Biopharmaceutical Manufacturing Technology: Vision for the ...

1 Biopharmaceutical Manufacturing Technology: Vision for the FutureJim Thomas, Vice PresidentProcess & Product DevelopmentHuman Genome ProjectNearly 5 million acres of biotech crops grown Development of leadingbiotech products Biotech has a rich history and bright futureFirst generic markers for human disease are found Recombinant human insulinCommercial biotechcompanies formedStem cell applicationsGene therapyProbioticsEfficient bioconversion of cellulose into fuelDNA moleculedeciphered by Watson & CrickEnzyme involved in the synthesis of nucleic acid isolated Harris & Watkins fuse mouse and human cellsAutomatic protein sequencer is perfectedCohen &

2 Boyer reproduce DNA in bacteriaYeast genes are expressed in E. coli bacteria1980sTomorrow1990s Today1950s1960s1970s More competitive business environment More challenging reimbursement environment More conservative regulatory industry is changing rapidlyThe molecules of biotechnology are complexSmall MoleculesBiochemistryRecombinant DNA TechnologyAcetylsalicylic Acid(Aspirin)Organic ChemistryMW: 180 DaMW: 150 kDaPDB identifier 1hzhScience(2001) 293:1155 Proteins(including therapeutic antibodies)Many patients have been served due to advances in Biopharmaceutical Biopharmaceutical Manufacturing is a very complex and expensive businessOpportunity for measurements and standards in the future Manufacturing of biotherapeuticsDesign the ProcessDesign the ProcessDesign the PlantMeasure quality / Safety / ProductivityThe MoleculeChallenges and Trends in MeasurementSSSSSSSSSSSSSSSSSSSSSSSSVLVHC LCH1CH2CH3 Heterogeneity can be influenced bythe Manufacturing processC-terminal lysine or argininecleavageN-linked glycoformsN-terminal glutamine cyclization to pyre-gluteic

3 AcidDisulphide scrambling misfoldsProduct variants from upstream (cell culture) processingmAbs IgG II subclassChallenges and trends in measuring protein therapeutics Greater sensitivity, more specificity Peak profile ( CEX) does not identify specific chemical changes Move toward more informative assays Elimination of subjectivity Visual inspection- presence of particles, color Move toward automated instrument-based inspection Focus on Critical quality Attributes (CQA) Move toward knowing the specific chemical change and the site ofmodification Move toward understanding the biological importance of chemical changesTrends in analytical tools Measuring host cell protein Measuring molecule fragmentation Measuring protein microheterogeneity-SDS-PAGE-ELISA-Chroma tography-Some MS1990MS based analysis.

4 -Host Protein Fingerprint- Top Down Analysis- Routine analysis of CQA 2020 Trends in mass spectrometer (MS) sensitivity*YearDetection limit of peptide (pmol)19901001993 10 1997 1 2000 2003 2005 2008 * typical industrial laboratoryProteins can form aggregates or particlesDissociable DimerNon Dissociable DimerNon Dissociable AggregateSubvisible ParticlesVisible Protein ParticlesVisible Extraneous Particlesnm mmmcm (107nm)1010010100monomers & oligomerssubvisible particlesvisible (HIAC)Visual InspectionMicroscopeAUCDLS*Laser Diffraction** qualitativeMeasuring protein aggregation/particulationPotency measurements In Vitro Binding Assays Cell Based Reporter GeneTargetFcRnSAbiotinTargetProLuciferas eTFSpecific Signaling CascadeProductTargetDeveloping the Manufacturing ProcessHow will biotherapeutics be manufactured in the future?

5 Transgenic animals Transgenic plants Cell free systems Cell based systemsThe Host CellMammalian Cells: Core Manufacturing TechnologyMOLECULAR CELL BIOLOGY 2ed by Lodish et al. (c) 1990 by Scientific American Books. Used with permission by Freeman and Company. Illustration by Tomo NarashiomaIsolating the best cell clones is challenging and time consuming96well plate~250 Clones24 Clones10 20 ClonesScale Up24well plateT7525mL shake flasksDay 9 Imaging11 Day Fed Batch AssayPoor growerseliminated Pool of stable transfected cells Individual encapsulation into gel microbead Capture and staining of secreted products FACS quantitationand single cell sorting of high producing subcloneT25(HTRF)

6 Greater productivity requires improved fundamental scientific understanding nutrients & metabolitesMEDIA DESIGNENVIRONMENTAL DESIGN bioreactor conditionsIncreased product titerCELLSMore cells / volume / timeMore product / cell / timeMOLECULAR DESIGN metabolic engineeringGene transcriptionDe-bottleneck expression pathway Anti-apoptosisMapping the design space of critical unit operations is a significant challenge Response surfaces represent titer Green dots represent conditions confirmed in 2L will be important for building quality into the Manufacturing processHigh throughput process development is needed5 incubatorsTemp.

