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ANALYTICALBIOCHEMISTRY
Analytical Biochemistry 347 (2005) 262–274
www.elsevier.com/locate/yabio
Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD
John S. Kim ¤, Erin R. Laskowich, Rasappa G. Arumugham, Raymond E. Kaiser, Gregory J. MacMichael
Wyeth Vaccine, Research & Development, 4300 Oak Park, Sanford, NC 27330, USA
Received 14 July 2005Available online 11 October 2005
Abstract
A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsularpolysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3 N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selec-tive detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of allsamples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and aldi-tols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, andchromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneu-mococcal polysaccharides and conjugates with conWrmation of peak assignments by retention time and mass spectral analysis. Thismethod allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and Wnal drug products with low-level detec-tion (10 pg) and peak purity. 2005 Elsevier Inc. All rights reserved.
Keywords: Streptococcus pneumoniae; Pneumococcal; Polysaccharide; Monosaccharide; Conjugate vaccine; Gas chromatography; GC–MSD; Methanol-ysis
Streptococcus pneumoniae is a major cause of invasivedisease, meningitis, and otitis media worldwide aVecting, inparticular, young children, the elderly, and immunocom-promised individuals. More than 90 pneumococcal sero-types have been identiWed, each possessing animmunologically distinct capsular polysaccharide. Vaccinesconsisting of a mixture of these polysaccharides haveproven to be eVective against pneumococcal disease inadults but are insuYciently immunogenic in young children[1,2]. Conjugated pneumococcal vaccines, where polysac-charides (or their derivatives) are covalently attached tocarrier proteins [2–5], have been successfully developed
* Corresponding author. Fax: +1 919 991 9011.E-mail address: [email protected] (J.S. Kim).
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.09.022
with proven eVectiveness in young children [6,7]. One suchvaccine is Prevenar, which contains seven serotypes individ-ually conjugated to a carrier protein and subsequentlymixed for Wnal vaccine formulation. Since the introductionof Prevenar, the incidence of pneumococcal disease in chil-dren under 5 years of age has decreased signiWcantly [8–10].
Vaccines containing additional serotypes are beingdeveloped to protect against invasive pneumococcal diseasein children living in other parts of the world, includingthose living in developing areas. Vaccine formulations toaccommodate regional or epidemiological factors, or theaddition of serotypes for extended coverage, are leading todevelopment of newer generations of pneumococcal vac-cines [11]. Consequently, it has become clear there is a needto evaluate newer technologies of vaccine characterizationin comparison with the existing technologies.
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 263
Characterization and testing of polysaccharides is criti-cal at every stage of the conjugate vaccine developmentprocess to conWrm product qualities such as identity,purity, and potency. Testing is typically performed by col-orimetric assays for speciWc classes of monosaccharides orsubstituents (e.g., hexoses, uronic acids, phosphorus, O-ace-tyl). More recently, assays have been developed using chro-matographic or spectroscopic instrumentation such ashigh-performance anion exchange chromatography withpulsed amperometric detection (HPAEC–PAD),1 reverse-phase HPLC with Xuorescence detection, and NMR [12–14]. The liquid chromatographic methods have proven tobe useful for routine testing of pneumococcal polysaccha-rides and conjugates [12]; however, polysaccharide-speciWchydrolysis schemes might be needed to eVect completerelease of carbohydrate residues, and speciWc standardsneed to be produced in some cases for peak assignments. Inaddition, the degradation of certain monosaccharide resi-dues under aqueous hydrolysis conditions may be a limitingfactor [12]. NMR has also proven to be extremely beneWcialfor characterization and testing of pneumococcal polysac-charides [14] but is limited to very pure saccharide samples.Currently, a suitable quantitative one-dimensional 1HNMR method for routine analysis of polysaccharide–pro-tein conjugates has not yet been realized due to sample preprequirements and the interfering protein content. An idealtest method would employ a single set of conditions thatcould be applied to neat samples from numerous pneumo-coccal polysaccharides at multiple stages of the conjugatevaccine process.
Gas chromatography (GC) has been used as a quantita-tive tool for carbohydrate analysis for several decades.Excellent resolution and robustness are typically associatedwith GC applications due to the large number of theoreti-cal chromatographic plates and the inherent purity of theWnal derivatized sample solubilized into organic solvent.Typical detectors used for carbohydrate analysis includethe Xame ionization detector (FID) and the mass selectivedetector (MSD). The latter provides additional conWrma-tion of peak assignments by analysis of mass spectra as wellas peak purity. These additional beneWts are unique to GC–MSD and are not realized with the current liquid chroma-tography or one-dimensional 1H NMR methods.
Anhydrous methanolysis is commonly used to depoly-merize polysaccharides into methyl glycosides, which canbe subjected to subsequent derivatization and GC [15–18].
