Posters & Publications


Glycobiology Publications

An Integrated Solution for High-throughput, User-friendly Glycoanalysis Using Rapid Separation by Capillary Electrophoresis

Poster presented at Bioprocessing Summit, Boston, MA, August 15-19, 2016

Glycan characterization is becoming necessary in the earliest stages of biotherapeutic cell line development, to the point where cell culture screening often requires glycan profiling. This entails significantly increased throughput for sample preparation, analytical instrumentation, data processing and expertise in glycan characterization. Unfortunately, these factors can cause a bottleneck to results. Here we present a glycan analysis solution that provides rapid sample preparation and analysis combined with a simplified data processing approach. The sample preparation includes a 5-minute deglycosylation step to release N-glycans, followed by glycan labeling and cleanup, and may completed in under 1 hour. Labeled N-glycans are separated using a small and user-friendly capillary electrophoresis (CE) instrument, with a run time of 2 minutes per sample. This process enables relative N-glycan quantification for up to 96 cell culture samples within a single workday.


Development of a 5-Minute Deglycosylation Method and Instant Labeling Dye for High Throughput N-Glycan Analysis by Mass Spectrometry

Poster presented at International Carbohydrate Symposium, New Orleans, LA, July 17-21, 2016

The structure of N-linked glycans can play a critical role in the pharmacology of therapeutic proteins, potentially affecting immunogenicity, pharmacokinetics and pharmacodynamics. This makes the characterization of N-glycans an essential part of the biotherapeutic development process. Analysis of N-glycans typically involve the labeling of enzymatically released glycans with a tag to allow for fluorescence detection; a process that often requires numerous hours or days to complete. In addition to fluorescence (FLR) detection, mass spectrometry (MS) is also often utilized. Unfortunately, many of the commonly-used fluorescent tags are limited with regard to MS sensitivity. We present a novel N-glycan sample preparation workflow, Gly-X ™, which uses a 5-minute in-solution digestion, instant labeling, and cleanup of excess label and denaturant prior to analysis.

• The Gly-X N-glycan sample preparation workflow can be completed in as little as 45 minutes

• The workflow includes InstantPC™, a new instant glycan labeling reagent that provides markedly increased MS and FLR sensitivity

• The InstantPC glycan label is suitable for hydrophilic interaction liquid chromatography (HILIC) utilizing both FLR and MS detection, allowing flexibility for screening applications as well as in-depth characterization of N-glycans


Automated N-Glycan Sample Preparation with an Instant Glycan Labeling Dye for Mass Spectrometry 

Poster presented at ASMS, San Antonio, TX, June 5-9, 2016

The characterization of glycans is an essential part of the biotherapeutic development process. Analysis typically involves the labeling of released glycans with a tag to allow for fluorescence detection; a process that often requires numerous hours or days to complete. In addition to fluorescence detection, mass spectrometry is also often utilized. Presented here is the use of an automated N-glycan sample preparation platform with a glycosylamine-reactive dye (InstantPC™) that provides increased fluorescence and MS sensitivity. Automated N-glycan sample preparation provides reproducible data between replicates and reduced operator involvement.


N-Glycan Analysis: From High-Throughput Screening to In-Depth Characterization

Aled R. Jones, Ph.D.  Seminar presented at CASSS WCBP, Washington, DC, Jan 26-28, 2016


Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

Szekrényes Á et al., including ProZyme co-authors.  2016;2(8):56-64. International multi-laboratory study utilizing GlykoPrep APTS kits and APTS-labeled glycan standards.

Abstract: An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.


An Integrated Solution for High-throughput, User-friendly Glycoanalysis Using Rapid Separation by Capillary Electrophoresis

Poster presented at CASSS CE Pharm, Brooklyn, NY, Sept 20-25, 2015

Abstract: Glycan characterization is becoming necessary in the earliest stages of biotherapeutic cell line development, to the point where cell culture screening often requires glycan profiling. This entails significantly increased throughput for sample preparation, analytical instrumentation, data processing and expertise in glycan characterization. Unfortunately, these factors can cause a bottleneck to results. Here we present a glycan analysis solution that provides rapid sample preparation and analysis combined with a simplified data processing approach, enabling relative N-glycan quantification for 96 cell culture samples within a single workday. 


