Lee, Tim

English

Engineering Conferences International

11Trouble-shooting Fermentation and Primary recovery manufacturing issues in order to optimize antigen expression for the Vaccine businessTim Lee, Ph.D.2AgendaFermentation manufacturing issues in antigen expressionParameters (i.e. physical & nutritional) to considerLarge-scale limitationsScale-down methodology in finalizing a large-scale processAntigen recovery issues in manufacturingProcess parameters for considerationLarge-scale limitations and solutions for antigen recoveryFuture considerations23Process considerations during fermentation scale-upPhysical parameters to maintainShear rate – turbulenceBulk flow – mixing timeKLa – mass transfer of oxygenPower/Volume ratiopHTemperatureNutritional requirements to controlSubstrate feeding concentrationOutput: Productivity, dissolve oxygen and pH profile4Problems associated with scaling-up to large bioreactorsOxygen transfer is less effective at the larger scale.To achieve similar mixing time of nutrients as the smaller scale bioreactor is not achievable Bioreactor ConstraintsAeration and AgitationSolutions• Increase the number of impellers (i.e. Rushton impeller) during operation to increase power and improve oxygen transfer• Try different combination of impellers (ie. Rushton and Hydrofoil impellers) to improve mixing35The effect of KLa on protein yields upon scale-up0204060801001200.1 0.21 0.3 0.4KLa (s-1)% Antigen expressed2 Rushton impellers (large-scale)2 Hydrofoil impellers (large-scale)3 Rushton impellers (large-scale)Small-scale fermentation reference( ) 2.0g0.7gLa UVP0.002K ⎟⎟⎠⎞⎜⎜⎝⎛=• Hydrofoil impellers didn’t improve protein expression• Improving mass-transfer by additional impeller improve the protein yield in large-scale reactorKLa relates to gas velocity and power input to stirrer for stirred fermentors containing non-coalescing non-viscous media (Doran, 1995)6Optimizing substrate feed and temperature to improve productivity (Large scale)05001000150020002500300035000 5 10 15 20 25 30 35Fermentation Time (hrs)Units of Antigen expressioncontrol (2 impellers, high feed rate, low temperature)3 impellers, High feed rate, lower temperature3 impellers, lower feed rate, High temperature• Optimization at large-scale: 6 batches & 2 months • Costly in time and resources47Scale-down process to Millilitres to determine key process parametersBenchtop, computer controlled fermentation systemmassive screening/testingKey Media components, Process conditions: Temperature, pH, Dissolved oxygen concentration, aerationsingle use 24-reactor cassetteindependently control and monitoredGas supply, temperature, pH, Dissolve oxygen24 simultaneous experimentsReduce cost Improve productivity8Where the µ-reactor fits59Temperature and pH  ControlIPTG InductionTemperature10Soluble protein Expression27°C Reproducible expression30°CReproducible expression↓ Induction Temp.↑ soluble protein expression27oC 30oC 30oC 27oCSolubleproteinInduction Temperature1         2        3        4 611Lessons Learnt:“ Scale-down” your final manufacturing processNutrient requirements/critical processParameters can be screened and determined at bench-scaleEngineering effects: Identify KLa (agitation/aeration)range achieved at manufacturing scaleScale-up process to small-scale bioreactor (taking into account large-scale constraints)Test process at large scale12Upstream recovery optimization for bacterial proteins713Considerations in protein recovery for Intracellular proteinsE.coli cells grow in fermentor → Cell broth Separation→ Homogenize → Clarification → Purification•Parameters affecting Homogenization• Pressure •Number of passes• Cell concentration •Process fluid variables (viscosity, temperature)•Cell broth Separation / Clarification•Clarification method (filtration/ centrifugation)•Processing time, unit operation and product recovery•Process