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A new, integrated, continuous purification process template for monoclonal antibodies Alex Xenopoulos* Alison Dupont, Christopher Gillespie, Ajish Potty,

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Presentation on theme: "A new, integrated, continuous purification process template for monoclonal antibodies Alex Xenopoulos* Alison Dupont, Christopher Gillespie, Ajish Potty,"— Presentation transcript:

1 A new, integrated, continuous purification process template for monoclonal antibodies Alex Xenopoulos* Alison Dupont, Christopher Gillespie, Ajish Potty, Michael Phillips Processing Technologies Merck Millipore Bedford, MA (USA) Integrated Continuous Biomanufacturing A new ECI conference Castelldefels, Spain October 20-24, 2013 Integrated Continuous Biomanufacturing A new ECI conference Castelldefels, Spain October 20-24, 2013

2 CONFIDENTIAL Highlights flow-through purification train We developed a flow-through purification train that enables an integrated, continuous process clarification capture We have novel solutions for continuous clarification and capture proof of principle Bench-scale proof of principle for several mAbs shown new technologies Breakthrough improvements not possible unless you look at new technologies AX | ECI Castelldefels | 21Oct20132

3 CONFIDENTIAL Monoclonal antibody production A mature, robust industry Templated process Protein A chromatography Yet, several issues remain Stability Capital and utilities Large footprint Frequent bottlenecks Sterility Cleaning validation AX | ECI Castelldefels | 21Oct20133

4 CONFIDENTIAL New alternative template AX | ECI Castelldefels | 21Oct20134 ClarificationCapturePurification/polishing Current template Alternative template BioreactorCentrifuge 2° depth filtration Protein A b/e chrom CEX b/e chrom AEX f/t chrom Bioreactor w/ precipitation 1° depth filtration Protein A b/e chrom continuous Carbon f/t device AEX f/t device CEX f/t device Virus filtration UF/DF

5 CONFIDENTIAL Comparison of templates – icons sized by device volume AX | ECI Castelldefels | 21Oct20135 ClarificationCapturePurification/polishing Current template Alternative template 3.3 m 2 4.4 m 2 14.1 L 19.3 L 0.6 L each 5 L0.4 L3 L 1,000 L @ 2 g/L

6 CONFIDENTIAL Comparison of templates – pool tanks AX | ECI Castelldefels | 21Oct20136 ClarificationCapturePurification/polishing Current template Alternative template 1000 L 500 L 50 L 250 L

7 CONFIDENTIAL Clarification assisted by precipitation and using novel Clarisolve™ filters results in post-Protein A benefits AX | ECI Castelldefels | 21Oct20137 Status Three launched Clarisolve™ filters optimized for particle size Portfolio of flocculants Continuous harvesting and loading of protein A column successful and beneficial Benefits Elimination of centrifuge up to 6,000 L Increased throughput (<3x membrane area) DNA removal (1-2 LRV) Advantages persist post protein A  Reduced turbidity  Enhanced HCP clearance  Reduced resin cleaning

8 CONFIDENTIAL Capture with continuous multicolumn chromatography and incompressible Protein A resins offers savings 8AX | ECI Castelldefels | 21Oct2013 RT (min) Effective DBC (g/L) Productivity (g/L/hr) 1-column batch4397 1-column batch0.22719 3-column continuous 0.2237136 Effective DBC (g/L) RT (min) Consumed resin (L) Consumed buffer (L) Batch394212646 Continuous450.52.82009 Savings87%24% Status Two incompressible resins available  Prosep® Ultra Plus  Eshmuno® A Continuous loading from clarified harvest and continuous loading to purification train successfully shown Benefits Higher productivity, especially at low residence times Resin and buffer savings

9 CONFIDENTIAL Protein A capture cannot be beaten as part of a holistic process evaluation Why not CEX chromatography?  Cheaper resin  Cheaper unit operation  Two dilution steps – volume increase  Longer processing time  Higher water/buffer use  Lower selectivity  Less virus removal  Lower yield  Increased process development  Less templatable  More expensive Why not precipitation?  Single-use  Buffer consumption  Processing time  More materials  Additional unit operations  Precipitant removal  No product concentration  Dilution steps  No purification  Increased process development  More expensive at commercial scale AX | ECI Castelldefels | 21Oct20139

10 CONFIDENTIAL Purification in flow-through mode using novel adsorbers, minimum interventions, fewer pool tanks and one skid Proposed Process Low pH VI Pool VF Pool Carbon + AEX f/t CEX f/t + VF In-line pH Low pH VI Pool CEX Pool AEX Pool VF Pool Traditional Process CEX b/eAEX f/t VF with prefiltration 10AX | ECI Castelldefels | 21Oct2013

11 CONFIDENTIAL Novel flow-through adsorber functionalities work synergistically to remove several classes of impurities MAb acidic pI basic Low MW high Larger acidic HCP, DNA, viruses AEX mAb Aggregates CEX Low MW impurities (leached Protein A, HCP, fragments) Carbon Cell culture components  Insulin, methotrexate, Pluronic F68®, hygromycin, antifoam C Process-related impurities  DNA, HCP, leached Protein A, viruses Product-related impurities  Aggregates, fragments 11AX | ECI Castelldefels | 21Oct2013

