Glycosylated fusion protein de novo sequencing method based on mass spectrum

Abstract

The invention provides a de novo sequencing method for a glycosylated fusion protein. The method comprises the following steps: S1, treating glycosylated fusion protein by using a solution containing tris (2-carbonyl ethyl) phosphorus hydrochloride (TCEP) and chloroacetamide so as to synchronously complete denaturation, disulfide bond opening through reduction and alkylation closing through a one-step method; s2, adding a glycosidase combination comprising PNGase F glycosidase, sialic acid glycosidase and O-glycosidase into the protein obtained in the step S1, and cutting off an N-carbohydrate chain and an O-carbohydrate chain on the glycopeptide so as to synchronously remove the N-carbohydrate chain and the O-carbohydrate chain; s3, performing enzyme digestion on the protein obtained in the step S2 and subjected to synchronous removal of the N-carbohydrate chain and the O-carbohydrate chain into peptide fragments by adding different mass spectrum sequencing grade proteases; s4, performing secondary fragment ion detection on the peptide fragment obtained in the step S3 by using a mass spectrum to obtain mass spectrum original data; and S5, performing analysis and protein splicing on the mass spectrum original data obtained in the step S4 to obtain a protein amino acid de novo sequencing result.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein de novo sequencing methods, and in particular, relates to a mass spectrometry-based de novo sequencing method for glycosylated fusion proteins. Background Art

[0002] De novo protein sequencing is a new technology that directly determines the amino acid sequence without relying on any known sequence or protein database information. The principle of de novo protein sequencing is based on the regular fragmentation pattern of peptide molecules after protease cleavage in mass spectrometry detection. The de novo sequencing software algorithm parses the corresponding peptide amino acid sequence information and post-translational modifications on the amino acids according to the mass difference between mass spectrometry peaks. Peptides are digested with a variety of proteases, and peptides of varying lengths are parsed and spliced ​​based on the overlapping information at the beginning and end. The protein sequence is assembled from the N-terminus to the C-terminus to obtain a complete protein sequence. De novo protein sequencing can not only be used to analyze the protein sequences of species whose genomes have not been sequenced, but also plays a pivotal role in the research and development of biopharmaceuticals and industrial production.

[0003] Fusion protein (FP) refers to the expression product of two or more gene coding regions obtained by DNA recombination technology. The fusion of two or more protein functional domains enhances biological activity or produces a new functional combination with wide biotechnology and biopharmaceutical applications. In recent years, a variety of therapeutic fusion proteins have been approved for marketing, showing good application prospects in the treatment of tumors and acute and chronic inflammatory and immune system diseases.

[0004] Since the production process of therapeutic fusion proteins mostly adopts eukaryotic expression systems, protein products have a variety of complex glycosylation modifications (N-linked glycosylation modification and O-linked glycosylation modification). However, glycosylation modification, especially negatively charged glycosides such as sialic acid, will significantly inhibit the ionization efficiency of peptides, causing insufficient fragmentation of the peptide chain amino acid backbone, resulting in the inability to obtain high-quality mass spectrometry secondary spectra for amino acid sequence analysis. Secondly, since glycosylation modification increases the complexity of mass spectrometry fragment ion spectra, the same secondary fragmentation spectrum contains both peptide fragment ions and sugar chain fragmentation products, which is still a challenge for the analysis of glycopeptide secondary fragmentation spectra. Third, the fusion protein products caused by glycosylation modification are not uniform, and the complexity of the sample enzymatic peptides increases. Finally, the fusion protein contains two or more protein functional domains and hinge regions, and the complexity and variability of the amino acid sequence are greater. Therefore, the conventional de novo sequencing methods of monoclonal antibodies and monomeric proteins cannot be directly applied to de novo sequencing of therapeutic fusion proteins. Therefore, it is necessary to develop a mass spectrometry de novo sequencing detection method for glycosylation-modified therapeutic fusion proteins, which is conducive to the characterization of key quality attributes of fusion proteins and meets the urgent requirements of biopharmaceutical research and development and production. Summary of the invention

[0005] The present invention aims to provide a method for de novo sequencing of glycosylated fusion proteins, which first uses a deglycosylation enzyme to deglycosylate the glycosylated fusion protein, then uses a combination of multiple proteases to enzymatically hydrolyze the protein to form overlapping complementary peptides, then uses mass spectrometry to sequence the peptides generated by different enzymatic hydrolysis schemes, and finally forms protein sequencing results by splicing overlapping peptides. The method has simple steps, convenient operation, rapidity and high efficiency, and can achieve highly sensitive and accurate de novo amino acid sequencing analysis of glycosylated therapeutic fusion proteins.

