Agilent Biopharma Portfolio Overview and Application Examples · Agilent Biopharma Portfolio...
Transcript of Agilent Biopharma Portfolio Overview and Application Examples · Agilent Biopharma Portfolio...
Agilent BiopharmaPortfolio Overview
andApplication Examples
Dr. Michael KraftPharma Industry Marketing Manager
Waldbronn, Germany
Agenda
Short Introduction that we have the same understanding:
• Biopharma industry: Key Market trends
• The biopharma production process
• Legal requirements
• Analytical procedures to ensure product quality
Agilent in Biopharma: The tools and technologies (one page summary)
• 1290 Infinity LC – Peptide maps and AAA
• 7100 CE in Biopharma
• 2100 Bioanalyzer
• LC/MS – BioConfirm SW
• Stratagene Quantitative PCR
• Informatics solutions with ECM, ELN and GeneSpring
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Biopharma: Key Market Changes
•The pharma industry is moving from NCE to NBE
•There are no blockbuster NCE on the horizon to close the revenue gap for small molecules, which go out of patent
•About 2,500 biotech drugs are in the discovery phase, 900 in preclinical trials and over 1,600 in clinical trials.
•Anti-Cancer drugs: more than 1500.
•Especially in Asia: bio-similars and bio generics
Overview: The biopharma scale-up process
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Source: A Practical Guide to Biopharmaceutical Manufacturing, © Informa UK Ltd, December 2006
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Industry Overview and Opportunities
ManufacturingPurificationDevelopment StabilityScale-up
Amino Acid Analysis
Amino acid sequence • Composition • Terminal AA sequence
Physicochemical Characterization
Isoform pattern • Peptide map • Sulfhydryl group(s), S-S bridges • Carbohydrate structure
Product related impurity determination
Truncated forms • Aggregates • Other modifications (deamidated, isomerized, mismatched S-S linked, oxidized or altered conjugated forms)
Fermentation process related impurity determination
Cell substrate-derived • Cell culture-derived • Downstream-derived
Fermentation
Activeingredient
Overview: What are the legal requirementsInternational Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH) quality guidelines (Quality of Biotechnology Products Q5A-D and especially Specifications : Test Procedures and Acceptance Criteria for Biotechnological/Biological Products Q6B)
www.ich.org/cache/compo/363-272-1.html
Eudralex: The Rules Governing Medicinal Products in the European Union http://ec.europa.eu/enterprise/pharmaceuticals/eudralex), especially the following:
- Volume 3, Medicinal Products for Human Use: Guidelines
- Volume 4, Good Manufacturing Practice, Medicines for Human and Veterinary Use (with special emphasis on Annex 1, Manufacture of Sterile Medicinal Products, and Annex 2, Manufacture of Biological Medicinal Products for Human Use)
- Part II, Basic Requirements for Active Substances Used as Starting Materials (with particular emphasis on Chapter 18, specific guidance for APIs manufactured by fermentation or cell culture)
US FDA Code of Federal Regulations, Title 21, Parts 210,211 and especially 600 (www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=600&showFR=1)
EU and national Pharmacopoeias for specific products and general analytical testing requirements
“Well-Characterized Biotechnology-derived Products”
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Analysis of complex glycoproteinsCombo of multiple analytical methods
HPLCElectrophoresisMass SpectrometryLectin BindingPotency
Mass spectrometry
HPLC, CE, Glycan mapping
Isolation of individual glycans
Whole Protein
HPLC, GC
Glycan pools
De-N/O-glycosylated protein
Monosaccharides
Enzymatic or chemical cleavage
Structural charaterization:
MS, Enzymatic, NMR
Enzymatic or chemical cleavage
HPLC, CE
Mass Spectrometry
Glycopeptides
HPLC
Peptide mapping
Electrophoresis, CE
Source – EGA presentation at WHO, Geneva in April 2007
Example: Biopharma application spaces for Mab´s
Research and Development
• Detailed protein Characterization• High performance• Link to Protein ID/ Proteomic procedures• Minor Component interest• Stress Analysis• Modification Analysis
QA/QC
• Routine Protein Characterization• Quick• Cost conscious• Ease of use• Compliance issues, SOPs• Reproducibility• Regulations
Bioanalyzer
CE
1290
Agilent in biopharma: The tools and technologies you need to bring your molecule to market.
