Detection and Quantitation of Low Abundance Oligosaccharides in

Feb 3, 2015 - Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is ...
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Detection and Quantitation of Low Abundance Oligosaccharides in Recombinant Monoclonal Antibodies Gomathinayagam Ponniah, Christine Nowak, Nidia Gonzalez, Dino Miano, and Hongcheng Liu* Product Characterization, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, Connecticut 06410, United States S Supporting Information *

ABSTRACT: Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics.

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recombinant mAbs because of its potential biological impact. The absence of core fucose (Fuc) increases antibody binding affinity to FcγRIIIa and results in enhanced antibody-dependent cellular cytotoxicity (ADCC).21,22 Thus, depending on the therapeutic target, ADCC functions can be either enhanced or reduced by manipulating the levels of core fucosylation. High mannose oligosaccharides, although present in human IgGs, are detected at relatively higher levels in recombinant mAbs.16 Compared to core-fucosylated complex oligosaccharides, antibodies with high mannose showed decreased binding affinities to FcγR1 and FcγRII as well as decreased complement activity,23−25 decreased thermal stability,26 and faster clearance.12,23,25,27−29 Antibodies with high mannose also show increased ADCC compared to core-fucosylated complex oligosaccharides.24,25,27 Hybrid oligosaccharides are present at very low levels in recombinant mAbs.13,14 Their impact on structure, stability, and functions are less well understood. Conflicting results have been reported for hybrid oligosaccharides on IgG effector functions including no-impact on30 or decreased27,31 effector functions. Because of their impact on stability, efficacy, and safety, various techniques have evolved to detect, identify, and quantify different oligosaccharides. Oligosaccharides are commonly analyzed while they are still attached to proteins or peptides or as free glycans released from glycoproteins. Reversed-phase

ecombinant monoclonal antibodies (mAbs) are glycoproteins with oligosaccharides attached to the conserved asparagine residues in the CH2 domain. Oligosaccharides are critical for maintaining antibody structural integrity, stability, and effector functions.1−5 The glycoforms of recombinant mAbs expressed in the most commonly used cell line, Chinese hamster ovary (CHO) cell line, are mainly core-fucosylated complex oligosaccharides with zero (Gal 0), one (Gal 1), or two (Gal 2) terminal galactose (Gal) and low levels of afucosylated complex oligosaccharides,6−12 high mannose oligosaccharides,6−12 and hybrid oligosaccharides.13,14 Those oligosaccharides are also found in human IgG molecules.15,16 Typically, oligosaccharides occurring in recombinant mAbs, which are also found at similar levels in endogenous IgG, are of less concern with regard to potential immunogenicity. Nevertheless, a thorough characterization and close monitoring of oligosaccharides is important for the development of recombinant mAb therapeutics. Several types of oligosaccharides have drawn close attention for the development of recombinant mAb therapeutics, including nonhuman, afucosylated, and high mannose oligosaccharides. Low levels of nonhuman oligosaccharides, α1,3-Gal and N-glycolylneuraminic acid (Neu5Gc), have been observed in recombinant mAbs expressed in murine but not CHO cell lines. Those nonhuman moieties could potentially cause immunogenicity. For example, IgE antibodies specific to α1,3-Gal in the Fab region of cetuximab have been detected in patients with hypersensitivity.17 Anti-Neu5Gc antibodies have also been detected in humans.18−20 The level of fucosylation is also an important consideration for the development of © XXXX American Chemical Society

Received: October 22, 2014 Accepted: February 3, 2015

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Analytical Chemistry liquid chromatography and mass spectrometry have been the major approaches for analyzing glycoproteins and glycopeptides.32−34 Free glycans have been directly analyzed using high pH anion exchange chromatography with pulsed-amperometric detection.32,35 Mass spectrometry has been applied to analyze free glycans with or without chemical derivatization.32,34,36 Derivatization with 2-aminobenzoic amine (2-AB) followed by normal-phase high performance liquid chromatography (NPHPLC) is a commonly used approach to analyze oligosaccharides in the regulatory environment.37,38 However, it is challenging to detect low abundance oligosaccharides including high mannose and hybrid oligosaccharides. High mannose (Man) oligosaccharides typically exist as multiple species such as five (Man-5), six (Man-6), or seven (Man-7) mannose residues, and each is present at a very low percentage. Terminal heterogeneity with either Gal or GlcNAc can also decrease the detectability of hybrid oligosaccharides. The improved methods described in this study increase the detectability of low abundance oligosaccharides and simplify quantitation. To accomplish this, 2-AB labeled oligosaccharides were digested using various glycosidases to consolidate high mannose oligosaccharides, hybrid oligosaccharides, or complex oligosaccharides.

