Science Webinar Series PLEASE STAND BY… the webinar Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development will begin shortly… Change the size of any window by dragging the lower right corner. Use controls in top right corner to close or maximize each window. What each widget does: shows the audio media player shows slide window opens the Ask a Question box shows speaker bios download slides and more info search Wikipedia Facebook login LinkedIn login Twitter (#ScienceWebinar) to login login to Twitter and send tweets if you need help Science Webinar Series Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development 14 November, 2012 Brought to you by the Science/AAAS Custom Publishing Office Participating Experts: Joey Studts, Ph.D. Boehringer Ingelheim Pharma GmbH & Co. KG Biberach, Germany Timothy Blanc ImClone Systems Branchburg, NJ Bahram Fathollahi, Ph.D. PerkinElmer San Francisco, CA Sponsored by: Microfluidic Assays for High Throughput Screening of Protein Quality in Bioprocess Development Bahram Fathollahi Microfluidics R&D November 14, 2012 Science/AAAS Microfluidics Webinar © 2009 PerkinElmer What is a Microfluidic Device? An integrated device with controlled transport of sample and reagents in microchannels to carry out bio/chemical analysis Advantages Miniaturization Fast analysis/high throughput Small sample volume Integration Automation 4 Microfluidic Chip Format and Fabrication Planar Chip Single use Samples added to wells manually Sipper Chip Reusable Samples introduced automatically from microtiter plate Sipper (autosampler) Chip Fabrication Chips manufactured using photolithography and wet etching technology Quartz and glass substrate Highly reproducible and precise control of channel geometry Typical depth - 5 µm to 25 µm Typical width - 10 µm to 100s µm 5 Electrophoretic Separation in Microfluidic Chips Buffer Reservoirs SAMPLE LOAD AND INJECTION SEPARATION DETECTION B C D A Sample Reservoir Sample Load B ∆V A 6 Waste Reservoir LIF detection Sample Separation Sample Injection ∆V ∆V ∆V Commercial Microfluidic Electrophoresis Platforms LabChip |DX/GX/GXII Bioanalyzer 2100 Higher Throughput • RNA & DNA analysis • Proteins • N-Glycans (LabChip Only) Experion • Digital information • Easy-to-use • Time and cost savings Planar chip format 7 Sipper chip format Protein Characterization Assays on LabChip GX II Multiple HT Screening Assays on a Single Microfluidic Platform 1) Microchip CE-SDS – 40 sec/sample Protein expression optimization Assess purity Reduced and non-reduced Titer Quantify impurities Product- and process-related Monitor fragmentation and Ab assembly 2) N-Glycan Profiling – 60 sec/sample LabChip GX II Measure relative amounts of major N-linked glycans 3) Charge Variant Profiling – 68 to 90 sec/sample Identify and measure relative amounts of basic, main, acidic variants 8 Microchip Microchip CE-SDS Assay Workflow Crude or purified protein sample (2µL) Add Sample Buffer Heat denature ~15min Reusable chip Up to 400 samples Prepare Gel/DyeSolution and Ladder Add Reagents to chip Place Ladder and Buffer on GXII Identified expected protein peaks Plate View ~ 40 sec per sample E-gram ~ 75 min for 96-well plate Protein Analysis Sizing, Conc., and Purity 9 Gel View Microchip CE-SDS vs. Conventional CE-SDS Reducing Non-reducing HC Microchip CE-SDS LC 30 seconds Monomer 1250 In-process sample 1000 750 HHL Fluorescence NGHC LM 500 Aggregates (covalent) HL 250 0 30 28 26 24 22 20 18 16 14 12 10 8 13 18 23 28 33 38 43 48 Time (seconds) Conventional CE-SDS 10 Attributes Assay Instrument time Microchip CE-SDS Concentration ProA HPLC RP HPLC 2-5 min ~20 min < 1 min Assembly, covalent