[0006] Therefore, the present invention provides a method for de novo sequencing of a glycosylated fusion protein, characterized in that it comprises the following steps:

[0007] S1, treating the glycosylated fusion protein with a solution containing tris(2-carbonylethyl)phosphine hydrochloride (TCEP) and chloroacetamide, thereby simultaneously completing denaturation, reduction to open the disulfide bond, and alkylation to close the glycosylated fusion protein in one step;

[0008] S2, adding a glycosidase combination including PNGase F glycosidase, sialidase (α2-3,6,8Neuraminidase) and O-glycosidase (O-Glycosidase) to the protein obtained in step S1, which has been simultaneously denatured, reduced to open disulfide bonds and alkylated to block, to remove the N-sugar chains and O-sugar chains on the glycopeptide, so as to simultaneously remove the N-sugar chains and O-sugar chains;

[0009] S3, by adding mass spectrometry sequencing grade protease to enzymatically cleave the protein obtained in step S2 from which N-sugar chains and O-sugar chains have been simultaneously removed into peptide fragments;

[0010] S4, using a mass spectrometer to perform secondary fragment ion detection on the peptide obtained in step S3 to obtain mass spectrometry raw data;

[0011] S5, the mass spectrometry raw data obtained in S4 are analyzed and protein spliced ​​to obtain the protein amino acid de novo sequencing results.

[0012] In step S1, the glycosylated fusion protein may refer to a glycosylated fusion protein. A fusion protein refers to an expression product obtained by DNA recombination technology after two genes are recombined, and examples thereof include but are not limited to Fc fusion proteins (a novel protein produced by the fusion of the Fc segment of an immunoglobulin (IgG, IgA, etc.) and a functional protein molecule with biological activity, such as a receptor extracellular domain, a cytokine, an enzyme, a peptide, etc.). In some embodiments, the glycosylated fusion protein may be a therapeutic glycosylated fusion protein, for example, Etanercept, dulaglutide, asfotaseα, aflibercept, and the like.

[0013] In step S1, the glycosylated fusion protein can be treated with a solution containing tris (2-carbonylethyl) phosphohydrochloride (TCEP) and chloroacetamide, thereby completing denaturation, reduction to open disulfide bonds and alkylation closure simultaneously in a one-step method. In some embodiments, tris (2-carbonylethyl) phosphohydrochloride (TCEP) and chloroacetamide can be used in a molar ratio of 1-3:4-12. In a further embodiment, the glycosylated fusion protein can be reacted in a solution containing a final concentration of 20g / L sodium deoxycholate, 10-30mmol / L tris (2-carbonylethyl) phosphohydrochloride (TCEP), 40-120mmol / L chloroacetamide, 50-100mmol / L, preferably 100mmol / L Tris, to complete denaturation, reduction to open disulfide bonds and alkylation closure simultaneously in a one-step method. In a preferred embodiment, the glycosylated fusion protein can be reacted at 95° C. for 5 to 15 min (e.g., 10 min) in a solution containing a final concentration of 20 g / L sodium deoxycholate (w / v), 10-30 mmol / L tris(2-carbonylethyl)phosphine hydrochloride (TCEP), 40-120 mmol / L chloroacetamide, 50-100 mmol / L, preferably 100 mmol / L Tris. Preferably, in the solution, the final concentration of tris(2-carbonylethyl)phosphine hydrochloride is 10 mmol / L, and the final concentration of chloroacetamide is 40 mmol / L.

[0014] In some embodiments, in step S1, as the sample to be tested, the glycosylated fusion protein is in the form of a solution dissolved in phosphate buffered saline (PBS) (particularly 1×PBS). Further, in some embodiments, the concentration of the glycosylated fusion protein in 1×PBS is 0.5-2μg / μL, the starting amount of the glycosylated fusion protein reaction is 10-100μg / tube, and the reaction system is 30-100μL; preferably, the concentration of the glycosylated fusion protein is 1μg / μL, the starting amount of the glycosylated fusion protein is 30μg, and the reaction system is 30μL.

[0015] In some embodiments, when the fusion protein is in the form of being included in a preparation, the method of the present invention further comprises: before S1, a step (S0) of removing excipients and replacing solvents is performed. Step S0 can be performed by ultrafiltration, dialysis, size exclusion chromatography, affinity chromatography, solid phase extraction, etc. Preferably, the solvent after replacement is a phosphate buffer. In some embodiments, step S0 can use a size exclusion Zeba Spin column (2 ml, 40K MWCO, Thermo) to remove excipients and replace solvents. For example, the specific steps may include:

[0016] 1) Place the column into a 15-mL collection tube and centrifuge at 1000 g for 2 min to remove the storage solution;

[0017] 2) Discard the storage solution and place the column back into the 15m collection tube;

[0018] 3) Add 1 ml of wash / equilibration buffer to the top of the resin. Centrifuge at 1000 g for 3 min and discard the flow-through. Repeat 2 times. Centrifuge for 3 min for the last time.

