Method for measuring molecular weight of IgG-like antibody

Abstract

The invention discloses a method for determining the molecular weight of an IgG-like antibody, which comprises the following steps: carrying out denaturation treatment on the IgG-like antibody by using a Tris buffer solution containing guanidine hydrochloride to obtain a first mixed solution; carrying out liquid change treatment on the first mixed liquid by adopting a Tris buffer solution containing urea to obtain a second mixed liquid; adding N-glycosidase F into the second mixed solution for enzyme digestion treatment to obtain an enzyme digestion solution; performing liquid chromatographic separation on the enzyme digestion liquid to obtain target protein, performing mass spectrometry on the target protein, and determining the molecular weight of the target protein by combining a charge deconvolution algorithm; according to the determination method disclosed by the invention, through selection of a specific process and raw materials, N-glycosylation modification of an Fc end and an Fab end of the complex IgG-like antibody can be removed only by adopting N-glycosidase F, and accurate determination of the molecular weight of the IgG-like antibody is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for determining the molecular weight of IgG-like antibodies. Background Technology

[0002] Protein molecular weight determination is of great significance in antibody process development, characterization and other quality studies. It can be used to assess the correctness of amino acid sequence expression, sequence integrity, disulfide bond pairing, identification of degradation products, batch-to-batch consistency analysis, post-translational modifications, etc.

[0003] IgG antibodies exhibit high heterogeneity due to N-glycosylation modifications, leading to multiple peak overlap or signal splitting in mass spectra, directly affecting the accuracy of molecular weight determination. High-resolution mass spectrometry (HRMS), with its ultra-high resolution and mass precision, has become the "gold standard" for antibody molecular weight determination. However, for complex IgG-like antibodies (such as bispecific antibodies, Fc fusion proteins, engineered Fc variants, etc.), de-glycosylation is necessary to simplify the mass spectrometry signal, thereby improving analytical reliability.

[0004] In the prior art, the molecular weight determination of IgG-like antibodies usually follows the following procedure: (1) Enzymatic desugaring: N-glycosidase F (PNGase F) is used to specifically cleave the β-glycosidic bond between N-glycan chain and asparagine (Asn); (2) Liquid chromatography separation and high-resolution mass spectrometry analysis: Orbitrap or Q-TOF mass spectrometer coupled with electrospray ionization (ESI) is used to determine the molecular weight, and the molecular weight is analyzed by combining charge deconvolution algorithm; (3) Data verification: The integrity and modification status of the antibody are confirmed by comparing the theoretical molecular weight (based on amino acid sequence) with the measured value. However, the existing technology mainly targets the N-glycosylation modification sites at the Fc end, such as typical N-glycosylation sites like Asn-297, which are located in the hydrophilic domain of the antibody epitope, with open glycan spatial arrangement, good accessibility of N-glycosidase F, and high desugaring efficiency. However, in structurally complex N-glycosylation sites (such as regions with dense glycan branches and significant steric hindrance on the protein surface), the binding barrier between N-glycosidase F and glycan is significantly increased, leading to a masking effect at the cleavage site and lower desugaring efficiency.

[0005] To address the aforementioned issues, existing literature has developed a method for rapidly cleaving the Fab segment of glycans using β-N-acetylglucosidase (EndoF2). However, Endo F2 can only cleave the N-glycan at the Fab end, and it still needs to be combined with N-glucosidase F to cleave the N-glycosylation modification at the Fc end to reduce heterogeneity, resulting in higher costs. In addition, Endo F2 does not completely cleave the glycan, but rather shortens it, still retaining an N-acetylglucosamine at the Asn glycosylation site. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the molecular weight of IgG-like antibodies. This method, through specific processes and the selection of raw materials, uses only N-glycosidase F to remove the N-glycosylation modification of the Fc and Fab ends of complex IgG-like antibodies, thereby achieving accurate determination of the molecular weight of IgG-like antibodies.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a method for determining the molecular weight of IgG-like antibodies, the method comprising the following steps: (a) The IgG-like antibody was denatured using Tris buffer containing guanidine hydrochloride to obtain the first mixture; (b) The first mixture was replaced with a Tris buffer solution containing urea to obtain the second mixture; (c) Add N-glycosidase F to the second mixture for enzymatic digestion to obtain the digestion solution; (d) The enzyme digestion solution was separated by liquid chromatography to obtain the target protein. The target protein was then analyzed by mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

