NEW...Suppression of peeling during the release of O-glycans by hydrazinolysis

Oxford January, 2012

Ludger Ltd is delighted to announce the publication of a paper in the journal Analytical Biochemistry. The paper, written by Radoslaw P. Kozak at Ludger and colleagues addresses the problem of degradation or ‘peeling’ of O-glycans when they are released from glycoproteins using hydrazinolysis. Peeling is a general problem found with chemical release methods for O-glycans and results in poor repeatability and stoichiometry for analytical glycan profiles.The paper describes a new method for cleanup of samples prior to hydrazinolysis which greatly reduces peeling and therefore enhances O-glycan analysis.

Citation:

R.P. Kozak, L. Royle, R.A. Gardner, D.L. Fernandes, M. Wuhrer. Suppression of peeling during the release of O-glycans by hydrazinolysis, Analytical Biochemistry (2012) In Press.


NEW...High Throughput Clean-Up of Glycan Samples following Exoglycosidase Treatment

Oxford January, 2012

Exoglycosidase sequencing is a glycoprofiling technique often used to characterise glycoproteins. It provides detailed information on glycan structures and their relative proportions.

However following exoglycosidase digestions, the enzymes (particularly sialidase) remain in the glycan samples and can be detrimental to HPLC columns. If injected onto your HPLC column, there is the possibility of it contaminating the column and then it could act on other samples that are injected afterwards. For mass spectrometry, proteins and peptides can suppress the MS signal or have masses which interfere with the data.

The LudgerClean™ PBM Post-Exoglycosidase Clean-Up plate (Cat # LC-PBM-96) is a 96-well plate system that has been developed for removal of exoglycosidase enzymes from 2-AA or 2-AB labelled N-glycans after glycan sequencing. It can also be used for unlabelled 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.

To request a quote or place an order please contact info@ludger.com


High Throughput Post-Labeling Cleanup of Biopharmaceutical Glycans

Oxford November, 2011

HPLC analysis of fluorescently labeled oligosaccharides is a widely used tool for biopharmaceutical glycoprofiling. In this process, glycans are released from the therapeutic glycoprotein then tagged with a fluorescent dye such as 2-AB (2-aminobenzamide) or 2-AA (2-aminobenzoic acid) by reductive amination prior to chromatography. Excess labeling reagents such as dye and reducing agent interfere with the HPLC analysis - so need to removed without disturbing the relative proportions of the various glycan species.

One of the most reliable methods for achieving post-labeling cleanup is use of LudgerClean S-cartridges. These employ a hydrophilic membrane to separate the labeled glycans from non-glycan contaminants and are routinely used in the biopharma industry during glycoprofiling QC for lot release of FDA and EMA approved biopharmaceuticals. They are also the method of choice in our laboratories for detailed characterisation of drug glycosylation patterns for regulatory submissions. S-cartridges work superbly well but they are slow and not suited for high throughput (HT) work.

Ludger has developed a fast, high-throughput system for post-labeling cleanup of glycan samples. The LudgerClean T1 cartridge system is designed for drug glycoprofiling analysts who want the reliability of S-cartridges but also want to process larger numbers of samples easily and get the results more quickly. The cartridges work with a Ludger-Velocity™ SPE vacuum manifold.www.ludger.com/t1

To request a quote or place an order please contact info@ludger.com

Quantitative monosaccharide standards

November 08, 2011

Quantitative monosaccharide analysis is important for developers and manufacturers of biologic drugs. Regulators are putting increasing pressure on companies to perform accurate glycoprofiling on their biopharmaceuticals and the FDA in particular have insisted that companies need to perform monosaccharide composition analyses to demonstrate consistency of their glycosylated therapeutics.

Such monosaccharide composition analyses fall within ICH guidelines Q6B and Q5E for comparability studies during product development and after major manufacturing changes. Furthermore, the recent EMA monograph on mAbs and forthcoming USP chapters <1084> and <1094> on glycosylation analysis reinforce the need to perform glycoprofiling throughout the drug life cycle.

However in order to achieve accuracy when quantifying monosaccharides you need to calibrate using accurate quantitative standards

Ludger now sells a quantitative monosaccharide standard comprised of NIST-F and USP traceable glucosamine (GlcN), galactosamine (GalN), galactose (Gal), mannose (Man), glucose/dextrose (Glc) and fucose (Fuc) monosaccharides. This standard (Cat# CM-MONOMIX-10) is prepared using NIST-F traceable laboratory equipment. The amount of each monosaccharide in a vial is quantified against USP standards. We quote the amount of each monosaccharide as the average values obtained from a random sample of 12 monomix vials per batch ± the 95% confidence interval, and verify bulk concentrations independently using quantitative nuclear magnetic resonance (NMR).

