Anal. Chem. 1997, 69, 2517-2524
Quantitative Comparison of Global Carbohydrate Structures of Glycoproteins Using LC-MS and In-Source Fragmentation Istvan Mazsaroff,* Wen Yu, Brian D. Kelley, and James E. Vath†
Genetics Institute, Inc., One Burtt Road, Andover, Massachusetts 01810
A comparative method for the quantitative analysis of the ratio of oxonium fragment (reporter) ions derived from sialic acid and N-acetylhexosamine residues on a large intact glycoprotein, the B domain of recombinant human factor VIII (rhFVIII), was developed. The method utilized liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) on a single-quadrupole instrument. During development, systematic approaches such as full-matrix and simplex strategies were used for the optimization of the signal-to-noise ratio by controlling source temperature and cone voltage. The method was found to be precise (RSD ) 0.84%), sensitive (capable of differentiating 1 sialic acid residue change among at least 29 sialic acids on a 103-kDa glycoprotein that is 38% carbohydrate), applicable to a wide range of loading (11.6-372 µg of FVIII), and accurate according to a comparison to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Combining the method with enzymatic removal of N-glycans, selective O-glycan analysis was also performed leading to differential fragment ion analysis ascribed to N- and O-glycans. Quantitative ESI in-source dissociation MS combined with LC can generally be used for glycoproteins, as one of the indicators, to compare the distribution of carbohydrate residues over N- and O-glycans, to investigate their isoforms, and compare batch-to-batch characteristics of biopharmaceuticals. In recent years, there has been an increased emphasis on structural or biochemical equivalence in the biopharmaceutical industry.1 Establishing biochemical equivalence requires the industry to develop analytical methods that are simple, reproducible, and at least semiquantitative and preferably quantitative. Methods focused on in-depth characterization are often laborious and time-consuming and can be subjective. Once the detailed structure is known, additional, relatively simple assays may be needed to assess biochemical equivalence. Therefore, one of the most effective strategies would include two levels of investigations. In the first level, the batches are compared and screened with a small number of simple methods. When differences are observed by these screening methods, detailed biochemical methods developed for product characterization can be used to find the exact source of difference. † Present address: Millennium Pharmaceuticals, 640 Memorial Drive, Cambridge, MA 02139. (1) Proceedings of Characterization of Biotechnology Pharmaceutical Products; conference sponsored by FDA; Washington, DC, December 11-13, 1995.
S0003-2700(96)01116-X CCC: $14.00
© 1997 American Chemical Society
Mass spectrometry is a rapid, versatile, and highly informative analytical method, which has great potential for detecting structural changes and demonstrating structural equivalence for biopharmaceuticals. Since the molecular mass of a protein is a direct link to its chemical composition including the amino acid sequence and posttranslational modifications, it was proposed to use molecular mass as an important benchmark for assessing protein biochemical equivalence.2 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)3 is a primary candidate technique for establishing biochemical equivalence because it is more applicable to large glycosylated proteins and the precision of molecular mass measurement is suitable to detect subtle changes occurring from amino acid or glycosylation variants. We will be investigating factor VIII, a glycoprotein having a molecular mass so large (∼280 kDa) that the domains resulting from thrombin digestion rather than the intact molecule are characterized by MALDI-TOF MS, for a simple assay for biochemical equivalence. The largest domain is the B domain (amino acids 741-1313) with a molecular mass of ∼103 kDa. Approximately 38% of this mass is carbohydrate related. Due to the microheterogeneity of carbohydrate isoforms, it is not surprising that the B-domain peak in the MALDI mass spectrum is ∼20 kDa wide. While mass spectra generated by MALDI-TOF MS of all the other domains (43, 50, and 73 kDa) are selective enough to detect changes in carbohydrate or peptide structures, in the case of B domain, due to the naturally wide heterogeneity this glycoprotein, another screening tool is required to address the global characterization of carbohydrate structures. There are elegant methods for the investigation of carbohydrate structures from glycoproteins; some of them employ simple mass measurements before and after glycosidase treatment of glycopeptides4 and free oligosaccharides.5 Other methods utilize tandem mass spectrometry in combination with fast atom bombartment6-9 or electrospray ionization collision-induced dissociation (CID) MS/MS of permethylated carbohydrates.10 Re(2) Yu, W.; Mazsaroff, I.; Vath, J. E.; Rouse, J. C.; Scoble, H. A. Assessing the Structural Equivalence of Complex Recombinant Glycoproteins. Proceedings of the 44th ASMS Conference on Mass Spectroscopy and Allied Topics, Portland, OR, May 12-16, 1996. (3) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A. (4) Sutton, C. W.; O’Neill, J. A.; Cottrell, J. S. Anal. Biochem. 1994, 218, 34. (5) Rouse, J. C.; Vath, J. E. Anal. Biochem. 1996, 238, 82. (6) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G. Anal. Chem. 1990, 62, 279. (7) Dongre, A. R.; Wysocki, V. H. Org. Mass Spectrom. 1994, 29, 700. (8) Domon, B.; Muller, D. R.; Richter, W. J. Int. J. Mass Spectrom. 1990, 19, 390.
