Advanced Applications of CE to Biotech Product Development: Monitoring quality from clone selection to final release

Transcription

1 Advanced Applications of CE to Biotech Product Development: Monitoring quality from clone selection to final release Dr Flavie ROBERT Biotech Process Sciences, Merck Serono SA, CH CEPharm, Vancouver, 2010

2 Early product development stages DNA Vector construction Transient transfection Cell line establishment, Clone selection, Expansion conditions, have major impacts on final product Quality attributes Gene insertion in host cell and amplification Screening and selection The earlier Quality is monitored, the better Small scale culture Manufacturability at large scale

3 Process Validation: a new regulatory framework Recent observations at the FDA: Increasing manufacturing defects, low manufacturing and QA process efficiency, resistance to Innovation and slow modernization The 21st century GMPs initiative ICH Guidelines Q8, Q9, Q10 (Q11) Process design Development Process Starting Materials Q9: Quality Risk Management: Regulatory control Support of operational efficiency Decision making: Development and Manufacturing Quality Systems Manufacturing Distribution Risk Management Q8: Pharmaceutical Development Enhanced process knowledge Characterization and control of variability Design Space Q10: Pharmaceutical Quality System Facilities (control strategy concept) Applicable to the entire product life cycle Monitoring of product and process variations Continuous process improvement Enhance process/product knowledge Control and monitor what is relevant to Safety and Efficacy 3

4 Our Quality by Design approach Selection Selection of of Critical Critical Process Process Parameters Parameters Process Process characterization characterization by by DOE DOE Scale-down model Scale-down model qualification qualification Large Large scale scale manufacturing manufacturing Process step characterization Definition Definition of of Design Design Space Space (DS) (DS) Process Process Development Development Prior process knowledge Quality Quality Risk Risk Management Management Selection Selection of of Critical Critical Quality Quality Attributes Attributes Continuous Continuous Verification Verification Requirements for Ana lab: Very large analytical panel High reproducibility High throughput Process validation Process Process Validation Validation (robustness) (robustness) Quality systems QTPP QTPP Expanded Expanded Change Change Protocol Protocol Definition Definition of of control control strategy strategy 4

5 Potential quality Attributes are many C-terminal Lysine Clipping Fragmentation Glycation Fucosylation Galactosylation High Mannose Glycans Pyroglutamate Formation Methionine Oxidation Deamidation Aggregation Conformation Sialylation Oligomerization Disulfide Bond Pattern Determining and monitoring variants is a challenge along product development and manufacturing process development 5

6 Reshaping the analytical lab What are the necessary technological adaptation in the analytical laboratory? Quality to be monitored at early stages, critical attributes to be linked to critical process parameters Broader analytical panel Immunoassays Robotics High Content analytics, faster technologies PAGE HPLC SPR MS Bioassays Capillary Electrophoresis: Imaging CE (ice280) pre-purified entire protein Spectroscopy High resolution CE (CGE-LIF) Labeled N-oligosaccharides 6

7 Imaging Capillary Electrophoresis as a profiling tool for process development 7

8 Imaging Capillary Electrophoresis for charge profiling (ice280) Electrophoregram Electrode Voltage H+ Ampholytes - ph Gradient OH- Fusedsilica capillary UV Light Beam 280 nm 8

9 Comparison of ice280 to orthogonal method IgG, 4 N-glycosylation sites, expected Cterm-lys variants Comparison to reference method for charge isoforms profiling (IEX-HPLC) : Fractionation on IEX-HPLC and major peaks relative quantitation on ice % % 20 0 Fr Fr 5 ice280 ice280 peaks Fr 1 Fr 3 IE-HPLC fractions IE-HPLC Order of elution correlates with pi order, relative surface areas are very similar. 9

10 cief Profile characterization for process knowledge (1/2) Native Partially deglycosylated Peak shift can be linked to protein deglycosylation Native Carboxypeptidase t ed Absence of last peaks: variability linked solely to Cterm K 10

11 cief Profile characterization for process knowledge (2/2) Native Deaminated Distribution change towards acidic forms could indicate deamination Charge heterogeneity mainly caused by sialylation levels Native Desialylated Effect of process changes on charge profile can be interpreted 11

