Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
•
0 likes•489 views
The document discusses strategies and considerations for the scalable purification of plasmid DNA for use in vaccine manufacturing. It covers the key upstream steps of cell harvest, lysis, and clarification. Cell harvest is typically done using centrifugation or tangential flow filtration. Lysis is usually an alkaline lysis process that must be carefully optimized to avoid damaging the plasmid DNA. Clarification can involve various pretreatment and filtration steps to reduce impurities before downstream purification. No single platform solution currently exists and processes vary between manufacturers.
Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
Similar to Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing (20)
More from Merck Life Sciences
More from Merck Life Sciences (20)
Recently uploaded
Recently uploaded (20)
Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
- 1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing Thomas Parker, Global Focal Point – Vaccines and Viral Therapies, Technology Management 29 October 2020
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. 1. Plasmid Production Overview 2. Cell Harvest, Lysis, and Clarification 3. Purification Considerations 4. Tangential Flow Filtration 5. Chromatography 6. Sterile Filtration 7. Summary Clarification Sterile filtrationFermentation Alkaline Lysis ChromatographyTangential flow filtration
- 5. • Circular double helix DNA molecules, naturally found in bacteria, intracellular replicated • Molecular characteristics: − Large size 1.5 – 150 kb − Poly anion, highly negatively charged − Sensitive to mechanical stress − Various topological forms ccc form I: covalentely closed circles, supercoiled, fully intact, wound around itself oc form: one strand nicked/broken, less compact, totally relaxed linear form: both strands broken, free ends Introduction What are Plasmids? Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing5 MW Size Plasmid 4 MD* 200 nm** mAB 0.2 MD* 10 nm** * Mega Dalton (6 kb Plasmid) ** Dynamic range Supercoiled plasmid is recognized by FDA as the most therapeutically effective Carsten Voß, 2007
- 6. Importance of Plasmid DNA The key-role of pDNA for viral vector and vaccine production Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing6 Plasmid DNA (Vector) Therapeutic transgene, structural genes CAGR 18% Other 14% Adeno 15% LV 49% pDNA 22% Viral Vectors Clinical trials by vector types Bioprocess International, December 17, 2019 DNA vaccines ▪ - prophylactic use, e.g. infections - therapeutic applications, e.g. cancer, allergy mRNA vaccines
- 7. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing7 ✓ Capable of inducing strong immune response ✓ Require appropriate formulation (buffers, stabilizers, and inorganic or organic matrices) for protection from degradation and enable efficient transfer and delivery Plasmid DNA as a Vaccine Basic Mechanism Delivery methods: lipofection, gene-gun (DNA coated gold particle, micro-particles/capsules) 1) Viral protein gene transferred into a plasmid 2) Plasmid purified and delivered to patient skin or muscle cells 3) Patient cells produce the viral protein, inducing an immune response
- 8. Advantages Challenges Downsides Characteristics Plasmid DNA as a Vaccine Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing8 • Simple design • Easy to manipulate/optimize • Production: Reproducible, rapid, scalable • Requires only BL1 safety level • No significant adverse events • Temperature stable, long shelf life • Similar immune response as other platforms • Removal of impurities and non desired pDNA forms need careful process design • High concentration needed (mg level) • Delivery and protection of DNA from degradation • Poor gene transfer efficiency • Estimated that for every 1000 plasmid molecules only one reaches the cell and is expressed. • Low immunogenic potential of bacterial DNA • Possible insertional mutagenesis
