Integrated Continuous Biomanufacturing A new ECI conference 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

Highlights We developed a flow-through purification train that enables an integrated, continuous process We have novel solutions for continuous clarification and capture Bench-scale proof of principle for several mAbs shown Breakthrough improvements not possible unless you look at new technologies AX | ECI Castelldefels | 21Oct2013

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 | 21Oct2013

New alternative template 2° depth filtration Protein A b/e chrom CEX b/e chrom AEX f/t chrom Virus filtration Current template UF/DF Clarification Capture Purification/polishing Bioreactor Centrifuge Protein A b/e chrom continuous 1° depth filtration Carbon f/t device AEX f/t device CEX f/t device Bioreactor w/ precipitation Alternative template AX | ECI Castelldefels | 21Oct2013

Comparison of templates – icons sized by device volume Current template Clarification Capture Purification/polishing 3.3 m2 14.1 L 14.1 L 19.3 L 0.6 L each 4.4 m2 5 L 0.4 L 3 L Alternative template 1,000 L @ 2 g/L AX | ECI Castelldefels | 21Oct2013

Comparison of templates – pool tanks Current template 1000 L 500 L Clarification Capture Purification/polishing 250 L Alternative template 50 L AX | ECI Castelldefels | 21Oct2013

Clarification assisted by precipitation and using novel Clarisolve™ filters results in post-Protein A benefits 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 AX | ECI Castelldefels | 21Oct2013

Productivity (g/L/hr) Capture with continuous multicolumn chromatography and incompressible Protein A resins offers savings 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 RT (min) Effective DBC (g/L) Productivity (g/L/hr) 1-column batch 4 39 7 0.22 19 3-column continuous 37 136 1000L feedstock, 1 g/L Effective DBC (g/L) RT (min) Consumed resin (L) Consumed buffer (L) Batch 39 4 21 2646 Continuous 45 0.5 2.8 2009 Savings 87% 24% AX | ECI Castelldefels | 21Oct2013

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 | 21Oct2013

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

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

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 | 21Oct2013

Internal bench-scale experimental case studies: Robustness of flow-through purification train (3 mAbs) mAb Monomer Yield (%) Aggregates ProtA  VF pool HCP ProA VF pool (ppm) VF Capacity (kg/m2) mAb04 88 N/A 250  2 > 3.5 mAb05 92 5.0  1.0 591  1 >3.6 mAb07 91 1.4  ~0 82  1 >3.7 AX | ECI Castelldefels | 21Oct2013

Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L External trials: Robustness of flow-through purification train (7 mAbs) # Monomer yield (%) Aggregates (%) Fragments (%) HCP (ppm) 1 91 5.1  0.8 1.2 à 0.1 688 à 4 2 83 1.0 à <0.1 0.3 à 0 64 à <1 3 87 1.6 à 0.6 n/a 80 à 3 4 86 2.0 à 0.8 0.2 à 0 350 à 7 5 84 0.13 à 0 155 à <1 6 85 9.2 à 2.7 600 à 6 7 3.0 à 0.8 1468 à 7 Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L AX | ECI Castelldefels | 21Oct2013

Internal case studies: Product quality   Current process Alternative process Yield 92% 87% Process-related impurities HCP: 11 ppm Leached ProtA: 10 ppm DNA: < 10 ppb HCP: 2 ppm Leached ProtA : 4 ppm Product-related impurities (% HMW/Main/LMW) 1/98/1 0.5/99/0.5 Charge variants (% Acidic/Main/Basic) 15/71/13 13/72/15 Glycan profile (% Gal: 0/1/2) 79/19/2 79/20/2 Higher order structure (CD) No change AX | ECI Castelldefels | 21Oct2013

Cost of Goods: where is the advantage? % cost savings for DSP process 5 g/L @ 5,000 L commercial 1 g/L @ 1,000 L clinical Old batch  New continuous 24% 35% AX | ECI Castelldefels | 21Oct2013

Process modeling: advantages of proposed template Parameter for DSP portion Units Current process Alternative process % change Equipment cost $M 6.9 3.1  55% Footprint m2 87 59  32% Water use (incl cleaning) L/g of mAb 24.2 1.4  94% Buffer use (excl WFI) 2.4 1.0  58% Processing time hrs 55 30  45% Cost $/g of mAb 219 109  50% 1,000 L @ 2 g/L | 2 kg batch | ~70% yield AX | ECI Castelldefels | 21Oct2013

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: Novel downstream purification process for mAbs – from bioreactor through formulation Connected unit operations – continuous operation, minimal interventions Novel unit operations developed – leverage continuous nature Clarification toolbox – novel depth filters, precipitating agents Product capture with continuous multicolumn protein A affinity chromatography – efficient use of resin and buffer Flow-through polishing – no bind/elute steps, improved simplicity and economics Virus filtration and ultrafiltration/diafiltration – no changes Proof of concept and feasibility data generated – performance equivalent to current, advantages in overall operational flexibility AX | ECI Castelldefels | 21Oct2013

Acknowledgments Downstream Technologies, MM Analytical Technologies, MM Kevin Galipeau Rong-Rong Zhu Meghan Higson Michael Bruce Jad Jaber Mikhail Kozlov Team Supply, MM Matthew Stone Michael McGlothlen William Cataldo Patricia Kumpey Romas Skudas Paul Hatch Jeff Caron Jonathan Steen Business Development, MM Scott Bliss Fred Mann Dennis Aquino Wilson Moya BioPharm Services, Inc Andrew Brown AX | ECI Castelldefels | 21Oct2013

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 | 21Oct2013