Improving characterization of monoclonal antibodies

Transcription

1 Webinar Series Improving characterization of monoclonal antibodies Making a better biotherapeutic November 19, 2014 Instructions for Viewers To share webinar via social media: To share webinar via e mail: To see speaker biographies, click: View Bio under speaker name Sponsored by: To ask a question, click the Ask A Question button under the slide window

2 Improving characterization of monoclonal antibodies Making a better biotherapeutic November 19, 2014 Webinar Series Brought to you by the Science/AAAS Custom Publishing Office Participating Experts Niomi Peckham, M.Sc. Alexion Pharmaceuticals Cheshire, Connecticut I-Jane Chen, Ph.D. PerkinElmer Hopkinton, MA Sponsored by:

3 N Linked Oligosaccharide Profiling and Exo glycosidase Sequencing Using a Labon a Chip Format Niomi Peckham, Scientist, Analytical Sciences Alexion Pharmaceuticals, Cheshire CT

4 Glycosylation of Antibodies N-Linked Oligosaccharide linked to asparagine at Asn-X-Ser, Asn- X-Thr, or Asn-X-Cys O-Linked Oligosaccharide linked to serine or threonine (a) Introduction to Protein Structure: Second Edition, Branden & Tooze 4

5 N Linked Oligosaccharides Sialic Acid Asn Sources of heterogeneity Bisecting GlcNAc Galactosylation Sialylation/Fucosylation High Mannose Galactose N-acetylglucosamine Mannose Fucose Commercial importance Bioactivity Pharmacokinetics Immunogenicity 5

6 N Linked Oligosaccharide Structures Asn G2F Asn MAN 5 Asn G1F Galactose Asn G0F GN (Biantennary Hybrid) Asn G0F N-acetylglucosamine Mannose Fucose 6

7 Caliper (PerkinElmer) LabChip GXII System Components LabChip GXII Instrument LabChip GXII Software LabChip Reagent/Chips: DNA (1K, 5K, and 12K) RNA ( nucleotides) HT Protein (14 to 350+ kda) HT Pico Protein (10 pg/μl 100 ng/μl) N Linked Glycan Profiling Charge Variant (New! pi 7.0 to 9.5) 7

8 Caliper ProfilerPro Glycan Profiling Kit Analogous to HPLC or CE Glycan Profiling Complement to MALDI ToF MS for mass profiling Developed for neutral N linked glycans (e.g. Man5, G0F, G1F, G2F) Separation by molecular weight High Throughput 96 well plate format, reagents pre aliquoted Measures relative amount of N Linked neutral glycans estimated LoQ of 1% Sample Requirements Minimum one step purified (e.g. Protein A), ph ~7.0 Normalized to 5 mg/ml, as low as 2 mg/ml 8

9 N Linked Oligosaccharide Profiling: Method Denaturation of protein with SDS βme solution at 70 C PNGase digestion removes N Linked glycans specifically cleaves the N acetyl glucosamine asparagine linkage Asn PNGase Label glycans with fluorophore Needed to visualize glycans Reductive amination Dye Separate electrophoreticaly by size on chip Detect by laser induced fluorescence 9

10 Workflow of the Glycan Assay Sample Preparation < 4 hours Chip Preparation < 15 minutes Reconstitute ladder and marker Clean chip and add gel and marker Place chip, ladder, wash buffer and samples in GXII 96 wells separation in ~1.5 hour 45 samples (duplicates) in < 6 hours 10

11 Separation of Glycan Standards 11

12 mab #1 Profile 12

13 Profiles of Other mabs mab #2 mab #3 13

14 mab #1 Sample Data Average Man 5 *G0F GN *G0 G0 F *G'1 F *G1 F G2 f Reference Sample *Peaks have been tentatively assigned. 14

15 mab #1 Sample Data Process Change Relative Percent Man 5 G0F GN G0 G0 F Reference Sample (G1'F + G1F) G2 F mab #1 Reference Clone xyz, New supplement

16 Protein Assay; Monitoring Deglycosylation May be required in USP Chapter <212> Oligosaccharide Analysis Evaluated with Caliper Protein Assay May be performed during Labeling step (2 hours) 16

17 Peak Identification Limitation of Lab on a Chip; peak identification Caliper data on human IgG glycans, standards Experimental Approach Compare to standards (G0F, G1F, G2F, Man 5, 6, 7, 8, 9) Utilize MALDI ToF MS data (N Linked Oligosaccharide Mass Profiling) as a guide Exo glycosidase Sequencing as an orthogonal technique 17