[0019] 4) Blot the bottom of the column or plate to remove excess liquid. Transfer the column to a new 15m collection tube;

[0020] 5) Load 350 μL of sample onto the resin, followed by 100 μL of buffer solution;

[0021] 6) Centrifuge at 1000 g for 3 min, retain the flow-through containing the sample, discard the spin column, and obtain 450 μL of effluent.

[0022] In step S2, the PNGase F glycosidase, sialidase (α2-3,6,8 Neuraminidase) and O-glycosidase (O-Glycosidase) are all commercial enzymes that can be obtained from commercial channels.

[0023] In step S2, in some embodiments, the N-sugar chains and O-sugar chains can be removed simultaneously according to the following parameters:

[0024] Enzyme dosage: 0.25-4U PNGF enzyme per 20μg glycosylated fusion protein; 0.25-4U sialidase; 0.5-4U O-glycosidase;

[0025] Preferably, the reaction adds an activating ion reagent: an equal volume of phosphate buffer;

[0026] Preferably, the reaction temperature is 37°C;

[0027] Preferably, the reaction time is 2 h.

[0028] In step S3, the mass spectrometry sequencing grade protease can be used alone or in combination, including but not limited to the following proteases, trypsin, chymotrypsin, elestase, Glu-C, Lys-C, Pepsin, Asp-N, etc.

[0029] In some embodiments, in step S3, enzyme digestion is performed according to the following parameters:

[0030]

[0031] In some embodiments, in step S3, after the enzyme cleavage is completed, a peptide desalting step is further performed. Preferably, the following parameters can be followed:

[0032] The pH is adjusted to <3 (for example, using formic acid), the sodium deoxycholate precipitate is removed by centrifugation (for example, 4°C, 15000g for 5 minutes), and the supernatant is desalted using a C18 solid phase extraction method to obtain peptide fragments.

[0033] In some embodiments, in steps S1, S2 and / or S3, the reaction environment further contains 50-100 mmol / L Tris to maintain the solution pH at a weak alkaline level (preferably, pH 7.6-pH 8.5, more preferably pH 8.0).

[0034] In step S4, the peptide obtained in step S3 is subjected to secondary fragment ion detection using a mass spectrometer to obtain mass spectrometry raw data. In some embodiments, the mass spectrometer may be a liquid chromatography tandem mass spectrometer (LC-MS / MS), and the peptides are analyzed using LC-MS / MS. In some embodiments, the chromatographic conditions of the LC-MS / MS are:

[0035] a. Use C18 reverse phase nanoflow liquid phase to separate peptides;

[0036] b. The mass spectrometer adopts data-dependent acquisition mode to collect parent ion and secondary fragment ion spectra respectively. The fragmentation modes of peptides include but are not limited to: HCD, ETD, EtHcd, UVPD, CID, etc.

[0037] In step S5, an algorithm / software can be used to perform database-independent de novo peptide sequencing analysis on the mass spectrometry raw data, and overlapped peptide fragments can be spliced ​​to obtain a de novo protein amino acid sequence sequencing result.

[0038] In some embodiments, the de novo sequencing algorithm / software includes but is not limited to: pNovo, Deep novo, pepNovo, PEAKS AB.

[0039] In some embodiments, the protein splicing method can be manual splicing, ALPS, pTA, MuCS, PEAKS AB, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a graph showing the molecular weight distribution of samples before and after deglycolysis on polyacrylamide gel electrophoresis. Ctrl is the control group, deglyco is the deglycolysis treatment group, Etan is etanercept, and T1, T2, and T3 correspond to three types of recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection: TNFR: Fc 1, TNFR: Fc 2, and TNFR: Fc 3.

[0041] Figure 2 It shows the molecular weight test results of the etanercept sample after denaturation and reduction treatment. A. Mass spectrum deconvolution result of the intact molecular weight of the sample without desugaring; B. Mass spectrum deconvolution result of the intact molecular weight of the desugaring sample; C. Mass spectrum deconvolution result of the molecular weight of the sample without desugaring after reduction; D. Mass spectrum deconvolution result of the molecular weight of the desugaring sample after reduction.

