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Titerless Infected-cellsPreservation and Scale-up
(TIPS)
David J. Wasilko & S. Edward LeePfizer Global Research & Development
Groton, CT
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TIPS Advantages
1. Titerless system
2. Stable baculovirus stocks
3. Rapid, streamlined process
4. Readily scaleable
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Using TIPS (2-3 weeks to 100-L scale)
Using the standard method (9-10 weeks to 100-L scale)
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Presentation Overview
• Compare typical baculovirus stock to the TIPS stock
• Baculovirus stock preparation• Protein expression using TIPS• Long term stability of the TIPS baculovirus
stock• Process scale-up
– From a 1-mL baculovirus stock to 10-L or 100-L in one easy step
– Scale-up in 7-L (5-L liquid) bioreactor
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Baculovirus Stock: Standard Method
Time (months/years)
Titered Virus Stock (4°C)(Passage 1, 2, 3…)- Titer- Evaluate Expression- Amplify
Re-titer Before Use
Degradation:– Proteases– Nucleases
Drop in titer
/ Amplification
Start over from bacmid stage
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TIPS Stock: Baculovirus Infected Insect Cells (BIIC)
Infected Sf9 cell (≤ -80°C)
Virus titer is not needed with TIPS!
Time (months/years)
Baculovirus stored and cryoBaculovirus stored and cryo--preserved within the cell.preserved within the cell.
Thaw the infected Sf9 cells• No Stability Issues• Small Volume
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The Key to the TIPS Process: Baculovirus-Infected Insect Cell (BIIC) Stocks.
• Infect Sf9 cells with baculovirus – Estimate the MOI (eMOI ≥ 3.0) – In general, infected cells produce ~100 pfu/cell
• Allow the cells to become infected– Monitor diameter increase (~2-3 µm)– High cell viability (>90% preferred)
• Cryo-preserve the “healthy” infected cells
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TIPS Process and Cell Diameter
• One of the most important parameter in the TIPS process is cell diameter.
– Monitor the infection process in real time– Used to prepare the BIIC stock– Used to predict protein expression
• Cell Counters:– Cedex (Innovatis)– Vi-CELL™ (Beckman Coulter)– Cellometer™ Auto T4 (Nexcelom Bioscience)
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Virus Stock Comparison
EliminatedNecessaryTiteringNo issuesQuestionableStability
≤ -80 oC4-8 oCStorage Temp.
Native host cellsSpent mediaEnvironment≤1 mL>100 mLVolume
TIPSInfected Cell Stocks
StandardVirus Stocks
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Evaluation of Baculovirus Storage in Sf9 Insect Cells
• Model protein to evaluate baculovirus storage in the cell– Secreted, his-tagged
• Infected an Sf9 culture with baculovirus– (MOI = 3)
• Monitored cell growth parameters
• Prepared BIICs every 6 hours
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0 1 2 2 4 3 6 4 8 6 05 .0 x 1 0 5
1 .0 x 1 0 6
1 .5 x 1 0 6
2 .0 x 1 0 6
2 .5 x 1 0 6
3 .0 x 1 0 6
V ia b le C e ll D e n s ity (m L -1) V ia b ility (% )
T im e (H o u rs p o s t in fe c tio n )
V ia b le C e llD e n s ity m L -1
5 0
6 0
7 0
8 0
9 0
1 0 0
V ia b ility(% )
Cell Growth and Viability During the Infection Process
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0 1 2 2 4 3 6 4 8 6 0
1 5 .0
1 6 .0
1 7 .0
1 8 .0
1 9 .0
A v e ra g e C e ll D ia m e te r (u m )
T im e (H o u rs p o s t in fe c t io n )
A v e ra g e C e llD ia m e te r (u m )
Cell Diameter IncreasedDuring the Infection Process
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1x10 6
1x10 7
1x10 8
1x10 9
0 12 24 36 48 60
15
16
17
18
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A verage C e ll D iam eter (um ) B acu lov irus T ite r (P FU /m L)
T im e (H ours post in fec tion )
A verage C e llD iam ete r (um )
B acu lov irusT ite r (P FU /m L)
Change in Cell Diameter is an Indicator of Virus Infection/Release
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1x10 6
1x10 7
1x10 8
1x10 9
50
60
70
80
90
100
0 12 24 36 48 60
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16
17
18
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C ell V iab ility (% ) A verage C e ll D iam ete r (um ) B acu lov irus T ite r (P FU /m L)
T im e (H ours post in fection )
A verage C e llD iam e te r (um )
B acu lov irusT ite r (P FU /m L)
Viability (%
)
Change in Cell Diameter is an Indicator of Virus Infection/Release
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1. Thaw the 6-hpi and 12-hpi BIIC stocks2. Transfer the cells to fresh medium3. Recovery cell density similar to the original
culture at the time of cryopreservation4. Monitor the infection process by changes in
cell diameter
Baculovirus Survival in the Cryopreserved Infected Cells
Will the infection process continue after freezing and thawing the BIIC?
