Deglycosylation method for electrophoresis of glycosylated proteins

Abstract

To provide a high-speed, accurate, and highly reproducible high-throughput method with which glycoprotein can be analyzed.SOLUTION: A method for analyzing a sample including target protein includes: (a) a step of denaturing the sample; (b) a labeling step of labeling the sample with a fluorescent label to generate a labeled sample; (c) a step of quenching an unreacted fluorescent label in the labeled sample; (d) a step of deglycosylating the labeled sample with endoglycosidase; and (e) executing electrophoresis of the labeled sample. Before the deglycosylation in the step (d), the sample is denatured, labeled, and quenched in the steps (a)-(c).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 963,646, filed January 21, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present disclosure relates to the fields of biochemistry, molecular biology, and analysis of proteins via electrophoresis.

[0003] INCORPORATION BY REFERENCE The contents of the text file submitted electronically herein are incorporated herein by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: REGE-019-001WO_SeqList_ST25.txt, Recorded on: December 22, 2020, File Size: 1 KB). [Background technology]

[0004] background Capillary-based electrophoresis (CE) and microchip-based capillary electrophoresis (MCE) are common analytical methods in the pharmaceutical industry used to characterize the integrity and purity of therapeutic proteins based on protein size and provide quality control. Although standard industry-recommended sample preparation methods are effective for many proteins, heavily glycosylated proteins are problematic due to poor separation and quantification by CE and MCE. In addition, partially glycosylated and unglycosylated peaks in the MCE profile may overlap with impurity peaks and interfere with quantification. Thus, there is a need in the art for additional sample preparation methods that can overcome the challenges of working with glycosylated proteins. The present invention provides methods for labeling heavily glycosylated proteins that can be used to prepare proteins for analysis by electrophoretic methods such as CE and MCE. Summary of the Invention

[0005] overview The present disclosure relates to a method of analyzing a sample containing a protein of interest, comprising denaturing, fluorescently labeling, quenching, and deglycosylating the sample, where the denaturing, labeling, and quenching steps occur prior to deglycosylation. The method of analyzing a sample of the present disclosure can reduce or eliminate electropherogram peaks due to endoglycosidases, reduce free dye interference, and thereby provide a fast, accurate, and highly reproducible high-throughput method through which glycoproteins may be analyzed.

[0006] The present disclosure provides a method of analyzing a sample containing a protein of interest, comprising the steps of: (a) denaturing the sample; (b) labeling the sample with a fluorescent label to generate a labeled sample; (c) quenching unreacted fluorescent label in the labeled sample; (d) deglycosylating the labeled sample with an endoglycosidase; and (e) performing electrophoresis of the labeled sample, wherein the sample is denatured, labeled, and quenched in steps (a)-(c) prior to deglycosylation in step (d).

[0007] In some embodiments of the methods of the present disclosure, the protein of interest comprises at least one glycosylation site. In some embodiments, the protein of interest is a glycosylated protein. In some embodiments, the glycosylated protein comprises at least one attached glycan. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 10% of the total weight of the glycosylated protein comprises glycan (10% w / w).

[0008] In some embodiments of the methods of the present disclosure, the protein of interest comprises an antigen binding domain. In some embodiments, the protein of interest comprises an antibody, an antibody fragment, or an scFv. In some embodiments, the protein of interest comprises an Fc domain. In some embodiments, the protein of interest comprises a receptor fusion protein. In some embodiments, the receptor fusion protein is a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein. In some embodiments, the receptor fusion protein is a trap protein or a mini-trap protein. In some embodiments, the protein of interest is a trap protein or a mini-trap protein. In some embodiments, the protein of interest is a recombinant human protein.

[0009] In some embodiments of the disclosed methods, the glycosylation site comprises an Asn-X-Ser / Thr consensus sequence. In some embodiments, at least one attached glycan is N-linked. In some embodiments, at least one attached glycan is N-linked to an asparagine in the glycosylated protein. In some embodiments, the endoglycosidase catalyzes the deglycosylation of the N-linked glycan. In some embodiments, the endoglycosidase is selected from the group consisting of Peptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2, and Endoglycosidase F4. In some embodiments, the endoglycosidase is PNGase F. In some embodiments, the PNGase F is Rapid PNGase F. In some embodiments, the Rapid PNGase F is non-reducing. In some embodiments, the PNGase F is reducing.

[0010] In some embodiments of the disclosed methods, deglycosylating the sample comprises heating the sample to about 35° C. for 30 minutes. In some embodiments, deglycosylating the sample comprises heating the sample to about 50° C. for 10-30 minutes. In some embodiments, deglycosylating the sample comprises heating the sample to about 50° C. for 10 minutes. In some embodiments, deglycosylating the sample comprises a reaction mixture comprising 0.2-1.5 mg of the labeled protein of interest and 1-5 μL of Rapid PNGase F in a reaction volume of 10 μL excluding the volume of Rapid PNGase F. In some embodiments, the reaction mixture comprises 0.2 mg of the labeled protein of interest. In some embodiments, the reaction mixture comprises 5 μL of Rapid PNGase F. In some embodiments, the reaction mixture comprises a buffer.

[0011] In some embodiments of the methods of the present disclosure, at least one glycan is an O-linked glycan. In some embodiments, the endoglycosidase catalyzes the deglycosylation of the O-linked glycan. In some embodiments, the endoglycosidase comprises an endo-α-N-acetylgalactosaminidase (O-glycosidase).

[0012] In some embodiments of the methods of the present disclosure, the step of labeling the sample with a fluorescent label comprises heating the sample to about 35° C. for 10-30 minutes. In some embodiments, the step of labeling the sample with a fluorescent label comprises heating the sample to about 35° C. for 15 minutes.

[0013] In some embodiments of the disclosed methods, the sample is denatured using a reducing solution. In some embodiments, the reducing solution comprises dithiothreitol (DTT). In some embodiments, the sample is denatured using a non-reducing solution. In some embodiments, the non-reducing solution comprises iodoacetamide (IAM). In some embodiments, denaturing the sample comprises heating the sample to between 40° C. and 99° C. for 1 minute to 5 hours. In some embodiments, denaturing the sample comprises heating the sample to between 50° C. and 99° C. for 1 minute to 60 minutes.

[0014] In some embodiments of the disclosed methods, the step of quenching unreacted fluorescent label comprises adding a stop solution.

[0015] In some embodiments of the methods of the present disclosure, the method further comprises analyzing a reference standard simultaneously with the sample.

[0016] In some embodiments of the methods of the present disclosure, the electrophoresis is selected from the group consisting of gel electrophoresis, isoelectric focusing, capillary electrophoresis (CE), or microchip capillary electrophoresis (MCE). In some embodiments, the electrophoresis is MCE. In some embodiments, the MCE is performed using an MCE instrument.

[0017] In some embodiments of the disclosed method, the method results in a reduction of free dye interference in the range of less than 20 kDa and a reduction or absence of endoglycosidase peaks in the electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation. In some embodiments, the endoglycosidase peak is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when compared to an electropherogram generated using a sample labeled after deglycosylation. In some embodiments, the endoglycosidase peak is absent in the electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation.

[0018] The present disclosure provides a method for determining stability of a protein of interest, comprising the steps of: (a) applying stress to a sample comprising the protein of interest; (b) denaturing the stressed sample and an unstressed sample comprising the protein of interest; (c) labeling the stressed sample and the unstressed sample with a fluorescent label to generate labeled stressed and labeled unstressed samples; (d) quenching unreacted fluorescent label in the labeled stressed and labeled unstressed samples; and (e) deglycosylating the labeled stressed and labeled unstressed samples with an endoglycosidase. (f) performing microchip capillary electrophoresis (MCE) of the labeled stressed sample and the labeled unstressed sample to generate electropherograms of the stressed and unstressed samples; and (g) comparing the electropherograms from the stressed and unstressed samples, thereby determining the stability of the protein of interest, wherein the stressed and unstressed samples are denatured, labeled and quenched in steps (b)-(d) prior to deglycosylation in step (e).

[0019] In some embodiments of the disclosed methods, the step of stressing the sample comprises thermally stressing the sample, hi some embodiments, thermally stressing the sample comprises holding the sample at about 30° C. to about 45° C. for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks.

[0020] In some embodiments of the disclosed methods, the step of subjecting the sample to stress comprises at least one freeze / thaw cycle.

[0021] In some embodiments of the disclosed methods, the step of subjecting the sample to stress comprises exposing the sample to storage conditions. In some embodiments, the storage conditions comprise a temperature of about -80°C to -30°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. In some embodiments, the storage conditions comprise a temperature of about 2°C to 8°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, or at least 18 months.

[0022] In some embodiments of the disclosed methods, the step of applying a stress to the sample comprises mechanically agitating the sample.

[0023] In some embodiments of the disclosed methods, the step of subjecting the sample to stress comprises freeze-drying and rehydrating the sample.

[0024] Stressing the sample can include exposing the sample to light, radiation, singlet oxygen species, free radicals, high pH conditions, or low pH conditions.

[0025] In some embodiments of the disclosed methods, the protein of interest comprises at least one glycosylation site. In some embodiments, the protein of interest is a glycosylated protein. In some embodiments, the glycosylated protein comprises at least one attached glycan. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 10% of the total weight of the glycosylated protein comprises glycan (10% w / w). In some embodiments, at least 10% of the total weight of the glycosylated protein comprises glycan (10% w / w).

[0026] In some embodiments of the methods of the present disclosure, the protein of interest comprises an antigen binding domain. In some embodiments, the protein of interest comprises an antibody, an antibody fragment, or an scFv. In some embodiments, the protein of interest comprises an Fc domain. In some embodiments, the protein of interest comprises a receptor fusion protein. In some embodiments, the receptor fusion protein is a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein. In some embodiments, the receptor fusion protein is a trap protein or a mini-trap protein. In some embodiments, the protein of interest is a trap protein or a mini-trap protein. In some embodiments, the protein of interest is a recombinant human protein.

[0027] In some embodiments of the disclosed methods, the glycosylation site comprises an Asn-X-Ser / Thr consensus sequence. In some embodiments, at least one attached glycan is N-linked. In some embodiments, at least one attached glycan is N-linked to an asparagine in the glycosylated protein. In some embodiments, the endoglycosidase catalyzes the deglycosylation of the N-linked glycan. In some embodiments, the endoglycosidase is selected from the group consisting of Peptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2, and Endoglycosidase F4. In some embodiments, the endoglycosidase is PNGase F. In some embodiments, the PNGase F is Rapid PNGase F. In some embodiments, the Rapid PNGase F is non-reducing. In some embodiments, the PNGase F is reducing.

[0028] In some embodiments of the disclosed method, deglycosylating the stressed and unstressed samples comprises heating the samples to about 35° C. for 30 minutes. In some embodiments, deglycosylating the stressed and unstressed samples comprises heating the samples to about 50° C. for 10-30 minutes. In some embodiments, deglycosylating the stressed and unstressed samples comprises heating the samples to about 50° C. for 10 minutes. In some embodiments, deglycosylating the stressed and unstressed samples comprises a reaction mixture comprising 0.2-1.5 mg of the labeled protein of interest and 1-5 μL of Rapid PNGase F in a reaction volume of 10 μL excluding the volume of Rapid PNGase F. In some embodiments, the reaction mixture for each of the stressed and unstressed samples comprises 5 μL of Rapid PNGase F. In some embodiments, each of the stressed and unstressed samples comprises 0.2 mg of the labeled protein of interest. In some embodiments, the reaction mixture for each of the stressed and non-stressed samples comprises a buffer.

[0029] In some embodiments of the methods of the present disclosure, at least one glycan is an O-linked glycan. In some embodiments, the endoglycosidase catalyzes the deglycosylation of the O-linked glycan. In some embodiments, the endoglycosidase comprises an endo-α-N-acetylgalactosaminidase (O-glycosidase).

[0030] In some embodiments of the disclosed methods, the step of labeling the stressed and unstressed samples with a fluorescent label comprises heating each sample to about 35° C. for 30 minutes.

[0031] In some embodiments of the disclosed methods, the stressed and unstressed samples are denatured using a reducing solution. In some embodiments, the reducing solution comprises dithiothreitol (DTT). In some embodiments, the stressed and unstressed samples are denatured using a non-reducing solution. In some embodiments, the non-reducing solution comprises iodoacetamide (IAM). In some embodiments, the step of denaturing the stressed and unstressed samples comprises heating the samples to between 40° C. and 99° C. for 1 minute to 5 hours. In some embodiments, the step of denaturing the stressed and unstressed samples comprises heating the samples to between 50° C. and 99° C. for 1 minute to 60 minutes.

[0032] In some embodiments of the disclosed methods, the step of quenching unreacted fluorescent label comprises adding a stop solution.