To request a quote or place an order please contact info@ludger.com

UHPLC column for fast sialic acid analysis

October 24, 2011

Ludger is very pleased to announce the launch of a UHPLC column for the analysis of sialic acids. This will be of interest to those monitoring the ratio of N-acetylneuraminic acid (NeuAc) to N-glycolyneuraminic acid (NeuGc) in biotherapeutics.

The LudgerSep™ uR2 column (Cat# LS-UR2-2.1x100) is designed for use with the latest generation of UHPLC instruments capable of withstanding high flow pressures and fast sample analyses. Ludger have optimised a gradient for analysis of DMB (1,2-diamino-4,5-methylenoxybenzene)-labelled sialic acids using this column. Run times are under 10 minutes per sample, so the time required for analysis of 96 samples is only 16 hrs. Furthermore, solvent usage is also reduced five-fold compared to our standard HPLC run.

Release and labelling of sialic acids can be performed using the LudgerTag™ sialic acid release and labelling kit, (Cat # LT-KDMB-A1).

To request a quote or place an order please contact info@ludger.com

Launch of NeuAc and NeuGc Quantitative Standards

October 03, 2011

Controlling the ratio of N-acetylneuraminic acid (NeuAc) to N-glycolyneuraminic acid (NeuGc) is critical for biomanufacturers. The reason is that NeuAc is the desired, normal human-type sialylation, while NeuGc is found in non-human glycoproteins and is considered an undesired, aberrant form of sialylation for therapeutic glycoproteins. A widely used method for determining the NeuAc:NeuGc ratio is HPLC analysis of sialic acids labelled with fluorescent DMB (1,2-diamino-4,5-methylenoxybenzene).

Ludger is very pleased to announce the launch of two new quantitative standards, NeuAc (Cat# CM-NEU-AC-01) and NeuGc (Cat# CM-NEU-GC-01). These are quantitative standards handled using NIST-F calibrated apparatus and calibrated against USP traceable Neu5Ac and Neu5Gc monosaccharides respectively using DMB analysis kits (Cat# LT-KDMB-A1) and UHPLC. Serial dilutions of these standards can be used to obtain a standard concentration-fluorescence response curve on the HPLC.

To order or request a quote for either of these standards please email info@ludger.com

UHPLC column available for monosaccharide analysis

September 06, 2011

Ludger Ltd now sell a column for analysis of monosaccharides using a UHPLC system. The LudgerSep™ uR2 column (Cat# LS-UR2-2.1x50) is designed for use with the latest generation of UHPLC instruments capable of withstanding high flow pressures and fast sample analyses. Ludger have optimised a gradient for analysis of 2AA-labelled monosaccharides using this column, with run times of only 8 mins, so the time required for analysis of 96 samples is less than 13 hrs. Furthermore, solvent usage is also reduced ten-fold compared to our standard HPLC run.

To order a column or get a quote, please contact info@ludger.com

Ludger appoints US Technical Sales Consultant

July 25, 2011

Ludger is delighted to announce the appointment of Dr. Mark T. Johnson as its U.S. Technical Sales Consultant.

Mark joins Ludger to support its Sales Department and provide key technical assistance to customers. Previously Mark worked in the same capacity for Associates of Cape Cod (ACC), East Falmouth, MA. Mark has a valuable knowledge of GMP validation requirements for U.S. customers who are setting up glycoprofiling methods. Mark's technical background includes biochemistry, enzymology, protein structure, and HPLC. He has a broad understanding of structures, functions, and analysis of therapeutic glycoproteins and antibodies.

Gal-alpha-1,3-Gal Standard

December 14, 2010

2-AB Labeled Gal-alpha-1,3-Gal Standard is now available.

Our 2-AB labelled alpha-1,3-Gal standard (Cat No. CAB-ALPHAGAL-01) is now available. This glycan standard can be used during glycoprofiling as a positive control for sequencing experiments utilising alpha1-3 galactose specific exoglycosidase.

The presence of Gal-alpha-1,3-Gal structures within biopharmaceuticals is of huge importance to the pharmaceutical industry. Gal-alpha-1,3-Gal (also known as Alpha Gal) is a potentially immunogenic glycan which has been reported to be present in recombinant biological pharmaceuticals (Chung et al, 2008). It is found in the meat of non-primate mammals, cows milk and dog and cat dander. Humans cannot synthesize the glycan but can express antibodies against this structure (Galili et al, 1985). Anti-Gal antibodies comprise 1% of circulating IgG antibodies in all humans. An IgE-mediated allergic/hypersensitivity response to this glycan was observed in patients taking Cetuximab as a cancr treatment (Chung et al, 2008). Cetuximab is a chimeric murine-human cell monoclonal antibody. The cause for developing IgE antibodies specific to Alpha-Gal has been reported to occur after being bitten by certain types of tick; cases have been concentrated predominantly in the Southeast United States and parts of Australia. (Commins et al 2009; Van Nunen et al, 2009).

