Mass spectrum-based rapid analysis method capable of simultaneously carrying out multi-attribute on antibody drugs

Abstract

The invention provides a mass spectrum-based rapid analysis method capable of simultaneously carrying out multi-attribute analysis on antibody drugs, Endo F2 and IdeS glycosidase are used for carrying out enzyme digestion on a sample, DTT is used for reduction, and liquid phase separation and mass spectrum analysis are carried out, so that simultaneous monitoring of quality related attributes of saccharified fucose, core-removed fucose and other products is realized; the methods were evaluated using two therapeutic antibody drugs having different glycosylation site numbers. The method has the characteristics of broad spectrum, rapidness and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mass spectrometry-based rapid analysis method capable of simultaneously performing multiple attributes on antibody drugs. Background Art

[0002] Therapeutic antibody drugs have become a major new drug class developed in recent years due to their outstanding specificity and affinity, as well as their low adverse reaction profile. With the increasing number of antibody drugs approved for the treatment of cancer, metabolic diseases, autoimmune diseases, and infectious diseases, the ability to efficiently and comprehensively assess their product quality attributes (PQAs) has become increasingly important. During biosynthesis, purification, storage, and administration, therapeutic antibody drugs undergo various post-translational modifications (PTMs). Common PTMs include oxidation, deamidation, glycosylation, glycation, and isomerization, which can affect their efficacy and safety. Glycation and glycation are two different concepts. Glycation is an enzymatic modification in which sugar molecules are attached to amino acid residues of proteins by the action of glycosyltransferases, forming glycosidic bonds. Glycation can be divided into N-linked and O-linked glycosylation based on the linkage mechanism. Glycation, on the other hand, is a non-enzymatic reaction similar to oxidation. It occurs when the aldehyde groups of reducing sugars (such as glucose) undergo Maillard condensation with primary amino groups of biomacromolecules such as proteins to form unstable Schiff bases. This then undergoes molecular rearrangement to form ketoamines, known as Amadori products. These ketoamines undergo a series of molecular rearrangements, forming irreversible polymers known as advanced glycation end products (AGEs), which are associated with diseases such as atherosclerosis, diabetes, and Alzheimer's disease. Glycation is common in antibody therapeutics and is influenced by multiple factors, including the pH, temperature, and incubation time of the culture medium during cell fermentation, as well as the formulation type and storage temperature during storage.

[0003] It is reported that glycation can affect the structure, physicochemical properties, stability and functional activity of antibody drugs. Some studies have found that glycation can affect the charge heterogeneity and aggregate formation of antibody drugs, thereby affecting batch consistency and functional activity. The effect of glycation on the activity of different antibody drugs varies, mainly depending on the site of modification. Studies have shown that a certain antibody drug is highly glycated at lysine 100 in the complementarity determining region (CDR), which directly affects the interaction with the antigen. Therefore, it is crucial to thoroughly evaluate glycation during the research and development of therapeutic antibody drugs.

[0004] With advances in mass spectrometry (MS) performance and the development of semi-automated data processing software, MS-based multi-attribute methods (MAM) have rapidly developed. They can be used to rapidly characterize Fc-containing antibodies, enabling the quantitative analysis of multiple quality attributes (PQAs) in a single assay. Glycosylation of lysine residues on therapeutic antibodies results in a molecular weight increase of 162 Da, making it amenable to quantitative analysis. Subunit-level MAM, due to its relatively simple sample preparation and operational procedures, is well-suited for rapid and simultaneous monitoring of multiple product quality attributes. However, at the intact protein and subunit levels, glucose-induced glycation and galactose in glycosylation have the same relative molecular mass, resulting in interference between the detection of both. To overcome this issue, PNGase F is commonly used to remove N-glycans, but this method completely loses glycosylation information and often fails to effectively remove glycans hidden within the molecular structure. Detecting glycation at the peptide level typically requires enzymatic digestion of protein molecules into smaller peptides. While this approach offers site-specificity, complex sample preparation, lengthy chromatographic separations, and time-consuming data processing limit its routine application in product quality monitoring. Consequently, relatively few reports exist on methods for rapidly monitoring MAMs, including glycation. Summary of the Invention

[0005] To address the above problems, the present invention provides a rapid mass spectrometry-based analysis method that can simultaneously perform multi-attribute analysis on antibody drugs, and can simultaneously monitor glycation and other product quality-related attributes, such as de-core fucose.

