Strategies for the purification of high titre, high volume mammalian cell culture batches Martin P. Smith. LONZA Biologics plc, 228 Bath Road, Slough, SL1 4DX. Presented at, Recovery & Purification. BioProcess International European Conference and Exhibition. Berlin, April 2005.
Overview Review of Fermentation process productivity increases Current status Challenges presented by high titre, high volume batches Strategies available for dealing with high-titre, high-volume batches Opportunities within current downstream processing technologies Chromatography Buffer supply Ultrafiltration Importance of timely process development Simple tools & methods for process development and scale-up 15/04/2005 / 2
LONZA Biologics manufacturing capacity LONZA currently operates monoclonal antibody facilities in both the US and UK UK Manufacturing-focused on rapid supply of clinical phase material Various disposable units (Wave ) 2x2000L ALF 2x200L ALF Additional capacity planning exercise underway US Manufacturing-focused on large-scale late phase & in-market supply 2x5000L ALF 1x2000L ALF 2x1500L perfusion (dedicated) 3x20,000L STR growing to 4 in 2006 15/04/2005 / 3
Biopharmaceutical Production Challenges Increased demand for cgmp manufacturing capacity at all scales. Increase in product approvals and marketing extensions driving a demand for large scale capacity (>5000L reactor volume) Strong demand for small scale capacity driven by full customer pipelines and need for early phase toxicology & clinical study material. Cell Line Construction cgmp Cell Banking Scale-Up & Process Pilot Prod. Development cgmp Manufacture Growing political and ethical pressures to control and reduce drug development time-to-market and production costs. 15/04/2005 / 4
Advances in Mammalian Cell Culture Process Productivity Product Accumulation (mg/l) 6000 5000 4000 3000 2000 1000 Iteration 1 (22H11) Iteration 2 (22H11) Iteration 2 (LB01) Iteration 3 (LB01) Iteration 4 (LB01) Iteration 5 (LB01) Iteration 5 (CY01) 0 5 10 15 20 25 Fermentation Time (days) Fermentation productivity is rising RAPIDLY. Advances in cell culture realising higher titres Already experiencing 4-5g/L batches after 14days in cgmp reactors 15/04/2005 / 5
Current Capacity Crunch Much of today s production capacity for in-market supply designed for low to modest productivities of monoclonal antibodies Resulted in requirement for multiple large volume reactors (4-6 x 12-20kL) for single purification trains. In-house BioPharma capacity can be optimised for specific medium to low risk products Contract Manufacturing capacity must remain reflective of wider industry status to capture current and near term business Rate of technology development in mammalian expression outpacing design-and-build timelines for new facilities Industry requires low risk, low capital and immediate solutions to purifying high productivity batches across all scales. 15/04/2005 / 6
Identifying bottlenecks in downstream processing Situation for CMO s is more complicated than single product facilities Business risk managed through large customer base Each product differs widely despite generic/platform technologies Fermentation productivities column capacities, number of column steps Differing degrees of process intensification Increasing titres poses significant challenges for: Throughput (speed of purification) Economics (Batch cost, cost of goods) 15/04/2005 / 7
Strategies to alleviate downstream processing bottlenecks 1. Invest in development of novel technologies Membrane Adsorbers (PnA capture and contaminant removal) Mimetic ligands Monolithic chromatography supports UV for virus inactivation 2. Invest in development of conventional technologies Centrifugation 2 phase separations Solvent extraction Precipitation Crystallisation 15/04/2005 / 8
Factors complicating selection of strategy 5g/L titres are here now! Novel technology and re-development of conventional technologies may not help in the near term Both involve significant investment of capital to refit existing facilities Need to sell technology improvements Internally across company Externally to clients Technology solutions must be scalable and generic 15/04/2005 / 9
