Perfusion and Beyond The XCell TM ATF System

Perfusion and Beyond The XCell TM ATF System Earl Pineda Field Application Scientist Web ads May 9, 2018

Inspiring advances in bioprocessing since 1985 Repligen Corporation: one of the fastest-growing companies in North America Most of the world s monoclonal antibodies are purified on Repligen s Protein A Upstream and Downstream Applications Chromatography 19% Proteins 38% Filtration 43% AN INDUSTRY LEADER IN Pre-packed Chromatography Columns Cell Culture process intensification Single-use Tangential Flow Filtration Protein A Affinity ligands manufacturing Multiple global GMP manufacturing sites 2 20 of the Top 25 Pharma companies use Repligen products

Rapid growth through innovative launches and strategic acquisitions 1985 1988 2010 2011 2012 2014 2015 2016 2017 Repligen is one of the fastest growing companies in North America Protein A ligands commercial manufacturing Launched Immobilized Protein A resins Acquired BioFlash technology: Pre-packed columns Acquired Novozymes Biopharma: Native Protein A, growth factors Launched OPUS prepacked columns Acquired Refine XCell ATF technology: XCell ATF Launched OPUS 45/ 60 Acquired Atoll: OPUS PD Acquired Spectrum HF TFF Systems Launched XCell Singleuse Systems Launched OPUS R Launched XCell 10 Single-use 3 Acquired TangenX TFF Cassettes

Repligen products used in PD workflow 4

Repligen products used in Commercial bioprocessing 5

Agenda Perfusion Basics XCell ATF Introduction XCell ATF Applications Perfusion CFB HD Cell Banking N-1 6

Perfusion Basics Traditional bioreactor processes Perfusion Process Benefits Drivers 7

Traditional Bioreactor Processes CELL RETENTION DEVICE Batch Fed Batch Continuous Chemostat Perfusion The traditional forms of cell culture processes are Batch, Fed-batch, Chemostat and Perfusion. Fed-batch and Perfusion are typically used as a manufacturing platform. 8

Continuous Process - Perfusion What is Perfusion? A cell culture process involving the continuous replenishment of cell culture media, removing of waste products, and harvesting of the product of interest, all while retaining the cells within the bioreactor system. Result = higher cell densities, longer runs, and higher productivity. 9

Perfusion Process What are the benefits? Perfusion Benefits: Economic benefits Higher cell densities -> Higher productivity Longer runs -> Less turnaround Smaller bioreactors -> Lower capital investment costs -> Single-use friendly Smaller facilities -> Lower operating costs R&D scale is commercial scale Benefits to cell culture and product quality Removal of waste products Replenishment of nutrients, fresh environment Stabilized cell environment Steady State Product is available almost immediately Fedbatch Perfusion Typical Bioreactor size 5,000 20,000 500 2000L Single-use friendly No Yes Typical Run length ~2 weeks 1-2 months Estimated runs per year 20-30 5-10 Steady state environment No Yes Typical Cell Density 5-25 e 6 50-100e 6 Productivity per run 1x 5-20x 10 Product Quality Consistency + ++

Drivers Product Quality Drivers Product Quality drivers Market drivers Trends Driving Change Process intensification / Capex reduction High density cell cultures from cell bank to final bioreactor Reduction in size and number of unit operations Capacity increases within existing infrastructure CoG now a process consideration Drive to improve facility productivity Small, modular, facilities can now provide equivalent drug quantities using ATF Technology Drive to towards mindset of Continuous Processing 12

Xcell TM ATF System What is ATF Backflush action System sizing Bioreactor connection 13

ON OFF XCell TM ATF system Overview FLUID INLET LEVEL CONTROL QUICK CONNECT ADDITION PUMP VALVE FILTRATE PUMP FI LTRATE HF MODULE OR SCREEN MODULE LIQUID LEVEL HOUSING CONTROLLER PROCESS VESSEL DI APHRAGM EXHAUST AIR INLET ATF PUMP STAND FILTER A cell retention device based on Alternating Tangential Flow (ATF) Filtration Cross flow is derived from a diaphragm pump. Filter is based on hollow fiber technology. Dynamic back flushing effect amounts to a practically self-cleaning filter! 14