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Comparison of the Original Infection to the 6-Hour BIIC Stock
0 24 48 72 9614
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18
20
Average CellDiameter (um)
Time (Hours post infection)
Exp No. 1: Cell Diameter (um) Exp No. 2: Cell Diameter (um) (6-Hour BIIC)
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Comparison of the Original Infection to the 12-Hour BIIC Stock
0 24 48 72 9614
16
18
20
Average CellDiameter (um)
Time (Hours post infection)
Exp No. 1: Cell Diameter (um) Exp No. 2: Cell Diameter (um) (12-Hour BIIC)
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Initial Cell Densities:0.50 x 106 Sf9 cells/mL
(eMOI ~ 0.10)
0.75 x 106 Sf9 cells/mL(eMOI ~ 0.07)
1.00 x 106 Sf9 cells/mL(eMOI ~ 0.05)
* Initial cell densities kept low to minimize nutrient limitation effects.
Can the infected cells be used for protein expression?
1-mL
1-mL
1-mL
1-mL100-mL(1:100)
100-mL/Flask(1:100)
Total Dilution(10- 4)
24-hpi BIIC Vial107 Cells/mL(109 PFU/mL)
Flask 1
Flask 2
Flask 3
*
1-BIIC Vial/10-L
Dilution Flask
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0 24 48 72 96 120 14410 5
10 6
10 7
105
106
107
V iable Cell Density m L-1
T im e (hp i)
SF1 V ia. Cell Density (#/m L) SF2 V ia. Cell Density (#/m L) SF3 V ia. Cell Density (#/m L)
Infection Kinetics were Similarin all Three Cultures
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0 24 48 72 96 120 1440
20
40
60
80
100
0
20
40
60
80
100
Cell V iability(% )
T im e (hp i)
SF1 V iability (% ) SF2 V iability (% ) SF3 V iability (% )
Similar Drop in Cell Viability
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0 24 48 72 96 120 144
15
16
17
18
19
20
15
16
17
18
19
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Average CellD iam eter (um )
Tim e (hpi)
SF1 Diam eter (um ) SF2 Diam eter (um ) SF3 Diam eter (um )
Cell Diameter Indicated that the Cultures were Equally Infected
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(Hours post infection)24 48 72 96 123 M 24 48 72 96 123 FB/C M
5.0 x10e5 cells/mL 7.5 x 10e5 cells/mL
Flask 1 Flask 2
(Cell density when infected)
Expression Profile of the Secreted, His-tagged Protein
in Flasks 1 and 2 (Western blot)
FB/C:Optimized control sample from a standard infection.
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Expression Profile of the Secreted, His-tagged Protein
in Flasks 3 (Western blot)
(Hours post infection)24 48 72 96 123 FB/C M
1.0 x10e6 cells/mL
Flask 3
(Cell density when infected)
FB/C:Optimized control sample from a standard infection.
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• New, larger BIIC stock prepared (18-vials)
• Evaluate BIIC stability:– Periodically thaw one BIIC vial.– Infect Sf9 cells and conduct a time course study
in shake flasks.– Evaluate protein expression by Western blot.
