Tools for Glycan Analysis

Transcription

1 Tools for Glycan Analysis

2 Enzyme Quality & Purity Endoglycosidases QA-Bio enzymes are highly-stable, pure preparations. Enzymes remain active for several days under reaction conditions. QA-Bio enzymes are proven free of contaminating protease and exoglycosidase activity. No glycerol or BSA additives. Endo F1 recombinant Elizabethkingia meningosepticum Selective release of high mannose and some hybrid type N-glycans Specific 16 U/mg 17 U/ml E-EF01 1 Unit 60 µl Endo F2 recombinant Elizabethkingia meningosepticum Selective release of biantennary N-glycans from peptides and proteins Specific 20 U/mg E-EF Unit 60 µl Endo F3 recombinant Elizabethkingia meningosepticum Release of triantennary and biantennary (at a 50x reduced rate) N-glycans Specific 25 U/mg E-EF Unit 60 µl Endo F Multi-Kit recombinant Elizabethkingia meningosepticum 20 µl each of Endo F1, Endo F2, and Endo F3 : Deglycosylation of native proteins resistant to PNGase F cleavage Determination of glycan type (high mannose, biantennary, tri/tetrantennary) Deglycosylating proteins which normally precipitate after deglycosylation X-Ray crystallography Endo F1 Endo F2 Endo F3 E-EF Unit 60 µl Tools For Glycan Analysis

3 Endoglycosidases PNGase F Elizabethkingia meningosepticum native and recombinant enzyme available PNGase F removes all N-glycans from mammalian glycoproteins 25 U/mg: 100% of the published PNGase F activity level No salts, BSA, EDTA, glycerol, or other additives Stable several days under reaction conditions Recombinant and native enzymes show no detectable differences Specific 25 U/mg E-PNG Unit 60 µl E-PNG05 1 Unit 200 µl Recombinant E-RPNG Unit 60 µl Endo H recombinant Elizabethkingia meningosepticum Release of asparagine-linked hybrid or high mannose oligosaccharides but not complex oligosaccharide Specific 30 U/mg E-EH Unit 60 µl O-Glycosidase Recombinant Streptococcus pneumoniae Cleaves only unsubstituted Gal-ß(1-3)GalNAc-alpha disaccharides attached to the serine or threonine residues of proteins or peptides Specific 12 U/mg 1.2 E-G U 60 µl Endo-Beta-Galactosidase recombinant Bacteroides fragili Cleaves internal β(1-4) galactose linkages in unbranched, repeating poly-nacetyllactosamine structures. Specific 150 U/mg 1 E-XBG Unit 60 µl

4 Exoglycosidases ß-(1-4)-Galactosidase recombinant S. pneumoniae Sialidase Au Alpha-(2-3,6,8,9) recombinant A. ureafaciens Specific 6 U/mg 3 U/ml Specific 135 U/mg E-BG Unit 60 µl E-S Unit 60 µl ß-(1-3,4,6)-Galactosidase Bovine testes Sialidase Cp Alpha-(2-3,6) recombinant Clostridium perfringens Specific 10 U/mg 2. Specific 250 U/mg 1 E-BG Unit 200 µl E-S Unit 60 µl Alpha(1-3,6)-Galactosidase recombinant E. Coli Specific 30 U/mg 400 U/ml E-AG01 24 Units 60 µl Sialidase Sp Alpha-(2-3) Streptococcus pneumoniae Specific 150 U/mg E-S Unit 60 µl Alpha-(1-2,3,6)-Mannosidase Jack Bean Glucosaminidase recombinant S. pneumoniae Specific 10 U/mg Specific 80 U/mg 40 U/ml E-AM Unit 60 µl E-GL Unit 60 µl Alpha-(1-6)-Mannosidase recombinant E. coli Alpha-(1-6)-Fucosidase Elizabethkingia meningosepticum Specific 1 U/mg 1 U/ml Specific 1.5 U/mg 1 U/ml E-AM Unit 60 µl E-F Unit 60 µl Tools For Glycan Analysis