[0042] Figure 3 The figure shows the molecular weight detection results of TNFR: Fc 1 samples after denaturation and reduction. A. Mass spectrum deconvolution results of intact molecular weight of undeglycosylated samples; B. Mass spectrum deconvolution results of intact molecular weight of deglycosylated samples; C. Mass spectrum deconvolution results of molecular weight of undeglycosylated samples after reduction; D. Mass spectrum deconvolution results of molecular weight of deglycosylated samples after reduction.

[0043] Figure 4 It shows the molecular weight detection results of TNFR: Fc 2 samples after denaturation and reduction. A. Mass spectrum deconvolution results of intact molecular weight of undeglycosylated samples; B. Mass spectrum deconvolution results of intact molecular weight of deglycosylated samples; C. Mass spectrum deconvolution results of molecular weight of undeglycosylated samples after reduction; D. Mass spectrum deconvolution results of molecular weight of deglycosylated samples after reduction.

[0044] Figure 5 The figure shows the molecular weight detection results of TNFR: Fc 3 samples after denaturation and reduction. A. Mass spectrum deconvolution results of intact molecular weight of undeglycosylated samples; B. Mass spectrum deconvolution results of intact molecular weight of deglycosylated samples; C. Mass spectrum deconvolution results of molecular weight of undeglycosylated samples after reduction; D. Mass spectrum deconvolution results of molecular weight of deglycosylated samples after reduction.

[0045] Figure 6 It is a diagram showing the sequence assembly results of de novo sequencing of the original drug etanercept, a recombinant human type II tumor necrosis factor receptor-antibody fusion protein for injection, after desugaring treatment (n=3).

[0046] Figure 7It is a diagram showing the results of de novo sequencing sequence splicing of three recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection, TNFR: Fc1, TNFR: Fc 2 and TNFR: Fc 3, after desugaring treatment.

[0047] Figure 8 It is a diagram showing the sequence similarity of recombinant human type II tumor necrosis factor receptor-antibody fusion protein drug for injection.

[0048] Figure 9 These are the peptide MS spectra (M->R) of the sequence difference sites of four recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection. A is the MS spectra of the peptide of etanercept sample; B is the MS spectra of the peptide of TNFR: Fc 1 sample; C is the MS spectra of the peptide of TNFR: Fc2 sample; D is the MS spectra of the peptide of TNFR: Fc 3 sample.

[0049] Figure 10 These are the peptide MS spectra (EM->DL) of the sequence difference sites of four recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection. A is the MS spectra of the peptide of etanercept sample; B is the MS spectra of the peptide of TNFR: Fc1 sample; C is the MS spectra of the peptide of TNFR: Fc 2 sample; D is the MS spectra of the peptide of TNFR: Fc 3 sample.

[0050] Figure 11 It is a graph showing the peptide coverage of de novo sequencing of the original drug etanercept, a recombinant human type II tumor necrosis factor receptor-antibody fusion protein for injection, in Comparative Example 1 without desugaring treatment (n=3).

[0051] Figure 12 The figure shows the results of middle-down subunit molecular weight detection of four recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection after desugaring treatment and IdeZ enzyme digestion. A. Deconvolution results of mass spectrum of molecular weight of TNFR end of etanercept; B. Deconvolution results of mass spectrum of molecular weight of TNFR end of TNFR: Fc 1; C. Deconvolution results of mass spectrum of molecular weight of TNFR end of TNFR: Fc 2; D. Deconvolution results of mass spectrum of molecular weight of TNFR end of TNFR: Fc 3; E. Deconvolution results of mass spectrum of molecular weight of Fc end of etanercept; F. Deconvolution results of mass spectrum of molecular weight of Fc end of TNFR: Fc 1; G. Deconvolution results of mass spectrum of molecular weight of Fc end of TNFR: Fc 2; H. Deconvolution results of mass spectrum of molecular weight of Fc end of TNFR: Fc 3. DETAILED DESCRIPTION

[0052] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Experimental materials and instruments: Etanercept was from Pfizer (Batch No. GA5492); three new recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection, TNFR: Fc 1 was from Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. (Batch No. 301-C2031); TNFR: Fc 2 was from Hisun Biopharmaceutical Co., Ltd. (Batch No. 0500222005); TNFR: Fc 3 was from Shanghai Saijin Biopharmaceutical Co., Ltd. (Batch No. A201222U). Sodium deoxycholate (w / v), tris(2-carbonylethyl)phosphine hydrochloride, chloroacetamide, Tris, formic acid, and Pepsin were purchased from Sigma Aldrich; phosphate buffer was purchased from Bio-Tech; PNGF enzyme, sialidase, O-glycosidase, and IdeZ protease were purchased from New England Biolabs; mass spectrometry sequencing grade proteins Trypsin, Chymotrpsin, and Elestase were purchased from Promega; Lys-C was purchased from Wako Chemistry; and Glu-C was purchased from Roche. Zeba TM Spin desalting column was purchased from Thermo; Nanodrop spectrophotometer, orbitrap Eclipse three-in-one mass spectrometer; vanquish u3000 nanoflow liquid phase was purchased from Thermo Fisher Scientific. 6545Q-TOF time-of-flight mass spectrometer, 1290 high performance liquid chromatograph were purchased from Agilent.

