fetuin CuCl 2
  • dexamethasone galactose
  • manganese chloride uridine
  • zinc zinc
  • hydrocortisone LongR3, or any combination thereof
  • a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours.
  • a) to f) take about 2 hours.
  • a) to f) take about 5 hours.
  • the method further comprises quantifying the titer of the polypeptides in the sample prior to a).
  • the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide- binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer.
  • the analytical device is a UV spectrophotometer or a fluorescence spectrophotometer.
  • a specific amount of polypeptides in the sample is collected in a second sample.
  • the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to f) (“repeat steps a) to f)”). In some aspects, any one of i) to iii) are automated. In some aspects, two or three of i) to iii) are automated. In some aspects, all of i) to iii) are automated. [0106] In some aspects, the method further comprises determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. In some aspects, the polypeptides are harvested when the polypeptides have a desired sialic acid to protein ratio.
  • the method further comprises harvesting the polypeptides from the cell culture media when the polypeptides contain the desired sialic acid content.
  • harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column, and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column.
  • the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a Flag column. [0109] In some aspects, one, two, or three of aa) to cc) are automated. In some aspects, all of aa) and cc) are automated.
  • the method comprises: a) moving a portion of a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column, to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labele
  • two, three, four, five, six, seven, eight, nine, or more of a) to j) are automated.
  • two of a) to j) are automated.
  • three of a) to j) are automated.
  • four of a) to j) are automated.
  • five of a) to j) are automated.
  • six of a) to j) are automated.
  • seven of a) to j) are automated.
  • eight of a) to j) are automated.
  • nine of a) to j) are automated.
  • all of a) to j) are automated.
  • the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; f) quantifying the amount N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues in the sample; g) moving a second sample
  • two, three, four, five, six, seven, eight or more of a) to i) are automated.
  • two of a) to i) are automated.
  • three of a) to i) are automated.
  • four of a) to i) are automated.
  • five of a) to i) are automated.
  • six of a) to i) are automated.
  • seven of a) to i) are automated.
  • eight or more of a) to i) are automated.
  • all of a) to i) are automated.
  • the method comprises: a) moving a portion of a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labeled
  • two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of a) to m) are automated.
  • two of a) to m) are automated.
  • three of a) to m) are automated.
  • four of a) to m) are automated.
  • five of a) to m) are automated.
  • six of a) to m) are automated.
  • seven of a) to m) are automated.
  • eight of a) to m) are automated.
  • nine of a) to m) are automated.
  • ten of a) to m) are automated.
  • polypeptides [0117] Any protein can be used in the disclosed method. In some aspects, the polypeptide is a fusion protein. In some aspects, the polypeptide is a therapeutic protein.
  • the therapeutic proteins that can be prepared by using the methods disclosed herein comprise, for example, antibodies, antibody fragments, Fc portions of antibodies and fusions thereof, antigen binding portions of antibodies, fusion proteins, naturally occurring proteins, recombinant proteins, chimeric proteins, immunoadhesins, enzymes, growth factors, receptors, hormones, regulatory factors, cytokines, or any combination thereof.
  • the therapeutic protein is produced in mammalian cells.
  • the mammalian cell line is a Chinese Hamster Ovary (CHO) cells, or baby hamster kidney (BHK) cells, murine hybridoma cells, or murine myeloma cells.
  • Any therapeutic protein that is expressible in a host cell may be assessed for its sialic acid content in accordance with the present disclosure and may be present in the compositions provided.
  • the therapeutic protein may be expressed from a gene that is endogenous to the host cell, or from a gene that is introduced into the host cell through genetic engineering.
  • the therapeutic protein may be one that occurs in nature, or may alternatively have a sequence that was engineered or selected by the hand of man.
  • An engineered therapeutic protein may be assembled from other polypeptide segments that individually occur in nature, or may include one or more segments that are not naturally occurring.
  • the methods provided in the present disclosure employ any cell that is suitable for growth and/or production of a therapeutic protein in a culture medium, including animal, yeast or insect cells.
  • the cell is any mammalian cell or cell type suitable to cell culture and to expression of polypeptides.
  • the methods provided herein e.g., methods of measuring a sialic acid content
  • the methods can therefore employ any suitable type of cell, including an animal cell.
  • the methods employ a mammalian cell.
  • methods can also employ hybridoma cells.
  • the mammalian cell is a non-hybridoma mammalian cell, which has been transformed with exogenous isolated nucleic acid encoding a desired therapeutic protein.
  • the methods employ mammalian cells selected from the group consisting of human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK. ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather. Biol.
  • human retinoblasts PER.C6 (CruCell, Leiden, The Netherlands)
  • monkey kidney CV1 line transformed by SV40 COS-7, ATCC CRL 1651
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
  • the methods employ CHO cells.
  • the culturing of CHO cell lines and expression of therapeutic proteins from CHO cell lines is employed.
  • therapeutic protein can be secreted into the culture medium from which the therapeutic protein may be isolated and/or purified or the therapeutic protein may be released into the culture medium by lysis of a cell comprising an isolated nucleic acid encoding the therapeutic protein.
  • the therapeutic protein is a CTLA4-Ig molecule, e.g., abatacept or belatacept.
  • CTLA4-Ig or “CTLA4-Ig molecule” are used interchangeably, and refer to a protein molecule that comprises at least a polypeptide having a CTLA4 extracellular domain or portion thereof and an immunoglobulin constant region or portion thereof.
  • the extracellular domain and the immunoglobulin constant region can be wild- type, or mutant or modified, and mammalian, including human or mouse.
  • the polypeptide can further comprise additional protein domains.
  • a CTLA4-Ig molecule can also refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers.
  • a CTLA4-Ig molecule also is capable of binding to CD80 and/or CD86.
  • CLA4Ig refers to a protein molecule having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:1, (ii) 26-382 of SEQ ID NO:1; (iii) 27- 383 of SEQ ID NO:1, or (iv) 27-382 of SEQ ID NO:1, or optionally (v) 25-382 of SEQ ID NO:1, or (vi) 25-383 of SEQ ID NO:1.
  • SEQ ID NO:1 monomers or monomers “having a SEQ ID NO:1 sequence”.
  • dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (v); (v) and (vi); (v) and (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (v); (vi); and, (
  • SEQ ID NO:1 proteins proteins or proteins “having a SEQ ID NO:1 sequence”.
  • CTLA4Ig as shown in SEQ ID NO:1 was deposited on May 31, 1991 with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, Va.20110-2209 under the provisions of the Budapest Treaty, and has been accorded ATCC accession number ATCC 68629; a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig as shown in SEQ ID NO:1 was deposited on May 31, 1991 with ATCC identification number CRL-10762).
  • Abatacept refers to SEQ ID NO:1 proteins.
  • L104EA29Y-Ig has the functional extracellular binding domain of modified human CTLA4 and the Fc domain of human immunoglobulin of the IgG1 class.
  • SEQ ID NO:2 depict a amino acid sequence of L104EA29YIg comprising a signal peptide; a mutated extracellular domain of CTLA4 starting at methionine at position +27 and ending at aspartic acid at position +150, or starting at alanine at position +26 and ending at aspartic acid at position +150; and an Ig region.
  • DNA encoding L104EA29Y-Ig was deposited on Jun.20, 2000, with the American Type Culture Collection (ATCC) under the provisions of the Budapest Treaty. It has been accorded ATCC accession number PTA-2104. L104EA29Y-Ig is further described in U.S. Pat.
  • L104EA29YIg in mammalian cells can result in the production of N- and C-terminal variants, such that the proteins produced can have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2; (iii) 27- 383 of SEQ ID NO:2 or (iv) 27-382 of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2.
  • these proteins can be referred to herein as “SEQ ID NO:2 monomers,” or monomers “having a SEQ ID NO:2 sequence.”
  • These proteins can dimerize, such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (ii); (ii) and (iv); (ii) and (v); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (iv) and (iv); (iv) and (vi); (iv) and (iv); (iv) and (vi); (v) and (v); (iv) and (vi); (v) and (v); (iv) and (vi); (v) and (v); (v)
  • SEQ ID NO:2 proteins proteins “having a SEQ ID NO:2 sequence”.
  • Belatacept refers to SEQ ID NO:2 proteins.
  • HER receptor family proteins including HER2, HER3, HER4, and the EGF receptor.
  • Cell adhesion molecules for example, LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v/beta 3 integrin.
  • VEGF vascular endothelial growth factor
  • growth hormone such as vascular endothelial growth factor (“VEGF”), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF ⁇ and TGF- ⁇ , insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-1), and osteoinductive factors.
  • VEGF vascular endothelial growth factor
  • TGF insulin-like growth factors-I and -II
  • Insulins and insulin-related proteins including insulin, insulin A- chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins.
  • Coagulation and coagulation-related proteins such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens.
  • Colony stimulating factors and receptors thereof including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor ( c-fms).
  • Receptors and receptor-associated proteins including, for example, flk2/flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors ("TPO-R,” "c-mpl”), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors.
  • Receptor ligands including, for example, OX40L, the ligand for the OX40 receptor.
  • Neurotrophic factors including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6).
  • Interleukins and interleukin receptors including IL-I to IL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, among others.
  • Viral antigens including an AIDS envelope viral antigen.
  • Lipoproteins Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies.
  • BMP bone morphogenetic protein
  • DAF decay accelerating factor
  • TALL proteins including TALL-I
  • amyloid proteins including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins ("TSLP"), RANK ligand ("OPGL”), c-kit
  • TNF receptors including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.
  • Exemplary therapeutic polypeptides and antibodies include ACTIVASE® (Alteplase); alirocumab, ARANESP® (Darbepoetin-alfa), EPOGEN® (Epoetin alfa, or erythropoietin); AVONEX® (Interferon ⁇ -Ia); BEXXAR® (Tositumomab); Betaseron® (Interferon- ⁇ ); bococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see US8080243); CAMPATH® (Alemtuzumab); DYNEPO® (Epoetin delta); VELCADE® (bortezomib); MLN0002 (anti-a4 ⁇ 7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); ENBREL® (etanercept); EPREX® (Epoetin alfa);
  • anthracis Protective Antigen mAb ABthraxTM; VECTIBIX® (Panitumumab); XOLAIR® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fe portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRl fused to IgGl Fe), ZENAPAX® (Daclizumab ); ZENAPAX® (Daclizumab), ZEVALIN® (lbritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-Ig), anti-a4 ⁇ 7 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc (huBR3 / hu
  • the polypeptide is an antibody or an antibody fragment.
  • the antibody or an antibody fragment is a human, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, or grafted, antibody or antibody fragment.
  • the antibody or an antibody fragment is a monoclonal or polyclonal antibody or antibody fragment.
  • the antibody or an antibody fragment is a IgG, IgM, IgA, IgD, or IgE antibody or antibody fragment.
  • the antibody or an antibody fragment is a IgG1, IgG2, IgG3, or IgG4 antibody or antibody fragment.
  • the antibody or antibody fragment is a Fab, F(ab')2, VHH, or ScFv.
  • the polypeptides are bispecific or multi-specific polypeptides.
  • Exemplary antibodies include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocili
  • the present disclosure also provides a method to treat a disease or condition comprising administering to a subject a therapeutic protein manufactured by the methods disclosed herein. Also provided is a pharmaceutical composition manufactured by the methods disclosed herein.
  • EXEMPLARY ASPECTS [0131] The disclosed invention includes the following non-exhaustive listing of aspects. This listing should not be construed to be in anyway limiting, and a person skilled in the art will appreciate that various modifications can be made to these embodiments without changing the essence and scope of the invention disclosed herein. 1.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column.