5 Sialic Acid Analysis SialiQuant Sialic Acid Quantitation Kit Kit contains all the reagents to quickly and accurately quantitate sialic acids, including N-acetylneuraminic acid (NANA), N-glycolylneuraminic acid (NGNA). This kit includes reagents for 25 assays, each assay measuring from nmoles of sialic acids. Most forms of sialic acid found in nature are complexed in glycoconjugates. Sialic acid can be released through the action of sialidase and can be detected as free sialic acid. The kit includes Sialidase Au and procedures for the quantitation of total sialic acid in: Glycoproteins Cell surface glycoproteins Polysialic acids Capsular polysaccharides consisting only of polysialic acid In the past, release of sialic acid from glycoconjugates has been limited by the effectiveness of the sialidase used and has led to under reporting of total sialic acid yields. Sialidase Au cleaves all sialic acid linkages, including α(2-8) and α(2-9) linkages, as well as branched sialic acids. Variants of sialic acid such as N-glycolylnueraminic acid or O-acylneuraminic acid are also cleaved. Digestion with Sialidase Au is therefore the only reliable method of generating total free sialic acid from glycoconjugates without introducing losses typically associated with chemical hydrolysis. In this method, N-Acetylneuraminic acid aldolase catalyzes the reversible reaction: N-Acetylneuraminic acid N-Acetylmannosamine + Pyruvic acid The pyruvic acid can be reduced to lactic acid by β-nadh and lactic dehydrogenase: Pyruvic Acid + β-nadh Lactic Acid + β-nad Under the proper conditions, the first forward reaction predominates, and when coupled with β-nadh reduction of pyruvic acid, the reaction goes to completion. β-nadh oxidation can be accurately measured spectrophotometrically. KE-SIALQ 25 Assays of nmoles of Sialic Acid LudgerTag TM Sialic Acid Labeling Kit DMB 1,2-diamino-4,5- methylenedioxybenzene.2hcl This kit contains the reagents for the release of all sialic acids followed by labeling with the DMB dye Includes a sialic acid reference panel (pictured here) Notes Can detect from 20 pmol to 2.5 nmol of sialic acids Labeled sialic acids can be detected by either fluorescence detection or UV absorbance LT-KDMB-A1 Sialic Acid Aldolase recombinant E. Coli Specific 15 U/mg 100 U/ml E-ALD01 6 Units 60 µl N-Acetylmannosamine + Pyruvic acid CMP-Sialic Acid Synthetase recombinant E. Coli Specific 2.5 U/mg 9 U/ml E-CMP Unit 60 µl

