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Introduction to NIBRT Why choose NIBRT contract research? Supporting ICH Requirements Bioanalytical techniques World class facilities Frequently Asked Questions NIBRT Scientists Key NIBRT research papers Client testimonials What’s new in 2016? Bioanalytical training Contact information
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NIBRT
National Institute for Bioprocessing Research and Training www.nibrt.ie
State-of-the-art facility funded (€57 million) opened in June 2011
Clients include Amgen, Lilly, Merck Serono, Regeneron, Janssen, United Therapeutics, FDA etc.
Awards include:
Winner of Manufacturing Collaboration of the Decade, Bioprocessing International 2012
Winner of Facility of the Year Awards 2012, ISPE-Interphex 2012
Center of Innovation Award, Waters Corp.
Multiple Irish Pharma Awards
Why choose NIBRT?
Detailed bioanalytical characterisation of biologics including post-translational modifications
with a specialist focus on glycosylation. Direct access to leading NIBRT scientists throughout the project lifetime. NIBRT reports provide a high level of detail and scientific understanding that is unrivalled. Access to world class bioanalytical facilities.
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Supporting ICH Requirements TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS Q6B
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2.1 Characterisation “Characterisation of a biotechnological or biological product (which includes the determination of physiochemical properties, biological activity, immunochemical properties, purity and impurities) by appropriate techniques is necessary to allow relevant specifications to be established”. “Extensive characterisation is performed in the development phase and, where necessary, following significant process change”.
COMPARABILITY OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS SUBJECT TO CHANGES IN THEIR MANUFACTURING PROCESS Q5E
2.2.2 Characterisation “When a manufacturing process change has been made that has the potential to have an impact on quality attributes, a complete or limited (but rationalised) repetition of the characterisation activity conducted for the market application is generally warranted to directly compare the pre-change and post change product”.
*NIBRT supports the highlighted ICH characterisation requirements
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Bioanalytical Techniques
N-glycan analysis
• Characterisation and structural elucidation
• Profiling • Occupancy site analysis
O-glycan analysis • Characterisation and structural
elucidation • Profiling
Peptide sequencing Post Translational Modification
analysis including Disulfide Bond Analysis
Post Translational Modification profiling
Intact protein analysis Host Cell Protein analysis
• Identification • Quanitfication-Hi3 LC-MSe
Analytical Ultra Centrifugation Other; HPLC, UPLC, mass
spectrometry, capillary electrophoresis based analysis
Method development and consultancy
New in 2016 • HDX-MS • O-glycan occupancy site analysis
NIBRT can provide the following analysis to support ICH-Q6B and Q5E requirements:
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World Class Facilities Equipment Capabilities
HPLC/UPLC Glycan profiling, glycan structural elucidation, Size Exclusion Chromatography, Ion Exchange Chromatography IEX, Chromatography-other.
LC-MS/MS Protein, glycan, small molecule characterisation and quantification.
Ce-Lif Capillary electrophoresis–Laser induced fluorescence provides and orthogonal means of separation of glycans to enable the glycan profiling and structural elucidation of a wider range of products.
CE-MS Capillary electrophoresis provides an orthogonal means of separation to enable MS analysis of a wider range of samples e.g. High mannose containing glycans.
LC-MRM Robust, specific and highly sensitive compound quantification.
HDX-MS Hydrogen Deuterium exchange mass spectrometry enables protein confirmation analysis, epitope mapping, protein-protein interaction, protein drug binding.
Analytical Ultra Centrifuge
Molecular weight determination, aggregation analysis, purity analysis and sedimentation analysis.
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What are the specific ICH requirements for N-glycan characterisation?
The minimal requirements as indicated by ICH-Q6B subsection 6.11f state that the carbohydrate content (neutral sugars, amino sugars and sialic acids) is determined and the structure of the carbohydrate chains, the oligosaccharide pattern and the glycosylation site(s) of the polypeptide is analysed.
NIBRT delivers this information thought a combination of complimentary techniques:
Level 1 UPLC-HILIC-FLR and/or Ce-Lif - glycan profile
Level 2 Exoglycosidase digestions - structural elucidation
Level 3 LC-MS - mass confirmation of structures
Frequently Asked Questions
Farrell, A et al. "Quantitative Host Cell Protein Analysis Using Two Dimensional Data Independent LC–MS E". Analytical Chemistry 87.18 (2015): 9186-9193. DOI: 10.1021/acs.analchem.5b01377
Frequently Asked Questions How does the NIBRT LC-MS Host Cell Protein (HCP) platform match existing analytical techniques?
Current HCP tests rely on the use of immunoassays (ELISA, Western Blot) however, such tests can be limited with the ability to develop polyclonal antibodies to detect the wide variety of HCP creating significant difficulty. In addition such tests are limited to providing quantitative information only.
The NIBRT Contract Research LC-MS platform can be applied as a powerful “orthogonal” technique to support immunoassay approaches currently employed in industry. The LC-MSe approach developed at NIBRT* enables Hi-3 quantification to support immunoassay based results and qualitative data to identify the individual HCP providing an insight to the potential immunogenic risk.