In humans the production of anti-Gal antibodies has presented a unique opportunity for developing methods that harness the immunologic potential of this antibody for clinically beneficial outcomes. Anti-Gal IgG molecules bound to -gal epitopes on particulate or soluble vaccines will target them to APC at the vaccination site, and induce effective uptake of the vaccine by APC (Galili, 2005).

References:


  • Chung CH, Mirakhur B, Chan E, et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. New Engl J Med. 2008;358:1109-1117.

  • Commins SP, Satinover SM, Hosen J, et al. Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-±-1,3-galactose. J Allergy Clin Immunol. 2009;123(2):426-433.

  • Commins SP and Platts-Mills TA. Anaphylaxis syndromes related to a new mammalian cross-reactive carbohydrate determinant. J Allergy Clin Immunol. 2009;124(4): 652-657.

  • Galili et al (1985) J. Exp. Med. 162: 573

  • Van Nunen S A, O’Connor, KS, Clarke LR, Boyle RX, Fernando SI. An association between tick bite reactions and red meat allergy in humans. Med J Aust. 2009;190:510-511.

  • Galili The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunology and Cell Biology (2005) 83, 674–686

For more details e-mail sales@ludger.com

Ammonium Formate x40 Buffer for Glycan Amide HPLC

Sept 02, 2010

The LudgerSep Ammonium Formate pH 4.4 Buffer x40 Concentrate for amide HPLC analysis of glycans is now available.

Preparation of ammonium format buffer for amide HPLC of glycans is laborious. Furthermore, variations in buffer pH between batches can result in significant variations in peak retention. The x40 concentrate makes preparation of clean, consistent ammonium formate buffer easy.

For more details e-mail sales@ludger.com

New method for LS-R1 HPLC Analysis of Glycans

Oct 10, 2009

Ludger have developed a method for analysis of 2AB labelled glycans using the LudgerSep R1 HPLC column as an orthogonal procedure to glycan analysis by hydrophilic chromatography on amide columns.

This method is particularly well suited for the following applications:

  • Determination of G0, G1, G2 ratios of monoclonal antibody (mAb) glycans
  • separation of fucosylated and non-fucosylated glycans (e.g.
  • bisecting GlcNAc/non-bisecting GlcNAc glycan separations
  • separating Man 5 from neutral complex bi-antennary glycans

In addition this method reduces analysis time by a factor of two (compared to the original method). Two columns joined with a zero dead volume connector are required for this application.

For more details e-mail sales@ludger.com

New Quantitative Monosaccharide Analysis Kit

Sept 14, 2009

Ludger Ltd is delighted to announce the launch of a new system for quantitative monosaccharide analysis of biopharmaceuticals. This is comprised of two parts; a kit for release and fluorescent labelling of monosaccharides and an HPLC column for analysis.

The kit (Catalogue No. LT-MONO-96) can be used in either of two modes:

  • Full quantitative monosaccharide analysis (e.g. for regulatory submissions) or
  • Routine monosaccharide analysis (e.g. for optimisation of glycosylation during clone screening and scale-up).

Samples for analysis can be intact therapeutics or oligosaccharides pre-released from the glycoprotein. The kit contains reagents for release and fluorescent labelling of neutral and amino monosaccharides alongside quantitative reference standards. Analysis of the labelled monosaccharide residues is then performed using the LudgerSep R1 column (Cat No. LS-R1-4.6x150) and quantitation is achieved using calibration curves obtained with the standards.

For more details e-mail sales@ludger.com

Ludger to launch two Ludger Liberate kits for release of N- and O-glycans

July 27, 2009

Ludger will soon be launching two new kits for releasing glycans from glycoproteins. These Ludger Liberate kits have been developed as tools for biopharmaceutical glycoprofiling and can be used for monitoring glycosylation throughout all the stages of the Drug Life Cycle.

Ludger Liberate Hydrazinolysis Kit (catalog number LL-HYDRAZ-A2)

Hydrazinolysis is a very well established method for the non-selective release and recovery of intact glycans from glycoproteins. The hydrazinolysis method is used in specialist laboratories for glycosylation analysis of approved biopharmaceuticals and follow up biosimilars.

The Ludger Liberate™ Hydrazinolysis Kit makes this practical method accessible to general R&D analytical labs to enable glycan characterisation during drug development, batch to batch comparability and support for regulatory submissions and for drug patents.

Allows release of both N- and O-linked glycans. Reaction conditions can be chosen for the release of either O-glycans, N-glycans or N+O glycans.

Standardized and qualified reaction components ensure consistent and reliable assay performance.

Released glycans have free reducing termini that are suitable for fluorescent labelling prior to analysis by HPLC, CE and MS. Fluorescent labelling allows relative quantitation of glycan species by HPLC.

Ludger Liberate™ Orela Kit (catalog number LL-ORELA-A2)

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 labelling prior to analysis by HPLC and MS. Fluorescent labelling allows relative quantitation of glycan species by HPLC.
  • Faster and lower cost than hydrazinolysis.
  • No special handling techniques.

For more details e-mail sales@ludger.com

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