[0006] To achieve the above object, the present invention is as follows: a using a glycosidase-containing composition to digest the sample to obtain a digestion solution; b. Add DTT to the enzyme digestion solution obtained in step a to obtain a reduced solution; c. The reducing solution is separated by liquid phase and detected by mass spectrometry; The glycosidase-containing composition contains EndoF2 and IdeS.

[0007] Preferably, in step a, the mass ratio of glycosidase EndoF2 to sample is 1:50, and the mass ratio of IdeS to sample is 1:50.

[0008] Preferably, the pH range of the enzymatic digestion in step a is 4.5-6.6.

[0009] Preferably, DTT is added in step b to a final concentration of 50 mM, and the mixture is incubated at 37° C. for 10 minutes.

[0010] Preferably, the reducing buffer in step b is a 50 mM bicarbonate buffer with a pH of 8.0.

[0011] The liquid phase separation method described in step c is: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% acetonitrile formate solution, column temperature is 60 ° C; gradient elution conditions are as follows

[0012] Preferably, the mass spectrometry detection method in step c is:

[0013] This method can be used to monitor the degree of glycosylation of antibodies.

[0014] This method can be used to monitor the de-core fucosylation level of antibodies.

[0015] This method can be used to monitor the levels of various quality attributes of antibodies, including N-terminal pyroglutamate cyclization, C-terminal lysine truncation, and oxidation.

[0016] The specific implementation is as follows: 1. Add the appropriate amount of IdeS at a mass ratio of 1:50 (enzyme: sample); add the appropriate amount of Endo F2 at a mass ratio of 1:50 (enzyme: sample); the pH range of enzymatic digestion is 4.5-6.6. Vortex to mix and incubate the sample at 37°C for 30 minutes.

[0017] 2. Replace the digested sample with 50 mM ammonium bicarbonate (pH 8.0) to a final concentration of 0.5 mg / mL. Use a pipette to add the required volume of reducing agent DTT to the sample to a final concentration of 50 mM. Incubate at 37°C for 10 minutes.

[0018] 3. Centrifuge the above samples at 17000g for 10 minutes, carefully pipette the supernatant into a clean sample bottle and wait for the instrument to detect.

[0019] 4. The methods for loading the machine include liquid chromatography method, mass spectrometry method and sample loading amount.

[0020] 5. The specific method is as follows: The liquid phase separation method is: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% acetonitrile formate solution, column temperature is 60 ° C; gradient elution conditions are as follows .

[0021] 6. Mass spectrometry method parameters are as follows .

[0022] 7. Sample volume: single sample volume is between 0.5 and 2.5 µg. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Spectrum of infliximab enzymatic digestion using Endo F2 at the intact protein level.

[0024] Figure 2 : Spectrum of cetuximab after enzymatic digestion with Endo F2 for 15 minutes at the intact protein level.

[0025] Figure 3 : Evaluation of the enzymatic digestion efficiency of IdeS and Endo F2 under different pH conditions.

[0026] Figure 4 : Product-related quality attribute assessment of a therapeutic antibody (infliximab) with a single glycosylation site on its heavy chain was performed by rapid MS-based MAM.

[0027] Figure 5 : Product-related quality attribute assessment of a therapeutic antibody (cetuximab) with two glycosylation sites on its heavy chain by rapid MS-based MAM.

