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HPLC '89Poster Presentation

Preparative Scale Separations of Oligosaccharideson Polymeric HPLC Packing Materials

G. Vella and K. JonesWaters Chromatography Division of Millipore

S. Hirani and D. McNeillyGenzyme Corporation, Boston, MA

D. Cummmg, R. Camphausen and D. BenjaminGenetics Institute, Andover, MA

/Water,. The absolute essentia I WatersMill,poreChromatographYcorporationDivision _A/'_ tersfor bioresearch. 34 Maple St. Divisionof MILLIPORE

Milford, MA O1757(508) 478-2000

PREPARATIVE SCALE SEPARATIONS OF OLIGOSACCHARIDES ONPOLYMERIC HPLC PACKING MATERIALS

C. Phoel_ 1 G. Vella 1 K. Jones 1 S. Hirani 2 D. McNeilly 2, D. Cumming 3I , ! ,

R. Camphausen 3, and D. Benjamin 3

1 Millipore Corporation, Waters Chromatography Division, 34 MapleStreet, Milford, MA 01757

2 Genzyme Corporation, 75 Kneeland Street, Boston, MA 021113 Genetics Institute, One Burtt Road, Andover, MA 01810

Objective: To demonstrate multi-mode chromatographic approaches usingpolymeric HPLC packing materials in the preparative purification ofoligosaccharides.

In elucidating oligosaccharide structures, identification of an unknownstructure based on chromatographic retention time alone, is not convincingbecause of the immense number of possible structural isomers which mayce-elute with the compound of interest. Unequivocal structuredetermination of oligosaccharides requires the use of high field NMRand/or FAB-mass spectrometry. One drawback of these techniques is thatthey require a large quantity of a homogeneous sample. High-performanceliquid chromatography (HPLC) has been used to purify large quantities ofoligosaccharides in high purity from complex mixtures. Described in thispaper are multi-mode chromatographic approaches, using stable polymericcolumns, which demonstrate the usefulness of HPLC in the isolation andpurification of large quanitities of oligosaccharides. Example onedemonstrates the use of the Prep Glyco-Pak TM N column for the isolationand purification of neutral complex and high mannose oligosaccharides.Example two illustrates a loading optimization study on prototypeGlyco-Pak II DEAE material packed in a glass AP column for thepurification of sialylated oligosaccharides from fetuin. Example threeillustrates the purification of chitin oligomers using the Glyco-Pak Nand Ultrahydrogel TM DP columns.

INTRODUCTION:

In oligosaccharide structure elucidation work, noidentification of an unknown structure based on

chromatographic co-migration is convincing becauseof the immense number of possible structuralisomers which might have identical retention times.Unequivocal structural determination ofoligosaccharides requires the usage of high fieldNMR and/or FAB-mass spectrometry. One draw-back of these techniques is that they require a largequantity of a homogeneous sample. High-performance liquid chromatography (HPLC) hasbeen used to purify large quantities ofoligosaccharides in high purity from complexmixtures. Described in this paper are severalexamples demonstrating the usefulness of HPLCwith stable polymeric columns for the isolation andpurification of large quanities of homogeneousoligosaccharides. Example one demonstrates theuse of the Prep Glyco-Pak TM N column for theisolation and purification of neutral complexand high mannose oligosaccharides. Example twoillustrates a loading optimization study on aprototype Glyco-Pak II DEAE in a glass AP columnfor the purification of sialylated oligosaccharidesfrom fetuin. Example three uses a multi-chromatographic mode approach (using twopolymeric columns) for the purification of chitinoligomers.

Prep Glyco-PakTMN

Purification of high mannoseand complex oligosaccharides

Hydrazine released ovomucoid DETECTION: UV at 200 nm

oligosaccharides INJECTION:: 20 mg of mixture

COLUMN: Prep Glyco-Pak TM N (22 x 600 mm)

ELUENT: Acetonitrile/Water (70:30)FLOWRATE: 7.25 ml/min.

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The maximum load on the analytical (7.8 x 300 mm)Glyco-PakTM N column is 1 to 2 mg. Illustrated arescale-up isolations using the Prep Glyco-Pak Ncolumn (22 x 600 mm) at a flow rate of 7.25ml/min, for neutral complex oligosaccharides(ovomucoid, Figures 1-3) and high mannoseoligosaccharides (Figure 4). Based on scale upstrategies, the load on the Prep Glyco-Pak Ncolumn is approximately 30 mg. Two injectionsof ovomucoid neutral complex oligosaccharidesat sample loads of 20 mg and 40 mg show littledeterioration of the analytical separation. Figure4 shows the baseline separation of high mannoseoligosaccharides on the Prep Glyco-Pak N columnutilizing a sample load of 23 mg.