Development of an Instant Glycan Labeling Dye for High Throughput Analysis by Mass Spectrometry

Poster presented at ASMS, St. Louis, MO, May 31 - June 4, 2015

Abstract: Glycosylation of biotherapeutic proteins is frequently a critical quality attribute; therefore the characterization of glycans on biotherapeutics is an important activity in the development process. A common approach is to release and label N-glycans with a tag to allow for fluorescence detection; a process that often requires numerous hours to complete. Unfortunately, many of the most commonly used fluorescent tags have poor MS sensitivity. • We present a novel instant glycan labeling reagent, InstantPC™ (IPC) that provides markedly increased MS and FLR sensitivity. The workflow utilizes a rapid in-solution digestion, instant labeling, and cleanup of excess IPC label. IPC is shown to lend itself to both rapid (5-minute) and high-resolution (60-minute) HILIC methods for N-glycan separation, allowing flexibility for screening applications and in-depth characterization.


Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods.

Reusch D et al. (Roche), including ProZyme co-authors. 2015;7(1):167-79.


Characterizing Low-Abundance Glycans on Therapeutic Proteins

Poster presented at ASMS, Baltimore, MD, June 15-19, 2014

Abstract: A strategy is presented for characterizing low-abundance N-glycans using single exoglycosidase digestions with LC-MS and LC-MS/MS. N-glycans from Rituxan® (Rituximab) and Enbrel® (Etanercept) were released and labeled with Procainamide (PCA), treated with a single exoglycosidase, and the products analyzed. Using a threshold of 0.01%, forty-one (41) N-glycans were identified on Rituxan and seventy-one (71) on Enbrel. Minor species included N-Glycolylneuraminic acid (NGNA), hybrid glycoforms and triantennary N-glycans (Enbrel only).


Glycan Remodeling of Therapeutic Proteins using Galactosyltransferase and Sialyltransferases

Abstract: Glycosylation of therapeutic proteins can play critical roles in product quality and efficacy; controlling or optimizing glycosylation has many potential benefits. Terminal galactosylation has been shown to directly impact complement-dependent cytotoxicity (CDC); terminal sialylation can affect serum half-life; core fucosylation modulates antibody-dependent cellular cytotoxicity (ADCC); terminal and bisecting N-acetylglucosamines and high mannose glycans can impact ADCC as well as rate of clearance. An alternative strategy for modifying glycosylation is through the use of selected glycosyltransferase enzymes in a post-expression setting. In this way, glycomodified products can be quickly generated for various applications, such as structure-activity relationship (SAR) studies and quality-bydesign (QbD) experiments. There is even potential for the use of glycosyltransferases as a means for generating glycoopitimized therapeutics. Presented here are the results from glycomodifications performed on Rituxan® and Enbrel® using β(1–4)-Galactosyltransferase (GalT) and two sialyltransferases, α(2–6)-Sialyltransferase (ST6) and α(2–3)-Sialyltransferase (ST3). Analysis of 2-AB labeled N-glycans by UPLC®-FLR demonstrates the ability to generate biotherapeutics with varying degrees of galactosylation and sialylation.


Automated Sample Preparation for N-Glycan Analysis by Orthogonal Methods: UPLC and CE

Poster presented at CASSS WCBP, Washington, DC, Jan 28-30, 2014.


Secondary N-Glycan Remodeling of Therapeutic Proteins: α(2-6) Sialyltransferase

Poster presented at Society for Glycobiology, St. Petersburg, FL, Nov 17-20, 2013.

Sialylation plays important roles in the development and function of biotherapeutic proteins; in particular it can have a large impact on product stability, efficacy and pharmacokinetics. An α(2‑6) linkage-specific sialyltransferase (ST) was used to increase sialylation of Galß1-4GlcNAc units on Enbrel® and Rituxan® in vitro. The activity of ST was examined via a timecourse, and N-glycan analysis was performed to determine the level of hypersialylation achieved at each time point. N-glycan samples were prepared using ProZyme's GlykoPrep® Rapid N-Glycan Sample Preparation with 2-AB and APTS; analysis was performed using a Waters ACQUITY UPLC®-FLR system and Beckman Coulter® PA 800 plus-LIF system. Additionally, linkage specificity was confirmed by treatment of the hypersialylated glycans with Sialidase A™ and Sialidase S™ exoglycosidases.


Orthogonal Methods for Glycoanalysis: CE and UPLC

Poster presented at CASSS CE Pharm, Arlington, VA, Oct 6-10, 2013.
Method details available here.