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labeled glycans were eluted off the cartridges using water three times, 0.5 mL each. The eluted samples were stored at −20 °C. Endo H Digestion. 2-AB labeled oligosaccharides were digested using Endo H. Each aliquot of 100 μL 2-AB labeled oligosaccharides was mixed with 100 μL of 20 mM citric acid, pH 5.5, and then digested using 5 μL of Endo H at 37 °C for overnight. Digestion by β-Galactosidase and β-N-Acetylhexosaminidase. 2-AB labeled oligosaccharides were also digested by a combination of β-galactosidase and β-N-acetylhexosaminidase. Each aliquot of 100 μL 2-AB labeled oligosaccharides was mixed with 100 μL of 10 mM sodium phosphate, pH 6.5. To optimize the digestion conditions, various amounts of the combination of β-galactosidase and β-N-acetylhexosaminidase containing 1 μL of each, 2 μL of each, 5 μL of each, or 10 μL of each was added to each 50 μL of the 2-AB labeled oligosaccharides. The samples were incubated at 37 °C overnight and then analyzed by NP-HPLC. Digestion by Endo H, β-Galactosidase, and β-NAcetylhexosaminidase. The 2-AB labeled oligosaccharides were also digested using all three enzymes. To do that, 2-AB labeled oligosaccharides were first digested using β-galactosidase and β-N-acetylhexosaminidase. The digested samples were incubated at 100 °C for 10 min to inactivate those two enzymes. Then, Endo H was added to the samples after mixing with an equal volume of 20 mM citric acid buffer, pH 5.5, and incubated at 37 °C overnight. Digestion Using Either Mannosidase or Fucosidase. Mannosidase was used to identify an oligosaccharide structure containing terminal mannose residues. The samples were prepared using the 5× reaction buffer from Prozyme and then digested using various amounts of mannosidase to determine the conditions that resulted in complete digestion. Fucosidase was used to identify the presence of core fucose. The samples were prepared using the 5× reaction buffer from Prozyme and then digested using various amounts of fucosidase to determine the conditions that resulted in complete digestion. Normal-Phase HPLC with Fluorescence Detection. A Waters Alliance HPLC with a Glycosep N column was used to analyze 2-AB labeled oligosaccharides. Mobile phase A was acetonitrile, and mobile phase B was 50 mM ammonium formate, pH 4.4. The sample was injected at 90% mobile phase A, 10% mobile phase B. After running 10% mobile phase B for 5 min, mobile phase B was increased to 30% in 100 min, to 35% in 5 min, and then to 45% in 70 min. The column was washed using 90% mobile phase B for 10 min and then equilibrated for 10 min using the initial condition. The flowrate was set at 0.4 mL/mins. The column was kept at room temperature. Elution was monitored using a fluorescence detector with an excitation wavelength of 330 nm and emission of 420 nm. A different method was also used to determine the identity of an oligosaccharide structure. All the parameters are the same except the use of a shorter gradient. In brief, samples were loaded at 34% mobile phase B. After 5 min, oligosaccharides were eluted by increasing mobile phase B to 44% in 80 min. The column was then washed and equilibrated before the next injection. MALDI-TOF Analysis. HPLC fractions collected from NPHPLC were concentrated using a speed-vacuum. The concentrated samples were mixed with Super DHB (a mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (90:10 ratio) prepared in acetonitrile with 0.1%TFA) and