  • the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column.
  • the affinity column is a protein A column.
  • the desialylation agent is an acid or an exoglycosidase.
  • the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof.
  • the method of aspect 12, wherein the exoglycosidase is a sialidase.
  • the method of aspect 15, wherein the labeling of the sialic acids is automated.
  • the method of aspect 15 or 16, wherein the labeling of the sialic acid takes about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour or about 0.5 hours.
  • sialic acid labeling agent is 1,2- diamino-4,5-methylenedioxybenzene (DMB) or o-phenylenediamine.
  • the analytical device is a UPLC, uHPLC or HPLC.
  • the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide-binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer.
  • the analytical device is a UV spectrophotometer or a fluorescence spectrophotometer.
  • any one of aspects 1-35, wherein the polypeptides are fusion proteins. 37. The method of any one of aspects 1-36, wherein the polypeptides are antibodies. 38. The method of any one of aspects 1-37, further comprising determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. 39. The method of any one of aspects 1-38, further comprising harvesting the polypeptides from the cell culture media when the polypeptides contain the desired sialic acid content. 40.
  • harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column; and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides.
  • the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 42.
  • a method for measuring an amount of sialic acid in polypeptides in near- real time comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated.
  • the method of aspect 60 wherein the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof.
  • the exoglycosidase is a sialidase.
  • the sialic acid labeling agent is 1,2-diamino-4,5-methylenedioxybenzene (DMB) or o-phenylenediamine.
  • the analytical device is a UPLC, uHPLC or HPLC.
  • any one of aspects 49-69 wherein a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours.
  • the method of any one of aspects 49-70, wherein a) to f) take about 2 hours.
  • the method of aspect 86, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column.
  • the method of aspect 87, wherein the affinity column is a protein A column.
  • the method of any one of aspects 85-88, wherein one, two, or three of aa) to cc) are automated.
  • the method of aspect 89, wherein all of aa) and cc) are automated.
  • the method of aspect 85, wherein the wash buffer is prepared based on the sialic acid content. 92.
  • a method for measuring an amount of sialic acid in polypeptides in real time comprising: a) moving a portion of a sample comprising the polypeptides to a polypeptide- binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column, to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the
  • a method for measuring an amount of sialic acid in polypeptides in real time comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; f) quantifying the amount N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues
  • a near real time system comprising an automated sampling system, a sequence injection system, and an analyzer was created.
  • the near real time system is a fully automated platform, configured to achieve online sampling, inline sample preparation, and subsequent online HPLC/UPLC analysis.
  • the near real time system can also be used for titer measurement of samples.
  • the system includes drawing samples from a bioreactor and loading the samples on to a Protein A column and eluting the proteins off in fixed volume of elution buffer to obtain protein titer in the bioreactor.
  • Picomoles of NANA and NGNA per injection is calculated and from that mole/mole ratio of sialic acid to protein is determined.
  • the protein A purification, desialylation, sialic acid labeling, and NGNA and NANA steps were optimized as provided in Examples 2-4.
  • Example 2 Optimization of Protein-A Purification [0136] During protein purification with a protein A column, an issue was encountered with the appearance of an interfering shoulder on the chromatogram that resulted from the stroke of the syringe during the delivery of the elution buffer. This artifact impacted the accuracy of titer determination.
  • the labeled sialic acids were quantified by HPLC. As depicted in FIG.2, the peak intensity is relatively higher for 3-hour labeling in comparison to 2 and 4 hours labeling.
  • [0141] Next, studies were conducted to determine the optimal combination and timing of desialylation and sialic acid labeling. The following conditions were tested in combination: 2 hour sialidase digestion, 1 hour acid hydrolysis, 2 hour acid hydrolysis, 2 hour DMB labeling, 3 hour DMB labeling, and 4 hour DMB labeling. Results show that the combination of 2 hour acid hydrolysis and 3 hour DMB labeling generated the highest yield of NANA with a low yield of NGNA (Table 1). Table 1.
  • the list of columns subjected for evaluation included Agilent C18 column (Infinity Lab Poroshell 120 EC-C18, 2.1 x 75 mm, 2.7 ⁇ m, narrow bore LC column), Supelco C-18 column and Waters X-bridge column along with Sigma/Aldrich Ascentis Express RP-Amide column (2.7 ⁇ m , 10 cm X 2.1 mm). All columns were evaluated under different gradients and column temperatures. [0145] NANA and NGNA were separated using a Ascentis RP-amide column with mobile phase 0.1% FA/10% acetonitrile under isocratic conditions for 10 minutes at a flow rate of 0.2 ml/min. Column temperature was maintained at 30 ⁇ C.
  • FIGs.3A-3B Chromatographic separation of DMB-labeled NANA and NGNA is shown in FIGs.3A-3B.
  • Ascentis RP-amide column outperformed other columns and separation by the Ascentis RP-amide column was further optimized.
  • a gradient elution instead of isocratic run was carried out at 0.2 ml/minute by maintaining the column temperature at 30 ⁇ C.
  • Mobile phases A and B are 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively.
  • Initial gradient of 6% was maintained for 1 minute followed by a ramp up to 20% for 3 minutes followed by a 2-minutes isocratic run during which NANA and NGNA were eluted.
  • Example 5 Sialic Acid Quantitation Using a Near Real Time System [0147] Based on the results from Examples 1-4, an optimized workflow has been established (FIG.5). For online sampling from bioreactors, the ⁇ SIA system from FIA Lab was interfaced with a SegFlow autosampler to draw samples from the bioreactor and deliver the samples to the designated port of the ⁇ SIA module.
  • Such on-line sampling technology allows rapid and accurate sampling from up to eight bioreactors or process streams and sample delivery to up to 4 analyzers and/or fraction collectors.
  • PAT on-line process analytical technology
  • FIAlab SIAsoft software.
  • the SIAsoft software simultaneously acquires and exports all integrated instrument data to any OPC-enabled SCADA or DCS for enhanced process monitoring and control.
  • Custom-scripts were written to establish communication between the SegFlow autosampler and the ⁇ SIA analyzer such that sample withdrawal and subsequent workflow can be scheduled and coordinated with minimal human intervention.
  • a Flownamics SegFlow 4800 autosampling system was interfaced with a ⁇ SIA device.
  • a schematic of the ⁇ SIA system architecture is provided in FIGs.6A-6B.
  • the autosampler drew cell-free sterile samples from the cell-free culture medium of bioreactors using a 310 nm F-series FISP probe with a 0.2mm pore size ceramic membrane (Flownamics Inc., Madison, WI) to deliver samples to the ⁇ SIA through a SegMod-SampleMod. Samples were drawn 6, 7, 8, 10, or 12 days after inoculation of the culture.
  • the received samples were then loaded on to a Protein A column.
  • the glycoproteins in the sample were eluted off in a fixed volume of elution buffer to obtain the protein titer via a UV-Vis spectrometer.
  • a fixed quantity of protein was loaded on to a Protein A column at neutral pH and subjected to inline Protein A purification to remove process impurities and extract the products of interest using a low pH buffer (e.g., mAbs and fusion proteins).
  • the purified glycoproteins were then subjected to acid hydrolysis for two hours with phosphoric acid to release the sialic acids.
  • sialic acids were then labeled with DMB for three hours.
  • the DMB-labeled sialic acid samples were then injected into an ACQUITY Classic UPLC to separate NANA from NGNA and other components of the reaction mixture.
  • a representative chromatogram of DMB labeled sialic acid released from the reference glycoprotein using the optimized workflow is shown in Figure 7.
  • the mole/mole ratio of sialic acid/protein was calculated after the full automated protocol (online) or an offline method either 6, 7, 8, 10, or 12 days after inoculation of the culture as shown below. Table 2.

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Abstract

Systems and methods for the automatic quantification of sialic acid of polypeptides in near real time.

Description

NEAR REAL TIME SIALIC ACID QUANTITATION OF GLYCOPROTEINS CROSS-REFERENCE TO RELATED TO APPLICATIONS [0001] The present application claims the priority benefit of U.S. Provisional Application No.63/508,228, filed June 14, 2023, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] Therapeutic glycoproteins such as erythropoietin, cytokines, antibodies, glycosyltransferases, and glycosidases are typically produced using recombinant DNA (rDNA) technology, which allows for control of glycosylation during drug development. Glycosylation has been shown to have an effect on protein activity, antigenicity, stability and pharmacokinetic/pharmacodynamic properties. [0003] In a lot of cases, sialylation needs to be optimized to ensure the prolonged circulatory half-life of glycoproteins in serum. For example, natural and recombinant forms of erythropoietin carry three sialylated N-glycans and one sialylated O-glycan. Engineering of erythropoietin to a hypersialylated form has shown to exhibit better pharmacokinetic properties, especially a longer half-life in the bloodstream (Varki A, et al. Essentials of Glycobiology.2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 14.). [0004] To maintain desired sialic acid contents across multiple batches of glycoprotein therapeutics, engineering design and control of sialylation levels of biopharmaceuticals is important in biotechnology (Varki A, et al. Essentials of Glycobiology.2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 14.). If the sialic acid content of production culture doesn’t meet the established sialic acid specification, the batch might have to be discarded, unless an enrichment can be performed downstream (Dahotre S, et al. J Chromatogr A.2022 Apr 15;1672:463067). The FDA and other health authorities have recognized the significance of reducing batch- to-batch variability and maintaining consistent product quality throughout the manufacturing. To maintain batch-to-batch reproducibility, manufacturers of biotherapeutics dedicate enormous effort to ensure that their approved drugs fall within the defined specifications (Bertozzi CR, et al. Glycans in Biotechnology and the Pharmaceutical Industry. In: Varki A, et al. Essentials of Glycobiology.2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 51.). [0005] Current practice typically involves sialic acid quantitation towards the end of a fed-batch culture by submitting samples for offline analysis. This method exhibits a bias due to the lag time associated with the lengthy analysis time. Similar difficulty occurs at the downstream ion exchange purification step at which the conductivity of the wash buffer is determined based on the sialic acid content of the feed material. BRIEF SUMMARY OF THE INVENTION [0006] Certain aspects of the disclosure are directed to methods for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated. [0007] In some aspects, the released sialic acids are mixed with a sialic acid labeling agent, thereby labeling the sialic acids, prior to d). [0008] Certain aspects of the disclosure are directed to methods for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated. [0009] In some aspects, two, three, four, or five or more of the steps are automated. In some aspects, all of the steps are automated. [0010] In some aspects, one, two, three, four, or five or more of the steps are performed in a closed system. In some aspects, all of the steps are performed in a closed system. [0011] In some aspects, the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected automatically. [0012] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. [0013] In some aspects, the desialylation agent is an acid or an exoglycosidase. In some aspects, the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof. In some aspects, the exoglycosidase is a sialidase. [0014] In some aspects, the sialic acid labeling agent is 1,2-diamino-4,5- methylenedioxybenzene (DMB) or o-phenylenediamine. In some aspects, the analytical device is a UPLC, uHPLC or HPLC. [0015] In some aspects, the amount of total sialic acid per polypeptide is calculated by Equation 1; ^^ ^^ ^^^^^^^^ ^^^ௗ ∑ ∑ ∑ ^ ^^ = ^^ௌ^ + ^^ௌଶ × 2 + ^^ௌଷ × 3 + ∑ ^^ௌସ × 4 ^^ ^
Figure imgf000004_0001
^^௧^^^ௗ௬ 100 wherein the amount of NANA per polypeptide in the sample is calculated by Equation 2;
Figure imgf000004_0002