Abstract: The biopharmaceutical drug development process often requires the application of orthogonal methods to show method selectivity and specificity, confirm and identify components, and show that the appropriate level of due diligence has been performed for full product characterization. For N-glycan analysis, the predominant methods are liquid chromatography and capillary electrophoresis. The method ultimately chosen for routine analysis or product release has largely been a matter of preference, but the importance of utilizing orthogonal analyses in the decision-making process should not be overlooked. Presented here are the analyses of different analyte mixtures by both liquid chromatography (Ultra-Performance LC, UPLC®) and capillary electrophoresis (CE with laser-induced fluorescence, CE-LIF); multiple separation programs were performed for each method. By comparing these results, it becomes clear that each method has its strengths and weaknesses. These differences highlight the importance of performing orthogonal analyses for product characterization. 


Assessing the Variability of an Innovator Molecule N-Glycan Profile

High-throughput Screening for a Biosimilar N-Glycan Profile

N-Glycan Profiling Using Orthogonal Methods: UPLC® and CE

An Integrated Strategy for N-Glycan Sample Preparation and Analysis Suitable for All Stages of Therapeutic Protein Discovery, Characterization, Manufacture and Quality Release

Feasibility of GlykoPrep™ Sample Preparation for Glycoanalysis on the AssayMAP® Bravo® Liquid Handling Workstation

Rapid Sample Prep for N-Glycan Analysis Using High Throughput Micro Chromatography

Rapid, High Throughput, High Sensitivity Determinations of MAb Concentration for Cell Line Screening and Process Optimization

A New Platform for High Throughput N-Glycan Sample Preparation

A New Platform for High Throughput Micro Chromatography Analysis

Poster presented at WCBP, Washington, DC, January 2010


Screening Sialic Acid Content to Optimize Pharmacokinetics and Pharmacodynamics of Glycoprotein Therapeutics

Poster presented at WCBP, Washington, DC, January 2010


On the Path to Biobetter Therapeutic Glycoproteins: Simple and Rapid Domain-Specific Screening to Target and Control Optimal Glycan Profiles

Improving Standard N-Glycan Sample Preparation with Manual Automation Using Microchromatography to Improve Efficiency, Accuracy, and Reproducibility

High-Throughput Platform for Preparation of APTS-Labeled N-Glycans: Improving the Accuracy, Reproducibility and Time-to-Results of N-Glycan Profiling

Rapid Sample Preparation of Biologics to Support High-throughput and High-resolution Glycan Analysis by Capillary Electrophoresis

Improved Sialic Acid Quantitation Assay Suitable For Robotics And Process Analytical Technology Applications

Poster presented at Society for Glycobiology, Fort Worth, TX, November, 2008


Qualification of a Process Analytical Technology for Quantifying Sialic Acid On Therapeutic Proteins Using Two Instrument Platforms

Poster presented at Society for Glycobiology, Boston, MA, November, 2007


An Enzyme-based Sialic Acid Quantitation Assay for Rapid Screening of Therapeutic Glycoproteins During Process Development: A Potential Process Analytical Technology 

Poster presented at WCBP, Washington, DC, January 29-31, 2007 


Phycobiliprotein Publications


Evaluation of anti-Human IgG Phycoerythrin Reporter Conjugate in a Model Human IgG Immunoassay

Poster presented at Luminex Planet xMAP, 2010


Room Temperature Stability Study of Streptavidin-Phycoerythrin Reporter Conjugates Using a Model TSH Immunoassay

Poster presented at Luminex Planet xMAP, 2009


Evaluation of Streptavidin-Phycoerythrin Reporter Conjugates in a Model TSH Immunoassay

Poster presented at Luminex Planet xMAP, 2009


Optimizing Nucleic Acid Detection by Altering Detection Chemistry

Poster presented at Luminex Planet xMAP, 2008


New SA-PE Conjugates Improve Critical Assay Parameters in Sandwich Immunoassays Performed using Luminex® xMAP® Technology

Poster presented at  SBS, 2008


New SA-PE Conjugates Reduce Capture-antibody-specific Background in Sandwich Immunoassays

Poster presented at Luminex Planet xMAP, 2008


Pushing the Limit of Detection: Signal Amplification and Screening for the Optimal Reporter Achieves Sub-attomole Sensitivity

Poster presented at Luminex Planet xMAP, 2007


Choice of Streptavidin-Phycoerythrin Conjugate is a Critical Element for Success in Assay Development

Poster presented at Luminex Planet xMAP, 2007


Lot-to-Lot Comparison of Streptavidin-PE Conjugates in Two Commercial Assays

Poster presented at Luminex Planet xMAP, Europe Symposium, 2006