fluid variables (cell and protein characteristics, viscosity)14Gel Polarization (Darcy’s law) for X-flow MicrofiltrationGelLayerRgMembrane RmSolution flowCbBoundarylayerCg Flux JFlux α (Driving Force)/(resistance)Rg = gel resistance, Rm = membrane resistance, J= flux, ΔPTM = Transmembrane pressure)R(RΔP JmgTM+∝μ815Parameters affecting the build-up of the gel layer (Rg)TMPAc22αμ=gRSpecific cake resistance (α)Membrane (A)Transmembrane Pressure (PTM)Crossflow RateCell concentration (c)Viscosity (μ)Filtration flux (LMH)Product recovery(product permeation)16Clarification efficiency using cross-flow filtration020406080100120protein releasedafterhomogenization  proteinclarified duringconcentration proteinclarified duringdiafiltration  proteinretained Amount of protein recovered %917Things to consider:  Irreversible fouling during concentration/Diafiltration using E.coli cells051015202530350 2 4 6 8 10 12StepFlux (LMH)Cell concentration/cell broth separationg Diafiltration using buffer• Subsequent diafiltration doesn’t restore flux due to lysis of cells onto the membrane• Age of the fermentation cells does play a role in filtration• Need to stop fermentation before death phaseCell concentration Diafiltration)R(RΔP JmgTM+∝μ18Problems associated using cross-flow filtration for product clarificationProtein characteristics may affect adhesion to membrane and thereby decrease recoverySurface charge densities ( i.e pH, solution ionic strength) (Baruah & Belfort, Biotech Bioeng., Vol.87, 2004)Cell surface chemistry– Cell surface adhesion causing membrane foulingNature of the proteinStatic Filtration1019Difficulty in static filtrationHigh product recovery at 20 L scaleNot scalable at the large scale ( 200 L and beyond)Difficult in handlingDisposable costs may be high when scaled to manufacturing scaleNot as robust as cross-flow filtration where the process is dependent on the upstream fermentation and homogenization conditionsDirect Adsorption method20. .. . .. ...... . .... . .Washing and Elution Buffer portsprotein binds Big BeadsHomogenate debris and proteins80 µm filter meshHomogenateBinding and washing steps of the batch clarification process1. Bind homogenate with beads2. Wash beads with buffer3. Wash beads with low-salt buffer4. Elute protein1121Large-scale process results Protein 1 Protein 2 Lot# 1 2 1 2 Amount protein produced at fermentation (%) 100 100 100 100 % Clarification recovery 90% 90% 75% 73%  1      2     3      4     5      6Lane 1: markerLane 2: Homogenized supernatant Lane 3: Unbound debrisLane 4: Wash with low salt bufferLane 5 : Eluted proteinLane 6: High salt strip 22Future of ClarificationMetal Affinity/ Ion Exchange MembranesAvailable in strong Anionic and Cationic forms, as well as in a metal ion complex formDisposable (single-use)Increase availability of affinity ligands will help increase its useMulti-modal adsorbents More selectivity (i.e. directly bind proteins from high ionic strength feedstocks) and higher capacityStreamline Direct CST – ion exchanger (Biotech BioEng Vol.94, no.6, 2006,1155-1163)Used for Expanded bed chromatographyPossibility remains for stirred tank applications1223AcknowledgmentsTony D’AmoreXuefeng Yu Yan-Ping YangTao Zhu Tao Yuan Kimberly Matheson Mathilde LucianiTammy Conrad Ernst BraendliCathy Hulcio Bruce CarpickIsabel Metelo-Almeida Davinder ChawlaJoyce Ni Estela JimenezPatricia Gomes Melanie MichelbergerKareen Lawson Chrysta Mayhew

Trouble-Shooting Fermentation and Primary Recovery Manufacturing Issues in Order to Optimize Antigen Expression for the Vaccine Business

https://dc.engconfintl.org/cgi/viewcontent.cgi?article=1013&amp;context=vaccine

Trouble-Shooting Fermentation and Primary Recovery Manufacturing Issues in Order to Optimize Antigen Expression for the Vaccine Business

Abstract

Similar works

Full text

Available Versions

Engineering Conferences International