process development and large-scale gmp production for ... · bulk post-clarification final u...
TRANSCRIPT
Process Development and Large-Scale GMP Production for Lentiviral (LV) vectors
Luca Alberici,
CBO
Margherita Neri,
USP DEV Manager
Francesca Bellintani
DSP DEV Manager
Leading the way in Cell & Gene therapy
MolMed Webinar, June 2020
This document may contain forward-looking statements that reflect the current views of the Company on future events based
on information available as of today’s date. Forecasts and estimates are generally identified by words such as "possible",
"should", "forecast", "expected", "estimated", "believe", "intend", "plan", "objective" or by the negative form of these
expressions or other variations thereof or by the use of comparable terminology. Although the Company believes that its
expectations are based on reasonable assumptions, these forward-looking statements are subject to numerous risks and
uncertainties that are beyond Managers’ control, including scientific, business, economic and financial factors, which could
cause actual results to differ materially from those projected in the forward-looking statements. The Company assumes no
obligation to publicly update and revise forecasts and estimates following the availability of new information, future events or
other factors, without prejudice to compliance with applicable laws. All subsequent forecasts and estimates, whether oral or
written, attributable to the Company or any persons acting on its behalf, are expressly qualified, in their entirety, by these
cautionary statements. This document does not constitute an offer or invitation to subscribe for or purchase any securities of
MolMed S.p.A The official manager responsible for preparing the Company’s financial reports, Salvatore Calabrese, herewith
attests, pursuant to Article 154-bis, paragraph 2 of the Legislative Decree 58/1998 (“Testo Unico della Finanza”), that the
accounting disclosure contained in this press release matches documentary evidence, corporate books, and accounting records.
2
Disclaimer
MolMed Webinar, June 2020
1. Company Presentation
2. LV Upstream production in CFs and bioreactor
3. LV Downstream processing
3
Agenda
MolMed Webinar, June 2020
MolMed is a pure player in the Cell&Gene arena
4
Milan, Italy
Focusing on innovative cell and gene therapies that can meet the therapeutic needs in the treatment of
tumors and rare diseases, with a clear and solid
industrial project based on research, developmentand production excellence
CDMO Business, with 35+ Programs developed with our Partners
R&D Business on our Autologous Product CAR-T CD44v6
MolMed Webinar, June 2020 5
CDMO business experience in manufacturing of vector and modified cells
Client Client Patient
Vectors deliveredto the Client
Vectors for Cell Transduction in MolMed
MolMed Webinar, June 2020 6
Viral Vector Manufacturing
Cell Engineering
Development/Feasibility Tech transfer/Engineering GMP manufacturing
LV/RV 48L Cell Factories – adhesion
LV/RV 200L bioreactor - adhesion
Development/Feasibility Tech transfer/Engineering GMP manufacturing
LV/RV T-Cell Transduction
LV CD34+ Cell Transduction
Current manufacturing platforms
MolMed Webinar, June 2020 7
Milan Site (San Raffaele)
Bresso Site
Excellent GMP capacity with more than 230 scientists and support staff
1,500 SQM (16,000 SQF) and 6 grade B/C suites
2003: Authorized GMP manufacturing facility for Clinical programs
2015: Authorized GMP manufacturing facility for Commercial products
3,300 SQM (36,000 SQF) and >20 Grade B/C suites
Authorized for GMP manufacturing and QC for the production of clinical and commercial products
Recently authorized Stream#2, for further services and new collaborations