aggregation nrCE-SDS 30-50 min < 1 min Fragmentation, Degree of N-glycosylation rCE-SDS 30-50 min < 1 min X. Chen et al .Electrophoresis 2008, 29, 4993-5002 53 HT N-Glycan Profiling Sample Prep Workflow LabChip GXII Denature and Reduce mAb in Denaturing Buffer Add Denatured Reduced mAb to Enzyme Plate Denaturing plate Heat (Denatured mAb + Enzyme) at 37 °C for ~1h microchip Transfer to Dye Plate PNGase-F Plate Heat Assay Plate at 55 °C for ~2h G0F Dilute with separation Buffer Read 96-well plate on LabChip GXII ~1.5h G1F’ Glycan Labeling Plate G2F 11 G1F G0 M5 Digested and Labeled N-Glycan Profile G0F G1’F G1F G2F Man5 UNK 12 G0 G1 G2 UNK UNK Methods of Characterizing Charge Heterogeneity Three methods of separation used in the biotherapeutics industry: Ion Exchange Chromatography (IEC) Extension of HPLC systems Proteins interact with charged resin, based on pI and hydrophobicity Capillary Zone Electrophoresis (CZE) Based on Capillary Electrophoresis (CE) systems Proteins migrate at different speeds, based on pI and hydrodynamic drag Isoelectric Focusing (icIEF) Based on imaged CE systems Proteins migrate to a particular position within a pH gradient, based on pI HT Protein Charge Variant Assay Limitation of current methods: Lack of speed Analysis Time 13 IEC icIEF CZE Microchip-CZE Per Sample 30 - 90 min 15 - 20 min 8 - 30 min 1 - 2 min Per 96-Well Plate 48 - 144 hrs 24 - 32 hrs 16 - 48 hrs 1.8 - 2.9 hrs Variant Detection Conventional methods use absorbance at 280 nm LabChip detects via LIF Dye must be conjugated to protein variants Dye must absorb & emit at wavelengths that are compatible with instrument optics Dye must conserve charge of protein variants From icIEF analysis Relative Amount (%) Before Labeling After Labeling Charge profile does not change Acidic Variants 14 Main Variant Basic Variants Direct Comparison With Alternative Methods HT PCV Microchip-CZE 60 sec Basic Main Isoform - 8.810 icIEF 15 min Acidic Conventional CZE ~10min Basic Acidic 8.948 9.044 8.712 8.585 0 8.00 15 8.20 8.40 8.60 Acidic Basic 8.80 9.00 9.20 9.40 9.60 1.60 1.80 2.00 2.20 2.40 2.60 Minutes 2.80 3.00 3.20 3.40 Small-Scale High Throughput Process Development Higher demand of product quality requires multi-factorial DOE studies in upstream and downstream process development Sample prep/ µscale purification Clone selection/cell culture optimization BioTx workstation Daughter Plate Mother Plate Screening high number of cell clones in smallscale bioreactors 16 Analytics for determination of critical quality attributes µscale chromatographic purification Daughter Plate Daughter Plate Informatics Purity Assessment N-Glycan profiling Charge Variant Process parameter optimization Optimization and Scale-up Process understanding and decisions Acknowledgements Microfluidics R&D Tobias Wheeler Lucy Sun Rajendra Singh Mai Ho Hui Xu Melissa Takahashi Roger Dettloff Marketing & App Support Rick Bunch Krystyna Hohenauer Nate Cosper Seth Cohen Linda Gary Marsha Paul Chip and Reagent Fabrication Joan McAuliffe Ken Summers Trang Le Khushroo Gandhi Software Advit Bhatt Dan Camporese Renil Das 17 Thank you! 14 November 2012 Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development Process Science Germany, 2012 Project Timelines Focus on Critical Bottlenecks in Development Overall Project Timeline Exploratory Research Preclinical Development Clinical Dev. Development Phase I,II Bottleneck to Clinic Cell line Process Development/ optimization non GMP scale up, Tox material GMP Pilot Plant Ph I - Scale up • Fast track development by applying “platform technologies” • Material supply: 1 g - 2 kg Phase IIb/ III Registration Market Supply • Critical bottleneck is time to the clinic • Reduce time to clinic to shorten time to market • Often is data on the critical path in development • Early process understanding critical to efficiency and safety More data Better Decisions Efficient Development Better Products 19 BI-PurEx: Lean Development Strategy Understanding the product & the process Cell Line Development Process Development USD & DSD Scale-up USD & DSD Pilot Scale GMP Product & Process are critical • • • • • Cell line development is on critical path Rate limiting step is cell growth, very little material with which to gain knowledge Choosing the correct cell line for each product is most critical Impacts product quality, functionality, expression levels, comparability… Early knowledge and correct decisions will have an significant impact on efforts and timeline throughout the development process. 25 Product Understanding Glyco Patterns 4A 6A 7 2 S:/BPQC/QC/QCAS/LG7/FOB/Caliper Datenabgleich/T1-HEX-110-09.opj/13.01.2011 Zug / Em420 nm Ex330 nm HPLC: 20 min/run MS: >> 20 min/run 8 4 1 20 30 3 40 5 6 50 60 70 • Initial challenges in Glycosylation Retention Time [min] • Mainly controlled by cell line decision • Purification normally has very little impact on glycans • Impacts comparability and functionality of molecule • Critical to get early knowledge and impact cell line development decisions! 26 Product Understanding High Throughput Glyco Analysis 20 18 % CGU (Caliper Glucose Units) 16 14 12 10 8 6 4 2 0 Man5 T9 Std. 5 T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101-T9-HEX2-101Standard Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 mg/mL 01 02 03 04 05 06 07 08 10 7,48 4 6,3 6,15 8,39 3,24 3,1 4,28 4,83 3,07 NGA2 3,09 1,94 1,23 0,92 6,65 1,73 1,16 1,19 2,54 2,31 NGA2F 16,95 14,71 17,45 0,37 0,73 12,03 9,15 12,17 14,2 11,52 NA2G1F 3,66 1,79 1,89 0 0,73 1,54 0,73 1,21 1,55 1,61 NA2 0,06 0,1 0 0 0 0 0,07 0,08 0,04 0,15 NA2F 0,24 0,26 0,14 0,37 0,22 0,17 0,1 0,17 0,17 0,26 • Using Microfluidic Assay – all pools are analyzed for glycan map prior to cell line decision. 27 Product Understanding High Throughput Glyco Analysis HT- Microfluidic Assay • High Throughput • Very reproducible • Comparable to standard Methods. DS01 [5mg/mL] Inj. 1 Inj. 2 Inj. 3 Mean SD VK Std. Method Man5 4,05 4,27 4,24 4,19 0,12 2,85 4,52 NGA2 2,93 2,89 2,83 2,88 0,05 1,75 2,73 NGA2F 59,61 57,5 59,1 58,79 1,10 1,87 60,48 NA2G1F 14,2 13,8 13,82 13,94 0,23 1,62 20,6 NA2F 1,7 1,83 1,43 1,65 12,34 12,3 1,9 28 Product Understanding Product Based Impurities Electorpherogram from reduced mAb sample Standard 97.1 % Purity Clone 1 94.1 % Purity Clone 2 94.6 % Purity Clone 3 95.1 % Purity Clone 4 94.3 % Purity Clone 5 94.2% Purity • Product purity influenced by purification process development • Microfluidic Assay helps point out critical impurities to focus later development efforts and to track in cell line decisions. 29 Product Understanding Product Purity – Charge Heterogeneity Qualitative analysis no longer enough • Impact on safety and efficacy MP Critical to Comparability • Impacted by cell line • Impacted by fermentation process • Impacted by purification process BPG APG High throughput method required to look at all samples WCX 7.417 19.120 MP iCE MP 14.00 14.50 15.00 15.50 16.00 17.00 17.50 18.00 Minutes 18.50 19.00 19.50 20.50 21.00 21.50 22.00 2 6.90 7.00 7.10 7.20 7.40 7.50 7.763 7.555 7.30 7.842 7.655 7.304 20.00 7.178 7.030 20.913 16.50 BPG 19.760 18.279 17.766 16.555 16.136 13.50 APG BPG APG 7.60 7.70 7.80 7.90 Minutes 30 Product Understanding Product Purity – Charge Heterogeneity 7.20 7.50 Minutes 7.60 7.70 13.00 14.00 15.00 16.00 17.00 18.00 Minutes 