1 Abbreviations used: HPAEC–PAD, high-performance anion exchangechromatography with pulsed amperometric detection; GC, gas chroma-tography; FID, Xame ionization detector; ATCC, American Type CultureCollection; Pn, pneumococcal serotype; EI, electron impact; TMS, trim-ethylsilyl; Glc, glucose; Gal, galactose; Rha, rhamnose; ManNAc, N-ace-tyl-mannosamine; GlcA, glucuronic acid; FucNAc, N-acetyl-fucosamine;GalA, galacturonic acid; GalNAc, N-acetyl-galactosamine; GlcNAc,N-acetyl-glucosamine; Gro, glycerol; Rib-ol, ribitol; PneNAc, N-acetyl-pneumosamine; Sug1, 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose;Sug2, 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose; LOD, limit of detec-tion; SIM, selective ion mode.
Methyl glycosides are relatively stable products comparedwith monosaccharides released under aqueous conditionsand can be subjected to harsher hydrolyses with minimaldegradation [19,20]. Pneumococcal polysaccharide andpolysaccharide conjugates were subjected to methanolysisas the sole hydrolysis step, followed by derivatization andchromatography on a GC–MSD. The results presentedherein are from a single quantitative procedure that can beapplied to pneumococcal puriWed, activated, and conju-gated polysaccharides.
Materials and methods
Polysaccharides and conjugates
PuriWed pneumococcal polysaccharides were obtainedfrom Wyeth Vaccine Development or from American TypeCulture Collection (ATCC, Manassas, VA, USA). Polysac-charide conjugate samples were obtained from Wyeth Vac-cine Development. The amounts of polysaccharide testedtypically ranged from 0.1 to 0.5 mg. Quantitation was basedon total recovered monosaccharides relative to authenticmonosaccharide standards (Sigma–Aldrich, St. Louis, MO,USA). The only exception was for pneumococcal serotype(Pn) 1, where an ATCC reference standard was qualiWedand used for quantitation purposes.
Methanolysis
Samples were transferred to TeXon screw cap tubes(13 £ 100 mm). Then 20 �g of myo-inositol was added as aninternal standard, and the samples were dried under asteady stream of nitrogen at 40 °C. To each tube, approxi-mately 0.5 ml of methanolic 3 N HCl (Supelco, Bellefonte,PA, USA) was added, and the tubes were sealed. Sampleswere incubated in a preheated block set at 121 °C for 2 h(unless otherwise noted). After incubation, the tubes wereremoved from the heat block, allowed to cool to room tem-perature, and centrifuged at 3500 rpm (»1 min) to collectsample to the bottom of the tubes. Caps were removed andthe samples were dried under a steady stream of nitrogen at40 °C. Approximately 0.1 ml of methanol was added to eachtube and was dried an additional three times to removeresidual HCl.
Derivatization
Postmethanolysis samples were re-N-acetylated by thesequential addition of 10 drops of methanol, 1 drop of pyri-dine, and 1 drop of acetic anhydride with vortexing aftereach addition. The samples were sealed and allowed to sit atroom temperature for 30 min. Caps were removed and sam-ples were dried under a steady stream of nitrogen at 40 °C.
Approximately 0.2 ml of Trisil reagent (Pierce, Rockford,IL, USA) was added to each sample, and tubes were sealedand incubated in a preheated block set at 80 °C for 20 min.After incubation, the tubes were removed from the heat
264 Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274
block, allowed to cool to room temperature, and centrifugedat 3500 rpm (»1 min) to collect sample to the bottom of thetubes. Caps were removed and samples were subjected to asteady stream of nitrogen at 40 °C until just dry.
Hexane (1 ml) was added to each sample, followed byvortexing and centrifugation at 3500 rpm (»1 min) to pelletany debris. Approximately 0.1 ml of clear supernatant wasadded to autosampler vials with inserts for injection intothe gas chromatograph.
GC–MSD
The system used was an Agilent 6890 gas chromato-graph/5973 mass selective detector with a 30-m HP-5 capil-lary column. Helium was used as the carrier gas at aconstant Xow rate of 1 ml/min. The oven conditionsincluded an initial temperature of 50 °C and an initial timeof 2 min, 30 °C/min to 150 °C, 3 °C/min to 220 °C, andWnally 30 °C/min to 300 °C for a 10-min bakeout. In thecase of Pn 1, the bakeout time was increased to 30 min. Theinlet temperature was kept constant at 250 °C, and the MStransfer line was set at 300 °C. MS acquisition parametersincluded scanning from m/z 50 to 550 in the electron impact(EI) mode for routine analysis.