XCell ATF System How it works Unique self cleaning action - Animation 15

XCell ATF Flow Pressure cycle (P-cycle) 16

XCell ATF Flow Exhaust cycle (E-cycle) 17

XCell ATF System Backflush Transmembrane pressure was measured at both ends of the filter. Higher XCell ATF rate results in greater TMP changes. Higher XCell ATF rate = stronger backflush. Increasing ATF rate increases backflush Backflush occurs on both P and E cycles. In exhaust cycle, back flush was observed in bottom part of the filter as TMP (P2-P3B) was negative. In pressure cycle, back flush was observed in top part of the filter as TMP (P1-P3A) was negative. BACK FLUSH BACK FLUSH BACK FLUSH 18

XCell ATF Perfusion System CELL BLEED BLEED WASTE PUMP PUMP HARVEST HARVEST/SPENT MEDIA PUMP FRESH MEDIA Overview MEDIA-IN LEVEL CONTROL kg ATF SYSTEM AIR VAC 19

ON OFF XCell ATF System Connection LEVEL CONTROL FLUID INLET QUICK CONNECT ADDITION PUMP VALVE FILTRATE PUMP FILTRATE HF MODULE OR SCREEN MODULE LIQUID LEVEL PROCESS VESSEL DIAPHRAGM HOUSING CONTROLLER EXHAUST AIR INLET Bioreactor connection ATF PUMP STAND FILTER Only a one connection required. Easily swappable if configured properly. Small scale connection is typically via bioreactor headplate. Large scale is via bioreactor sideport. 20

XCell ATF System Sizing Guideline bioreactor volume for each XCell System for Perfusion System Filter Effective Surface Area (m 2 ) Repligen filter Pump Displacement Volume (L) ATF Flow Range (LPM) Typical Bioreactor Sizes (L) ATF2 0.13 0.1 0.3-1.5 2-10 L Single-Use available Sizing Guidelines ATF4 0.77 0.4 2-10 10-50 L ATF6 2.5 1.3 5-20 50-200 L ATF10 11 6 20-90 200-1000 L + Single-Use available Single-Use available NOTE: These numbers are just guidelines, actual capacity and vessel size depend upon process conditions. Systems are already used in both clinical manufacture and commercial manufacturing and come with validation packages. 21

XCell ATF System Family The XCell ATF Single-use uses the same filters 60cm 60cm Similar fiber lengths, just increasing fiber count to increase SA 60cm 60cm 30cm XCell ATF 2 XCell ATF 6 XCell ATF 10 XCell ATF 4 22 NOTE The XCell ATF 4 is 30cm and a little more attention is required to scale-up accurately

The XCell ATF System Bioreactor Connection SS bioreactor 23

The XCell ATF System Bioreactor Connection Single-use bioreactor 24

The XCell ATF System Bioreactor Connection SU-ATF to SUB SU ATF6 SU ATF10 25

XCell ATF Applications Perfusion Continuous Processing High Density Cell Banking N-1 Extended Harvest 26

ATF Perfusion Microfiltration (0.2-0.5µ) filter retains cells inside bioreactor while protein passes through the filter ATF using Microfilter What is a batch in perfusion? scale Continuous harvest, batches determined by DSP 27

ATF Perfusion vs. Fedbatch Fed-Batch ATF Perfusion Medium cell density Fedbatch was a typical 14 day culture, while ATF Perfusion was run for 25 days. Maximum VCD in Fed-Batch culture was 14e6 cells/ml whereas in ATF Perfusion the VCD was maintained at 40-60e6 cells/ml at higher viability. Drop in viability during ATF Perfusion was due to bioreactor issue, and not typical. 28