- 9. Importance of Plasmid DNA Vaccines Fighting COVID 19 Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing9 Four pDNA based vaccines in clinical evaluation 12 pDNA vaccines in preclinical evaluation WHO report, 02 October 2020 COVID-19 Vaccine developer/manufacturer Type of candidate vaccine Number of doses Timing of doses Route of Administration Clinical Stage Inovio Pharmaceuticals/ International Vaccine Institute DNA plasmid vaccine with electroporation 2 0, 28 days Intradermal I/II Osaka University/ AnGes/ Takara Bio DNA plasmid vaccine + Adjuvant 2 0, 14 days intramuscular I/II Cadila Healthcare Limited DNA plasmid vaccine 3 0, 28, 56 days Intradermal I/II Genexine Consortium DNA Vaccine (GX-19) 2 0, 28 days Intramuscular I/II
- 10. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing10 Plasmid Production Unique challenges of pDNA purification Adequate technologies and application expertise for providing solutions for Plasmid manufacturing needed. • Low productivity of microbial fermentation due to copy number limitations in the relevant strains. • Similarity of product and contaminants (gDNA, Endotoxin, RNA, Plasmid isoforms) leads to low resolution separation. • Feed often highly viscous, complicating downstream processing. • Low flowrate needed for chromatography • Difficult to achieve desired concentrations at the final TFF step. • Shear sensitivity. • Need to eliminate multiple undesirable components from the process. • Lack of platform process and integrated solutions. From: Xenopoulos, A. and Pattnaik, P. (2014), Expert Rev. Vaccines 13, 1537-1551
- 11. Unit Operation BI LONZA Cobra bio Merck & Co. Inovio GSK GEHC BIA Plasmid pRZ-hMCP1 pTX0161 pVAX1 Pegfp-N1 Host K12 JM108 Clean Genome DH1 DH1 OB DH1 DH5a Cell Harvest Continuous centrifugation centrifugation centrifugation TFF (0.45 UM) Cell lysis Continuous alkaline lysis Continuous alkaline lysis Alkaline lysis with RNase Heat lysis with Lysozyme Continuous alkaline lysis Alkaline lysis Alkaline lysis Alkaline lysis Primary Clarification Flocculation Woven nylon bag filter Bubble column with 3/0.1 um depth filters Centrifugation>membr ane (0.45/0.2 um) concentration Ultrafiltration (hollow fiber) TFF Ultrafiltration (hollow fiber, 100 kD) Contaminant Precipitation Ammonium sulphate Chemical CTAB Fractional Calcium chloride Calcium chloride Calcium chloride Sec.Clarification filtration Depth filter Filtration with DE Depth filtration 1.2/0.45/0.2 um membranes Concentration Hollow fiber TFF TFF 100 KD Chromatography HIC-Butyl 650 M, B/E. AEX: Fractogel DEAE B/E SEC-Sepharose 6FF HIC-hexyl 650 C F/T. HIC-Butyl 650 S, B/E. SEC-Sepharose 6FF AEX; DEAE Expanded bed SEC S500HR AEX : Q Membrane, B/E HIC: Resin B/E AEX: Fractogel TMAE SEC: Sepharose 6FF media Thiophilic aromatic plasmid Select B/E AEX: Source 3 0Q, B/E AEX: DEAE Monolith HIC: monolith Concentration TFF TFF (Spiral device, 20 kD) Ethanol Precipitation and TFF,RC, 100 Kda TFF-PES, 50 kD Final filtration PVDF 0.22 PVDF 0.22 PES No industry platform solution has been accepted Diversity of existing process schemes Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing 11 Large-Scale Production Processes for Plasmid DNA From: Xenopoulos, A. and Pattnaik, P. (2014), Expert Rev. Vaccines 13, 1537-1551
- 12. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing12 Generic Production Process for Plasmid DNA Plasmid Overview Fermentation Harvest Lysis Alkaline Lysis or Heat, Mechanical, Enzymatic Clarification/Primary recovery Purification Capture, Polishing Sterile Filtration Centrifugation Filtration Flocculation, ATPS, Selective Precipitation (PEG, salt detergent), Expanded Bed Optional Feed pre-treatment Centrifugation TFF or hollow fiber TFF Concentration, Diafiltration AEX -> HIC HIC -> AEX SEC -> HIC Affinity 1-2 Chromatography steps By CaCl2-Preciptitation or RNAse Concentration Buffer exchange by TFF Adjustment for Capture RNA removal
- 13. Integrated strength from harvest to final fill Our Capabilities for pDNA Purification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing13 Fermentation Clarification Purification with Chromatography(1-2 steps) Concentration and Diafiltration (UF/DF) Final filtrationStorage Concentration and diafiltration (UF/DF) Thaw cells Cell harvest Cell lysis Prostak®/Pellicon® Mobius® Mixer Clarisolve®/Millistak+® Pellicon® 2 100 or 300 kDaFractogel® DEAE Fractogel® DMAE Natrix® Q Pellicon® 2 100 kDaMillipore Express® Validation and Testing Services Mobius® Single Use Solutions