12 CONFIDENTIAL Benefits of flow-through purification Disposable chromatography devices connected without pool tanks No bind/elute chromatographic steps Minimal interventions Orthogonal mechanisms for impurity removal Needed pH adjustments incorporated in skid One skid (protein A elution  TFF) is possible Enables integrated, continuous process template AX | ECI Castelldefels | 21Oct201312

13 CONFIDENTIAL Internal bench-scale experimental case studies: Robustness of flow-through purification train (3 mAbs) AX | ECI Castelldefels | 21Oct201313 mAb Monomer Yield (%) Aggregates ProtA  VF pool (%) HCP ProA  VF pool (ppm) VF Capacity (kg/m 2 ) mAb0488N/A250  2> 3.5 mAb05925.0  1.0591  1>3.6 mAb07911.4  ~082  1>3.7

14 CONFIDENTIAL External trials: Robustness of flow-through purification train (7 mAbs) AX | ECI Castelldefels | 21Oct201314 #Monomer yield (%) Aggregates (%)Fragments (%)HCP (ppm) 1915.1  0.81.2  0.1688  4 2831.0  <0.10.3  064  <1 3871.6  0.6n/a80  3 4862.0  0.80.2  0350  7 5841.6  0.60.13  0155  <1 6859.2  2.7n/a600  6 7913.0  0.8n/a1468  7 Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L

15 CONFIDENTIAL Internal case studies: Product quality AX | ECI Castelldefels | 21Oct201315 Current processAlternative process Yield92%87% Process-related impurities HCP: 11 ppm Leached ProtA: 10 ppm DNA: < 10 ppb HCP: 2 ppm Leached ProtA : 4 ppm DNA: < 10 ppb Product-related impurities (% HMW/Main/LMW) 1/98/10.5/99/0.5 Charge variants (% Acidic/Main/Basic) 15/71/1313/72/15 Glycan profile (% Gal: 0/1/2) 79/19/279/20/2 Higher order structure (CD) No change

16 CONFIDENTIAL Cost of Goods: where is the advantage? AX | ECI Castelldefels | 21Oct201316 % cost savings for DSP process 5 g/L @ 5,000 L commercial 1 g/L @ 1,000 L clinical Old batch  New continuous24%35%

17 CONFIDENTIAL Process modeling: advantages of proposed template AX | ECI Castelldefels | 21Oct201317 Parameter for DSP portionUnits Current process Alternative process % change Equipment cost$M6.93.1  55% Footprintm2m2 8759  32% Water use (incl cleaning)L/g of mAb24.21.4  94% Buffer use (excl WFI)L/g of mAb2.41.0  58% Processing timehrs5530  45% Cost$/g of mAb219109  50% || 1,000 L @ 2 g/L | 2 kg batch | ~70% yield

18 CONFIDENTIAL Key features of the alternative template An alternative templated process for downstream purification of mAbs is proposed It matches performance of current templates, provides operational advantages Features: NovelprocessNovel downstream purification process for mAbs – from bioreactor through formulation ConnectedConnected unit operations – continuous operation, minimal interventions Novel unit operationsNovel unit operations developed – leverage continuous nature precipitatingClarification toolbox – novel depth filters, precipitating agents continuous multicolumnProduct capture with continuous multicolumn protein A affinity chromatography – efficient use of resin and buffer Flow-throughFlow-through polishing – no bind/elute steps, improved simplicity and economics Virus filtration and ultrafiltration/diafiltration – no changes feasibilityProof of concept and feasibility data generated – performance equivalent to current, advantages in overall operational flexibility AX | ECI Castelldefels | 21Oct201318

19 CONFIDENTIAL Acknowledgments Downstream Technologies, MM Kevin Galipeau Meghan Higson Jad Jaber Mikhail Kozlov Matthew Stone William Cataldo Romas Skudas Jeff Caron Jonathan Steen Scott Bliss Dennis Aquino Wilson Moya Analytical Technologies, MM Rong-Rong Zhu Michael Bruce Team Supply, MM Michael McGlothlen Patricia Kumpey Paul Hatch Business Development, MM Fred Mann BioPharm Services, Inc Andrew Brown AX | ECI Castelldefels | 21Oct201319


21 Abstract We have developed a new monoclonal antibody purification template comprised of precipitation-based clarification, protein A capture chromatography and flow- through polishing that offers a robust, single-use manufacturing solution while significantly reducing overall cost of goods (COGs). Modeling studies verify that the individual clarification, capture and flow-through polishing solutions offer significant advantages as stand-alone unit operations. Additionally, these technologies were designed to be integrated in a continuous purification process template. Proof of principle for single-batch, continuous operation was obtained at bench scale. Performance of the integrated process matched or surpassed that of the traditional batch process. Advantages of pairing unit operations will be presented and discussed. Extensive process modeling both supported and guided the experimental work and documented cost and operational advantages of the new process template. AX | ECI Castelldefels | 21Oct201321

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