Further Strategies to alleviate downstream processing bottlenecks 3. Maximise utilisation of existing assets. Process Optimisation Downstream Yield Improvement Programs Process Intensification Reduce processing time Eliminate non-processing time Reduce start-up and turn around/change over times 4. Drive process excellence across business LEAN/Six Sigma/Quality systems 15/04/2005 / 10
Typical Development Program Construct expression vector Construct Cell Line Develop Manufacturing Process Non GMP Pilot Run GMP Production Material Supply Typical Quantities Cell culture supernatants Protein A purified product Research grade fully purified product Material for toxicology studies, reference standard, stability studies 10-50mls 10-50mgs 0.1-1g 10-100g Clinical Trial Supply 0.1kg to multi-kgs Speed to Clinic for Early Phase material ensured through use of generic processes Fast Track Downstream development characterised by aggressive timelines 15/04/2005 / 11
Focus of presentation Bottlenecks and Production Economics change markedly as a function of scale. Key bottlenecks and areas for cost/throughput optimisation: Protein A Capture Buffer Supply Ultrafiltration Optimisation 15/04/2005 / 12
Effect of fermentation titre on intermediate process volumes Example of 2000L reactor titre increase. For same purification process: 1g/L Chromatography elution tank volume constraints Ultrafiltration tank volume capacity 2500 Virus filtration throughput constraints 2000 1500 1000 Increased demand for buffers 500 0 Process Volume vs Concentration 30 25 20 15 10 5 0 15/04/2005 / 13 Harvest Process Vol (L) CCS PnA Eluate VI Eluate UF 1 Product Q FT UF 2 Product VRF Product SP Product UF 3 Product Process conc (g/l) 2500 2000 1500 1000 500 0 L g/l Process Volume vs Concentration 30 25 20 15 10 5 0 Harvest Process Vol (L) CCS PnA Eluate VI Eluate UF 1 Product Q FT UF 2 Product VRF Product SP Product UF 3 Product Process conc (g/l) L g/l 5g/L
Process Development Case Study 1 Reducing Buffer loading during downstream processing 2000L reactor titre increase. Total Buffer Demand (L) 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 0 Effect of Fermentation Titre on Buffer Demand 1 2 3 4 5 Fermentation Titre (g/l) 10-15 % of buffer Demand is derived from Protein A step 15/04/2005 / 14
Large Scale Build Out Facility, Buffer Hold-Upper Level 15/04/2005 / 15
Buffer Concentrates and In-Line Dilution Possible to minimise Buffer Make-up and Hold requirements through in-line dilution Example: Protein A Equilibration, Post Load Wash Buffer 50mM Glycine Glycinate, 250mM Sodium Chloride, ph 8.0 Cl - H 3 N + COOH C H - COO pka =2.35 pka =9.78 1 H 3 N + 2 C H H 2 N COO - Na + C H H Glycine Hydrochloride (AA +1 ) H Isoelectric Glycine (free base) (AA 0 ) Implementing in-line dilution requires In-depth understand of buffer chemistry Solubility and stability of buffer concentrates Effect of temperature on ph and ms/cm Appropriate equipment for in-line dilution H Sodium Glycinate (AA -1 ) 15/04/2005 / 16
Protein A Equilibration Buffer Chemistry Effect of temperature on atstrength buffer ph & ms/cm Static dilution of buffer concentrates ph (-) 8.30 8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 5 10 15 20 25 30 Temperature ( C) 26 24 22 20 18 16 Conductivity (ms/cm) ph (-) 8.30 8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 0 0 2 4 6 8 10 Concentrate Strength (-) 160 140 120 100 80 60 40 20 Conductivity (ms/cm) Establish process tolerance for ph and ms/cm (as wide as possible) Check ability to control concentrate and WFI temperature Ensure buffer concentrate is soluble at required strength Check Salt Strength at ph for compatibility with MOC s 15/04/2005 / 17
Demonstrating potential for in-line dilution % B 100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 80 100 120 Volumetric Flowrate (L/h) 5x Dilution 10x Dilution 15/04/2005 / 18
10x Concentrate dilution at 20L/h ms/cm 150 100 CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark ph 9.0 8.0 7.0 6.0 % B 100 90 80 70 60 50 40 30 20 10 0 5x Dilution 10x Dilution 0 20 40 60 80 100 120 50 Water rinse 1x baseline 10x_concentrate Feedback enabled 5.0 4.0 3.0 Volumetric Flowrate (L/h) 0 0.0 2.0 4.0 6.0 8.0 10.0 min 2.0 10x concentrate: 500mM Gly-Gly, 2500mM NaCl, 8.03pH, 166.8 ms/cm Controller set point = 15%B for 10x dilution to 25.1mS/cm 15/04/2005 / 19