- 14. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing14 Poll Question #1
- 16. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing16 Plasmids are produced intracellularly • Multiple unit operations are needed before downstream purification Cells harvest by centrifugation or tangential follow filtration (TFF) microfiltration Cell lysis by physical or chemical treatment Clarification by depth filtrating or centrifugation Harvest overview Cell Harvest, Lysis, & Clarification
- 17. • Plasmids are produced intracellularly in a batch and/or fed-batch using E. coli host • Minimal mineral salt media commonly used - low price, autoclavable • Productivity and specific productivity (amount of pDNA per unit of cell mass) are proportional to the final cell density in a fermenter • Fast production (~1 day) relative to mammalian cell culture Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing17 Fermentation overview and output Cell Harvest, Lysis, & Clarification Defining the harvest input: • Wide range of volume possible • 30-200L common, but 6000L proven • OD600nm of 100-120 • 100-250 mg/L, yields up to 2 g/L reported • Specific yield of 50 mg pDNA/g DCW, up to 75% of total DNA is Plasmid
- 18. Microfiltration Tangential Flow Filtration (MF-TFF) High solids count favor open-channel formats Flat-sheet TFF devices work well in this application − Linear scalability − Wide range of formats and installation options Centrifugation Traditional approach is cost effective at large scale (>500L) or very small scale with swinging bucket (<5L) Special attention is needed due to high shear generation in large scale centrifugation Centrifugation vs Tangential Flow Filtration Cell Harvest, Lysis, & Clarification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing18 Parameters Value Device Prostak™ or Pellicon® 2 with Durapore® 0.1, 0.22 µm, or 0.45 µm Pellicon® 2 Biomax® 1000 kDa, V screen Pellicon® 2 Ultracel® 1000 kDa, V screen Volumetric loading 10 – 60 L/m2 Feed flow 7 - 9 L/min/m2 TMP < 0.5 bar Average flux 20 - 30 LMH Volumetric concentration factor 2 to 5 Diafiltration volumes 3 to 5 MF-TFF Typical Operating Parameters
- 19. Principle • Alkaline condition plus detergent solubilizes the cell walls and the alkaline environment denatures genomic DNA. • Sodium dodecyl sulfate and Triton X-100 common • Reaction neutralized with acid after short incubation Challenges • Both alkaline and shear from mixing will damage supercoiled plasmid DNA • Sudden increase of viscosity will impact mixing efficiency Solutions • Optimize mix speed, incubation time, and chemical concentrations • Mobius® single use mixers Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing19 Key considerations for lysis Cell Harvest, Lysis, & Clarification • Lysis affects pDNA quality and impurity level, directly impacting subsequent down stream steps. • Continuous lysis approach may be needed for large scales
- 20. Aims at reducing lysate impurity level for improving clarification and purification by use of: • PEG/PEI for selective precipitation of gDNA • Divalent cations (2M Ca2+) for precipitation of RNA • RNAse for enzymatic digestion RNA (cost and regulatory concerns) • Anionic detergent for lowering endotoxin levels Watch Outs • Changes of lysate pH, salt conc. conductivity, viscosity • Additives might not be compatible with subsequent chromatography capture and require additional TFF for buffer exchange • Additives might be difficult to remove Cell Harvest, Lysis & Clarification Optional Pretreatment Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing20 Pretreatment impacts clarification performance and can affect subsequent chromatography capture step. Ca+2 mediated RNA precipitation AEX Capture Clarification AEX Capture vs. LysateLysate TFF Clarification
