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

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

Source: A Practical Guide to Biopharmaceutical Manufacturing, © Informa UK Ltd, December 2006

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”

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.

• 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

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

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.

14

Experimental

15

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.

16

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).

17

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.

19

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

0

5

10

15

20

25

Rs= 2.4

4.6 x 250 mm, 5 µm on an Agilent 1200 (quaternary pump)PN 959990-902

1 2 3 4 567

89

12

10 11 13 14

15 1617 19

1820

21 22 23

min2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

Rs= 2.6

4.6 x 100 mm, 1.8 µm on an Agilent 1200SLPN 959964-902

12 3 4

67

8

9 1210 11 1314

15

16 19

20

2122 23

min2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

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

mAU

0

2

4

6

8

10

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

2

4

6

8

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

1

2

3

4

5

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

0

10

20

30

40

50

60

70

80

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

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

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

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

Peptide Mapping by CE/MS

min5 10 15 20 25 30

mAU

0

20

40

60

80

100

120

1 mg/ml tryptic digest of human haemoglobin

UV detection at 195nm

min10 20 30 40 50 60

0

2500

5000

7500

10000

12500

15000

17500

20000

22500

m/z500 550

0

1000

3000

5000

541

.3 5

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

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

Page 30

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

LC/MS application: Biomarker ID

LC/MS application: Nucleotides by LC/MS

Stratagene Quantitative PCR

Group/Presentation TitleAgilent Restricted

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


Summary: Agilent tools and technologies


• 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.


Agilent Biopharma Portfolio Overview and Application Examples · Agilent Biopharma Portfolio...

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