[0008] Preferably, in step (a), the concentration of guanidine hydrochloride in the Tris buffer containing guanidine hydrochloride is 5-7 M, the concentration of Tris is 45-55 mM, and the pH value of the Tris buffer containing guanidine hydrochloride is 7.2-7.8.

[0009] Preferably, in step (b), the urea concentration in the urea-containing Tris buffer is 1.5~2M, the Tris concentration is 45~55mM, and the pH value of the urea-containing Tris buffer is 7.2~7.8.

[0010] Preferably, in step (c), the enzyme digestion treatment temperature is 22~28℃ and the time is 18~22h.

[0011] Preferably, in step (a), the denaturation process includes: Add IgG-like antibody and Tris buffer containing guanidine hydrochloride to an ultrafiltration tube and centrifuge. After centrifugation, add Tris buffer containing guanidine hydrochloride to the ultrafiltration tube and continue centrifugation. Repeat this process 2-4 times to obtain the first mixture.

[0012] Preferably, the mass-to-volume ratio of the IgG-like antibody to the Tris buffer containing guanidine hydrochloride is 1:(6~10). The Tris buffer containing guanidine hydrochloride is replenished to 0.9 to 1.5 times the original volume.

[0013] Preferably, in step (b), the fluid replacement treatment includes: Add the first mixture and Tris buffer containing urea to the ultrafiltration tube, mix well and centrifuge. After centrifugation, add Tris buffer containing urea to the ultrafiltration tube and continue centrifugation. Repeat this process 4 to 6 times to obtain the second mixture.

[0014] Preferably, the volume ratio of the first mixture to the urea-containing Tris buffer is 1:(3~5). Replenish with Tris buffer containing urea to 0.9 to 1.5 times the original volume.

[0015] Preferably, in step (c), the amount of N-glycosidase F added per mg of IgG-like antibody is ≥15 U.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following: The method of this invention first denatures the IgG-like antibody protein with Tris buffer containing guanidine hydrochloride before desugaring to reduce the influence of the protein's higher-order structure on the accessibility of glycosidases and substrates. Then, the buffer is changed with Tris buffer containing urea to increase the solubility and stability of the IgG-like antibody protein and help maintain the denatured state of the protein. Then, N-glycosidase F is used for enzymatic digestion to simultaneously remove the N-glycosylation modification of the Fc and Fab ends of the complex IgG-like antibody. Finally, the molecular weight of the IgG-like antibody is accurately determined by liquid chromatography and mass spectrometry. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1 These are mass spectrometry analysis diagrams from different methods used to determine the molecular weight of IgG-like antibodies in Experimental Example 2 of this invention. Figure 2 These are mass spectrometry analysis diagrams from different methods used to determine the molecular weight of IgG-like antibodies in Experimental Example 3 of this invention. Figure 3 These are mass spectrometry analysis diagrams from different methods used to determine the molecular weight of IgG-like antibodies in Experimental Example 4 of this invention. Figure 4 These are mass spectrometry analysis diagrams from different methods for determining the molecular weight of IgG-like antibodies in Experimental Example 5 of this invention. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] This invention provides a method for determining the molecular weight of IgG-like antibodies, the method comprising the following steps: (a) The IgG-like antibody was denatured using Tris buffer containing guanidine hydrochloride to obtain the first mixture; (b) The first mixture was replaced with a Tris buffer solution containing urea to obtain the second mixture; (c) Add N-glycosidase F to the second mixture for enzymatic digestion to obtain the digestion solution; (d) The enzyme digestion solution was separated by liquid chromatography to obtain the target protein. The target protein was then analyzed by mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