[0033] In some embodiments of the methods of the present disclosure, the method further comprises analyzing a reference standard simultaneously with the stressed and unstressed samples, in some embodiments, comparing the electropherograms of the stressed and unstressed samples comprises comparing peak number, height, location, area, or a combination thereof. [The present invention 1001] 1. A method for analyzing a sample containing a protein of interest, comprising the steps of: The method further comprising: (a) denaturing the sample; (b) labeling the sample with a fluorescent label to produce a labeled sample; (c) quenching any unreacted fluorescent label in the labeled sample; and (d) deglycosylating the labeled sample with an endoglycosidase; (e) performing electrophoresis of the labeled sample; Including, The sample is denatured, labeled and quenched in steps (a)-(c) prior to deglycosylation in step (d); method. [The present invention 1002] The method of claim 10, wherein said protein of interest comprises at least one glycosylation site. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein said protein of interest is a glycosylated protein. [The present invention 1004] The method of claim 1003, wherein said glycosylated protein comprises at least one attached glycan. [The present invention 1005] The method according to any one of claims 1001 to 1004, wherein the target protein comprises an antigen-binding domain. [The present invention 1006] The method of claim 1005, wherein said protein of interest comprises an antibody, an antibody fragment, or an scFv. [The present invention 1007] The method according to any one of claims 1001 to 1006, wherein the target protein comprises an Fc domain. [The present invention 1008] The method according to any one of claims 1001 to 1007, wherein the target protein comprises a receptor fusion protein. [The present invention 1009] The method of claim 1008, wherein said receptor fusion protein is a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein. [The present invention 1010] 1009. The method of claim 1008 or 1009, wherein said receptor fusion protein is a trap protein or a mini-trap protein. [The present invention 1011] The method according to any one of claims 1001 to 1010, wherein the target protein is a recombinant human protein. [The present invention 1012] The method of any of claims 1002 to 1011, wherein the glycosylation site comprises an Asn-X-Ser / Thr consensus sequence. [The present invention 1013] The method of any of claims 1004 to 1012, wherein said at least one attached glycan is N-linked. [The present invention 1014] The method of claim 1013, wherein said at least one attached glycan is N-linked to an asparagine in said glycosylated protein. [The present invention 1015] The method of any of claims 1001 to 1014, wherein the endoglycosidase catalyzes deglycosylation of an N-linked glycan. [The present invention 1016] Any of the methods of the present invention 1001 to 1015, wherein the endoglycosidase is selected from the group consisting of peptide-N-glycosidase F (PNGase F), endoglycosidase H (Endo H), endoglycosidase S (Endo S), endoglycosidase D, endoglycosidase F1, endoglycosidase F2, and endoglycosidase F4. [The present invention 1017] The method according to any one of claims 1001 to 1015, wherein the endoglycosidase is PNGase F. [The present invention 1018] The method of the present invention, wherein the PNGase F is Rapid PNGase F. [The present invention 1019] The method of any one of claims 1 to 8, wherein the Rapid PNGase F is non-reducing. [The present invention 1020] 1019. The method of any one of claims 1017 to 1019, wherein the step of deglycosylating the sample comprises heating the sample to about 50°C for 10 minutes. [The present invention 1021] Deglycosylating the sample comprises: A reaction mixture containing 0.2-1.5 mg of labeled protein of interest and 1-5 μL of Rapid PNGase F in a reaction volume of 10 μL excluding the volume of Rapid PNGase F. Any of the methods of the present invention 1001 to 1020, comprising: [The present invention 1022] The method of any one of claims 1 to 21, wherein said reaction mixture comprises 0.2 mg of the labeled protein of interest. [The present invention 1023] 1021. The method of claim 1021, wherein the reaction mixture comprises 5 μL of Rapid PNGase F. [The present invention 1024] The method of any one of claims 1021 to 1023, wherein the reaction mixture comprises a buffer solution. [The present invention 1025] The method of any one of claims 1004 to 1011, wherein the at least one glycan is an O-linked glycan. [The present invention 1026] The method of claim 1025, wherein said endoglycosidase catalyzes the deglycosylation of an O-linked glycan. [The present invention 1027] The method of any one of claims 1025 to 1026, wherein the endoglycosidase comprises endo-α-N-acetylgalactosaminidase (O-glycosidase). [The present invention 1028] Any of the methods according to claims 1001 to 1027, wherein the step of labelling the sample with a fluorescent label comprises heating the sample to about 35°C for 10 to 30 minutes. [The present invention 1029] The method of any one of claims 1001 to 1027, wherein the step of labeling the sample with a fluorescent label comprises heating the sample to about 35°C for about 15 minutes. [The present invention 1030] The method of any of claims 1001 to 1029, wherein said sample is denatured using a reducing solution. [The present invention 1031] The method of claim 1030, wherein the reducing solution comprises dithiothreitol (DTT). [The present invention 1032] The method of any of claims 1001 to 1029, wherein said sample is denatured using a non-reducing solution. [The present invention 1033] The method of claim 1032, wherein the non-reducing solution comprises iodoacetamide (IAM). [The present invention 1034] The method of any one of claims 1001 to 1033, wherein the step of denaturing the sample comprises heating the sample to between 40°C and 99°C for 1 minute to 5 hours. [The present invention 1035] Any of the methods of claims 1001 to 1033, wherein the step of denaturing the sample comprises heating the sample to between 50°C and 99°C for 1 to 60 minutes. [The present invention 1036] The method of any one of claims 1001 to 1035, wherein the step of quenching unreacted fluorescent label comprises adding a stop solution. [The present invention 1037] The method of any one of claims 1001 to 1036, further comprising the step of analyzing a reference standard simultaneously with said sample. [The present invention 1038] The method according to any one of claims 1001 to 1037, wherein said electrophoresis is selected from the group consisting of gel electrophoresis, isoelectric focusing, capillary electrophoresis (CE), or microchip capillary electrophoresis (MCE). [The present invention 1039] The method according to any one of claims 1001 to 1037, wherein the electrophoresis is MCE. [The present invention 1040] The method of the present invention 1039, wherein the MCE is performed using an MCE device. [The present invention 1041] Any of the methods of claims 1001 to 1040, which results in reduced free dye interference in the range below 20 kDa and reduced or absent endoglycosidase peaks in the electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation. [The present invention 1042] The method of the present invention 1041, wherein the endoglycosidase peak is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when compared to an electropherogram generated using a sample labeled after deglycosylation. [The present invention 1043] The method of claim 1041, wherein said endoglycosidase peak is absent from the electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation. [The present invention 1044] 1. A method for determining the stability of a protein of interest, comprising: The method further comprising: (a) applying a stress to a sample containing the protein of interest; (b) denaturing the stressed sample and a non-stressed sample containing the protein of interest; (c) labeling the stressed sample and the unstressed sample with a fluorescent label to generate labeled stressed sample and labeled unstressed sample; (d) quenching unreacted fluorescent labels in the labeled stressed sample and the labeled unstressed sample; (e) deglycosylating the labeled stressed sample and the labeled unstressed sample with an endoglycosidase; (f) performing microchip capillary electrophoresis (MCE) of the labeled stressed sample and the labeled unstressed sample to generate electropherograms of the stressed sample and the unstressed sample; (g) comparing the electropherograms from the stressed and unstressed samples, thereby determining the stability of the protein of interest. Including, The stressed and unstressed samples are denatured, labeled and quenched in steps (b) to (d) prior to deglycosylation in step (e); method. [The present invention 1045] The method of any one of claims 10 to 15, wherein the step of stressing the specimen comprises thermally stressing the specimen. [The present invention 1046] The method of any one of claims 1 to 8, wherein thermally stressing the sample comprises maintaining the sample at about 30°C to about 45°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks. [The present invention 1047] The method of any one of claims 10 to 15, wherein the step of subjecting the sample to stress comprises at least one freeze / thaw cycle. [The present invention 1048] The method of any one of claims 10 to 15, wherein the step of subjecting the sample to stress comprises exposing the sample to storage conditions. [The present invention 1049] The method of claim 1048, wherein the storage conditions include a temperature of about -80°C to -30°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. [The present invention 1050] The method of claim 1048, wherein the storage conditions include a temperature of about 2°C to 8°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, or at least 18 months. [The present invention 1051] The method of any one of claims 10 to 15, wherein the step of applying stress to the sample comprises mechanically agitating the sample. [The present invention 1052] The method of any one of claims 10 to 15, wherein the step of subjecting the sample to stress comprises freeze-drying and rehydrating the sample. [The present invention 1053] The method of any one of claims 10 to 15, wherein the step of subjecting the sample to stress comprises exposing the sample to light, radiation, singlet oxygen species, free radicals, high pH conditions, or low pH conditions. [The present invention 1054] The method of any of claims 1044 to 1053, wherein said protein of interest comprises at least one glycosylation site. [The present invention 1055] The method according to any one of claims 1044 to 1053, wherein the target protein is a glycosylated protein. [The present invention 1056] The method of claim 1055, wherein said glycosylated protein comprises at least one attached glycan. [The present invention 1057] The method according to any one of claims 1044 to 1056, wherein the target protein comprises an antigen-binding domain. [The present invention 1058] The method of claim 1057, wherein said protein of interest comprises an antibody, an antibody fragment, or an scFv. [The present invention 1059] The method according to any one of claims 1044 to 1058, wherein the target protein comprises an Fc domain. [The present invention 1060] The method of any one of claims 1044 to 1059, wherein the target protein comprises a receptor fusion protein. [The present invention 1061] The method of claim 1060, wherein said receptor fusion protein is a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein. [The present invention 1062] The method of any one of claims 1060 to 1061, wherein said receptor fusion protein is a trap protein or a mini-trap protein. [The present invention 1063] The method according to any one of claims 1044 to 1062, wherein the target protein is a recombinant human protein. [The present invention 1064] The method of any of claims 1054 to 1063, wherein the glycosylation site comprises an Asn-X-Ser / Thr consensus sequence. [The present invention 1065] The method of any one of claims 1056 to 1064, wherein said at least one attached glycan is N-linked. [The present invention 1066] The method of claim 1065, wherein said at least one attached glycan is N-linked to an asparagine in said glycosylated protein. [The present invention 1067] The method of any of claims 1044 to 1066, wherein the endoglycosidase catalyzes the deglycosylation of an N-linked glycan. [The present invention 1068] Any of the methods of claims 1044 to 1067, wherein the endoglycosidase is selected from the group consisting of peptide-N-glycosidase F (PNGase F), endoglycosidase H (Endo H), endoglycosidase S (Endo S), endoglycosidase D, endoglycosidase F1, endoglycosidase F2, and endoglycosidase F4. [The present invention 1069] The method according to any one of claims 1044 to 1067, wherein the endoglycosidase is PNGase F. [The present invention 1070] 1069. The method of the present invention, wherein the PNGase F is Rapid PNGase F. [The present invention 1071] The method of claim 1070, wherein the Rapid PNGase F is non-reducing. [The present invention 1072] 10. The method of any one of claims 1044 to 1071, wherein the step of deglycosylating the stressed and unstressed samples comprises heating the samples to about 50°C for 10 minutes. [The present invention 1073] Deglycosylating the stressed and non-stressed samples comprises: A reaction mixture for each sample containing 0.2-1.5 mg of labeled protein of interest and 1-5 μL of Rapid PNGase F in a reaction volume of 10 μL excluding the volume of Rapid PNGase F. Any of the methods of claims 1044 to 1072. [The present invention 1074] 1073. The method of claim 1073, wherein the reaction mixture for each of the stressed and unstressed samples comprises 5 μL of Rapid PNGase F. [The present invention 1075] 1075. The method of claim 1073 or 1074, wherein each of said stressed sample and said unstressed sample comprises 0.2 mg of the labeled protein of interest. [The present invention 1076] The method according to any one of claims 1073 to 1075, wherein the reaction mixture for each of the stressed sample and the non-stressed sample comprises a buffer solution. [The present invention 1077] The method of any one of claims 1044 to 1063, wherein the at least one glycan is an O-linked glycan. [The present invention 1078] The method of claim 1077, wherein said endoglycosidase catalyzes the deglycosylation of an O-linked glycan. [The present invention 1079] The method of any one of claims 1077 to 1078, wherein the endoglycosidase comprises endo-α-N-acetylgalactosaminidase (O-glycosidase). [The present invention 1080] Any of the methods of claims 1044 to 1079, wherein the step of labeling the stressed sample and the unstressed sample with a fluorescent label comprises heating each sample to about 35°C for 10 to 30 minutes. [The present invention 1081] Any of the methods of claims 1044 to 1079, wherein the step of labeling the stressed sample and the unstressed sample with a fluorescent label comprises heating each sample to about 35°C for about 15 minutes. [The present invention 1082] The method according to any of claims 1044 to 1081, wherein said stressed sample and said non-stressed sample are denatured using a reducing solution. [The present invention 1083] The method of claim 1082, wherein the reducing solution contains dithiothreitol (DTT). [The present invention 1084] The method according to any of claims 1044 to 1081, wherein said stressed sample and said non-stressed sample are denatured using a non-reducing solution. [The present invention 1085] The method of claim 1084, wherein the non-reducing solution comprises iodoacetamide (IAM). [The present invention 1086] Any of the methods of claims 1044 to 1085, wherein the step of denaturing the stressed sample and the unstressed sample comprises heating the samples to between 40°C and 99°C for 1 minute to 5 hours. [The present invention 1087] Any of the methods of claims 1044 to 1085, wherein the step of denaturing the stressed sample and the unstressed sample comprises heating the samples to between 50°C and 99°C for 1 to 60 minutes. [The present invention 1088] The method of any one of claims 1044 to 1087, wherein the step of quenching unreacted fluorescent label comprises adding a stop solution. [The present invention 1089] The method of any of claims 1044 to 1088, further comprising the step of analyzing a reference standard simultaneously with said stressed sample and said unstressed sample. [The present invention 1090] Any of the methods of claims 1044 to 1089, wherein the step of comparing the electropherograms of the stressed sample and the unstressed sample comprises comparing peak number, height, position, area, or a combination thereof. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows protocols for method A without deglycosylation, method B with deglycosylation before labeling, and method C with deglycosylation after labeling. NR: non-reduced, R: reduced, MC: microchip capillary electrophoresis. [Diagram 2] Electropherograms generated using protein 1 under non-reducing conditions using a protocol without deglycosylation (Method A, shown in red) and with deglycosylation prior to protein labeling (Method B, shown in blue). Numeric peak labels indicate the molecular weight of the protein as measured by microchip capillary electrophoresis (MCE). Rapid PNGase F (PNGase) peaks appear in the electropherograms where indicated. [Diagram 3] Three electropherograms generated with protein 1 under non-reducing conditions using method B (deglycosylation before labeling) are shown. Protein 1 samples were treated with heat stress prior to analysis at 37 °C for no time (0, red, top), 2 weeks (blue, middle), or 4 weeks (black, bottom). The low molecular weight 1 peak (LMW1) peak increased with stress and merged with the PNGase peak. [Figure 4]Electropherograms generated using protein 1 under reducing conditions using a protocol without deglycosylation (Method A, shown in red) and with deglycosylation prior to protein labeling (Method B, shown in blue). Numeric peak labels indicate the molecular weight of the protein as measured by MCE. PNGase F peaks appear in the electropherogram of Method B as shown. Grey shaded boxes indicate free dye peaks. [Diagram 5] A pair of electropherograms generated with Protein 1, where Protein 1 was deglycosylated after labeling (Method C). Top: Deglycosylation reactions were carried out with 1 μL Rapid™ PNGase at 50° C. for 10, 15, 20, and 30 minutes. Bottom: Deglycosylation reactions were carried out with 2 μL Rapid™ PNGase at 50° C. for 10, 15, 20, and 30 minutes. [Figure 6] Electropherograms generated using Method C and protein 1 showing the results of deglycosylation with 1, 2, 3, or 4 μL of Rapid™ PNGase F in reactions held at 50° C. for 10 min. The inset shows the incompletely deglycosylated peak of protein 1 (right shoulder of the main peak) with an arrow indicating the reduction of glycosylated protein with increasing amounts of Rapid™ PNGase. The free dye peak is indicated by the grey shaded box. MP: main peak, LMW1: low molecular weight 1 peak. [Figure 7] A series of four electropherograms generated using Method C (deglycosylation after labeling) and protein 1 deglycosylated with 1, 2, 3, or 4 μL of Rapid™ PNGase (top to bottom). Low molecular weight (LMW) peaks 1-5, major peak (MP), and high molecular weight peak (HMW) are indicated. [Figure 8]Electropherograms generated from method C (deglycosylation after labeling) using protein 1 that was thermally stressed by holding the protein at 37° C. for 4 weeks (37C 4w, black) and protein 1 that was not stressed (t=0, red). Deglycosylation was performed using Rapid™ PNGase F. [Figure 9] Electropherograms generated using Protein 2 comparing Protein 2 labeled with deglycosylation (Method C) and without deglycosylation (Method A) under non-reducing conditions. PNGase F has the expected size of 37 KDa and this peak is absent. The free dye peak is indicated by the shaded box. [Figure 10] Electropherograms generated using protein 3 comparing protein 3 treated with deglycosylation after labeling (blue, method C) and protein 3 without deglycosylation (red, method A) under non-reducing conditions. Numeric peak labels indicate the molecular weights of proteins and protein fragments as measured by MCE. [Figure 11] Electropherograms comparing protein 3 treated with deglycosylation after labeling (blue, method C) and protein 3 without deglycosylation (red, method A) under non-reducing conditions. Numerical peak labels indicate the molecular weights of proteins and protein fragments as determined by MCE. [Figure 12] Electropherograms comparing protein 4 without deglycosylation (Method A, red) and deglycosylated after labeling (Method C, blue). Protein 4 was denatured using non-reducing (NR) conditions. Numeric peak labels indicate molecular weights measured by MCE. LMW: low molecular weight, DGMP: major deglycosylated peak, GMP: major glycosylated peak. Free dye peaks are indicated by shaded boxes. [Figure 13]Electropherograms comparing protein 4 labeled without deglycosylation (Method A, red) and deglycosylated after labeling (Method C, blue). Protein 4 was denatured using reducing (R) conditions. LC: light chain, DHC: deglycosylated heavy chain, GHC: glycosylated heavy chain. Free dye peaks are indicated by shaded boxes. [Figure 14] Three electropherograms are shown assaying the effect of light stress on protein stability produced under non-reducing conditions using Method C and Protein 1. Protein 1 was light stressed under bluish white (CW) fluorescent lamp light with 1.2 million lux hours (MLH) cumulative exposure (blue, middle) and 2.4 MLH cumulative exposure (black, bottom) and compared to unstressed Protein 1 (red, top). Deglycosylation was performed using Rapid™ PNGase F. LMW: low molecular weight, MP: major peak, HMW: high molecular weight. [Figure 15] Three electropherograms are shown assaying the effect of light stress on protein stability produced under non-reducing conditions using Method C and Protein 1. Protein 1 was light stressed under integrated near ultraviolet (UVA) energy of 200 watt-hours / meter squared (blue, middle) and 400 watt-hours / meter squared (black, bottom) and compared to unstressed Protein 1 (red, top). Deglycosylation was performed using Rapid™ PNGase F. LMW: low molecular weight, MP: major peak, HMW: high molecular weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description The present disclosure provides a new method for preparing a sample containing a protein of interest for analysis via electrophoresis. In the methods provided herein, the protein of interest is denatured, followed by covalent labeling of the protein with a fluorescent dye, followed by quenching the labeling reaction. After labeling, the labeled protein is contacted with an enzyme, such as an endoglycosidase, to remove glycans from the protein of interest without further purification. Unlike previous methods of preparing glycosylated proteins for electrophoresis, which deglycosylate the protein before labeling, the methods described herein allow for clear separation of protein and peptide species based on mass. These methods also eliminate interference from enzymes used in deglycosylation and free dye from the labeling reaction within the microchip electrophoresis (MCE) electropherogram. The methods are fast, highly reproducible, high-throughput, and have been successfully used to analyze glycosylated proteins. Without wishing to be bound by theory, it is believed that the methods described herein are advantageous for heavily glycosylated proteins, since heavy glycosylation interferes with protein migration in MCE or capillary electrophoresis (CE) analysis platforms, resulting in inaccurate measurement of protein molecular weight and imprecise electropherogram peaks. The methods described herein can be used in platform approaches that are applicable to any glycosylated protein analyzed by methods such as CE and MCE, and to characterize proteins or for quality control purposes. For example, the methods described herein can be used to measure the stability of a protein of interest when subjected to various conditions, such as long holding times at various temperatures or different formulations.