MolMed Webinar, June 2020 8
Development and manufacturing partners in EU and US geographies
8+ International Service Partners
2 Commercial Cell-Engineered Products in EU
2 Commercial Viral Vectors in EU
15+ Cell-Engineering programs for EU&US
20+ Viral Vectors programs for EU&US
35+ Programs currently in Development and GMP
300+ Treated Patients (autologous)
220+ Manufactured GMP Vectors
30+ C&G Clinical Trials Supplied in EU&US
Track Record
MolMed Webinar, June 2020 9
Ready Proprietary
Processes for vectors
and cells engineering
25yrs Experience in
proprietary projects
now available for
CDMO collaborations
High GMP manufacturing
Capacity thanks to new
facility in Milan area
Recognized Flexibility in
accommodating Partners’
requests
1st Approved Facility for
C&G therapies
160 QC tests internalized, ensuring
reduction in time and cost
Strenghts of MolMed CDMO
MolMed Webinar, June 2020
1. Company Presentation
2. LV Upstream production in CFs and bioreactor
3. LV Downstream processing
10
Agenda
MolMed Webinar, June 2020 11
Development Tech transfer/engineering GMP manufacturing
48L CF LVV process - adhesion
Current vector manufacturing processes – Cell factories 48L
MolMed Webinar, June 2020 12
Cell line adherent HEK293T
Culture Area 24CF (15.2m2)
Harvest 48L
DSP Purification and concentration
Process currently in GMP for clinical and commercial application
More than 100 batches manufactured in GMP
LV production process – 48L Cell Factories
Cell thawing and expansion
Harvest
Transfection
Clarification/prefiltration
Benzonase addition
Anion exchange DEAE resin
TFF concentration
Gel filtration
Sterile filtration and filling
MolMed Webinar, June 2020 13
Bulk Final
TU/mL4.1x107
±2.2x107
6.5x108
±4.6x108
ngP24/mL481
±1857428
±4109
LV production process – 48L Cell Factories - Productivity
Infectious viral titer Physical Viral Titer
BULK
FINAL
MolMed Webinar, June 2020 14
LV production process – 48L Cell Factories - Residuals
RemovalContaminants
HCP >99%
Total DNA >99%
BSA >99%
HCP Total DNA BSA
BULK
FINAL
MolMed Webinar, June 2020 15
Current vector manufacturing processes – Bioreactor 200L
Development Tech transfer/engineering GMP manufacturing
200L disposable bioreactor - adehsion
iCellis system allows the development of fully cGMP compliant manufacturing processes for LV
vectors compliant with commercial vector application
Vectors produced in iCellis systems:
are qualitative comparable to CF clinical batches
batch size is cost-effective in terms of number of patients treated versus cost of production and QC
high reproducibility of genetically modified cells batches
MolMed Webinar, June 2020 16
LVV production in iCELLis 500: process flow chart
Adherent HEK293T
DNA Transfection
Cell thawing and expansion
Harvest
Benzonase addition
Clarification/prefiltration
Anion exchange chromatography
Concentration and diafiltration
Sterile filtration and filling
Full Scale
Scale down
ÄKTA Pilot and Axichrom column
KrosFlo System
ÄKTA Ready and pre-packed column
ÄKTA Ready FluxSystem
iCellis 500
iCellis Nano
MolMed Webinar, June 2020 17
LVV production in iCELLisnano: development data USP
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
3.0E+07
3.5E+07
4.0E+07
0.0E+00
1.0E+10
2.0E+10
3.0E+10
4.0E+10
5.0E+10
6.0E+10
7.0E+10
1 5 9 13 17 21 25 29 33 37 41 45 49 53 4 8 12 16 20 24 28 32 36 40 44
TU/m
L
To
tal T
U
run
TU tot TU/mliCELLis nano 0.53m2iCELLis nano 1.06m2
Productivity on bulk supernatant
Conditions optimization permitted to increase system productivity of 3-4 fold
MolMed Webinar, June 2020 18
LVV production in iCELLis 500: development data USP
Scale up in iCellis500 runs confirmed system scalabilityLinearity in production using different packed-bed surface areas