[0022] The method of this invention first denatures the IgG-like antibody protein with Tris buffer containing guanidine hydrochloride before desugaring to reduce the influence of the protein's higher-order structure on the accessibility of glycosidases and substrates. Then, the buffer is changed with Tris buffer containing urea to increase the solubility and stability of the IgG-like antibody protein and help maintain the denatured state of the protein. Then, N-glycosidase F is used for enzymatic digestion to simultaneously remove the N-glycosylation modification of the Fc and Fab ends of the complex IgG-like antibody. Finally, the molecular weight of the IgG-like antibody is accurately determined by liquid chromatography and mass spectrometry.

[0023] In some embodiments, in step (a), the concentration of guanidine hydrochloride in the Tris buffer containing guanidine hydrochloride is 5-7 M, the concentration of Tris is 45-55 mM, and the pH value of the Tris buffer containing guanidine hydrochloride is 7.2-7.8.

[0024] In some embodiments, in step (b), the urea concentration in the urea-containing Tris buffer is 1.5-2 M, the Tris concentration is 45-55 mM, and the pH value of the urea-containing Tris buffer is 7.2-7.8.

[0025] In some embodiments, in step (c), the enzyme digestion treatment temperature is 22~28℃ and the time is 18~22h.

[0026] In this invention, the method of denaturation processing is not strictly limited. Those skilled in the art can use conventional methods in the field. In one embodiment, step (a) includes denaturation processing: Add IgG-like antibody and Tris buffer containing guanidine hydrochloride to an ultrafiltration tube and centrifuge. After centrifugation, add Tris buffer containing guanidine hydrochloride to the ultrafiltration tube and continue centrifugation. Repeat this process 2-4 times to obtain the first mixture.

[0027] In some embodiments, the mass-to-volume ratio of the IgG-like antibody to the Tris buffer containing guanidine hydrochloride is 1:(6-10). The Tris buffer containing guanidine hydrochloride is replenished to 0.9 to 1.5 times the original volume.

[0028] In this invention, the method of fluid replacement is not strictly limited, and those skilled in the art can use conventional fluid replacement methods. In one embodiment, step (b) includes: Add the first mixture and Tris buffer containing urea to the ultrafiltration tube, mix well and centrifuge. After centrifugation, add Tris buffer containing urea to the ultrafiltration tube and continue centrifugation. Repeat this process 4 to 6 times to obtain the second mixture.

[0029] In some embodiments, the volume ratio of the first mixture to the urea-containing Tris buffer is 1:(3~5). Replenish with Tris buffer containing urea to 0.9 to 1.5 times the original volume.

[0030] In some embodiments, in step (c), the amount of N-glycosidase F added per mg of IgG-like antibody is ≥15 U.

[0031] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0032] Example This embodiment describes a method for determining the molecular weight of IgG-like antibodies, which includes the following steps: (a) Load a 10K ultrafiltration tube into a suitable centrifuge tube, add 400 μL of Tris buffer containing guanidine hydrochloride to the ultrafiltration tube, add 50 μg of IgG-like antibody protein, centrifuge at 12000 rpm for 8 min, and the remaining solution in the ultrafiltration tube is about 100 μL. Add 400 μL of Tris buffer containing guanidine hydrochloride to it. Repeat this process 3 times. After that, the ultrafiltration tube will have about 100 μL of solution left. Let it stand at room temperature for 15 min to obtain the first mixture. The concentration of guanidine hydrochloride in the Tris buffer containing guanidine hydrochloride is 6 M, the concentration of Tris is 50 mM, and the pH of the Tris buffer containing guanidine hydrochloride is 7.5. (b) Transfer the first mixture to a 10K ultrafiltration tube, add 400 μL of urea-containing Tris buffer, mix well, and centrifuge at 12000 rpm for 8 min. The remaining solution in the ultrafiltration tube is about 100 μL. Add another 400 μL of urea-containing Tris buffer to the ultrafiltration tube and continue centrifugation. Repeat this process 4 times. The remaining solution in the ultrafiltration tube is about 100 μL. Transfer the solution to a 1.5 mL EP centrifuge tube to obtain the second mixture, in which the urea concentration in the urea-containing Tris buffer is 1.5 M and the Tris concentration is 50 mM; the pH of the urea-containing Tris buffer is 7.5. (c) Add 1.5 μL of N-glycosidase F solution (purchased from Hanhai New Enzyme, catalog number HH6901) to the second mixture and mix well. Then, perform enzymatic digestion at 25°C for 20 h to obtain the digestion solution. (d) The enzyme digestion solution was separated by reverse liquid chromatography to obtain the target protein. The target protein was then analyzed by high-resolution mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