MATERIALS AND METHODS

Materials. The recombinant monoclonal antibodies A (mAb-A) and B (mAb-B) were expressed in the Chinese hamster ovary (CHO) cell line and purified at Alexion (Cheshire, CT). The third recombinant monoclonal antibody (mAb-C) is commercially available and has been on the market for more than ten years. Acetonitrile, ammonium formate, and sodium phosphate were purchased from Sigma (St, Louis, MO). PNGase F, endoglycosidase H (Endo H), β-galactosidase, β-N-acetylhexosaminidase, mannosidase, and fucosidase were purchased from Prozyme (Hayward, CA). PNGaseF Digestion. The recombinant mAbs were digested using PNGase F at 2 mg/mL (1 μL of PNgaseF for each 100 μg of antibody) in phosphate buffered saline including 0.5% Noctylglucoside at 37 °C for approximately 18 h. After digestion, the samples were incubated at 100 °C for 5 min followed by centrifugation to precipitate proteins. Free glycans in the supernatant were collected and dried using a Speed-vacuum. 2-AB Labeling. Glycans released from the mAbs were labeled by 2-aminobenzoic amine by reductive amidation using a 2-AB labeling kit from Ludger (Oxfordshire, UK) and following the manufacturer’s instructions. In brief, 100 μL of acetic acid was added to a vial of DMSO and mixed. Then, 100 μL of the acetic acid and DMSO mixture was added to a vial of 2-AB dye. The entire contents of the 2-AB dye were transferred to a vial of reductant. After a brief incubation at 65 °C to dissolve all the solid content in the reductant vial, 5 μL of the final mixture was added to each sample containing glycans from 200 μg of antibodies followed by incubation at 65 °C for 3 h. Residual reagents were removed using GlycoClean S cartridges (Prozyme). The cartridges were washed first using 1 mL of water for each, followed by five washes using 30% acetic acid in water, 1 mL for each wash. The cartridge was then washed using acetonitrile. Each of the labeled samples was applied to the cartridge immediately after acetonitrile flow through. The samples were allowed to bind to the cartridge for 5 min. The cartridge was washed with 1 mL of acetonitrile and then 5 times with 96% acetonitrile in water, 1 mL each. Finally, B

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Analytical Chemistry Table 1. Methods to Determine the Levels of High Mannose and Hybrid Oligosaccharidesa

a

Method 1 uses peak areas determined from direct integration of 2-AB labeled peaks. Method 2 uses Endo H to convert high mannose oligosaccharides to a single peak containing GlcNAc only. Method 3 uses two enzymes, β-galactosidase and β-N-acetylhexosaminidase, to convert complex oligosaccharides to either GlcNAc2Man3 or GlcNAc2Man3Fuc and hybrid oligosaccharides to GlcNAc2Man4Fuc. Method 4 uses all three enzymes to convert high mannose oligosaccharides to GlcNAc, complex oligosaccharides to GlcNAc2Man3 or GlcNAc2Man3Fuc, and hybrid oligosaccharides to GlcNAc2Man4Fuc.

the detectability of hybrid oligosaccharides by removing their terminal heterogeneity. The fourth method is a combination of the second and the third method. The samples were first digested using β-galactosidase and β-N-acetylhexosaminidase and then by Endo H. The chromatogram from the analysis of the samples after three enzyme digestion is expected to contain only four peaks corresponding to GlcNAc, GlaNAc2Man3, GlcNAc2Man3Fuc, and hybrid oligosaccharide. Method 1: Direct Quantitation of 2-AB Labeled Oligosaccharides. A typical NP-HPLC chromatogram of the 2-AB labeled oligosaccharides from mAb-A is shown in Figure 1. The peaks are assigned on the basis of the common oligosaccharide structures reported in the literature for recombinant mAbs expressed in CHO cell lines. To further identify peaks containing high mannose oligosaccharides, high mannose standards were analyzed. Peaks with retention times matching peaks in the high mannose standards are identified as Man-5, Man-6, Man-7, and Man-8. Man-8 was detected as two peaks in the high mannose standards and in the sample, probably representing two isoforms of Man-8. The relative percentage of high mannose oligosaccharides determined by this method is 2.62%, 1.31%, and 8.50% for mAb-A, mAb-B, and mAb-C, respectively (Table 2), as calculated by dividing

deposited on a MALDI target and air-dried. MALDI-TOF spectra were recorded on an Ultraflextreme mass spectrometer (Bruker, Billerica, MA). MALDI-CID spectra were acquired in the positive ion mode.