- 21. • Lysis output can be challenging for clarification • Complex mixture: • Large particles, cell debris, plasmid DNA, genomic DNA, RNA and host cell proteins • Very high solids load • Highly viscous • Two-phase separation • Froth phase (top) – cell debris and gDNA • Lysate phase (bottom) – pDNA, RNA, HCP • Manufacturers utilize a wide range of pre- clarification methods to enhance unit operation efficiency Cell Harvest, Lysis & Clarification No platform solution for lysis and conditioning Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing21 Internal Data 25% 10% 10% 15% 25% 15% Pre-clarification conditioning Sieving/Filtration Centrifugation Polymer Flocculation Natural Floating Separation No Pre-clarification Proprietary
- 22. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing22 Filtration based clarification preferred up to 500L+ fermentation scale Modern approach, use size exclusion and absorption mechanisms Can be used in series Attention: positive charged filtration aid may interact with plasmid Centrifugation vs Filtration Cell Harvest, Lysis & Clarification Traditional approach, which is capable of handling high %solids May require secondary clarification Attention: high shear rate can damage the structure of supercoiled plasmid DNA Centrifugation Normal flow filtration
- 23. Depth filters at a glance Cell Harvest, Lysis & Clarification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing23
- 24. Depth filters at a glance Cell Harvest, Lysis & Clarification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing24 Media type and density impact adsorptive properties Lower adsorption Higher adsorption
- 25. Capacity range for non-conditioned lysateCapacity range for pre-conditioned lysate Data summary for clarification using depth filtration Cell Harvest, Lysis & Clarification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing25 0 50 100 150 200 250 300 350 Clarisolve 20MS Clarisolve 40MS Clarisolve 60 HX Millistak+ D0HC Millistak+ C0HC Capacity(L/sqm) 0 50 100 150 200 250 300 350 400 450 Clarisolve 40MS Clarisolve 60HX Millistak+ D0HC Millistak+ C0HC Millistak+ D0SP Millistak+ CE50 Capacity(L/sqm) High yields at >90% • Millistak+® D0HC or C0HC: >80% • Millistak+® CE: >90% • Clarisolve®: >90% • Salt chase recommended • Bioburden reduction membrane capacity post depth filtration varies – >500L/m2 typical
- 26. Case Study #1 Clarisolve® 60HX Alkaline lysis + CaCl2 precipitation − gDNA and RNA being removed Case Study #2 Millistak+® D0HC Alkaline lysis without pretreatment − gDNA removed Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing26 For 500L, Case #1 needs two Process Scale Pod Racks and Case #2 needs one Case studies Cell Harvest, Lysis & Clarification Filter Flux (LMH) Volumetric Throughput at 15psi dP (L/m2) Installation for 500L with 1.5x safety factor Case #1 Clarisolve® 60HX 200 160 14 x 0.55m2 Case #2 Millistak+® D0HC 100 150 8 x 1.1m2
- 28. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing28 Poll Question #2
- 29. General Considerations Plasmid Purification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing29 Polish Chromatograph HIC AEX Ultrafiltration/ Diafiltration Final filtrationFill in assemblies Optional Ultrafiltration Capture Chromatography AEX HIC Plasmid purification requires a combination of more than one unit operation. Tangential Flow Filtration (TFF) at 1-2 steps • Can remove RNA, small sized genomic DNA and protein • Essential for concentration to reduce loading time and complete buffer exchange prior chromatography steps • After clarification and before chromatography Chromatography • Selectively separates supercoiled Plasmid from oc-/linear isoforms and residual impurities (host cell protein and nucleic acid, endotoxin) by charge, size or hydrophobicity • Anion Exchange (AEX) and Hydrophobic interaction (HIC) typically used • Sometimes size exclusion (SEC) for RNA removal Sterile filtration to ensure process safety or Biggest Challenge: Separation of Plasmid isoforms
- 30. Plasmid TFF
- 31. Ultrafiltration and Diafiltration Overview TFF 30, 100 or 300 kD membranes can be used at two different steps First TFF step, after clarification and before chromatography − Remove residual impurities and protein − Concentrate plasmid to reduce loading time and buffer exchange for chromatography steps − 4-25x concentration and 5-10 diavolumes Second TFF step, after chromatography and before sterile filtration of drug substance − Achieve final concentration and exchange into formulation buffer − 10x concentration and ~10 diavolumes typical 31 1 2 Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