6 LudgerTag TM Fluorescent Labeling Kits Glycan Labeling Kits Acetic Acid Promotes acid catalyzed ring opening of the glycan reducing terminus prior to the formation of an imine intermediate between the sugars and the unprotonated form of the dye. DMSO An anhydrous solvent used to dissolve all the components of the reductive amination labeling reaction. Sodium Cyanoborohydride This reductant stabilizes the imine produced by the reaction of the dye and glycans to produce stable fluorescently labeled glycans. Fluorescent Dye Analytical grade fluorescent dye. The comprehensive range of LudgerTag dyes include 2-AB, 2-AA, and DMB to cover different types of glycoanalysis by HPLC and MS. The LudgerTag kit reagents are purified to analytical grade and are dispensed and sealed under clean, inert atmospheres. The ampoules are pre-cleaned by pyrolysis at 500 C then opened just before dispensing and sealing under oxygen-free dry nitrogen. These controls ensure that your analyses work properly each and every time. LudgerTag TM 2-AB Kit 2-Aminobenzamide LudgerTag TM 2-AA Kit 2-Aminobenzoic acid Quantitative glycoprofiling of therapeutic glycoproteins by HPLC and MS Glycan characterization in proteomics studies Notes LudgerTag 2-AB labeling technology is now widely used for quantitative glycoprofiling analyses as part of biopharmaceutical lot release QC. 2-AB labeling significantly enhances sensitivity for mass spectrometric analysis of glycans by MALDI-TOF (enhancements of S/N for oligosaccharides are typically in the range of 5-40 fold). LT-KAB-A2 λ ex =320 nm λ em =420 nm Mass= gmol -1 Quantitative glycoprofiling of therapeutic glycoproteins by HPLC, MS, CE and gel electrophoresis Glycan characterization in proteomics studies Monosaccharide analysis by HPLC Notes For most applications 2-AA can replace 2-AB labeling with equivalent results, often times with increased sensitivity. 2-AA labeling significantly enhances sensitivity for mass spectrometric analysis of glycans by MALDI-TOF by localizing the charge distribution during laser desorption. λ ex =320 nm λ em =420 nm LT-KAA-A2 Mass= 137 gmol -1 LudgerTag TM DMB Sialic Acid Labeling Kit LudgerTag TM 2-AA Monosaccharide Analysis Kit Qualitative sialic acid analysis Notes Contains all the reagents for the release and DMB labeling of sialic acids. Included in the kit are a sialic acid reference panel and both NeuAc and NeuGc quantitative standards. LT-KDMB-A1 λ ex =295 nm λ em =352 nm Mass= gmol -1 Quantitative monosaccharide analysis for biopharmaceuticals Notes Contains all the reagents for the release and 2-AA labeling of monosaccharides. Kit includes the release reagents, labeling reagents and monosaccharide standards LT-MONO-96 λ ex =320 nm λ em =420 nm Mass= 137 gmol -1 Tools For Glycan Analysis

7 Purification and Separation LudgerClean TM T1 Cartridges Fast, high-throughput cleanup of glycan samples after fluorescent labeling T1 cartridges give significant reduction in levels of excess dye while giving excellent glycan recovery. The cartridges have been validated following ICH Q2(R1) guidelines for cleanup of 2-AA and 2-AB labeled glycans. Precision and repeatability CVs over a range of glycan concentrations were <3%. LC-T1-A6 6 Cartridges LudgerClean TM S Cartridges Purification of labeled glycans after fluorescent labeling Purification of non-labeled glycans from peptides Protocol Prime cartridge with a sequence of solvent washes Apply sample (typically 5 μl of LudgerTag labeling reaction mix) Wash unbound unreacted dye using high acetonitrile wash Elute glycans with water LC-S-A6 6 Cartridges LudgerClean TM EB10 Cartridges Purification of glycans after glycosidase (e.g PNGase F) treatment Removal of salts and detergent from samples prior to fluorescent labeling or MS Protocol Prime cartridges with a sequence of solvent washes Apply sample (typically up to 0.5 mg glycoprotein) Wash off unbound non-glycan contaminants (e.g. salts) Elute glycans with water/acetonitrile/trifluoroacetic acid mix LC-EB10-A6 6 Cartridges LudgerClean TM PBM Glycosidase Cleanup Plates For the removal of exoglycosidase enzymes from 2-AA or 2-AB labeled N-glycans after glycan sequencing Also works with unlabeled glycans The plate is designed for use with the Ludger-Velocity SPE vacuum manifold system to give excellent clean up of up to 96 samples simultaneously. LC-PBM Well plate

