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Benjamin Ross-Johnsrud, Rui Tostoes, John Cushman, Robert Scott, Jack Saloio,
Shirae Leslie, Kedar Chitale, Bart Lipkens
FloDesign Sonics Inc., 380 Main Street, Wilbraham, MA 01095
T-Cell Concentration, Washing and Affinity Cell Selection using
Multi-Dimensional Standing Wave Acoustic Technology
ACW:• 85% average T-cell recovery• 95-99% protein wash• Input volume 1-2L,• Output volume 5-120mL• Input VCD 1-40M/mL• Output VCD 100-500M/mL• No viability loss• No phenotype change• Closed, GMP-ready instrument and
disposable by Q1 2019Fluidic Control Unit
System Control Unit
Disposable Kit
PROCESS OVERVIEW
20 min30 rpm
3 ml/ min, 2.25 W40 min
3 ml/ min, 2.25 W40 min
Negative Selection - Depletion of Target Cells
Outflow
SampleFlush 1
Sample
Flush
1
Buffer
Edge
Effect
CaptureAntibody – Cell Incubation
Cell LabelingFlush
Elution
SampleFlush 2
Sample
Elution
Flush
Buffer
Edge
Effect
Elution Flush
Elution
Buffer
Positive Selection - Enrichment of Target Cells
BACKGROUNDBACKGROUNDPROCESS OVERVIEW
Acoustic Concentrate-Wash (ACW) Acoustic Affinity Cell Selection (AACS)
BSA Wash Results Methods 1,2, and 3 compared (BCA assay)Method # Feed (mg/mL) Concentrate (mg/mL) Wash efficiency
1 1841.8 108.5 94%2 1908.1 18.3 99%3 1896.4 91.7 95%
Method #1 -2.5 wash cycles through inlet (265 seconds), 2.5 wash cycles through collector (265 seconds)
Method #2 - 30 Second wash through inlet, 2.5 wash cycles through supernatant (30 sec inlet + 235 supernatant), 2.5 wash cycles through collector (265 seconds)
Method #3 - 30 Second wash through inlet, 5.0 wash cycles through collector (30 sec inlet + 500 sec)
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
0.0 1.0 2.0 3.0 4.0 5.0 6.0
C/C
0
Wash Volumes
BSA Wash Method 1,2 and 3 (supernatant BCA)
Wash Method #1 Wash Method #2 Wash Method #3
94-99% wash in concentrate
RESULTSRESULTS
1E6/mL
2E6/mL
3E6/mL
5E6/mL
7E6/mL
9E6/mL
12E6/mL
20E6/mL
40E6/mL
0%
50%
100%
750 mL 1000 mL 1200 mL 1250 mL 2000 mL
Robust viable cell recovery (VCR) from 1-2L and 1-40E6/mL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
750 1500 2250 3000 3750 4500 5250 6000 6750 7500 8250 9000 9750 10500 11250 12000 12750 13500 14250 15000 15750 16500 17250 18000 18750 19500 20250 21000 21750 22500 23250 24000 24750 25500 26250 27000 27750 28500 29250 30000
800 1600 2400 3200 4000 4800 5600 6400 7200 8000 8800 9600 10400 11200 12000 12800 13600 14400 15200 16000 16800 17600 18400 19200 20000 20800 21600 22400 23200 24000 24800 25600 26400 27200 28000 28800 29600 30400 31200 32000
850 1700 2550 3400 4250 5100 5950 6800 7650 8500 9350 10200 11050 11900 12750 13600 14450 15300 16150 17000 17850 18700 19550 20400 21250 22100 22950 23800 24650 25500 26350 27200 28050 28900 29750 30600 31450 32300 33150 34000
900 1800 2700 3600 4500 5400 6300 7200 8100 9000 9900 10800 11700 12600 13500 14400 15300 16200 17100 18000 18900 19800 20700 21600 22500 23400 24300 25200 26100 27000 27900 28800 29700 30600 31500 32400 33300 34200 35100 36000
950 1900 2850 3800 4750 5700 6650 7600 8550 9500 10450 11400 12350 13300 14250 15200 16150 17100 18050 19000 19950 20900 21850 22800 23750 24700 25650 26600 27550 28500 29450 30400 31350 32300 33250 34200 35150 36100 37050 38000
1000 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000 31000 32000 33000 34000 35000 36000 37000 38000 39000 40000
