Abstract Silkworm–baculovirus expression systems are efficient means for the production of recombinant proteins that provide high expression levels and post-translational modifications. Here, we characterized the stability, glycosylation pattern and antibody-dependent cell-mediated cytotoxicity activity of anti-HER2 monoclonal antibodies containing native or glycoengineered mammalian-like N-glycans that were produced by using a silkworm–baculovirus expression system. Compared with a monoclonal antibody produced by using a Chinese hamster ovary cell expression system, the glycoengineered monoclonal antibody had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity activity. These results suggest that silkworm–baculovirus expression systems are next-generation expression systems potentially useful for the cost-effective production of therapeutic antibodies. baculovirus, monoclonal antibody, N-glycosylation, silkworm, trastuzumab The global market for therapeutic monoclonal antibodies (mAbs) continues to grow steadily. More than 300 therapeutic mAbs are currently under development, and the market is expected to further expand in the near future (1). Most mAbs currently on the market are produced by using Chinese hamster ovary (CHO) cell expression systems (2, 3). It is partly because the accumulated knowledge regarding this production process and the characteristics of the antibodies produced, including their post-translational modifications and how to deal with viral safety issues, making it easier to establish effective quality control strategies. However, mAb production systems using yeasts (4, 5), plants (6, 7) and insects (8) are being developed as lower cost alternatives to CHO cell expression systems. Silkworm–baculovirus expression systems provide recombinant proteins with high expression levels and are widely used for the production of functionally active recombinant eukaryotic proteins (9–12). In addition, the system can be easily scaled up and can also modify the amount of production (13). Although the post-translational modifications of proteins that occur in insect cells are similar to those that occur in mammalian cells, it has been reported that the glycosylation patterns of recombinant glycoproteins expressed by insect cells are different from those expressed by mammalian cells (14–16), which could affect their stability and biological activity (17, 18). Recently, methods using the co-expression of glycosyltransferases to modify the N-glycan patterns of recombinant proteins produced by using silkworm–baculovirus expression systems have been reported (19, 20). However, since this technology is relatively new, little is known about the biophysical and functional characteristics of these glycoengineered antibodies. Therefore, to commercialize the antibodies produced by using glycoengineering protocols and silkworm–baculovirus expression systems, more detailed information on the effects of glycosylation pattern on the stability and activity of antibodies produced by using these technologies is needed. Here, we used a silkworm–baculovirus expression system to produce three anti-HER2 mAbs—one with native N-glycans and two with mammalian-like N-glycans. Mammalian-like N-gycans are GlcNAc-terminated or galactose-terminated N-glycans, which are major glycoforms in polyclonal human antibodies (21, 22) and therapeutic mAbs produced by using CHO cell expression systems (5, 8). Human GnT2 and mouse GalT3 were co-expressed with anti-HER2 mAb to produce glycoengieered anti-HER2 mAb. In the silkworm larvae, BmFDL (23) and BmGlcNAcase2 (24), which are GlcNAcases cleaving terminal GlcNAc residues from N-glycans, exist in the fat body and hemolymph, respectively (25). Terminal GlcNAc residues of N-glycans contained recombinant mAbs produced by using