- 32. Two-pump control adds operational consistency TFF Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing32 Use 2-pump control for 100kD and higher When low TMP causes Critical Permeate Flux, 2-pump control is recommended TMP control has difficulties maintaining a stable system at low TMP (~5psi or less) Very tight window of operation below ~5psi TMP Deviation from TMP setpoint could result in permeate flux near or above critical flux 2-pump control allows consistent operation Ensures permeate flux does not approach critical flux Minimize fouling Consistent impurity clearance and yield 0 2 4 6 8 10 12 14 30kD 100kD 300kD 1000kD TMP(psi) TMP @ Critical Flux 4LMM 5.5LMM 7LMM
- 33. Parameter Value Feed Flux (LMM) 4 or less Permeate Flux (LMH) – for two pump 20-50 TMP (psi) – for TFF control <10 Loading (L/m2) 70-140 Concentration Factor 5x-50x TFF Recommended operating parameters Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing33 Watch out for: • High viscosity • Shear D.R. Latulippe, A.L. Zydney / Journal of Colloid and Interface Science 357 (2011) 548–553 Operating parameters can change retention properties:
- 35. Common Approaches at Large Scale Chromatography 35 Anion Exchange Chromatography (AEX) • Well suited for binding polyanionic DNA • Applicable for capture, intermediate and polishing • Weak AEX resins (DEAE, DMAE) give highest recovery and selective impurity removal o Resins with higher ionic capacity can show recovery issues • Charge similarity of Plasmid and impurities poses a challenge • Capable of separating plasmid from proteins, RNA and gDNA and removing Endotoxin • Separation of Plasmid isoforms difficult Hydrophobic Interaction Chromatography (HIC) • Works by salt promoted binding (≈ 2.5 M NH4SO4) • Supercoiled pDNA is less hydrophobic than RNA, oc- and linear- Plasmid forms and denatured gDNA • Conveniently used for intermediate purification after AEX capture due to high salt AEX eluate pool o Can also be used for primary capture Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
- 36. Membrane Chromatography • Improved hydraulic performance due to large convective pores and low bed height • Large accessible surface for Plasmids enabling for improved binding capacity (5 - 10 mg/mL) at very short residence times < 0.2 min • High productivity due to short cycling time • Single use format, principally scalable Two technologies to consider Chromatography Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing36 Resin Chromatography • Low binding capacity due to large Plasmid size limiting diffusion into beads restricting binding to the resin surface only • High pressure drops due to elevated viscosity feed stream resulting in flow limitations and long processing times (2-8min RT typical) • Re-use needed for economic feasibility • Flexible installation – pack as much as needed • Generally better selectivity than membranes
- 37. Solutions Chromatography Products for Plasmid Purification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing37 Single-use solution providing superior productivity and flexibility to meet the challenges of multi-product / multi-purpose manufacturing of Plasmids Ideally suited for the capture of small and mid-sized Plasmids (<20kbp) Versatile solution offering unique selectivity and best in class capacity Efficient and cost-effective operation, however, require optimal column sizing (in order to accomplish rapid cycling and realize low hardware costs) Fractogel® DEAE/DMAE (wAEX)Natrix® Q
- 38. • DMAE and DEAE ligands exhibit unique binding selectivity • Moderate binding capacity of 2 – 4 mg/mL • High yield: 80% to 95% of ccc-form • Removal of residual Endotoxin • Good resolution due to moderate bead size (d50: 48 - 60 µm) • Moderate flow: 4-8 min residence time Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing38 ✓ High surface area of tentacle resin offer improved capacity relative to conventional anion exchange resins due to tentacles ✓ Proven resins for reliable Plasmid purification Plasmid Purification Fractogel® DEAE and DMAE Chromatography Resin Ideal for intermediate purification / polishing but still frequently used for capture.