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RESULTS AND DISCUSSION Workflow of the Method. Four methods were tested and compared in the current study to detect and quantify oligosaccharides from three recombinant mAbs with a focus on high mannose and hybrid oligosaccharides. These methods are summarized in Table 1. The first method is the one that has been commonly used, where 2-AB labeled oligosaccharides are analyzed directly by NP-HPLC with fluorescence detection. The second method is to digest 2-AB labeled oligosaccharides using EndoH, which converts all high mannose oligosaccharides to a monosaccharide with GlcNAc only. Digestion with Endo H helps one to identify high mannose peaks and simplifies integration. The third method is to digest 2-AB labeled oligosaccharides using β-galactosidase and β-N-acetylhexosaminidase and thus converts all complex oligosaccharides to GlcNAc2Man3 with or without core fucose. Digestion using those two enzymes identifies complex oligosaccharides by merging peaks and shifting the peaks to different retention times. More importantly, these two enzymes can also increase C

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Figure 1. Chromatograms of 2-AB labeled oligosaccharides from mAb-A (A) and high mannose oligosaccharide standards (B). Peaks are labeled according to the common oligosaccharide structures of recombinant mAbs reported in the literature. Peaks containing high mannose oligosaccharides are labeled on the basis of comparison of the retention times to high mannose standards. The original high mannose standard containing Man-5, Man-6, Man-7, Man-8, and Man-9 was digested using mannosidase to generate the standard used in (B).

high mannose oligosaccharides disappeared (Supporting Information, Figure 1). The percentage of peak areas corresponding to GlcNAc is 1.78%, 0.74%, and 6.11% for mAb-A, mAb-B, and mAb-C, respectively (Table 2), as calculated by dividing the peak area of GlcNAc by the sum of the peak areas of all the oligosaccharides. Presumably, high mannose oligosaccharides with fewer than 5 and greater than 9 residues that are not detected should become detectable after converting to the same single peak. However, because the percentage from EndoH digestion for all three mAbs is lower than the percentage determined from direct calculation using Method 1, it is likely that peak overlapping is the major issue. Method 3: β-Galactosidase and β-Acetylhexosaminidase. Quantitation of High Mannose Oligosaccharides after Two Enzyme Digestion. As a complementary approach, 2-AB labeled oligosaccharides were digested using a combination of β-galactosidase and β-N-acetylhexosaminidase and then analyzed by NP-HPLC with fluorescence detection. β-Galactosidase removes terminal galactose. β-N-Acetylhexosaminidase removes terminal GlcNAc. Thus, complex oligosaccharides with or without core fucose were converted into GlcNAc2Man3Fuc or GlcNAc2Man3, respectively, while high mannose structures remain unchanged. A typical fluorescence chromatogram is shown in Figure 3. Because complex oligosaccharides are the major glycoforms for mAb-A, the major peak at the retention time of approximately 127 min was assigned to GlcNAc2Man3Fuc from digestion of core-fucosylated complex oligosaccharides. The small peak at the retention time of 122 min was assigned to GlcNAc2Man3 from digestion of complex oligosaccharides without core fucose. The peak identities of

the total peak areas of high mannose peaks by the peak areas of all peaks. Table 2. Percentage of High Mannose and Hybrid Oligosaccharides of Three Antibodies mAb-A Method Method Method Method

