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-Confidential-
One and Done – Ge+ng Biopharmaceu5cal Produc5on Processes Right the First Time
Ying Huang, Ph.D. Associate Director, Process Development
KBI Biopharma, Durham NC
Presented at: World Orphan Drug Congress, Washington DC, April 25, 2013
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Pre-Clinical Phase I Phase II Phase III
Process Development Process
Characterization Process
Validation Process Monitoring &
Improvement
FIH Process • Deliver clinical process
quickly • Platform process • Clinical Supply
Submission & Approval
Lifecycle management
BLA Prep & PAI
Commercial Process • Deliver manufacturing process for
registrational trials and market • Design keeping large-scale manufacturing
in mind • Improve productivity, efficiency, robustness,
manufacturability, COGs • Analytical characterization and method
development
Process Characterization and Validation • Develop IPC strategy through understanding of process inputs and
outputs (design space) • Scale-down characterization and validation studies • Large-scale process validation to demonstrate process consistency • BLA preparation • Supporting documents for licensure inspections • Post-commercial process improvements (CI) • Post-commercial process monitoring
FIH process Commercial process
Gottschalk U., Brorson K., Shukla A. Nature Biotechnology, 30(6), 489-491, 2012
Biologics Commercializa5on
2
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Approaches to Get Every Step Right at the 1st Time? • Screen the best protein candidate construct by transient transfec3on. • Iden3fy the stable high producing clones within a short 3meline • Pool enrichment with FCAS to achieve a good heterogeneous pool • Single cell cloning with ClonePix to improve selec3vity
• Define the op3mized cell culture process by using high throughput microbioreactor with rapid, accurate 3ter and product quality assessment. • Develop an efficient downstream purifica3on processes using selec3ve column washes.
3
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Cell Line Development – Ini5a5on of Biologic Produc5on
4
Vector Construc5on
Transfec5on & Selec5on (Amplifica5on)
Pool Enrichment
Clone Isola5on & Screening
Stability Assessment
Strong vector w/ enhance element
Transient Transfec5on
Gene
Stable Pool
FACS
High throughput cloning (FACS or ClonePix)
Earlier material generaHon
Stable Cell Line GeneraHon
Gene codon op5miza5on
Valid Clone Screening Process
CSI
Process Development
Process Development
Cell Bank
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Decide the Right Molecule with Transient Gene Expression (TGE)
5
7 to 14 days
Large Amount of Star5ng Cells (K-‐Sep)
Large Scale FB Culture for Produc5on (Wave)
Benefit of TGE Ø Screening candidate molecules – lock the best construct Ø Fast material genera3on – analy3c method development
0 1 2 3 4 5 6 7 8
Titer
Culture Days
GOI_1 GOI_2 GOI_3
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Pool Enrichment with FACS
6
#3. 2nd Round of Enrichment Sorting
#2. 1st Round of Enrichment Sorting
#1. Original Population Gate1
Using the FACS Jazz instrument to sort the top 5% of the popula5on
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Original Round 1 Enriched
Round 2 Enriched
Fold In
crease
Pools
Pool Produc5vity Assessment ( 7-‐day batch culture)
Time: ~ 2 weeks
Ø FACS allows sor5ng of high producing cells to an enriched pool, which provides a be_er pool for earlier material genera5on and/or cloning.
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• Automated Colony Picking Robot • High throughput • Asep3c
• Fluorescent Technology for Selec3ve Cloning • Ranks and selects only the highest
producing clones
0
5000
10000
15000
20000
25000
30000
35000
40000
1G7
1E10
1C12
1F10 2B6
2A8
1D12
1B12
1A8
1A4
1G5
1F7
2B1
2B9
2A1
2A6
1G4
1G12 1B5
2A2
2A4
1B8
1H7
2G8
2A9
1F1
2B5
1B9
2G11 1A2
1A5
1F3
2D2
1C4
2F12 1F9
2B2
2E11
2G10 1F2
2A10
1E5
2E10
1H5
1E8
1F8
1C2
1B11
1E11
1A6
2F10 2E4
2C12
2E8
1F4
1H8
2F1
1F5
2G12 2F5
2G7
2G2
1F12 1B4
2A12
2A11
2E3
1E6
2C2
1C10 2F9
2F4
1G8
2F3
1E9
1E2
1C5
1H9
2F2
2B10
2E5
2E12
1G2
2E2
1E1
1E4
2E1
1H4
2F8
2E9
2B8
2F11 1A9
2A3
2H8
1C11
Colony
Tite
r (PD
U/m
LL)
Semi-‐Solid Matrix with Conjugated An3body
Colony (White light)
Halo (Florescent light)
Image Plane
High throughput Cloning with ClonePixTM
7
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100% is the 1ter of best clone from ClonePixTM ClonePix vs LDC
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Clone
Rel
ativ
e R
anki
ng (%
)
ClonePix Top 30
LDC Top 30
ClonePixTM Can Isolate Be_er Clones with Less Effort than LDC
Clones
Rel
ativ
e Titer (
%)
ClonePixTM Top 30 Clones
LDC Top 30 Clones
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Run specific assays for protein glycan and protein charge variant for harvest samples with turnaround of 1-‐2 days.