Analytical Chemistry, Vol. 69, No. 13, July 1, 1997 2517
cently, MALDI postsource decay TOF11 was used for the isomeric differentiation of asparagine-linked oligosaccharides.12 Due to the information obtained and the complexity of the assays, all of these techniques belong to the group of second-level characterization techniques. There is no appropriate, direct, quantitative method available to assess the global compositional properties of carbohydrate moieties of glycoproteins. Alternatively, the types of carbohydrate moieties on glycoproteins may be selectively characterized in the form of glycopeptides of proteolytic digests using LC-MS/MS on a triple-quadrupole instrument with fragmentation from collision-induced dissociation with the advantage that coeluting glycopeptides could be resolved.13-18 The same carbohydrate fragment ions can also be formed in a nonselective manner as a result of collisions in the declustering region of the electrospray ionization source, which extends this capability to single-quadrupole instruments.14,15,17 In this paper, the use of these carbohydrate-related fragment ions for comparative glycoprotein analysis is presented utilizing a single-quadrupole MS. This in-source fragmentation involves stepping of the collision energy during the LC-ESI MS analysis which can indicate the peptides carrying carbohydrate moieties through the detection of low-mass carbohydrate fragment ions in selective ion recording (SIR) or scan mode. During these analyses the oxonium ions of carbohydrates such as hexose (Hex+, m/z 163), N-acetylhexosamine (HexNAc+, m/z 204), sialic acid (Nacetylneuraminic acid, SA+, m/z 292), and hexose-N-acetylhexosamine (HexHexNAc+, m/z 366) were generated. Since HexNAc+ and HexHexNAc+ ions can be derived not only from terminal sugars but also by two-bond cleavages from internal sugars, these are the most frequently detected sugar-related ions.14 The development, optimization, and evaluation of a comparative method are described, based on the generation of carbohydrate oxonium fragment ions from a large intact glycoprotein (the B domain of recombinant human factor VIII). The quantification of these fragment ions in selective ion recording mode is also assessed. EXPERIMENTAL SECTION Materials. Glycerol, sodium acetate, HPLC-grade 2-propanol, acetonitrile, and water were purchased from Fisher Scientific (Fair Lawn, NJ). Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL). Sodium iodide (99.99+%) and sinapinic acid were obtained from Aldrich (Milwaukee, WI). Thrombin was purchased from Haematologic Technologies (Essex Junction, VT), N-Glycosidase F (PNGase F) from Boehringer Mannheim (Indianapolis, IN) and D-phenylalanyl-prolyl-arginyl-chloromethylketone (PPACK) from Calbiochem (San Diego, CA). Neuramidase, thioredoxin, and bovine serum albumin (BSA) were obtained from (9) Gillece-Castro, B. L.; Burlingame, A. L. In Mass Spectrometry; McCloskey, J. A., Ed.; Methods in Enzymology 193; Academic: San Diego, CA, 1990; Chapter 37. (10) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772. (11) Rouse, J. C.; Yu, W.; Martin, S. A. J. Am. Soc. Mass Spectrom. 1995, 6, 822. (12) Rouse, J. C.; Strang, A.-M.; Yu, W.; Vath, J. E., submitted for publication. (13) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877. (14) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183. (15) Bean, M. F.; Annan, R. S.; Hemling, M. E.; Mentzer, M.; Huddleston, M. J.; Carr, S. A. In Techniques in Protein Chemistry VI; Crabb, J. W., Ed.; Academic Press: San Diego, CA, 1995, 107. (16) Hunter, A. P.; Games, D. E. J. Rapid Comm. Mass Spectrom. 1995, 9, 42. (17) Conboy, J. J.; Henion, J. D. J. Am. Soc. Mass Spectrom. 1992, 3, 804. (18) Greis, K. D.; Hayes, B. K.; Komer, F. I.; Kirk, M.; Barnes, S.; Lowary, T. L.; Hart, G. W. Anal. Biochem. 1996, 234, 38.