- 39. Optimal amount of salt supplement: • depends on resin type • varies with initial lysate conditions (protocol specific lysis buffer) • gives higher binding capacity and improved Plasmid eluate purity • should be determined for each specific lysate condition by screening Optimal Conditions for direct AEX Capture Fractogel® DEAE and DMAE Chromatography Resin Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing39 0.0 1.0 2.0 3.0 4.0 5.0 0 100 200 300 400 500 PlasmidSBC(mg/mL) NaCl supplement (mM) FG DEAE (M) FG DMAE (M) 1 Marker 2 0 mM NaCl 3 100 mM NaCl 4 120 mM NaCl 5 150 mM NaCl Purity of Fractogel® DEAE capture eluates from lysates with varying NaCl supplement Binding of Plasmid from lysate Salt supplementation of lysate is a simple and efficient method to prevent RNA binding and reduce interference with plasmid binding on AEX
- 40. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing40 Commercial Plasmid Production Preferred Chromatography Approach • Lysate with 120 mM NaCL supplement • Capacity of ~2.5 mg Plasmid / mL CV • > 95% RNA in flow through • > 95% pDNA purity in eluate • > 90% pDNA yield by salt step gradient • Pre-elution wash step including alcohols (e.g. 2-Propanol) required for improved endotoxin removal AEX Capture Fractogel® DEAE or Fractogel ® DMAE Removes RNA, endotoxin, gDNA, host cell proteinSupplementation of NaCl HIC Polishing Separates Plasmid isoforms Clarified lysate pH 5.0, 67 mS/cm, 1M K-acetate adjust to 2.4M NH4SO4 Plasmid Capture with Fractogel® DEAE (M) Salt supplementation for AEX direct capture eliminates need for pre-treatment and TFF
- 41. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing41 Alternative Chromatography Approach Commercial Plasmid Production pDNA binding to Fractogel® AEX resins tolerates presence of elevated concentration of (NH4)2SO4 Precipitate formation upon lysate adjustment to HIC conditions is an issue, involves additional effort for clarification AEX Polishing Fractogel® DEAE or Fractogel ® DMAE Removes residual RNA, endotoxin, gDNA, host cell protein HIC Capture Clarified lysate pH 5.0, 67 mS/cm, 1M K-acetate Adjustment to HIC binding conditions *Data using purified pDNA, 4.7kbp
- 42. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing42 Natrix® Q Chromatography Membrane in Single-Use Format High-throughput Capture Plasmid Application • High capacity of 5-10 mg/mL at only 0.1 min residence time possible with small (5.7 kb) and mid-sized Plasmids (13kb) • Yield: ≥80 % of ccc-form • > 95 % RNA removal • Short cycle time: 35 min • Caustic stability enables for efficient cleaning (1M NaOH +2M NaCl) • Re-use in rapid-cycling operation mode allows for cost reduction • Narrow pore size poses challenge for larger Plasmids Material Properties • Composite membrane • High mechanical strength and hydraulic permeability • Nominal pore size: 0.4 µm • Permits high flow • Recommended 10MV/min, (6 sec. residence), range of 5-25 MV/min
- 43. Performance Overview - Examples Plasmid Purification with Natrix® Q Chromatography Membrane Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing43 Feed used: • Original E. coli lysates from alkaline lysis, clarified by centrifugation/depth filtration, supplemented with optimal NaCl to eliminate RNA interference, pH 5.0, finally 0.22 µm filtered • Plasmids ranging from small to large size • Varying initial pDNA purity ranging from 0.7% - 4% For Plasmids with 20 kb size or larger, a collapse of membrane permeability was encountered with loadings greater than 2 mg/mL. Plasmid Size Plasmid Titer (µg/mL) Initial pDNA Purity (A260 based) Initial Lysate Conductivity (mS/cm) Optimal NaCl Supplement (mM) Final Lysate Conductivity (mS/cm) Residence Time (min) Operating Capacity (mg/mL MV) Yield (%) pDNA Eluate Purity (A260 based) 5.7 kb 45 4.0 % 69 160 82 0.1 10 ≥ 80 ≥ 80 % pDNA 13 kb 33 2.4 % 72 130 82 0.1 4 ≥ 77 ≥ 90 % pDNA 20 kb 25 0.7 % 79 100 86 0.2 1 ≥ 65 ≥ 62 % pDNA
