Professional Documents
Culture Documents
Large Scale Stir To Air Transfer
Large Scale Stir To Air Transfer
a stirred tank reactor to an air-lift fermenter” This poster examines how a fed-batch transfectoma process operated in a
stirred tank reactor was successfully transferred to an air-lift fermenter.
Cell growth and metabolism, productivity and product quality are
compared. Optimisation of both modes of operation to ensure a similar
by S.Abraham(1), H.Bland(1), A.Westlake(2), L.Smith(2), M.Brown(2), D.Velez(3), J.Goldstein(3), B.Hornberger(3), A.Daus(3), Q.Zhou(3) physicochemical environment for the cells maximises the ability to
maintain equivalent product quality and yield. The process of transferring
technology from customer to contract manufacturer and between
Address : (1) : Lonza Biologics Inc, 101 International Drive, Portsmouth NH 03801. development and manufacturing is described with particular reference to
areas that require careful consideration to ensure seamless process
(2) : Lonza Biologics plc, 228 Bath Road, Slough SL1 4DY, United Kingdom. (3) : ImClone Systems, 22 Chubb Way, Somerville NJ 08876. transfer to manufacturing scale.
Results
Introduction & Aim Program
• Objectives : Cell Growth 1.E+07
IEF Analysis
• Process review
1200L STR (n=4)
10L ALF (n=3) 1 2 3 4 Lane Sample
– To transfer and scale-up a fed-batch mammalian cell culture process from 1200 L 1 pI Markers
(cell.mL )
130L ALF (n=1)
-1
Stirred Tank Reactor (STR, ImClone) to 5000 L Air-Lift Fermenter (ALF, Lonza) • Raw materials qualification 5000L ALF (n=4)
2 Product ImClone 1200 L
STR
– Maintain process and product equivalence • Laboratory evaluation in flask culture and 10 L ALF 1.E+06
3 Product Lonza 5000 L ALF
Product Accumulation
Lane S ample
– Minimise changes to the process 1 Molecular Weight Markers
500
2 Blank
– Evaluate impact of any process modifications
– Control critical raw materials during scale-up
Materials & Methods 400
1200L STR (n=4)
10L ALF (n=3)
130L ALF (n=1)
5000L ALF (n=4)
3 Product ImClone 1200 L
S TR
4 Product Lonza 5000 L ALF
• Cell line : Mouse SP2/0 AG14 Transfectoma 5 Blank
Product Concentration
6 Molecular Weight Markers
• Product : Murine/Human Chimeric IgG1 300
Comparison of STR vs. ALF • Serum-free medium with several concentrated nutrient feeds 200
• Control parameters : Temp = 36.7°C, pH 7.1 to 6.8, DO 25% saturation with air
Stirred Tank Reactor Air-Lift Fermenter
• Low aspect ratio (typically 1:1) • High aspect ratio (typically 5:1)
• Product concentration determined by Protein A HPLC
100
Conclusions
• Greater flexibility in terms of • Restricted flexibility in operating volume • Glucose, glutamine and lactate concentrations determined by autoanalyser 0
operating volume • Ballast gas ensures CO2 does not accumulate • Ammonia determined by colorimetric assay Elapsed Time (hours)
• CO2 accumulation may be an issue to toxic levels • pCO2 determined by Blood Gas Analyser process from STR to ALF
• Mixing time : 3 minutes (Ref 1) • Mixing time : 5 min • Product quality : Isoelectric Focusing (IEF) and Sodium Dodecyl Sulfate
Carbon Dioxide Partial Pressure 140
• kLa typically up to 10 hr-1 (Ref 1) • kLa typically up to 20 hr-1 Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis (Data shown) 120
10L ALF (n=3)
130L ALF (n=1)
5000L ALF (n=1)
• Tip speed » 0.5m.sec-1 • Liquid velocity » 0.1m.sec-1 • More extensive product characterisation performed (Data not shown) 100
• Impeller requires mechanical seals, • No moving parts or mechanical seals for pCO 2 (mm Hg)
80
• Cell specific productivity and nutrient metabolism were
maintenance impeller statistically equivalent between 1200 L STR and 5000 L ALF
Process Flow Process Transfer
60
Ampoule
20
Ampoule
• Raw Materials
Spinner Flask Spinner Flask
0.5 L to 8 L 3L q Expertise in cell q Raw materials
culture and testing
q Cell Line process scale up q Product Testing
q Product
Acknowledgements
Manufacturing Scale (n=3) Scale (n=1) Manufacturing
– (NB : vvm = maximum total gas flow rate
knowledge,
REQUIREMENTS q cGMP expertise
– Vendor Qualification
understanding and
data
30 L Seed STR 80 L Seed ALF
q Product Quantity SALES AND Scale (n=4) Scale (n=4)
per unit liquid volume)
LOGISTICS
q Product Quality MARKETING
• Inoculum Methodology
q Timeline q Contract Management q Procurement of cGMP
q Budget
q Project Co-ordination
q
raw materials
Scheduling
qproduct 100 ± 18 90 ± 9 79 82 ± 4 • Dr.Jerry Tong • Lonza Quality Services Laboratories
– potential differences in pCO2
Proposal
q Regulatory Package Data Reporting
• Subculture Ratio than STR qglutamine 6.0 ± 0.7 5.9 ± 3.7 8.0 6.5 ± 0.5
– 1 in 6 (STR) vs 1 in 9 (ALF)
9 -1
References
mg(10 cells.hr)
subculture ratio
4
qammonia 0.7 ± 0.1 0.7 ± 0.0 0.7 0.6 ± 0.1 “Homogenisation and oxygen transfer rates in large agitated and sparged
animal cell bioreactors : Some applications for growth and production”
9 -1
mg(10 cells.hr)