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Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
Sigma (St. Louis, MI). Recombinant human antihemophilia factor (rhAHF, rhFVIII, or factor VIII) was produced by Genetics Institute. Thrombin Digestion. Factor VIII (FVIII) was digested in 1-mL aliquots at a final concentration of 0.31 mg/mL. A 16-µL aliquot of thrombin (100 unit/mL) was mixed with 288 µL of 50% glycerol and added to 1 mL of protein solution. The mixture was incubated at 37 °C for 1 h; 8.2 µL of 8.8 mM PPACK was added to the solution to stop the reaction. Neuraminidase Digestion. A 50-µL aliquot of neuraminidase (10 unit/100 µL) in 50 mM sodium acetate, pH 5.0 was added to 1.3 mL of 0.31 mg/mL factor VIII thrombin digest. The mixture was incubated at 37 °C for 4 h. After the completion of digestion, the sample was kept frozen (-80 °C) until use. Partial digestion was performed on a series of aliquots of thrombin digest (500 µL) using identical conditions as above except incubation time was varied between 0.5 and 12 min. Digestion was stopped by adding 2 µL of TFA to the digest on ice. N-Glycosidase F (PNGase F) Digestion. A 100-µL (20 units) PNGase F aliquot was added to 1.3 mL of 0.31 mg/mL factor VIII thrombin digest. The mixture was incubated at 37 °C for 3 h and kept frozen (-80 °C) until use. Desalting Thrombin-Digested Samples. Desalting of digested samples were performed based on similar methods19 on a TSK G2000 SWXL 7.8 × 300 mm column (TosoHaas, Montgomeryville, PA) using 50% acetonitrile in 0.1% TFA as mobile phase at 1 mL/min flow rate on an Integral Micro-Analytical Workstation (PerSeptive Biosystems, Framingham, MA). Effluent was monitored at 214 and 230 nm. (Sample buffer of factor VIII carried Tween, which had high absorbance at 230 nm.) A fraction containing all domains was collected and vacuum-dried using SpeedVac (Model SVC 100, Savant Instruments, Farmingdale, NY). For flow-injection studies in temperature and cone voltage optimization, samples were resolubilized in 40% 2-propanol in 0.1% TFA. MALDI-TOF MS of B Domain. The B domain of factor VIII (typically 200 pmol) was isolated using reversed-phase liquid chromatography according to the description in the section Liquid Chromatography. The lyophilized fraction of B domain was resolubilized in 20 µL of saturated sinapinic acid in 67% acetonitrile in 0.1% TFA. A 2-µL aliquot of the solution was applied to the probe and air-dried. Escherichia coli thioredoxin and BSA as external standards were used for mass calibration of the instrument. Based on the measurement of the standards, mass accuracy was expected to be 0.12%. Measurements were performed on a time-of-flight mass spectrometer (Bruker Reflex) equipped with a matrix-assisted laser desorption ion source and a UV laser (337 nm). The instrument was operated in linear and positive ion modes. Typically, 50-200 shots were collected for each spectrum. Mass values were calculated from multiple spectra taken from multiple areas of the sample surface. Liquid Chromatography-In-Source Dissociation-Mass Spectrometry. (a) Liquid Chromatography. Reversed-phase liquid chromatographic separation of FVIII thrombin domains was performed on a POROS R1/H 4.6 × 50 mm poly(styrenedivinylbenzene) column (PerSeptive Biosystems) utilizing a BioCAD Workstation (PerSeptive Biosystems), a Waters 717 autosampler (Waters, Milford, MA), and an Advantec SF-2120 fraction collector (ToyoKaisha, Tochigi, Japan). Typically (19) Porter, T., in preparation.
∼60 µg (∼0.2 nmol) of digested factor VIII was injected. An elution gradient consisted of three segments, the first spanned from 14.25 to 36% 2-propanol in 0.1% (w/v) TFA over 2.9 min, the second to 40.9% over 5 min, and the third up to 66.5% 2-propanol over 2.7 min. Volumetric flow rate was 2 mL/min. After the column, the flow line was split and a 12 µL/min stream was introduced to the probe of an electrospray quadrupole mass spectrometer. (The split ratio was 2000:12 ) 167). The rest of the effluent passed through the UV detector (214 or 280 nm), and peaks were collected by the fraction collector for further analysis on MALDI-TOF MS. The chromatography column was cleaned and regenerated with a thorough procedure consisting of two gradients from 14.3 to 95% 2-propanol in 0.1% TFA and a 33-column-volume equilibration. (b) Electrospray Mass Spectrometry. The fragment ions were recorded on an electrospray single-quadrupole mass spectrometer (Micromass Platform, Altrincham, England) which was calibrated using sodium iodide clusters (1 mg/mL) in the mass range of 100-500 u. (In this study, determination of the molecular mass of B domain was not a concern.) Gas flow rates, probe position, and a number of source and MS parameters were optimized with sodium iodide flow injection. Optimized mass spectrometric conditions are summarized in the section Optimum Conditions in Results and Discussion. Optimization of Temperature and Cone Voltage Using Flow Injection. During the studies aimed to identify the optimum range for the two variables, temperature (T) and cone voltage (CV) were optimized with a desalted thrombin digest of factor VIII that was introduced to the mass spectrometer via flow injection. BioCAD pump provided a 12 µL/min flow of 40% 2-propanol in 0.1% TFA through a flow splitter. (Flow was measured at the tip of the probe.) Sample was injected through a Rheodyne injector (model 5217, Cotati, CA) having a 100-µL loop attached. For every injection, the source temperature was adjusted to a new value. Cone voltage was varied during data collection at each temperature. The rest of the parameters were kept constant. Data were evaluated using the response surface analysis of JMP, Statistical Discovery Software Package (SAS Institute, Inc., Cary, NC). Optimization of SIR and Mass Resolution Using LC-ISD MS. During LC-ISD MS operation, the instrument was used either in scan mode (100-400 u) or SIR mode, depending on the task performed. In scan mode, typical conditions were as follows: positive ion mode, temperature, 115 °C; cone voltage, 120 V; mass resolution, 12.5 (or varied if it was under investigation); scan time, 2 s; inter scan time, 0.1. The rest of the parameters were set the same as in the case of flow injection. In SIR mode, conditions were as follows: inter channel delay, 0.01 s; span, 2.5 m/z; dwell, 0.2 s (or varied if it was under investigation). The rest of the parameters were set the same as in scan mode. In both scan and SIR modes, fragment ion chromatograms of oxonium ions were generated and peaks corresponding to the retention of B domain were integrated. In the optimization of temperature and cone voltage, peak area values were used as response. During the optimization of mass resolution, using the settings of variables optimized so far (see above), first, peak width in mass spectrum was measured at different resolution settings with scanning masses from 100 to 400 u at a rate of 150 u/s. Mass resolution values were adjusted together. The scanning measure-
Figure 1. Reversed-phase chromatogram of recombinant factor VIII thrombin digest. A 60-µg aliquot of digested FVIII was injected. Chromatographic conditions are described in the Experimental Section.