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• Liquid chromatography (reversed phase, ion exchange and size exclusion) incl. Evaporative light scattering• Capillary electrophoresis• Ion analysis (CE and HPLC)• Isoelectric focusing• Micro fluidics: 2100 Bioanalyzer• Gas chromatography • GC/MS• LC/MS• qPCR• Informatics solutions with ECM, ELN and GeneSpring
Application example: Tryptic digest analysis
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Tryptic digest analysisusing theAgilent 1290 Infinity LC System
Products: 1290 infinity LC System
Industry: Life science, biopharma
Author:Gerd Vanhoenacker, Frank David,and Pat SandraResearch Institute for ChromatographyKennedypark 26B-8500 Kortrijk, BelgiumKoen SandraMetablysKennedypark 26B-8500 Kortrijk, BelgiumBernd Glatz and Edgar NaegeleAgilent Technologies R&D andMarketing GmbH and Co. KGHewlett-Packard-Str. 876337 Waldbronn, Germany
Application note publication number: 5990-4031ENPublication date May 1, 2009
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Peptide mapping: why
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Peptide mapping, a widely used tool for identifying proteins and determining protein modification, is routinely performed with reversed phase liquid chromatography (RP-HPLC).
•Protein identification•Purity checking of synthetic peptides•Re-analysis of peptide fractions collected by HPLC prior to sequencing
Sample complexity is enormous with typically hundreds of species encountered in biopharmaceutical preparations and many thousands of peptides in proteomics samples. Evidently, the chromatographer is confronted with an enormous separation challenge.
Abstract
This Application Note demonstrates:
• The applicability of the Agilent 1290 Infinity LC System to resolve peptide mixtures of higher complexity.
• A bovine serum albumin (BSA) tryptic digest was separated on a 250 mm ×2.1 mm id × 1.7 μm dp RP-LC column using different gradient slopes and flow rates.
• The maximum pressure applied was 900 bar. Peak capacities from 188 to 851 within total analysis times of 8 and 260 min, respectively, were obtained.
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Comparison
In this application note:
•The resolving power of ultra-high pressure LC (UHPLC) using the 1290 Infinity LC system is demonstrated.
•BSA tryptic digest was separated on a 250 mm × 2.1 mm × 1.7 μm dp column.
•Peak capacity and peak capacity productivity, two powerful metrics used to evaluate the separation, were determined at different gradient slopes and flow rates.
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Experimental
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Tryptic digestion of BSA was carried out in an ammonium bicarbonate buffer at pH 8. Trypsin enzyme/substrate ratio of 1/50 and the mixture was incubated overnight at 37 °C. Another BSA sample (called BSA RA) was reduced and alkylated prior to digestion. A peptide standard mixture, used to aid in the calculation of the peak capacity, was dissolved in mobile phase A and contained bradykinin 1–5 (5 nmol/mL), angiotensin II (3 nmol/mL), neurotensin (2 nmol/mL), ACTH clip [18-39] (2.5 nmol/mL), and bovine insulin chain B (12.5 nmol/mL).
High speed analysis of the peptide standard mixture (upper trace), BSA digest (middle trace) and BSA RA digest (lower trace).Flow rate: 0.4 mL/min, gradient: 0–50% B in 6.25 min.
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A peak capacity of approximately 190 was generated with this short gradient time. This corresponded to a peak capacity production rate of over 30 peaks/min.
Analyses of the BSA digest with different gradients.Flow rate: 0.4 mL/min, gradient: 0-50%B in 6.25 min (8%/min), in 12.5 min (4%/min), in 25 min (2%/min), and in 50 min (1%/min).
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The peak capacity tripled from 188 to 567 when the gradient time was increased from 6.25 min (8% B/min) to 50 min (1% B/min), respectively. If only the elution window of the BSA digest is taken into account for the 50 min gradient, the peak capacity is 375 in 39 min.
Peak capacity and peak capacity production rate in function of gradient time.
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It can be deduced that the best compromise between peak capacity and analysis time is obtained with a gradient time of 100 to 150 min.
Conclusion
This work demonstrates the versatility of Agilent 1290 Infinity LC system for separating peptide mixtures of high complexity.
Protein digests were analyzed on a 250 mm long column packed with 1.7-μm particles and operated at a pressure up to 900 bar.
Depending on the need, high productivity (peak capacity of 188 in less than 10 min) or high resolution (peak capacity exceeding800 in 3h) can be obtained.