[0028] Figure 6 : Comparison of product-related quality attributes of infliximab and cetuximab after MS-based rapid MAM and conventional PNGase F digestion, respectively. Implementation Method Example 1

[0029] Add the appropriate amount of Endo F2 at a mass ratio of 1:50 (enzyme:sample); vortex to mix thoroughly, and incubate the sample at 37°C for 30 minutes. Replace the digested sample with 50 mM ammonium bicarbonate (pH 8.0) to a final concentration of 0.5 mg / mL. Use a pipette to add the required volume of the reducing agent DTT to a final concentration of 50 mM, and incubate at 37°C for 10 minutes. Centrifuge the sample at 17,000 g for 10 minutes, and carefully transfer the supernatant to a clean loading vial for analysis. The analysis procedure includes liquid phase separation: mobile phase A is 0.1% formic acid in water, mobile phase B is 0.1% acetonitrile formate, and the column temperature is 60°C. Gradient elution conditions are as follows:

[0030] The mass spectrometry detection method is: .

[0031] Infliximab was used as a sample and digested with Endo F2 for 8 hours. The main peak mass after enzymatic digestion was 146325 Da, with two C-terminal lysine residues removed and GnF ( Figure 1), the monosaccharification rate of infliximab measured by this method was 16.73%, and the disaccharification rate was 2.12% (Table 1).

[0032] Using cetuximab as the sample, after digestion with Endo F2 for 15 min, cetuximab produced three main peaks, corresponding to mass numbers of 146673.92 Da (Fab: GnF, GnF; Fc: GnF, GnF), 147770.29 Da (Fab: GnF, GnF; Fc: GnF, G0F) and 148867.67 Da (Fab: GnF, GnF; Fc: G0F, G0F) ( Figure 2 The monosaccharide rate of cetuximab measured by this method was 14.42% (Table 1).

[0033] After the two antibodies were digested with two different glycosidases, the detected proportions of each modification were essentially identical. Therefore, total glycation levels can be quantified at the intact protein level after glycan release by Endo F2, without interference from retained glycans. However, PNGase F was unable to remove glycans from the cetuximab Fab fragment under native conditions, which affected the detection of Fab glycans.

[0034] Table 1: Comparison of product quality attribute data at the intact protein level for infliximab and cetuximab after treatment with different glycosidases ND: relevant modifications not captured; N / A: omitted.

[0035] Example 2 Add the appropriate amount of IdeS at a mass ratio of 1:50 (enzyme:sample); add the appropriate amount of Endo F2 at a mass ratio of 1:50 (enzyme:sample); vortex to mix thoroughly, and incubate the sample at 37°C for 30 minutes. Replace the digested sample with 50 mM ammonium bicarbonate (pH 8.0) to a final concentration of 0.5 mg / mL. Use a pipette to add the required volume of the reducing agent DTT to the sample to a final concentration of 50 mM. Incubate at 37°C for 10 minutes.

[0036] We further explored the enzymatic cleavage effects of these two enzymes at pH 4.5 and 6.6. The results showed that at pH 5.0, the enzymatic efficiencies of IdeS and Endo F2 were both over 97% ( Figure 3 ).

[0037] The method was evaluated using two monoclonal antibodies with different numbers of glycosylation sites. For infliximab, which has only one glycosylation site on the heavy chain, the main peaks for LC, Fc, and Fd were 23,434 Da, 24,104 Da, and 25,642 Da, respectively. The Fc fragment we detected had a molecular weight of 23,882 Da, indicating that it retained GnF and had a C-terminal lysine truncation ( Figure 4 ). A glycation peak 162 Da larger than the main peak was detected in all three subunits. The glycation ratios on the three subunits were calculated by the formula to be 2.77%, 2.66%, and 2.42%, respectively (Table 2). For cetuximab, which has two glycosylation sites on its heavy chain, the results showed that this method can effectively remove glycans at both sites while retaining GnF ( Figure 5 ), the glycation ratios for Fc, LC, and Fd were 1.67%, 2.08%, and 2.05%, respectively (Table 2). Furthermore, the detected glycation ratios were generally consistent with those observed using conventional methods that use PNGase F for glycan removal and subsequent quantification of glycation.