Polysaccharide quantitation
Polysaccharide levels were calculated based on theresponse of known amounts of authentic monosaccharidestandards. Each standard set included 50�g of each mono-saccharide, and 20�g of myo-inositol was added to all stan-dards and samples as an internal reference. Responsefactors for each monosaccharide standard were calculatedand applied for quantitation of each sample. Standardswere run with each set of experiments, yielding responsefactors of approximately 0.51 (galactose), 0.31 (galact-uronic acid), 0.13 (N-acetyl-galactosamine, 0.58 (glucose),0.20 (glucuronic acid), 0.18 (N-acetyl-glucosamine), 0.16 (N-acetyl-mannosamine), 0.45 (rhamnose), and 0.57 (ribitol).In the case of ribitol, under the current method, the stan-dard yielded two main peaks corresponding to the open-chain trimethylsilyl (TMS) derivative and a ring-formadduct; both peak areas were combined to calculate theresponse factor for ribitol. For all other monosaccharidestandards, only the peak areas for the major TMS methylglycosides were included for response factor calculations.Because authentic monosaccharide standards were unavail-able for N-acetyl-fucosamine and N-acetyl-pneumosamine,the value generated from N-acetyl-galactosamine was usedas a generic amino sugar response factor for these tworesidues.
Results
Pneumococcal polysaccharide structures for the sero-types used in this study are provided in Fig. 1. A total of 13serotypes were subjected to analysis at various stages of the
conjugate vaccine process, including puriWed, activated,and conjugated polysaccharides. Table 1 lists all of themonosaccharide components contained in these pneumo-coccal serotype polysaccharides. The most common mono-saccharides include glucose (Glc), galactose (Gal), andrhamnose (Rha), followed by N-acetyl-mannosamine(ManNAc), glucuronic acid (GlcA), N-acetyl-fucosamine(FucNAc), galacturonic acid (GalA), N-acetyl-galactos-amine (GalNAc), N-acetyl-glucosamine (GlcNAc), glycerol(Gro), ribitol (Rib-ol), N-acetyl-pneumosamine (PneNAc),2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (Sug1),and 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose (Sug2).
Excellent resolution is typically associated with GC,even with a mixture of monosaccharides that can each yieldseveral methyl glycoside forms due to variation in ringform or anomeric conWguration. Fig. 2 shows resultingchromatograms from monosaccharide analysis of serotypes3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Sero-types 6A and 6B have the same monosaccharide composi-tion and yield very similar chromatograms; likewise, the19A and 19F chromatograms appear to be identical. Theremaining serotypes each yielded a unique distribution ofmonosaccharides. The labeled peaks indicate the majorTMS methyl glycoside peaks for each monosaccharide;these peaks were routinely used for quantitation. Minorpeaks were also identiWed based on their retention times rel-ative to standards and mass spectra but were not includedfor quantitation. Slight coelution was seen for severalminor peaks, but the only signiWcant overlap of majorpeaks was for the GlcNAc and the internal standard inserotypes 7F and 14. For these two serotypes, manual inte-gration or analysis by peak purity was used to apportionthe peak areas prior to quantitation. If necessary, furtherdevelopment could include the use of another internal stan-dard (e.g., an epimer of myo-inositol) for these two sero-types.
All monosaccharides from each serotype could be quan-titatively released, with the following two exceptions. First,in Pn 5, Sug2 (a keto sugar) is presumably destroyed duringmethanolysis and does not yield TMS methyl glycosides.Second, both 19A and 19F do not release their ManNAcdue to linkage to an acid-resistant phosphodiester bond.Even with extensive treatment (24 h), only trace amounts ofManNAc could be detected in serotypes 19A and 19F (datanot shown). For these serotypes, multiplication factors of1.26 [(180.2 + 205.2 + 204.2 + 194.1 + 205.2)/(180.2 + 205.2 +194.1 + 205.2)] for Pn 5 and 1.92 [(221.2 + 180.2 + 164.2 +96.0)/(180.2 + 164.2)] for Pn 19 were applied to calculatetotal saccharide content. For the remaining serotypes, themonosaccharide content was totaled to equate the amountof each polysaccharide.
Fig. 3 shows chromatographic results from analysis ofPn 1 polysaccharide. Complete hydrolysis down to themonosaccharide level was unattainable with Pn 1 usingthe current conditions. This was not surprising given thatthe polysaccharide contains a unique combination of acid-resistant glycosidic bonds from GlcA and the amino sugar,
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 265
Fig. 1. Repeating unit structures for the 13 pneumococcal polysaccharides used in this study. Sug1, 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose; Sug2,2-acetamido-2,6-dideoxy-D-xylo-heos-4-ulose; PneNAc, N-acetylpneumosamine (2-acetamido-2,6-dideoxy-L-talose). Structures are referenced from thefollowing: Pn 1 [21], Pn 3 [22], Pn 4 [23], Pn 5 [24], Pn 6A [25], Pn 6B [26], Pn 7F [27], Pn 9V [28], Pn 14 [29], Pn 18C [30], Pn 19A [31], Pn 19F [32], and Pn23F [33].