- 44. Performance Overview - Examples Chromatography Products for Plasmid Purification Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing44 Recommended Process Step Resin / Membrane Adsorber Dynamic Capacity (mg/mL) Cycle Time (min) Residence Time (min) RNA Remov al Yield ccc- Form Purity (A260 based) High- throughput capture Natrix® Q 10 0.5 h 0.1 – 0.03 >95 % ≥80 % >80 % pDNA Polishing Purification Fractogel® DEAE (M) 2.5 3 h 4 >95 % ≥80 % >95 % pDNA Fractogel® DMAE (M) 3 3 h 4 >95 % ≥95 % >95 % pDNA Feed used: Original E. coli lysate, clarified by centrifugation and subsequent depth filtration, directly supplemented with NaCl (120 – 250 mM, depending on resin or membrane type) to eliminate RNA interference, pH 5.0, 74 – 82 mS/cm pDNA size 5.7 kbp, pDNA titer 45 µg/mL * 10 cm bed height
- 46. Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing46 Sterile Filtration Overview of pDNA Sterile Filtration Recommendations ✓Millipore Express® SHC (PES) over Durapore® 0.22um (PVDF) ✓Optimize conditions for best yield and capacity Attributes Parameters Issues Sterility assurance Membrane pore size Large size of pDNA Particulate reduction Membrane chemistry Shear sensitivity of pDNA Filtration capacity and flux Driving force Viscosity of pDNA solution (especially at high concentration, >5mg/mL) pDNA yield Formulation Bacterial retention for adjuvanted pDNA solutions Filtration endpoint
- 47. • Internal data and literature search show that pDNA size, purity, and concentration have significant effect on sterile filtration • Effective size of pDNA can be altered by changing the ionic concentration of background buffer solution • Yield issues (<90%) are common with >10kbp plasmid and can be significant (<80%) with >20kbp Sterile Filtration pDNA Sterile Filtration Optimization Suggestions Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing47 Optimization Parameter Yield Capacity Product integrity Salt concentration X X Supercoiled pDNA content (purity) X X Filtration endpoint X Membrane type – PVDF or PES X – PES X - PES pDNA concentration X X Feed flux or pressure X doi:10.1002/bit.21575
- 48. Summary
- 49. Clarification Sterile filtrationFermentation Alkaline Lysis ChromatographyTangential flow filtration Integrated solutions for pDNA purification Capabilities Summary 49 Lysis – Mobius® Mix - extract DNA, denature gDNA, potentially precipitate or degrade RNA Clarification – Clarisolve® 60HX or Millistak+® D0xx - remove cell debris and precipitated impurities (gDNA, RNA) TFF – Pellicon® 30kD, 100kD, 300kD – concentrate and remove small impurities Chromatography – Natrix® Q or Fractogel® DEAE or DMAE – remove RNA, gDNA, HCP, and endotoxin Sterile Filtration – Millipore Express® SHC – sterility assurance Data driven strategies and considerations for scalable purification of Plasmid DNA for use in vaccine manufacturing
- 50. Acknowledgements • Andreas Stein • Andre Kiesewetter • Niddhivinayak Mishra • Youness Cherradi • Josselyn Haas Thomas Parker thomas.parker@milliporesigma.com
- 51. Thank You Merck, Millipore, Biomax, Bioreliance , Clarisolve, Durapore, Fractogel, Millistak, Millistak+, Millipore Express, Mobius, Natrix, Pellicon, Prostak, Ultracel and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Brought to you by Merck KGaA, Darmstadt, Germany and its portfolio brands: For more information about vaccine production, please visit: sigmaaldrich.com/vaccines