ments were followed by determinations in SIR mode, where span values for each resolution setting were set as peak width ( 0.25 u (for possible peak shifts). Then, the peak widths in SIR were substituted from the previous scan measurements having the corresponding resolution values. In the optimization calculations, signal-to-noise ratio was determined as the ratio of peak height to baseline noise height in fragment ion chromatogram. Methods of Evaluation. During evaluation, LC-ISD MS in the mode of SIR were used at optimum conditions. In order to overcome UV detection limitation problems at high loads, both 214 and 280 nm were measured. Peak area values at 214 nm for high loads were calculated from peak areas detected at 280 nm. From fragment ion chromatograms of SA and HexNAc oxonium ions of the B domain, peak area, peak height, and noise height were recorded. Fragment ion ratio (FIR) was calculated from the peak area ratio of sialic acid to N-acetylhexosamine. The yield of fragment ions was calculated as peak area ratio of fragment ion to B domain at 214 nm. The accuracy of changes in FIR and sensitivity were assessed for sialic acid. Sialic acid residues of the B domain were removed by partial digestion using neuraminidase (see above). The fragment ion ratio was measured for the partially digested samples with LC-ISD MS. Fractions of partially digested B domain samples were collected (200 pmol) during FIR measurements and subsequently analyzed by MALDI-TOF MS. Fragment ion ratio and molecular mass values of samples were compared. RESULTS AND DISCUSSION Chromatography. Thrombin digestion of factor VIII results in five domains: N-Terminal peptide of light chain (∼5 kDa), B domain (∼103 kDa), and 50, 73, and 43 kDa peptides (Figure 1). Despite its huge molecular mass and heterogeneity, B domain elutes as a narrow peak at relatively low organic concentrations. Most likely, the high carbohydrate content is responsible for the relatively weak binding to the hydrophobic stationary phase. The presence of Tween 80 in the sample buffer resulted in multiple Tween peaks in the chromatogram (Figure 1). The HPLC separation of these peaks from the protein peaks was necessary to avoid the interference of large Tween signals in MS. Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
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Optimization of MS Parameters. Instrument Setup and Initial Survey of Analytical Conditions. As previously reported, the formation of oxonium carbohydrate marker ions from relatively small glycopeptides can be achieved at moderate temperature and elevated cone voltage values.14 To determine the optimum range of source temperature and cone voltage for the generation of oxonium ions from a large glycoprotein, a 5 × 5 full-matrix experiment was designed for response surface analysis of signalto-noise ratio of fragment ions utilizing flow injection of aliquots of desalted thrombin digest of factor VIII. Since B domain carries ∼81% of the total carbohydrate content of factor VIII, it was feasible to search for the optimum range of parameters by analyzing the entire digest. For every injection, the source temperature was adjusted to a new value. Temperature values were set to 60, 95, 130, 165, and 200 °C. The ∼8-min resident time of the sample made it possible to set the cone voltage to five different values for 1-1.5 min each. Cone voltage values applied were set to 10, 50, 100, 150, and 200 V. Fragment ion chromatograms were generated for the oxonium ions of N-acetylhexosamine using the sum of the net peak intensities of (HexNAc - 2H2O)+, (HexNAc - H2O)+, HexNAc+, and HexHexNAc+ at the following m/z values: (168.2-176) + (186.3-192) + (204.1-210) + (366-376), respectively. For the oxonium ions of the sialic acid (SA) fragment, ion chromatograms were generated using the sum of the net peak intensities of (SA - H2O)+ and SA+, at the following m/z values: (274.2-280) + (292.3-306), respectively. Every second m/z value (with negative sign) represents the baseline level for the preceding peak. (No oxonium ion of fucose was detected.) From the fragment ion chromatogram (net fragment ion intensity vs time), the cumulative signal intensity and noise were determined for both HexNAc and SA at each temperaturecone voltage combination. Fragment ion chromatograms of sialic acid and N-acetylhexosamine were generated from the scan data. Signal intensity and noise values for all the 25 conditions were tabulated and analyzed. Using the response surface analysis package of JMP, Statistical Discovery Software Package, contour plots of signal-to-noise ratios generated for the oxonium ions of HexNAc and SA were depicted in Figure 2. The optimum temperature yielding the highest signal-to-noise ratio for both fragment ions was 125 °C. The optimum cone voltages varied for the oxonium ions of the two sugars, and was 155 V for HexNAc and 100 V for SA. The contour plot of the HexNAc signal to noise showed a gently sloping optimum while SA’s signal-to-noise optimum was close to the edge of a steep slope. The broad plateau containing the optimum signal-to-noise value of HexNAc is depicted as a box in Figure 2. Response surface analysis of the assay noise for sialic acid helped to trace the origin of the steep slope in the response surface for the signal-to-noise ratio of SA. Increasing temperature increased the noise for all cone voltage values (data not shown). In the case of SA, noise was most intense in the cone voltage range of 50-100 V. This high-intensity noise is responsible for the steep breakdown of signal to noise below 100 V. A slightly lower cone voltage optimum for sialic acid may be the result of the unusually labile nature of the sialic acid bond.20 During the selection of a common optimal range, the magnitude of signal-to-noise values (especially that of SA) and shallowness of the surface in the vicinity (ruggedness) were considered (20) Kobata, A.; Furukawa, K. In Glycoconjugates: Composition, Structure and Function; Allen, H. J., Kisaiws, E. C., Eds.; Marcel Dekker: New York, 1992, p 34.
