Overview of Waters Solutions for Biopharmaceutical Analysis and Characterization

Transcription

1 Overview of Waters Solutions for Biopharmaceutical Analysis and Characterization Denis Calnan Biopharma Business Development Manager (Northern Europe) 2011 Waters Corporation 1

2 About Waters Facts and Figures Est s Direct sales/service in 97 offices 54 countries worldwide 5000 employees Local Expertise: European MS Headquarters in Manchester Research & Development Manufacturing: Manchester, UK. Wexford, Ireland Milford, Massachusetts Corporate Headquarters Manchester, England MS Technologies Centre 2011 Waters Corporation 2

3 Our Product Portfolio 2011 Waters Corporation 3

4 Biotherapeutics have varying size and complexity Increasing Molecular Weight, Complexity and Heterogeneity Insulin 6kDa GCSF 19kDa PEG-Interferon 20kDa EPO 34kDa Complex Glycosylation mab 150kDa Fusion Proteins Conjugated mab 150kDa+ Blood Factors 250kDa 2011 Waters Corporation 4

5 Biopharmaceutical Analysis Involves Many Application Areas Common biopharma applications: HIGHER ORDER STRUCTURE (IMS, HDX) ANTIBODY CHARACTERISATION INTACT PROTEIN MASS PEPTIDE MAPPING GLYCAN ANALYSIS GLYCOPROTEIN ANALYSIS VACCINE ANALYSIS PROTEIN VARIANT ANALYSIS (IEX, SEC) OLIGONUCLEOTIDE ANALYSIS SYNTHETIC PEPTIDE ANALYSIS HOST CELL PROTEIN ANALYSIS LEACHABLES AND EXTRACTABLES AMINO ACID ANALYSIS 2011 Waters Corporation 5

6 Instrument and Solutions Portfolio Acquity UPLC ACQUITY Xevo TQD and Xevo TQ-S High Sensitivity Analysis Exact mass [UPLC-TOF] 2011 Waters Corporation 6

7 2011 Waters Corporation 7

8 PATROL UPLC 2011 Waters Corporation 8

9 PATROL UPLC 2011 Waters Corporation 9

10 UPLC Introduction: High Productivity % UPLC/MS HPLC/MS Time Waters Corporation 10

11 ACQUITY UPLC H-Class Bio System 2011 Waters Corporation 11

12 ACQUITY UPLC H-Class Bio A UPLC system for biomolecules Ultimate chromatographic performance for biomolecules Maximum flexibility, robustness & ease of use Bio-compatible flow path Suitable for RP, IEX, SEC, HILIC, HIC Auto-Blend Plus for dial-up ph buffer formation Needle in flow path design Total volume sample injection Low carryover performance 2011 Waters Corporation 12

13 Why UPLC for LC/MS? HPLC 60 min gradient 2.1x150 mm 3.5 µm C18 ultra-pure silica-based HPLC column (0-50% acetonitrile, 0.1% formic acid) 37 peptides identified with >=8 b/y ions 52 peptides identified with >=5 b/y ions 2011 Waters Corporation 13

14 Why UPLC for LC/MS? UPLC 20 min gradient 2.1x50 mm 1.7 µm C18 ultra-pure silica-based HPLC column (0-50% acetonitrile, 0.1% formic acid) 74 peptides identified with >=8 b/y ions 111 peptides identified with >=5 b/y ions 2011 Waters Corporation 14

15 Charged Surface Hybrid (CSH) Technology patent pending Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography. P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper EN 2011 Expands upon the robust BEH particle technology CSH130 C18 = BEH130 base particle + low level of basic moieties + trifunctional C18/end cap Acidic ph Positive Surface Charge 2011 Waters Corporation 15