10x Concentrate dilution at 120L/h ms/cm 150 100 CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark ph 9.0 8.0 7.0 6.0 % B 100 90 80 70 60 50 40 30 20 10 0 5x Dilution 10x Dilution 0 20 40 60 80 100 120 50 Water rinse 1x baseline 10x_concentrate Feedback enabled 5.0 4.0 3.0 27.0 26.0 Volumetric Flowrate (L/h) 27.0 26.0 0 0.0 2.0 4.0 6.0 8.0 10.0 min 2.0 25.0 25.0 24.0 23.0 22.0 Feedback enabled 24.0 23.0 22.0 21.0 6.0 7.0 8.0 9.0 10.0 min 21.0 15/04/2005 / 20
Importance of in-line dilution for addressing demands of high titre batches. Careful selection of buffers during early phase development can alleviate serious headaches at scale later Select buffers (especially equilibration/wash) that can be prepared in concentrate form Design capability for in-line dilution into chromatography and ultrafiltration rigs Possible to obtain a 30-40% reduction in total buffer prep and hold requirements for equilibration buffers alone 15/04/2005 / 21
Process Development Case Study 2 Throughput optimisation-protein A capture Recent advances in Protein A matrix design High throughput matrices High capacity matrices Alkali stable options Explore relationship between titre increase and matrix selection Examine impact of column diameter on process design and operation 15/04/2005 / 22
Effect of fermentation titre on column capacity 100 80 PnA Sepharose, 0.5g/L 10x increase in fermentation titre PnA Sepharose, 5g/L 83% increase in binding capacity under identical conditions Breakthrough (C/Co) % 60 40 20 0 0 10 20 30 40 50 IgG loaded (mg/ml.matrix) Despite increased binding capacity, compressibility limits attainable throughput and scale-up potential Pressure Drop (psi) 40 35 30 25 20 15 10 5 0 0 100 200 300 400 500 600 700 Linear velocity (cm/h) Column Diameter 1.6cm 2.6cm 5.0cm 10cm 20cm 28cm 40cm 140cm 15/04/2005 / 23
Addressing chromatographic throughput 100 80 MabSelect, 0.5g/L MabSelect, 5g/L Incompressible matrices offer benefit of higher throughput. Essential for high volume fermenters Breakthrough (C/Co) % 60 40 20 Breakthrough curves generated at 450cm/h (3x faster than Sepharose maximum ) 23% increase in binding capacity at 5% C/Co under identical conditions. 0 0 10 20 30 40 50 IgG loaded (mg/ml.matrix) 15/04/2005 / 24
Importance of matrix selection on process throughput at different titres 2000L Reactor 0.5g/L Titre 5g/L Titre Rmp Protein A Sepharose FF MabSelect Conclusion High throughput matrices can deliver 50% improvement in throughput at high fermentation titres 15/04/2005 / 25
Influence of column diameter on batch and campaign costs. 2000L reactor at 5g/L MabSelect for initial capture Small PnA Column Large PnA Column No difference in campaign cost over 15 batches 15/04/2005 / 26
Factors to consider when selecting a column diameter Range of fermentation titres across all products in portfolio (Flexibility) A wide range in titre drives higher utilisation of narrower column diameters Campaign length (number of batches) longer campaigns may favour large diameter columns Risk Bioburden contamination, operating close to re-use limits Capital equipment requirements more chromatography skids, larger elution tanks) Facility design, operation, logistics floor space-processing/storage, packing-unpacking 15/04/2005 / 27
Adressing capacity challenges with larger columns Small diameter columns Large diameter columns Lower capital costs Columns and skids Lower one-off batch costs Higher capital costs Columns (stainless?) Skids Increased flexibility across range of titres Extremely high first batch and replacement PnA costs Potential issues with high utilisation rates of equipment Maintenance Column Re-packing Large elution volumes per cycle High exposure to risk through contamination (e.g. bio-burden) or other process failure 15/04/2005 / 28
Stay abreast of latest vendor offerings Amersham Matrix launches. 1991 Protein A Seph 4FF 1996 rprotein A Seph 4FF 2000 rmp Protein A Seph 4FF 2001 MabSelect 2005.MabSelect SuRe, Xtra Immunoglobulin binding domains Ss E D A B C XM Gly29Ala mutation Z domain 15/04/2005 / 29