266 Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274
Sug1. The resistance to hydrolysis of the serotype 1 poly-saccharide has been reported previously [12]. Using the cur-rent methanolysis scheme, the only monosaccharide
released from Pn 1 was GalA, with signiWcant amounts ofhigher molecular weight species remaining intact. Thesenonmonosaccharide TMS derivatives were presumed to be
Fig. 1. (continued)
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 267
di- and trisaccharides given that their retention times com-pared well with those from model di- and trisaccharidecompounds (data not shown). Attempts to subject thisresistant polysaccharide to more extreme conditions(longer methanolysis) resulted in signiWcant degradationand low recovery. It appeared that under severe methanoly-sis treatment, the Pn 1 di- and trisaccharides were gettingdestroyed before hydrolyzing to monomeric units, as seenby GC–MSD (data not shown). However, the currentmethanolysis condition resulted in a reproducible distribu-tion of the monomer/dimer/trimer, allowing for subsequentquantitation. Thus, Pn 1 was analyzed and quantitatedbased on a qualiWed Pn 1 reference standard, whereas quan-titation of the other remaining 12 serotypes studied wasbased on authentic monosaccharide standards.
Pn 1 was the only serotype in which residual trisaccha-rides could be detected. However, residual disaccharidescould be detected in all serotypes tested. Table 2 lists theamounts of detected residual di- and trisaccharidesexpressed as percentages of total saccharide peak area. Inthe case of Pn 1, 11 and 34% of the total peak area was dueto disaccharides and trisaccharides, respectively. Serotypes3, 4, 5, and 9V contained between 3.7 and 9.5% residualdisaccharide and is not surprising given that these serotypescontain intermediate amounts of the acid-resistant uronicacids and amino sugars; quantitation of these four sero-types did not appear to be signiWcantly impacted so far ascomparison with results obtained from colorimetric assays.All other serotypes gave less than 1% residual disaccharide.
Mass spectra for the various peaks can be analyzed todetermine characteristics such as identity and peak purity.Even epimers such as GalA and GlcA fragment diVeren-tially on electron impact, facilitating identiWcation of eachunique residue. All peaks were evaluated for both identityand peak purity during the development of this method.Fig. 4 shows mass spectra for the major TMS methyl glyco-sides of the 14 monosaccharide components comprising thepneumococcal serotypes used in this study. Rib-ol, foundonly in serotypes 6A and 6B, was released on methanolysisentirely as the ring-form adduct and gave a mass spectrum
that was distinct from Rib-ol that had been treated withTMS alone (data not shown). As mentioned earlier, Pn 1yielded GalA as the sole monosaccharide released on meth-anolysis. Because the Sug1 residue is not released in mono-meric form, the Sug1 mass spectrum was taken from themajor disaccharide peak in Pn 1. Analysis of this mass spec-trum revealed the fragment mass m/z 173, indicative of anamino sugar TMS methyl glycoside. The m/z 173 mass wasfound only in the di- and trisaccharide peaks of Pn 1 andnot in the monosaccharide region. The Sug2 (from Pn 5)TMS methyl glycoside is not formed, and a resulting massspectrum was not produced.
Methanolysis conditions were optimized to achieve max-imum polysaccharide recovery for as many serotypes aspossible. Fig. 5 shows results for serotypes 19A and Pn 1, asan example, to evaluate the methanolysis condition overtime. Samples were methanolyzed in the methanolic 3 NHCl from 0.5 to 24 h, followed by the current derivatizationand chromatography procedure. Values obtained fromGC–MSD analysis were compared with the knownamounts of sample tested. The percentages of polysaccha-ride recovery gradually increased when the methanolysistreatment went from 0.5 to 2 h and then dropped oV when itwent from 2 to 24 h. In the case of Pn 1, the recovery ofpolysaccharide dramatically reduced after 2 h as the sam-ples were apparently getting destroyed (samples charredincreasingly dark over the extended treatment). In thesetwo cases, the 2-h methanolysis was optimal and was simi-larly applied to the other serotypes.