12 Application to clone selection and process adaptation Follow-on IgG in preclinical phase, adaptation to a generic process and final manufacturing clone selection: 100% 75% 50% 25% 0% no feed Feed 1 no feed no feed no feed Feed 1 Feed 1 Feed 1 Feed 2 Feed 4 no feed no feed Feed 2 Feed 1 Feed 3 Feed 5 Clone 1 Clone 2 Clone 3 Harvest day ice280 is a relevant tool for clone screening and early process development, with a focus on Quality attributes. 12

13 Imaging Capillary IEF Pro s and Con s Quick and easy Optimization Quick sample Preparation Highly resolutive Non Specific Isoform identification indirect Need large amount Robusteness to improve 13

14 Capillary Gel Electrophoresis for N-glycosylation identification and monitoring 14

15 Capillary Gel Electrophoresis for Glycoanalysis Introduction and principles Protein Purification glyxtools processing Glycan release Glycan extraction APTS Laroy et al., Nature Protocols 2006, Vol 1 N 1 15 Glycan labeling (APTS) Capillary Gel Electrophoresis DNA Sequencer 4 or 16 Capillaries E. Rapp, J. Schwarzer, U. Reichl, C. Bohne : European patent: EP ( ) E. Rapp, J. Schwarzer, U. Reichl, C. Bohne : US patent: US 12/428, 003 ( ) J. Schwarzer, E. Rapp, U. Reichl, Electrophoresis, 2008, 29,

16 Capillary Gel Electrophoresis Data processing DNA ladder injected with each glycan preparation Glycan to DNA bp conversion glyxtools Glycan identification Calibration data points to DNA bp Software and Database are resulting from a collaborative work with E. RAPP and R. HENNIG, Max Planck Institute, Magdeburg E. Rapp, J. Schwarzer, U. Reichl, C. Bohne : European patent: EP ( ) E. Rapp, J. Schwarzer, U. Reichl, C. Bohne : US patent: US 12/428, 003 ( ) J. Schwarzer, E. Rapp, U. Reichl, Electrophoresis, 2008, 29,

17 Capillary Gel Electrophoresis Assay performance in HT mode Fc fusion protein 96W MTP preparation - 16 capillaries 2 days for 96 samples Peak 1 A2F Peak 2 A2FG1(1-6)F Peak 3 A2FG1(1-3)F Peak 4 A2FG2F Base pairs Average StDev CV% Relative Intensities Average StDev CV% Assay Performances on 4 main peaks Relative Intensities precision < 3% Base pairs precision < 0.05% Peak capacity

18 Capillary Gel Electrophoresis Strategy for oligosaccharides annotation and library construction Man5 A1FG0 A1FG1 A1FG1αG1M1 A2G0 A2FG0 A2G1 A2FG1 A2FG1S1 A2G2 A2FG2 A2FG2S1 A2FG2S2 A2FG2αG1 A2FG2αG2 A2FG2αG1S1 A3FG3αG1 A3FG3αG3. 1 Characterized and commercially available Direct migration time measurement CGE-LIF measurement 2 LC Prep-LC MS/MS In-house generated CE-LIF MS/MS Candidates oligosaccharides Oligosaccharide isolation Structure identification 18

19 Capillary Gel Electrophoresis Strategy 1 for oligosaccharide identification: spiking Purified oligosaccharide, spiked in prepared glycans from in-house product Purified A2FG1 Determination of ref. migration time (bp) for the analyzed oligosaccharide A2FG1 + prepared glycans Prepared glycans Higher resolution compared to 2AB-HPLC methodology Migration time (bp) 19

20 Capillary Gel Electrophoresis Strategy 2 for oligosaccharide identification: LC MS/MS F 9 CGELIF NP-HPLC F 12 APTS-labeled glycans EU F 13 F 23 F 22 F 16 F 18 F Minutes T: FTM S - p ESI Full ms [ ] R=0 z=1 MS/MS (structure) Relative Abundance R=0 z= R=0 R=0 z=1 z= R=0 R=0 z=1 z= R= R= z=4 R=0 R=0 z= R= z= z=? R=0 z=? R=0 R= R= z=? z=? z=? R=0 R=0 R=0 z=? R=0 R=0 z=? z=? z=? z=? z=? m/z MSMS fragmentation + CGE measurement migration time / structure correlation 20