[0033] Comparative Example 1 This comparative example illustrates a method for determining the molecular weight of IgG-like antibodies, the method comprising the following steps: (a) Load a 10K ultrafiltration tube into a suitable centrifuge tube, add 400 μL of Tris buffer to the ultrafiltration tube, add 50 μg of IgG-like antibody protein, centrifuge at 12000 rpm for 8 min, and the remaining solution in the ultrafiltration tube is about 100 μL. Add 400 μL of Tris buffer to it. Repeat this process 3 times. After that, the ultrafiltration tube will have about 100 μL of solution. Let it stand at room temperature for 15 min to obtain the first mixture. The Tris concentration in the Tris buffer is 50 mM and the pH of the Tris buffer is 7.5. (b) Transfer the first mixture to a 10K ultrafiltration tube, add 400 μL of urea-containing Tris buffer, mix well, and centrifuge at 12000 rpm for 8 min. The remaining solution in the ultrafiltration tube is about 100 μL. Add another 400 μL of urea-containing Tris buffer to the ultrafiltration tube and continue centrifugation. Repeat this process 4 times. The remaining solution in the ultrafiltration tube is about 100 μL. Transfer the solution to a 1.5 mL EP centrifuge tube to obtain the second mixture, in which the urea concentration in the urea-containing Tris buffer is 1.5 M and the Tris concentration is 50 mM; the pH of the urea-containing Tris buffer is 7.5. (c) Add 1.5 μL of N-glycosidase F solution to the second mixture and mix well. Then, perform enzymatic digestion at 25°C for 20 h to obtain the digestion solution. (d) The enzyme digestion solution was separated by reverse liquid chromatography to obtain the target protein. The target protein was then analyzed by high-resolution mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

[0034] Comparative Example 2 This comparative example illustrates a method for determining the molecular weight of IgG-like antibodies, the method comprising the following steps: (a) Load a 10K ultrafiltration tube into a suitable centrifuge tube, add 400 μL of Tris buffer containing urea to the ultrafiltration tube, add 50 μg of IgG-like antibody protein, centrifuge at 12000 rpm for 8 min, and the remaining solution in the ultrafiltration tube is about 100 μL. Add 400 μL of Tris buffer containing urea to it. Repeat this process 3 times. After that, the ultrafiltration tube will have about 100 μL of solution left. Let it stand at room temperature for 15 min to obtain the first mixture. The urea concentration in the Tris buffer containing urea is 8 M, the Tris concentration is 50 mM, and the pH of the Tris buffer containing urea is 7.5. (b) Transfer the first mixture to a 10K ultrafiltration tube, add 400 μL of urea-containing Tris buffer, mix well, and centrifuge at 12000 rpm for 8 min. The remaining solution in the ultrafiltration tube is about 100 μL. Add another 400 μL of urea-containing Tris buffer to the ultrafiltration tube and continue centrifugation. Repeat this process 4 times. The remaining solution in the ultrafiltration tube is about 100 μL. Transfer the solution to a 1.5 mL EP centrifuge tube to obtain the second mixture, in which the urea concentration in the urea-containing Tris buffer is 1.5 M and the Tris concentration is 50 mM; the pH of the urea-containing Tris buffer is 7.5. (c) Add 1.5 μL of N-glycosidase F solution to the second mixture and mix well. Then, perform enzymatic digestion at 25°C for 20 h to obtain the digestion solution. (d) The enzyme digestion solution was separated by reverse liquid chromatography to obtain the target protein. The target protein was then analyzed by high-resolution mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