where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. and wherein the amount of NGNA per polypeptide in the sample is calculated by Equation 3; ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ∗ 1 ^^ ^^ ^^ ^^ ∗ 10^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. [0016] In some aspects, a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. [0017] In some aspects, the method further comprises determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES [0018] FIG.1 are overlaid chromatograms showing the amount of sialic acids released from glycoproteins after either enzymatic digestion with sialidase (black line) or acid hydrolysis (blue line). [0019] FIG.2 is a chromatogram showing the amount of labeled sialic acids after 2 hours (black line), 3 hours (blue line), or 4 hours (green line) of labeling with DMB. [0020] FIGs.3A-3B are chromatograms showing UPLC separation of DMB labeled NANA and NGNA from either a mixture of NANA and NGNA (blue line) (FIG.3A) or isolated NANA (blue line) and NGNA (red line) (FIG.3B). The reagent peak is shown in red (FIG.3A) or green (FIG.3B). [0021] FIGs.4A-4B are chromatograms showing UPLC separation of NANA and NGNA. FIG.4A is a chromatogram showing UPLC separation of 1.9 pmol (blue line), 3.9 pmol (orange line), 7.85 pmol (dark green line), 15.6 pmol (dark blue line), 31.25 pmol (brown line), 62.5 pmol (pink line), 125 pmol (light blue line) 250 pmol (bright green line), 500 pmol (blue line), or 1000 pmol (black line) of NANA and NGNA. FIG. 4B is a chromatogram showing UPLC separation of NANA and NGNA (blue line). Acid was run as a control (black line). [0022] FIG.5 is a workflow schematic for online sialic acid quantitation. [0023] FIGs.6A-6B are schematics showing the top module (FIG.6A) and bottom module (FIG.6B) of the µSIA system architecture. [0024] FIG.7 is a chromatogram showing the UPLC separation of NANA and NGNA from a reference protein. DETAILED DESCRIPTION OF THE INVENTION [0025] Sialic acid moieties of certain glycoprotein therapeutics are influencing the biological and physiochemical properties as well as playing an important role in maintaining serum half-life. For certain molecules, slight variation in sialic acid content can impact pharmacokinetic and/or pharmacodynamics properties significantly. Hence, sialic acid is designated as critical quality attributes (CQA) for such biotherapeutic glycoproteins and is an essential regulatory requirement to monitor and control the sialic acid to a specified level. To maintain consistent levels of sialic acid with reduced variability, the harvest decision is often based on the sialic acid content. The current paradigm of prolonged offline analysis for sialic acid quantitation conflicts with the requirement of getting rapid sialic acid results for the timely harvest decision. The online method described in this manuscript is intended to acquire near-real-time sialic acid data. With this approach the harvest decision can be made with better precision and accuracy. With the deployment of such a novel integrated platform, no human intervention is required. [0026] In order to acquire near-real-time sialic acid data, the present disclosure provides a method for measuring an amount of sialic acid in polypeptides in near real time, the method comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated. In some aspects, the method of a) to e) can occur near time, i.e., within or less than about five hours. In some aspects, the method of a) to e) can occur within or less than about four hours. In some aspects, the method of a) to e) can occur within or less than about three hours. In some aspects, the method of a) to e) can occur within or less than about two hours. Terms [0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including the definitions, will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [0028] Throughout this disclosure, the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. [0029] Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [0030] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower), unless indicated otherwise. [0031] The term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures). [0032] The term "polypeptide" as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As used herein, a "protein," "peptide," "peptide fragment," "amino acid chain," "amino acid sequence," or any other term used to refer to a chain or chains of two or more amino acids, are generically included in the definition of a "polypeptide”. The term further includes proteins, which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, or amidation. [0033] The terms "neuraminic acid" or "5-amino-3,5-dideoxy-D-glycero-D-galacto-non- 2-ulosonic acid" refer to an acidic amino sugar with a backbone formed by nine carbon atoms. [0034] The terms "sialic acid" or "sialic acid residue" refer to any member of a family of nine-carbon carboxylated sugars. Sialic acids include a group of ~50 chemical variants of the nine carbon sugar neuraminic acid that are strategically placed at the tip of glycans. The most common member of the family is N-acetyl-neuraminic acid (also known as NANA, Neu5Ac, Neu5Ac, and 2-keto-5-acetamido-3,5-dideoxy-D-glycero-D- galactononulopyranos-l-onic acid). A second member of the family is N-glycolyl- neuraminic acid (also known as NGNA, Neu5Gc, NeuGc). [0035] The term "near real time" means events in real time or nearly in real-time, without significant or meaningful delay that would affect results. “Real-time” data is data that's collected, processed, and analyzed on a continual basis. It's information that's available for use immediately after being generated. Near real time pertains “to the timeliness of data or information which has been delayed by the time required for analysis, data processing and communication. The term "real time" means as an event occurs, i.e., the actual time and event or process occurs. The term “near real time” includes two events occurring in time within or less than about five hours, about four hours, about three hours, or about two hours. In some aspects, “near real time” means two or more events in time occurring within or less than five hours. In some aspects, “near real time” means two or more events occurring in time within or less than four hours. In some aspects, “near real time” means two or more events occurring in time within or less than three hours. In some aspects, “near real time” means two or more events occurring in time within or less than two hours. In some aspects, “near real time” means two or more events occurring in time between five hours and two hours. [0036] The term "elution buffer" refers to the buffer, which is typically used to remove (elute) the polypeptide from the purification device (e.g. a chromatographic resin, polypeptide binding column) to which it was applied earlier. Typically, the elution buffer is selected so that separation of the polypeptide of interest from unwanted aggregates/impurities can be accomplished. [0037] The term "desialylation agent" refers to an agent that removes sialic acids from a polypeptide. Desialylation agent allows a silyl group on a polypeptide to be exchanged for a proton. Various fluoride salts (e.g. sodium, potassium, tetra-n-butylammonium fluorides) are known for this purpose [0038] The term "sialic acid labeling agent" refers to a detectable moiety which can be attached to sialic acid. [0039] The terms "polypeptide-binding column," "binding column," or "column" refers to a filtration and/or elution column that can be used for separating components of a sample or derivatives thereof. [0040] The term "binding complex" refers to the complex formed when a polypeptide binds to a polypeptide-binding column. [0041] The terms "automated" or "automatic" refer to a system that operates, i.e., proceeds step-by-step through various mechanical movements and actions, with little or no intervention or control from a human operator. In general, the online system described herein is controlled via computer or other suitable control module. [0042] The term "closed system" refers to a system that is closed to the outside environment. [0043] The term "cell culture" refers to the cultivation of cells in a liquid medium, in the present context, for the purpose of producing polypeptides. [0044] The terms "cell culture media," "culture media," "cell culture medium," or "culture medium" refers to any liquid, semi-solid or solid media that can be used to support the growth of a cell or microorganism. [0045] The term "bioreactor" refers to a vessel within which cells are grown under controlled conditions. The vessel may comprise a stainless steel tank, or tank of other material, or a lined tank, or a polymer bag of dimensions suitable to grow the number of cells required to produce a target amount of the desired polypeptide; equipped with sensors and inputs to permit monitoring of critical process parameters and adjustments as necessary to maintain ideal conditions for cell growth and polypeptide production. [0046] The term "exoglycosidase" refers to glycoside hydrolase enzymes that cleave the glycosidic linkage of a terminal monosaccharide in an oligosaccharide or polysaccharide. [0047] The terms "sialidase" or "neuraminidase" refer to a class of enzymes that are glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids and sialic acids on glycosylated molecules. Near Real Time Quantitation Methods of Sialic Acids [0048] In some aspects, the present disclosure provides methods for measuring an amount of sialic acid in polypeptides in near real time. Sialic acids are negatively charged monosaccharides present as terminal oligosaccharide residues on many glycans. Among the two most common sialic acids in biopharmaceuticals are N-acetylneuraminic acid (Neu5Ac or NANA) and N-glycolylneuraminic acid (Neu5Gc or NGNA). While NANA is found in both human and non-human cells, NGNA is synthesized by all mammalian cells except human cells. The presence of terminal NANA has been shown to impact various key properties of glycosylated proteins, including protein stability, circulatory half-life, solubility, and thermal stability (Lewis, A. M., et al. PloS one, 11(6), e0157111). NGNA has been shown to be highly immunogenic in humans. Hence, maintaining a desired level NANA and low levels of NGNA in protein therapeutics is important to achieve better pharmacokinetic properties and longer half-life in the bloodstream. [0049] In order to control the sialic acid content, the present disclosure provides measuring or assessing the sialic acid content during the upstream process as well as during downstream purification processes. In some aspects, the conductivity of the wash buffer is adjusted to achieve a desired sialic acid to protein ratio. To decide on the strength of wash buffer, the sialic acid content at the termination of the preceding unit operation can be a pre-requisite. Considering the prolonged analysis time of offline sialic acid methods, the timely synchronization of data generation and harvest execution and downstream processing steps are inefficient and imperfect tasks to carryout. Monitoring and controlling of sialylation, especially maintaining the levels of NANA and NGNA in biotherapeutics during all stages of the product life cycle are critical to ensure pharmacokinetics/pharmacodynamics, safety and efficacy of these drugs. The pharmacokinetics or serum half-life of protein therapeutics is influenced by the terminal sialylation, which masks the galactose moiety from the hepatocyte asialoglycoprotein receptor, reducing glomeruli clearance in the kidneys (Buettner, M., et al. Front. Immunol., 02 November 2018 Sec. T Cell Biology. Vol. //doi.org/10.3389/fimmu.2018.02485). Sialic acid for instance, increases the serum half- life of numerous recombinant glycoproteins including erythropoietin (EPO), interferon γ, interferon α, and IgG antibodies by masking the terminal galactose and GlcNAc residues from the hepatocyte ASGP receptor, preventing endocytosis to extend prolong circulatory lifetime (See id.). Therefore, the present disclosure provides obtaining sialic acid results with quick turn-around-time. [0050] Continuous bioprocesses can increase production flexibility, overall reduction of facility footprints and increased productivity, securing reduced overall production cost. One key challenge in implementing continuous bioprocessing is the real-time or near real- time acquisition of product quality information to facilitate timely control of the process. Developing appropriate PAT tools to monitor CQAs that influence the efficacy and safety of the drug can ensure desired final quality. The shift toward quality-by-design (QbD) is intended to improve product quality and has been the focus of the biopharmaceutical industry to achieve high productivity with >10 g/L mAb titers (Gyorgypal and Chundawat, Anal. Chem.94, 19, 6986-6995 (2022)). [0051] Shifting from batch process to QbD enabled continuous bioprocessing with the present disclosure can provide an opportunity to enhance process and product understanding as well as to facilitate cost-effective manufacturing, to meet increased demand for achieving process robustness, to improve process understanding during bioprocess, and to offer efficient delivery of high-quality products at a reduced cost. [0052] Unlike other product quality assays, however, sialic acid quantitation method along with some of the convoluted analytical methods including peptide mapping, multi- attribute methods, amino acid analysis and N-linked glycan analysis are requiring lengthy and complex manual sample preparation. These methods are causing a setback to the recombinant protein production process to develop tools to generate real-time/near-real- time CQA measurements. To support QbD driven next generation continuous bioprocessing, developing QbD enabled technologies such as online PAT tools with reduced analysis time is essential for real-time or near-real-time data generation such that appropriate control strategies can be deployed to maintain the process in a steady sate. This in turn can support QbD paradigm to modernize the pharmaceutical/biopharmaceutical industry and enable the deployment of inline, online, or at-line tools to align with the strategy to monitor real-time or near-real-time product quality and control the process with the goal of achieving a desired product quality. [0053] Therefore, the present disclosure provides an online analysis of complex techniques such a glycan analysis and peptide mapping. The prsent disclosure thus provides methods to maintain sialic acid content by making harvest decision based on the levels of sialic acid. In some aspects, the present disclosure provides use of µSIA system as a sample preparation platform coupled with SegFlow sampling device and an online ultra-performance liquid chromatography (UPLC) system. In some aspects, online sampling using segFlow and automatic sample preparations using µSIA system along with chromatographic separation using UPLC facilitates near-real-time monitoring of sialic acid contents in the bioreactors. In some aspects, the approach presented in the present disclosure provides the opportunity to make the harvest decision purely based on the sialic acid content of cell culture harvest to maintain consistent mole/mole ratio of sialic acid to protein from batch-to-batch. [0054] In some aspects, the method of the present disclosure comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated. [0055] In some aspects, two, three, four, or more of a) to e) are automated. In some