ATF Perfusion vs. Fedbatch Cumulative Productivity 2L wv Cell Specific Productivity Medium cell density The cumulative amount of IgG produced in ATF Perfusion culture is 3 times higher than in Fed-Batch culture after 14 days of cultivation Fed-Batch: 1.42g (14 days) ATF Perfusion: 4.41g (14 days) 10.8g (25 days) The cell specific productivity in ATF Perfusion culture is slightly higher than in Fed-Batch culture Fed-Batch: 6.3 pg/cell/day ATF Perfusion: 7.5 pg/cell/day 29

High Cell Density Perfusion VCD and Viability High cell density High density perfusion run was maintained at steady state at high VCD of approx. 100e6 cells/ml and consistent viability >90%. 30

High Cell Density Perfusion Cell Specific Productivity High cell density No change in cell specific productivity through out the run, even at high 100e6 cells/m cell densityl 31

Continuous Processing Media Production bioreactor 1000L Buffer Wash Steps X L/min 1vvd 0.7 L/min 1g/L product Clarified Harvest 0.2 um Surge Tank 0.14 L/min Product Concentrate 5g/L product Linking Upstream and Downstream unit operations. 200+L 50 kda 0.56 L/min + X L/min UF Waste Chromatography Perfusion can result in larger harvest volumes, depending on perfusion rates. 32 A continuous concentration step can be implemented to minimize volume and control desired concentration for DSP.

Process Intensification X X X HD seed transfers remove whole unit operations, increasing efficiency Concentrating a process to produce higher cell densities and higher product yields in shorter time. Reducing the number of upstream steps through increased cell concentration at each step Cell banking High density seed transfers N-1 perfusion Concentrated perfusion Concentrated fed-batch X X 33

HD Cell Banking Procedure Overview Expand existing bank into a reactor Run perfusion process desired cell concentration, e.g. 50e6/ml, in 4-7 days Cool down bioreactor contents to room temperature Concentrate cells through the ATF System in 5-15 minutes Add freezing medium into the reactor, while mixing Fill bag manifolds with cell suspension directly from reactor Freeze bags in blood bank type freezer Advantages All steps are performed within the sterile reactor environment No centrifuge required Same cell concentration & volume in each bag First & last bag from the same environment Reduced manual handling of cells Faster bank creation: complete <2 hours from harvest Using the ATF to create HD Cryo-Seed Intermediate (HDCSI) bags 34

HD Cell Banking Industry case study Genentech FASTEC Frozen Accelerated Seed Train for Execution of a Campaign Source: Gargi Seth, Development of a New Bioprocess Scheme Using Frozen Seed Train Intermediates to Initiate CHO Cell Culture Manufacturing Campaigns, Biotechnology and Bioengineering, 2012 35

N-1 Perfusion Seed or N-1 Perfusion Reducing the number of upstream steps by eliminating one or more steps through high cell density seed transfers N-1 or N-2 Benefits Reduced seed expansion bioreactors Reduced of operational risk Minimize time in production bioreactor Reduced bioreactor footprint & cost Using the ATF to reduce number and size of expansion bioreactors X X 36

N-1 Perfusion with high seed in FB Seeding density high vs low Intensified N-1 used to inoculate Fedbatch bioreactor at higher seeding density Using cells from the N-1, cells were individually inoculated into low seeding density (0.5E6 cells/ml) and high seeding density (10E6 cells/ml) fed-batch production cultures. The low seed culture reached a max VCD of 19E6 cells/ml in a two week run, whereas the high seed culture reached 25E6 cells/ml in a one week run. Note: A 3L bioreactor (2L wv) was used to mimic a 2000L production fed batch culture 39

N-1 Perfusion with high seed in FB Total Protein Production Cell Specific Productivity Intensified N-1 used to inoculate Fedbatch bioreactor at higher seeding density The high seed culture was able to produce same amount of protein in 7 days as the low seed in 14 days. No difference was observed in cell specific productivity between low and high seed cultures 40