[0056] Example 1: Evaluation of the desugaring effect of therapeutic fusion protein drugs (SDS-PAGE gel electrophoresis)

[0057] The SDS-PAGE method was used to analyze the original drug of a therapeutic glycosylated fusion protein injection recombinant human type II tumor necrosis factor receptor-antibody fusion protein (Etanercept, sequence information see https: / / go.drugbank.com / drugs / DB00005 ) and three new drugs / similar drugs with similar sequences (TNFR: Fc 1, TNFR: Fc2, TNFR: Fc 3) were evaluated for deglycosylation effects. These four samples were expressed in Chinese hamster ovary cells and carried a variety of glycosylation modification types including N-sugar and O-sugar.

[0058] 10 μg of therapeutic glycosylated fusion protein, 0.5 U PNGF enzyme, 0.5 U sialidase, and 0.5 U O-glycosidase were added to make up the volume to 20 μL with phosphate buffer, and reacted at 1200 rpm, 25°C for 1 hour, and then at 37°C overnight.

[0059] The above samples, together with the control group samples that were not desugared, were subjected to 12% SDS-PAGE. Prepare a 12% SDS-PAGE gel and add an equal amount of 5μg of protein solution to each lane. Turn on the electrophoresis instrument, and keep the constant voltage at 60V for 30min in the first stage and 110V for 60min in the second stage. After the electrophoresis is completed, carefully remove the electrophoresis gel and place it in a 15cm culture dish. Add Coomassie Brilliant Blue staining solution and incubate on a shaker at room temperature for 30min. Discard the staining solution and add destaining solution for multiple destaining until the gel background becomes colorless. The results are as follows: Figure 1 shown.

[0060] Figure 1 What is shown is the molecular weight distribution of samples before and after desugaring on polyacrylamide gel electrophoresis. It can be seen that the molecular weight of the protein decreases after desugaring treatment.

[0061] Example 2: Evaluation of the desugaring effect of therapeutic fusion protein drugs (mass spectrometry molecular weight determination)

[0062] The desugaring effect of the recombinant human type II tumor necrosis factor receptor-antibody fusion protein for injection (Etanercept) was evaluated by protein mass spectrometry molecular weight determination.

[0063] 10 μg of therapeutic glycosylated fusion protein, 0.5 U PNGF enzyme, 0.5 U sialidase, and 0.5 U O-glycosidase were added to make up the volume to 20 μL with phosphate buffer, and reacted at 1200 rpm, 25°C for 1 hour, and then at 37°C overnight.

[0064] 20 μL of 6M guanidine hydrochloride and 100 mM dithiothreitol were added, and the mixture was reacted at 1200 rpm and 57°C for 45 minutes to open the disulfide bonds and denature the protein, so that Fc and TNFR form monomers. The protein molecular weight was determined using electrospray ionization-time of flight mass spectrometry (ESI-Q-TOF). The sample loading amount was 1 μg. The protein was separated by Agilent 1290 high performance liquid chromatography and entered 6545 Q-TOF through an electrospray ionization source to determine the molecular weight. The results are shown in Figures 2 - 5 shown.

[0065] Figures 2 - 5 It is the molecular weight mass spectrometry test result after the sample has been denatured and reduced. The sample without desugaring has high complexity and poor uniformity, and the complete molecular weight cannot be measured. After desugaring, the uniformity is significantly improved, and the accurate molecular weight can be measured, which is consistent with the theoretical calculated value.

[0066] Example 3: De novo sequencing of therapeutic fusion protein drugs

[0067] Four recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection (Etanercept, TNFR: Fc 1, TNFR: Fc 2, TNFR: Fc 3) were respectively administered to the patients with Zeba TM Spin desalting column (40KMWCO, 2mL) was used for solvent replacement to remove excipients and replace the solvent with phosphate buffer (PBS). 180 μg of the above therapeutic glycosylated fusion protein was dissolved in 180 μL of 2% sodium deoxycholate (w / v), 10 mmol / L tris (2-carbonylethyl) phosphate hydrochloride, 40 mmol / L chloroacetamide, 100 mmol / L Tris (pH = 8) at a final concentration of 2%, and reacted at 95°C for 10 min for denaturation, reduction of disulfide bonds and alkylation of disulfide bonds.