Hydrazine released ovomucoid nc DETECTION: UV at 197 nm

oligosaccharides INJECTION:: 200 pg of mixtureCOLUMN: Glyco-Pak TM N (7.8 x 300 mm)ELUENT: Acetonitrile/Water (70:30)FLOW RATE: 1 ml/min.

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Reduced Endo H released high manno_ DETECTION: UV at 200 nmoligosaccharides INJECTION: 23 mg of mixture

C.,OLUMN: Prep Glyco-Pak TM N (22 x 600 mm) PEAK ID'S: 1. Man 3 GN 1ELUENT: Acetonitrile/Water (70:30) 2. Man 4 GN 1FLOWRATE: 7.25 ml/min. 3. Man 5 GN 1

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Hydrazine released ovomucoid DETECTION: UV at 200 nmoligosaccharides INJECTION:: 40 mg of mixture

COLUMN: Prep Glyco-Pak TM N (22 x 600 mm)ELUENT: Acetonitrile/Water (70:30)FLOWRATE: 7.25 ml/min.

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Loading optimization studyon a prototype Glyco-PakTMIIDEAE in a glass AP column

Purification of sialylatedoligosaccharides from fetuin

In order to scale the sample load to larger columnsfor preparative isolation work, a loading study mustbe done on an ana!ytical column. Illustrated(Figures 5-8) is a loading optimization studyutilizing fetuin oligosaccharides on a prototypeGlyco-Pak Ii DEAE_in a glass AP column (10 x100 mm). Figure 5 shows the analytical separationat 150 _g sample load. Figures 6 and 7 showsample loads of 5 and 10 mg respectively, with the10 mg load showing signs of overload but stillmaintaining an acceptable separation. Figure 8shows a sample overload of 50 mg with loss ofresolution. From this study it is now known thata 10 mg load on the 10 x 100 mm column can bescaled to a 40 mg load on the 20 x 100 mm columnand to 250 mg on the 50 x 100 mm column.

Hydrazine released fetuin oligosacch_llWes FLOW RATE: 1.6 ml/minCOLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm

column (10 x 100 mm) INJECTION: 5 mg of mixtureELUENTA: Water PEAK ID'S: 1. Mono-sialylatedELUENTB: 100 mM KH2PO4, pH 5.4 2. Di-sialylatedGRADIENT: Hold for 5 minutes at 0 mM B, then linear from 3. Tri-sialylated

2 mM B to 50 mM B for 60 minutes, hold for 4. Tetra-sialylated

30 minutes. 3 5. Penta-sialylatedmV

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Hydrazine releasedfetuinoligosac( FLOW RATE: 1.6 ml/minC(_UMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 210 nm

column (10 x 100 mm) INJECTION: 150 lu.g of mixtureELUENTA: Water PEAK ID'S: 1. Mono-sialylated

ELUENTB: 100 mM KH2PO4, pH 5.4 2. Di-sialylatedGRADIENT: Hold for 5 minutes at 0 mM B, then linear from 3. Tri-sialylated

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SAMPLE: Hydrazine released fetuin oligosaccharides FLOW RATE: 1.6 ml/minCOLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm

column (10 x 100 mm) INJECTION: 10 mg of mixtureELUENTA: Water PEAK ID'S: 1. Mono-sialylatedELUENTB: 100 mM KH2PO4, pH 5.4 2. Di-sialylatedGRADIENT: Hold for 5 minutes at 0 mM B, then linear from 3. Tri-sialylated

2 mM B to 50 mM B for 60 minutes, hold for 4. Tetra-sialylated30 minutes. 5. Penta-sialylated

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SAMPLE: Hydrazine released fetuin oligosaccharides FLOW RATE: 1.6 ml/minCOLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm

column (10 x 100 mm) INJECTION: 50 mg of mixtureELUENTA: Water PEAK ID'S: 1. Mono-sialylated

ELUENTB: 100 mM KH2PO4, pH 5.4 2. Di-sialylatedGRADIENT: Hold for 5 minutes at 0 mM B, then linear from 3. Tri-sialylated