[0036] Thus, the present disclosure provides a method of preparing a sample containing a protein of interest for analysis using electrophoresis, comprising the steps of (a) denaturing a sample, (b) labeling the sample with a fluorescent label to produce a labeled sample, (c) quenching unreacted fluorescent label in the labeled sample, (d) deglycosylating the labeled sample with an endoglycosidase, and (e) performing electrophoresis of the labeled sample, wherein the sample is labeled and quenched in steps (b) and (c) prior to deglycosylation in step (d). In some embodiments, the electrophoresis is microchip capillary electrophoresis (MCE) and the output is an electropherogram.

[0037] definition Unless otherwise indicated herein or clearly contradicted by context, use of the terms "a," "an," "the," and similar referents in the context of describing the invention claimed herein (particularly in the context of the claims) are intended to be construed to cover both the singular and the plural.

[0038] Unless otherwise indicated herein, the recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within that range, and each separate value is incorporated within this specification as if it were individually recited herein.

[0039] Use of the term "about" is intended to describe values ​​that are either above or below within approximately ±10% of the stated value. In other embodiments, values ​​may range in values ​​that are either above or below within approximately ±5% of the stated value. In other embodiments, values ​​may range in values ​​that are either above or below within approximately ±2% of the stated value. In other embodiments, values ​​may range in values ​​that are either above or below within approximately ±1% of the stated value. The preceding ranges are intended to be clear in context, and no further limitation is implied.

[0040] As used herein, "protein" refers to a molecule that comprises two or more amino acid residues joined together by peptide bonds. Proteins include polypeptides and peptides, and may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs, and proteins include enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins, among others. Proteins are produced by various types of recombinant cells using well-known cell culture methods, and are generally introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intronless sequences, etc.), where they may exist as episomes or be integrated into the cell genome.

[0041] Unless otherwise indicated herein or clearly contradicted by context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0042] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0043] Denaturation The present disclosure provides methods for denaturing a protein of interest in a sample. Denaturing a protein involves the disruption of secondary and tertiary protein structure under conditions insufficient to break peptide bonds, leaving the primary structure intact.

[0044] Methods for denaturing a protein of interest under both reducing and non-reducing conditions are within the scope of this disclosure.

[0045] Protein reducing agents are agents that break disulfide bonds. These disulfide bonds can be within a single polypeptide or between multiple subunits of a protein that are encoded on separate polypeptides. Breaking the disulfide bonds between subunits allows the analysis of individual subunits of a multi-subunit protein that are analyzed separately. Reducing agents will be known to those skilled in the art. Exemplary reducing agents include, but are not limited to, dithiothreitol (DTT, CAS 3483-12-3), beta-mercaptoethanol (BME, 2BME, 2-ME, b-mer, CAS 60-24-2), 2-aminoethanethiol (2-MEA-HCl, also called cystamine-HCl, CAS 156-57-0), tris(2-carboxyethyl)phosphine hydrochloride, (TCEP, CAS 5961-85-3), cysteine ​​hydrochloride (Cys-HCl, CAS 52-89-1), or 2-mercaptoethanesulfonic acid sodium salt (MESNA). Other methods for reducing protein bonds are known in the art, such as immobilized reducing agent columns, which contain a resin on which a thiol-based reducing agent is immobilized to allow for solid-phase reduction of peptide and protein disulfide bonds. Reducing agents, including oxidizing agents, that are suitable for reducing chemical interactions between polypeptides are also contemplated.

[0046] In some embodiments, the protein of interest is denatured using a reducing solution. In some embodiments, the reducing solution contains 135-155 mM dithiothreitol (DTT). In some embodiments, the reducing solution further contains sodium phosphate and lithium dodecyl sulfate. In some embodiments, the reducing solution contains or consists essentially of 0.69% lithium dodecyl sulfate (LDS), 69 mM sodium phosphate, and 142 mM dithiothreitol. In some embodiments, the reducing solution contains 40-120 mM DTT, 40-80 mM sodium phosphate, and 0.5%-2.0% LDS. In some embodiments, the reducing solution contains 60-100 mM DTT, 50-70 mM sodium phosphate, and 0.75%-1.5% LDS. In some embodiments, the reducing solution contains about 80 mM DTT, about 60 mM sodium phosphate, and about 1.2% LDS. In some embodiments, the reducing solution is added to a sample containing the protein of interest in a ratio of about 1:4 by volume.

[0047] In some embodiments, the protein of interest is denatured using a non-reducing solution, i.e., under conditions that preserve disulfide bonds in the protein of interest. In some embodiments, the non-reducing solution comprises iodoacetamide (IAM). In some embodiments, the non-reducing solution comprises 100-200 mM iodoacetamide. In some embodiments, the non-reducing solution further comprises sodium phosphate and lithium dodecyl sulfate (LDS). In some embodiments, the non-reducing solution comprises 166 mM iodoacetamide (IAM). In some embodiments, the non-reducing solution comprises or consists essentially of 166 mM iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate. In some embodiments, the non-reducing solution comprises 100-300 mM iodoacetamide, 40-80 mM sodium phosphate, and 0.5%-2.0% LDS. In some embodiments, the non-reducing solution comprises 150-250 mM iodoacetamide, 50-70 mM sodium phosphate, and 0.75%-1.5% LDS. In some embodiments, the non-reducing solution comprises about 200 mM iodoacetamide, about 60 mM sodium phosphate, and about 1.2% LDS. In some embodiments, the non-reducing solution is added to a sample containing the protein of interest in a ratio of about 1:4 by volume.