[0068] The samples with reduced disulfide bonds and alkylated disulfide bonds were cooled on ice, and 153 μL of phosphate buffer was added and mixed, 9U of PNGF enzyme; 9U of sialidase; 9U of O-glycosidase. The control group samples without desugaring were added with 180 μL of phosphate buffer and mixed. The mixture was shaken at 1200 rpm and 37°C. The reaction time was 2 h.

[0069] Each group of samples was divided into 6 tubes, each with a volume of about 60 μL, and 40 μL of 50 mM Tris (pH=7.6) was added to maintain a weakly alkaline pH. A mass spectrometry sequencing grade protease was added to each tube according to the parameters in the table below for protein enzymatic hydrolysis. The protease enzymatic reaction conditions are shown in Table 1 below.

[0070] Table 1. Protease enzymatic reaction conditions

[0071]

[0072] The enzymatically hydrolyzed peptides were mixed with formic acid at a final concentration of about 1% by volume, and the pH was <3. The mixture was centrifuged at 15,000 g for 5 minutes at 4°C to remove the sodium deoxycholate precipitate. The supernatant was desalted using a C18 solid phase extraction method to obtain the peptides.

[0073] Liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to analyze peptides and collect secondary fragmentation spectra to obtain raw mass spectrometry data;

[0074] PEAKS AB software was used to analyze the original mass spectrometry data and achieve de novo peptide sequencing;

[0075] Peptides with Average Local Confidence (ALC) scores greater than or equal to 50 were selected and assembled into proteins using MuCS. Figures 6 - 10 shown.

[0076] After desugaring, the samples can be assembled to obtain the complete protein sequence ( Figure 6 ). Comparing the protein sequences obtained by de novo sequencing with the known sequences in the database, the sequence coverage of this method is about 98.9%, and the sequencing accuracy is greater than 99.5% (Table 2).

[0077] Table 2. Accuracy of de novo sequencing of the original drug etanercept, a recombinant human tumor necrosis factor receptor type II-antibody fusion protein for injection

[0078]

[0079] The results of three technical repetitions were reproducible, indicating that the sequencing effect of this method is comparable to that of the conventional de novo protein sequencing method without glycosylation modification (Zhi-Biao Mai et al., Highly Robust de Novo Full-Length Protein Sequencing, Anal. Chem. 2022, 94, 3467-3475).

[0080] The method of the present invention was further applied to the de novo sequencing of three recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection with unknown sequences. The sequence information obtained was as follows: Figure 7 As shown. The amino acid sequence similarity of the samples was analyzed. The TNFR: Fc 1 and etanercept sample sequences were more similar, and the TNFR: Fc 2 and TNFR: Fc 3 sample sequences were more similar ( Figure 8 ). The results of de novo sequencing showed that there were three significant sequence differences among the four protein samples, namely: M->R( Figure 9 ),E->D and M->L( Figure 10 ), the secondary mass spectrometry spectrum corresponding to the mutation site, the secondary mapping quality is good and the positioning is clear, which can confirm that the de novo sequencing results have a high accuracy.

[0081] Comparative Example 1

[0082] The drug Etanercept, a recombinant human type II tumor necrosis factor receptor-antibody fusion protein for injection, was used with Zeba TMSpin desalting column (40K MWCO, 2mL) was used for solvent replacement to remove excipients and replace the solvent with phosphate buffer (PBS). 180 μg of the above therapeutic glycosylated fusion protein was dissolved in 180 μL of 2% sodium deoxycholate (w / v), 10 mmol / L tris (2-carbonylethyl) phosphate hydrochloride, 40 mmol / L chloroacetamide, 100 mmol / L Tris (pH = 8) at a final concentration of 2%, and reacted at 95°C for 10 min for denaturation, reduction of disulfide bonds and alkylation to block disulfide bonds.

[0083] The sample was divided into 6 tubes, each with a volume of about 60 μL, and 40 μL of 50 mM Tris (pH=7.6) was added to maintain a weakly alkaline pH. A mass spectrometry sequencing grade protease was added to each tube according to the parameters in the table below to perform protein enzymatic hydrolysis. The protease enzymatic reaction conditions are shown in Table 2 below.