2 mM B to 50 mM B for 60 minutes, hold for 4. Tetra-sialylated30 minutes. 5. Penta-sialylated

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Illustrated is the isolation and purification of chitinoligosaccharides (Figures 9-15) utilizing a multiplechromatographic separation mode approach.UltrahydrogeFMDP ( aqueous size exclusionchromatography) and Glyco-PakTM N(acetonitrile/water partition chromatography)columns have been used to evaluate chitin samplesprior to and after purification on a Prep Glyco-PakN column. Figures 9 and 10 respectively show theseparation of crude chitin and a fraction (FractionA) of the higher oligomers using a bank of threeUltrahydrogel DP columns and on an analyticalGlyco-Pak N column. With the Ultrah]drogel

columns, the larger oligomer elutes first (Peak 6 isa larger oligomer than Peak 5). With the Glyco-PakN column the reverse is true, the larger oligomer(Peak 6) elutes later than the smaller oligomer(Peak 5). Because the resolution of the oligomers isbest on the Giyco-Pak N column, 8 mg of Fraction Achitin oligomers were purified on a Prep Glyco-PakN column (Figure 11). Inorder to determine thepurity of the materials isolated, Figures 12 - 15show the analysis of the fractions pooled torepresent peaks 5 and 6 on the Ultrahydrogel DPcolumns and on the Glyco-Pak N column. Figure 12shows that on the Ultrahydrogei DP columns, Peak 6appears to be pure. However, the analysis of Peak

6 on the Glyco-Pak N column (Figure 13) shows thatthere is a small amount of Peak 5 still present.Figure 14 shows the analysis of Peak 5 with theUltrahydrogel DP columns which is stillcontaminated with a small quantity of a largeroligomer (possibly Peak 6). Figure 15 shows thatPeak 5 is also contaminated with a small quantity ofa smaller oligomer (present as a shoulder). Thismultiple column approach, which utilizes twodifferent selectivities (size exclusion andpartition), illustrates the strength of using morethan one chromatographic mode for thefractionation and purification of oligosaccharidesprior to structure elucidation.

Preparative isolation ofchitin oligosaccharides

Chitin Oligosaccharides

15COLUMN: UItrahydrogelTM DP (3 columns-7.8 x 300 mm)

ITIV ELUENT: Water_ FLOW RATE: 1 ml/min.

:I0 COL.TEMP.: 50° CDETECTION: R ISENSITIVITY: 16

- INJECTION: 100 ug of mixture

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0 15 FLOW RATE: 1 ml/min.DETECTION: UV at 200 nm

INJECTION: 150 ug of mixture

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Chitin Olig)saccharidesFraction A

S

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mVC(1Ub_: UltrahydrogelTM DP (3 columns-7.8 x 300 ram)

- ELUENT: Water_.0 FLOWRATE: 1 ml/min.

COL TEMP.: 50° CDETECTION: RISENSITIVITY: 16

5 - INJECTION: 60 ug of mixture

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Chitin Oligosaccharides Fraction A DETECTION: UV at 200 nmCOLUMN: Prep Glyco-Pak TMN (22 x 600 mm) INJECTION:: 8 mg of mixtureELUENT: Acetonitrile/Water (72:28)FLOWRATE: 7.25 ml/min.

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C(_UMN: Ultrahydrogel m DP (3 columns-7.8 x 300 mm) DETECTION: RIELUENT: Water SENSITIVITY: 16FLOW RATE: 1 ml/min.COL. TEMP.: 50 ° C

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10 / INJECTION: 60 _g of mixture

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- INJECTION: 20 pg'10

COLUMN: Glyco-Pak TM N (7.8 x 300 mm) DETECTION: UV at 200 nmELUENT: Acetonitrile/Water (72:28)FLOW RATE: 1 ml/min.

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: Ultrahydrogel TM DP (3 columns-7.8 x _)mm) DETECTION: RIWater SENSITIVITY: 1 6FLOW RATE: 1 ml/min.COL. TEMP.: 50 ° C

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15 - SAMPLE: ChilinOligosaccharidesINJECTION: 100lig of mixture

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'10 - INJECTION: 60 lig of mixture

mvI A15 SAMPLE: Chitin Oligosaccharide - Peak 5INJECTION: 20 p.g

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