The methanolysis conditions established could also beused for polysaccharide at other stages of the conjugate vac-cine process, including activated polysaccharides and conju-gates. The carrier protein used for conjugation isnonglycosylated and does not contribute to the monosaccha-ride signals. Fig. 6 shows a comparison of the results for puri-Wed, activated, and conjugated polysaccharides from Pn 7F.Because the activation chemistry is very mild, aVecting only asmall percentage of residues, all three samples appear to bevery similar in monosaccharide composition. Furthermore,the analysis is not hampered by detection of the protein
Table 1Monosaccharide composition for the 13 pneumococcal serotype polysaccharides
Pn Serotype Monosaccharide
FucNAc Gal GalA GalNAc Glc GlcA GlcNAc Gro ManNAc PneNAc Rha Rib-ol Sug1 Sug2
1 2 13 1 14 1 1 1 15 1 1 1 1 1
6A 1 1 1 16B 1 1 1 17F 2 1 1 1 29V 1 2 1 114 2 1 1
18C 1 3 1 119A 1 1 119F 1 1 123F 1 1 1 2
268 Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274
content in the conjugate and is speciWc for the carbohydratecomponents. The GlcNAc peak area in the Pn 7F samplewas determined by peak purity analysis prior to quantitation.
Table 3 shows a comparison of colorimetric values ver-sus GC–MSD for the 13 serotypes at various stages of theprocess. The numbers vary from 92 to 123% over the entirerange of samples tested. GC–MSD quantitation of Pn 1was performed against a reference standard, whereas allother serotypes were quantitated against authentic mono-saccharide standards.
Linear response was evaluated for the GC–MSDmethod. Fig. 7 shows the results from testing of a Pn 19A
sample ranging from 5 to 500 �g. The 5- to 50-�g samplescontained 2 �g of myo-inositol as the internal standard, andthe Wnal derivatized samples were brought up in 0.1 ml ofsolvent for injection. The 50- to 500-�g samples contained20 �g internal standard and were worked up as outlined inthe current method. Then 1 �l of each sample was injectedby autosampler for GC–MSD analysis. The inset in Fig. 7shows the region spanning the 5- to 50-�g samples revealingslightly less linearity (R2 D 0.9743), which is not unexpectedat lower sample concentrations where assay variability maybe more pronounced. However, the overall R2 value was0.9987 for the unusually high range of samples covering
Fig. 2. GC–MSD total ion chromatograms for the 13 pneumococcal polysaccharides. The amounts of polysaccharide tested ranged from 0.1 to 0.5 mg,with 20 �g of myo-inositol included as an internal standard (ISTD).
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
Glc
Glc
10 15 20 25
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To
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on
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Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GlcGlc
GlcGlc
Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GalGalGal
Gal
GalGal
GalGalGal
Gal
GalGal
Gal
GalGal
GalGalGal
Gal GalGal
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
GlcA GlcA
GlcA GlcA
GlcA GlcA
ManNAc
ManNAc
GalNAc
GalNAc
GalGalGal
FucNAc
FucNAcPneNAc
GlcNAc
GlcNAc
Rib-ol
Rib-ol
3
4
5
6A
6B
7F
9V
14
18C
19A
19F
23F
(GalNAc)(FucNAc)
(PneNAc)
(Glc)
(ManNAc)
(Glc)(Glc)
Gal-pyruv
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
Glc
Glc
10 15 20 25
Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GlcGlc
GlcGlc
Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GalGalGal
Gal
GalGal
GalGalGal
Gal
GalGal
Gal
GalGal
GalGalGal
Gal GalGal
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
GlcA GlcA
GlcA GlcA
GlcA GlcA
ManNAc
ManNAc
GalNAc
GalNAc
GalGalGal
FucNAc
FucNAcPneNAc
GlcNAc
GlcNAc
Rib-ol
Rib-ol
(GalNAc)(FucNAc)
(PneNAc)
(Glc)
(ManNAc)
(Glc)(Glc)
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
ISTD
Glc
Glc
10 10 1515 2020 2525
Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GlcGlc
GlcGlc
Glc
Glc
Glc
Glc
Glc
Glc
GlcGlc
GalGalGal
Gal
GalGal
GalGalGal
Gal
GalGal
Gal
GalGal
GalGalGal
Gal GalGal
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
Rha
GlcA GlcA
GlcA GlcA
GlcA GlcA
ManNAc
ManNAc
GalNAc
GalNAc
GalGalGal
FucNAc
FucNAcPneNAc
GlcNAc
GlcNAc
Rib-ol
Rib-ol
(GalNAc)(FucNAc)
(PneNAc)
(Glc)
(ManNAc)
(Glc)(Glc)
Gal-pyruv
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 269
from 1£ to 100£ concentrations. For routine analysis, thetypical sample load for polysaccharide was 100 to 500�g;these polysaccharide samples routinely provided R2 valuesgreater than 0.99.