§ Use 24 miniaturized Bioreactors (10-‐15mL working volume).
§ DOE design of factors (pH, DO, temperature, feeds volume and frequency).
§ Perform daily analysis to monitor cell growth and key metabolites.
ambrTM
ProA-‐based 3ter analysis of in process cell culture samples with quick turnaround of same day.
Ø Cell growth, cell viabili3es, metabolite profiles
For each tested condi5on
Ø Titers and Product Quality
+
Selec3on of best process condi3on(s) with respect to cell growth, produc3vity and product quality.
Integrated High throughput Process Development and Analy5cal Support
9
forteBIO Octet Caliper LabChip GX II
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Cell Culture Process Op5miza5on with ambrTM – Growth Profile
10
Ø Real 5me monitor of cell growth profiles by tes5ng mul5ple culture condi5ons like pH, temperature, DO level and feeds.
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Cell Culture Process Op5miza5on with ambrTM – Produc5vity
11
Ø Produc5vity and protein quality (data not shown) reported by high throughput assays with a quick turnaround 5me.
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A CHO cell line producing a recombinant glycoprotein
Cell Growth
Titers Product Quality Attributes
Scalable Model Micro-‐bioreactor : ambrTM
12
Ø The process decisions and results from ambrTM were reproducible to other tradi5onal bioreactor scales (10 L and 200 L).
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Taking Advantage of Modulators in Downstream Purifica5on • Incorpora3on of modulators into process can help increase selec3vity and purity of product
• Combina3ons of modulators can further enhance process step
• Goal: U3lize mobile phase modulators to decrease HCP levels during Capto MMC process step for an3body purifica3on
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Incorpora5ng Modulators into Wash steps
• Case Study: » Target molecule: E. coli derived
recombinant protein » Process step: Phenyl Sepharose FF » Ini1al product yield: 88.8%
» Result: – Individual modulators showed some
selec3vity enhancement but also product loss
– A combina3on of urea, sodium thiocyanate and glycerol in the wash step increased product purity to >95%
Shukla AA, et al., 2002. Biotechnol Prog 18: 556–564.
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0.50
0.70
0.90
1.10
1.30
1.50
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%
Normalized
HCP
Recovery
HCP vs. Recovery aier Intermediate Wash for Capto MMC Capture
baseline
During Capture step
Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.
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0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%
Normalized
HCP
Recovery
HCP vs. Recovery aier Intermediate Wash for Capto MMC Polishing
baseline
During Polishing step
Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.
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Process Impact on HCP Clearance of Using Selec5ve Column Washes • Inclusion of an intermediate wash using Tris, 0.1M NaCl, 50mM arginine, 5% ethylene glycol, pH 7.0 resulted in 2-‐fold lower HCP levels when compared to process where a modulator was not u3lized.
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Conclusions
• Orphan biopharmaceu3cal development needs par3cular emphasis on • Genera3ng a high producing cell line with short 3meline
» Earlier material genera1on » Iden1fying high producer clones with an integrated approach
• Developing a process with the end in mind (licensure filing) to avoid mul3ple changes along the way » Op1mizing cell culture process with high throughput technologies
• U3lizing mixed mode chromatography to improve product purity and maintain process step yield
• A dedicated CDMO with the right knowledge and capabili3es can help smooth the development pathway
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Acknowledgments
• Abhinav Shukla, Ph.D. Vice President, Process Development & Manufacturing
• Michael Cavanaugh Vice President, Business Development
• Sigma Mostafa, Ph.D. Director, Process Development
• Shahid Rameez, Ph.D. Process Development Scien3st II, Upstream Process Development
• Leslie Wolfe, Ph.D. Process Development Scien3st II, Downstream Process Development
• Rich Harper, B.S. Process Development Scien3st I, Cell Line Development