2520 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
Figure 2. Response surface analysis of the optimization of source temperature and cone voltage in terms of signal-to-noise ratio for (A) N-acetylhexosamine (HexNAc) and (B) sialic acid using flow injection analysis of desalted FVIII thrombin digest. See text for experimental details.
important. The selected range spanned from 100 to 130 °C and from 100 to 150 V. Final Optimization of Temperature and Cone Voltage. In the second step, to find the optimum values for temperature and cone voltage for SA’s signal, a simple, constant step size simplex optimization strategy21 was used utilizing LC-ISD MS with scanning masses between 100 and 400 u. Since the signal-to-noise values for sialic acid were low and constant (only half of that of HexNAc), optimization was performed exclusively on SA’s signal intensity. From scan data, fragment ion chromatograms of oxonium ions were generated and peaks corresponding to the retention of the B domain were integrated. Peak area values were used as response. In seven steps, the optimum was identified at 115 °C and 120 V (Figure 3). In the vicinity of the optimum, the SA intensity surface was relatively flat, ensuring rugged operational conditions. A change of a few degrees or volts resulted in an intensity change less than 10%. The observed surface in the LC-ISD MS analysis of B domain showed minor differences compared to the surface obtained with flow injection of the entire FVIII digest, which may be explained by the difference between the two sets of experiments. For the sake of comparison, the (21) Beveridge, G. S. G.; Schechter, R. S. Optimization: Theory and Practice; McGraw-Hill: New York,1970, pp 290-322.
Table 1. Mass Resolution Optimization for Fragment Ions of FVIII B Domain Rs 5 8 9 10 12.5 15
Figure 3. Sialic acid peak area in the vicinity of optimum conditions. Results of the constant step size simplex optimization of source temperature and cone voltage in terms of sialic acid intensity using LC-ESI(ISD) MS. Optimization used seven steps: (1) 125 °C, 115 V; (2) 125 °C, 125 V; (3) 120 °C, 120 V; (4) 115 °C, 115 V; (5) 115 °C, 125 V; (6) 110 °C, 120 V; (7) 115 °C, 120 V. Sialic acid intensity values are displayed next to the measured points.
Figure 4. Fragment ion spectrum of the B domain at 60-µg load of FVIII. Scan: 150 u/s scan speed from 100 to 400 u. Oxonium carbohydrate ions are labeled. For the SIR mode, the mass values of baselines selected for the preceding signals are indicated with arrows. See Experimental Section for details.
parameter range of cone voltage and temperature that was covered by simplex optimization of sialic acid signal is depicted as a small box in Figure 2. Selection of Baseline for Fragment Ion Peaks. In SIR mode, in order to measure the net intensity of sialic acid and N-acetylhexosamine fragment ions originating from the B domain and to eliminate the effect of baseline drift caused by eluting Tween molecules originating from sample buffer, the intensity of the reference mass values of the baseline in the vicinity of the peaks had to be subtracted. Since the spectrum baseline decreased with increasing mass, the mass values of the selected reference baseline were higher than that of the ions (Figure 4). In this way, negative net intensity was avoided. Mass Resolution. The proper setting of resolution in mass spectrometry was important since too narrow a value can cut peak intensity while too wide a window would include extra noise. Either way, the intensity ratio intended to be determined would be altered; furthermore, the signal-to-noise ratio would suffer also. In the first part of the optimization procedure, using scans from m/z 100 to 400 u, peak width value was determined as a function of resolution. Six different settings were investigated (Table 1). As was expected, opening the window (lowering resolution) increased peak width. Increasing the mass of ions increased peak width slightly. An average peak width was always considered. In
Wpeaka HSAb HHexNAcb noiseSAb noiseHexNAcb S/NSAc S/NHexNAcc 4 3.1 2.6 2.4 2 1.5
327 407 315 287 217 101
935 1190 956 859 765 442
1.63 1.36 1.02 0.89 0.59 0.51
3.52 3.22 2.54 2.22 1.72 1.52
201 299 309 322 368 198
266 370 376 387 445 291
a Average peak width of fragment ions in mass spectra, u. b Peak height (H) of fragment ions and noise of baseline in mass chromatograms, ×10-3. c Signal-to-noise ratios for fragment ions in mass chromatograms.