7 CO2, O2. Humidity, Mixing1260 mini-reactors8 sources for liquid addition ( )pH control (< )OD measurementIntegrated databaseLink to other systemsTECAN, Guava high throughput purification developmentStack Filterplate on ELISA plateCentrifugeCollect 300 L SupernatantWells: 800 LResin: 100 LLiquid: 365 LBatch-bindingFilterplates with 96 conditionsStack Filterplate on ELISA plateCentrifugeCollect 300 L SupernatantWells: 800 LResin: 100 LLiquid: 365 LBatch-bindingFilterplates with 96 conditions/ Vacuum filter HTS batch binding and elution narrows choice of conditions column runs for further optimizationHigh throughput analytics are needed to support real time data acquisition and analysisExamples of implemented HT assays.

8 Titer Aggregate High mannose Amino acid analysis CHOP ElisaThe Manufacturing OperationProcess DesignPlant DesignMeasuring the EnvironmentQuality of Raw MaterialsManufacturing operations will be more efficient in the future Higher yielding processes Greater plant flexibility Better utilization of capital Significant reduction in operating costsHolistic approach to optimizationProcessDesignPlantDesignProc essControlThe Manufacturing operation -opportunities for measurements and standardsPROCESSRaw MaterialsProductProductivityQuality / SafetyEnvironmentPLANTP rocess DesignProductivity and QualityUpstream parameters for potential measurement and control O2, CO2, pH, temperature Nutrient composition Depletion of nutrients Metabolic byproducts Amino acids Carbohydrates Cell mass, cell number, cell viability Product titre Product quality Aggregation Clipped species GlycosylationExamples of monitoring and control pointsEnvironmentalWaterRaw materialsProcess aidsBioreactorpH, OD, temp, pO2, pCO2 Product titreViability, metabolitesTMP, flow, conductivity UVpH, conductivity C, time, concnpH, conductivity UV, flow, TADrying time.

9 TemperatureOpportunities for monitoring and control EnvironmentalWaterRaw materialsProcess aidsBioreactorpH, OD, temp, pO2, pCO2 Product titreViability, metabolitesTMP, conductivity UVpH, conductivity C, timepH, conductivity UV, BioreactorOn-line HPLC, NIR, MS, CZEV iral detection metabolitesProduct variantsHPLC/CZEA ggregates variants, contaminantsHPLC/CEHPLC/CZE/MSBiosensor Drying time, temperatureNIR, Moisture contentPlant DesignTrends in Manufacturing plant designFlexibility for optimizing plant capacityCapital Cost engineering construction materialsOperating Cost utilities maintenance environmental control /monitoringPlug n Play concept assemblyUTILITY HEADERREMOVABLE off the headers and connections to the skids would be made only as needed for each specific skid wall panels allow skid replacement Comprehensive Prefab integrated prefab structure / facility / process Box-in-a-Box integrated pre-fab facility within a stick-built structure Partial Prefab prefab

10 Assemblies for insertion in stick-built structure and facilityModular pre-fabrication approachesDegree of IntegrationDisposable technology could improve plant utilization and decrease utility cost Top mounted magnetic-drive agitator Disposable optical pH and DO sensors Sparge, overlay, and exhaust filter lines Weldable media addition and sample lines Fully customizable bag design Touch screen PLC controller Movable platform skidMeasuring the EnvironmentMycoplasmaBacteriaVirusesHigh throughput methods for detecting microbes will improve environmental control Detects all infectious threat agents Bacteria, mycoplasma, viruses, fungi, protozoa Resolves mixtures of organisms Quantitative Sub-species resolution (strain genotyping)Universal Pathogen DetectorContamination turn-around time*Assumes a detection assay exists for the contaminant**Best case scenario.