aspects, two of a) to e) are automated. In some aspects, three of a) to e) are automated. In some aspects, four of a) to e) are automated. In some aspects, all of a) to e) are automated. [0056] In some aspects, one, two, three, four, or more of a) to e) are performed in a closed system. In some aspects, one of a) to e) are performed in a closed system. In some aspects, two of a) to e) are performed in a closed system. In some aspects, three of a) to e) are performed in a closed system. In some aspects, four of a) to e) are performed in a closed system. In some aspects, all of a) to e) are performed in a closed system. [0057] In some aspects, the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected from a cell lysate. In some aspects, the sample is collected automatically. In some aspects, the sample is collected after protein purification. In some aspects, the sample is collected from downstream after protein-A purification. [0058] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, a hydrophilic interaction column, a reverse phase column, or an ion exchange column. In some aspects, the polypeptide binding column is a size exclusion column. In some aspects, the polypeptide binding column is a hydrophobic interaction column. In some aspects, the polypeptide binding column is an ion exchange column. In some aspects, the polypeptide binding column is a hydrophilic interaction column. In some aspects, the polypeptide binding column is a reverse phase column. [0059] In some aspects, the polypeptide binding column is an affinity column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a FLAG column. [0060] In some aspects, the desialylation agent is an acid or a proteolytic enzyme. [0061] In some aspects, the desialylation agent is a proteolytic enzyme. In some aspects, the proteolytic enzyme is a exoglycosidase. In some aspects, the exoglycosidase is a sialidase. [0062] In some aspects, the desialylation agent is an acid. In some aspects, the acid is phosphoric acid, acetic acid, sulfuric acid, or any combination thereof. In some aspects, the acid is phosphoric acid. In some aspects, the acid is acetic acid. In some aspects, the acid is sulfuric acid. [0063] For quantification, free sialic acid is often derivatized with sialic acid labeling agents (e.g., chromophores and/or fluorophores) to achieve stabilization and significant enhancement in detection sensitivity. In some aspects, the sialic acid is labeled with a sialic acid labeling agent. In some aspects, the sialic acid labeling agent is 1,2-diamino- 4,5-methylenedioxybenzene (DMB) or o-phenylenediamine. In some aspects, the sialic acid labeling agent is 1,2-diamino-4,5-methylenedioxybenzene (DMB). In some aspects, the sialic acid labeling agent is o-phenylenediamine. [0064] In some aspects, the labeling of the sialic acids is automated. In some aspects, the labeling of sialic acid takes about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour, or about 0.5 hours. In some aspects, the labeling of sialic acid takes about 3.5 hours, 3 hours, or 2.5 hours. In some aspects, the labeling of sialic acid takes about 3 hours. [0065] Various methods have been established for quantification of sialic acids including HPLC, microtiter plate based colorimetric/fluorometric assays as well as mass spectrometry (MS) based methods. For the quantitation of non-derivatized sialic acid, sialic acids may be quantified by LC/MS, HPLC, or UPLC. The HPLC and UPLC can be interfaced with NQAD detector or other universal detectors such as charge aerosol detector (CAD), Evaporative Light Scattering Detector (ELSD) and Refractive Index (RI) detector can be employed. Over the past decade, several bio-affinity-based approaches for direct detection of sialic acids and sialylglycans have been developed, including lectins, antibodies, and recombinant sialic acid-binding proteins (Zhou X, et al. Cells.2020; 9(2):273). Lectins are sugar-binding proteins that can specifically recognize glycans on glycoconjugates. Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL) are commonly used to preferentially bind to sialic acid (Zhou X, et al. Cells.2020; 9(2):273). In some aspects, the sialic acid is not labeled. [0066] In some aspects, the analytical device is a UPLC, HPLC, uHPLC, or LC/MS. In some aspects, the analytical device is a UPLC or a HPLC. In some aspects, the analytical device is a UPLC. In some aspects, the analytical device is a HPLC. In some aspects, the analytical device is a uHPLC. In some aspects, the analytical device is LC/MS. [0067] In some aspects, the amount of total sialic acid per polypeptide is calculated by Equation 1. Asx represents the normalized area of x = 1 mono-, x = 2 di-, x = 3 tri-, and x= 4 tetrasialylated species in percentage and N the number of N-glycan sites on the polypeptide. This calculation assumes that all N-glycan sites are 100% occupied (no macro-heterogeneity). Equation 1. ^^ ^^ ^^^^^^^^ ^^^ௗ ∑ ^^ௌ^ + ∑ ^^ௌଶ × 2 + ∑ ^^ௌଷ × 3 + ∑ ^^ௌସ × 4 ^^ ^^ ^^ = × ^^ ^^^௬^^^௧^ௗ^ 100 [0068] In some aspects, the amount of NANA per polypeptide in the sample is calculated by Equation 2. X.XX μg is the amount of NANA injected and 309.27 μg is the molecular weight of NANA. Equation 2.
Figure imgf000015_0001
20 ^^ ^^( ^^ ^^ ^^. ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^) where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. [0069] In some aspects, the amount of NGNA per polypeptide in the sample is calculated by Equation 3. X.XX μg is the amount of NGNA injected and 325.27 μg is the molecular weight of NGNA. Equation 3. ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ∗ 1 ^ ^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^ ^ ^^ ^^ ^^ ∗ 10 ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. [0070] In some aspects, the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture. The metrics are based on the sialic acid specification range, which vary from molecule to molecule. [0071] In some aspects, the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture. In some aspects, DANA, N-acetylmannosamine (ManNAc), fetuin, CuCl2, dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture. [0072] In some aspects, a) to e) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. In some aspects, a) to f) take about 5 hours. In some aspects, a) to e) take about 2 hours. [0073] In some aspects, the method further comprises quantifying the titer of the polypeptides in the sample prior to a). In some aspects, the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide- binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer. [0074] In some aspects, the analytical device is a UV spectrophotometer, a fluorescence spectrometer, UPLC, HPLC, uHPLC, or LC/MS. In some aspects, the analytical device is a UV spectrophotometer. In some aspects, the analytical device is a fluorescence spectrometer. In some aspects, the analytical device is a UPLC or a HPLC. In some aspects, the analytical device is a UPLC. In some aspects, the analytical device is a HPLC. In some aspects, the analytical device is a uHPLC. In some aspects, the analytical device is a LC/MS. [0075] In some aspects, after determining the titer of the polypeptides, a specific amount of polypeptides in the sample is collected in a second sample. In some aspects, the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to f). [0076] In some aspects, one of i) to iii) are automated. In some aspects, two or three of i) to iii) are automated. In some aspects, two of i) to iii) are automated. In some aspects, all of i) to iii) are automated. [0077] In some aspects, the polypeptides are fusion proteins. In some aspects, the polypeptides are therapeutic proteins. In some aspects, the polypeptides are antibodies. In some aspects, the polypeptides are bispecific or multi-specific polypeptides. [0078] In some aspects, the method further comprises determining when to harvest polypeptides from a cell culture based on the sialic acid content of the polypeptides. In some aspects, the method further comprises harvesting the polypeptides from the cell culture when the polypeptides contain the desired sialic acid content. In some aspects, the polypeptides are harvested when the polypeptides have a desired sialic acid to protein ratio. The current harvest NANA range for Abatacept process J is between 7.9 to 9.1, but in some cases it is preferential to harvest when the predicted NANA is around 8.8. In manufacturing, the NANA prediction was based on many factors (scale-down model, large scale daily data, etc.), therefore an exact metric for NANA harvest might not be accurate all the time. The harvest NANA of recent Abatacept process J lots has been falling between 8.3 and 9.0. [0079] In some aspects, the method further comprises determining when to harvest polypeptides from a bioreactor based on the sialic acid content of the polypeptides. In some aspects, the method further comprises harvesting the polypeptides from the bioreactor when the polypeptides contain the desired sialic acid content. In some aspects, the polypeptides are harvested when the polypeptides have a desired sialic acid to protein ratio. [0080] In some aspects, harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column; and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides. [0081] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, a reverse phase column, a hydrophilic interaction column, or an ion exchange column. In some aspects, the polypeptide binding column is a size exclusion column. In some aspects, the polypeptide binding column is a hydrophobic interaction column. In some aspects, the polypeptide binding column is a reverse phase column. In some aspects, the polypeptide binding column is a hydrophilic interaction column. In some aspects, the polypeptide binding column is an ion exchange column. [0082] In some aspects, the polypeptide binding column is an affinity column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a FLAG column. [0083] In some aspects, one, two, or three of aa) to cc) are automated. In some aspects, all of aa) to cc) are automated. [0084] In some aspects, wash buffer is moved through the polypeptide binding column. In some aspects, the conductivity of the wash buffer is adjusted to achieve a desired sialic acid to protein ratio. [0085] In some aspects, the wash buffer is prepared based on the sialic acid content of the polypeptides. In some aspects, the wash buffer is modified based on the sialic acid content of the polypeptides. In some aspects, the wash buffer comprises NaCl, KCl, Na2SO4, K2SO4, Na3PO4 NaOAc, or any combination thereof. In some aspects, the wash buffer comprises NaCl, KCl, Na2SO4, K2SO4, or any combination thereof. In some aspects, the wash buffer comprises Na3PO4, NaOAc, or any combination thereof. [0086] In some aspects, the amount of Na3PO4, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified based on the sialic acid content of the polypeptides. [0087] In some aspects, the amount of Na3PO4, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified to increase the sialic acid content of the polypeptides. [0088] In some aspects, the amount of Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, in the wash buffer is modified to increase the amount of NANA on the polypeptides. [0089] In some aspects, 20 mM to 70 mM Na3PO4 (e.g., 40 mM, 50 mM, or 60 mM), 200 mM to 400 mM NaOAc (e.g., 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM), or any combination thereof, is added to the wash buffer to increase sialic acid content of the polypeptides. In some aspects, the pH of the wash buffer is pH 7.0. In some aspects, a conductivity range of the wash buffer is between about 7 and 13 mS/cm. [0090] In some aspects, 20 mM to 70 mM Na3PO4 (e.g., 40 mM, 50 mM, or 60 mM), 200 mM to 400 mM NaOAc (e.g., 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM), or any combination thereof, is added to the wash buffer to increase the amount of NANA on the polypeptides. In some aspects, the pH of the wash buffer is pH 7.0. In some aspects, a conductivity range of the wash buffer is between about 7 and 13 mS/cm. [0091] In some aspects, the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated. [0092] In some aspects, the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein two, three, four, or five or more of a) to f) are automated. In some aspects, two or more of a) to f) are automated. In some aspects, three or more of a) to f) are automated. In some aspects, four or more of a) to f) are automated. In some aspects, five or more of a) to f) are automated. In some aspects, all of a) to f) are automated. [0093] In some aspects, the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one, two, three, four, or five or more of a) to f) are performed in a closed system. In some aspects, one or more of a) to f) are performed in a closed system. In some aspects, two or more of a) to f) are performed in a closed system. In some aspects, three or more of a) to f) are performed in a closed system. In some aspects, four or more of a) to f) are performed in a closed system. In some aspects, five or more of a) to f) are performed in a closed system. In some aspects, all of a) to f) are performed in a closed system. [0094] In some aspects, the sample is collected from the media of cultured cells. In some aspects, the sample is collected from a bioreactor. In some aspects, the sample is collected automatically. In some aspects, the sample is collected after protein purification. In some aspects, the sample is collected after protein-A purification. [0095] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a Flag column. [0096] In some aspects, the desialylation agent is an acid or an exoglycosidase. In some aspects, the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof. In some aspects, the acid is phosphoric acid. In some aspects, the acid is sulfuric acid. In some aspects, the acid is acetic acid. In some aspects, the exoglycosidase is a sialidase. [0097] In some aspects, the sialic acid labeling agent is 1,2-diamino-4,5- methylenedioxybenzene (DMB) or o-phenylenediamine. In some aspects, the analytical device is a UPLC, uHPLC, or HPLC. [0098] In some aspects, the amount of total sialic acid per polypeptide is calculated by Equation 1. Asx represents the normalized area of x = 1 mono-, x = 2 di-, x = 3 tri-, and x= 4 tetrasialylated species in percentage and N the number of N-glycan sites on the polypeptide. This calculation assumes that all N-glycan sites are 100% occupied (no macro-heterogeneity). Equation 1.