Limit of detection (LOD) levels for the GC–MSD areknown to be extremely low for semivolatile compounds,including monosaccharide TMS derivatives. Fig. 8 showsan overlay of chromatograms for a 10-�g Pn 19A samplefocusing on the main Rha TMS methyl glycoside peak. Thechromatograms were collected in the selective ion mode(SIM) for mass m/z 204, the major fragment mass ion gen-erated on electron impact ionization of the Rha TMSmethyl glycoside. The sample was serially diluted in theWnal solvent for injection from 10 �g down to 0.0001 �g.
Table 2Residual disaccharides and trisaccharides released on methanolysis forthe 13 pneumococcal serotypes expressed as percentages of total saccha-ride peak areas
Note. Pn 1 was the only serotype to release trisaccharides.
Pn Percentage residuals
Disaccharides Trisaccharides
1 11 343 5.3 —4 3.8 —5 9.5 —6A 0.04 —6B 0.92 —7F 0.11 —9V 3.7 —14 0.19 —18C 0.11 —19A 0.68 —19F 0.32 —23F 0.13 —
The Rha peak could be detected at the lowest level sampleof 0.0001 �g; however, the mass integrator was able to inte-grate the peak only for the samples greater than or equal to0.01 �g. Thus, the LOD/Q was set roughly set at 10 ng forthe amount of sample tested (theoretical). The amount ofsample on-column (1�l injected from a 1-ml sample)equates to 10 pg, consistent with the detection limits of theMSD.
Discussion
Both Pn 6 and Pn 19 contain phosphodiester bonds intheir primary polysaccharide structures. Interestingly, onlyserotypes 6A and 6B showed cleavage on both sides of thephosphodiester bond releasing Rib-ol and Gal, whereasserotypes 19A and 19F only cleaved at the Rha glycosidicbond, presumably leaving the phosphate–ManNAc intact,leading to subsequent incomplete derivatization by TMS.In the case of Pn 18C and Pn 23F, both of which containphosphoglycerol side chains, the branching residue contain-ing these side chains appeared to be released during meth-anolysis, indicating that this bond is acid labile. Thecomplete release of glycerol from the phosphate using thecurrent method is unclear at this time given that quantita-tion of glycerol is diYcult due to the high volatility of thederivative and the ubiquitous nature of glycerol as a con-taminant. Based on the work done by Talaga and co-work-ers [12], it is presumed that the phosphate–Gro remainsintact and would not be seen by GC–MSD analysis.
To this point, we have encountered no interference fromproteins or nucleic acids and only minimal interference fromcommon pneumococcal carbohydrate contaminants such asC-polysaccharide. Because contaminant levels are typicallylow in vaccine intermediates and conjugates, minimal
Fig. 3. GC–MSD total ion chromatogram for Pn 1. The saccharide content was released as GalA, disaccharides (30–35 min), and trisaccharides (44–51 min). ISTD, internal standard.
Minutes
To
tal I
on
Ab
un
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15 20 25 30 35 40 45 50
ISTD
GalA
GalA
GalAGalA
Disaccharides
Trisaccharides
(GalA)
Minutes
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Ab
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15 15 20 20 25 25 30 30 35 35 40 40 45 45 50 50
ISTD
GalA
GalA
GalAGalA
Disaccharides
Trisaccharides
(GalA)
270 Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274
interference was seen in the chromatography and data analy-sis. For example, C-polysaccharide will partially release Rib-ol when subjected to the current method (data not shown);however, at low levels, the presence of C-polysaccharidewould aVect quantitation for serotypes 6A and 6B only mini-mally. For all other serotypes, quantitation would be based onserotype-speciWc monosaccharides and would be unaVectedby the presence of contaminating C-polysaccharide.
Interestingly, the quantitation of saccharide contentby GC–MSD closely matches the results attained fromcolorimetric assays (for hexose or uronic acid), with arange of 92 to 123% for the samples tested. Even thoughthe colorimetric assays, such as the anthrone and phenolsulfuric acid methods for neutrals and the phenylphenoland carbazole methods for uronic acid, are not individualsugar-speciWc assays (e.g., Glc vs. Gal), these assays
Fig. 4. Mass spectra for the main TMS methyl glycosides for the 12 monosaccharides, plus Gro and Rib-ol, comprising the 13 pneumococcal polysaccha-rides. A mass spectrum for the Sug2 TMS derivative was unavailable (see Results). All spectra were collected for the mass range of 50 to 550u in the EI ion-ization mode.