[0038] Table 2: Comparison of product quality attribute data at the subunit level for infliximab and cetuximab after processing under different conditions N / A: Omitted.

[0039] Example 4 Endo F2, because it retains N-acetylglucosamine or GnF after enzymatic cleavage, can additionally measure the critical quality attribute of de-core fucose. We detected 3.24% de-core fucose in this batch of infliximab (Table 2). When either Fc chain retains N-acetylglucosamine, the de-core fucose ratio is similar to that of the intact protein (6.4% / 2 ≈ 3.24%). Similar results were obtained for cetuximab, with varying proportions of de-core fucose modifications detected in the Fd and Fc / 2 subunits of the antibody.

[0040] Quantifying the fraction of de-core fucose after PNGase F cleavage is difficult because the enzyme completely removes glycans from the Fc region. However, for cetuximab, since PNGase F cannot cleave glycans from the Fd region, we calculated the fraction of de-core fucose from the main peaks bearing Pyr Q N-TERM and G2F+SA and the peaks bearing Pyr Q N-TERM and G2+SA. The results were generally consistent with those of MS-based rapid MAM (Table 2).

[0041] Table 2: Comparison of product quality attribute data at the subunit level for infliximab and cetuximab after processing under different conditions N / A: Omitted.

[0042] Example 5 For infliximab, 2.71% of Pyr E N-TERM and 89.04% of C-terminal lysine truncation were detected in the Fc region. Similarly, for cetuximab, 90.81% of Pyr Q N-TERM and 93.62% of C-terminal lysine truncation were detected on the Fd region, and the oxidation modification rate was 5.65%. The above modification ratios are basically consistent with the ratios measured after PNGase F digestion and without any enzyme digestion treatment ( Figure 6 and Table 1).

[0043] Table 1: Comparison of product quality attribute data at the intact protein level for infliximab and cetuximab after treatment with different glycosidases ND: relevant modifications not captured; N / A: omitted.

Claims

1. A rapid mass spectrometry-based analysis method for simultaneous multi-attribute analysis of antibody drugs, characterized by: a. using a composition containing glycosidase to enzymatically digest the sample to obtain an enzymatic digestion solution; b. Add DTT to the enzyme digestion solution obtained in step a to obtain a reduced solution; c. Use liquid phase separation and mass spectrometry to detect the reduced solution; The glycosidase-containing composition contains EndoF2 and IdeS.

2. The rapid analysis method according to claim 1, wherein In step a, the mass ratio of the glycosidase EndoF2 used for enzymatic digestion to the sample is 1:50, and the mass ratio of IdeS to the sample is 1:

50.

3. The rapid analysis method according to claim 1, wherein The pH range of the enzymatic digestion in step a is 4.5-6.

6.

4. The rapid analysis method according to claim 1, wherein In step b, add DTT to a final concentration of 50 mM and incubate at 37°C for 10 minutes.

5. The rapid analysis method according to claim 1, wherein The reducing buffer in step b is a 50 mM bicarbonate buffer with a pH of 8.

0.

6. The rapid analysis method according to claim 1, wherein The liquid phase separation method described in step c is: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% acetonitrile formate solution, column temperature is 60 ° C; gradient elution conditions are as follows 。 7. The rapid analysis method according to claim 1, wherein This method is used to monitor the degree of glycation of antibodies.

8. The rapid analysis method according to claim 1, wherein This method was used to monitor the de-core fucosylation level of antibodies.

9. The rapid analysis method according to claim 1, wherein This method is used to monitor the levels of multiple quality attributes of antibodies, including N-terminal pyroglutamate cyclization, C-terminal lysine truncation, and oxidation.

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