silkworm–baculovirus expression system are likely to be cleaved by these GlcNAcases because they are mainly expressed in the fat body and secreted into the hemolymph. Although terminal galactose residues of N-glycans are also likely to be cleaved by the activity of β-galactosidase, which exists in the hemolymph of silkworm larvae (26), it was expected that GlcNAc-terminated N-glycans increase in glycoengineered mAbs by co-expression of GalT3 which transfers galactose residue to terminal GlcNAc. We then characterized these mAbs with respect to their stability and activity. The glycoengineered mAb produced by using the silkworm–baculovirus expression system had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity (ADCC) activity compared with the mAb produced by using a CHO cell expression system, suggesting that glycoengineered mAbs produced by using silkworm–baculovirus expression systems may be useful as biopharmaceuticals. Materials and Methods Construction of recombinant baculovirus expressing anti-HER2 mAbs An Eco81I recognition sequence was inserted into the cysteine proteinase gene deletion site of a hybrid baculovirus DNA (27). This novel baculovirus DNA has two homologous recombination sites in which two foreign target genes can be simultaneously expressed. Originally, the hybrid baculovirus DNA had only one homologous recombination site; however, the DNA was reconstructed to contain a second recombination site. Next, a transfer vector, designated pCPM, was constructed that would cause homologous recombination at the cysteine proteinase gene deletion site. The pCPM vector contained a polyhedrin promoter (28). cDNAs encoding the heavy and light chains of trastuzumab, an anti-HER2 antibody (Herceptin monograph, www.rochecanada.com), were artificially synthesized. The gene encoding the heavy chain was inserted into transfer vectors pMVPLR or pM (28), and the gene encoding the light chain was inserted into transfer vector pCPM. All of the vectors contained the 30K signal sequence (29). Silkworm BmN cells were cultured at 25°C in TC-100 medium supplemented with 10% fetal bovine serum. Then, 5 × 105 BmN cells were co-transfected with linearized baculovirus DNA (100 ng) and the two transfer vectors for heavy and light chain expression (250 ng each) by using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH), and the cells were incubated at 25°C. The culture supernatant was collected 7 days after transfection. Recombinant baculovirus was isolated by using a limiting dilution method. Construction of expression plasmids for use with a CHO cell expression system The genes encoding the heavy and light chain in the transfer vectors were subcloned into the pcDNA3.4 vector, respectively (Thermo Fisher Scientific). Production of anti-HER2 antibodies containing a native N-glycan pattern (Mab-Bac) and mammalian-like N-glycan patterns (Mab-BacG EP and Mab-BacG PP) by using a silkworm–baculovirus expression system Three mAbs (Mab-Bac, Mab-BacG EP, Mab-BacG PP) were prepared by using a silkworm–baculovirus expression system. Mab-Bac contained a native N-glycan pattern, and Mab-BacG EP and Mab-BacG PP contained a mammal-like N-glycan pattern. Two different combinations of promoters were used in these mAbs. In Mab-Bac and Mab-BacG EP, E-vp39 promoter, a late promoter (28), was used to express the heavy chain, and polyhedrin promoter, a very late promoter, was used to express the light chain, whereas in Mab-BacG PP, polyhedrin promoter was used to express both the heavy and light chains. For Mab-Bac production, silkworm larvae were infected with the antibody-expressing recombinant baculovirus produced by using the pMVPLR-heavy chain and the pCPM-light chain transfer vectors. For Mab-BacG EP and Mab-BacG PP production, silkworm larvae were infected with a