[0048] In some embodiments, denaturing the sample includes adding a reducing or non-reducing solution to the sample and heating the combined sample and reducing or non-reducing solution. In some embodiments, the sample is denatured by heat. For example, the combined sample and reducing or non-reducing solution can be heated to between 30°C and 99°C, between 30°C and 90°C, between 30°C and 80°C, between 30°C and 70°C, between 30°C and 60°C, between 30°C and 50°C, between 30°C and 40°C, between 40°C and 99°C, between 40°C and 90°C, between 40°C and 80°C, between 40°C and 70°C, between 40°C and 60°C, between 40°C and 50°C, between 50°C and 99°C, between 50°C and 90°C, between 50°C and 80°C, between 50°C and 70°C, or between 50°C and 60°C. In some embodiments, the combined sample and reducing or non-reducing solution may be heated for 1 minute to 12 hours, 1 minute to 10 hours, 1 minute to 5 hours, 1 minute to 4 hours, 1 minute to 3 hours, 1 minute to 2 hours, 1 minute to 60 minutes, 1 minute to 30 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 5 minutes to 60 minutes, 5 minutes to 30 minutes, 5 minutes to 15 minutes, 5 minutes to 10 minutes, 10 minutes to 60 minutes, 10 minutes to 45 minutes, 10 minutes to 30 minutes, or 10 minutes to 15 minutes. In some embodiments, the combined sample and reducing or non-reducing solution may be heated to between 40°C and 99°C for 1 minute to 60 minutes. In some embodiments, the combined sample and reducing or non-reducing solution may be heated to between 50°C and 99°C for 1 minute to 60 minutes. As a further example, the combined sample and reducing or non-reducing solution may be heated to between 60° C. and 85° C. for 5 minutes to 30 minutes. Alternatively, the combined sample and reducing or non-reducing solution may be heated to 75° C. for 10 minutes. In some embodiments, the combined sample and reducing or non-reducing solution may be heated to 70° C. for 10 minutes.

[0049] Deglycosylation The present disclosure provides a method for deglycosylating a protein of interest in a sample. In some embodiments, the protein of interest is deglycosylated after being labeled with a fluorescent label using the methods described herein. Deglycosylation can be performed using an enzyme such as an endoglycosidase.

[0050] Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to amino acid side chains. These oligosaccharide chains are attached to the protein in a co-translational or post-translational manner.

[0051] As used herein, the term "glycan," which may be used interchangeably with "polysaccharide" and "oligosaccharide," refers to a compound that contains or consists of glycosidically linked monosaccharides. The term glycan may also be used to refer to carbohydrates attached to glycoproteins or glycolipids, even when the carbohydrate is a monosaccharide. https: / / en.wikipedia.org / wiki / Carbohydratehttps: / / en.wikipedia.org / wiki / Glycolipidhttps: / / en.wikipedia.org / wiki / OligosaccharideGlycans may contain O-glycosidic bonds of monosaccharides. Glycans may be homo- or heteropolymers of monosaccharides and may be linear or branched. Exemplary glycans may contain monomers of mannose, N-acetylglucosamine (GlcNAc), N-glycolylneuraminic acid (Neu5Gc), galactose, sialic acid, and fucose, among others.

[0052] Glycans can be attached to a protein of interest via either an N-linked or an O-linked linkage, and a protein of interest can include N-linked glycans, O-linked glycans, or a combination of N-linked and O-linked glycans. As referred to herein, "N-linked glycan" or "N-linked glycosylation" refers to the attachment of a sugar monomer or polysaccharide to a nitrogen atom, such as the amide nitrogen of an asparagine (Asn) amino acid of a protein. As used herein, "O-linked glycan" or "O-linked glycosylation" refers to the attachment of a sugar monomer or polysaccharide to an oxygen atom of a serine (Ser) or threonine (Thr) amino acid of a protein. Exemplary O-linked glycans include, but are not limited to, ON-acetylgalactosamine (O-GalNAc), ON-acetylglucosamine (O-GlcNAc), O-mannose, O-galactose, O-fucose, and O-glucose.

[0053] Endoglycosidases are enzymes that hydrolyze internal glycosidic bonds in oligosaccharides, thereby releasing the oligosaccharides from the glycoprotein if they are part of the glycoprotein.

[0054] As used herein, "endoglycosidase" refers to an enzyme that releases glycans from glycoproteins or glycolipids. Endoglycosidases can cleave polysaccharide alterations between non-terminal residues, thus releasing long-chain carbohydrates from their cognate protein conjugates. Exemplary endoglycosidases include, but are not limited to, peptide-N-glycosidase F (PNGase F), endoglycosidase H (Endo H), endoglycosidase S (Endo S), endoglycosidase D, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, O-glycosidase, and endo-β-galactosidase.

[0055] In some embodiments, the endoglycosidase catalyzes the deglycosylation of N-linked glycans. Exemplary endoglycosidases that target N-linked glycans include, but are not limited to, peptide-N-glycosidase F (PNGase F), endoglycosidase H (Endo H), endoglycosidase S (Endo S), endoglycosidase D, endoglycosidase F1, endoglycosidase F2, and endoglycosidase F4. In some embodiments, for example, in embodiments where the protein of interest comprises N-linked glycans, the endoglycosidase is PNGase F.

[0056] In some embodiments, the endoglycosidase catalyzes the deglycosylation of O-linked glycans. Exemplary endoglycosidases that target O-linked glycans include, but are not limited to, endo-α-N-acetylgalactosaminidase (O-glycosidase).

[0057] In some embodiments, the endoglycosidase is PNGase F. PNGase F is an amidase that cleaves between the innermost N-acetyl-D-glucosamine (GlcNAc) and asparagine residues of high mannose hybrid complex oligosaccharides in N-linked glycoproteins. In some embodiments, PNGase F is recombinant. In some embodiments, PNGase F is Rapid™ PNGase F. Rapid™ PNGase F is known in the art and available from New England Biolabs and other vendors. In some embodiments, Rapid™ PNGase F is a non-reduced form that preserves disulfide bonds in the protein of interest. In some embodiments, Rapid™ PNGase F is a reduced form that does not preserve disulfide bonds in the protein of interest.

[0058] In some embodiments, deglycosylating the sample comprises a reaction mixture comprising 0.1-3.0 mg of the labeled protein of interest. In some embodiments, the reaction mixture comprises 0.1-2.0 mg of the labeled protein of interest. In some embodiments, the reaction mixture comprises 0.1-1.5 mg of the protein of interest. In some embodiments, the reaction mixture comprises 0.5-1.5 mg of the protein of interest. In some embodiments, the reaction mixture comprises 0.2 mg of the labeled protein of interest. In some embodiments, the reaction mixture comprises 1-7 μL of Rapid™ PNGase F enzyme in a 10 μL reaction volume excluding the enzyme volume. In some embodiments, the reaction mixture comprises 1-5 μL of Rapid™ PNGase F enzyme in a 10 μL reaction volume excluding the enzyme volume. In some embodiments, the reaction mixture comprises 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, or 7 μL of Rapid™ PNGase F enzyme added to a volume of 10 μL containing the labeled protein of interest. In some embodiments, the reaction mixture comprises 5 μL of Rapid™ PNGase F enzyme in a reaction volume of 10 μL excluding the volume of the enzyme. In some embodiments, the reaction mixture comprises 5 μL of Rapid™ PNGase F enzyme added to a volume of 10 μL containing the labeled protein of interest. In some embodiments, the reaction mixture comprises an additional buffer, e.g., a reaction buffer that facilitates the action of the PNGase F enzyme. In some embodiments, the reaction mixture does not comprise an additional buffer.

[0059] In some embodiments, e.g., embodiments where the endoglycosidase is PNGase F, deglycosylating the sample comprises heating the sample to 25° C.-65° C. for 100-60 minutes. In some embodiments, deglycosylating the sample comprises heating the sample to 30° C.-50° C. for 20-40 minutes. In some embodiments, deglycosylating the sample comprises heating the sample to 35° C. for 30 minutes.

[0060] In some embodiments, e.g., embodiments in which the endoglycosidase is Rapid™ PNGase F, deglycosylating the sample comprises heating the sample to 50° C. for 10 to 30 minutes. In some embodiments, deglycosylating the sample comprises heating the sample to 50° C. for 10 minutes.

[0061] Protein labeling The present disclosure provides a method for labeling a protein of interest. In some embodiments, the protein of interest is labeled before deglycosylation. In some embodiments, the protein of interest is labeled with a fluorescent label, such as a fluorescent dye. Any suitable label is contemplated to be within the scope of the present disclosure.

[0062] As used herein, a "detectable label" or "label" refers to a chemical used to facilitate the identification and / or quantification of a target substance, such as a protein of interest. Exemplary labels include labels that can be directly observed or measured, or indirectly observed or measured. Such labels include, but are not limited to, radioactive labels that can be measured with a radiation counting device, dyes, dyes, or other chromogens that can be visually observed or measured with a spectrophotometer, chemiluminescent labels that can be measured with a photomultiplier-based instrument or photographic film, spin labels that can be measured with a spin label analyzer, and fluorescent moieties where the output signal is generated by excitation of a suitable molecular adduct and can be visualized by excitation with light that is absorbed by a dye or can be measured with a standard fluorometer or imaging system. The label can be a luminescent substance, such as a phosphor or fluorophore, a bioluminescent substance, a chemiluminescent substance where the output signal is generated by chemical modification of the signal compound, a metal-containing substance, or an enzyme, resulting in an enzyme-dependent second generation signal, such as the formation of a colored product from a colorless substrate or the formation of a spontaneous chemiluminescent product from a suitable precursor. The term label can also refer to a "tag" or hapten that can selectively bind to a labeling molecule, such that the labeling molecule is subsequently added and used to generate a detectable signal.

[0063] Numerous labels are known to those of skill in the art, including, but not limited to, microparticles, fluorescent dyes, haptens, enzymes and their chromogenic, fluorescent and chemiluminescent substances, and other labels described in Molecular Probes Handbook Of Fluorescent Probes And Research Chemicals 6th Ed. (1996) by Richard P. Haugland, the contents of which are incorporated by reference, and its subsequent 7th and 8th edition updates published on CD Rom in November 1999 and May 2001, respectively.

[0064] Exemplary fluorescent labels include, but are not limited to, fluorescent dyes.As used herein, "fluorescent dye" refers to a non-protein molecule that absorbs light and emits light at longer wavelengths.Exemplary fluorescent dyes include, but are not limited to, Alexa Fluor® dyes, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), DyLight fluorescent, Cy dyes, IRDye, HiLyte dyes, sulfonated and / or PEGylated coumarin dyes, sulfonated and / or PEGylated xanthene dyes, sulfonated or PEGylated cyanine dyes, and sulfonated and / or PEGylated pyrene dyes.

[0065] Additional detectable labels include, but are not limited to, Dyomics DY-631 NHS ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots, and the like, as well as those disclosed in U.S. Patent No. 6,924,372, which is incorporated herein by reference in its entirety.

[0066] Exemplary fluorescent labels also include, but are not limited to, biological fluorophores, such as green fluorescent protein, and nanoscale crystals, such as quantum dots.

[0067] Further exemplary fluorescent labels are available from Perkin Elmer as part of the Pico Protein Reagent Kit (also referred to as Protein Pico Assay Reagent Kit, part number 760498). In some embodiments, the fluorescent label comprises a Perkin Elmer Pico labeling dye. In some embodiments, labeling the protein of interest comprises adding a 4-20 μM Pico dye solution to a sample containing the protein of interest in a ratio of about 1:1 by volume. In some embodiments, labeling the protein of interest comprises adding a 4 μM, 5 μM, 6 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM, 20 μM, or 25 μM Pico dye solution to a sample containing the protein of interest. In some embodiments, the Pico dye solution is added to the sample containing the protein of interest in a ratio of about 1:5 dye to sample by volume, 1:4 dye to sample by volume, 1:3 dye to sample by volume, 1:2 dye to sample by volume, 1:1 dye to sample by volume, 2:1 dye to sample by volume, 3:1 dye to sample by volume, 4:1 dye to sample by volume, or 5:1 dye to sample by volume. In some embodiments, labeling the protein of interest comprises adding 16 μM Pico dye solution to the sample containing the protein of interest in a ratio of about 1:1 by volume. In some embodiments, the method comprises heating the sample and the dye.

[0068] In some embodiments, the fluorescent label or dye is covalently attached to the protein of interest. In some embodiments, the fluorescent label contains an amine-reactive group and is covalently attached to a free amine in the protein of interest. In some embodiments, the label is non-covalently attached to the protein of interest through a high affinity interaction.

[0069] Additional suitable kits for protein labeling will be known to those skilled in the art. Exemplary kits include, but are not limited to, the Antibody / Protein Labeling Kit-FITC from MedChemExpress, and the (Fast) Alexa Fluor® Conjugation Kit.

[0070] Any covalently attached fluorescent label, and any method of attaching the fluorescent label, is contemplated within the scope of the present methods.

[0071] In some embodiments, the sample and dye are heated to between about 30° C. and 40° C. for about 5-40 minutes. In some embodiments, the sample and dye are heated to between about 30° C. and 40° C. for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the sample and dye are heated to about 35° C. for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the sample and dye are heated to about 35° C. for about 15 minutes. This heating step can generate a sample that contains a denatured, labeled protein of interest. Excess label can optionally be removed from the sample, for example, by using a spin filter.