[0084] Table 2. Protease enzymatic reaction conditions

[0085]

[0086] The enzymatically hydrolyzed peptides were mixed with formic acid at a final concentration of about 1% by volume, and the pH was <3. The mixture was centrifuged at 15,000 g for 5 minutes at 4°C to remove the sodium deoxycholate precipitate. The supernatant was desalted using a C18 solid phase extraction method to obtain the peptides.

[0087] Liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to analyze peptides and collect secondary fragmentation spectra to obtain raw mass spectrometry data;

[0088] PEAKS AB software was used to analyze the original mass spectrometry data and achieve de novo peptide sequencing;

[0089] Peptides with Average Local Confidence (ALC) scores greater than or equal to 50 were screened and spliced ​​using MuCS. Due to the presence of a large number of O-sugar modifications in etanercept, the overlapping peptide fragments digested by multiple enzymes could not effectively cover the full length of the protein, especially the middle region (150-240AA) including the hinge region, resulting in the inability to complete the automatic splicing process based on the MuCS software, resulting in the inability to achieve complete protein sequencing. The experimental results are shown in Figure 2. Figure 11 As shown (3 experiments repeated).

[0090] Example 4: Molecular Weight Analysis of Mutated Subunits of Therapeutic Fusion Protein Drugs (Molecular Weight Determination)

[0091] Four recombinant human type II tumor necrosis factor receptor-antibody fusion protein drugs for injection (Etanercept, TNFR: Fc 1, TNFR: Fc 2, TNFR: Fc 3) were respectively administered to the patients with Zeba TMThe solvent was replaced by phosphate buffer using a spin desalting column (40KMWCO, 2 mL) to remove the auxiliary materials.

[0092] 10 μg of therapeutic glycosylated fusion protein, 0.5 U PNGF enzyme, 0.5 U sialidase, and 0.5 U O-glycosidase were added to make up the volume to 20 μL with phosphate buffer, and reacted at 1200 rpm, 25°C for 1 hour, and then at 37°C overnight.

[0093] 40 U of IdeZ protease was added, and the mixture was reacted at 1200 rpm and 37° C. for 1 hour to cut the recombinant human type II tumor necrosis factor receptor-antibody fusion protein into two parts, Fc and type II tumor necrosis factor receptor (TNFR).

[0094] 20 μL of 6 M guanidine hydrochloride and 100 mM dithiothreitol were added and reacted at 1200 rpm and 57° C. for 45 minutes to open the disulfide bonds and denature the protein, so that Fc and TNFR formed monomers for protein molecular weight determination.

[0095] The protein molecular weight was determined using electrospray ionization-time of flight mass spectrometry (ESI-Q-TOF). The sample loading amount was 1 μg. The protein was separated by Agilent 1290 HPLC and entered 6545Q-TOF through the electrospray ionization source to determine the molecular weight. The results are shown in Figure 12 And as shown in Table 3.

[0096] Figure 12 The results in Table 3 show that the molecular weight change results are consistent with the difference value of the intact protein and are less than the mass deviation threshold (1 Da) allowed by the Q-TOF mass spectrometer, confirming the accuracy of the sequencing amino acid mutation.

[0097] Table 3. Calculation of molecular weight of drug domain subunits of recombinant human type II tumor necrosis factor receptor-antibody fusion protein

[0098]

[0099]

[0100]

[0101]

Claims

1. A method for de novo sequencing of glycosylated fusion proteins. It is characterized in that The following steps are involved: S1, treating the glycosylated fusion protein with a solution containing tris(2-carbonylethyl)phosphine hydrochloride and chloroacetamide, thereby simultaneously completing denaturation, reduction to open the disulfide bond, and alkylation to close the protein in one step; S2, adding a glycosidase combination including PNGase F glycosidase, sialidase and O-glycosidase to the protein obtained in step S1, which has been simultaneously denatured, reduced to open disulfide bonds and alkylated to block, to cleave the N-sugar chains and O-sugar chains on the glycopeptide, so as to simultaneously remove the N-sugar chains and O-sugar chains; S3, by adding mass spectrometry sequencing grade protease to enzymatically cleave the protein obtained in step S2 from which N-sugar chains and O-sugar chains have been simultaneously removed into peptide fragments; S4, using a mass spectrometer to perform secondary fragment ion detection on the peptide obtained in step S3 to obtain mass spectrometry raw data; S5, the mass spectrometry raw data obtained in S4 are analyzed and protein spliced ​​to obtain the protein amino acid de novo sequencing results.