7.90 Clone Z Clone X 19.00 20.00 8.00 Main Peak - 7.417 6.85 6.90 6.95 7.00 7.05 7.15 7.20 7.25 7.30 21.00 7.35 7.40 7.45 7.50 Minutes 7.55 7.60 Basic Peak 4 - 7.841 Basic Peak 3 - 7.759 Basic Peak 2 - 7.635 Basic Peak 1 - 7.554 Acidic Peak 3 - 7.304 Acidic Peak 2 - 7.180 7.10 Ability to detect differences to reference material. basic Peakgroup - 19.772 acidic Peakgroup - 18.332 WCX 7.80 Acidic Peak 1 - 7.035 Basic Peak 2 - 7.633 Basic Peak 1 - 7.543 7.40 7.30 Ability to detect differences between clones iCE 7.65 7.70 7.75 7.80 7.85 7.90 WCX 22.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 7.95 R&D 1 Clone Z basic Peakgroup - 19.821 7.10 Sensitivity very high R&D 1 Clone Z Mainpeak - 19.212 7.00 R&D 1 Clone Z acidic Peakgroup - 18.324 6.90 Clone Z Clone X Mainpeak - 19.235 .80 Acidic Peak 3 - 7.308 Acidic Peak 2 - 7.191 Acidic Peak 1 - 7.036 iCE Main Peak - 7.409 Clone Z Clone X 20.00 Minutes 21.00 22.00 23.00 24.00 31 Process Understanding RAPPTor® Rapid Automated Protein Purification Technology Separate but Seamless Tecan 150 – HTScreeningTool Tecan 200 - HTAnalytical Tool Fully automated purification screening platform From resin and plate preparation to step elution Screen 96 variables and generate of up to 500 sample per day Fully automated sample preparation and analytical assays Automated homogeneous assays to screen critical output parameters 32 Process Understanding High-Throughput Parallel Chromatography robustness study (11 runs) initial screening (96 runs) Filterplate run 4 4 RoboColumn runs 9 72 33 Serial Column runs 288 0 100 200 300 time (h) gaining process knowledge earlier in a shorter timeframe with less material demand • Microfluidic Assay allows analysis of impurities profile for each of the samples generated. 33 Process Understanding Critical Impurities Sample Name • • • • % Purity Type Standard 0,22 Unknown AT Prod Pool 0,88 Unknown DF Prod Pool 0,87 Unknown AEX Prod Pool 0,88 Unknown CEX Prod Pool 0,76 Unknown Bulk Prod Pool 0,79 Unknown Assay set-up not complex, carried out within development lab Data delivered within hours, not days Can take next step to test fractionation of individual process steps IMPACTon real time decsions! 34 Process Understanding Determination of aggregates Acquity UPLC H-Class: • Shorten analysis time by factor of 2.5 Size exclusion chromatography Molecular weight standard • Increase resolution through smaller particle diameter • Lower injection volumes • Low carry over • Applicable to WCX, RP…. ~ 8 min ACQUITY UPLC BEH200 SEC, 4.6 x 150 mm, 1.7 μm,4 µL ~ 20 min HPLC TSK G3000 SWXL, 7.8 x 300 mm, 5 µm, 20 µL 35 Process Understanding Analytical Throughput: Method portfolio UPLC- SEC 96-well UV detection Lab Chip GXII CHO HCP AlphaScreenTM (768 data points overnight) Excitation 680 nm Emission 615 nm 1O 2 Streptavidin -coated Donor bead Host cell proteins Anti-CHO HCP conjugated acceptor bead Automated ELISA iCE280 Analytical techniques with increased throughput. Understand impact of each step on product quality and contaminant removal. 36 Boehringer Ingelheim Biopharma Process Science Thanks to many colleagues at Boehringer Ingelheim especially: to H. Kaufmann, D. Ambrosious to, J. Stolzenberger, S. Combe, O. Haas, S. Müller, D. Dieterle Boehringer Ingelheim, Bibearch, Germany Thanks for your attention!!! 