This document discusses implementing single-use technologies for a clinical drug supply pilot run. It summarizes: 1) A template process and pre-selected operating parameters were used to minimize process development work and reduce timelines. 2) A 100L pilot scale run was conducted using commercially available single-use systems and assemblies to scale up a downstream process developed at bench scale. 3) Comparison of bench and pilot scale runs showed similar impurity clearance, charge variant distribution, and overall yields, demonstrating successful scale up using single-use technologies.

1. Pragmatic implementation of Single-use
technologies to deliver clinical supply
Priyabrata Pattnaik, PhD
Director – Strategic Initiative

2. Agenda
1 Market and need assessment
2 PD Activities
3 Pilot Scale Run
4 Process Integration using Single use technology
5 Comparison of Bench and Pilot scales
6 Summary
7 Conclusions

3. MAb Market – Trends, Characteristics
 mAbs ($48 bil) continue to represent the ‘growth’ driver for the
biopharmaceutical market
• Total ~ 1400 biologics projects in R&D and clinical phases
• ~ 50% in preclinical and Phase I
 Biomanufacturing capacity demand is ‘uncertain’
– Success rate of Ph1 to approval is <30%
– Not many easily accessible large manufacturing facilities
– A Flexible concept for approaching manufacturing is desirable
 Significant capital investment required for commercializing a mAb
– > 100-300 $ Mil installed cost for a traditional large scale SS production facility
 Time to clinic is still a key driver especially for smaller and newer biotech
firms
3
Data from Evaluate Pharma

4. “Commoditization” of mAb Processes
 Developing a downstream (DSP) process for monoclonal antibody
(MAb) purification is essentially a “solved” problem
 Time to clinic is still a key driver especially for smaller and newer
biotech firms
 Facilitated by template and single-use approach
“ … it is unlikely that non-conventional downstream unit
operations would be needed to replace conventional
chromatographic and filtration separation steps, at least for
recombinant antibodies”
- Brian Kelley, Biotechnol. Prog. 2007, 23, 995-1008
4

5. The Journey to Clinic
Challenge - Reduce timelines with limited resources
A pragmatic approach minimizes time and effort
 Template process
 Pre-select operating parameters to minimize PD work
 “Pre-package” devices/systems/ancillaries to reduce
specification, procurement and installation effort
 Use of ClinicReady Process Template
 Single-use technology
5

6. Single-Use Technology in DSP of MAbs
Drivers Current Situation
 Reduce/eliminate cleaning, utility and  Single-use technology
validation costs most widely used in
 Eliminate concerns of carryover holding and preparing
 Reduce turnaround time between buffers
batches/campaigns  Single-use flow paths for
 Facilitate duplication of manufacturing certain unit operations at
suites in multiple locations pilot scale
 Ability to use same equipment with
various MAbs
Evidence for single use flow paths for entire DSP train
has been sparse
6

7. What’s needed to produce material for
clinical supply
Technology &
Knowledge
Product Resources
& Expertise
Innovation
Process Template Single Use GMP Facility
7
Technologies

8. ClinicReady MAb Process Template
AFFINITY CEX
CLARIFICATION CHROMATOGRAPHY CHROMATOGRAPHY
Bioreactor Millistak+ Pod – ProSep Ultra Plus Fractogel SO3
D0HC / X0HC
AEX
ChromaSorb
CHROMA-
TOGRAPHY
Pellicon 3
Express Viresolve Pro+
Ultracell
Bulk
Drug
Substance STERILE
FILTRATION ULTRAFILTRATION VIRUS REMOVAL
8

9. Process development space
Effort Risk
# Unit
# Devices # Process Execution X Operations
# Vendors X
options
X
parameters + Protocols? Data?
Analysis? Scale up?
2-3 2-3 2-3 7
56 – 189 trials
Pre-select operating parameters
# Unit
< X Operations
1 X 1 X 1-2 + Protocols, Data
collection, Analysis
tools & Scale up tools
9 14 trials

10. Proof of Principle
Pilot Scale
[100L bioreactor]
Bench Scale
Millistak
D0HC + X0HC
ProSep Ultra Plus
Selection
Millistak X0HC
Tool
e
Fractogel SO3
y
e
y
ChromaSorb
Sizing
Viresolve Pro+
Tool
P3 UltraCel
Template
10

