Sabrina Pelz IDT Biologika GmbH

1. Evaluation of Different Manufacturing Technologies
for Manufacturing of rMVA Vectors on DF-1 Cell Line
Sabrina Pelz, 15-09-2022

2. 2
Introduction to IDT Biologika
CDMO Development Sites
IDT Biologika
Dessau (Germany)
Manufacturing License and
GMP Certificate granted by
the Saxony-Anhalt state
administrative office.
IDT Biologika
Rockville (USA)
Manufacturing according US FDA
regulations for development and
manufacturing of phase I/II
investigational medicinal products.
Approved by US CDC
EHS/ ISO-certified.
IDT Biologika / ZENIT
Magdeburg (Germany)
Approved for development of
biological products according to
biosafety and GMO regulations for
BSL 2.
Process development for viral vector
and cell and gene therapy
technologies.
Owner
Klocke Holding GmbH
Carsten Klocke and Stefan Klocke
(CEOs)
1,900
employees in
2020
€ 293 m
turnover in 2021
€ 550 m
invested by IDT
since 1993,
Vaccine Technology Summit 2022

3. 3
Modified Vaccinia Virus Ankara Vaccines
 MVA has been used as a vaccine vector in many clinical trials (e.g. Malaria, Tuberculosis, HBV,
HIV, Smallpox, Filoviruses, Mers CoV, SARS); the vaccines are well tolerated in those clinical
studies.
 MVA has been tested in several ‘prime-boost’ regimens with high immunogenicity.
 Both humoral and cell-mediated responses have been induced by vaccination.
 Smallpox (Bavaria Nordic) and Ebola (Janssen) vaccines are approved MVA based vaccines.
 IDT started MVA vector production in 1997
 Major published projects with Oxford University, Bavarian Nordic, Geovax and Ludwig
Maximilian University Munich, CEPI
 IDT evaluated and developed several technologies and scales for manufacturing of rec. MVA
 Major achievement – High resolution large scale DSP technology for purification of IMV virus
particles
Vaccinia- 4 virus configurations
Ref: Journal of General Virology
(2002), 83, 2915-2931
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4. 4
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DF-1 Chicken Cell Line
Inventors: Douglas N. Foster; Linda K. Foster
UMNSAH/DF-1 ATCC® CRL-12203™ / continuous Chicken Embryo Fibroblasts cell line /
Origin: East Lansing Chicken Line (ELL-0)
Chicken cell substrate, none tumorigenic
- genetic stability for CEF adapted vaccine viruses
- Adherent cell line, spontaneously immortalized, not transformed
- Free of endogenous retroviral activity
- Permissive for a range of avian viruses, Vaccinia viruses, Morbilliviruses, VSV ,
Orthomyxoviruses and …
 Current Status
- Exclusive license from Minnesota State University
- Clone purified
- Qualified according Pharm. Eu. Cell bank (MCS; WCS; EoP), none tumorigenic
- Phase 1, IND for Phase 2 submitted, Phase 3 and PPQ ready
DF-1, 72h p. seeding

5. 5
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Technology Platforms for rMVA-X Production
1. CEF Technology
2. CellStak Technology
3. Fixed Bed Technology
4. Micorcarrier Technology
With respect to the size of MVA virus a
sterile filtration (0.2µm) is not feasible
Aseptic processing is mandatory over
complete production process
Aseptic process validation is a
regulatory requirement independent
from the clinical phase
All product contact materials have to be
sterile with a validated of SAL10-6
1 2
3 4
Adherent DF-1 Cell Line

6. 6
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Days 15-21
Days 3
Days 1+1
Hold time
at -80C
Hold time
at 2-8C
Process Time
DF-1_MVA-X CellStack Platform
Generic Process Flow CS10/CS40

