Comparative assessment of glycosylation of recombinant human ...（二）
Experimental section
Chemicals and reagents
One lot of PuregonR-HP of 50 IU/0.5mL and two lots of PuregonR-HP of 100 IU/0.5mL (rhFSH) (Organon, Oss, Netherlands) were purchased in China. All strengths were presented as liquor. One lot of high purity uhFSH (>98%) was obtained directly from the manufacturer, Shanghai Techwell Biopharmaceutical Company (Shanghai, China), as active pharmaceutical ingredient. Clean gel IEF was obtained from Amersham Biosciences (Piscataway, NJ). Dithiothreitol (DTT), iodoacetamide (IAM), NH4HCO3, trypsin, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) were purchased from Sigma-Aldrich (St. Louis, MO). Chymotrypsin was obtained from Roche (Penzberg, Germany). PNGase F, from New England Biolabs (Ipswich, MA); 10kDa MWCO centrifugal filters, from Millipore (Bedford, MA); and porous graphitized carbon (PGC) columns, from Grace (Columbia, MD). Glycoclean column and 2-AB labeling kit were obtained from Prozyme (San Leandro, CA). All other reagents were purchased from Sigma (St. Louis, MO) or Fluka (Bucho, Switzerland).
Purification of rhFSH
The rhFSH isolation by SEC column was performed as previously described.16 For this study, three lots of PuregonR injection were dispensed and combined, then loaded on to a Sephadex G-25 column (GE Healthcare life science) at a flow rate of 2 mL/min under isocratic condition of buffer A (2 mM phosphate, pH 7.4). The baseline of the flow through was monitored at 215 and 280 nm until a return to baseline was observed. The eluted rhFSH was collected and applied to a 10 kDa MWCO Centrifuge device (50 mL). The centrifuge was spun for 1 h at 3,000 rpm at 4°C and washed with buffer of 5 mM phosphate containing 150 mM NaCl pH 7.4. After concentration, fractions were combined and stored at -80°C. The isolated rhFSH concentration was determined by SEC-HPLC (superdex 75, GE Healthcare life science), and the protein purity, quality and recovery were compared to PuregonR.
Intact protein and α/β-subunit mass analysis
For intact molecular weight assay, the hFSH samples were subjected to 10% SDS-PAGE under non-reducing condition. Gels were stained by Coomassie R-250. The subunits of hFSH were analyzed in linear mode by the 4800 Plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA).14 The reduced hFSH subunits were further analyzed by the Acquity UPLC system connected on-line to a quadrupole time-of-flight tandem mass spectrometer (Xevo G-2S, Waters Corporation, Milford, MA). The column was a Waters Acquity UPLC BEH C4 column (2.1 mm × 100 mm, 1.7 μm particle). The flow rate was 0.2 mL/min using a gradient from 15% to 25% solvent B (100% acetonitrile with 0.1% formic acid) in 25 min at a column temperature of 80°C. Solvent A was 0.1% formic acid in water. For mass spectrometric analysis, a MS data acquisition method was employed with a scan range m/z 500-2500.
Isoelectric focusing (IEF) for hFSH isoform pattern distribution
A commercial dried polyacrylamide gel, Clean gel IEF, was rehydrated in a solution containing an appropriate composition of carrier ampholytes necessary to create the desired pH gradient (3–6). The separated proteins and pI marker were stained by Coomassie blue.
Quantitative determination of sialic acid
The sialic acid contents of both hFSHs were quantitatively estimated using a colorimetric resorcinol-hydrochloric acid method. The method was followed ChP 2010 Vol III: Appendix VI C, 35. The mean relative content and SD were determined for two replicates per sample.
Protease and PNGase F digestion
For N-glycosylation site identification and O-glycopeptide analysis, both hFSHs were reduced by incubating with 10 mM DTT for 30 min at 57°C and alkylated with 20 mM IAA at room temperature for 1h in the dark. One molar NH4HCO3 was diluted in the sample to make its final concentration to 50 mM. Deglycosylation was performed by adding 300IU PNGase F at 37°C overnight. Trypsin was added at 1:50 w/w and incubated at 37°C for 14 h. Digestion was quenched by adding 10% TFA.For N-glycopeptide analysis, samples of each hFSH were reduced and alkylated.Chymotrypsin was added at 1:25 w/w in 100 mM Tris-HCl, 10mM CaCl2 (pH 7.8).Enzymatic digestion was performed overnight at 25°C.
Releasing and labeling of N-glycans
The oligosaccharides of hFSH were released by PNGase F and desalted on a non-porous graphitized carbon. Then 2-AB labeling and purification of N-glycans were performed as described in previous study.17
Profiling and relative quantification of N-glycans
Analysis of 2-AB labeled N-glycans was performed on a Waters Acquity UPLC BEH glycan column (2.1 mm × 150 mm, 1.7 μm particle) using an Acquity UPLC with a fluorescence detector (Waters Corporation, Milford, MA).18 The glycans were analyzed using mobile phase A, 50 mM ammonium formate (pH 4.4), and mobile phase B,acetonitrile. The gradient was 70-53% B in 35 min with a flow rate of 0.4 mL/min and
column temperature of 40°C. Samples were injected in 80% acetonitrile. The fluorescence detection was carried out using an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The eluted positions of the N-glycans were determined in glucose units (GU) by comparison with a standard dextran hydrolyzate 2-AB labeled (dextran ladder).19 The LC-MS data were acquired on Xevo G-2S (Waters Corporation, Milford, MA). The interested parent ions were selected for MS2 data acquisition.20
Identification of N-glycan sites
A multiplexed data acquisition method (MSE) was employed for mass spectrometric analysis of tryptic digest of hFSHs. The instrument and analyzed method were described previously.20
Intact N-glycopeptide analysis
N-Glycopeptides were analyzed by the Acquity UPLC system connected on-line to Xevo G-2S. The column was also a Waters Acquity UPLC BEH glycan column (2.1 mm × 150 mm, 1.7 μm particle). The experiment was performed as described elsewhere.21 The flow rate was 0.2 mL/min using a gradient of 1% to 40% solvent B (100% acetonitrile with 0.1% formic acid) in 63 min, followed by an increase to 90% B in 4 min, and then to 90% B in 6 min. Solvent A was 0.1% formic acid in water.
Intact O-glycopeptide analysis
O-Glycopeptides were analyzed by nano LC-MS/MS on an Orbitrap Fusion Tribrid MS (Thermo Fisher Scientific, San Jose, CA) coupled to an EASY-nLC System (Thermo Fisher Scientific, San Jose, CA). Peptide mixtures were loaded onto a Magic C18 spray tip 15 cm × 75 μm i.d. column (Michrom Bioresources) and separated at a flow rate of 350 nL/min using a gradient of 8% to 22% solvent B (100% acetonitrile with 0.1% formic acid) in 54 min, followed by an increase to 35% B in 15 min, and then to 90% B in 10 min and held for another 6 min. Solvent A was 0.1% formic acid in water.Data acquisition was performed under data dependent acquisition (DDA) with HCD-pd-ETD. The parameters settings were: top speed mode with 3 s cycle time; FT MS: scan range (m/z) = 350−2000; MS resolution = 120K; MS2 resolution=30K; other parameters followed a previous report.22 To restrict the ETD MS2 data acquisition to true O-glycopeptide precursors, the preceding HCD can be applied in producing diagnostic glyco-oxonium ions (138.0545, 204.0867 and 366.1396) using as a filtering criterion to trigger ETD in the HCD-pd-ETD mode.
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项目成果