[0035] Experimental Example 1 The molecular weight of the symmetrical bispecific IgG-like antibody (theoretical molecular weight of 198088.6 Da) produced by Beijing Zhaoyan Biotechnology Co., Ltd. was determined using the determination method of the above embodiment. The result was 198095.6 Da, with an error within 50 ppm. As can be seen, the molecular weight measured by the method in this application embodiment is consistent with the theoretical molecular weight, and the mass peak is single; therefore, the method in this application embodiment has high accuracy and can be used for the molecular weight determination of IgG-like complex antibodies.

[0036] Experiment Example 2 This experimental example illustrates the effects of non-denaturing and different denaturing reagents on the molecular weight determination of IgG-like antibodies: The molecular weight of the symmetrical bispecific IgG-like antibody was determined using the methods described in Examples 1 and 2, respectively. The mass spectrometry chromatograms for each method are shown below. Figure 1 As shown, Figure 1 In the figures, (a) is the mass spectrometry chromatogram of the determination method in Comparative Example 1 (i.e., without denaturation treatment); (b) is the mass spectrometry chromatogram of the determination method in Comparative Example 2 (i.e., using urea as a denaturant); and (c) is the mass spectrometry chromatogram of the determination method in the example.

[0037] Depend on Figure 1 It can be known that: Denaturation treatment can better remove N-glycan modification, simplify mass spectrometry signals and improve measurement accuracy. Different denaturants affect the removal effect of N-glycan modification. In this application, guanidine hydrochloride is used as a denaturant, which has a better effect on removing N-glycan modification.

[0038] Experimental Example 3 This experimental example illustrates the effect of different urea addition amounts on the molecular weight determination of IgG-like antibodies. Based on the examples, in step (b), experiments were conducted with urea concentrations of 0 M, 1 M, 1.5 M, and 2 M in the Tris buffer containing urea, while all other conditions remained the same. Mass spectrometry analysis chromatograms at different urea concentrations are shown below. Figure 2 As shown, Figure 2 In the image, (a) is the mass spectrometry analysis chromatogram of urea at 0M (i.e., urea-free); (b) is the mass spectrometry analysis chromatogram of urea at 1M; (c) is the mass spectrometry analysis chromatogram of urea at 1.5M; and (d) is the mass spectrometry analysis chromatogram of urea at 2M. Depend on Figure 2 It can be known that: There is a significant difference in mass spectrometry response with and without urea; the mass spectrometry response in Tris buffer without urea is only about one-third of the response with 1-2M urea; within the range of 1-2M urea in the digestion solution, adding 1.5-2M urea can obtain a more stable mass spectrometry response.

[0039] Experiment Example 4 This experimental example illustrates the effect of different enzyme digestion temperatures on the determination of the molecular weight of IgG-like antibodies. Based on the previous example, the enzyme digestion temperature in step (c) was set to 25°C, 30°C and 37°C respectively for enzyme digestion treatment, while other conditions were the same; Mass spectrometry analysis at different enzyme digestion temperatures is shown below. Figure 3 As shown, Figure 3 In the image, (a) is the mass spectrometry analysis at an enzyme digestion temperature of 25℃; (b) is the mass spectrometry analysis at an enzyme digestion temperature of 30℃; and (c) is the mass spectrometry analysis at an enzyme digestion temperature of 37℃. Depend on Figure 3 It can be known that: Molecular weight results obtained at the optimal enzyme activity temperature of 37°C generally showed significant heterogeneity, with the dominant molecular weight containing one or more carbamylated modifications. Molecular weight results obtained at incubation temperatures of 25°C and 30°C were more homogeneous. Since incubation conditions above 30°C easily lead to urea decomposition and the generation of isocyanate, which can covalently bind to the α-amino (N-terminus), lysine ε-amino, arginine amino, and cysteine ​​sulfhydryl groups of proteins, forming carbamylated modifications, 25°C was chosen as the robust incubation temperature.