16 A New Column Chemistry CSH130 C18 Competitor s Industry Standard C18 Porous (300Å) 5 µm 2.1 x 250 mm Competitor s Superficially Porous Peptide C18 SPP (100Å) 1.7 µm 2.1 x 150 mm BEH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm CSH130 C18 Porous (130Å) 1.7 µm 2.1 x 150 mm TFA UV absorbance (214 nm) Formic Acid Time (min) Time (min) Time (min) Time (min) 2011 Waters Corporation 16

17 Mass Spectrometer Positioning Xevo: High Performance for Routine Biotherapeutic Characterization Synapt: Flexibility for Advanced Biotherapeutic Characterization 2011 Waters Corporation 17

18 Intact Protein Analysis Workflow Sample Preparation + + UPLC Xevo G2 Tof MS Bioinformatics 2011 Waters Corporation 18

19 MaxEnt1 Deconvoluted Spectrum (Intact IgG) + 51 Charge state G0F/G1F G1F/G1F G0F/G2F GOF/G0F G1F/G2F G2F/G2F G0/GOF 2011 Waters Corporation 19

20 Why Intact Mass? Has the molecule been expressed correctly by the cell? What does this tell me? BiopharmaLynx did most of the work Are the expected glycoforms present? BiopharmaLynx automatically processed the data Are all the batches similar? BiopharmaLynx automatically compared samples Do these batches satisfy the regulator that I made them correctly? 2011 Waters Corporation 20

21 BiopharmaLynx Difference plot for Referecne vs. Unknown Batch Intact protein glycosylation profile of a monoclonal Antibody in difference mode Quantitative measure of glycoforms 2011 Waters Corporation 21

22 Injections of Two Less Similar Antibody Batches Appearance of new glycoforms easily seen in BiopharmaLynx 2011 Waters Corporation 22

23 Total Ion Chromatograms of Reduced mabs LC HC Innovator mab LC Biosimilar mab HC Biosimilar HC shows two peaks 2011 Waters Corporation 23

24 Deconvoluted Mass Spectra of the Heavy Chain G0F G1F Innovator mab G0 G2F Biosimilar mab (Seq Variant) 2 Oxidations (+32 Da) 1 Oxidation (+16 Da) 2011 Waters Corporation 24

25 HPLC to UPLC SEC Comparison ACQUITY UPLC BEH200 SEC,1.7 µm 4.6 x 300mm HPLC 100% Silica-Diol SEC 250Å 5µm 7.8 x 300 mm AU AU % Aggregate % Aggregate Minutes Minutes Murine monoclonal antibody - Scaled load Conditions: 0.4 ml/min; 25mM Sodium Phosphate, ph 6.8, 0.15 M NaCl 2011 Waters Corporation 25

26 System Solutions for Peptide Mapping 2011 Waters Corporation 26

27 Confirming primary structure data acquisition Digested sample Generic UPLC gradient Generic MS E method Comprehensive unbiased MS/MS acquisition of exact masses of all precursor ions and fragment ions from a single injection 2011 Waters Corporation 27

28 Equivalent protein coverage was obtained for innovator and biosimilar Innovator N-terminal Peptide Innovator HC Biosimilar N-terminal Peptide Biosimilar HC N-terminal Peptide Innovator LC N-terminal Peptide Biosimilar LC 2011 Waters Corporation 28

29 Peptide Mapping: Zeroing in on Biosimilar Protein Differences INNOVATOR (TIC) BIOSIMILAR (TIC) Peptide T34-35 (missed cleavage) in INNOVATOR not in BIOSIMILAR INNOVATOR (spectrum) BIOSIMILAR (spectrum) New Peak in BIOSIMILAR sample at m/z Waters Corporation 29

30 Built on decades of analytical data and informatics experience Waters Corporation 30

31 Unifi 5 Key Messages 1. Focus on Routine Characterization Efficiencies of automation and workflow 2. Workgroup Benefits Data/Instrument Access and Management 3. High resolution analytics across an organization NonCompliant, GxP, Compliant-Ready, Qualification, QC? 4. Reporting, Reporting, Reporting Goal is to communicate results, not collect/process data 5. Universal Interface for Chromatography and MS Reduce Training Costs and Increase Accessibility 21CFR Waters Corporation 31