8 A2 & A2F Glycans Biantennary Standards Glycan Mass Label Part Number Amount A2F 2370 Daltons none SA-A2F 20 µg 2-AA CAA-A2F pmol 2-AB CAB-A2F pmol A1F 2078 Daltons none SA-A1F 20 µg 2-AA CAA-A1F pmol 2-AB CAB-A1F pmol NA2F (G2F) 1787 Daltons none SA-NA2F 20 µg 2-AA CAA-NA2F pmol 2-AB CAB-NA2F pmol FA2G1 (G1F) 1624 Daltons none SA-FA2G1 20 µg 2-AA CAB-FA2G pmol 2-AB CAA-FA2G pmol NGA2F (G0F) 1463 Daltons none SA-NGA2F 20 µg 2-AA CAA-NGA2F pmol 2-AB CAB-NGA2F pmol A Daltons none SA-A2 20 µg 2-AA CAA-A pmol 2-AB CAB-A pmol A Daltons none SA-A1 20 µg 2-AA CAA-A pmol 2-AB CAB-A pmol NA2 (G2) 1641 Daltons none SA-NA2 20 µg 2-AA CAA-NA pmol 2-AB CAB-NA pmol NGA2 (G0) 1317 Daltons none SA-NGA2 20 µg 2-AA CAA-NGA pmol 2-AB CAB-NGA pmol Tools For Glycan Analysis

9 Mannose Glycans Oligomannose Standards Glycan Mass Label Part Number Amount M3N2 911 Daltons none SA-M3N2 20 µg v 2-AA CAA-M3N pmol 2-AB CAB-M3N pmol Oligomannose Daltons none SA-MAN5 20 µg 2-AA CAA-MAN pmol 2-AB CAB-MAN pmol Oligomannose Daltons none SA-MAN6 20 µg 2-AA CAA-MAN pmol 2-AB CAB-MAN pmol Oligomannose Daltons none SA-MAN7 20 µg 2-AA CAA-MAN pmol 2-AB CAB-MAN pmol Oligomannose-7D Daltons none SA-MAN7D1 20 µg 2-AA CAA-MAN7D pmol 2-AB CAB-MAN7D pmol Oligomannose Daltons none SA-MAN8 20 µg 2-AA CAA-MAN pmol 2-AB CAB-MAN pmol Oligomannose Daltons none SA-MAN9 20 µg 2-AA CAA-MAN pmol 2-AB CAB-MAN pmol Hybrid 1642 Daltons none SA-HYBRID 20 µg 2-AA CAA-HYBRID pmol 2-AB CAB-HYBRID pmol

10 Glycan Standards MAb4 TM Glycan Reference Panel a mixture of glycans commonly found on monoclonal antibodies includes G2F (NA2F), G0F(NGA2F), and both G1F isomers SA-MAB4 10 µg Misc. Standards Glycan Mass Label Part Number Amount O-Linked Core Daltions none SA-CORE1 20 µg Gal α-(1-3) Gal 666 Daltons none SA-AGAL 20 µg A Daltons none SA-A3 20 µg 2-AA CAA-A pmol 2-AB CAB-A pmol NA Daltons none SA-NA3 20 µg 2-AA CAA-NA pmol 2-AB CAB-NA pmol NGA Daltons none SA-NGA3 20 µg 2-AA CAA-NGA pmol 2-AB CAB-NGA pmol NA Daltons none SA-NA4 20 µg 2-AA CAA-NA pmol 2-AB CAB-NA pmol NGA Daltons none SA-NGA4 20 µg 2-AA CAA-NGA pmol 2-AB CAB-NA pmol Hybrid 1642 Daltons none SA-HYBRID 20 µg 2-AA CAA-HYBRID pmol 2-AB CAB-HYBRID pmol Tools For Glycan Analysis