As recently discussed in US Pharmacopeial Forum (PF40(4) <1132>) HCP should be measured during process characterisation, in pre-clinical lots, in lots during clinical development and process validation samples from the final manufacturing process.
NIBRT Scientists
Prof Rudd’s research focuses on the development of robust glyco-technology for quantitative, detailed structural N- and O-glycan analysis. In 2012, Prof Rudd was awarded the Waters Centers of Innovation Programme which recognizes and supports the efforts of scientists facilitating breakthroughs in health and life science research
Prof Pauline Rudd Dr. Jonathan Bones
Dr. Bones research focuses on the development and application of liquid phase separations and mass spectrometry (LC-MS and CE-MS) for the analysis of complex biological systems. Recent work of note includes: the development of an LC-MS based platform for Host Cell Protein identification and quantification. Development of advanced LC-MS based platforms for quantitative glycomics.
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Key publications
Farrell, A et al. "Quantitative Host Cell Protein Analysis Using Two Dimensional Data Independent LC–MS E". Analytical Chemistry 87.18 (2015): 9186-9193. DOI: 10.1021/acs.analchem.5b01377. Millán Martín, S et al. "Comparative Analysis Of Monoclonal Antibody N-Glycosylation Using Stable Isotope Labelling And UPLC-Fluorescence-MS". The Analyst 140.5 (2015): 1442-1447. DOI: 10.1039/C4AN02345E. Houel, S et al. "N- And O-Glycosylation Analysis Of Etanercept Using Liquid Chromatography And Quadrupole Time-Of-Flight Mass Spectrometry Equipped With Electron-Transfer Dissociation Functionality". Analytical Chemistry 86.1 (2014): 576-584. DOI: 10.1021/ac2007784. Bones, J et al. "Identification Of N -Glycans Displaying Mannose-6-Phosphate And Their Site Of Attachment On Therapeutic Enzymes For Lysosomal Storage Disorder Treatment". Analytical Chemistry 83.13 (2011): 5344-5352. DOI: 10.1021/ac2007784. Bones, J et al. "2D-LC Analysis Of BRP 3 Erythropoietin N -Glycosylation Using Anion Exchange Fractionation And Hydrophilic Interaction UPLC Reveals Long Poly- N -Acetyl Lactosamine Extensions". Analytical Chemistry 83.11 (2011): 4154-4162. DOI: 10.1021/ac200406z. Shahrokh, Zahra et al. "Erythropoietin Produced In A Human Cell Line (Dynepo) Has Significant Differences In Glycosylation Compared With Erythropoietins Produced In CHO Cell Lines". Mol. Pharmaceutics 8.1 (2011): 286-296. DOI: 10.1021/mp100353a. 11
“NIBRT Contract Research has enabled Levicept to access a wide range of analytical services delivered through the provision of top specification analytical instrumentation. In addition to providing protein and glycan analytical services their ability to develop new methods to meet our specific needs enables us to quickly resolve our analytical queries in timely and cost effective manner. Working with NIBRT Contract Research has enable us to gain a greater understanding of our clinical candidate”. Dr. Simon Westbrook, CEO Levicept.
Client Testimonials
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New in 2016
o Epitope mapping o Protein-drug binding o Protein-protein interactions o Aggregation o Effects of mutation on protein
conformation and dynamics o Localization of conformational
changes as a result of formulations and stability testing
Hydrogen Deuterium Exchange Mass Spectrometry
O-Glycan occupancy site studies
o Structural elucidation to identify O-Glycan core type o Targeted ETD-MS to identify occupancy site.
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Bioanalytical Training
Award winning hands-on practical training in state-of-the-art facilities with leading instructors.
Choose from publically available courses or contact us to discuss a course customised to your
particular requirements.
Public courses available include: Glycan Analysis, Introduction to Bioanalytics, Advanced
Bioanlaytics.
New for 2016: Small to Large Molecule Analytical Converter Course.
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New Training for 2016
Small to Large Molecule Analytical Converter Course
This course is designed for those currently working in small molecule active pharmaceutical ingredient quality or technical roles who wish to learn more about high end analytical methods for mAb characterisation or those who are interested in entering the mAb analysis arena but who require an in depth immersion in the technology required. The training will address several current challenges to complete mAb characterisation and will include an introduction to best practices and hands on exercises using state-of-the-art bioanalytical instrumentation located in the NIBRT facility. Key Topics Covered: A recap of small molecule API QC testing to identify transferrable and non-transferrable skills for therapeutic protein characterisation. An overview of industrial bioprocessing, protein structure, protein chemistry and post translational modifications to facilitate an
understanding of the complexity of large molecule QC. An introduction into bioanalytical methods for protein purity and quantitative assessment including SDS-PAGE, ELISA and protein
estimation assays. Strategies for complete mAb characterisation based on intact protein analysis using chromatography, electrophoresis, mass
spectrometry and other orthogonal techniques. Strategies for complete mAb characterisation based on peptide centred methods for stability and post translational modification
identification. An introduction to glycosylation and the quantitative structural characterisation of N-glycans.
Date: July 5-6 2016
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Further Information
Please contact:
Dr. Brian Morrissey, NIBRT Contract Research Manager
Email: [email protected]
Phone: + 353 1 215 8178
www.nibrt.ie/contractresearch
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