18 N Linked Oligosaccharide Sequencing Traditionally done on single peak or excised band Exo glycosidase Sequencing PNGase Digest mab, ethanol precipitate Digest with Exoglycosidases (Prozyme) Label with ProfilerPro Analyze with GXII Enzyme R1 R2 R3 R4 R5 R6 Sialidase A - x x x x - β(1-4) Galactosidase - - x x x - β-n-acetylhexosaminidase x x - α-mannosidase (Jack Bean) x x α-mannosidase (X. manibotis) x x For molecules with few species present (mab) all species may be sequenced simultaneously Data interpretation can be challenging 18

19 Exo glycosidase Digestions Exo glycosidase Sequencing of an N Linked Biantennary Oligosaccharide Neuraminidase (Sialidase) α Mannosidase (α1 6) β Galactosidase α Mannosidase (α1 2,3,6) β N Acetylhexosaminadase 19

20 N Linked Oligosaccharide Sequencing; G2F Sequencing of G2F standard as a control Use enzyme specificity and digest mobility to generate linkage Galactose = 1 GU Mannose = 0.8 GU Fucose = 0.5 GU GlcNac = 1.1 GU α mannosidase digest of Man 5 as a control 20

21 N Linked Oligosaccharide Sequencing; Step 1 Digest Control No change in profile No significant sialyation of mab#1 Sialidase Digest 21

22 N Linked Oligosaccharide Sequencing; Step 2 G0F Sialidase Digest G1? G1'F + G1F G2F G1 may not resolve from G1F and G0F Man 7 elutes with G1F All forms with terminal Gal shift to G0F Confirms G2F, G1F G0F GN? G0? Sialidase + β Galactosidase Man 7? 22

23 N Linked Oligosaccharide Sequencing; Step 3 Sialidase + Β Galactosidase G0F G0? And G0F GN? shift with Hexosaminidase Preliminary assignment based on abundance and size G0F digested by Hexosaminidase only Confirms G0F G0F GN? G0? Sialidase + β Galactosidase + β N Acetylhexosaminidase 23

24 N Linked Oligosaccharide Sequencing; Step 4 Man 5 at 10.8 GU digested by Mannosidase only Sialidase + β Galactosidase + β N Acetylhexosaminidase Man 5 Sialidase + β Galactosidase + β N Acetylhexosaminidase + α Mannosidase All digested by mannosidase Some incomplete digests possible 24

25 Summary and Future Direction Method appears to be reproducible and offers a highthroughput option for process development samples Has the potential to be used for release and stability Many biopharmaceutical companies adopting USP Chapter <1084> Glycoprotein and Glycan Analysis Introduction and Choice of Analysis Methods; cites CE separations 25

26 Improving characterization of monoclonal antibodies Making a better biotherapeutic November 19, 2014 Webinar Series Brought to you by the Science/AAAS Custom Publishing Office Participating Experts Niomi Peckham, M.Sc. Alexion Pharmaceuticals Cheshire, Connecticut I-Jane Chen, Ph.D. PerkinElmer Hopkinton, MA Sponsored by:

27 LabChip Capillary Electrophoresis: The Next Generation of High Throughput Protein Characterization I-Jane Chen, Ph. D. Sr. R&D Engineer, Microfluidics November 19, PerkinElmer

28 Content What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance 28

29 29 What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance

30 Lab-on-a-Chip Technology - a.k.a. micro total analysis system mixing detection Sample separation chemical rxn/labeling sample loading heating Miniaturization Integration Automation Detection Analysis sample purification 30 For research use only. Not intended for diagnostic procedures.

31 Capillary electrophoresis in lab-on-a-chip Separation Channel Detection Window Automatic sampling from microplates On-chip non-covalent FL staining and destaining Sample loading Integrates the entire SDS- PAGE process onto a microfluidic chip Fast sample analysis - 40 seconds per sample 31 For research use only. Not intended for diagnostic procedures.

32 32 What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance

33 Photolithography to make glass chips 1. Expose Light Mask 5. Bond Glass or Quartz Well Plate Glass or Quartz 2. Develop Photo resist 6. Sipper Etched Channel Plate 3. Etch 4. Strip 33 For research use only. Not intended for diagnostic procedures.

34 Microchannels- after etching & bonding Grooves on glass substrate 34 Bonded Channels Substrate forms the ceiling 2 substrates totally fused Into one Substrate with channels etched in For research use only. Not intended for diagnostic procedures.

35 35 What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance

36 Surface property matters Siloxane Silanol At ph 7.5 Si Si O Hydrolysis Si Si OH OH OH Si Si O- O- O- Si O Heat Si O Si O Si O Si OH OH Si O- O- Si O Si OH OH Si O- O- pk a of silanol = For research use only. Not intended for diagnostic procedures.

37 Electrokinetic Fluid Actuation FLOW v electrokinetic = v electroosmotic + v electrophoretic 37 For research use only. Not intended for diagnostic procedures.