Higher through, higher capacity, alkali stability 100 80 rmppna Seph 4 FF Mabselect Prototype SuRe Rmp Protein A 4 FF XK16, 15cm Ho, 150cm/h, 6min RT 5% C/Co = 26g/L Op.DBC = 20.8g/L Breakthrough (C/Co) % 60 40 20 0 0 10 20 30 40 Antibody Loaded (mg/ml) MabSelect TC5, 20cm Ho, 500cm/h, 2.4min RT 5% C/Co = 19g/L Op.DBC = 15.2/L MabSelect SuRe TC5, 20cm Ho, 500cm/h, 2.4min RT 5% C/Co = 26g/L Op.DBC = 20.8g/L 15/04/2005 / 30
Strategies for Chromatography Process Development Ensure process development teams understand manufacturing requirements: High throughput per batch? Low batch costs? Operate closer to 1% Breakthrough to minimise cycling requirements Operate as close to matrix throughput limits as manufacturing capabilities permit (minimise residence time) Employ latest matrix technologies Ensure processes have detailed cost models to justify/defend process decisions 15/04/2005 / 31
Individual Reactor Volume established at 20,000L LONZA Biologics LSBO Facility 3x20,000L Reactors Increasing to 4 Reactors in 2006 Purified through a Single Purification train 15/04/2005 / 32
Do ultra-large columns adequately meet high titre challenge? LSBO Facility designed to handle high-titre batches Protein A Capture in either a 1.4 or 2.0 m diameter Chromatography column. Chromatographic capture of 20,000L at 5g/L feasible given facility design basis 15/04/2005 / 33
Process Development Case Study 3 Ultrafiltration Optimisation Early Phase fast track development may not allow sufficient time to optimise UF operations Diafiltration conditions may be selected based on Prior experience with antibody class/pi Prior experience with particular cassette type/vendor Manufacturing equipment capabilities Antibody stability UF optimisation can substantially improve purification process performance 15/04/2005 / 34
Ultrafiltration Optimisation Flux - J ctmp High Q Med Q Low Q TMP Bulk Flow (C b ) Crossflow Flux - J Boundary Layer Gel Layer (C g ) Membrane C b Increasing Concentration C g C df = C e g ln C C g 15/04/2005 / 35
Results from an Ultrafiltration optimisation Membrane Area = 40 m2 Initial Volume = 7,300 L Initial concentration = 5.24 g/l, concentrated to membrane Cg Membrane Un-Optimised Optimised Gel Concentration (Cg) (g.l -1 ) 44.6 72.1 Optimum Process Conc (Cg/e) (g.l -1 ) 16.4 26.5 Flux at Cg/e (L.m -2.h -1 ) 31.1 59.3 Diafiltration Volume (L) 2,332 1,443 5 x Diafiltration Volume (L) 11,660 7,215 Concentration Time (h) 2 h 32 min 1 h 22 min Diafiltration Time (h) 9h 22 min 3h 2 min Total Process Time (h) 11 h 54 min 4 h 24 min Un-optimised Pin = 20 psig Pout = 5 psig Cross flow = 1x Optimised Pin = 35 psig Pout = 12 psig Cross flow = 1.33x 15/04/2005 / 36
Ultrafiltration Optimisation Influence of Cassette type and vendor Total Process Time (h) 14 12 10 8 6 4 2 Omega Biomax Hydrosart Alpha 5 x DF Volume 0 0 0 10 20 30 40 50 60 70 80 90 100 110 Product Concentration at DF (g.l -1 ) 20k 18k 16k 14k 12k 10k 8k 6k 4k 2k Diafiltrate Volume (L) Model: 40kg batch Membrane Area = 40 m2 Initial Volume = 7,300 L Initial concentration = 5.24 g/l, concentrated to Cg, 5 diavolumes. Key Polyethersulfone Vendor 1 Polyethersulfone Vendor 2 Regen. Cellulose Vendor 2 Regen. Cellulose Vendor 3 Wide variation in optimised performance across different UF cassettes, especially at extremities of protein concentration 15/04/2005 / 37
Large Scale Ultrafiltration-80m 2 capacity 15/04/2005 / 38
Ultrafiltration Optimisation Optimisation of UF operations can deliver reduced processing times and considerable reduction in buffer volumes Simple ultrafiltration optimisation can be performed in a single day with tremendous benefits Avoid mid-range operating conditions-lead to un-optimised processes Product stability at elevated concentrations should be assessed with appropriate stability studies 15/04/2005 / 39
Summary Maximise utilisation of existing assets for as long as possible Optimise performance of unit operations early in development life cycle Employ high throughput chromatography matrices Select buffers with in-line dilution in mind Strive for purification at elevated protein concentration wherever possible However above strategy may have only a limited lifespan Parallel track with investment into novel and conventional technologies 15/04/2005 / 40