37 The Use Of Microfluidics Technology in the Pharmaceutical Industry: MicroChip Electrophoresis as a High Throughput Process Analytical Platform to support the Development of Therapeutic Monoclonal Antibodies Tim Blanc and Qinwei Zhou ImClone Systems. Branchburg, NJ 08876 Company Confidential Copyright © 2010 ImClone LLC Introduction Rapid analytical separations is an important application stemming from the development of Microfluidics. One such separation technique is MicroChip Electrophoresis (MCE). MCE can be performed in several modes to provide valuable insights to the relationship between bioprocess parameters and the quality attributes of biopharmaceutical products. Recombinant Monoclonal antibodies commonly exist as heterogeneous mixtures of product variants. Several factors contribute to product variants, one such factor is glycosylation. Using Glycosylation as the example, It is important to understand what the relationship is between process parameters and glycan distribution expressed on the antibody. An effective Quality by Design (QbD) approach uses design of experiments (DOE) to determine the most important process parameters, termed Critical Process Parameters (CPPs). However the number of samples required for the experimental design can be high. Testing this number of sample to gain the most thorough process understanding is time and cost prohibitive by standard methods. Discussed will the regulatory and biopharmaceutical issues that have accelerated the need for rapid analytical separation. Then, will be discussed application on MCE that have been developed to address those needs. Company Confidential Copyright © 2010 ImClone LLC 39 The New Quality Paradigm • ICH Q8 (R2) Pharmaceutical Development • ICH Q9 Quality Risk Management • ICH Q10 Pharmaceutical Quality Systems • ICH Q11 Development and Manufacture of Drug Substance ICH Q11: “Risk management, and scientific knowledge are used more extensively to identify and understand process parameters and unit operations that impact critical quality attributes (CQA’s) and develop appropriate control strategies applicable over the lifecycle of the drug substance Company Confidential Copyright © 2010 ImClone LLC 40 Quality Risk Management: Linking Process Knowledge, Product Knowledge and Quality Systems Product Knowledge Critical Quality Attributes Process Knowledge Critical Process Parameters Quality Risk Management Control Strategy Quality Systems Ref.- Quality Attributes of Recombinant Thereapeutic Proteins: An Assessment of Impact on Safety and Efficacy as part of a Quality by Design Development Approach Eon-Duval, A. et.al BIOTECHNOL. PROG., 2012, Vol.00, No. 00 Company Confidential Copyright © 2010 ImClone LLC 41 Good Business too Phase I IND Phase II Phase III BLA Tox & PK Studies Process Development and Scale-up (Changes) Comparability Studies- Linking Commerical scale product back to original material use in Tox. & PK Studies Assure QCA’s are maintained within acceptable ranges (QTPP), Quality Target Product Profile, throughout Process Development. Maintain link to Tox. & PK studies. Company Confidential Copyright © 2010 ImClone LLC 42 Heterogeneity of Monoclonal Antibodies Therapeutic Mab Product Contents Product Variants Process Related Impurities Trace amounts (ppb-ppm) • Residual Host Cell DNA, • Residual Host Cell Protein, • Residual Protein-A. ProductRelated Impurities 1-5% • Antibody Fragments, • Aggregates Product Variants Post-Translational Modifications: • Glycan Variants, • C-Terminal Truncation, • Deamidation, etc. Critical Quality Attributes (CQA’s) Company Confidential Copyright © 2010 ImClone LLC 43 Heterogeneity of Monoclonal Antibodies Therapeutic Mab Product Contents Product Variants Multiple Sources give rise to Product Variants Company Confidential Copyright © 2010 ImClone LLC 44 Critical Process Paramenters (CPP) CPPs are independent process parameters most likely to affect the quality attributes of a product or intermediate CPPs are determined by analytical