[0072] In some embodiments, the labeling reaction is stopped (quenched) before the deglycosylation reaction. For example, in embodiments where the fluorescent label is a Perkin Elmer Pico labeling dye, the labeling reaction can be stopped by adding an equal volume of Perkin Elmer Pico stop buffer to the labeling reaction. In some embodiments, the labeling reaction is quenched by adding 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, or 20 μL of an appropriate stop solution to the labeling reaction. In some embodiments, the dye is a Perkin Elmer Pico labeling dye, and the labeling reaction is quenched by adding 5 μL of Perkin Elmer Pico stop solution to the labeling reaction. A further exemplary stop buffer includes 1.5 M hydroxylamine (pH 8.5), for example, when the label includes an amine-reactive fluorescent dye. Those skilled in the art will be able to select appropriate stop buffers for various dye labeling reactions. Without wishing to be bound by theory, it is believed that quenching the labeling reaction prevents labeling of the endoglycosidase enzyme used in the subsequent deglycosylation step. This prevents or reduces the labeled endoglycosidase peak in the electropherogram used to visualize the labeled sample.

[0073] Protein of interest The present disclosure provides a method for preparing a sample comprising a protein of interest for analysis using electrophoresis. In some embodiments, the protein of interest is glycosylated. In some embodiments, the method includes labeling the protein of interest, followed by deglycosylation.

[0074] All proteins of interest, including post-translational modifications such as N-linked or O-linked glycosylation, are contemplated to be within the scope of this disclosure. In some embodiments, the protein of interest is a therapeutic protein, such as a therapeutic antibody, which may be a drug substance, a formulated drug substance, or a drug product.

[0075] In some embodiments, the protein of interest comprises an antigen-binding domain. In some embodiments, the protein of interest is a fusion protein. In some embodiments, the protein of interest comprises an antibody, an antibody fragment, or a single chain variable fragment (scFv). In some embodiments, the protein of interest is an antibody, an antibody fragment, or an scFv.

[0076] In some embodiments, the protein of interest comprises a recombinant human protein. For example, the protein of interest can comprise a human antibody or antibody fragment, or a humanized antibody or antibody fragment.

[0077] As used herein, an "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, i.e., CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. The term antibody also includes bispecific antibodies, including heterotetrameric immunoglobulins capable of binding to more than one epitope. Bispecific antibodies are generally described in U.S. Pat. No. 8,586,713, which is incorporated herein by reference.

[0078] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antibody-binding portion" include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) an isolated CDR; and (vii) an scFv consisting of the two domains of the Fv fragment, i.e., VL and VH, joined by a synthetic linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule. Other forms of single chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1 121-1 123).

[0079] Still further, an antibody or its antigen-binding portion may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as via papain or pepsin digestion of whole antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques generally known in the art (see Sambrook et al., 1989).

[0080] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0081] The term "humanized antibody," as used herein, includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences or otherwise modified to increase their similarity to antibody variants naturally produced in humans.

[0082] In some embodiments, the protein of interest is an antibody. In some embodiments, the antibody is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication US2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication US2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent Application Publication US9,402,898), an anti-angiopoietin-like 3 antibody (e.g., an anti-Dll4 antibody as described in U.S. Patent Application Publication US9,018,356), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent Application Publication US9,018,356), an anti-Dll4 antibody, an anti-angiopoietin-like 3 antibody (e.g., an anti-Dll4 ...Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 No. 9,302,015), anti-complement C5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-AngPtl3 antibodies as described in U.S. Patent No. 9,132,192), anti-platelet derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement C5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-TNF antibodies as described in U.S. Patent No. 9,132,192), anti-TNF antibodies, ... or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication US2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibodies (e.g., anti-PCSK9 antibodies as described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449), anti-growth and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies as described in U.S. Pat. No. 8,871,209 or U.S. Pat. No. 9,260,515), anti-glucagon receptor (e.g., Anti-GCGR antibodies, such as those described in Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., anti-IL4R antibodies, such as those described in Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibodies (e.g., those described in U.S. Patent Application Publication No. 7,582,298, 8,043,617 or 9,173,No. 9,447,173), anti-IL6R antibodies such as those described in U.S. Pat. No. 880, anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., anti-IL33 antibodies such as those described in U.S. Pat. No. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies such as those described in U.S. Pat. App. No. 9,447,173), anti-cluster of differentiation 3 (e.g., U.S. Pat. No. 9,447, 173 and 9,447,173, and U.S. Patent Application No. 62 / 222,605), anti-cluster of differentiation 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Nos. 9,657,102 and US20150266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of differentiation 48 (e.g., anti-CD48 antibodies as described in U.S. Patent No. 9,228,014), anti-Fel No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibodies as described in U.S. Patent Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication No. US2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibodies or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., anti-NGF antibodies as described in U.S. Patent Application Publication No. US2016 / 0017029 and U.S. Patent Nos. 8,309,088 and 9,353,176), and anti-protein Y antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Muc16 bispecific antibody), and an anti-CD3 x anti-prostate specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody).

[0083] In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, and the like. vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansine alirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomab tiuxetantiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab ecitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab anitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, and vedolizumab.

[0084] The protein of interest can be made or isolated by any means known in the art. These include recombinant means, such as proteins (e.g., antibodies) expressed using recombinant expression vectors transfected into host cells. The antibody protein of interest can be isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or from recombinant combinatorial human antibody libraries prepared, expressed, made, or isolated by any other means involving splicing of human immunoglobulin genes to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0085] In some embodiments, the protein of interest comprises a fragment crystallizable (Fc) domain. For example, the protein of interest can be a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein. In some embodiments, the receptor-Fc fusion protein is a trap protein.

[0086] A fusion protein comprises two or more portions of a protein that are not found together otherwise. For example, an "Fc fusion protein" can comprise the Fc portion of an immunoglobulin molecule fused to another heterologous domain, such as a receptor ligand binding domain. The preparation of fusion proteins, comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains), is described, for example, in Ashkenazi et al., Proc. Natl. Acad. ScL USA 88: 10535,1991; Byrn et al., Nature 344:677,1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl.4, pages 10.19.1 - 10.19.11,1992. A "receptor Fc fusion protein" comprises one or more extracellular domains of a receptor bound to an Fc portion, which in some embodiments includes a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, an Fc fusion protein contains two or more distinct receptor chains that bind to one or more ligands. For example, an Fc-fusion protein is an interleukin 1 (IL-1) trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the IL-1 R1 extracellular domain fused to the Fc of hIgG1, see U.S. Patent No. 6,927,004), or a vascular endothelial growth factor A (VEGF) trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1, see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0087] In some embodiments, the protein of interest is a fusion protein, such as a receptor fusion protein. Receptor fusion proteins can include trap proteins and mini-trap proteins, among others.

[0088] The term "fusion protein" refers to a molecule comprising two or more proteins or fragments thereof covalently linked via their respective peptide backbones, optionally produced through genetic expression of a polynucleotide molecule encoding the fusion protein.

[0089] In some embodiments, the protein of interest is a trap protein or a mini-trap protein. In some embodiments, the trap protein is an engineered therapeutic protein that can bind to a target protein and act as a decoy receptor to antagonize or regulate its activity. An exemplary trap protein comprises one or more receptor components that mimic the binding domain of the receptor for its target protein (e.g., VEGF receptor Ig domain 2 and Ig domain 3 of Flt-1) fused to a human IgG constant region, optionally including additional domains such as linkers, dimerization or multimerization domains, and cleavage sites. In some embodiments, the trap protein is cleaved or reduced in size (mini-trap), for example, through proteolytic cleavage, which can aid in the tissue penetration of the mini-trap. Non-limiting examples of trap proteins include an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the IL-1R1 extracellular domain, which in turn is fused to the Fc of hlgGl) (e.g., SEQ ID NO: 1) (see U.S. Pat. No. 6,927,004), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl, which in turn is fused to the Fc of hlgGl. See, e.g., U.S. Pat. Nos. 7,087,411, 7,279,159, the contents of each of which are incorporated herein by reference in their entireties; also see U.S. Pat. No. 5,610,279 for etanercept (TNF trap).

[0090] Protein production The protein of interest assayed by the methods described herein can be produced by any method known in the art. For example, the protein of interest can be produced by cell culture. The cell culture can be a "fed-batch cell culture" or "fed-batch culture" in which cells and culture medium are initially fed into a culture vessel, and additional culture nutrients are slowly fed to the culture in discrete increments during the culture, with or without periodic cell and / or product harvesting before the end of the culture. Fed-batch culture includes "semi-continuous fed-batch culture" in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. While fed-batch culture is distinct from simple "batch culture", all components for cell culture (including animal cells and all culture nutrients) are fed into the culture vessel at the beginning of the culture process in batch culture. Fed-batch culture may differ from "perfusion culture" insofar as during standard fed-batch culture processes, the supernatant is removed from the culture vessel, whereas in perfusion culture, the cells are left in the culture, e.g., by filtration, and culture medium is continuously or intermittently introduced and removed from the culture vessel. However, removal of samples for validation purposes during fed-batch cell culture is contemplated. The fed-batch process is continued until it is determined that maximum operating volume and / or maximum protein production has been reached, after which the protein is harvested.

[0091] The cell culture may be a "continuous cell culture", which is a technique used to continuously grow cells, usually in a specific growth phase. For example, if a constant supply of cells is required, or if the production of a specific protein of interest is required, the cell culture may need to be maintained in a specific growth phase. Therefore, conditions must be continuously monitored and adjusted accordingly to maintain the cells in that specific phase.

[0092] Cells are cultured in cell culture media. The terms "cell culture medium" and "culture medium" typically refer to a nutrient solution used to grow mammalian cells that provides the nutrients necessary to enhance the growth of cells, such as carbohydrate energy sources, essential amino acids (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and non-essential amino acids (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine), trace elements, energy sources, lipids, vitamins, etc. Cell culture media may contain extracts, such as serum or peptones (hydrolysates), that provide raw materials to support cell growth. The media may contain yeast-derived extracts or soybean extracts instead of animal-derived extracts. Chemically defined media refers to cell culture media in which all chemical components are known (i.e., have a known chemical structure). Chemically defined media are completely free of animal-derived components, such as serum or animal-derived peptones. In one embodiment, the medium is a chemically defined medium.

[0093] A "cell line" refers to one or more cells derived from a particular lineage through successive passage or subculture of cells. The term "cell" is used synonymously with "cell population." The term "cell" includes any cell suitable for expression of a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells, such as bacterial cells, mammalian cells, human cells, non-human animal cells, avian cells, insect cells, yeast cells, or cell fusions, such as, for example, hybridomas or quadromas. In certain embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, the cell is selected from the following cells: Chinese Hamster Ovary (CHO) (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocytes such as Jurkat (T lymphocytes) or Daudi (B lymphocytes), A431 (epidermal), U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) that expresses a viral gene. In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0094] Cells can be transformed with a heterologous polynucleotide encoding a protein of interest using any method known in the art, including but not limited to transformation, transfection, electroporation, and the like.

[0095] The term "heterologous polynucleotide" refers to a polynucleotide sequence that encodes a heterologous nucleotide sequence not found in a wild-type cell, which may include a sequence encoding a protein of interest. Exemplary heterologous polynucleotides include vectors that include a sequence encoding a protein of interest, including but not limited to plasmids, phages, and viral particles. Optionally, the vector allows for the transfer of a particular nucleic acid molecule into a cell. When introduced into an appropriate cell, the expression vector contains the necessary genetic elements to direct the expression of the protein of interest. Exemplary vectors may include a transcription promoter element (i.e., expression control sequence) operably linked to the sequence encoding the protein of interest. Vectors may be composed of either DNA or RNA, or a combination of the two (e.g., DNA-RNA chimeras). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, and one or more selectable markers, such as phosphotransferase or hygromycin phosphotransferase. In addition, depending on the cell type selected and the vector employed, other genetic elements such as origins of replication, additional nucleic acid restriction sites, enhancers, and sequences that confer inducibility of transcription may also be incorporated into the vector. The selection of an appropriate vector and transformation method will be apparent to one of skill in the art.

[0096] Glycosylation In some embodiments, the protein of interest is glycosylated. Glycosylation can include N-linked glycosylation, O-linked glycosylation, or a combination thereof. Many proteins and polypeptides of interest produced in cell culture are glycoproteins, containing covalently linked carbohydrate structures including oligosaccharide chains (glycans). These oligosaccharide chains are attached to proteins in the endoplasmic reticulum and Golgi apparatus via either N- or O-linkages. Oligosaccharide chains can comprise a significant portion of the mass of glycoproteins. Oligosaccharide chains can play roles including promoting correct folding of glycoproteins, mediating protein-protein interactions, conferring stability, conferring favorable pharmacodynamic and / or pharmacokinetic properties, inhibiting protein digestion, targeting glycoproteins to the appropriate secretory pathway, and targeting glycoproteins to one or more specific organs.

[0097] In some embodiments, the protein of interest comprises N-linked glycosylation. Generally, N-linked oligosaccharide chains are added to nascent translocation proteins in the lumen of the endoplasmic reticulum. The oligosaccharides are added to amino groups on the side chains of asparagine residues contained within a target consensus sequence, such as Asn-X-Ser / Thr or, in some cases, Asn-X-Cys, where X can be any amino acid except proline. The nascent oligosaccharide chain is usually trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short branched core oligosaccharide consisting of two N-acetylglucosamine and three mannose residues.