recombinant baculovirus mixture. The mixture contained antibody-expressing virus (produced by using either the pMVPLR-heavy chain and the pCPM-light chain transfer vectors, or the pM-heavy chain and the pCPM-light chain transfer vectors), human GnT2 (accession no. NM_002408)-expressing virus containing polyhedrin promoter, and mouse GalT3 (accession no. NM_020579)-expressing virus containing vp39 with HR3 (E-vp39) promoter (Suganuma et al., in preparation). The silkworm larvae were raised at 25°C. After 6 days of infection, the hemolymph was collected and centrifuged at 20,000 × g for 60 min. The supernatant was passed through a filter with a pore size of 0.22 µm. Expression of an anti-HER2 antibody by using a CHO cell expression system (Mab-CHO) The plasmids for anti-HER2 antibody were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific) by using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. After 9 days of transfection, the medium containing the recombinant anti-HER2 mAbs was harvested and centrifuged at 2,130 × g for 15 min. The supernatant was passed through a filter with a pore size of 0.22 µm. Purification of mAbs Supernatants were first passed through a MabSelect SuRe column (GE Healthcare). The mAb solutions that had been eluted with 50 mM citrate sodium (pH 3.0) were equilibrated with 1 M Tris–HCl at pH of 9.0. The mAbs were then subjected to size-exclusion chromatography (SEC) with a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) and phosphate-buffered saline (PBS; pH 7.4). SDS-PAGE Purified antibodies were subjected to SDS-PAGE using a 10–20% Tris–glycine gradient gel (ATTO Corporation). For SDS-PAGE under reducing conditions, DTT was added to the samples at a final concentration of 50 mM. The samples were then mixed with the loading buffer and heated for 5 min at 95°C. A total of 2 µg of the antibody solutions were loaded per lane. Precision Plus Protein Standards (Bio-Rad) were used as molecular mass markers. The gels were stained with Coomassie Brilliant Blue by using standard protocols. Size-exclusion chromatography Samples (100 µl) containing 100 µg of antibody were passed through a Superdex 200 Increase 10/300 GL column (GE Healthcare) with PBS as the mobile phase. The column was run at 0.5 ml/min at room temperature. The column effluent was monitored by means of UV detection at 280 nm. The purity of the samples was assessed in their peak areas. Antigen-binding assay The antigen-binding activity of the anti-HER2 mAbs was measured by means of an enzyme-linked immunosorbent assay. Briefly, the wells of a 96-well microtiter plate (Nunc Maxisorp; Thermo Fisher Scientifics) were coated with 50 µl c-erbB-2 Human (ProSpec-Tany TechnoGene) diluted with sodium phosphate buffer (1 µg/ml) and the plate was incubated overnight at 4°C. The wells were washed three times with PBS containing 0.05% Tween-20 (PBS-T) after each of the following steps of the process. After incubation, the wells were blocked with 1% BSA-PBS for 1 h at room temperature. Purified anti-HER2 mAb (50 µl per well; 2.0–0.00012 µg/ml) was added to the wells and the plate was incubated for a further 1 h at room temperature. After washing, horseradish peroxidase-conjugated anti-human kappa light chain antibody (Bethyl Laboratories) was added to the wells to detect the mAbs bound to c-erbB-2 Human. Finally, 100 µl of TMB substrate solution (Kirkegaard & Perry Laboratories) was added to the wells, and after addition of 100 µl of stop solution, the absorbance at 450 nm was measured by using an iMark plate reader (Bio-Rad). Glycan analysis by using liquid chromatography–tandem mass spectrometry The N-glycan patterns of the anti-HER2 mAbs were determined by using liquid chromatography–tandem mass spectrometry (MS/MS). Lyophilized mAbs (20 µg) were dissolved in 0.5 