7. 7
Fast Track Upscale Approach between CTM PhaseⅠ CT and CTM PhaseⅡ CT
Process parameter CTM PhaseⅠCT CTM PhaseⅡCT Comment
Cell cultivation systems 20 CS 10 16 CS 40 Scale up
Cell cultivation surface 12.7 m2 40.7 m2 Scale up
Virus harvest / cell lysis 1 virus harvest 1 virus harvest divided
into 2 sub-batches
Scale up
Purification of the DS One harvest is purified
in 1 TFF rig by 2 consecutive TFF
processes
Each sub-batch is purified in 1
separate TFF rig by 2 consecutive
TFF processes
Scale up
Formulation of the DS batch 1 harvest is formulated as 1 DS
batch
Each separate purified sub-batch
(ca. 3300 ml) is pooled and
formulated as one DS batch
Scale up
Target volume of
concentrated and purified
final DS batch
1200 ml 6600 ml Scale up
DP filling as single dose
presentation in 2 R vials
Filling of 2 R vials on aseptic RABS
filling line 4 (IDT Biologika)
Fill volume 0.7 ml/vial
Filling of 2 R vials on aseptic
RABS filling line 3 (IDT Biologika)
Fill volume 0.7 ml/vial
Scale up
Both filling lines are validated for aseptic
performance. Line 4 operates with pre sterilized vials
at small scale; Line 3 operates with inline heat
sterilized / depyrogenized vials for large scale
DF-1_MVA-X CellStack Platform
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8. 8
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Visual analysis of cell monolayer,
relies on secondary infection
Subjective plaque counting
 pfu/mL
Test duration: 4 to 14 days
Analysis on the single cell level during
primary infection
Objective counting of infected cells
 IU/mL
Test duration: 48 h up to 4 days
Plaque Assay Flow Cytometrie
DF-1_MVA-X
New assay‘s required to accelerate up the process development
Plaque Assay Flow Cytometry
Assay
performance
Repeatability Similar repeatability
Intermediate
Precision
Less precise
( CV = 22.0 %)
Twice as precise
(CV = 11.0 %)
Accuracy
Worse recovery
(worst recovery =
166.2 %)
better recovery
(worst recovery = 112.3 %)
Linearity linear (R = 0.996) Strongly linear (R = 1.000)
Principle of
titer
determination
Manual plaque
counting
(operator
dependent)
Equipment-based
(operator independent)
Sensitivity
Highly sensitive –
depending on
chosen dilution
scheme
Quantification limit
3x105 IU/mL
Specificity
Non-specific or
specific
specific