[0098] After initial processing in the endoplasmic reticulum, glycoproteins may undergo further processing before being secreted to the cell surface. N-linked oligosaccharide chains may be modified by the addition of mannose residues, resulting in high-mannose oligosaccharides. Alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the core mannose subunit to form complex oligosaccharides. Galactose may be added to the N-acetylglucosamine subunit, and sialic acid subunits may be added to the galactose subunit, resulting in chains that terminate in either sialic acid, galactose, or N-acetylglucosamine residues. In addition, fucose residues may be added to the N-acetylglucosamine residues of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0099] In addition to being modified by the N-linked glycosylation pathway, glycoproteins can also be modified by the addition of O-linked oligosaccharide chains to specific serine or threonine residues as they are processed in the Golgi apparatus. The residues of the O-linked oligosaccharides are added one at a time, and the addition of each residue is catalyzed by a specific enzyme. In contrast to N-linked glycosylation, the consensus amino acid sequence for O-linked glycosylation is less well-defined. In some embodiments, the protein of interest comprises O-linked glycosylation. In some embodiments, O-linked glycosylation comprises the attachment of a sugar molecule to a serine (Ser) or threonine (Thr) amino acid of the protein of interest.

[0100] In some embodiments, the protein of interest is a glycosylated protein. In some embodiments, the glycosylated protein comprises at least one attached glycan. In some embodiments, the protein of interest comprises at least one attached glycan, at least two attached glycans, at least three attached glycans, at least four attached glycans, at least five attached glycans, at least six attached glycans, at least seven attached glycans, at least eight attached glycans, at least nine attached glycans, at least ten attached glycans, at least eleven attached glycans, at least twelve attached glycans, at least fifteen attached glycans, at least twenty attached glycans, or at least twenty-five attached glycans. In some embodiments, the protein of interest has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 attached glycans. In some embodiments, the glycan is N-linked. In some embodiments, the glycans are O-linked. In some embodiments, the protein of interest comprises both N- and O-linked glycans.

[0101] In some embodiments, the protein of interest is a glycosylated protein. In some embodiments, the protein of interest comprises at least one glycosylation site. In some embodiments, the protein of interest comprises at least one glycosylation site, at least two glycosylation sites, at least three glycosylation sites, at least four glycosylation sites, at least five glycosylation sites, at least six glycosylation sites, at least seven glycosylation sites, at least eight glycosylation sites, at least nine glycosylation sites, at least ten glycosylation sites, at least ten glycosylation sites, at least eleven glycosylation sites, at least twelve glycosylation sites, at least fifteen glycosylation sites, at least twenty glycosylation sites, or at least twenty-five glycosylation sites. In some embodiments, at least the glycosylation site is an N-linked glycosylation site, for example, an asparagine in an N-linked glycosylation consensus sequence. In some embodiments, the at least one glycosylation site is an O-linked glycosylation site, e.g., a serine or threonine. In some embodiments, the protein of interest contains both at least one N-linked glycosylation site and at least one O-linked glycosylation site.

[0102] In some embodiments, the glycans comprise at least 0.5%, 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 75% (5% weight / weight or w / w) of the total weight of the glycosylated protein. In some embodiments, the glycans comprise at least 5% (5% w / w) of the total weight of the glycosylated protein. In some embodiments, the glycans comprise at least 10% (10% w / w) of the total weight of the glycosylated protein. Methods for determining the percentage of a protein weight that is composed of glycans are readily apparent to one of skill in the art and include, but are not limited to, comparing the predicted weight derived from the amino acid sequence to the actual weight determined by the electrophoretic analysis methods described herein.

[0103] reference standard In some embodiments, the reference standard undergoes the same preparation method as the sample containing the target protein and is analyzed at the same time as the sample containing the target protein.In some embodiments, the method comprises comparing one or more characteristics of the target protein with the reference standard.For example, the method comprises comparing the electropherograms of the target protein and the reference standard.

[0104] As used herein, a "reference standard" refers to a sample containing a protein that has been previously analyzed using methods known in the art and whose characteristics are known. The known characteristics can be determined from the amino acid sequence of the reference standard (e.g., predicted molecular weight) or experimentally determined (e.g., electropherogram profile). These characteristics can include, but are not limited to, predicted and experimentally determined molecular weights, electropherograms generated using methods described herein or known in the art, isoelectric point, extinction coefficient (a measure of how strongly the protein of interest absorbs light at a given wavelength), number of glycosylation sites, and molecular weight of the attached glycan. The reference standard can be similar to the protein of interest in one or more characteristics. For example, both the protein of interest and the reference standard can be monoclonal antibodies, or contain Fc domains, or be of similar molecular weight, or equivalent.

[0105] In some embodiments, the reference standard comprises a protein of interest, for example, the reference standard may be derived from a batch of the protein of interest separate from the sample that has been previously characterized and stored under controlled conditions to prevent degradation.

[0106] In some embodiments, the disclosure provides methods of preparing samples containing proteins of interest and reference standards for analysis using electrophoresis, comprising the steps of: (a) denaturing a sample and a reference standard; (b) labeling the proteins of interest and the reference standard with a fluorescent label to generate a labeled sample and a labeled reference standard; (c) quenching the labeling reaction of the proteins of interest and the reference standard; (d) deglycosylating the labeled samples and the labeled reference standard with an endoglycosidase; and (e) performing electrophoresis of the labeled samples and the labeled reference standard, wherein the samples and reference standards are labeled and quenched in steps (b) and (c) prior to deglycosylation in step (d).

[0107] In some embodiments, the electrophoresis is microchip capillary electrophoresis (MCE) and the output is an electropherogram. In some embodiments, the method comprises determining a major peak intensity of the protein of interest and the reference standard and comparing the intensity values ​​of the major peak of the protein of interest and the major peak of the reference standard. In some embodiments, the major peak of the protein of interest or the reference standard is glycosylated. In some embodiments, the major peak of the protein of interest or the reference standard is non-glycosylated, i.e., deglycosylated after labeling using the methods described herein. In some embodiments, determining the major peak comprises determining a height of the major peak. In some embodiments, determining the major peak comprises determining an area of ​​the major peak. In some embodiments, determining the major peak comprises determining a time-corrected area of ​​the major peak, which is the peak area divided by its migration time. In some embodiments, the major peak intensity of the protein of interest is between 50% and 150%, 50% and 140%, 50% and 130%, 50% and 120%, 50% and 110%, 50% and 100%, 50% and 90%, 60% and 150%, 70% and 150%, 80% and 150%, 90% and 150%, 100% and 150%, 110% and 150%, 12 ...50%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 50% and 150%, 5 Within 50%, 130%-150%, 140%-150%, 60%-140%, 70%-140%, 70%-130%, 70%-120%, 70%-110%, 80%-140%, 80%-130%, 80%-120%, 80%-110%, 80%-100%, 90%-140%, 90%-130%, 90%-120%, 90%-110%, or 90%-100%. In some embodiments, the major peak intensity of the protein of interest is within 60%-140%, 70%-130%, 80%-120%, or 90%-110% of the major peak intensity of the reference standard. In some embodiments, the major peak intensity of the protein of interest is within 70%-130% of the major peak intensity of the reference standard. Determining the major peak intensity of the protein of interest relative to the reference standard can ensure proper separation and data quality by the CE or MCE instrument.

[0108] Electrophoresis Provided herein are methods for analyzing a sample containing a protein of interest prepared using the methods described herein using electrophoresis.

[0109] Electrophoresis-based methods for analyzing proteins include, but are not limited to, gel-based methods such as sodium dodecyl (lauryl) sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis in the presence of lithium dodecyl sulfate, free-flow electrophoresis, isoelectric focusing, capillary gel electrophoresis, capillary electrophoresis (CE), and microchip capillary electrophoresis (MCE).

[0110] In some embodiments, the electrophoresis is CE. In some embodiments, the CE comprises a lithium dodecyl sulfate (LDS) buffer.

[0111] In some embodiments, the electrophoresis is MCE. The terms "MCE" or "microchip capillary electrophoresis" and "capillary electrophoresis (CE)" refer to capillary electrophoresis (CE) and its microfluidic counterpart (MCE), which are used to separate analytes in a sample. MCE technology can be used to separate, identify, and quantify proteins of interest, impurities in protein samples, and analyze degradation products of proteins of interest, such as protein fragments. CE and MCE separate analytes based on their electrophoretic mobility when a voltage is applied to the sample. The presence of a gel matrix (e.g., gel electrophoresis) will separate analytes based on size as well as charge. Impurities in a sample include, but are not limited to, protein aggregates, protein fragments, protein multimers, and assay contaminants.

[0112] In MCE, denatured, labeled proteins of interest are diluted and subjected to MCE to separate the diluted protein samples on a microchip capillary electrophoresis system and generate electropherograms. Because multiple samples can be run simultaneously on the same microchip, MCE-based methods are easily adaptable to high-throughput approaches. Furthermore, MCE is rapid and uses minimal sample volumes.

[0113] As used herein, an electropherogram is a plot resulting from an electrophoretic method, such as CE or MCE. The electropherogram contains peaks corresponding to the protein of interest and impurities.

[0114] Methods for analyzing electropherograms are known in the art and include comparing the position, size and area under each peak.Methods for calculating the peak area (area under the peak) of an electropherogram are known in the art and include, for example, integrating to estimate the area under the peak.Peak area can be calculated using software such as Empower.

[0115] Instruments for performing the disclosed MCE assays are commercially available. In some embodiments, the disclosed MCE assays are performed using the LabChip GXII, LabChip GXII Touch™, LabChip GXII Touch™ HT, and Protein Express Assay LabChip (LabChip® HT Protein Express Chip).

[0116] Instruments for performing the disclosed CE assays are also commercially available. For example, the CE assays can be performed using a Beckman Coulter capillary electrophoresis system, such as the PA800 Plus Pharmaceutical Analysis System.

[0117] In some embodiments of the methods described herein, the method further comprises labeling and running a protein standard molecular weight ladder to assess the size of the protein of interest.Protein molecular weight ladders are known to those skilled in the art and include PageRuler, Mark12, BenchMark, PageRuler High Range, and PageRuler Low Range available from ThermoFisher, and HT PICO Protein Express ladder from Protein Pico Assay Reagent Kit available from PerkinElmer.The selection of an appropriate ladder based on the size of the protein of interest will be apparent to those skilled in the art.

[0118] Purpose The present disclosure provides methods for characterizing proteins of interest using the labeling, deglycosylation, and electrophoresis methods described herein.

[0119] Analyzing a protein of interest can include, but is not limited to, characterizing the number, location, height, width, intensity, size, or area of ​​one or more peaks in an electropherogram generated by CE or MCE.

[0120] Characterizing the number and position of the peaks in the electropherogram can determine whether degradation products of the protein of interest are present in the sample, for example as peaks with molecular weights lower than that of the main peak.Comparing the peaks generated from the non-deglycosylated protein of interest and the protein of interest that has been deglycosylated and labeled using the methods described herein can determine whether a glycosylated form of the protein of interest is present in the sample, since the deglycosylated main peak of the protein of interest has a lower molecular weight than the glycosylated main peak of the protein of interest.

[0121] The method of the present disclosure can be used to assay the stability of target protein under various conditions.These include the storage conditions for target protein formulated as drug substance or pharmaceutical.For example, the comparison of peak number and peak area between the reference sample containing target protein and the stressed sample can be used to determine the stability of target protein over time and under various conditions, such as exposure to high or low pH or light.

[0122] Thus, the present disclosure provides a method for determining the stability of a protein of interest using the methods for labeling and deglycosylating a protein of interest described herein. In some embodiments, the method includes (a) applying stress to a sample containing the protein of interest; (b) denaturing the stressed sample and an unstressed sample containing the protein of interest; (c) labeling the stressed sample and the unstressed sample with a fluorescent label to generate a labeled stressed sample and a labeled unstressed sample; (d) quenching unreacted fluorescent label in the labeled stressed sample and the labeled unstressed sample; and (e) denaturing the labeled stressed sample and the labeled unstressed sample. with an endoglycosidase; (f) performing microchip capillary electrophoresis (MCE) of the labeled stressed sample and the labeled unstressed sample to generate electropherograms of the stressed and unstressed samples; and (g) comparing the electropherograms from the stressed and unstressed samples, wherein the stressed and unstressed samples are labeled and quenched in steps (c) and (d) prior to deglycosylation in step (e).

[0123] Any method of applying stress to a protein of interest in a sample is contemplated to be within the scope of the disclosed methods, including but not limited to chemicals, pH, radiation, light, freeze-thaw cycles, lyophilization, and heat.

[0124] In some embodiments, stressing the protein of interest comprises thermally stressing the sample. Thermally stressing the sample comprises simulating storage conditions for the protein of interest formulated as a drug substance or a formulated pharmaceutical, i.e., the stressed sample is kept at about -80°C to -30°C or about 2°C to about 8°C, respectively. In other embodiments, thermally stressing the sample comprises simulating handling and shipping conditions for the sample. In other embodiments, thermally stressing the sample comprises inducing forced degradation of the sample, for example, by increasing the temperature to which the sample is exposed.