11. Two different mAbs – MAb04 and MAb08
30
CEX Elution Cond (mS/cm)
25
MAb04
20
MAb08
15
10
30 40 50 60 70 80 90 100 110
HIC Elution Cond (mS/cm)
11

12. Reduction of PD Parameter Space
Operational Parameters Fixed Operating Operating Parameter to be
Unit Operation to be established Parameter determined by PD
Clarification Flux, Capacity Flux Capacity of depth filters
Capacity of sterile filter
Protein A Residence Time, Capacity, Residence Time Capacity
Elution Buffer pH Elution buffer pH
Viral Inactivation Capacity of sterile filter No pH adjustment to lower pH Capacity of sterile filter
CEX Residence Time, Capacity, Residence Time Capacity
Loading pH, Elution pH and Constant pH Elution Conductivity
conductivity
AEX Flow rate, Capacity, loading pH Flow Rate Capacity
and conductivity pH relative to pI
Conductivity range
Virus Filtration Flow rate, Capacity, loading pH Flow rate Capacity
and conductivity pH
UF/DF Feed flux, # of diavolumes, Feed flux TMP
concentration for DF, process Diavolumes Concentration for DF
time
Process time
12

13. PD Parameter Space
Operational Parameters Fixed Operating Operating Parameter to be
Unit Operation to be established Parameter determined by PD
Clarification Flux, Capacity 100 LMH Capacity of depth filters
Capacity of sterile filter
Protein A Residence Time, Capacity, 3 minute Residence Time Capacity
Elution Buffer pH Elution buffer pH
Viral Inactivation Capacity of sterile filter No pH adjustment to lower pH Capacity of sterile filter
CEX Residence Time, Capacity, 6 minute Residence Time Capacity
Loading pH, Elution pH and Constant pH operation (5-5.5) Elution Conductivity
conductivity
AEX Flow rate, Capacity, loading pH Flow Rate = 12.5 MV/min Capacity
and conductivity pH 1 unit below pI
Conductivity < 12 mS/cm
Virus Filtration Flow rate, Capacity, loading pH Constant flux operation – 200 Capacity
and conductivity LMH
pH 5 – 5.5
UF/DF Feed flux, TMP, # of Feed flux = 5 LMM TMP
diavolumes, concentration for Diavolumes = 10 Concentration for DF
DF, process time
Process time = 3-6 hrs
13

14. PD Data
Parameter Expected Range MAb04 MAb08
Clarification – Primary 50 – 125 L/m2 85 L/m2 300 L/m2
depth filter capacity
Clarification – Secondary 150 – 400 L/m2 250 L/m2 374 L/m2
depth filter capacity
Protein A > 40 g/L @ 3 min residence 58 g/L 45 g/L
time
CEX > 50 g/L @ 6 min residence 68 g/L > 100 g/L
time
AEX > 3 kg/L 3 kg/L > 5 kg/L
Virus Filtration 2 – 5 kg/m2 ~ 3 kg/m2 > 5 kg/m2
UF/DF 50-150 g/m2/hr 75 g/m2/hr 89 g/m2/hr
Note: In the case of MAb08, the cell culture process is a low titer, low density process. Hence, the depth filter
capacities are higher than expected
14

15. Impurity Clearance – Bench Scale (MAb04)
1000000 HCP < 10 ppm (ng HCP/mg MAb) 1000000 DNA < 50 ppb (pg DNA/mg MAb)
LRV = 0.4
DNA (ppb)
HCP (ppm)
LRV = 2.8
LRV = 0.8
LRV = 0.9
1 1
Harvest Clarif ied Protein A CEX AEX Harvest Clarif ied Protein A CEX AEX
Harvest Harvest
4.5 16 < 10 ppm (ng HCP/mg MAb)
< 2%
Leached Protein A (ppm)
Aggragate %
0 0
Clarif ied Protein A CEX AEX VF UFDF Harvest Clarif ied Protein A CEX AEX
Harvest Harvest

16. Disposable bioreactor for PD
 Cell Growth similar between Small
scale (3L, 50L) disposable or glass
bioreactor (3L).
 Viability is maintained and consistent
whatever the scale
 Productivity is comparable to
stainless steel system
16