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Improved Amino Acid Analysis using Eclipse Plus C18
New method uses Eclipse Plus C18 instead of Eclipse AAA columns
Benefits of this protocol over previous iterations include:
• Better retention of the first two eluting amino acids, aspartic and glutamic acid.
• Higher resolution of several closely eluting amino acid pairs, depending on the column configuration used.
• Several configurations including three particle sizes and several column lengths and diameters, allowing the analyst to customize the separation to his specifications and constraints (e.g. available Agilent HPLC model, desired throughput, desired resolution, mobile phase consumption).
• Of course, the previous protocols’ benefits are still retained especially the automated online OPA/FMOC derivatization.
High Resolution Amino Acid Analyses can be achieved with ZORBAX Eclipse Plus C18 in different column dimensions and different LCs
min5 10 15 20 25 30
mAU
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4.6 x 250 mm, 5 µm on an Agilent 1200 (quaternary pump)PN 959990-902
1 2 3 4 567
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min2 4 6 8 10 12 14
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4.6 x 100 mm, 1.8 µm on an Agilent 1200SLPN 959964-902
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min2.5 5 7.5 10 12.5 15 17.5 20
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402.1 x 150 mm, 3.5 µm on an Agilent 1200 (binary pump)PN 959763-902 Rs= 2.0
1. Aspartic acid2. Glutamic acid3. Asparagine4. Serine5. Glutamine6. Histidine7. Glycine8. Threonine9. Arginine10. Alanine11. Tyrosine12. Cystine13. Valine14. Methionine15. Norvaline16. Tryptophan17. Phenylalanine18. Isoleucine19. Leucine20. Lysine21. Hydroxyproline22. Sarcosine23. Proline
Month ##, 200X
Longevity of ZORBAX Eclipse Plus C18, 2.1 x 150mm, 3.5µm
Run #10, Pmax = 186 bar
Run #300, Pmax = 190 bar
Run #500 Pmax = 190 bar
min2.5 5 7.5 10 12.5 15 17.5 20 22.5
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min2.5 5 7.5 10 12.5 15 17.5 20 22.5
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min2.5 5 7.5 10 12.5 15 17.5 20 22.5
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Rs= 2.22
Rs= 1.80, all other pairs >2.0
Rs= 1.8610 days (24h)
nonstop operation
Method Reproducibility, Overlay of back-to-back injections, ZORBAX Eclipse Plus C18, 2.1 x 150, 3.5µm, 1 nmol/ul sample
Month ##, 200X
min2 4 6 8 10 12 14 16 18
mAU
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DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0010.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0009.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0008.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0007.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0006.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0005.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0004.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0003.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0002.D)
Peak Areaamino acid Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Injection 6 Injection 7 Injection 8 Injection 9 StDEV Mean %RSD
glu 90.7 93.7 92.4 93.1 93.8 94.7 92.5 95.7 93.2 1.43 93.3 1.5ala 109.9 113.2 111.8 112.1 113.3 114 111.6 116 113.3 1.72 112.8 1.5cy2 153.2 157 155 157 157.8 159 154.4 162.6 158 2.79 157.1 1.8lys 142.3 142.5 137.1 144.4 141.9 143.5 137.9 140.7 138.9 2.55 141.0 1.8pro 60.8 62.9 61.8 65.8 60.9 63.2 61.2 70.2 61 3.11 63.1 4.9
Five Point Calibrations of Early, Middle and Late Eluting Amino Acids on the 2.1x150 mm, 3.5 µm Eclipse Plus C18 Column
Month ##, 200X
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Capillary Electrophoresis Applications in biopharma
CE, CEC & CE-MS capabilities with a single instrument offers a widerange of applications as well as an orthogonal technique to LC or LC-MS
Antibody purity
Oligonucleotides
Protein separations
Peptidemapping
Bio - Molecules
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Buffer: 20mM borate pH 9.3Capillary: 64cm (56cm eff)Detection: HSC, 225nm / 20nmInjection: 200mbar*sRun: 20°C, 30kV
impurities reported as % area/area of main peak
min0 1 2 3 4 5 6 7 8 9
mAU
-50510152025303540
0.036 %
0.09 %
Use of Agilent High sensitivity Cellprovides accurate main component and low level impurity determination in a Regulated Environment.
Impurities Determination by CE
Rapid high resolution separation with a different selectivity than LC methods.
Data taken from Application Note 5965-9034E
Biopharma purity check: Heparin
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High speed – high resolution impurity check for pharmaceuticals
Data taken from Application Note 5990-3517E
CE is the ideal tool for separation of biomolecules (proteins, peptides, nucleic acids and carbohydrates) and encouraged by regulatory authorities as an accepted orthogonal technique
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Peptide Mapping by CE/MS
min5 10 15 20 25 30
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1 mg/ml tryptic digest of human haemoglobin
UV detection at 195nm
min10 20 30 40 50 60
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m/z500 550
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52.3
Abundance
MS detection
Agilent CE/MS solution is fully integrated at software and hardware level.The most robust Electrospray-MS solution on the market
CE-MS analysis + DAD data providing maximum information per run
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Agilent 2100 BioanalyzerInstrumentsG2939A for Electrophoresis applicationsG2938C for Electrophoresis and Cell application
PC and SoftwareSoftware only, no PC included Desktop PC and SoftwareDesktop PC and SoftwareLaptop PC and SoftwareSecurity Pack for 21 CFR part 11
Accessories (icluded)Chip priming stationChip vortex instrument
Assay + KitsMethods in Software includedKits includeChips all Reagents
3 different RNA Kits3 different DNA Kits3 different Protein Kits2 Cell Kits
ServicesWarranty extensionsPreventive MaintenanceIQ / OQ ServicesOperational Services
2009
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High Sensitivity Protein 250 Kit (HSP-250)
Highest sensitivity:
Labeled proteins can be measured down to pg/µL concentrations loaded on Chip
Direct comparison of samples run on SDS-PAGE with Silver staining and on 2100 Bioanalyzer.
Extra wide linear dynamic range:
4 orders of magnitude linear dynamic range assuring excellent determination of impurities
BioConfirm Software: Mapping Peptide Peaks From MS Analysis to Theoretical Protein Sequence
Coverage:95% of heavy chain85% of light chain
LC/MS applications: Characterization of MAB’s
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LC/MS application: Biomarker ID
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LC/MS application: Nucleotides by LC/MS
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Stratagene Quantitative PCR
Group/Presentation TitleAgilent Restricted
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With the addition of Stratagene real-time PCR ,Agilent is able to offer complete workflow solutions to address contamination of host cell DNA and viruses.
Industry Leading Informatics Portfolio & ProductsCustomer Centric Value Proposition
• Installation & Training• IQ/OQ• Consulting• Maintenance & Asset Protection
Professional Services
• ChemStation• EZChrom Elite• OpenLAB ICM• MassHunter• Feature Extraction
Chromatography / Instrumentation /
Spectrometry
• OpenLAB ECM
Enterprise Content Management
Bioinformatics
• GeneSpring• DNA Analytics• eArray* Partnering with Strand
LIMS
Partnering withLIMS vendors
• ECM API / SDK* Partnering with PSI
Electronic Lab Notebook
• Kalabie ELN
Reduce the time, effort & cost to go from raw data to final insight
Agilent Software and Informatics
Specific Informatics offering for BiopharmaKalabie : Electronic Lab Notebook (ELN) for paperless documentation of BioPharma
Research Experiments and Results. Client/Server application based on Oracle DB for multiple users, ideally across sites. Demo video: http://www.chem.agilent.com/en-US/products/software/kalabieeln/pages/gp66976.aspx
OpenLAB ECM: Enterprise Content Management System managing any file in any format secure, centrally, and compliant. Can manage any BioPharma record including images, spreadsheets and Word documents and make them searchable. More info: http://www.chem.agilent.com/en-us/products/software/datasystems/openlab/ecm/pages/default.aspx
GeneSpring: Data mining application for statistical evaluation from large volume gene expression, protein and MS/MS analysis. Data are generated from Agilent Microarrays or Agilent high-end LC-MS instruments (TOF and Q-TOF).Application Example: Identify significant differences between diseased vs normal tissue in a gene expression experiment. http://www.chem.agilent.com/en-us/products/software/lifesciencesinformatics/genespringms/pages/default.aspx
Group/Presentation TitleAgilent Restricted
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Summary: Agilent tools and technologies
Group/Presentation TitleAgilent Restricted
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• Liquid chromatography (reversed phase, ion exchange and size exclusion) incl. Evaporative light scattering• Capillary electrophoresis• Ion analysis (CE and HPLC)• Isoelectric focusing• Micro fluidics: 2100 Bioanalyzer• Gas chromatography • GC/MS• LC/MS• qPCR• Informatics solutions with ECM, ELN and GeneSpring
Thanks for your attention.
Group/Presentation TitleAgilent Restricted
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