1050 1050 2100 3150 4200 5250 6300 7350 8400 9450 10500 11550 12600 13650 14700 15750 16800 17850 18900 19950 21000 22050 23100 24150 25200 26250 27300 28350 29400 30450 31500 32550 33600 34650 35700 36750 37800 38850 39900
1100 1100 2200 3300 4400 5500 6600 7700 8800 9900 11000 12100 13200 14300 15400 16500 17600 18700 19800 20900 22000 23100 24200 25300 26400 27500 28600 29700 30800 31900 33000 34100 35200 36300 37400 38500 39600
1150 1150 2300 3450 4600 5750 6900 8050 9200 10350 11500 12650 13800 14950 16100 17250 18400 19550 20700 21850 23000 24150 25300 26450 27600 28750 29900 31050 32200 33350 34500 35650 36800 37950 39100
1200 1200 2400 3600 4800 6000 7200 8400 9600 10800 12000 13200 14400 15600 16800 18000 19200 20400 21600 22800 24000 25200 26400 27600 28800 30000 31200 32400 33600 34800 36000 37200 38400 39600
1250 1250 2500 3750 5000 6250 7500 8750 10000 11250 12500 13750 15000 16250 17500 18750 20000 21250 22500 23750 25000 26250 27500 28750 30000 31250 32500 33750 35000 36250 37500 38750 40000
1300 1300 2600 3900 5200 6500 7800 9100 10400 11700 13000 14300 15600 16900 18200 19500 20800 22100 23400 24700 26000 27300 28600 29900 31200 32500 33800 35100 36400 37700 39000
1350 1350 2700 4050 5400 6750 8100 9450 10800 12150 13500 14850 16200 17550 18900 20250 21600 22950 24300 25650 27000 28350 29700 31050 32400 33750 35100 36450 37800 39150
1400 1400 2800 4200 5600 7000 8400 9800 11200 12600 14000 15400 16800 18200 19600 21000 22400 23800 25200 26600 28000 29400 30800 32200 33600 35000 36400 37800 39200
1450 1450 2900 4350 5800 7250 8700 10150 11600 13050 14500 15950 17400 18850 20300 21750 23200 24650 26100 27550 29000 30450 31900 33350 34800 36250 37700 39150
1500 1500 3000 4500 6000 7500 9000 10500 12000 13500 15000 16500 18000 19500 21000 22500 24000 25500 27000 28500 30000 31500 33000 34500 36000 37500 39000
1550 1550 3100 4650 6200 7750 9300 10850 12400 13950 15500 17050 18600 20150 21700 23250 24800 26350 27900 29450 31000 32550 34100 35650 37200 38750
1600 1600 3200 4800 6400 8000 9600 11200 12800 14400 16000 17600 19200 20800 22400 24000 25600 27200 28800 30400 32000 33600 35200 36800 38400 40000
1650 1650 3300 4950 6600 8250 9900 11550 13200 14850 16500 18150 19800 21450 23100 24750 26400 28050 29700 31350 33000 34650 36300 37950 39600
1700 1700 3400 5100 6800 8500 10200 11900 13600 15300 17000 18700 20400 22100 23800 25500 27200 28900 30600 32300 34000 35700 37400 39100
1750 1750 3500 5250 7000 8750 10500 12250 14000 15750 17500 19250 21000 22750 24500 26250 28000 29750 31500 33250 35000 36750 38500
1800 1800 3600 5400 7200 9000 10800 12600 14400 16200 18000 19800 21600 23400 25200 27000 28800 30600 32400 34200 36000 37800 39600
1850 1850 3700 5550 7400 9250 11100 12950 14800 16650 18500 20350 22200 24050 25900 27750 29600 31450 33300 35150 37000 38850
1900 1900 3800 5700 7600 9500 11400 13300 15200 17100 19000 20900 22800 24700 26600 28500 30400 32300 34200 36100 38000 39900
1950 1950 3900 5850 7800 9750 11700 13650 15600 17550 19500 21450 23400 25350 27300 29250 31200 33150 35100 37050 39000
2000 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000
LVLD LVHD
VCD (E6cells/mL)
Vo
lum
e (
mL)
Vo
lum
e (m
L)
Viable Cell Density (E6/mL)
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12
Fold
ce
ll ex
pn
sio
n
Process time (days)
Post Acoustic Processing T-cell Expansion and Viability
Fold expansion Viability