38 38 What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance

39 Sample loading using 6-way valves Position A: Sample loop is filled Position B: Contents of the loop are injected onto the column At least 25 nl sample needs to be loaded 39

40 Sample loading using cross junctions- Electrokinetic or hydrodynamic Gated Injection sample waste buffer column sample as small as 1 nl is loaded 40 For research use only. Not intended for diagnostic procedures.

41 Sample loading using cross junctions Pinched injection buffer sample waste column Plug size is determined by 1. Channel width 2. Currents applied For a 10 µm wide channel, the plug size is << than CE! 41 For research use only. Not intended for diagnostic procedures.

42 Electrophoretic separations E Separate molecules based on their mobility under applied electric field Mobility, µ, is velocity per unit applied electric field signal x 42 For research use only. Not intended for diagnostic procedures.

43 Electrophoretic separations E Separate molecules based on their mobility under applied electric field Mobility, µ, is velocity per unit applied electric field signal x 43 For research use only. Not intended for diagnostic procedures.

44 Electrophoretic separations E DL Separate molecules based on their mobility under applied electric field Mobility, µ, is velocity per unit applied electric field w 1 w 2 signal Ds x 44 For research use only. Not intended for diagnostic procedures.

45 Electrophoretic separations: performance Resolution: the ability to differentiate between multiple species often quoted as the smallest mobility (or size) difference that can be measured Sensitivity the ability to detect the presence of a species often described as the concentration or mass that yields a peak of some minimum signal-to-noise ratio signal E Ds x w 1 DL w 2 45 For research use only. Not intended for diagnostic procedures.

46 Resolution signal Dt w 1 w 2 R t 2 w 1 w 2 Gaussian distribution: w 4 t 1. 7FWHM t Time separation: t t 2 t 1 L v 2 L v 1 L E R t ( 4 t, 1 4 t, 2) / 2 46 For research use only. Not intended for diagnostic procedures.

47 Resolution: general expression Adding dispersion (band-broadening) contributions: Injector dispersion Detector dispersion 2 inj 2 det 2 w inj E w det E 2 12 Diffusion dispersion 2 2 diffusion DL E 3 tot 2 inj 2 det ector 2 diffusion 2 other 47 For research use only. Not intended for diagnostic procedures.

48 Sample plug size impact on resolution: R t 2 w 1 w 2 R L 1 1 E tot,1 tot,2 R 2 12 w 2 inj,1 w 2 det,1 L E 1 2 2D L inj, E1 12 E1 E1 12 E2 12 E2 E2 w injection plug size w 2 det,2 2D 2 L e.g. loading 2 nl sample into a 50 µm diameter capillary, the plug is 100 µm long! shrink, reduce required L (mm vs. cm) 48 For research use only. Not intended for diagnostic procedures.

49 Automated sample loading and separation 50

50 51 What is lab-on-a-chip Micro fabrication Surface properties and Electrokinetics Sample injection and resolution Automated system and high throughput performance

51 Work flow of labchip 52 For research use only. Not intended for diagnostic procedures.

52 Work flow of labchip 53 For research use only. Not intended for diagnostic procedures.

53 Work flow of labchip staining 54 For research use only. Not intended for diagnostic procedures.

54 Work flow of labchip 55 For research use only. Not intended for diagnostic procedures.

55 Work flow of labchip 56 For research use only. Not intended for diagnostic procedures.

56 Work flow of labchip 57 For research use only. Not intended for diagnostic procedures.

57 Work flow of labchip destaining 58 For research use only. Not intended for diagnostic procedures.

58 Work flow of labchip 59 For research use only. Not intended for diagnostic procedures.

59 Raw Profile of Reduced mab HC System Peaks (SDS) Lower Marker LC NGHC 60 For research use only. Not intended for diagnostic procedures.

60 Raw Profile of Non-Reduced mab Intact mab Fragments 61 For research use only. Not intended for diagnostic procedures.

61 Antibody Percent Glycosylation: LabChip vs. CE-SDS LabChip LabChip Produces Comparable Data Faster PA800 Microchip CE-SDS provides sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s vs 50 min per sample) than conventional CE separation Comparison of microchip and conventional CE-SDS. Antibody samples were denatured, reduced and analyzed with LabChip or ProteomeLab PA800. Analytical & Formulation, Thousand Oaks, Amgen Inc. CA, USA Chen, X. et al, Electrophoresis. 29, 2008, For research use only. Not intended for diagnostic procedures.

62 Low Level Impurity Detection Impurities of as low as 0.5% are quantified reproducibly Lysozyme was spiked into the sample at 1% of total protein and was readily identified, with a S:N of 9:1 In the Amgen publication in Electrophoresis the authors reported the LOD and LOQ of the method to be 1 and 3.3. mg/ml 63 For research use only. Not intended for diagnostic procedures.