1 2 3 4

Method 3 Method 4

2.62 1.78 1.84 1.81

± ± ± ±

1.07 ± 1.05 ±

mAb-B

High Mannose 0.08 1.31 ± 0.03 0.74 ± 0.08 0.91 ± 0.11 0.68 ± Hybrid 0.06 0.93 ± 0.04 1.03 ±

mAb-C ± ± ± ±

0.1 0.04 0.03 0.01

8.50 6.11 6.81 5.89

0.18 0.09 0.08 0.11

0.01 0.06

1.02 ± 0.003 1.03 ± 0.05

Method 2: Endo H Digestion. The 2-AB labeled oligosaccharides were digested by Endo H and then analyzed by NP-HPLC. Chromatograms of the 2-AB labeled oligosaccharides before and after EndoH digestion are shown in Figure 2. Because Endo H cleaves high mannose oligosaccharides between the two primary GlcNAc residues, high mannose peaks disappeared, while peaks corresponding to complex oligosaccharides remain unchanged. At the same time, a small peak immediately after the reagent peak appeared in the chromatogram obtained from analyzing the sample after Endo H digestion. This peak is assigned as containing GlcNAc as the product from digestion of high mannose oligosaccharides. The peak identity was confirmed by Endo H digestion of high mannose oligosaccharide standards, where a peak eluted at the same retention time was detected while peaks corresponding to D

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Figure 2. Chromatogram of 2-AB labeled oligosaccharides from mAb-A before (A) and after (B) Endo H digestion. The portion of the chromatogram showing oligosaccharide peaks before Endo H digestion is shown as the inset in (A). The portion of the chromatogram containing GlcNAc is shown as the inset in (B). The portion of the chromatogram showing peaks containing complex oligosaccharides after Endo H digestion is shown as another inset in (B).

Figure 3. Chromatogram of 2-AB labeled oligosaccharides from mAb-A after β-galactosidase and β-N-acetylhexosaminidase digestion. The major peak is GlcNAc2Man3Fuc from digestion of core-fucosylated complex oligosaccharides. The peak eluted in front of the major peak contains GlcNAc2Man3 from digestion of complex oligosaccharides without core fucose. High mannose peaks remained unchanged. Hybrid oligosaccharides became detectable as peak x.

GlcNAc2Man3Fuc and GlcNAc2Man3 were further confirmed by MALDI-TOF analysis (Supporting Information, Figure 2). There is a small peak (peak x) at the retention time of approximately 137 min. Increasing the amounts of the two enzymes has no impact on this peak, indicating it was not due to incomplete digestion of G0F (Supporting Information, Figure 3). In addition, this peak is not high mannose oligosaccharide because its retention time does not match the retention times of high mannose oligosaccharides.

The relative percentage of the high mannose structures is 1.84%, 0.91%, and 6.81% for mAb-A, mAb-B, and mAb-C, respectively (Table 2), as calculated by dividing the total peak area of high mannose oligosaccharides by the total peak areas of all oligosaccharides. This percentage is in good agreement with the percentage determined from Endo H digestion, supporting the notion that peak-overlapping resulted in overestimation of the percentage of high mannose oligosaccharides from direct measurement. E

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Figure 4. MALDI-TOF mass spectra of peak x. Four major peaks were observed corresponding to sodium and potassium adducts of peak x and peak x with or without the 2-AB tag as shown in the figure.

Figure 5. Chromatograms of peak x after fucosidase digestion and high mannose standards. The chromatograms were acquired from (A) the reanalysis of the collected peak x; (B) analysis of peak x after fucosidase digestion; (C) high mannose standards containing Man-3, Man-4, and Man5.

Identification and Quantitation of Peak x. Because peak x can only be detected after digestion using the combination βgalactosidase and β-N-acetylhexosaminidase, either it coeluted with the G0F peak or it was generated from digestion of other oligosaccharides. To test those two hypotheses, the G0F peak was collected from the analysis of 2-AB labeled oligosaccharides and then digested using the combination of β-galactosidase and β-N-acetylhexosaminidase. If peak x was present originally in the sample, it should be detected after converting G0F to

GlcNAc2Man3Fuc that was eluted at a different retention time. However, peak x was not detected (data not shown). On the basis of this observation, it was concluded that peak x was generated from other oligosaccharides probably from collapsing peaks containing terminal Gal or GlcNAc. To determine the peak x identity, it was collected and analyzed by MALDI-TOF. As shown in Figure 4, a peak with a molecular weight of 1361.5 Da was observed. On the basis of this molecular weight, the structure of peak x was tentatively F

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Figure 6. Chromatograms obtained from mannosidase digestion. (A) Reanalysis of the peak containing GlcNAc2Man3Fuc; (B) analysis of GlcNAc2Man3Fuc after mannosidase digestion; (C) reanalysis of peak x; (D) analysis of peak x after mannosidase digestion.