How Stable is the BIIC Stock?
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Time (hpi): M 72 96 72 96 72 96 72 96 72 96 72 96 M
BIIC Stability Determined by Protein Expression (Western blot)
Months of Storage0 1 4 11 37
The BIIC stock was stable for at least 37 months.
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Stability Study: The BIIC stock has been stable for at least 60 months.
8.0 x10e5 cells/mL 7.5x10e5 cells/mL 7.9x10e5 cells/mL
0 Months (2001)
(Cell density when infected)
49 Months (2005) 60 Months (2006)
Infection Time (hpi)24 50 72 96 124 M 24 48 72 96 120 M M 24 48 72 98 125
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How can the infected cells be used for scale-up?
1-mL
1-mL
1-mL100-mL(1:100) 100-mL
(10-5)BIIC Vial
107 Cells/mL(109 PFU/mL)
100-mL
100-mL
100-LScale-up
(10-5)
10-L Scale-up
(10-4)
100-mL(10-4)(1:100)
(1:1,000)
Test Expression
Test Expression
TIPS Direct Scale-up
From 1 mL to 10-L
TIPS Direct Scale-up
From 1 mL to 100-L
eMOI ~ 0.1
eMOI ~ 0.01
Evaluate• Cell lines• Cell densities• Culture media
Evaluate• Cell lines• Cell densities• Culture media
(1:100)(1:10)
1-mL
10-mL
Cells
Cells
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How can the infected cells be used for scale-up?
1-mL
1-mL
1-mL100-mL(1:100) 100-mL
(10-5)BIIC Vial
107 Cells/mL(109 PFU/mL)
100-mL
100-mL
100-LScale-up
(10-5)
10-L Scale-up
(10-4)
100-mL(10-4)(1:100)
(1:1,000)
Test Expression
Test Expression
TIPS Direct Scale-up
From 1 mL to 10-L
TIPS Direct Scale-up
From 1 mL to 100-L
eMOI ~ 0.1
eMOI ~ 0.01
(1:100)(1:10)
1-mL
10-mL
Cells
Cells
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Scale-up in 7-L Bioreactors (5-L Liquid) – 1-BIIC/10-L Volume Ratio
• Cells infected at approx. 8.5x10e5 cells/mL
– 1-BIIC/100-L Volume Ratio• Additional round of baculovirus amplification • Cells infected at approx. 5.5x10e5 cells/mL
– Evaluate protein expression by Western blot.
Bioreactor Scale-upUsing the 60 Month BIIC Stock
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Scale-up in 7-L Bioreactors(5-L Liquid)
1-mL
1.5-mL
10-mL100-mL(1:100) 100-mL
(10-3)
BIIC Vial107 Cells/mL(109 PFU/mL)
50-mL
50-mL
Bioreactor B5-L Scale-up
(1-BIIC/100-L)(10-5)
Bioreactor A5-L Scale-up(1-BIIC/10-L)
(10-4)
150-mL(10-4)(1:100)
(1:10)
Shaker Flask(10-4)
TIPSScale-up
1-BIIC/10-L Ratio
TIPSScale-up
1-BIIC/100-L Ratio
eMOI ~ 0.1
eMOI ~ 0.01(1:100)
(1:100)
Cells
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-24 0 24 48 72 96 120 1441.0x105
1.0x106
1.0x107
Bioreactor A (Cell Density) B ioreactor A (V iability)
T im e (Hours post infection)
V iable CellDensity m L-1
0
20
40
60
80
100
Viability(% )
Bioreactor Culture(1-BIIC/10-L Ratio)
(Cell Growth and Viability During the Infection Process)
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-24 0 24 48 72 96 120 1441.0x105
1.0x106
1.0x107
Bioreactor A (Cell Density) B ioreactor A (V iability) Shake Flask Culture (Cell Density) Shake Flask Culture (V iability)
T ime (Hours post infection)
V iable CellDensity m L-1
0
20
40
60
80
100
Viability(% )
Bioreactor Culture vs. Shake Flask Culture(1-BIIC/10-L Ratio)
(Cell Growth and Viability During the Infection Process)
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Bioreactor Culture (1-BIIC/10-L Ratio)
(Change in Cell Diameter During the Infection Process)
-24 0 24 48 72 96 120 14413.0
14.0
15.0
16.0
17.0
18.0
19.0
Bioreactor A
T im e (Hours post infection)
Cell D iam eter (um )
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Bioreactor Culture vs. Shake Flask Culture(1-BIIC/10-L Ratio)
(Change in Cell Diameter During the Infection Process)
-24 0 24 48 72 96 120 14413.0
14.0
15.0
16.0
17.0
18.0
19.0
Bioreactor A Shake Flask
T im e (Hours post infection)
Cell D iam eter (um )
Incubator temperature~ 2°C above the set point!