21 CGELIF: Illustration of Glycan Library (IgG) On-going library construction for future automated characterization of new products. 21

22 CGELIF: Agreement with alternative method Highly glycosylated protein, 20 kd Variable sialylation level: How to control it? Reference assay : ESI-MS on entire protein ESI-MS m/z CGE-LIF The technology is directly applicable to improve process understanding 22

23 CGELIF: Application to process development support Process scale-down models qualifications are mandatory for practical implementation of a QbD based validation 2 scales of a DSP step are compared on 8 independent runs (yield, aggregation, isoform profiles, Glycosalytion profile) 1.50 Ratio of % Normalized Intensities for Process scales A and B Glycan # 1 Glycan # 2 Glycan # 3 Glycan # 4 Glycan # 5 Glycan # 6 Glycan # 7 Glycan # 8 Glycan # 9 Glycan # 10 Some glycans relative abundances are different depending on the purification scale. Quick and systematic glycoprofiling using CGELIF becomes a key tool for QbD implementation. 23

24 Capillary Gel Electrophoresis Pro s and Con s Knowledge to build «Beta» software Instrument cost Versatile tool Small scale HT preparation Intensive library construction Fast turnover High resolution Direct identification 24

25 Conclusion Both Capillary Electrophoresis techniques have shown adequate capabilities to serve Biotherapeutics product development. They contributes to the pool of innovative and high throughput platforms supporting a quality and knowledge based development of product and processes. 25

26 Acknowledgements Merck Serono Tiburtina, Italy Horst Bierau Francesca Cutillo Christian Hunzinger Mara Rossi Max Planck Institute for Dynamic Complex Technical Systems Magdeburg, Germany Merck Serono Vevey, Switzerland Gianni Baer David Beattie Ana Krstanovic Marine Lacroix Zeynep Manco Arnaud Perilleux Emmanuel Rossy Jonathan Vesin René Hennig Erdmann Rapp and Franka Kálmán 26

27 27 Back up

28 Merck Serono Introduction Largest division of Merck KGaA, established in 2007 and dedicated to innovative small molecules and Biopharmaceuticals. Technical Operations: Manufacturing and Product Development Missions: Production, supply chain and quality management operations for Biopharmaceuticals New products development and process improvement in several pilot plants Merck Serono SA in Corsier-sur-Vevey, Switzerland: operating since 1999, under ISO since 2004 Large expansion project underway to increase capacity for production of Erbitux by 2012 and potential future AIID treatments currently in clinical development. 28 Introducing Merck Serono August 2010

29 Glycosylation affects product Safety and Efficacy Galactosylation Complement activation Inflammation Fucosylation ADCC High Mannose Glycans Sialylation + Non human glycoforms α-galactose and NGNA Immunogenicity Serum half life and inflammation Potential immunogenicity N-acetyl-glucosamine Mannose Galactose Fucose Sialic Acid N-glycolyl sialic acid Glycosylation is becoming the utmost complex quality attribute to control in the new Product development paradigm (QTPP) 29

30 N-Glycosylation Background High mannose glycoforms (neutral) Complex glycoforms (neutral + acidic) a1 -GlcNAc N-acetylglucosamine (GlcNAc) Mannose (Man) Galactose (Gal) Fucose (Fuc) Sialic acid (Neu5Ac / Neu5Gc) 30

31 Main Strategies for detailed Analysis of Glycostructures 31

32 Principles of CGE-LIF DNA Sequencer 1, 4, 16, 48, 96 capillaries Collaboration with E. Rapp MAX-PLANCK-INSTITUT DYNAMIK KOMPLEXER TECHNISCHER SYSTEME MAGDEBURG 32

33 Capillary Gel Electrophoresis Comparison to orthogonal method 2AB labeling HPLC, historical Quality control and characterization tool Glycoprotein Release of N-glycans (PNGase F) Cleanup 2-AB labeling HLPC-FLD LU Legend: GlcNAc Mannose Galactose Fucose Online MS (ESI-MS) Offline MS (MALDI-MS) Precision on relative Surface areas: 8% 33 CE Pharm CE in Biotech Process Dev - F. Robert