9. 9
Viral yield
DF-1_MVA-X CellStack Platform
Different rMVA yield in similar viral titers on DF-1_MVA-X CellStak
platform
Viral titer USP and DSP remain constant during scale-up
CS40 platform: output 6L BDS up to 1.3E+07 IU/ cm² at 60 - 80%
recovery, whereas the main losses occurred during the second
diafiltration step
1,0E+06
1,0E+07
1,0E+08
1,0E+09
Lysate Clarific. DF1_R DF2_R BDS
Infectious
titer
[pfu/mL]
16xCS10
#001
16xCS10
#002
1,0E+06
1,0E+07
1,0E+08
1,0E+09
Lysate KF DF1_1R DF2_1_R DF1_2_R DF2_2_R BDS Pool
Infectious
titer
[IU/mL]
16CS40#001
16CS40#002
1,0E+06
1,0E+07
1,0E+08
1,0E+09
Lysate KF DF1_R DF2_R BDS
Infectious
titer
[IU/mL]
16xCS10
pfu
16xCS10
IU
TFF1 TFF2
TFF1 TFF2
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10. 10
BDS characterization – Impurity profile
Test of DS
Method
Unit
per
2E+08
IU/mL
Batch
#001
#002
#003
#004
#005
#006
#007
#008
MVA_X1 MVA_X2
CS10 CS40
Residual
BSA
ng <7.73 <11.1 <2.7 <3.9 2.2 <0.7 <0.5 <0.8
Total Protein µg 204 182 305 103 62 54 53 57
Endotoxins EU <1.7 <2.5 4 3.03 4.16 1.5 1.3 <0.6
Residual
DNAse
ng <4.3 <6.2 <6.8 <2.2 <1.2 <1.9 <1.3 <2.0
Residual
trypsin
IU <0.0002 <0.0002 <0.0003 <0.0001 <0.00005 <0.0001 <0.0001 <0.0001
Host Cell
DNA
(>127 bp)
ng 6.30 3.27 9.81 4.60 2.85 2.77 3.71 2.02
DF-1_ MVA-X on CS-10/CS40 platform
All QC assay are validated
Consistent impurity data for
each vector and during scale-up
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11. 11
Current Status
DF-1_MVA-X CellStack Platform
- Smale scale development focusing
general process understanding of
critical process parameter and process
attributes
- Yield and impurity optimization
- Process optimization focusing
processing times and process
complexity and costs
- Process transfer into production
including scale up from 4 x CS10 to 16 x
CS 10
- Second scale-up from 16 x CS10 to
16xCS using CS40 manipulation system
- Assay Validation
- Qualification of raw & starting materials
- FMEA based process characterization
IDT Biologika has Phase 3 ready rMVA-X cell stack platform with
exceptional high understanding of rMVA processing (Drug Substance
& Drug product manufacturing)
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12. 12
DF-1_MVA-X
1 Stirred Bioreactor – Microcarrier
Not very suitable – weak cell growth and low
yields
2 Fixed bed bioreactor - PALL
Excellent cell growth of DF-1 also at low seeding
density 3-5E+03 c/cm²
Viral yield 1 log below expectations
Further Upscaling Options
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13. 13
Smale scale Proof of concept (PoC) - Comparison PALL vs Univercells Fixed Bed Bioreactor
Smale scale POC
4 x 4 small scale runs with 2.4m² UniverCells bioreactor performed
Parameter as seeding density, media consumption and lysis condition investigated
Achievements
 Same growth rates as in CS-10 an in iCellis nano achieved, Doubling time 30h
 Achieved virus yields one log higher as in iCELLis nano, comparable to CS-10 platform
Virus yield PALL CS10 CS40 Univercells
rMVA_X1 5.0E+05 pfu/cm² 8.4E+06 pfu/cm² n/a n/a
rMVA_X2 n/a 2.4E+07 IU/cm²
3.0E+06 pfu/cm²
1.2E+07 IU/cm² 1.5E+07 IU/cm²
4.0E+06 pfu/cm²
UniverCells fixed bed technology seems a suitable alternative to e.g. cell stack systems for DF-1_MVA-X
POC show same comparable virus yield to CS-10
DF-1_MVA-X Fixed Bed Bioreactor Platform
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14. 14
Proof of Concept – 200 m² Scale-Up
Direct scale up into Univercells
200m² fixed bed Bioreactor to
gain a better understanding of
potential scale-up risks
Two 200m² PoC runs performed
First run direct transfer of small
scale process to 200m² system
Second run with adaptions
based on 1rst run learnings
DF-1_ MVA-X Fixed Bed Bioreactor Platform
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15. 15
Days 15-21
Days 2 - 4
Process Time
Proof of Concept – 200 m² Scale-Up, Generic Process Flow
DF-1_ MVA-X Fixed Bed Bioreactor Platform
With respect to process comparability, process steps and if applicable process parameter kept constant
during PoC.
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16. 16
Proof of Concept – 200 m² Scale-Up – BR Cell Growth Phase
DF-1_ MVA-X Fixed Bed Bioreactor Platform
Significant higher glucose metabolism at 200m²
Lactate metabolism slope between 2.4m² and
200m“ similar
Lactate concentration above 30mM at harvest
Based on carrier cell count accelerated growth rate
at 200m² scale
2.4m² scale cell growth 4 day delayed
Glucose
[mM]
Lactate
[mM]
200m²
2.4 m²
200m²
2.4 m²
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17. 17
Proof of Concept – 200 m² Scale-Up – Virus Production Phase
DF-1_ MVA-X Fixed Bed Bioreactor Platform
1,0E+05
1,0E+06
1,0E+07
1,0E+08
1,0E+09
IU/
ml
Batch 1
Batch 2
Same distribution of extracellular and
intercellular virus in FB bioreactor compared
to CellStack
Due to the higher titer per volume in the
lysate the particle amount is higher and may
influence the downstream process, but also is
allowing a reduced concentration factor
reaching similar target titers between the two
platforms
The current 200m² reactor design leads to 1/3
(10L) dead volume = Lost Product
Multiple lysis steps enable product recovery
but increase the downstream starting volume,
processing times and production cost
residual volume
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18. 18
Proof of Concept – 2.4m² scale – Virus Harvest Impurities
DF-1_ MVA-X Fixed Bed Bioreactor Platform
Batch
Titer
impurities
volume
Total protein BSA*
IU/ ml IU/ cm² mg/ml ng/ml ml
1 1.6E+08 1.1E+07 0.17 100 1600
2 1.1E+08 0.7E+07 0.14 92 1600
3 1.1E+08 0.7E+07 0.13 110 1600
4 1.6E+08 1.0E+07 0.22 107 1600
5 1.5E+08 1.0E+07 0.17 78 1600
Further development activities were performed to streamline the process achieving a robust and manufacturing suitable
USP process for recombinant MVA – production.
Current USP fixed-bed platform is reaching similar yields and impurity profiles (except BSA*) comparing to
established cell stack platform on a development scale
Currently wash regime procedure for the current process to reduce BSA* further as well as a serum-free medium for DF-
1 cell line in development
1
10
100
1000
10000
BSA
[ng/ml]
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19. 19
DF-1_ MVA-X
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Summary and Outlook
IDT Biologika has Phase 3 ready rMVA_X cell stack platform with exceptional high understanding of rMVA
processing (Drug Substance & Drug product manufacturing)
Different cell stack scales are implemented – 16 x CS10; 16 x CS40 with scale out possibility to 32 x CS40
Virus production including adherent cells in fixed-bed reactors provides advantages concerning scalability
compared to process in cell stacks.
Current platform in development to offer scaled-up process in fixed-bed systems for feasibility batches (R&D),
CTM production & commercialization , USP development data available from 2.4m² up to 200m² fixed-bed
DSP in development, current data sets are showing similar titer recoveries known from the cell stack process
IDT Biologika development and validated a FACS titer method for fast and robust titer determination for
development and product release purposes
DownStream process will be further optimized and stabilized, focusing higher BDS titers of > 1E+09 IU/mL

20. Thanks to my colleagues who asking questions, frequently improve the process,
solved and explained the unexpected, being not afraid to change the direction, did
all the analytical assay‘s and 10000 of viral titrations, transferred and scaled
processes, created batch records and fight in the lab if things does not happen as
planed …
Thanks for being a part of this team !!!