17. Scaling of Single Use Bioreactors
17

18. Mobius Single Use Chromatography
Mobius FlexReady Smart System: Smart Flexware Assembly:
Modular & Automated
18

19. Scale-up of developed Downstream Process
Process from bench (~3-4 g) to 200L pilot scale (70-100g) using
commercially available, off-the-shelf systems and single use assemblies
First step
Cell thawing
7
weeks
USP
week 1 to 5 Cell amplification – 3 weeks Production – 2 weeks
DSP Depth Filtration Capture Step Virus Inactivation
week 6
Last Step
Drug Substance
DSP CEX Chromatography AEX Chromatography Viral Filtration Tangential Final Filtration
week 7
19

20. Single Use - Process Scale (MAb04)
1.00E+09
1000000
1000000
1.00E+08 Bench Scale 100g
Bench Scale
100g process 1.00E+07
HCP (ppb)
1.00E+06
DNA (ppb)
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
1 1
1.00E-01
Leached Protein A (ppm)
8 70
70
Bench Scale
60 Bench Scale 100 g
100g process
Aggregate %
50
40
30
20
10
0 0
0
Protein A CEX AEX UF/DF

21. Charge Variants
Bench Scale Process Scale
*M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B E HC R DH R E T1.D
CX A 04B NC LA IFIE A V S ) *M D1A S 280,16R
W , ig= ef=360,100(2011-09-13_W _TH R CE SC R E HA E T
CX P O S LA IFI D RV S .D)
*M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B E HB T HCE P O )
CX A 04B NC A C X O L1.D *M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B R CE SB T HCE P O )
CX A 04P O S A C X O L1.D
*M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B E HB T HCH O A O BP O )
CX A 04B NC A C R M S R O L1.D *M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B R CE SB T HCH O A O BP O )
CX A 04P O S A C R M S R O L1.D
*M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B E HB T HP O E AP O )
CX A 04B NC A C R T IN O L1.D *M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B R CE SB T HP O E AP O )
CX A 04P O S A C R T IN O L1.D
*M D1A S 280,16R
W , ig= ef=360,100(2011-09-13_W _TH E HU F )
CX B NC FD 1.D *M D1A S 280,16R
W , ig= ef=360,100(2011-09-01_W _M B R CE SB T HUF F D)
CX A 04P O S A C D 1.
mAU mAU
120 120
100 UFDF 100
UFDF
Protein A Protein A
80 Chromasorb 80 Chromasorb
CEX CEX
60 60
Clarified Harvest Clarified Harvest
40 40
20 20
0 0
0 5 10 15 20 25 30 35 40 min 0 5 10 15 20 25 30 35 40 min
M D A,Sig
W1 =280,16Ref=360,100 (2011 9-1 C _T BE C U D 1.D
-0 3_W X H N H F F )
Norm.
40
M D A,Sig
W1 =280,16Ref=360,100 (2011 9-0 C _M B04PR C S B C U D 1.D
-0 1_W X A O ES AT H F F )
• Distribution of charge variants
35 unaffected by unit operations at both
30
scales
25
20 • Distribution of charge variants very
Bench Scale
15
Process Scale similar in final pool from both scales
10
5
• Carboxypeptidase B digestion
0
0 5 10 15 20 25 30 35 40 45min
confirmed that basic peaks are same
as C terminal Lysine variations
21

22. Comparison of Yields
100
90
80
Bench Scale
70
Yield (%)
Process Scale
60
50
40
30
20
10
0
Overall yield at bench scale and 100g scale ~ 85%
22

23. Cost of Pilot Scale Runs - Summary
Units Utilized
Hardware
MIX sytems 2
Drum dollies 6
200L Bioreactor 1
Buffer systems 1
Chrom systems 1
Non-chrom systems [ CLF, VF, TFF ] 3
Systems/Hardware Cost ~ $2.0M
Disposables
MIX Bags 15
2D and 3D bags 22
Sterile filters 8
Devices 11
Single use flow paths 11
Total cost of disposables* ~ $50k
23 * Excludes chrom resins and TFF membranes

24. Resources at hand to produce the product
for clinical supply
Upstream
Downstream

25. Summary
 Demonstration of rapid scale-up of a MAb purification process
using streamlined PD activities
 Eliminated screening multiple devices
 Minimized process development (PD) space by fixing certain
operational parameters
 Successful scale-up of entire downstream process using
commercially available, off-the-shelf, largely single-use systems
and process containers
 Minimized engineering workload and start-up times by employing
pre-existing systems and assemblies
25

26. Thank you
26