GlcNAc
73
131147
173
204 259 314226
FucNAc73
131
147
173
204
247
316
Gal
73
133147
204
217
GalA73
133147
204217
159 234247 331
GalNAc
73
131147
173
204247
314218
Glc
73
133147
204
217
GlcA73
133147
204
217
159
234247
Gro73
103117
147205
218133
ManNAc
73
131147
173
204259217
PneNAc73
131
147
173
204
247
348
217
Rha
73
133147
204
217
Rib-ol73
103147
204
217
116
129
191247
350
Sug173
173
300
116
131
285
217
m/z
Fra
gm
ent
Mas
s Io
n A
bu
nd
ance
GlcNAc
73
131147
173
204 259 314226
FucNAc73
131
147
173
204
247
316
Gal
73
133147
204
217
GalA73
133147
204217
159 234247 331
GalNAc
73
131147
173
204247
314218
Glc
73
133147
204
217
GlcA73
133147
204
217
159
234247
Gro73
103117
147205
218133
ManNAc
73
131147
173
204259217
PneNAc73
131
147
173
204
247
348
217
Rha
73
133147
204
217
Rib-ol73
103147
204
217
116
129
191247
350
GlcNAc
73
131147
173
204 259 314226
GlcNAc
73
131147
173
204 259 314226
FucNAc73
131
147
173
204
247
316
FucNAc73
131
147
173
204
247
316
Gal
73
133147
204
217
Gal
73
133147
204
217
GalA73
133147
204217
159 234247 331
GalA73
133147
204217
159 234247 331
GalNAc
73
131147
173
204247
314218
GalNAc
73
131147
173
204247
314218
Glc
73
133147
204
217
Glc
73
133147
204
217
GlcA73
133147
204
217
159
234247
GlcA73
133147
204
217
159
234247
Gro73
103117
147205
218133
Gro73
103117
147205
218133
ManNAc
73
131147
173
204259217
ManNAc
73
131147
173
204259217
PneNAc73
131
147
173
204
247
348
217
PneNAc73
131
147
173
204
247
348
217
Rha
73
133147
204
217
Rha
73
133147
204
217
Rib-ol73
103147
204
217
116
129
191247
350
Rib-ol73
103147
204
217
116
129
191247
350
Sug173
173
300
116
131
285
217
Sug173
173
300
116
131
285
217
73
173
300
116
131
285
217
Fra
gm
ent
Mas
s Io
n A
bu
nd
ance
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 271
provide accurate quantitative measurements providedthat appropriate standards and controls are used.Although chromatographic (GC and HPLC) assays pro-vide quantitation of sugar-speciWc species present in thetest sample, these methods require hydrolysis of the testsample prior to chromatographic separation. The resultspresented here demonstrate that optimized hydrolysisconditions provide quantitative recovery of the saccha-ride content. Therefore, careful consideration should begiven to multiple factors depending on the intent of theassay requirements. If one needs to quantify the totalamount of saccharide content present in vaccine compo-nents, both methods will provide satisfactory results pro-
Fig. 5. Optimization of methanolysis in 3 N HCl–methanol for Pn 19Aand Pn 1 polysaccharides. Samples were treated for 0.5 to 24 h at 121 °C.PS, polysaccharide.
vided that these methods are appropriately qualiWed and/or validated. However, if one needs to look at the compo-sition of a particular lot or serotype for investigative and/or characterization purposes, GC–MSD would providesuperior speciWc information. For characterization andinvestigative purposes, the GC methodology is preferredover methods such as HPAEC–PAD due to the addi-tional information obtained from the peak purity andmass spectral analyses. More important, GC–MSD maycontribute to characterization, along with other physico-chemical techniques (e.g., linkage analysis, NMR charac-terization, molecular size distribution), to deWne thesevaccines as well-characterized biologicals. Such compos-ite characterization studies can be used to demonstrateproduct equivalencies of various intermediates duringvaccine development, manufacturing changes in the pro-duction processes, and/or multisite manufacturing ofvaccine components.
Final drug product, a diluted mixture of multiple sero-types, is often diYcult to analyze due to the low levels ofpolysaccharide in the formulation. In addition, speciWc sero-type quantitation might not be possible in some cases; forexample, monosaccharide composition analysis would notdistinguish between serotypes 6A and 6B or between sero-types 19A and 19F. However, the low-level and speciWcdetection capabilities of the MSD can allow estimation oftotal saccharide content using this method by quantitating allrecovered monosaccharides in the mixture (data not shown).Limited serotype-speciWc data can be obtained from the mix-ture by using serotype-speciWc markers such as GalA for
Fig. 6. GC–MSD total ion chromatograms for Pn 7F at the puriWed, activated, and conjugate stages.