the SIR mode, 12 m/z values were monitored: 168.2 (HexNAc 2H2O)+, 176, 186 (HexNAc - H2O)+, 192, 204.1 (HexNAc)+, 210, 274.3 (SA - H2O)+, 280, 292.4 (SA)+, 306, 366.3 (HexHexNAc)+, 376 u. Adjusting the span in SIR mode to the peak width obtained in scan mode, the signal-to-noise ratios of both fragment ions were determined in a wide range of mass resolution (Table 1). Span values for each resolution setting were set as peak width ( 0.25 u (for possible peak shifts) where peak widths were substituted from the previous scan measurements having the corresponding resolution values. The mass values of baselines were selected in a way that the mass windows of signal and baseline never overlapped. In the optimization calculations, signal-to-noise ratio was determined as the ratio of peak height to baseline noise height in the fragment ion chromatogram. Signal-to-noise data of both SA and HexNAc showed an asymmetric curve with a maximum resolution of ∼12.5 (Table 1). The consequence of this asymmetry is that opening the resolution window slightly wider than optimum and detecting some extra noise is less damaging in the case of a weak signal. For example, reducing mass resolution from 12.5 to 8 decreases the signal-to-noise ratio by ∼20% yet increases signal intensity by ∼90%. Optimum Conditions. The results of optimization work are the optimum conditions such as liquid flow rate, 12 µL/min; drying gas flow rate, 400 L/h; and nebulizing gas flow rate, 35 L/h. Source parameters: capillary, 4.5 kV; HV lens, 0.5 kV; cone voltage, 120 V; temperature, 115 °C. MS parameters: ionization energy, 2.0 V; mass resolution, 12.5 (2 m/z peak width). SIR parameters: interchannel delay, 0.01 s; span, 2.5; dwell, 0.2 s (∼50 data points/fragment ion peak). The m/z values of monitored fragment ions and their baselines to be subtracted are as listed in the Mass Resolution section. Figure 5 shows data collected at optimum conditions. The band broadening of chromatographic peaks in the interfacing tube between HPLC and the mass spectrometer was significant due to the tube length and the high viscosity of the mobile phase (Figure 5B,C). At these optimum conditions, the FIR was determined as the peak area ratio of the oxonium ions of sialic acid to N-acetylhexosamine. Performance Evaluation. The performance of the method was assessed by investigating linearity (dynamic range), reproducibility, accuracy of changes in FIR, and sensitivity under the optimal LC-MS conditions. Linearity and Dynamic Range. Since the concentration of samples to be investigated by this method may vary, it was imperative to demonstrate that change of load would not alter the value of the fragment ion ratio. The amount of digested factor VIII loaded was varied from 5.8 to 372 µg (Table 2). The fragment Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
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Table 3. Reproducibility of Optimized Method run no.
areaSA
areaHexNAc
area280
FIR
1 2 3 4 5 6
122 804 118 791 121 424 120 450 122 500 118 776
411 400 399 576 411 293 403 801 419 637 402 132
3 691 325 3 426 668 3 454 509 3 438 708 3 392 911 3 348 530
0.299 0.297 0.295 0.298 0.292 0.295
av SD % RSD
120 791 1 764 1.46
407 973 7 495 1.84
3 458 775 120 019 3.47
0.296 0.002 0.84
Table 4. Partial Neuraminidase Digestion of the B Domain
Figure 5. Typical chromatograms at optimal conditions: (A) 214nm signal of HPLC separation recorded by the mass spectrometric software (Masslynx); (B) fragment ion chromatogram of sialic acid; (C) fragment ion chromatogram of N-acetylhexosamine. See text for technical details. Table 2. Yield of Fragment Ions Normalized to 93-µg Load in the Range of 5.8-372-µg Loads and Averages from 11.6 to 186 µg Loads load (µg)
(areaSA/area214)i (areaSA/area214)93µg
(areaHexNAc/area214)i (areaHexNAc/area214)93µg
5.8 11.6 23.3 46.5 93 186 372
73.08 101.93 109.99 104.73 100.00 106.80 93.55
49.23 99.37 100.25 104.06 100.00 106.00 91.52
av (11-186 µg) SD (11-186 µg) % RSD (11-186 µg)
104.69 3.94 3.77
101.94 2.92 2.87
ion ratio was constant (FIR ) 0.285, RSD ) 3.5%) in the range of 11.6-372-µg load. The collected data made it possible to investigate the normalized yield of fragment ions at different loads (Table 2). The normalized yield of fragment ions was defined as follows: yield (sialic acid peak area/B domain peak area at 214 nm) at any load per yield at 93-µg load. Normalized yield is constant with an RSD ) 3.5% in the range of a 11. 6-186-µg load of factor VIII (Table 2). During this work, load was expressed in terms of glycoprotein mass; however, the method is carbohydrateconcentration sensitive. Therefore, at the determination of the sample’s optimum load range, carbohydrate amount should be considered. Reproducibility. The same-day reproducibility of the method was calculated from the results of six experiments at 60-µg loads (Table 3). The average FIR ) 0.296 and RSD ) 0.84%. The RSDs of reproducibility of fragment ion peak areas were in the range 2522 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
% RSD MWi - MWcda % digest FIRi/FIRo
time (min)
MW
0 0.25 0.5 1 2 4 6 9 12
103 744 103 109 102 250 102 118 100 114 98 318 97 745 96 912 96 279
0.47 0.57 0.45 0.50 0.37 0.39 0.39 0.25 0.20
8566 7931 7072 6940 4936 3141 2567 1734 1101
0.0 7.4 17.4 19.0 42.4 63.3 70.0 79.8 87.1
1.000 0.615 0.494 0.467 0.295 0.205 0.093 0.065 0.047
complt digstb
95 178
0.14
0
100.0
0.000
a Mass of sialic acid residues left on the B domain after partial neuraminidase digestion: mass of partially digested B domain minus completely digested B domain. b Represents complete digestion.