0
50
100
150
200
250
300
350
400
450
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+07
6.0E+07
66m2 133m2 #1 133m2 #2 Average nano runs
ngP
24
/mL
TU/m
L
TU/mL iCellis 500 TU/mL iCellis nano ctrl ngP24/mL iCellis 500 ngP24/mL iCellis nano ctrl
0.53m20.53m2
1.06m2
1.06m21.06m2
4.1x1012Total TU 1.1x1013 1.0x1013
MolMed Webinar, June 2020 19
From Upstream to Downstream
Adherent vs Suspension cell
culture
Transient transfection vs stable
producer
Serum containing vs serum free
culture medium
Addition of USP additives
Cell supernatant vs cell lysate
Residual HCPs and DNA levels
Residual BSA presence
Additives removal
Clarification strategy:
Centrifugation /TFF / NFF
Chromatography:
Ion exchange
Size exclusion
Affinity
Other ligands
TFF:
Ultrafiltration
Diafiltration
Culture Conditions Residual Levels Downstream Strategies
Upstream process parameters can impact the desing of Downstream processing
MolMed Webinar, June 2020 20
LVV purification from clarified bulk
Process designed to remove main contaminants and to have the same level of quality of CF vector
67% vector recovery98% HCP removal>88% DNA removal
Anion exchange chromatography• Captures and concentrates LV• Removes HCP • Removes DNA contaminants • Removes BSA
Concentration and diafiltration (TFF)• Concentrates LV• Reduces HCP• Reduces small DNA contaminants• Reduces BSA
Sterile filtration and filling• Bioburden reduction and sterile filtration• Filling in target vials/bottles/bags
57% vector step recovery>99% HCP removal44% DNA removal
75% vector step recovery>99% HCP removal74% DNA removal
MolMed Webinar, June 2020 21
LVV purification from clarified bulk
24% 30%*
*Process yield calculated applied downstream scale down model
45% 58%
0.0E+00
2.0E+12
4.0E+12
6.0E+12
8.0E+12
1.0E+13
1.2E+13
Bulk Post-clarification Final
tota
l TU
iCellis 66m2
0.0E+00
2.0E+12
4.0E+12
6.0E+12
8.0E+12
1.0E+13
1.2E+13
Bulk Post-clarification Final
tota
l TU
iCellis 133m2
Yield
Total Process yield is very high and consistent
MolMed Webinar, June 2020 22
Comparison LVV production in iCELLis500 vs standard CF process
0 20 40 60 80 100
CF
iCellis500
Trasduction effciency (%)
Transduction efficiency on T-Cells
VCN2.3 ± 0.3
VCN3.8 ± 0.1
Total TU/batchInfectious viral titer on Final Filled vector
Infectious viral titer on bulk supernatant
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+07
6.0E+07TU
/mL
0.0E+00
1.0E+08
2.0E+08
3.0E+08
4.0E+08
5.0E+08
6.0E+08
7.0E+08
8.0E+08
TU
/mL
0.0E+00
2.0E+11
4.0E+11
6.0E+11
8.0E+11
1.0E+12
1.2E+12
1.4E+12
1.6E+12
1.8E+12
2.0E+12
Tota
l TU
5 Fold increase in total vector TU and
same potency on primary cells
MolMed Webinar, June 2020 23
Characterization of LVV produced in iCellis – Impurities profile
0
500
1000
1500
2000
2500
3000
iCellis500 iCellis nano CF
ng/
mL
Total DNA
0
500
1000
1500
2000
2500
3000
3500
4000
iCellis500 iCellis nano CF
ng/
mL
Host Cell Proteins
0
200
400
600
800
1000
1200
1400
1600
1800
2000
iCellis500 iCellis nano CF
µg/
mL
Residual BSA
No
td
etec
tab
le
No
td
etec
tab
le
No
td
etec
tab
le
LLOQLLOQ
No
td
etec
tab
le
No
td
etec
tab
le
No
td
etec
tab
le
No
td
etec
tab
le
Improved impurities profile for vector produced in bioreactor
MolMed Webinar, June 2020 24
From vector to patients
Patients treated with 1 batch of manufactured vector:
Considering to transduce about 1X109 T cells/pt and 0.7X109 CD34+ cells/pt
~ 90 patientsT-cells transduction at MOI 4
~ 10 patientsCD34 cells transduction at MOI 50
~ 400 patients
~ 50 patients
CF Bioreactor
MolMed Webinar, June 2020
Thank you for your attention!
Luca AlbericiCBO
e-mail:
Margherita NeriUSP DEV Manager
e-mail:
Francesca BellintaniDSP DEV Manager
e-mail: [email protected]