[0040] Experimental Example 5 This experimental example illustrates the effect of different enzyme digestion times on the molecular weight determination of IgG-like antibodies. Based on the previous example, the enzyme digestion time in step (c) was set to 16h, 20h and 24h respectively, while other conditions were the same; Mass spectrometry analysis at different enzyme digestion times is shown below. Figure 4 As shown, Figure 4 In the image, (a) is the mass spectrometry analysis result after 16 hours of enzyme digestion; (b) is the mass spectrometry analysis result after 20 hours of enzyme digestion; and (c) is the mass spectrometry analysis result after 24 hours of enzyme digestion. Depend on Figure 4 It can be known that: Generally, extending the incubation time can increase the enzyme catalytic reaction rate, but it will also reduce the analytical efficiency. Among the three conditions, the mass spectrometry response of 20h and 24h incubation is relatively stable and is higher than that of 16h incubation. To improve the analytical efficiency, an incubation time of 20h is selected.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for determining the molecular weight of IgG-like antibodies, characterized in that, The determination method includes the following steps: (a) The IgG-like antibody was denatured using Tris buffer containing guanidine hydrochloride to obtain the first mixture; (b) The first mixture was replaced with a Tris buffer solution containing urea to obtain the second mixture; (c) Add N-glycosidase F to the second mixture for enzymatic digestion to obtain the digestion solution; (d) The enzyme digestion solution was separated by liquid chromatography to obtain the target protein. The target protein was then analyzed by mass spectrometry and the molecular weight of the target protein was determined by charge deconvolution algorithm.

2. The determination method according to claim 1, characterized in that, In step (a), the concentration of guanidine hydrochloride in the Tris buffer containing guanidine hydrochloride is 5-7 M, and the concentration of Tris is 45-55 mM; the pH value of the Tris buffer containing guanidine hydrochloride is 7.2-7.

8.

3. The determination method according to claim 1, characterized in that, In step (b), the urea concentration in the Tris buffer containing urea is 1.5~2M, and the Tris concentration is 45~55mM; the pH value of the Tris buffer containing urea is 7.2~7.

8.

4. The determination method according to claim 1, characterized in that, In step (c), the enzyme digestion treatment temperature is 22~28℃; the time is 18~22h.

5. The determination method according to claim 1, characterized in that, In step (a), the denaturation process includes: Add IgG-like antibody and Tris buffer containing guanidine hydrochloride to an ultrafiltration tube and centrifuge. After centrifugation, add Tris buffer containing guanidine hydrochloride to the ultrafiltration tube and continue centrifugation. Repeat this process 2-4 times to obtain the first mixture.

6. The determination method according to claim 5, characterized in that, The mass-to-volume ratio of the IgG-like antibody to the Tris buffer containing guanidine hydrochloride is 1:(6~10). The Tris buffer containing guanidine hydrochloride is replenished to 0.9 to 1.5 times the original volume.

7. The determination method according to claim 1, characterized in that, In step (b), the fluid replacement process includes: Add the first mixture and Tris buffer containing urea to the ultrafiltration tube, mix well and centrifuge. After centrifugation, add Tris buffer containing urea to the ultrafiltration tube and continue centrifugation. Repeat this process 4 to 6 times to obtain the second mixture.

8. The determination method according to claim 7, characterized in that, The volume ratio of the first mixture to the urea-containing Tris buffer is 1:(3~5). Replenish with Tris buffer containing urea to 0.9 to 1.5 times the original volume.

9. The determination method according to claim 1, characterized in that, In step (c), the amount of N-glycosidase F added per mg of IgG-like antibody is ≥15 U.

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