M Tris–HCl (pH 8.6) containing 8 M guanidine–HCl and 5 mM EDTA. After the addition of 1 µl of 0.5 M DTT, the mixture was incubated at 37°C for 90 min. Then, 1.2 µl of 1 M iodoacetic acid was added to the mixture, and the mixture was incubated at 37°C for a further 30 min in the dark. After carboxymethylation, 1 µl of 0.2 M DTT was added to the mixture to quench any unreacted iodoacetic acid. The reaction mixture was applied to a PD-25 column (GE Healthcare) to remove the reagents, and the carboxymethylated protein fraction was dried. The samples were dissolved in 20 µl of 50 mM ammonium bicarbonate buffer containing 0.5 µl of Trypsin Gold (Promega). Then, the mixtures were incubated at 37°C for 4 h. The tryptic digests (1 µl) were dissolved in 14 µl of water containing 2 µl of 2% acetonitrile and 0.1% trifluoroacetic acid solution, and were separated by using an Eksigent Nano LC system (SCIEX) and a NanoLC column (3 µm, ChromXP C18CL; SCIEX). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 90% acetonitrile (solvent B). The chromatography was performed with a gradient from 0% to 55% solvent B in 40 min at a flow rate of 0.3 µL/min. Mass spectrometric analyses were performed by using a TripleTOF 6600 mass spectrometer (SCIEX). Mass spectra were acquired over m/z 400–2, 000 for mass spectrometry (MS), and m/z 100–2,000 for MS/MS. The percentage distribution of the glycopeptides was determined from the areas of the peaks obtained. Differential scanning calorimetry Differential scanning calorimetry (DSC) analyses were performed by using a VP-capillary DSC system (MicroCal). Antibody concentrations were adjusted to 1 mg/mL prior to measurement. The samples were heated from 30 to 95°C at a rate of 1°C/min. The collected data were analyzed by using Origin 7.0 software (OriginLab Corporation). The temperature-induced unfolding profiles were corrected by subtraction of buffer scans and normalized to the molar concentration of the antibody. Characterization of low pH-induced aggregation of anti-HER2 antibodies Samples of mAb in PBS (10 mg/ml) were diluted with 50 mM citrate buffer (pH 3.0) to a final concentration of 1 mg/ml and then incubated for 0, 1, and 3 h at 40°C. After incubation, the samples were centrifuged at 2,300 × g for 4 min and then subjected to SEC or dynamic light scattering (DLS) analysis. SEC was performed by using a Superdex 200 Increase 5/150 GL column (GE Healthcare) with PBS as the mobile phase and a flow rate of 0.45 m/min. DLS measurements were performed by using a Zetasizer Nano (Malvern Instruments) at 25°C. Data were analyzed by using Zetasizer software 7.12 (Malvern Instruments), in accordance with the manufacturer’s instructions. ADCC reporter gene assay An FcγRIIIa activation assay was performed by using reporter cells that produce FcγRIIIa and harbour the response element-driven luciferase gene (30). The HER2-positive human carcinoma cell line SKBR-3 was used as the target cell line. The day before the assay, target cells were seeded at 10,000 cells/well in Opti-MEM I reduced serum medium (Thermo Fisher Scientific) and incubated at 37°C and 5% CO2. The next day, the supernatant was removed and Jurkat/FcγRIIIa/NFAT-Luc receptor cells suspended in Opti-MEM I reduced serum media were added to the target cells at 50,000 cells/well. Anti-HER2 mAb serially diluted in PBS was then added to the effector and target cell mix, and the cells and antibodies were incubated for 4 h at 37°C and 5% CO2. After incubation, luciferase activity was measured by using a ONE-Glo luciferase assay system (Promega) and an EnSight plate reader (PerkinElmer). Results Production of anti-HER2 mAbs by using a silkworm–baculovirus expression system The yields of Mab-Bac and Mab-BacG EP produced with the same breeding lot silkworm were 4.9mg and 4.8 mg