[0125] In some embodiments, stressing the sample comprising the protein of interest comprises thermally stressing the sample. In some embodiments, the thermal stress comprises holding the sample at 25°C to 45°C. In some embodiments, the thermal stress comprises holding the sample at 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 35°C, 37°C, or 40°C. In some embodiments, the thermal stress comprises holding the sample at 37°C. In some embodiments, the thermal stress comprises holding the sample at 22°C to 26°C. In some embodiments, the thermal stress comprises holding the sample at 30°C. In some embodiments, the thermal stress comprises holding the protein at about 25°C to 45°C. In some embodiments, the heat stress comprises holding the stressed sample for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year. In some embodiments, the stressed sample is held for 2 weeks. In some embodiments, the stressed sample is held for 4 weeks.

[0126] In some embodiments, thermally stressing the sample comprises holding the sample at about 25° C. to about 45° C. for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks. In some embodiments, thermally stressing the sample comprises holding the sample at about 30° C. to about 45° C. for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks.

[0127] In some embodiments, stressing the sample includes at least one freeze / thaw cycle, for example starting with a liquid sample, lowering the temperature until the sample freezes, and then returning the sample to a temperature that makes it liquid before analysis.

[0128] In some embodiments, subjecting the sample to stress comprises exposing the sample to storage conditions. In some embodiments, the storage conditions comprise a temperature of about -80°C to -30°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. In some embodiments, the storage conditions comprise a temperature of about 2°C to 8°C for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, or at least 18 months.

[0129] In some embodiments, applying stress to the sample comprises mechanically agitating the sample, for example using a vortex or a magnetic stirrer.

[0130] In some embodiments, subjecting the sample to stress comprises lyophilizing and rehydrating the sample. Methods of lyophilizing a sample containing a protein of interest will be known to those of skill in the art and include, for example, lyophilization and spray drying.

[0131] In some embodiments, stressing the sample includes exposing the sample to light, radiation, singlet oxygen species, free radicals, high pH conditions, or low pH conditions. Exemplary low pH conditions include, among others, exposing the sample to a pH below 7.0, such as a pH below 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.0, 1.5, or 1.0. Exemplary high pH conditions include, among others, exposing the sample to a pH above 7.0, such as a pH above 8.0, 8.5, 9.0, 9.5, or 10.0.

[0132] In some embodiments, applying stress to the sample includes exposing the sample to light. The exposure to light can include light of any wavelength, or range of wavelengths. In an exemplary embodiment, the sample is exposed to bluish white fluorescent light or near ultraviolet light. An exemplary bluish white fluorescent light includes mixed wavelengths of light having a correlated color temperature (CCT) of about 4,100 to about 4,500 Kelvin (K). In some aspects, the bluish white fluorescent light has a CCT of 4,100K. In some embodiments, exposing the sample to light comprises exposing the sample to about 500,000, 600,000, 700,000, 800,000, 900,000, 1,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000, 2,000,000, 2,100,000, 2,200,000, 2,300,000, 2,400,000, 2,500,000, 2,600,000, 2,700,000, 2,800,000, 2,900,000, or 3,000,000 lux-hours of cumulative exposure to bluish-white light. In some embodiments, exposing the sample to light comprises exposing the sample to about 1,200,000 or about 2,400,000 lux-hours of cumulative exposure to bluish-white light. Exemplary near-ultraviolet light has a wavelength of about 300 nm to about 400 nm. In some embodiments, the near-ultraviolet light has a cumulative energy of about 100 watt-hours per square meter to about 600 watt-hours per square meter. In some embodiments, the near-ultraviolet light has a cumulative energy of about 100, 200, 300, 400, 500, or 600 watt-hours per square meter.

[0133] The reduction of the main peak area between the reference sample and its stressed version may indicate, for example, the reduction of the protein of interest in the main peak through degradation.In some embodiments, the area of ​​the main peak of the protein of the stressed subject is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6% at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to the main peak of the protein of the non-stressed subject.Similarly, the increase of the area of ​​the low molecular weight peak in the stressed reference sample compared to the reference sample may indicate the degradation of the protein of interest, as the abundance of low molecular weight species representing the degradation products of the protein of interest increases. In some embodiments, the area of ​​at least one low molecular weight peak of a protein from a stressed subject is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6 at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to at least one low molecular weight peak of a protein from a non-stressed subject.

[0134] Kits and Manufactured Products The present disclosure provides a kit that includes one or more of the disclosed buffers, enzymes, dyes, and reference standards that are used in the deglycosylation and labeling methods described herein.The kit can include a container for the components.The buffer can be in solution or lyophilized form.In some embodiments, the kit includes a second container that contains a diluent or reconstitution solution for lyophilized formulations, and optionally includes instructions for use of the solution or reconstitution, and / or use of the lyophilized buffer or powder components.

[0135] The kits described herein may further include additional reagents necessary to perform the disclosed MCE assays, including one or more of buffers, diluents, and filters. The buffers and reagents may be in bottles, vials, or test tubes.

[0136] In some embodiments, the kit comprises instructions.

[0137] This description sets forth numerous example configurations, methods, parameters, and equivalents, however, it should be recognized that such description is not intended as a limitation on the scope of the disclosure, but instead is provided as a description of example embodiments. EXAMPLES

[0138] Example 1: Reagents Materials and Equipment

[0139] (Table 1) Materials (equivalent products can also be used) TIFF0007661428000001.tif111163

[0140] (Table 2) Chemical substances (equivalent products can also be used) TIFF0007661428000002.tif231163

[0141] (Table 3) Equipment TIFF0007661428000003.tif131166

[0142] Table 4: Reagent solutions TIFF0007661428000004.tif232167TIFF0007661428000005.tif103166

[0143] (Table 5) Overview of MCE method TIFF0007661428000006.tif72166

[0144] Table 6. Summary of proteins used in the examples TIFF0007661428000007.tif60164

[0145] Example 2: Protocol for microchip capillary electrophoresis without deglycosylation (Method A) This protocol describes preparation methods for analysis of test proteins by non-reducing (NR) and reducing (R) microchip capillary electrophoresis (MCE) using a GXII instrument to estimate purity and impurity levels. These methods are used for protein characterization or for determining the level of fragmentation in protein samples. These conventional methods are performed without deglycosylation.

[0146] procedure See Figure 1 for the information-only flow path for this procedure.

[0147] (1) Denaturation. Dilute protein reference standard or test substance with water to approximately 0.2-2.0 mg / mL. Add protein sample and non-reducing (NR) / reducing (R) solution in a 4:1 volume ratio (volumes can vary) in a 96-well plate. Seal the plate with a polypropylene seal and heat the plate at the protein-specific denaturing temperature (typically 50-99°C) for an optimized time (typically 1-60 min).

[0148] (2) Labeling. Prepare 5 μM dye as described in Table 4. Add 5 μM dye to the denatured protein solution in a 1:1 volume ratio (volumes can vary). Heat the 96-well plate in a thermocycler at 35 °C for 30 min. To quench the labeling reaction, add 105 μL of stop solution (prepared according to Table 4) and 5 μL of labeled protein to a new 96-well plate and mix well.

[0149] (3) Run on GX-II. Prepare the MCE instrument and microchip and perform the measurement according to the manufacturer's instructions.

[0150] Example 3: Protocol for microchip capillary electrophoresis with deglycosylation prior to protein labeling (Method B) This method applies to glycoproteins that need to be deglycosylated before undergoing MCE measurements. Unless otherwise specified, all protocols, chemicals, reagents, and analyses are the same as those described in Examples 1-2.

[0151] Table 7. Additional Reagents TIFF0007661428000008.tif34153

[0152] procedure (1) Deglycosylation. Dilute 100 μg total protein sample to 90 μL with 0.1% RapiGest SF. Protein weight can be determined by UV-based method. Add 10 μL of NEB PNGase F stock to make 100 μL deglycosylation mixture, vortex, and settle. Incubate the mixture at 37° C. for 3 hours on a heating block with shaking at 400 revolutions per minute (rpm).

[0153] (2) Denaturation. The denaturation of the above-mentioned deglycosylated sample is carried out as described in Example 2.

[0154] (3) Labeling. Proceed with labeling of the denatured samples as described in Example 2.

[0155] (4) Run on GX-II. Prepare the MCE instrument and microchips and perform the measurements according to the manufacturer's instructions.

[0156] Example 4: Protocol for microchip capillary electrophoresis with protein labeling followed by deglycosylation (Method C) This method applies to glycoproteins that need to be deglycosylated before undergoing MCE measurement. Unless otherwise specified, all protocols, chemicals, reagents, and analyses are the same as those described in Examples 1-3.

[0157] A diagram of the protocol for deglycosylation after protein labeling can be seen in FIG. 1.

[0158] Table 8: Additional Reagents TIFF0007661428000009.tif21153

[0159] procedure (1) Denaturation: Dilute and denaturate the sample as described in Example 2.

[0160] (2) Labeling. Prepare 5 μM dye as described in Table 4. Mix 5 μM dye and the above denatured protein solution in a 1:1 volume ratio. For example, if the sample volume is 10 μL, add 10 μL of 5 μM dye. Seal the 96-well plate with a polypropylene seal and heat in a thermocycler at 35 °C for 30 minutes. Prepare an unused 96-well plate to quench the labeling reaction. Add 2.5 μL of stop buffer (orange cap vial from Pico Protein Reagent Kit, use original solution from the kit) to the empty plate wells according to the sample run settings. Transfer 2.5 μL of labeled sample to the plate wells containing stop solution. Pipette the mixed sample into each well and hold for at least 3 minutes.

[0161] (3) Deglycosylation. To each well, add 3 μL of MilliQ water and 2 μL of 5x Rapid™ PNGase Buffer (non-reduced form from NEB) to make a reaction volume of 10 μL. To each well, add 1-4 μL of Rapid™ PNGase (non-reduced form from NEB). Seal the 96-well plate with a polypropylene seal and heat at 50°C in a thermocycler for 10-30 minutes. After deglycosylation, add 17 μL of sample buffer (white cap vial from Pico Protein Reagent Kit) and 80 μL of MilliQ water to each well.

[0162] (4) Run on GX-II. Prepare the MCE instrument and microchips and perform the measurements according to the manufacturer's instructions.

[0163] Example 5: Comparison of no deglycosylation and deglycosylation prior to protein labeling using a heavily glycosylated sialic acid-containing protein Protein 1 is a disulfide-linked recombinant fusion protein 49 kDa in size by peptide mass and with eight predicted N-glycosylation sites (Note: not all sites are predicted to be glycosylated).

[0164] One goal was to develop a microchip capillary electrophoresis-based method to characterize and monitor low molecular weight (LMW) fragments of heavily glycosylated sialic acid-containing proteins (e.g., protein 1) for research stability and quality control (QC) testing.

[0165] The properties of Protein 1 are shown in Table 9 below.

[0166] Table 9. Molecular characteristics of protein 1 TIFF0007661428000010.tif21154 Abbreviations: Intact MS, intact protein mass spectrometry; SEC-MALS, size exclusion chromatography multiple angle laser light scattering.

[0167] A comparison of a protocol without deglycosylation (Method A, Example 2) and a protocol with deglycosylation prior to labeling (Method B, Example 3) is shown in Figure 1. A comparison of electropherograms of protein 1 produced by Method A and Method B is shown in Figure 2 for non-reducing (NR) conditions and in Figure 4 for reducing (R) conditions, respectively. For the NR conditions, Method A (without deglycosylation) resulted in broad peaks in the electropherogram with no peak resolution (i.e., no separation of the main, high molecular weight (HMW), and low molecular weight (LMW) peaks). In addition, the peak positions appeared in a much higher molecular weight (MW) region (70-120 kDa) than expected at about 64 kDa based on the orthogonal method. The MCE assay may inaccurately estimate size with larger errors when the protein is glycosylated (as described by Engel et al. in Electrophoresis, 2015 Aug;36(15):1754-8). In addition, there was an interfering free dye peak at <20 kDa that could mask any LMW peaks below 20 kDa.

[0168] In contrast, as a result of Method B (Example 3), where deglycosylation occurs prior to labeling, there is peak resolution and baseline separation between peaks (major, HMW, LMW peaks). The major peak appeared close to the expected MW (~49 kDa). The protocol indicates stability and more accurately determines molecular size. However, the protocol of Method B also requires 3 hours of deglycosylation, limiting the overall throughput of the assay. In addition, the PNGase peak (~36 kDa) interferes with the LMW1 and 2 peaks (impurity peaks from fragments of protein 1). Notably, for the thermally stressed protein 1 sample, the LMW1 peak increases and expands, merging with the PNGase peak (Figure 3). Another nearby artifact is the free dye peak interference at <20 kDa. The combination of these artifacts can lead to inaccurate integration when quantifying impurities, limiting the ability to indicate stability of the assay.

[0169] Similar observations were found for reducing conditions (FIG. 4), where deglycosylation of glycoproteins is required for accurate sizing, separation, and resolution of the major, LMW, and HMW peaks.