11 HPLC Columns and Buffers LudgerSep TM N1 and N2 Amide Columns and Buffer Widely used for profiling of 2-AB and 2-AA labeled glycans as part of lot release QC for therapeutic glycoproteins and monoclonal antibodies. The mode of action is hydrophilic interaction chromatography using an acetonitrile/ammonium formate (aq) gradient. LS-N2-2.0x150 N2 2.0 mm Column 2.0 x 150 mm LS-N2-4.6x150 N2 4.6 mm Column 4.6 x 150 mm LS-N1-4.6x250 N1 Column 4.6 x 250 mm LS-N1-4.6x10 N1 Guard Column 4.6 x 10 mm LS-N-BUFFX40 LS-N Buffer 2.0 x 150 mm LudgerSep TM C2 and C3 Anion Exchange Columns A strong anion exchange column used for determining the charge profile of 2-AB and 2-AA labeled glycans as part of biopharmaceutical QC. A superior replacement to weak anion exchange columns giving reliable, reproducible separations. Samples are applied in water and are typically eluted with a sodium acetate or ammonium formate aqueous salt gradient. LS-C3-7.5x75 C3 Anion Exchange Column 7.5 x 75 mm LS-C2-4.6x50 C2 Anion Exchange Column 4.6 x 50 mm LS-C2-4.6x150 C2 Anion Exchange Column 4.6 x 150 mm LudgerSep TM R1 and R2 Reverse Phase Columns These reverse phase column resolves glycans based on hydrophobicity. : Analysis of sialic acid variants labeled with DMB Analysis of monosaccharides labeled with 2-AA Analysis of glycans labeled with 2-AB. LS-R1-4.6x250 R1 Column 4.6 x 250 mm LS-R2-4.6x150 R2 Column 4.6 x 150 mm LudgerSep TM ur2 Reverse Phase UHPLC Columns The LudgerSep ur2 50 mm column was developed for the analysis of monosaccharides labeled with 2-aminobenzoic acid (2-AA) using UHPLC. The 100 mm ur2 column was developed for the analysis of sialic acids labeled with DMB using UHPLC. LS-UR2-2.1x50 ur2 50 mm Column 2.1 x 50 mm LS-UR2-2.1x100 ur2 100 mm Column 2.1 x 100 mm

12 Deglycosylation Kits Enzymatic CarboRelease Kit The enzymes are packaged separately in individual 20 microlitre vials. Kit includes enzymes and buffers required to remove all N-linked oligos and many O-linked sugars from 2 mg of glycoprotein, via twenty 100 microgram samples. Contains 20 µl each of: PNGase F O-Glycosidase (S. pneumoniae) Sialidase Au (A. ureafaciens) ß-Galactosidase (S. pneumoniae) Glucosaminidase (S. pneumonia) PNGase F O-Glycosidase ß-Galactosidase KE-DG01 Enzymatic DeGlycoMx Kit Sialidase This kit includes our DeGlycoMx, a premixed cocktail of the enzymes required to remove all N-linked oligosaccharides and most O-linked sugars from 0.5 mg of glycoprotein, via 10 reactions of up to 50 micrograms of protein per reaction. Contains a 20 µl mix of: PNGase F O-Glycosidase (S. pneumoniae) Sialidase Au (A. ureafaciens) ß-Galactosidase (S. pneumoniae) Glucosaminidase (S. pneumonia) PNGase F Glucosaminidase KE-DGMX Endo F2 Endo F Multi-Kit 20 µl each of Endo F1, Endo F2, and Endo F3 recombinant Elizabethkingia meningosepticum Endo F1 Endo F3 : Deglycosylation of native proteins resistant to PNGase F cleavage Determination of glycan type (high mannose, biantennary, tri/tetrantennary) Deglycosylating proteins which normally precipitate after deglycosylation X-Ray crystallography E-EF03 Orela O-linked Glycan Release Kit The Ludger Liberate Orela Kit has been developed for the release of O-linked glycans from glycoproteins. Designed for easy, routine analysis of O-glycosylation by QC labs Straightforward and simple method Released glycans have free reducing terminii suitable for fluorescent labeling Fluorescent labeling allows relative quantitation of glycan species by HPLC Faster and lower cost than hydrazinolysis No special handling techniques The kit contains reagents for the release of O-linked glycans from glycoprotein biopharmaceuticals. Released glycans have free reducing terminii to allow fluorescent tagging by reductive amination. It includes reagents and materials for up to 12 glycoprotein samples analysed in parallel or two sets of 6 samples, typically up to 1 mg of glycoprotein per sample. LL-ORELA-A2 Orela Orela Tools For Glycan Analysis info@qa-bio.com fax