10 15 20 25
Minutes
To
tal I
on
Ab
un
dan
ce
ISTDGlc
Glc
Gal
GalGal
Rha
RhaGalNAc
GlcNAc
ISTD
Glc
Glc
Gal
GalGal
Rha
Rha GalNAc
ISTDGlc
Glc
Gal
Gal
Gal
Rha
RhaGalNAc GlcNAc
GlcNAc
10 10 1515 2020 2525
Minutes
To
tal I
on
Ab
un
dan
ce
ISTDGlc
Glc
Gal
GalGal
Rha
RhaGalNAc
GlcNAc
ISTD
Glc
Glc
Gal
GalGal
Rha
Rha GalNAc
ISTDGlc
Glc
Gal
Gal
Gal
Rha
RhaGalNAc GlcNAc
GlcNAc
272 Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274
serotype 1 and PneNAc for serotype 5 or Rib-ol for the sero-type 6A/6B family and GalNAc for serotypes 4 and 7F. Per-haps more appropriate, analysis of Wnal drug product by thecurrent method can be used as a unique proWling toolbecause chromatographic information can be obtained at themonosaccharide level for the most abundant monosaccha-ride (e.g., Glc) to the least abundant one (e.g., PneNAc) in asingle chromatogram. For serotype-speciWc information andWnal product characterization using GC–MSD, proceduressuch as monosaccharide linkage analysis may prove to bemore useful because this type of analysis would allow quanti-tation based on additional diagnostic signals (e.g., 3-linkedRib-ol in serotype 6A vs. 4-linked Rib-ol in serotype 6B).
The work presented here demonstrates the utility ofGC–MSD as a tool for analysis of pneumococcal polysac-charides at various stages of the conjugate vaccine process.Excellent resolution of monosaccharide derivatives withextremely speciWc, low-level detection makes this methodattractive for analysis of polysaccharides from varioussources and conWgurations, with the Xexibility of testing awide range of concentrations. The added beneWt of massspectral and peak purity capabilities allows increased conW-
dence in peak assignments and data analysis. GC–MSDmethods have been developed for the routine testing anddetailed characterization of pneumococcal polysaccharidesand conjugate vaccines.
Table 3Polysaccharide concentrations determined by colorimetrics versus GC–MSD
a The colorimetric values were generated by validated and/or qualiWed procedures for determination of hexose or uronic acid content.b Pn 1 samples were quantitated against a qualiWed Pn 1 reference standard. GC–MSD analysis of all other serotypes was based on authentic monosac-
charide standards.
Sample Colorimetrics (mg/ml polysaccharide)a GC–MSD (mg/ml polysaccharide) GC–MSD/Colorimetrics (%)
Pn 1 4.2 4.26b 102Pn 3 2.1 2.31 110Pn 4 1.59 1.64 103Pn 5 2.67 3.30 123Pn 6A 2.6 2.61 101Pn 6B 4.96 4.65 94Pn 7F 6.34 5.97 94Pn 9V 5.7 5.61 98Pn 14 8.1 9.47 117Pn 18C 2.6 2.45 94Pn 19A 8.1 7.76 95Pn 19F 4.5 4.50 100Pn 23F 2.7 2.90 108Activated Pn 1 7.1 7.80b 110Activated Pn 5 2.09 2.11 101Activated Pn 7F 5.0 4.80 96PnC 1 4.6 4.37b 95PnC 3 0.734 0.844 115PnC 5 3.2 3.35 105PnC 6A 0.54 0.543 101PnC 7F 1.9 1.77 93PnC 19A 0.62 0.575 93
Fig. 7. Linear responses of Pn 19A from 5 to 500 �g. The responses are measured by total polysaccharide detected and quantitated by GC–MSD.
y = 0.9894x - 5.9871
R2 = 0.9987
0
100
200
300
400
500
600
700
0 100 200 300 400 500
Sample Tested (µg)
Sam
ple
Det
ecte
d (
µg)
y = 0.8534x - 3.6901
R2 = 0.9743
0
10
20
30
40
50
0 10 20 30 40 50
y = 0.9894x - 5.9871
R2 = 0.9987
0
100
200
300
400
500
600
700
0 100 200 300 400 500
y = 0.8534x - 3.6901
R2 = 0.9743
0
10
20
30
40
50
0 10 20 30 40 50
y = 0.9894x - 5.9871
R2 = 0.9987
0
100
200
300
400
500
600
700
0 100 200 300 400 500
0
10
20
30
40
50
0 10 504020 30
Determination of saccharide content / J.S. Kim et al. / Anal. Biochem. 347 (2005) 262–274 273
Acknowledgment
We thank Vincent Turula and Eric Finley for their criti-cal reading of the manuscript.
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0.01
0.1
1
10 0.001
0.0001
11.00 11.10 11.20
Minutes
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(20
4.00
) A
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(Rha)
0.01
0.1
1
10 0.001
0.0001
11.00 11.10 11.20
Minutes
Sel
ect
Ion
(20
4.00
) A
bu
nd
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0.01
0.1
1
10 0.001
0.0001
0.01
0.1
1
10 0.001
0.0001
11.0011.00 11.10 11.10 11.20 11.20
Minutes
Sel
ect
Ion
(20
4.00
) A
bu
nd
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(Rha)
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