[0170] Example 6: Deglycosylation after protein labeling using heavily glycosylated sialic acid-containing proteins A comparison of protocols involving deglycosylation before (Method B) and after labeling (Method C) is shown in FIG.

[0171] To develop a protocol involving deglycosylation after labeling, the deglycosylation reaction conditions were optimized because incomplete removal of the glycosylated peak was initially observed in a 30 min deglycosylation reaction. Temperature, time, concentration, and buffer conditions were varied to determine the optimal deglycosylation of protein 1.

[0172] Optimization to remove the incompletely deglycosylated peak in the NR electropherogram of protein 1 resulted in several improvements to the protocol. These included using NEB Rapid™ PNGase F and performing deglycosylation at a higher temperature of 50° C. for 10 min (using conventional PNGase F requires a 3-h incubation at 37° C.), increasing the endoglycosidase concentration, and adding the glycoprotein buffer from the NEB PNGase kit.

[0173] Experiments showed that there was no clear improvement in deglycosylation with increasing reaction time. Figure 5 shows electropherograms generated using protein 1 with 1 μL or 2 μL of Rapid™ PNGase F (non-reduced form, NEB P0711) at 50° C., with reaction times varying from 10 to 30 minutes. As can be seen from Figure 5, there was no clear improvement (i.e., reduction in incompletely deglycosylated peaks) with reaction times greater than 10 minutes.

[0174] Increasing the Rapid™ PNGase F concentration improved deglycosylation. Using the protocol for deglycosylation after labeling, 1, 2, 3, or 4 μL of Rapid™ PNGase F was added to the deglycosylation reaction and the reaction proceeded for 10 minutes at 50° C. The results are shown in FIG. 6 and FIG. 7. As can be seen from the inset in FIG. 6, increasing the concentration of Rapid™ PNGase F reduces the shoulder peak of incomplete deglycosylation of protein 1.

[0175] Adding 1 to 4 μL of Rapid™ PNGase F provided robust results. Electropherograms generated using reference protein 1 deglycosylated using 1, 2, 3, or 4 μL of Rapid™ PNGase F are shown in FIG. 7. The areas under the indicated peaks were integrated using Empower and the results are provided below in Table 10.

[0176] Table 10. Integration of Protein 1 peaks generated using different amounts of Rapid™ PNGase F TIFF0007661428000011.tif50144

[0177] %RSD stands for percent relative standard deviation.

[0178] Integration results showed that the total integration percentage of the main peak (MP) had the highest value at approximately 3 μL, although there was a decrease in the shoulder peak after the main peak (incompletely deglycosylated protein 1) observed with higher Rapid™ PNGase F concentrations. From 1 μL to 4 μL of PNGase, the %RSD of MP was 0.33%, suggesting that using Rapid™ PNGase F concentrations of 1 to 4 μL per reaction provides robust results. Following this trend, using more Rapid™ PNGase is expected to provide similar results.

[0179] Method C, in which deglycosylation occurs after labeling, allows this MCE assay to be both precise and stable. Protein 1 was stressed by holding the protein solution at 37° C. for 4 weeks (compared to the stressed reference standard, i.e., “SRS”, “37° C. 4w”, in Tables 11 and 12, “RS”, i.e., unstressed reference standard) and assayed using the post-labeling deglycosylation protocol and MCE described herein. This stressed protein 1 was compared to unstressed protein 1 (time equal to 0 or t0, i.e., no 37° C. hold). Electropherograms were generated for stressed and unstressed protein 1 (FIG. 8) and the indicated peaks were integrated using Empower. The measurements were repeated in three replicates (S1-S3). The results are shown in Tables 11 and 12 below.

[0180] Table 11. Comparison of stressed (SRS) and unstressed (RS) protein 1 using deglycosylation after labeling protocol TIFF0007661428000012.tif53144

[0181] Table 12. Percent relative standard deviation (%RSD) of stressed and unstressed protein 1 (N=3) TIFF0007661428000013.tif44144

[0182] Three replicates of RS and SRS showed that multiple LMW and HMW peaks were consistently identified between runs and integrated with less than 1% RSD for LMW and MP peaks. All changes were significant from RS to SRS. Comparison of electropherograms of stressed and unstressed protein 1 prepared by method C (Figure 8) showed that the method is precise and robust.

[0183] When deglycosylation with PNGase F is performed prior to dye labeling (Method B), the PNGase F peak is visible in the electropherogram profile and interferes with the LMW1 and LMW2 peaks. Long deglycosylation times (3 h) are used and free dye interference (<20 kDa) is present.

[0184] When deglycosylation with Rapid™ PNGase F is performed after labeling with the dye (Method C), no PNGase peaks are visible in the electropherogram. There is rapid and complete deglycosylation (e.g., in 10 minutes). Resolution of MP, HMW, and LMW peaks is achieved with minimal free dye interference down to about the 10 kDa region (e.g., in Figure 8, the LMW5 peak is at 11 kDa and is baseline resolved from the free dye peak artifact).

[0185] In summary, MCE methods that use deglycosylation after dye labeling, such as method C, have good resolution, demonstrate stability, are high-throughput, reproducible, and avoid assay artifacts from PNGase F peak interference and free dye interference. The methods also demonstrate good precision, linearity, and robustness. These assays can be used in a plate-based high-throughput format, which is suitable for quality control purposes.

[0186] Example 7: Comparison of MCE results generated with and without deglycosylation using Protein 2 Protein 2 is a single-chain recombinant (Fab')2-like protein that comprises a single-chain fusion protein containing a ligand-binding domain joined by a linker of the sequence GGGGSGGGGSGGGGSGGGGGSGGGGS (SEQ ID NO: 1). The protein has a predicted molecular weight (peptide backbone) of 48 kDa. Protein 2 has eight N-glycosylation sites.

[0187] MCE electropherograms of protein 2 were generated under both non-reducing and reducing conditions using a protocol without deglycosylation (Method A, Example 2) and with deglycosylation after labeling (Method C, Example 4). As can be seen in Figure 9, without deglycosylation there was only a broad peak without resolution appearing in the MW region (90-140 kDa), much larger than the theoretical value (~48 kDa). In contrast, deglycosylation produced a major peak near the expected MW (48 kDa) and a clearly resolved LMW peak. Furthermore, the PNGase peak (~36 kDa) did not appear in the electropherogram.

[0188] Example 8: Comparison of MCE results generated with and without deglycosylation using protein 3 Protein 3 is a recombinant human IgG1 Fc subunit cleaved at a specific site by a recombinant cysteine ​​protease. The protein has a predicted molecular weight of 23 kDa. The protein has one N-glycosylation site.

[0189] MCE electropherograms were generated under non-reducing conditions using a protocol without deglycosylation (Method A, Example 2) and with deglycosylation after labeling (Method C, Example 4). The results can be seen in Figure 10. In the non-deglycosylated profile, two major peaks (MP) were found, representing the non-glycosylated (MP1, left) and glycosylated (MP2, right) populations in the original sample. In the deglycosylated profile, only the non-glycosylated peak was observed, with some HMW peaks resolved. The same comparison was also performed under reducing conditions (Figure 11).

[0190] Example 9: Stability evaluation of monoclonal antibodies Protein 4 is a human IgG4 monoclonal antibody with a molecular weight of 145 kDa and two N-linked glycosylation sites.

[0191] MCE electropherograms were generated for four protein samples prepared using a protocol without deglycosylation (Method A in Figures 12 and 13) and using a protocol with deglycosylation after labeling (Method C in Figures 12 and 13). Proteins were assayed using denaturation under both non-reducing (Figure 12) and reducing (Figure 13) conditions. As can be seen in Figure 13, deglycosylation shifts the glycosylated major peak (GMP) to a deglycosylated major peak (DGMP) at a lower molecular weight as a result of the removal of glycans. In Figure 13, deglycosylation reduces the size of the heavy chain (HC) peak as can be seen by comparing the deglycosylated heavy chain (DGHC) and glycosylated heavy chain (FGHC) peaks.

[0192] Example 10: Evaluation of the stability of protein 1 under light stress Protein 1 was photo-stressed by exposing the protein solution under bluish white (CW) fluorescent lamp light with cumulative exposure of 1.2 and 2.4 million lux hours (MLH) (Figure 14) or under integrated near ultraviolet (UVA) light with energy of 200 and 400 watt hours per square meter (Figure 15). Samples were assayed using the post-labeling deglycosylation protocol (Method C) and MCE as described in Examples 1 and 4. Stressed protein 1 samples were compared to unstressed protein 1 controls (incubated under the same conditions but covered with aluminum foil). Electropherograms were generated for stressed and unstressed protein 1 and the indicated peaks were integrated using Empower. The results are shown in Table 13. Both CW and UVA exposure led to a slight increase in the LMW peak and a significant increase in the HMW peak, which can be attributed to the photoinitiated formation of covalent dimers and multimers. The results show that the method is capable of demonstrating stability and assessing protein fragmentation and covalent HMW formation under stress conditions.

[0193] Table 14. Comparison of photostressed and non-photostressed protein 1 using deglycosylation after labeling protocol (Method C) TIFF0007661428000014.tif41128

[0194] Sequence information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> Deglycosylation Methods for Electrophoresis of Glycosylated Proteins <150> US 62 / 963,646 <151> 2020-01-21 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30

Claims

1. A method for analyzing a sample containing a protein of interest, the protein being a therapeutic protein comprising an Fc domain, comprising: The method further comprising: (a) denaturing the sample; (b) labeling the therapeutic protein with a fluorescent label to produce a labeled sample; (c) quenching any unreacted fluorescent label in the labeled sample; and (d) deglycosylating the labeled sample with an endoglycosidase to produce a deglycosylated labeled sample; (e) performing electrophoresis of the deglycosylated, labeled sample; Including, The sample is denatured, labeled and quenched in steps (a)-(c) prior to deglycosylation in step (d); method.

2. The method of claim 1, wherein the therapeutic protein comprises at least one glycosylation site.

3. The method of claim 1 or 2, wherein the therapeutic protein is a glycosylated protein comprising at least one attached glycan.

4. The method of any one of claims 1 to 3, wherein the therapeutic protein comprises an antigen-binding domain.

5. The method of claim 4, wherein the therapeutic protein is an antibody.

6. The method of any one of claims 1 to 4, wherein the therapeutic protein is a receptor fusion protein.

7. The method of claim 6, wherein the receptor fusion protein is a receptor-Fc fusion protein or a soluble TCR-Fc fusion protein.

8. The method of any one of claims 1 to 7, wherein the therapeutic protein is a recombinant human protein.

9. The method of claim 3 , wherein the at least one attached glycan is N-linked.

10. the endoglycosidase catalyzes the deglycosylation of N-linked glycans; and / or the endoglycosidase is selected from the group consisting of peptide-N-glycosidase F (PNGase F), endoglycosidase H (Endo H), endoglycosidase S (Endo S), endoglycosidase D, endoglycosidase F1, endoglycosidase F2, and endoglycosidase F4; The method according to any one of claims 1 to 9.

11. 11. The method of claim 10, wherein the PNGase F is Rapid PNGase F.

12. 12. The method of claim 11, wherein the Rapid PNGase F is non-reducing.

13. 13. The method of claim 11 or 12, wherein the step of deglycosylating the labeled sample comprises heating the sample at about 50° C. for 10 minutes.

14. Deglycosylating the labeled sample comprises: forming a reaction mixture comprising 0.2-1.5 mg of a labeled therapeutic protein and an endoglycosidase; The method according to any one of claims 1 to 13, comprising:

15. The method of any one of claims 3 to 8, wherein the at least one attached glycan is an O-linked glycan.

16. the endoglycosidase catalyzes the deglycosylation of O-linked glycans; and / or The endoglycosidase comprises endo-α-N-acetylgalactosaminidase (O-glycosidase). The method of claim 15.

17. 17. The method of any one of claims 1 to 16, wherein labelling the sample with a fluorescent label comprises heating the sample at about 35°C for 10 to 30 minutes.

18. The method according to any one of claims 1 to 17, wherein the sample is denatured using a reducing or non-reducing solution.

19. the sample is denatured using a reducing solution, and the reducing solution comprises dithiothreitol (DTT); or the sample is denatured using a non-reducing solution, and the non-reducing solution comprises iodoacetamide (IAM); The method of claim 18.

20. denaturing the sample comprises heating the sample to between 50°C and 99°C for 1 to 60 minutes; and / or the step of quenching unreacted fluorescent label comprises adding a stop solution; The method further comprises analyzing a reference standard simultaneously with the deglycosylated, labeled sample.

20. The method according to any one of claims 1 to 19.

21. 21. The method of any one of claims 1 to 20, wherein the electrophoresis is selected from the group consisting of gel electrophoresis, isoelectric focusing, capillary electrophoresis (CE), or microchip capillary electrophoresis (MCE).

22. 22. The method of any one of claims 1 to 21, which results in a reduction of free dye interference in the <20 kDa range and a reduction or absence of endoglycosidase peaks in the electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation.

23. 23. The method of claim 22, wherein the endoglycosidase peak is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when compared to an electropherogram generated using a labeled sample after deglycosylation.

Images