of 1.4-1.9%, which was better than that of 280 nm (3.47%) (Table 3). The level of FIR seems to slightly vary from day to day (data not shown) but the reproducibility in a day was excellent. Since this is planned to be a comparative method in which samples are compared to a reference material, i.e., FIRsample/FIRref is calculated, day-to-day variation does not play a role in the method and results should be normalized to the actual value of the reference material. Accuracy and Sensitivity of FIR in Measuring Compositional Change. Due to the lack of a B domain standard with a known amount of sialic acid and N-acetylhexosamine residues, the accuracy of changes in FIR reflecting the changes of overall carbohydrate composition in the glycoprotein was estimated by comparing its results to that of an independent method such as molecular mass determination by MALDI-TOF MS. A series of partial neuraminidase digestions was performed varying digestion time from 0.25 to 12 min. Additionally, the masses of nondigested and fully digested samples were also determined. Removal of sialic acid residues was followed by LC-ISD MS and MALDITOF MS (Table 4). Since in these determinations no molar concentrations of sialic acid residues were measured, determination of the rate law could not been performed. Subtracting the molecular mass of fully digested B domain from those of nondigested or partially digested, one can obtain the mass of sialic acid residues left on the B domain after partial digestion (Table 4). It should be pointed out that, in LC, the presence of TFA, and, in MALDI-TOF MS, the presence of sinapinic acid and the prompt dissociation of the B domain during the MALDI process potentially can result in a loss of sialic acid residues. In the range of 70-100% of complete digestion, the peak area of SA in the fragment ion chromatogram was below the limit of quantification (ASA < 5200 area unit) determined in the studies on the effect of
load on FIR. Therefore, these values were excluded from the accuracy of changes in FIR evaluation. Plotting normalized fragment ion ratio determined by LC-ISD MS against the mass of sialic acid residues left on the B domain after partial digestion determined by MALDI-TOF MS, data showed linear relationship [FIRi/FIRo ) 9 × 10-5 (MWpart. dig. - MWcompl. dig.) - 0.112]. The good correlation (r2 ) 0.97) between the two independent methods strongly suggests that the change in FIR reflects real changes in structure. The positive x-intercept suggests a greater loss of sialic acid residues during MALDI analysis. The nondigested sample was found to deviate from the straight line fit. This discrepancy will be investigated in a future study. The sensitivity of the method for detecting a change in the number of sialic acid residues, which provided a weaker signal than N-acetylhexosamine, could be assessed from the partial neuraminidase digestion study. Dividing the mass of sialic acid residues left on the B domain after partial digestion with the molecular mass of a sialic acid residue (292 Da), an estimate for the number of residues was obtained. Fitting a straight line on normalized FIR (FIRi/FIRo) vs number of sialic acid residues (#SA) provided a good correlation: FIRi/FIRo ) 0.0254(#SA) 0.111, r2 ) 0.9755. The slope value (0.025) expresses the sensitivity of the method. According to this result, one sialic acid residue change resulted in a 2.5% change in FIR. This is 3 times the RSD of reproducibility (0.84%); therefore, the method can determine a single sialic acid residue change. It is important to point out that the sensitivity determined for the FIR assay by comparing its results to those of MALDI-TOF MS represents the worst-case scenario due to the potential loss of sialic acid residues during MALDI-TOF MS analysis. O-Glycans. Measurement of PNGase F-Digested B Domain. Combining the method of reporter ion ratio determination with selective carbohydrate digestion can further enhance its usefulness. Selective removal of N-glycans then allows direct and selective measurement of the comparison of the O-glycan remaining on the protein. After PNGase F digestion of FVIII thrombin digest fragments, LC-ISD MS provided a fragment ion ratio for O-glycans of the B domain of FIRO-GLC/FIRN+O-GLC ) 1.133. It is known for the N-glycan map of FVIII that the N-glycans are relatively undersialylated (data not shown); therefore, the higher SA to HexNAc ratio determined for O-glycans is in accordance with expectations. Considering that PNGase F digestion is relatively short and a quite simple procedure, the fragment ion ratio for both N+O-glycans and O-glycans can further improve the quality of the comparison of glycoproteins on the screening level without significantly increasing the time or the complexity of the method. General Applicability of the the FIR Method. The method of fragment ion ratio measurement is not specific to the B domain of factor VIII; any glycoprotein can be analyzed in this way, though the optimum conditions may vary from protein to protein. For example, the fragment ion ratio was generated for the 50-and 73kDa domains of FVIII as well. However, due to their lower carbohydrate content, significantly larger load was necessary to obtain an acceptable quality of fragment ion peak integration. Similarly, the method was successfully tested on other wellcharacterized proteins, including ribonuclease B, recombinant human BMP-2,23 and recombinant factor IX (data not shown). The (22) Townsend, R. R.; Hardy, M. R.; Hindsgaul, O.; Lee, Y. C. Anal. Biochem. 1988, 174, 459-470.