2. The method for de novo sequencing of glycosylated fusion proteins according to claim 1, in, In step S1, in the solution containing tris(2-carbonylethyl)phosphine hydrochloride and chloroacetamide, tris(2-carbonylethyl)phosphine hydrochloride and chloroacetamide are used in a molar ratio of 1-3:4-12; Preferably, in step S1, the glycosylated fusion protein is reacted in a solution containing a final concentration of 20 g / L sodium deoxycholate, 10-30 mmol / L tris(2-carbonylethyl)phosphine hydrochloride, 40-120 mmol / L chloroacetamide, and 50-100 mmol / L, preferably 100 mmol / L Tris, so as to simultaneously complete denaturation, reduction to open the disulfide bond, and alkylation to close in one step; More preferably, in step S1, the glycosylated fusion protein is reacted at 95° C. for 5 to 15 min in a solution containing a final concentration of 20 g / L sodium deoxycholate, 10-30 mmol / L tris(2-carbonylethyl)phosphine hydrochloride, 40-120 mmol / L chloroacetamide, and 50-100 mmol / L, preferably 100 mmol / L Tris; Still more preferably, in step S1, in the solution containing tris(2-carbonylethyl)phosphine hydrochloride and chloroacetamide, the final concentration of tris(2-carbonylethyl)phosphine hydrochloride is 10 mmol / L, and the final concentration of chloroacetamide is 40 mmol / L.

3. The method for de novo sequencing of glycosylated fusion proteins according to claim 1, in, In step S1, as a sample to be tested, the glycosylated fusion protein is in the form of a solution dissolved in phosphate buffered saline (PBS) (particularly 1×PBS); Preferably, in step S1, the concentration of the glycosylated fusion protein in 1×PBS is 0.5-2 μg / μL, the starting amount of the glycosylated fusion protein reaction is 10-100 μg / tube, and the reaction system is 30-100 μL; More preferably, in step S1, the concentration of the glycosylated fusion protein is 1 μg / μL, the starting amount of the glycosylated fusion protein is 30 μg, and the reaction system is 30 μL.

4. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In the case where the glycosylated fusion protein is in the form of being contained in a preparation, the method further comprises: before S1, performing a step S0 of removing auxiliary materials and replacing solvents, Preferably, S0 adopts a method selected from ultrafiltration, dialysis, size exclusion chromatography, affinity chromatography, solid phase extraction, Preferably, the solvent after replacement is phosphate buffer.

5. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In step S2, N-glycans and O-glycans are removed simultaneously according to the following parameters: Enzyme dosage: 0.25-4U PNGF enzyme per 20μg glycosylated fusion protein; 0.25-4U sialidase; 0.5-4U O-glycosidase; Preferably, the reaction adds an activating ion reagent: an equal volume of phosphate buffer; Preferably, the reaction temperature is 37°C; Preferably, the reaction time is 2 h.

6. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In step S3, the mass spectrometry sequencing grade proteases are used alone or in combination. Preferably, the mass spectrometry sequencing grade protease is selected from trypsin, chymotrypsin, Elestase, Glu-C, Lys-C, Pepsin, Asp-N, More preferably, in step S3, enzyme digestion is performed according to the following parameters:

7. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In step S3, after the enzyme cleavage is completed, the peptide segment is further desalted. Preferably, the step of desalting the peptide segment complies with the following parameters: The pH was adjusted to <3, the sodium deoxycholate precipitate was removed by centrifugation, and the supernatant was desalted using C18 solid phase extraction to obtain peptide fragments.

8. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In steps S1, S2 and / or S3, the reaction environment also contains 50-100 mmol / L Tris to maintain the solution pH at a weak alkaline level, preferably maintaining the solution pH at pH 7.6-pH 8.5, more preferably maintaining the solution pH at pH 8.

0.

9. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In step S4 of the method of the present invention, the mass spectrometer is liquid chromatography tandem mass spectrometry (LC-MS / MS), and LC-MS / MS is used to analyze peptide segments. Preferably, the chromatographic conditions of the LC-MS / MS are: a. Use C18 reverse phase nanoflow liquid phase to separate peptides; b. The mass spectrometer adopts a data-dependent acquisition mode to collect parent ion and secondary fragment ion spectra respectively. Preferably, the fragmentation mode of the peptide is selected from HCD, ETD, EtHcd, UVPD, and CID.

10. The method for de novo sequencing of a glycosylated fusion protein according to claim 1, in, In step S5, the algorithm / software is used to perform database-independent de novo peptide sequencing analysis on the mass spectrometry raw data, and overlapping fragments of the peptides are spliced ​​to obtain the de novo sequencing results of the protein amino acid sequence. Preferably, the de novo sequencing algorithm / software is selected from pNovo, Deep novo, pepNovo, PEAKS AB, Preferably, the protein splicing method is selected from manual splicing, ALPS, pTA, MuCS, PEAKS AB.

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