per 100 silkworm larvae, respectively, revealing that co-transfection with two different kinds of glycosyltransferase expression virus did not adversely affect the yield of Mab-BacG EP obtained. The yield of Mab-Bac PP was 3.1 mg per 100 silkworm larvae. For comparison, the yield of Mab-CHO, which was produced by using a CHO expression system, was 1.8 mg per 100 ml of culture medium. The purity of the mAbs was confirmed by means of SDS-PAGE and SEC. SDS-PAGE gel analysis of the purified mAbs is shown in Fig. 1. All of the mAbs displayed typical antibody features both under reducing and non-reducing conditions. Two bands corresponding to the heavy chain (50 kDa) and light chain (25 kDa) were observed under reducing conditions. The SEC profiles of the anti-HER2 mAbs are shown in Supplementary Fig. S1. All four of the mAbs were eluted from the SEC column as a single prominent peak and had a purity of more than 96%. The peak for each mAb was observed at the same retention volume. An enzyme-linked immunosorbent assay showed that all of the mAbs had comparable antigen binding activities with respect to HER2 (Supplementary Fig. S2). Fig. 1 View largeDownload slide Characterization of the anti-HER2 mAbs by means of SDS-PAGE. mAbs (2 µg/lane) were subjected to SDS-PAGE under reducing and non-reducing conditions, and then stained with Coomassie Brilliant Blue. Fig. 1 View largeDownload slide Characterization of the anti-HER2 mAbs by means of SDS-PAGE. mAbs (2 µg/lane) were subjected to SDS-PAGE under reducing and non-reducing conditions, and then stained with Coomassie Brilliant Blue. Analysis of the glycosylation patterns of the mAbs The glycosylation patterns of the mAbs were determined by means of liquid chromatography–MS/MS (Fig. 2). Mab-Bac contained mainly insect-specific paucimannose N-glycans (e.g. M4, M3, M2, M3F or M2F [95.3%]) and few GlcNAc-terminated N-glycans (e.g. G0-N, G0, G0F-N, or G0F [1.7%]). In contrast, Mab-CHO contained mainly GlcNAc-terminated N-glycans (92.0%) and few paucimannose N-glycans (2.9%). Mab-BacG EP and Mab-BacG PP, which were co-transfected with two different kinds of glycosyltransferase expression virus, each contained more GlcNAc-terminated N-glycans than did Mab-Bac; in Mab-BacG EP, the percentages of paucimannose N-glycans and GlcNAc-terminated N-glycans were 53.6% and 43.1%, respectively, and in Mab-BacG PP, they were 22.3% and 66.3%, respectively. Mab-BacG PP contained the highest percentage of afucosylated N-glycans (31.9%), followed by Mab-BacG EP (9.4%), Mab-CHO (8.1%) and Mab-Bac (6.1%). Fig. 2 View largeDownload slide Glycosylation patterns of the anti-HER2 mAbs. Percentage distributions of the N-glycans were calculated by using the relative peak area average values. Total is not 100.0% because the percentages are rounded to one decimal place. M7, Man7GlcNAc2; M6, Man6GlcNAc2; M5, Man5GlcNAc2; M4, Man4GlcNAc2; M3, Man3GlcNAc2; M2, Man2GlcNAc2; M3F, Man3GlcNAc2Fuc; M2F, Man2GlcNAc2Fuc; G0-N, GlcNAcMan3GlcNAc2; G0, GlcNAc2Man3GlcNAc2; G0F-N, GlcNAcMan3GlcNAc2Fuc; G0F, GlcNAc2Man3GlcNAc2Fuc; G1F, GalGlcNAc2Man3GlcNAc2Fuc. Fig. 2 View largeDownload slide Glycosylation patterns of the anti-HER2 mAbs. Percentage distributions of the N-glycans were calculated by using the relative peak area average values. Total is not 100.0% because the percentages are rounded to one decimal place. M7, Man7GlcNAc2; M6, Man6GlcNAc2; M5, Man5GlcNAc2; M4, Man4GlcNAc2; M3, Man3GlcNAc2; M2, Man2GlcNAc2; M3F, Man3GlcNAc2Fuc; M2F, Man2GlcNAc2Fuc; G0-N, GlcNAcMan3GlcNAc2; G0, GlcNAc2Man3GlcNAc2; G0F-N, GlcNAcMan3GlcNAc2Fuc; G0F, GlcNAc2Man3GlcNAc2Fuc; G1F, GalGlcNAc2Man3GlcNAc2Fuc. Evaluation of thermal stability The thermal stability of the mAbs was evaluated by using DSC. The temperature-induced unfolding profiles of the four mAbs are shown