Figure imgf000020_0001
^௬^^^௧^ௗ^ [0099] In some aspects, the amount of NANA per polypeptide in the sample is calculated by Equation 2. X.XX μg is the amount of NANA injected and 309.27 μg is the molecular weight of NANA. Equation 2.
Figure imgf000021_0001
where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. [0100] In some aspects, the amount of NGNA per polypeptide in the sample is calculated by Equation 3. X.XX μg is the amount of NGNA injected and 325.27 μg is the molecular weight of NGNA. Equation 3. ^^ ^^ ^^ ^^ ^^ ∗ 1 ^^ ^^ ^^ ^^ ∗ 10^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. [0101] In some aspects, the method further comprises modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture media. In some aspects, DANA, N-acetylmannosamine (ManNAc), fetuin, CuCl2, dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture media. [0102] In some aspects, a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. In some aspects, a) to f) take about 2 hours. In some aspects, a) to f) take about 5 hours. [0103] In some aspects, the method further comprises quantifying the titer of the polypeptides in the sample prior to a). In some aspects, the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide- binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer. [0104] In some aspects, the analytical device is a UV spectrophotometer or a fluorescence spectrophotometer. In some aspects, after determining the titer of the polypeptides, a specific amount of polypeptides in the sample is collected in a second sample. [0105] In some aspects, the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to f) (“repeat steps a) to f)”). In some aspects, any one of i) to iii) are automated. In some aspects, two or three of i) to iii) are automated. In some aspects, all of i) to iii) are automated. [0106] In some aspects, the method further comprises determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. In some aspects, the polypeptides are harvested when the polypeptides have a desired sialic acid to protein ratio. [0107] In some aspects, the method further comprises harvesting the polypeptides from the cell culture media when the polypeptides contain the desired sialic acid content. In some aspects, harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column, and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides. [0108] In some aspects, the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. In some aspects, the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein A column. In some aspects, the affinity column is a protein G column. In some aspects, the affinity column is a protein L column. In some aspects, the affinity column is a His column. In some aspects, the affinity column is a GST column. In some aspects, the affinity column is a Myc column. In some aspects, the affinity column is a Flag column. [0109] In some aspects, one, two, or three of aa) to cc) are automated. In some aspects, all of aa) and cc) are automated. [0110] In some aspects, the wash buffer is prepared based on the sialic acid content of the polypeptides. In some aspects, the wash buffer is modified based on the sialic acid content of the polypeptides. In some aspects, the conductivity of the wash buffer is adjusted to achieve a desired sialic acid to protein ratio. In some aspects, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase sialic acid content of the polypeptides. In some aspects, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase amount of NANA on the polypeptides. [0111] In some aspects, the method comprises: a) moving a portion of a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column, to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and j) quantifying the amount N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues in the sample, wherein one or more of a) to j) are automated. [0112] In some aspects, two, three, four, five, six, seven, eight, nine, or more of a) to j) are automated. In some aspects, two of a) to j) are automated. In some aspects, three of a) to j) are automated. In some aspects, four of a) to j) are automated. In some aspects, five of a) to j) are automated. In some aspects, six of a) to j) are automated. In some aspects, seven of a) to j) are automated. In some aspects, eight of a) to j) are automated. In some aspects, nine of a) to j) are automated. In some aspects, all of a) to j) are automated. [0113] In some aspects, the method comprises: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; f) quantifying the amount N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues in the sample; g) moving a second sample comprising polypeptides to a second polypeptide-binding column to form a second binding complex; h) moving a wash buffer to and through the polypeptide- binding column; and i) moving an elution buffer to and through the second binding complex to elute the bound polypeptides, wherein one or more of a) to i) are automated. [0114] In some aspects, two, three, four, five, six, seven, eight or more of a) to i) are automated. In some aspects, two of a) to i) are automated. In some aspects, three of a) to i) are automated. In some aspects, four of a) to i) are automated. In some aspects, five of a) to i) are automated. In some aspects, six of a) to i) are automated. In some aspects, seven of a) to i) are automated. In some aspects, eight or more of a) to i) are automated. In some aspects, all of a) to i) are automated. [0115] In some aspects, the method comprises: a) moving a portion of a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labeled sialic acids through an analytical device to separate the N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; j) quantifying the amount N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues in the sample; k) moving a third sample comprising polypeptides to a third polypeptide-binding column to form a third binding complex; l) moving a wash buffer to and through the polypeptide-binding column; and m) moving an elution buffer to and through the third binding complex to elute the bound polypeptides, wherein one or more of a) to m) are automated. [0116] In some aspects, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of a) to m) are automated. In some aspects, two of a) to m) are automated. In some aspects, three of a) to m) are automated. In some aspects, four of a) to m) are automated. In some aspects, five of a) to m) are automated. In some aspects, six of a) to m) are automated. In some aspects, seven of a) to m) are automated. In some aspects, eight of a) to m) are automated. In some aspects, nine of a) to m) are automated. In some aspects, ten of a) to m) are automated. In some aspects, ten of a) to m) are automated. In some aspects, eleven of a) to m) are automated. In some aspects, twelve of a) to m) are automated. In some aspects, all of a) to m) are automated. Polypeptides [0117] Any protein can be used in the disclosed method. In some aspects, the polypeptide is a fusion protein. In some aspects, the polypeptide is a therapeutic protein. [0118] As disclosed above, in some aspects, the therapeutic proteins that can be prepared by using the methods disclosed herein comprise, for example, antibodies, antibody fragments, Fc portions of antibodies and fusions thereof, antigen binding portions of antibodies, fusion proteins, naturally occurring proteins, recombinant proteins, chimeric proteins, immunoadhesins, enzymes, growth factors, receptors, hormones, regulatory factors, cytokines, or any combination thereof. In some aspects, the therapeutic protein is produced in mammalian cells. In some aspects, the mammalian cell line is a Chinese Hamster Ovary (CHO) cells, or baby hamster kidney (BHK) cells, murine hybridoma cells, or murine myeloma cells. [0119] Any therapeutic protein that is expressible in a host cell may be assessed for its sialic acid content in accordance with the present disclosure and may be present in the compositions provided. The therapeutic protein may be expressed from a gene that is endogenous to the host cell, or from a gene that is introduced into the host cell through genetic engineering. The therapeutic protein may be one that occurs in nature, or may alternatively have a sequence that was engineered or selected by the hand of man. An engineered therapeutic protein may be assembled from other polypeptide segments that individually occur in nature, or may include one or more segments that are not naturally occurring. [0120] The methods provided in the present disclosure, in some aspects, employ any cell that is suitable for growth and/or production of a therapeutic protein in a culture medium, including animal, yeast or insect cells. In one aspect, the cell is any mammalian cell or cell type suitable to cell culture and to expression of polypeptides. The methods provided herein (e.g., methods of measuring a sialic acid content) can therefore employ any suitable type of cell, including an animal cell. In one aspect, the methods employ a mammalian cell. In some aspects, methods can also employ hybridoma cells. In some aspects, the mammalian cell is a non-hybridoma mammalian cell, which has been transformed with exogenous isolated nucleic acid encoding a desired therapeutic protein. In one aspect, the methods employ mammalian cells selected from the group consisting of human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK. ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather. Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some aspects, the methods employ CHO cells. In some aspects, the culturing of CHO cell lines and expression of therapeutic proteins from CHO cell lines is employed. In some aspects, therapeutic protein can be secreted into the culture medium from which the therapeutic protein may be isolated and/or purified or the therapeutic protein may be released into the culture medium by lysis of a cell comprising an isolated nucleic acid encoding the therapeutic protein. [0121] In some aspects, the therapeutic protein is a CTLA4-Ig molecule, e.g., abatacept or belatacept. The terms “CTLA4-Ig” or “CTLA4-Ig molecule” are used interchangeably, and refer to a protein molecule that comprises at least a polypeptide having a CTLA4 extracellular domain or portion thereof and an immunoglobulin constant region or portion thereof. The extracellular domain and the immunoglobulin constant region can be wild- type, or mutant or modified, and mammalian, including human or mouse. The polypeptide can further comprise additional protein domains. A CTLA4-Ig molecule can also refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule also is capable of binding to CD80 and/or CD86. [0122] In one aspect, “CTLA4Ig” refers to a protein molecule having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:1, (ii) 26-382 of SEQ ID NO:1; (iii) 27- 383 of SEQ ID NO:1, or (iv) 27-382 of SEQ ID NO:1, or optionally (v) 25-382 of SEQ ID NO:1, or (vi) 25-383 of SEQ ID NO:1. In monomeric form these proteins can be referred to herein as “SEQ ID NO:1 monomers,” or monomers “having a SEQ ID NO:1 sequence”. These SEQ ID NO:1 monomers can dimerize, such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These different dimer combinations can also associate with each other to form tetramer CTLA4Ig molecules. These monomers, dimers, tetramers and other multimers can be referred to herein as “SEQ ID NO:1 proteins” or proteins “having a SEQ ID NO:1 sequence”. (DNA encoding CTLA4Ig as shown in SEQ ID NO:1 was deposited on May 31, 1991 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 under the provisions of the Budapest Treaty, and has been accorded ATCC accession number ATCC 68629; a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig as shown in SEQ ID NO:1 was deposited on May 31, 1991 with ATCC identification number CRL-10762). As utilized herein “Abatacept” refers to SEQ ID NO:1 proteins. >SEQ ID NO:1 MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCA ATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD QEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK >SEQ ID NO:3 (SEQ ID NO:1 without signal peptide) MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQV NLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [0123] In another aspect, the therapeutic protein is CTLA4-L104EA29Y-Ig (sometimes known as “LEA29Y” or “L104EA29Y”), which is a genetically engineered fusion protein similar in structure to CTAL4-Ig molecule as shown in SEQ ID NO:1. L104EA29Y-Ig has the functional extracellular binding domain of modified human CTLA4 and the Fc domain of human immunoglobulin of the IgG1 class. Two amino acid modifications, leucine to glutamic acid at position 104 (L104E), which is position 130 of SEQ ID NO:1, and alanine to tyrosine at position 29 (A29Y), which is position 55 of SEQ ID NO:1, were made in the B7 binding region of the CTLA4 domain to generate L104EA29Y. SEQ ID NO:2 depict a amino acid sequence of L104EA29YIg comprising a signal peptide; a mutated extracellular domain of CTLA4 starting at methionine at position +27 and ending at aspartic acid at position +150, or starting at alanine at position +26 and ending at aspartic acid at position +150; and an Ig region. DNA encoding L104EA29Y-Ig was deposited on Jun.20, 2000, with the American Type Culture Collection (ATCC) under the provisions of the Budapest Treaty. It has been accorded ATCC accession number PTA-2104. L104EA29Y-Ig is further described in U.S. Pat. No.7,094,874, issued on Aug.22, 2006, and in WO 01/923337 A2, which are incorporated by reference herein in their entireties. [0124] Expression of L104EA29YIg in mammalian cells can result in the production of N- and C-terminal variants, such that the proteins produced can have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2; (iii) 27- 383 of SEQ ID NO:2 or (iv) 27-382 of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2. In monomeric form these proteins can be referred to herein as “SEQ ID NO:2 monomers,” or monomers “having a SEQ ID NO:2 sequence.” [0125] These proteins can dimerize, such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These different dimer combinations can also associate with each other to form tetramer L104EA29YIg molecules. These monomers, dimers, tetramers and other multimers can be referred to herein as “SEQ ID NO:2 proteins” or proteins “having a SEQ ID NO:2 sequence”. As utilized herein “Belatacept” refers to SEQ ID NO:2 proteins. >SEQ ID NO:2 MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCA ATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD QEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK >SEQ ID NO:4 (SEQ ID NO:2 without signal peptide) MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQV NLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [0126] In some aspects, exemplary polypeptides include CD proteins, including CD3, CD4, CDS, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding. HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor. Cell adhesion molecules, for example, LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v/beta 3 integrin. Growth factors, such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGFα and TGF-β, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-1), and osteoinductive factors. Insulins and insulin-related proteins, including insulin, insulin A- chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins. Coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens. Colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor ( c-fms). Receptors and receptor-associated proteins, including, for example, flk2/flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors ("TPO-R," "c-mpl"), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors. Receptor ligands, including, for example, OX40L, the ligand for the OX40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A-chain, relaxin B-chain, and prorelaxin; interferons and interferon receptors, including for example, interferon-a,-~, and -y, and their receptors. Interleukins and interleukin receptors, including IL-I to IL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, among others. Viral antigens, including an AIDS envelope viral antigen. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatins, TALL proteins, including TALL-I, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins ("TSLP"), RANK ligand ("OPGL"), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing. [0127] Exemplary therapeutic polypeptides and antibodies include ACTIVASE® (Alteplase); alirocumab, ARANESP® (Darbepoetin-alfa), EPOGEN® (Epoetin alfa, or erythropoietin); AVONEX® (Interferon ~-Ia); BEXXAR® (Tositumomab); Betaseron® (Interferon-~); bococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see US8080243); CAMPATH® (Alemtuzumab); DYNEPO® (Epoetin delta); VELCADE® (bortezomib); MLN0002 (anti-a4~7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); ENBREL® (etanercept); EPREX® (Epoetin alfa); ERBITUX® (Cetuximab); evolocumab; GENOTROPIN® (Somatropin); HERCEPTIN® (Trastuzumab); HUMATROPE® (somatropin [rDNA origin] for injection); HUMIRA® (Adalimumab ); INFERGEN® (Interferon Alfacon-1 ); N ATRECOR® (nesiritide); KINERET® (Anakinra), LEUKINE® (Sargamostim); LymphoCide® (Epratuzumab); BenlystaTM (Belimumab); METALYSE® (Tenecteplase); MIRCERA® (methoxy polyethylene glycol-epoetin beta); MYLOTARG® (Gemtuzumab ozogamicin); RAPTIVA® (efalizumab); CIMZIA® (certolizumab pegol); SolirisTM (Eculizumab); Pexelizumab (Anti-CS Complement); MEDI-524 (NUMAX®); LUCENTIS® (Ranibizumab); Edrecolomab (PANOREX®); TRABIO® (lerdelimumab); TheraCim hR3 (Nimotuzumab); Omnitarg (Pertuzumab, 2C4); OSIDEM® (IDM-I); OVAREX® (B43.13); NUVION® (visilizumab); Cantuzumab mertansine (huC242-DMl); NEORECORMON® (Epoetin beta); NEUMEGA® (Oprelvekin); NEULASTA® (Pegylated filgastrim, pegylated G-CSF, pegylated hu-Met-G-CSF); NEUPOGEN® (Filgrastim); Orthoclone OKT3® (Muromonab-CD3), PROCRIT® (Epoetin alfa); REMICADE® (Infliximab ), REOPRO® (Abciximab ), ACTEMRA® (anti-IL6 Receptor mAb), AVASTIN® (Bevacizumab), HuMax-CD4 (zanolimumab), RITUXAN® (Rituximab); TARCEVA® (Erlotinib); ROFERON-A®-(Interferon alfa-2a); SIMULECT® (Basiliximab); StelaraTM (Ustekinumab); PREXIGE® (lumiracoxib); SYNAGIS® (Palivizumab); 146B7- CHO (anti-IL15 antibody, see US7153507), TYSABRI® (Natalizumab); VALORTIM® (MDX-1303, anti-B. anthracis Protective Antigen mAb); ABthraxTM; VECTIBIX® (Panitumumab); XOLAIR® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fe portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRl fused to IgGl Fe), ZENAPAX® (Daclizumab ); ZENAPAX® (Daclizumab), ZEVALIN® (lbritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-Ig), anti-a4β7 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); SimponiTM (Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab, anti-a5β1 integrin mAb); MDX- 010 (Ipilimumab, anti-CTLA-4 mAb and VEGFR-1 (IMC-18Fl); anti-BR3 mAb; anti-C. difficile Toxin A and Toxin BC mAbs MDX-066 (CDA-1) and MDX-1388); anti-CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti- TSLP antibodies; anti-TSLP receptor antibody (US8101182); anti-TSLP antibody designated as A5 (US7982016); (anti-CD3 mAb (NI-0401); Adecatumumab (MT201, anti-EpCAM- CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAbs); MDX-1333 (anti- IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxinl mAb (CAT-213); antiFGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibodies (see, US8715663 or US7592429) anti-sclerostin antibody designated as Ab-5 (US8715663 or US7592429); anti-ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM- CSP Receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNa mAb); anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); antiIL12/IL23p40 mAb (Briakinumab); anti-IL-23p19 mAb (LY2525623); anti-IL13 mAb (CAT-354); anti-IL- 17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti-IL5 Receptor mAb; anti-integrin receptors mAb (MDX-O18, CNTO 95); anti-IPIO Ulcerative Colitis mAb (MDX- 1100); anti-LLY antibody; BMS-66513; anti-Mannose Receptor/hCG~ mAb (MDX-1307); anti- mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDlmAb (MDX-1106 (ONO- 4538)); anti-PDGFRa antibody (IMC-3G3); antiTGFβ mAb (GC-1008); anti-TRAIL Receptor-2 human mAb (HGS-ETR2); antiTWEAK mAb; anti-VEGFR/Flt-1 mAb; anti- ZP3 mAb (HuMax-ZP3); and an amyloid-beta monoclonal antibody comprising sequences, SEQ ID NO:8 and SEQ ID NO:6 (US7906625). [0128] In some aspects, the polypeptide is an antibody or an antibody fragment. In some aspects, the antibody or an antibody fragment is a human, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, or grafted, antibody or antibody fragment. In some aspects, the antibody or an antibody fragment is a monoclonal or polyclonal antibody or antibody fragment. In some aspects, the antibody or an antibody fragment is a IgG, IgM, IgA, IgD, or IgE antibody or antibody fragment. In some aspects, the antibody or an antibody fragment is a IgG1, IgG2, IgG3, or IgG4 antibody or antibody fragment. In some aspects, the antibody or antibody fragment is a Fab, F(ab')2, VHH, or ScFv. In some aspects, the polypeptides are bispecific or multi-specific polypeptides. [0129] Exemplary antibodies include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab. Methods of manufacture and methods of treatment [0130] The present disclosure also provides a method to treat a disease or condition comprising administering to a subject a therapeutic protein manufactured by the methods disclosed herein. Also provided is a pharmaceutical composition manufactured by the methods disclosed herein. EXEMPLARY ASPECTS [0131] The disclosed invention includes the following non-exhaustive listing of aspects. This listing should not be construed to be in anyway limiting, and a person skilled in the art will appreciate that various modifications can be made to these embodiments without changing the essence and scope of the invention disclosed herein. 1. A method for measuring an amount of sialic acid in polypeptides in near- real time, the method comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated. 2. The method of aspect 1, wherein two, three, four or more of a) to e) are automated. 3. The method of aspect 1, wherein all of a) to e) are automated. 4. The method of any one of aspects 1-3, wherein one, two, three, four or more of a) to e) are performed in a closed system. 5. The method of any one of aspects 1-3, wherein all of a) to e) are performed in a closed system. 6. The method of any one of aspects 1-5, wherein the sample is collected from the media of cultured cells. 7. The method of any one of aspects 1-6, wherein the sample is collected from a bioreactor. 8. The method of any one of aspects 1-7, wherein the sample is collected automatically. 9. The method of any one of aspects 1-8, wherein the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 10. The method of embodiment 9, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. 11. The method of aspect 10, wherein the affinity column is a protein A column. 12. The method of any one of aspects 1-11, wherein the desialylation agent is an acid or an exoglycosidase. 13. The method of aspect 12, wherein the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof. 14. The method of aspect 12, wherein the exoglycosidase is a sialidase. 15. The method of any one of aspects 1-14, wherein the released sialic acids are mixed with a sialic acid labeling agent, thereby labeling the sialic acids, prior to d). 16. The method of aspect 15, wherein the labeling of the sialic acids is automated. 17. The method of aspect 15 or 16, wherein the labeling of the sialic acid takes about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour or about 0.5 hours. 18. The method of aspect 17, wherein the labeling of the sialic acid takes about 3 hours. 19. The method of aspect 15, wherein the sialic acid labeling agent is 1,2- diamino-4,5-methylenedioxybenzene (DMB) or o-phenylenediamine. 20. The method of any one of aspects 1-19, wherein the analytical device is a UPLC, uHPLC or HPLC. 21. The method of any one of aspects 1-20, wherein the amount of total sialic acid per polypeptide is calculated by Equation 1. ^^ ^^ ^^^^^^^^ ^^^ௗ ∑ ^^ௌ^ + ∑ ^^ௌଶ × 2 + ∑ ^^ௌଷ × 3 + ∑ ^^ௌସ × 4 ^^ ^^ ^^ =
Figure imgf000036_0001
^^௧^^^ௗ௬ 100 22. The method of any one of aspects 1-20, wherein the amount of NANA per polypeptide in the sample is calculated by Equation 2.