testing, scientific judgment and on research, scale‐up or manufacturing experience CPPs are controlled and monitored to confirm that the impurity profile is comparable to or better than historical data from development and manufacturing. Quality attributes related to CPPs include: • Product Variants Distribution (Glycosylation is a major contributor) • Product-Related Impurities • Process-Related Impurities Company Confidential Copyright © 2010 ImClone LLC 45 Glycosylation Company Confidential Copyright © 2010 ImClone LLC 46 Implications of Glycosylation Variants • The greatest source of Mab Product Variants is Glycosylation • Glycosylation is very closely tied to process parameters • Aspects of glycosylation will frequently be CQA’s Glycan Structural Characteristic Implication Fucose: Effector Function (e.g. ADCC) Sialic Acids (NANA, NGNA) PK modulation (up or down) NGNA Immunogenicity α-Galactose Immunogenicity Company Confidential Copyright © 2010 ImClone LLC 47 MCE and Product Variants Sample prep designed in microtiter plate format with immobilized enzyme to reduce sample prep from 2 days to ½ day (Glycans). Rapid electrophoretic separation on Microchip with sensitive laserinduced fluorescence detection. (<60 seconds per sample) Analytical separations in under 60 seconds for determinations of: • Size Heterogeneity • Charge Heterogeneity • Glycosylation Heterogeneity Throughput and Accuracy for thorough QbD testing, CPP determination and Successful Control Strategy efforts Company Confidential Copyright © 2010 ImClone LLC 48 High Throughput Platform for PAT Charge Size Glycosylation MCE-CZE Charge-Based Evaluation MCE-SDS Size-Based Evaluation MCE N-Linked Glycan Analysis Relative Distribution Of Product Variants % APG % NPG % BPD % Purity (NR) % Aggregate % Fragment % Purity (Red.) % Non-Reducible % Aglyc HC % G0 F % G1F % G2F % α-Gal % Fucosylated % Sialylated (NGNA) Data is all numerical for statistical comparison Company Confidential Copyright © 2010 ImClone LLC 49 MCE Platform: Size Heterogeneity MCE-SDS < 60 SECONDS per Samples CE-SDS ≈ 60 Minutes Samples Company Confidential Copyright © 2010 ImClone LLC 50 MCE Platform For QbD: Charge Heterogeneity MCE < 60 SECONDS per Samples IEC >60 MINUTES per Sample Company Confidential Copyright © 2010 ImClone LLC 51 MCE Platform: Glycan Heterogeneity MCE < 60 SECONDS per Samples HPLC ≈ 60 Minutes Samples Company Confidential Copyright © 2010 ImClone LLC 52 Conclusions • Rapid Analysis time and easily automated. • Allows more samples tested and better insight to Impact of process parameters. • CPP’s determined and detailed process understanding. • Effective Control Strategy. • Relevant Drug Substance Specification (JOS). Company Confidential Copyright © 2010 ImClone LLC 53 Science Webinar Series AMicrofluidic DVANCINGElectrophoresis EPIGENETICS:Assays for Characterization of Protein NRapid OVEL DRUG DISCOVERY STRATEGIES FOR in ResearchTARGETS and Development EPIGENETIC 1417 November, October, 2012 Brought to you by the Science/AAAS Custom Publishing Office Participating Experts: Joey Studts, Ph.D. Boehringer Ingelheim Pharma GmbH & Co. KG Biberach, Germany Timothy Blanc ImClone Systems Branchburg, NJ Bahram Fathollahi, Ph.D. PerkinElmer San Francisco, CA To submit your questions, type them into the text box and click . Sponsored by: Science Webinar Series Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development 14 November, 2012 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 [email protected] Sponsored by: For related information on this webinar topic, go to: www.perkinelmer.com/biotherapeutics
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