use of a very steep chromatographic gradient and acetonitrile instead of 2-propanol resulted in a very sharp and concentrated peak of factor IX in the mass spectrometer. The high solute concentration ensured high oxonium ion concentrations and, concomitantly, an excellent signal-to-noise ratio. During a comparison of the calculated ratio of sialic acid to N-acetylhexosamine from the peak areas in the N-glycan high-pH anion-exchange maps to fragment ion ratio determined by LC-ISD MS for factor VIII and factor IX, a good correlation was found (factor IX was more rich in sialic acid than factor VIII); however, the actual values differed due to the bias of the pulsed amperometric detector toward sialic acid over N-acetylhexosamine,22 and O-glycans were not considered in the high-pH anion-exchange map either. Another shortcoming of high-pH anion-exchange chromatography of glycans was the relatively poor precision of quantification. In a recent application23 of this technique, quantitative ISD MS was used to analyze the different isoforms of two intact glycoproteins in terms of the Hex+/HexNAc+ area ratio. The success of this technique in answering a different analytical question for two wellcharacterized glycoproteins is another proof that the technique can be used universally. One of the great advantages of fragment ion ratio determination is that it does not require any derivatization or chemical/enzymatic release of glycans. The determination of the fragment ion ratio seems to complement the method of the molecular weight measurement by MALDI-TOF MS. The performance of quantitative LC-ISD MS increases with the abundance of carbohydrate (assuming constant glycoprotein concentration) while the quality of mass determination with MALDI-TOF MS increases with a reduction of carbohydrate heterogeneity. In this way, determination of FIR seems to be a good complementary method to the determination of molecular masses of domains in the characterization of factor VIII as a simple assay for biochemical equivalence. CONCLUSIONS In this paper, the development, evaluation, and application of a comparative (relative) quantitative LC-ISD MS of a very large glycoprotein fragment, the B domain of factor VIII, was presented. This work describes a method quantifying the ratio of fragment ions produced from a large glycoprotein in LC-ESI(ISD) MS. By providing the ratio of oxonium ions of sialic acid to Nacetylhexosamine, this comparative method can serve as an assay to assess structural equivalence for the carbohydrate content of complex glycoproteins in terms of their overall degree of glycosilation and sialylation, which are concerns for stability and purification equivalence. Using a systematic approach, the optimization of source temperature and cone voltage for the signal-to-noise ratios of fragment ions was performed in two steps. First, applying flow injection analysis of desalted thrombin digest of factor VIII, a response surface analysis of the two variables was used to determine the optimum operational range in terms of maximum signal-to-noise ratio for the two ions. Then, using LC-ISD MS, simplex optimization helped to locate the optimum conditions. The two-stage optimization provided a high signal-to-noise level and, most importantly, ensured the robustness of the method, which may be considered a crucial aspect of the quantification of fragment ions. The other advantage of the systematic optimization strategy applied over random (trial-and-error) optimization was the relatively short time it required to find the optimum. It is (23) Yeung, B.; Porter, T. J.; Vath, J. E. Anal. Chem. 1997, 69, 2510-2516.
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important to understand that the fragment ion peak area ratio at optimum conditions for signal-to-noise ratio might not reflect the absolute ratio of the fragment ions present in the glycoprotein molecule investigated, but the optimum conditions yield the best measure for relative quantification of fragment ion ratio in the comparison of the different batches of a biopharmaceutical product to its standard. In order to further maximize method sensitivity, all the other MS parameters were optimized for selective ion recording. Method performance was evaluated by assessing linearity (dynamic range), reproducibility, accuracy of changes in FIR, and sensitivity. The fragment ion ratio was constant (RSD ) 3.5%) in a very wide range, 11.6-372-µg load of FVIII digest. The short-term reproducibility of FIR was excellent, RSD ) 0.84%. The accuracy of changes in FIR was assessed for sialic acid by comparing fragment ion ratio results to molecular mass values determined by MALDI-TOF MS on partially neuraminidasedigested B domain. The correlation coefficient (r2) of the comparison was greater than 0.97, suggesting that the changes in FIR capture real changes in structural composition. The method was able to detect a change of 1 sialic acid residue out of 29 (3.4%). Combining the method with selective digestion of N-glycans, the fragment ion ratio was also determined for O-glycans of the B domain. Determination of fragment ion ratio for both N+Oglycans and O-glycans can further improve the quality of the
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comparison of glycoproteins at the screening level without significantly increasing the time or the complexity of the method. Comparative quantitative LC-ISD MS seems to be generally applicable for glycoproteins; however, the optimum MS conditions may vary from protein to protein. The application of comparative quantitative LC-ISD MS as a simple assay providing quantitative data, makes possible the establishment of parameter ranges of acceptance for well-characterized proteins rather than depending on the subjective interpretation of a complex trace of a qualitative method. The direct, quantitative nature of LC-ISD MS, assessing global properties of carbohydrate moieties of glycoproteins, seems to make it the appropriate tool to assess the biochemical equivalency of biopharmaceuticals containing carbohydrates. ACKNOWLEDGMENT We thank Jason Rouse, Stephen Koza, Bernice Yeung, Mark R. Hardy, and Anne-Marie Strang of Genetics Institute for their valuable suggestions.
Received for review November 1, 1996. Accepted April 4, 1997.X AC961116+ X
Abstract published in Advance ACS Abstracts, May 15, 1997.