Figure imgf000036_0002
where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. 23. The method of any one of aspects 1-20, wherein the amount of NGNA per polypeptide in the sample is calculated by Equation 3. ^^ ^^ ^^ ^^ ^^ ∗ 1 ^^ ^ ⁄ ^^ ^^ ^^ ∗ 10 ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. 24. The method of any one of aspects 1-23, further comprising modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture media. 25. The method of aspect 24, wherein DANA, N-acetylmannosamine (ManNAc), fetuin, CuCl2, dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture media. 26. The method of any one of aspects 1-25, wherein a) to e) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. 27. The method of aspect 26, wherein a) to e) take about 2 hours. 28. The method of any one of aspects 1-27, further comprising quantifying the titer of the polypeptides in the sample prior to a). 29. The method of aspect 28, wherein the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide-binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer. 30. The method of aspect 29, wherein the analytical device is a UV spectrophotometer or a fluorescence spectrophotometer. 31. The method of any one of aspects 28-30, wherein after determining the titer of the polypeptides, a specific amount of polypeptides in the sample is collected in a second sample. 32. The method of aspect 31, wherein the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to e) (“repeat steps a) to e)”). 33. The method of any one of aspects 29-32, wherein any one of i) to iii) are automated. 34. The method of aspect 33, wherein two or three of i) to iii) are automated. 35. The method of aspect 33 or 34, wherein all of i) to iii) are automated. 36. The method of any one of aspects 1-35, wherein the polypeptides are fusion proteins. 37. The method of any one of aspects 1-36, wherein the polypeptides are antibodies. 38. The method of any one of aspects 1-37, further comprising determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. 39. The method of any one of aspects 1-38, further comprising harvesting the polypeptides from the cell culture media when the polypeptides contain the desired sialic acid content. 40. The method of aspects 38 or 39, wherein harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column; and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides. 41. The method of aspect 40, wherein the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 42. The method of aspect 39, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. 43. The method of embodiment 42, wherein the affinity column is a protein A column. 44. The method of any one of aspects 40-43, wherein one, two, or three of aa) to cc) are automated. 45. The method of aspect 44, wherein all of aa) and cc) are automated. 46. The method of aspect 40, wherein the wash buffer is prepared based on the sialic acid content. 47. The method of aspect 46, wherein Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase sialic acid content of the polypeptides. 48. The method of aspect 46, wherein Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase amount of NANA on the polypeptides. 49. A method for measuring an amount of sialic acid in polypeptides in near- real time, the method comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated. 50. The method of aspect 49, wherein two, three, four, or five or more of a) to f) are automated. 51. The method of aspect 49, wherein all of a) to f) are automated. 52. The method of any of aspects 49-51, wherein one, two, three, four, or five or more of a) to f) are performed in a closed system. 53. The method of any one of aspects 49-52, wherein all of a) to f) are performed in a closed system. 54. The method of any one of aspects 49-53, wherein the sample is collected from the media of cultured cells. 55. The method of any one of aspects 49-54, wherein the sample is collected from a bioreactor. 56. The method of any one of aspects 49-55, wherein the sample is collected automatically. 57. The method of any one of aspects 49-56, wherein the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 58. The method of aspect 57, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. 59. The method of aspect 58, wherein the affinity column is a protein A column. 60. The method of any one of aspects 49-59, wherein the desialylation agent is an acid or an exoglycosidase. 61. The method of aspect 60, wherein the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof. 62. The method of aspect 60, wherein the exoglycosidase is a sialidase. 63. The method of any one of aspects 49-62, wherein the sialic acid labeling agent is 1,2-diamino-4,5-methylenedioxybenzene (DMB) or o-phenylenediamine. 64. The method of any one of aspects 49-63, wherein the analytical device is a UPLC, uHPLC or HPLC. 65. The method of any one of aspects 49-64, wherein the amount of total sialic acid per polypeptide is calculated by Equation 1.
Figure imgf000040_0001
^^௧^^^ௗ௬ 66. The method of any one of aspects 49-65, wherein the amount of NANA per polypeptide in the sample is calculated by Equation 2.
Figure imgf000040_0002
where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. 67. The method of any one of aspects 49-66, wherein the amount of NGNA per polypeptide in the sample is calculated by Equation 3. ^^ ^^ ^^ ^^ ^^ ^ ⁄ ∗ 1 ^^ ^^ ^^ ^^ ∗ 10 ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. 68. The method of any one of aspects 49-67, further comprising modifying the cell culture media to increase the sialic acid content of the polypeptides in the cell culture media. 69. The method of aspect 68, wherein DANA, N-acetylmannosamine (ManNAc), fetuin, CuCl2, dexamethasone, galactose, manganese chloride, uridine, zinc, hydrocortisone, LongR3, or any combination thereof, is added to the cell culture media to increase sialic acid content of the polypeptides in the cell culture media. 70. The method of any one of aspects 49-69, wherein a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. 71. The method of any one of aspects 49-70, wherein a) to f) take about 2 hours. 72. The method of any one of aspects 49-71, wherein a) to f) take about 5 hours. 73. The method of any one of aspects 49-72, further comprising quantifying the titer of the polypeptides in the sample prior to a). 74. The method of aspect 73, wherein the titer of the polypeptides is quantified by: i) moving the sample comprising the polypeptides to the polypeptide-binding column, thereby to form a binding complex; ii) moving an elution buffer to and through the binding complex to elute the bound polypeptides; and iii) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer. 75. The method of aspect 74, wherein the analytical device is a UV spectrophotometer or a fluorescence spectrophotometer. 76. The method of aspects 74 or 75, wherein after determining the titer of the polypeptides, a specific amount of polypeptides in the sample is collected in a second sample. 77. The method of aspect 76, wherein the amount NANA and NGNA sialic acid residues in the second sample are quantified by performing a) to f) (“repeat steps a) to f)”). 78. The method of any one of aspects 74-77, wherein any one of i) to iii) are automated. 79. The method of aspect 78, where any one of in two or three of i) to iii) are automated. 80. The method of aspects 78 or 79, wherein all of i) to iii) are automated. 81. The method of any one of aspects 1-80, wherein the polypeptides are fusion proteins. 82. The method of any one of aspects 1-81, wherein the polypeptides are antibodies. 83. The method of any one of aspects 1-82, further comprising determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides. 84. The method of any one of aspects 1-82, further comprising harvesting the polypeptides from the cell culture media when the polypeptides contain the desired sialic acid content. 85. The method of aspect 83 or 84, wherein harvesting the polypeptides comprises: aa) moving a second sample comprising polypeptides to a polypeptide-binding column, to form a binding complex; bb) moving a wash buffer to and through the polypeptide-binding column; and cc) moving an elution buffer to and through the binding complex to elute the bound polypeptides. 86. The method of aspect 85, wherein the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 87. The method of aspect 86, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. 88. The method of aspect 87, wherein the affinity column is a protein A column. 89. The method of any one of aspects 85-88, wherein one, two, or three of aa) to cc) are automated. 90. The method of aspect 89, wherein all of aa) and cc) are automated. 91. The method of aspect 85, wherein the wash buffer is prepared based on the sialic acid content. 92. The method of aspect 91, wherein Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase sialic acid content of the polypeptides. 93. The method of aspect 91, wherein Na3PO4, NaOAc, NaCl, KCl, Na2SO4, K2SO4, or any combination thereof, is added to the wash buffer to increase amount of NANA on the polypeptides. 94. A method for measuring an amount of sialic acid in polypeptides in real time, the method comprising: a) moving a portion of a sample comprising the polypeptides to a polypeptide- binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column, to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and j) quantifying the amount N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues in the sample, wherein one or more of a) to j) are automated. 95. The method of aspect 94, wherein two, three, four, five, six, seven, eight, nine, or more of a) to j) are automated. 96. The method of any one of aspects 94-95, wherein all of a) to j) are automated. 97. A method for measuring an amount of sialic acid in polypeptides in real time, the method comprising: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; f) quantifying the amount N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues in the sample g) moving a second sample comprising polypeptides to a second polypeptide- binding column to form a second binding complex; h) moving a wash buffer to and through the polypeptide-binding column; and i) moving an elution buffer to and through the second binding complex to elute the bound polypeptides, wherein one or more of a) to i) are automated. 98. The method of aspect 97, wherein two, three, four, five, six, seven, eight, or more of a) to i) are automated. 99. The method of aspect 97 or 98, wherein all of a) to i) are automated. 100. A method for measuring an amount of sialic acid in polypeptides in real time, the method comprising: a) moving a portion of a sample comprising the polypeptides to a polypeptide- binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) moving the eluted polypeptides through an analytical device to quantify the polypeptide titer; d) quantifying the amount of polypeptides present in the sample; e) moving a specific amount of the sample to a second polypeptide-binding column to form a second binding complex; f) moving an elution buffer to and through the second binding complex to elute the bound polypeptides; g) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; h) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; i) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; j) quantifying the amount N-acetylneuraminic acid (NANA) and N- glycolylneuraminic acid (NGNA) sialic acid residues in the sample; k) moving a third sample comprising polypeptides to a third polypeptide-binding column to form a third binding complex; l) moving a wash buffer to and through the polypeptide-binding column; and m) moving an elution buffer to and through the third binding complex to elute the bound polypeptides, wherein one or more of a) to m) are automated. 101. The method of aspect 100, wherein two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more of a) to m) are automated. 