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Continuous
BioManufacturing
of Mabs by QbD
Maurizio Cattaneo, PhD, CPIP
President
BioVolutions Inc.
Woburn, Massachusetts, USA
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Continuous Biomanufacturing of
Therapeutic Mabs Smaller equipment and facilities More flexible operation Reduced inventory Lower capital costs, less work-in-progress materials Smaller ecological footprint Integrated processing with fewer steps. No manual handling, increased safety Shorter processing times Increased efficiency
On-line monitoring and control for increased product quality assurance in real-time (PAT) Amenable to Real Time Release Testing approaches Consistent quality
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Continuous Manufacturing of Mabs Upstream and Protein A Downstream
(2L and 50L Scale Up)
(5mL and 500mL Scale Up)
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CONFIDENTIAL 4
Our Continuous Biomanufacturing Pilot Plant
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CQAs (eg. Glycosylation, HCP, DNA, Aggregation)
Glycosylation (eg. Galactosylation, Fucosylation)
IgG1 % HighMan % G0 % G1 % G2 % Sialic
acid
% Gal
Alpha
% Core
Fucose
A-Mab 7±5 71±10 19±10 3±10 1±5 0±5 91±20
B-Mab 1±5 78±10 17±10 4±10 2±5 0±5 94±20
R-Mab 2±5 46±10 40±10 12±10 3±5 0±5 95±20
CQA Acceptable Range
HCP 0-100ng/mg
DNA <10-3 ng/dose
Aggregates 0-5%
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Risk Assessment Mitigation Matrix (RAMM) for
selecting CPPs
Relative Importance of Output on CQA 9 1 1 9 3 3 3 3 3 3
pH
CO
2
VC
D
Via
bili
ty
Bio
bu
rden
End
oto
xin
Tite
r
Myc
op
lasm
In V
itro
vir
al
MM
V P
CR
Process Parameters Tot Impacts Proc
Perfusion Rate 1 1 9 9 1 1 1 1 1 1 127 Quality & Growth WC-CPP
Sparge Oxygen Flowrate 1 9 1 1 1 1 1 1 1 1 55
Agitation 110-125 RPM 3 9 3 3 1 1 3 1 1 1 99 Gas Evolution Rate WC-CPP
pH 0 3 3 3 1 1 3 1 1 1 138 Quality & Growth CPP
DO (>40%) 1 1 3 3 1 1 3 1 1 1 73 Titer
Seed Density (0.5 x 10E6) 1 1 3 9 1 1 3 1 1 1 127 Cell Growth WC-CPP
N-1 Inoculum Volume 1 1 1 1 1 1 1 1 1 1 47
Temperature 1 1 3 9 1 1 9 1 1 1 163 Quality &Growth CPP
Duration of Perfusion 1 1 1 3 1 1 1 1 1 1 83
Glucose Control 1 1 9 9 1 1 9 1 1 1 151 Quality & Growth CPP
Lactate Control 1 1 9 9 1 1 9 1 1 1 151 Quality & Growth CPP
Anti-Foam Control 3 1 1 1 1 1 1 1 1 1 65
Protein Load Protein A 1 1 1 1 3 3 9 3 1 1 161 Quality & Titer CPP
Elution pH Protein A 1 1 1 1 3 3 9 3 1 1 161 Quality & Titer CPP
Totals 17 32 48 62 18 18 62 18 14 14
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Design of Experiments (DoE)
(Taguchi L9)
Settings Temperature
(°C)
pH Glucose
1 36.5 7.2 0
2 36.5 7 1
3 36.5 6.8 3
4 36 7.2 1
5 36 7 3
6 36 6.8 0
7 35.5 7.2 3
8 35.5 7 0
9 35.5 6.8 1
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Continuous Culture Overview
• It can produce high titers and good quality product
• Equipment is utilized more efficiently and the
equipment footprint is much smaller
• The Continous System pushes productivity to the
next level, ~10x higher cell densities
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Upstream Continuous Cell
Culture
1.E+05
1.E+07
2.E+07
3.E+07
4.E+07
5.E+07
6.E+07
7.E+07
8.E+07
0 10 20 30
Via
ble
Cel
l D
ensi
ty (
vc/
mL
)
Run Time (Days)
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30
Tit
er (
g/L
)
Run Time (Days)
Run 1
Run 2
Run 3
Key
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Glucose and Lactate Control
-1
0
1
2
3
4
5
6
7
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
An
alyt
e (
g/L)
Time (Days)
Glucose Lactate
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Design Space (DS) based on
Glycosylation CQA
IgG1 % HighMan % G0 % G1 % G2 % Sialic
acid
% Gal
Alpha
% Core
Fucose
A-Mab (Test Article) 0 54 39 7 2 0 97
A-Mab (Reference) 7±5 71±10 19±10 3±10 1±5 0±5 91±20
B-Mab (Test Article) 0 85 15 1 0 0 96
B-Mab (Reference) 1±5 78±10 17±10 4±10 2±5 0±5 94±20
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Downstream Continuous
Purification Synchronize the protein load (g/mL of resin) with the
upstream rate of perfusion
Replace interim holding tanks with peristaltic pumps to
perform continuous downstream purification
Reduce the amount of Protein A resin by ~ 20-fold
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Continuous Purification of A-Mab
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Antibody production Perfusion Per Day * 60-Day Run 6 Runs/Yr
50L 25g 1.5Kg 9Kg
1000L 500g 30Kg 180Kg
Fed-Batch Per Run * 20 Runs/Yr
50L 25g 0.5Kg
1000L 500g 10Kg
* 50% Purfication Yield
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Assumptions Parameter FB CP
Production Bioreactor 10,000L 1,000L
Product Titer 2 g/L 2 g/L
Production Phase 10 days 10 days (1VVD)
Media Consumption 10,000L 10,000L
Protein A Resin
($11,400/L)
40L ($456,000) 2L ($22,400)
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Capital
(US$)
106.7M 33.1M Capital
(US$)
103.9M 44.1M
Floor
Area (m2)
4,012 2,447 Floor Area
(m2)
5,096 3,152
Results at 500Kg / year
• A comparison of fed-batch to perfusion at “large” scale:
– Perfusion utilizes a smaller footprint and
– Lower capital investment costs
– And has lower operating costs
• The advantage of disposable bioreactors reduces with increasing bioreactor size, as
required for large scale fed-batch operation for example
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KEY POINTS
• Making therapeutic antibodies can benefit from new
advances in continuous biomanufacturing such as
Continuous Perfusion (CP) and Continuous
Chromatography (CC).
• The Capex for a new facility is significantly lower than
conventional batch facilities
CONFIDENTIAL 18