N-1 Perfusion with high seed in FB Protein Quality Intensified N-1 used to inoculate Fedbatch bioreactor at higher seeding density Conditions (Seeding Density) Low Seed (0.5E 6 cells/ml) High Seed (10E 6 cells/ml) Glycan Analysis Size Distribution %G0F %G1F % Aggregation 79.7% 15.8% 2.3 82.2% 14.4% 1.9 Equivalent protein quality, no significant changes in protein aggregation and glycosylation Note: Small percentages of G0 and G2F were also observed in the protein samples. Total percentage of aggregates were measured by comparing to the monomer 41

Process Intensification HD seed transfers and N-1 remove whole unit operations, increasing efficiency Using HDCSI only Using HDCSI and N-1 Perfusion for high seeding 42

extended Harvest Conventional Clarification Fed-batch BR Centrifugation Depth Filtration DSP (Typically 14 days) Extended Harvest using the XCell ATF to increase final yields extended Harvest using XCell ATF Fresh Media Harvest XCell ATF 43 Fed-batch BR Day 1-10 Fed-batch BR Day 11-14 DSP

VCD (E 6 Cells/mL) Viability (%) extended Harvest 60 Viable Cell Density and Viability 100 50 Traditional FB extended Harvest_FB_ATF 1vvd 80 extended Harvest_FB_ATF 0.5vvd 40 60 30 40 20 extended Harvest 10 20 Extended Harvest using the XCell ATF to increase final yields 0 0 2 4 6 8 10 12 14 16 0 Time (days) Viable cell density increased by more than 3-fold in fed-batch cultures using extended Harvest compared to traditional FB. Viability in fed-batch cultures using extended Harvest stayed at >90% until the end of the run, a healthier environment for the cells and the product of interest. 44

Total Accu. Protein (ug) extended Harvest 2.0E+6 extended Harvest_FB_ATF 0.5vvd Specific Productivity y = 5.2235x + 49920 extended Harvest_FB_ATF 1vvd 1.6E+6 Traditional FB Linear (extended Harvest_FB_ATF 0.5vvd) y = 6.4333x + 23217 Linear (extended Harvest_FB_ATF 1vvd) y = 4.6555x + 67689 Linear (Traditional FB) 1.2E+6 8.0E+5 Extended Harvest using the XCell ATF to increase final yields 4.0E+5 0.0E+0 0.E+00 5.E+04 1.E+05 2.E+05 2.E+05 3.E+05 3.E+05 IVC (Cells E6*day) Cell specific productivity is higher in extended Harvest process compared to traditional FB process Higher perfusion rate (1vvd vs 0.5vvd) shows higher cell specific productivity. 45

Total Accu. IgG (mg) extended Harvest 2000 extended Harvest_FB_ATF 0.5vvd Specific Productivity 2X IgG extended Harvest_FB_ATF 1vvd (Clarified) 1600 Traditional FB 1200 extended Harvest 1.4X IgG (Clarified) 800 1X IgG 400 Extended Harvest using the XCell ATF to increase final yields 0 0 2 4 6 8 10 12 14 16 Time (days) FB using extended Harvest process produced up to 2X more IgG compared to traditional FB within the same culture time. Harvested product is 0.2µm filtered and ready for downstream processing. 46

extended Harvest extended Harvest using XCell ATF Fresh Media Harvest XCell ATF Fed-batch BR Day 1-10 Fed-batch BR Day 11-14 DSP Extended Harvest using the XCell ATF to increase final yields Benefits 2X increase in protein production w/o additional time Sterile, closed system and single step process Keeps cells healthy until the end of the run Healthy cells = Better control over product quality Reduction in residence time of product in reactor Harvested material is ready for DSP Limitations Expanded harvest volume Higher media consumption 47

Applications summary Where ATF is used today 48

Earl Pineda Field Application Scientist epineda@repligen.com www.repligen.com 2017 Repligen Corporation. All rights reserved. The trademarks mentioned herein are the property of Repligen Corporation and/or its affiliate(s) or their respective owners.