in Fig. 3. The profile of each mAb consisted of two transition peaks, with the second peak having a larger amplitude. The profiles observed for the anti-HER2 antibodies were similar to a previously reported profile for trastuzumab (31). In the profiles, the first transition peak represents unfolding of the CH2 domain, and the second transition peak represents unfolding of the Fab and CH3 domains. The values of Tm for CH2 domain (Tm1) of Mab-Bac and Mab-BacG EP were lower than that of Mab-CHO by 3.9°C and 1.6°C, respectively (Table I). The value of Tm1 of Mab-BacG PP was comparable with that of Mab-CHO. Table I. Differential scanning calorimetry measurement of the melting transition of the anti-HER2 mAbs Tm1 Tm2 Tm3 Mab-Bac 68.33 79.30 NA Mab-BacG EP 70.57 79.47 NA Mab-BacG PP 72.83 79.65 NA Mab-CHO 72.20 79.71 NA Tm1 Tm2 Tm3 Mab-Bac 68.33 79.30 NA Mab-BacG EP 70.57 79.47 NA Mab-BacG PP 72.83 79.65 NA Mab-CHO 72.20 79.71 NA NA, not available. Table I. Differential scanning calorimetry measurement of the melting transition of the anti-HER2 mAbs Tm1 Tm2 Tm3 Mab-Bac 68.33 79.30 NA Mab-BacG EP 70.57 79.47 NA Mab-BacG PP 72.83 79.65 NA Mab-CHO 72.20 79.71 NA Tm1 Tm2 Tm3 Mab-Bac 68.33 79.30 NA Mab-BacG EP 70.57 79.47 NA Mab-BacG PP 72.83 79.65 NA Mab-CHO 72.20 79.71 NA NA, not available. Fig. 3 View largeDownload slide Temperature-induced unfolding profiles of Mab-Bac, Mab-BacG EP, Mab-BacG PP and Mab-CHO, as measured by using DSC. In each thermogram the dashed line represents the profile for Mab-CHO. Fig. 3 View largeDownload slide Temperature-induced unfolding profiles of Mab-Bac, Mab-BacG EP, Mab-BacG PP and Mab-CHO, as measured by using DSC. In each thermogram the dashed line represents the profile for Mab-CHO. Acid-induced aggregation In the process of manufacturing of antibodies, low-pH treatment is used as a common virus inactivation after purification by affinity chromatography, but this treatment can also give a significant stress to antibodies. To examine the effects of low-pH treatment on the developed mAbs, samples of Mab-Bac and Mab-CHO in PBS (10 mg/ml) were diluted with 50 mM citrate buffer (pH 3.0) to a final concentration of 1 mg/ml and then incubated for 0, 1, and 3 h at 40°C. The degree of aggregation in samples collected at different incubation times was determined by means of SEC and DLS. The SEC profiles obtained are shown in Fig. 4. The percentages of oligomers and monomers are shown in Table II. Time-dependent oligomer formation was observed for both mAbs. The intensity-weighted particle-size distribution calculated from the DLS signal revealed that aggregate formation was limited (Supplementary Fig. S3). Table II. Percentages of oligomers and monomers Incubation time (h) Oligomers (%) Monomers (%) Mab-Bac 0 0.0 100.0 1 11.7 88.3 3 20.6 79.4 Mab-CHO 0 0.0 100.0 1 11.2 88.8 3 21.9 78.1 Incubation time (h) Oligomers (%) Monomers (%) Mab-Bac 0 0.0 100.0 1 11.7 88.3 3 20.6 79.4 Mab-CHO 0 0.0 100.0 1 11.2 88.8 3 21.9 78.1 Table II. Percentages of oligomers and monomers Incubation time (h) Oligomers (%) Monomers (%) Mab-Bac 0 0.0 100.0 1 11.7 88.3 3 20.6 79.4 Mab-CHO 0 0.0 100.0 1 11.2 88.8 3 21.9 78.1 Incubation time (h) Oligomers (%) Monomers (%) Mab-Bac 0 0.0 100.0 1 11.7 88.3 3 20.6 79.4 Mab-CHO 0 0.0 100.0 1 11.2 88.8 3 21.9 78.1 Fig. 4 View largeDownload slide Size-exclusion chromatograms recorded during aggregation of the anti-HER2 antibodies at pH 3.0 and 40°C for 0, 1 and 3 h. Fig. 4 View largeDownload slide Size-exclusion chromatograms recorded during aggregation of the anti-HER2 antibodies at pH 3.0 and 40°C for 0, 1 and 3 h. ADCC-reporter gene assay The level of FcγRIIIa activation in the presence of target cells was measured as a surrogate of ADCC activity by using a reporter assay. The reporter assay was performed by mixing