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Protein Expression: 1-BIIC/10-L Volume Ratio in a Flask and a 7-L (5-L Liquid) Bioreactor
7.9x10e5 cells/mL 8.6x10e5 cells/mL(Cell density when infected)
Shake Flask(2006)
Bioreactor A (2006)
Infection Time (hpi)M 24 48 72 98 125 24 48 72 98 125 M
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-24 0 24 48 72 96 120 144105
106
107 Bioreactor B (V iable Cell Density) B ioreactor B (V iability)
T im e (Hours post infection)
V iable CellDensity m L-1
0
20
40
60
80
100
Viability(% )
Bioreactor Culture(1-BIIC/100-L Ratio)
(Cell Growth and Viability During the Infection Process)
37
-24 0 24 48 72 96 120 144105
106
107
Bioreactor A (V iable Cell Density) B ioreactor A (V iability) B ioreactor B (V iable Cell Density) B ioreactor B (V iability)
T im e (Hours post infection)
V iable CellDensity m L-1
0
20
40
60
80
100
Viability(% )
Bioreactor A (1-BIIC/10-L Ratio) Bioreactor B (1-BIIC/100-L Ratio)
(Cell Growth and Viability During the Infection Process)
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-24 0 24 48 72 96 120 14413.0
14.0
15.0
16.0
17.0
18.0
19.0 B ioreactor B: Average Cell D iam eter
Tim e (Hours post infection)
Average CellD iam eter (um )
Bioreactor B (1-BIIC/100-L Ratio) (Change in Cell Diameter During the Infection Process)
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-24 0 24 48 72 96 120 14413.0
14.0
15.0
16.0
17.0
18.0
19.0
B ioreactor A: Average Cell D iam eter B ioreactor B: Average Cell D iam eter
T im e (Hours post infection)
Average CellD iam eter (um )
Bioreactor A (1-BIIC/ 10-L Ratio) Bioreactor B (1-BIIC/100-L Ratio)
(Change in Cell Diameter During the Infection Process)
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Protein Expression in 7-L (5-L Liquid) Bioreactors Bioreactor A: 1-BIIC/ 10-L Volume RatioBioreactor B: 1-BIIC/100-L Volume Ratio
7.9x10e5 cells/mL 8.6x10e5 cells/mL 5.5x10e5 cells/mL(Cell density when infected)
Bioreactor A (2006) 10-L
Infection Time (hpi)M 24 48 72 98 125 24 48 72 98 125 M 24 48 72 98 125 140 M
Bioreactor B(2006) 100-L
Shake Flask Study(2006)
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Summary
• Compared baculovirus stocks• Cell diameter:
– Monitor the infection process– Predict protein expression
• BIIC stocks are very stable• Process is readily scalable
– 1-mL to either 10-L or 100-L in one easy step!• Powerful tool
– Very flexible
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Reference
David J. Wasilko and S. Edward Lee. TIPS: TiterlessInfected-Cells Preservation and Scale-up. BioProcessing Journal 5(3): 29-32. (2006).
Available at: www.bioprocessingjournal.com
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