Figure 7. Chromatogram of 2-AB labeled oligosaccharides from mAb-A after three enzyme digestion. The portion of the chromatogram containing the peak of GlcNAc is shown as an inset. The portion of the chromatograms containing GlcNAc2Man3Fuc, GlcNAc2Man3, and peak x is shown as another inset.

mannosidase (Figure 6). As a positive control, the major peak from the two enzyme digested 2-AB labeled oligosaccharides, which has a structure of GlcNAc2Man3Fuc, was also collected and digested using mannosidase. Digestion of peak x and GlcNAc2Man3Fuc resulted in peaks with the same retention times, indicating the same digestion product of GlcNAc2Fuc. Taken together, the peak x structure assigned based on the molecular weight was confirmed. It was likely that

assigned as a structure containing four mannose and a core fucose. The structure of peak x was further analyzed by fucosidase digestion and mannosidase digestion. If peak x contains a structure with one Fuc and four Man residues, fucosidase digestion should remove Fuc and generate Man-4. As shown in Figure 5, as expected, a peak with a retention time matching the retention time of Man-4 was detected after fucosidase digestion. Peak x was also digested using G

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Analytical Chemistry Notes

various hybrid oligosaccharides were present and each at a very low level that cannot be easily detected and identified. However, hydrid oligosaccharides became detectable by collapsing multiple low abundance peaks into a single peak containing GlcNAc2Man4Fuc. The percentage of hybrid oligosaccharides was determined and shown in Table 1, where approximately 1% was detected for all three mAbs. Method 4: Three Enzyme Digestion. The samples were also analyzed by the fourth method where all three enzymes were used. 2-AB labeled oligosaccharides were first digested using β-galactosidase and β-N-acetylhexosaminidase and then End H. A typical chromatogram is shown in Figure 7. As expected, complex oligosaccharides were converted into GlcNAc2Man3Fuc and GlcNAc2Man3; high mannose oligosaccharides were converted to GlcNAc, and hybrid oligosaccharides were converted to GlcNAc2Man4Fuc. The relative percentage of the high mannose structures is 1.81%, 0.68%, and 5.89% for mAb-A, mAb-B, and mAb-C, respectively (Table 2), as calculated by dividing the total peak area of high mannose oligosaccharides by the total peak areas. This percentage is in good agreement with the percentage determined using Methods 2 and 3. The percentage of hybrid oligosaccharides for the three antibodies can also be determined from this method. The resulting percentage of hybrid oligosaccharides determined by this method is in good agreement with the values determined from Method 3.

The authors declare no competing financial interest.

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CONCLUSIONS Methods for detection and quantitation of low abundance oligosaccharides including high mannose and hybrid oligosaccharides were established. The levels of high mannose oligosaccharides can be determined from integration of all oligosaccharide peaks from routine analysis of 2-AB labeled oligosaccharides by NP-HPLC with fluorescence detection. To simplify the chromatogram and for ease of data analysis, digestion using EndoH, a combination of β-galactosidase and βN-acetylhexosaminidase, or a combination of all three enzymes were used. Endo H specifically converts high mannose oligosaccharides into a single peak of GlcNAc. Complementary to the Endo H digestion, β-galactosidase and β-N-acetylhexosaminidase convert complex oligosaccharide structures to GlcNAc2Man3 or GlcNAc2Man3Fuc, while high mannose oligosaccharide structures remain unchanged. The advantage of the enzyme digestion method is to quantify the levels of high mannose oligosaccharides without identification of each high mannose structures, which will be useful for comparisons of different molecules, different clones, or conditions. The disadvantage is the introduction of additional enzyme digestion steps. More importantly, the above-mentioned methods allowed the detection and thus quantitation of hybrid oligosaccharides, which are typically not identified and quantified.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: 203-271-8354. E-mail: [email protected]. H

DOI: 10.1021/ac504738c Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac504738c Anal. Chem. XXXX, XXX, XXX−XXX