effector cells, target cells and various concentrations of the anti-HER2 mAbs. The ADCC activity of Mab-BacG EP and Mab-BacG PP was higher than that of Mab-Bac and Mab-CHO (Fig. 5). The relative activity of each antibody was Mab-BacG PP > Mab-BacG EP > Mab-Bac, Mab-CHO. Fig. 5 View largeDownload slide Results of a reporter-gene assay to assess the ADCC of the anti-HER2 mAbs. Fig. 5 View largeDownload slide Results of a reporter-gene assay to assess the ADCC of the anti-HER2 mAbs. Discussion Here, we characterized three anti-HER2 mAbs produced by using a silkworm–baculovirus expression system (Mab-Bac, Mab-BacG EP and Mab-BacG PP), and compared the results with an antibody produced by using a CHO expression system (Mab-CHO). Two different promoters, polyhedrin and E-vp39, were used to express the heavy chain containing an N-glycosylation site. The results of a previous study examining the expression of various glycoproteins suggested that a combination of promoters (i.e. polyhedrin for human GnT2 and E-vp39 for mouse GalT3) was an effective means of modifying glycosylation pattern (our unpublished results). Therefore, we first evaluated the influence of promoter selection on the N-glycan pattern in the developed mAbs. Glycan analysis showed different N-glycan patterns in Mab-BacG EP and Mab-BacG PP, that is, more GlcNAc-terminated N-glycans were detected in Mab-BacG PP, where the polyhedrin promoter was used to express the heavy chain, than in Mab-BacG EP, where the E-vp39 promoter was used. This suggested that different promoters produce mAbs with different glycosylation patterns. While GlcNAc-terminated N-glycans were significantly increased in both Mab-BacG EP and Mab-BacG PP compared with in Mab-Bac, galactose-terminated N-glycans were detected only in Mab-BacG PP and their percentage was small (Fig. 2). Injection of galactosidase inhibitor to silkworm larvae has been effective in increasing percentage of galactose-terminated N-glycans in glycoengineered recombinant glycoproteins (our unpublished data). Galactose residues attached to N-glycans in this study were likely to be cleaved by β-galactosidase activity in the hemolymph. DSC analysis demonstrated that Mab-Bac was less thermally stable than Mab-CHO (Fig. 3 and Table I). Although the onset of the transition peak representing the CH2 domain of Mab-BacG EP was similar to that of Mab-Bac, the shape of the peak for Mab-BacG EP was broader, and the Tm1 of Mab-BacG EP was higher; a mixture of two types of antibodies—one containing paucimannose N-glycans and the other containing GlcNAc-terminated N-glycans—is thought to be the cause of the broad transition peak observed for Mab-BacG EP. Mimura et al. reported that glycosidase-mediated removal of terminal GlcNAc residues significantly reduces the stability of the CH2 domain (32). In the present study, the Tm1 of Mab-BacG PP, which contained more GlcNAc-terminated glycans (66.3%) than did Mab-BacG EP (43.1%), was larger than that of Mab-BacG EP and comparable with that of Mab-CHO. Therefore, attaching GlcNAc residues to the non-reducing end most likely increases the stability of glycoengineered mAbs produced by using a silkworm–baculovirus expression system. The glycosylation pattern is also known to affect the tendency of a protein to aggregate. Hristodorov et al. reported that deglycosylated mAbs were significantly more susceptible to aggregation at pH 3.0 than their glycosylated counterparts (33). In the present study, we showed that Mab-Bac had comparable tendency to aggregate at low pH compared to Mab-CHO (Fig. 4 and Table II). This suggests that the tendency of antibodies containing paucimannose N-glycans to aggregate at low pH is comparable with that of the antibodies containing GlcNAc-terminated N-glycans. The glycosylation pattern of the