63 Antibody Fragmentation: LabChip vs. CE-SDS CE-SDS LabChip Protein Fragmentation Analysis LabChip and conventional CE-SDS show comparable profiles LabChip profile was generated in 40 seconds vs. ~15 minutes by conventional CE-SDS Analytical & Formulation, Thousand Oaks, Amgen Inc. CA, USA Chen, X. et al, Electrophoresis. 29, 2008, For research use only. Not intended for diagnostic procedures.

64 How simple is it? Chip Prep Add dye to gel matrix, mix, pass through spin filter. Sample Prep Add DTT or IAM to sample buffer to create reducing or non-reducing buffers Load wells on chip with gel/dye, destain gel, and marker solution. Add 2 ul sample to 7 ul sample buffer. Heat to 70 o C for 10 min. Heat 12 ul ladder in parallel. Place chip on instrument for priming. Add 35 ul water to sample, mix, and spin. Add 120 ul water to ladder, mix, transfer to ladder tube. No dye crosslinking steps Automated staining and destaining steps 40 seconds/sample analysis, that is 400 sample analyses/5 hour! 65 For research use only. Not intended for diagnostic procedures.

65 Thorough Process Development leads to Success Target Discovery Process Development Clinical Manufacturing Commercial Manufacturing Clone Selection Cell Line Development Protein Purification Bioprocess Scale Up Formulation 66 For research use only. Not intended for diagnostic procedures.

66 High Throughput Process Development The potential design space is vast, the relationships between process parameters and critical quality attributes must be determined experimentally Parameter Choices Cell line Media components Feed strategy Control settings for ph, T, O 2, CO 2 Chromatography resin Binding and Elution Buffers Residence Time Process Outcome Growth Titer Purity Potency Glycosylation Charge heterogeneity Stability High throughput analytics are needed to address multi-factorial DOE studies in Upstream, Downstream and Formulations Development 67 For research use only. Not intended for diagnostic procedures.

67 Closing remarks Fluorescence label Automated Simple Time Actuators Cell culture Drug screening PCR Sensors Electrochemical SPR.. 68

68 Thank You for Your Attention! 69 PerkinElmer, Inc. All rights reserved. Some of the information contained in this presentation was obtained from third party sources, as cited. PerkinElmer, LabChip, and the LabChip logo are registered trademarks of PerkinElmer, Inc. and/or its parent, affiliates, and/or subsidiary companies (collectively PerkinElmer ). The PerkinElmer logo is a registered trademark of PerkinElmer, Inc. All references to other company names, products, trademarks and/or registered trademarks are the property of their respective holders

69 Miniaturization: How Small Can We Go? 1 L 1 nl 1 pl 1 fl Volume (10-6 L) (10-9 L) (10-12 L) (10-15 L) Cube dimension 1 mm 100 m 10 m 1 m Diffusion time across the cube* Number of molecules in 1 nm 1000 s 10 s 100 ms 1 ms 6x10 8 6x (*Assume a molecular diffusivity of 5x10-6 cm 2 /sec typical for small molecules) L = (2Dt) 1/2 70 For research use only. Not intended for diagnostic procedures.

70 Flow rate profiles- Pressure driven and electrokenitic driven Flow rate profile Sample plug profile (a) Pressure driven flow of bulk T i T d (b) Electrokinetic driven flow of ions/bulk T i T d 71

71 Reverse Pinch Injections buffer sample waste column 72 For research use only. Not intended for diagnostic procedures.

72 Double T Junction 73

73 Electrokinetic Dispersion Around a 90 Bend Electric field gradient around the corner induces an electrokinetic velocity gradient, causing sample band dispersion. J. Molho, PhD thesis, Stanford University, 2001 (Simulation by Microcosm s NetFlow CFD package) 74

74 Fluorescence Detection Fluorescence Molecules absorb light over a given wavelength range and emit light at a higher wavelength At low concentration the intensity of the emitted light is proportional to dye concentration Absorption Energy Loss Fluorescence - hn Molecule Excited Molecule Ground state Molecule 75

75 Double Layer and Electroosmotic Flow Ion distribution near a charged surface: Electric double layer Electroosmotic flow in an electric field 76

76 Improving characterization of monoclonal antibodies Making a better biotherapeutic November 19, 2014 Webinar Series Brought to you by the Science/AAAS Custom Publishing Office Participating Experts Niomi Peckham, M.Sc. Alexion Pharmaceuticals Cheshire, Connecticut To submit your questions, type them into the text box and click I-Jane Chen, Ph.D. PerkinElmer Hopkinton, MA Sponsored by:

77 Webinar Series Improving characterization of monoclonal antibodies Making a better biotherapeutic November 19, 2014 Brought to you by the Science/AAAS Custom Publishing Office Look out for more webinars in the series at: webinar.sciencemag.org To provide feedback on this webinar, please e mail your comments to webinar@aaas.org Sponsored by: For related information on this webinar topic, go to: