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

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Transcript of Agilent Biopharma Portfolio Overview and Application Examples .Agilent Biopharma Portfolio Overview

  • 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

    Page 2

  • Page 3

    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

    Page 4

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

  • Page 5

    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-derivedFermentation

    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

    Page 6

  • Page 7

    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 Mabs

    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.

    Page 9

    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

    Page 10

  • 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

    11

  • Peptide mapping: why

    Page 12

    Peptide mapping, a widely used tool for identifying proteins and determining protein modification, is routinely performed with reversed phase liquid chromatography (RP-HPLC).

    Protein identificationPurity checking of synthetic peptidesRe-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 m 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.

    13

  • 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 m 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 15 (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: 050% 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.

    18

    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-m 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 c