Fc region in IgG1 affects the antibody’s ADCC activity (34). Although the ADCC activity of Mab-Bac was comparable with that of Mab-CHO, both Mab-BacG EP and Mab-BacG PP had a higher ADCC activity than Mab-Bac and Mab-CHO. Antigen binding analysis revealed that the antigen binding activities of all four mAbs were comparable (Supplementary Fig. S2). Therefore, we hypothesized that the observed difference in ADCC activity among the mAbs was due to minor differences in the glycosylation patterns in the Fc region. We focused on two types of N-glycans in the Fc region. The first was afucosylated N-glycans. mAbs containing afucosylated N-glycans generally have a greater ADCC activity (8, 35–37). Most mammalian cell lines, including CHO cells, produce heavily fucosylated mAbs (38). Therefore, several methods for producing afucosylated antibodies have been developed. For example, CHO cells in which the FUT8 gene encoding α1,6-fucosyltransferase is disrupted to yield mAbs completely lacking core fucose (39, 40). Alternatively, afucosylated antibodies can be produced by using CHO cells overexpressing β-1,4-N-acetylglucosaminyltransferase III, which mediates the bisecting N-GlcNAc modification; attaching bisecting N-GlcNAc to the N-glycans inhibits the addition of fucose. The genetically engineered cells produce mAbs with an increased amount of bisecting GlcNAc content, and the ADCC activity in the mAbs is elevated (41). The percentage of afucosylated N-glycans in Mab-Bac, Mab-BacG EP, Mab-BacG PP and Mab-CHO was 6.1%, 9.4%, 31.9% and 8.1%, respectively. It might be possible to explain why Mab-BacG PP showed the highest ADCC activity from the viewpoint of the percentage of afucosylated N-glycans. Although a reduction of fucosylated N-glycans was observed in other glycoproteins co-transfected with glycosyltransferase expression virus (data not shown), the mechanism for the reduction in fucosylated N-glycans is unclear. The second type of N-glycan in the Fc region was GlcNAc-terminated N-glycans. Kurogochi et al., compared the ADCC activities among antibodies with afucosylated N-glycans, reported that antibodies containing GlcNAc-terminated N-glycans (G0) had a slightly higher ADCC activity than those containing paucimannose N-glycans (M3) (42). In the present study, the percentages of GlcNAc-terminated N-glycans of Mab-Bac, Mab-BacG EP, Mab-BacG-PP and Mab-CHO were 1.7%, 43.1%, 66.3% and 92.0%, respectively. The percentages of afucosylated N-glycans with terminal GlcNAc residues (G0-N, G0) in Mab-Bac, Mab-BacG EP, Mab-BacG PP and Mab-CHO were 0.0%, 4.6%, 18.5% and 1.1%, respectively. This suggests that a reduction in the relative amount of fucosylated N-glycans accompanied by an increase in the relative amount of afucosylated N-glycans with terminal GlcNAc residues was an underlying factor in the relatively higher ADCC activity of Mab-BacG EP compared with Mab-Bac and Mab-CHO. In conclusion, we demonstrated that although the CH2 domain of mAb produced by using a silkworm–baculovirus expression system was slightly less stable than that of mAbs produced by using a CHO cell expression system, there was no difference in antigen binding activity or tendency to aggregate at low pH. 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PLoS One 10, e0132848 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ADCC antibody-dependent cell-mediated cytotoxicity CHO Chinese hamster ovary DLS dynamic light scattering DSC differential scanning calorimetry GlcNAc N-acetylglucosaine mAb monoclonal antibody MS mass spectrometry MS/MS tandem mass spectrometry SEC size exclusion chromatography © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
The Journal of Biochemistry – Oxford University Press
Published: Feb 5, 2018
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