102. The method of aspect 100 or 101, wherein all of a) to m) are automated. EXAMPLES Example 1: Near Real Time System for Sialic Acid Quantitation [0132] Continuous bioprocesses can increase production flexibility, overall reduction of facility footprints and increased productivity, securing reduced overall production cost. One key challenge in implementing continuous bioprocessing is the real-time or near real- time acquisition of product quality information to facilitate timely control of the process. Currently deployed batch processes are costly, inefficient, and lack manufacturing flexibility. [0133] Therefore, to quantitate sialic acids content of glycoproteins, a near real time system comprising an automated sampling system, a sequence injection system, and an analyzer was created. The near real time system is a fully automated platform, configured to achieve online sampling, inline sample preparation, and subsequent online HPLC/UPLC analysis. The near real time system can also be used for titer measurement of samples. [0134] The system includes drawing samples from a bioreactor and loading the samples on to a Protein A column and eluting the proteins off in fixed volume of elution buffer to obtain protein titer in the bioreactor. Based on the titer information generated in the first step, predetermined quantity of protein is loaded on to the Protein A column, subjected to inline Protein A purification to remove process impurities and extract the products of interest such as mAbs and fusion proteins. The purified protein is then subjected to acid hydrolysis to release the sialic acid and subsequently to submit for DMB labeling. The DMB-labeled sialic acid samples are then injected into a classic UPLC for the separate NANA from NGNA and other components of the reaction mixture. [0135] Quantitation of sialic acid is carried out by interpolating the standard curve constructed using sialic acid standards derivatized with DMB. Picomoles of NANA and NGNA per injection is calculated and from that mole/mole ratio of sialic acid to protein is determined. The protein A purification, desialylation, sialic acid labeling, and NGNA and NANA steps were optimized as provided in Examples 2-4. Example 2: Optimization of Protein-A Purification [0136] During protein purification with a protein A column, an issue was encountered with the appearance of an interfering shoulder on the chromatogram that resulted from the stroke of the syringe during the delivery of the elution buffer. This artifact impacted the accuracy of titer determination. To overcome this issue, hardware and python script modifications were applied to have the elution buffer delivered from two syringes, one from the top and one from the bottom modules of the µSIA system. Example 3: Optimization of Sialic Acid Desialylation and DMB Labeling [0137] The efficiency of both acid hydrolysis and sialidase digestion to release sialic acids from the glycoproteins were evaluated. To accommodate acid hydrolysis, hardware reconfiguration was required because the acid can impact the integrity of the column and the hydrolysis would need to be performed at 80˚C for 2 hours. To overcome this challenge, hardware configuration was made such that the column heater was relocated to perform hydrolysis while protein A purification is being done at ambient temperature. [0138] For enzymatic digestion, an Agilent kit was used to release sialic acid using Sialidase. In the method, 18μl of sample + 4μl of Sialidase A + 8μl of reaction buffer were incubated at 37ºC for 30 minutes followed by 3 hours of labeling with DMB. For acid hydrolysis using acetic acid, 90μl of sample was treated with 10μl of acetic acid, incubated at 80ºC for 2 hours followed by 3 hours of DMB labelling. After labeling, water was added to bring the total volume to 1 mL (265μL of labelled product + 735μL of water). Results show that acid hydrolysis is more efficient than sialidase digestion (FIG. 1). [0139] To reduce the hydrolysis time from a lengthy two hours to make it comparable to the 30-minute incubation time suggested for sialidase digestion, a time-course study of acid hydrolysis for 30 minutes and 2 hours at 37˚C was performed. The two hour hydrolysis is more effective than the 30 minute hydrolysis since the intensities of NANA and NGNA peaks are significantly higher for 2-hour hydrolysis (data not shown). [0140] To optimize DMB derivatization, a time course study was carried out at 2, 3 and 4 hours. For DMB labeling, 30μL of sample and 10μL of DMB were vortexed and incubated at 50ºC for 2, 3, or 4 hours. After incubation, 160µL water was added to the vial. The labeled sialic acids were quantified by HPLC. As depicted in FIG.2, the peak intensity is relatively higher for 3-hour labeling in comparison to 2 and 4 hours labeling. [0141] Next, studies were conducted to determine the optimal combination and timing of desialylation and sialic acid labeling. The following conditions were tested in combination: 2 hour sialidase digestion, 1 hour acid hydrolysis, 2 hour acid hydrolysis, 2 hour DMB labeling, 3 hour DMB labeling, and 4 hour DMB labeling. Results show that the combination of 2 hour acid hydrolysis and 3 hour DMB labeling generated the highest yield of NANA with a low yield of NGNA (Table 1). Table 1. NGNA and NANA Yields After Desialylation and Labeling Digestion RP Amide column C18 column Conditions NGNA NANA NGNA NANA (mol/mol) (mol/mol) (mol/mol) (mol/mol) 2-hr sialidase digestion 0.5 7.9 0.4 6.9 3-hr labelling 1-hr acid hydrolysis 0.5 7.4 0.4 6.4 3-hr labelling 2-hr acid hydrolysis 0.6 8.7 0.5 7.6 2-hr labelling 2-hr acid hydrolysis 0.5 7.8 0.5 7.0 2-hr labelling 2-hr acid hydrolysis 0.6 9.0 0.5 7.9 3-hr labelling 2-hr acid hydrolysis 0.6 8.5 0.5 7.5 4-hr labelling Example 4: Chromatographic Conditions to Separate NANA and NGNA [0142] An issue with online chromatography was initially encountered as the synchronization between the µSIA and the UPLC was not in action. Changing the python script to trigger µSIA or modifying the instrument settings to synchronize the UPLC injection was not successful until an external valve was mounted to the UPLC system. The external valve served as a switch between the µSIA and the UPLC system accepting the command from the event table of the instrument method. With this hardware modification the communication between µSIA and UPLC was fully established. [0143] Separation of labelled sialic acids (NANA and NGNA), was initially evaluated using Ascentis Express 90A, RP-amide columns (10cm x 2.1mm, 2.7um; 10cm x 2.1mm, 2 um) under an isocratic condition using the mobile phase 0.1% FA/10% acetonitrile for 10 minutes at a flow rate of 0.2 ml/min. Column temperature was maintained at 30˚C. Before further optimization, a dry run was performed with a NANA and NGNA standard mixture at a 1:1 ratio. [0144] Separation of NANA and NGNA was optimized by evaluating columns with different chemistries and from different manufacturers. The list of columns subjected for evaluation included Agilent C18 column (Infinity Lab Poroshell 120 EC-C18, 2.1 x 75 mm, 2.7 µm, narrow bore LC column), Supelco C-18 column and Waters X-bridge column along with Sigma/Aldrich Ascentis Express RP-Amide column (2.7 μm , 10 cm X 2.1 mm). All columns were evaluated under different gradients and column temperatures. [0145] NANA and NGNA were separated using a Ascentis RP-amide column with mobile phase 0.1% FA/10% acetonitrile under isocratic conditions for 10 minutes at a flow rate of 0.2 ml/min. Column temperature was maintained at 30˚C. Chromatographic separation of DMB-labeled NANA and NGNA is shown in FIGs.3A-3B. [0146] As a mixed mode column, Ascentis RP-amide column outperformed other columns and separation by the Ascentis RP-amide column was further optimized. To achieve better separation between NANA and NGNA, a gradient elution instead of isocratic run was carried out at 0.2 ml/minute by maintaining the column temperature at 30˚C. Mobile phases A and B are 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Initial gradient of 6% was maintained for 1 minute followed by a ramp up to 20% for 3 minutes followed by a 2-minutes isocratic run during which NANA and NGNA were eluted. The eluate was detected with fluorescence detector (Excitation and Emission wavelengths are 373nms and 448nms, respectively). As illustrated in FIGs.4A-4B, the resultant chromatogram exhibited baseline separation of NANA and NGNA. Example 5: Sialic Acid Quantitation Using a Near Real Time System [0147] Based on the results from Examples 1-4, an optimized workflow has been established (FIG.5). For online sampling from bioreactors, the µSIA system from FIA Lab was interfaced with a SegFlow autosampler to draw samples from the bioreactor and deliver the samples to the designated port of the µSIA module. Such on-line sampling technology allows rapid and accurate sampling from up to eight bioreactors or process streams and sample delivery to up to 4 analyzers and/or fraction collectors. This way, the existing off-line and at-line analytics were seamlessly integrated into a multi-functional on-line process analytical technology (PAT) tool through FIAlab’s SIAsoft software. The SIAsoft software simultaneously acquires and exports all integrated instrument data to any OPC-enabled SCADA or DCS for enhanced process monitoring and control. Custom-scripts were written to establish communication between the SegFlow autosampler and the µSIA analyzer such that sample withdrawal and subsequent workflow can be scheduled and coordinated with minimal human intervention. [0148] To quantitate sialic acid content of a reference glycoprotein, a Flownamics SegFlow 4800 autosampling system was interfaced with a µSIA device. A schematic of the µSIA system architecture is provided in FIGs.6A-6B. The autosampler drew cell-free sterile samples from the cell-free culture medium of bioreactors using a 310 nm F-series FISP probe with a 0.2mm pore size ceramic membrane (Flownamics Inc., Madison, WI) to deliver samples to the µSIA through a SegMod-SampleMod. Samples were drawn 6, 7, 8, 10, or 12 days after inoculation of the culture. [0149] For the online method, the received samples were then loaded on to a Protein A column. The glycoproteins in the sample were eluted off in a fixed volume of elution buffer to obtain the protein titer via a UV-Vis spectrometer. Based on the titer information generated in the first step, a fixed quantity of protein was loaded on to a Protein A column at neutral pH and subjected to inline Protein A purification to remove process impurities and extract the products of interest using a low pH buffer (e.g., mAbs and fusion proteins). [0150] The purified glycoproteins were then subjected to acid hydrolysis for two hours with phosphoric acid to release the sialic acids. The sialic acids were then labeled with DMB for three hours. The DMB-labeled sialic acid samples were then injected into an ACQUITY Classic UPLC to separate NANA from NGNA and other components of the reaction mixture. [0151] A representative chromatogram of DMB labeled sialic acid released from the reference glycoprotein using the optimized workflow is shown in Figure 7. The mole/mole ratio of sialic acid/protein was calculated after the full automated protocol (online) or an offline method either 6, 7, 8, 10, or 12 days after inoculation of the culture as shown below. Table 2. Quantification of Sialic Acid Offline Online Days NGNA NANA TSA NGNA NANA TSA 6 0.02 24.01 24.03 0.02 24.59 24.61 7 0.03 27.43 27.45 0.02 23.45 23.47 8 0.02 25.24 25.26 0.03 27.18 27.21 10 0.02 22.15 22.17 0.02 18.20 18.21 12 0.02 22.40 22.43 0.02 19.59 19.61

Claims

WHAT IS CLAIMED IS: 1. A method for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) moving the released sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and e) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to e) are automated. 2. The method of claim 1, wherein the released sialic acids are mixed with a sialic acid labeling agent, thereby labeling the sialic acids, prior to d). 3. A method for measuring an amount of sialic acid in polypeptides in near-real time, the method comprising steps of: a) moving a sample comprising the polypeptides to a polypeptide-binding column to form a binding complex; b) moving an elution buffer to and through the binding complex to elute the bound polypeptides; c) adding a desialylation agent to the eluted polypeptides to release sialic acids from the eluted polypeptides; d) mixing the released sialic acids with a sialic acid labeling agent, thereby labeling the sialic acids; e) moving the labeled sialic acids through an analytical device to separate the N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acid residues; and f) quantifying the amount NANA and NGNA sialic acid residues in the sample, wherein one or more of a) to f) are automated. 4. The method of any of claims 1-3, wherein two, three, four, or five or more of the steps are automated. 5. The method of any of claims 1-4, wherein all of the steps are automated. 6. The method of any of claims 1-5, wherein one, two, three, four, or five or more of the steps are performed in a closed system. 7. The method of any one of claims 1-6 , wherein all of the steps are performed in a closed system. 8. The method of any one of claims 1-7, wherein the sample is collected from the media of cultured cells. 9. The method of any one of claims 1-8, wherein the sample is collected from a bioreactor. 10. The method of any one of claims 1-9, wherein the sample is collected automatically. 11. The method of any one of claims 1-10, wherein the polypeptide binding column is an affinity column, a size exclusion column, a hydrophobic interaction column, hydrophilic interaction column, reverse phase column, or an ion exchange column. 12. The method of claim 11, wherein the affinity column is a protein A, protein G, protein L, His, GST, Myc, or FLAG column. 13. The method of any one of claims 1-12, wherein the desialylation agent is an acid or an exoglycosidase. 14. The method of claim 13, wherein the acid is phosphoric acid, sulfuric acid, acetic acid, or any combination thereof. 15. The method of claim 14, wherein the exoglycosidase is a sialidase. 16. The method of any one of claim 2-3, wherein the sialic acid labeling agent is 1,2- diamino-4,5-methylenedioxybenzene (DMB) or o-phenylenediamine. 17. The method of any one of claims 1-16, wherein the analytical device is a UPLC, uHPLC or HPLC. 18. The method of any one of claims 1-17, wherein the amount of total sialic acid per polypeptide is calculated by Equation 1;
Figure imgf000054_0001
wherein the amount of NANA per polypeptide in the sample is calculated by Equation 2;
Figure imgf000054_0002
where X.XX μg is the amount of NANA injected, and 309.27 μg is the molecular weight of NANA. and wherein the amount of NGNA per polypeptide in the sample is calculated by Equation 3 ^^ ^^ ^^ ^^ ^^ ∗ 1 ^^ ^^ ^^ ^^ ∗ 10^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^ ^^ ^^ ^^ 325.27 ^^ ^^ 1 ^^ ^^ ^^ ^^ ^^ Where XXX μg is the amount of NGNA injected, and 325.27 μg is the molecular weight of NGNA. 19. The method of any one of claims 1-18, wherein a) to f) take about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6.5 hours, about 6 hours, about 5.5 hours, about 5 hours, about 4.5 hours, about 4 hours, about 3.5 hours, about 3 hours, about 2.5 hours, about 2 hours, or about 1.5 hours. 20. The method of any one of claims 1-19, further comprising determining when to harvest polypeptides from a cell culture media based on the sialic acid content of the polypeptides.
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