32 Simple Intact Antibody LCMS Analysis Report 2011 Waters Corporation 32

33 What is NEW in UNIFI 1.7? Application Solution for released glycan analysis Intact Protein Glycan Application Solution Biopharmaceutical Platform Solution Peptide Mapping Bioseparations Size Exclusion (UV) Ion Exchange (UV) DDA (Peptide & Glycan) Core Hardware: Xevo G2-S QTof ACQUITY UPLC H-Class and H- Class BIO TUV FLR 2011 Waters Corporation 33

34 UNIFI Biopharm v1.7 Integrating NIBRT Glycobase Benefits: Integration/Speed Regulatory Compliance Reporting 2011 Waters Corporation 34

35 Glycoprotein and Glycan Analytical Tools Separations Chemistries Informatics UPLC and HPLC Sample Preparation Mass Spectrometry Glycan Standards 2011 Waters Corporation 35

36 Higher Order Structural Analysis Tools 2011 Waters Corporation 36

37 SYNAPT G2: Flexibility for Advanced Analytics /(S) Routine Biotherapeutic Analysis + Advanced Analytical Applications: MALDI MS/MS (Peptides, Glycans) ETD (Proteins + Peptides) Ion Mobility (Protein Conformation) Ion Mobility enhanced Proteomics/HCP analysis 2011 Waters Corporation 37

38 Triwave Single ion m/z m/z Drift time Drift time Precursor ion fragmented Product ions separated by IMS Precursor and products share same drift time 2011 Waters Corporation 38

39 MS signal of Human Insulin monomer obscures the dimer NO MS Separation of monomeric insulin from dimeric insulin Monomer 3+/ Dimer Waters Corporation 39

40 IMS separation of monomeric insulin from dimeric insulin in minutes 6+ Intensity Å Å 2 Difference NOT shown in this dimension 3+ Drift Time (ms) Difference IS shown in this dimension 2011 Waters Corporation 40

41 Analysis of human insulin analogs by Synapt G2 HDMS Humulin Humalog Apidra 1MS O 1LP H No X-Ray data! IMS DOES provide data Multiple different forms can be compared directly thanks to comparable mobility conditions including relative proportions Lantus Novolog Levemir 2VJ Z 1ZE G 1XD A 2011 Waters Corporation 41

42 Ion Mobility: an additional dimension of separation 2011 Waters Corporation 42

43 IMS separates co-eluting peptides: example from HDX experiment No IMS separation Ion Mobility Enabled ASIQKFGERALKA with IMS-enabled separation AVEGPKL Waters Corporation 43

44 Deuterium Exchange HDX at Amide Hydrogen Exchange slowly Exchange fast D 2 O Solution added H s H s & D s 1 Da 2 Da at backbone amide positions Engen, J. R. (2009) Anal. Chem. 81(19), 7870 Exchange rate is determined using the solvent accessibility and # of hydrogen bonds 2011 Waters Corporation 44

45 Waters System Solution for HDX 2.0 nanoacquity UPLC with HDX technology Synapt G2-S HDMS E or Xevo G2-S QTOF ProteinLynx Global Server (PLGS) DynamX TM 2.0 Fast chromatography at 0 C Accurate mass measurement Reliable peptic peptide ID by MS E Automated HDX data processing for deuteration determination nanoacquity UPLC with auto-sampler for high throughput analyses 2011 Waters Corporation 45

46 Conclusion UPLC and Mass Spectrometry are powerful techniques provide: specificity, sensitivity, quantitation, flexibility and future proofing Waters Instruments and methods available for Biopharma applications Extensive applications support and dedicated service organisation Chemistry Consumables ISO, CE manufacturing, Single vendor complete solution Waters Corporation 46

47 2011 Waters Corporation 47