Current input map (A, volume and viable cell density) and color-matching process parameters (B) to enable acoustic concentrate-wash of 1-2L and a total viable cell density of 1-40E6/mL in less than 90 minutes
A B
The viable cell recovery (A) for the process inputs and parameters above (same color coding) averaged 85% with Jurkat T-cells, with higher VCR in the center of the volume-VCD space and lower in the extremes. When primary cultures of human T-cells are processed with the ACW they will grow at their typical growth rate (B, 30h doubling time),maintain high viability and yield a 30-fold expansion in a perfused Xuri bioreactor culture.
Wash efficiency: 1-2L volume, 1-40E6/mL T-cells
AACS:• Negative selection: 10-fold reduction in contaminant cell
population and 80% cell recovery• Positive selection: >40% recovery and 2.5-fold increase in the
target population• Process time < 2h• Non-magnetic cell selection• GMP-ready disposable expected 2020• Next steps: 30B cells, 100-fold TCR+ reduction in negative
selection
Manufacturing risk:• Concentrate and wash ubiquitous in cell
processing• Often an open process step with risk of
contaminationAcoustic processing enables:• Gentle, low shear cell processing• Closed and automated system• Robust recovery• High cell density output for flexible
downstream formulation
Manufacturing risk:• Allogeneic therapies require stringent
TCR+ removal• Magnetic selection single technology,
single supplier, non-scalable and slowAcoustic processing enables:• Low shear cell processing• Closed and automated system• Fast, scalable• Non-magnetic
Positive Selection – Target Receptor CD3
% Purity
A – Purity of samples before and after positive selection via acoustic processing of 100E6 cells (Jurkats and Jurkat 376) and B – Recovery of the target population.
A – Purity of samples before and after positive selection via acousticprocessing of 1000E6 Primary T cells and B – Recovery of the targetpopulation
A
BA
0%
20%
40%
60%
80%
100%
Initial Final
% Purity
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
Initial Final
0%
20%
40%
60%
80%
100%
Processing 100E6 cells
Processing 1000E6 cells
% RecoveryB
% Recovery
A B
60%
70%
80%
90%
100%
0.27x 0.638x 1x 5x
Initial Final
Optimal Parameters
A – The effect of antibody concentration on purity of samples before and after negative selection viaacoustic processing of 100E6 primary T cells and B The effect of the number of cycles through thecolumn on the purity of samples, after negative selection via acoustic processing of 100E6 primary Tcells
Antibody Concentration Number of Cycles through Column
% Purity
60%
70%
80%
90%
100%
1 2 3 4
Purity Recovery
% Purity and % Recovery
Negative Selection – Target Receptor TCR
50%
60%
70%
80%
90%
100%
Test 1 Test 2
% PurityInitial Final
B
0%
20%
40%
60%
80%
100%
1B 3B
0%
20%
40%
60%
80%
100%
Test 1 Test 2
A
A – Purity of samples before and after negative selection via acoustic processing of 1000E6 and 3000E6 primary T cells B –Corresponding plots of the cell population from flow cytometry and C – Recovery of target cell population
0%
20%
40%
60%
80%
100%
1B 3B
A – Purity of samples before and after negative selection via acoustic processing of 100E6 primary T cells, B – Corresponding plots of the cell population from flow cytometry and C – Recovery of target cell population
BA C
Processing 100E6 cells
Processing 1000E6 and 3000E6 cells
% Purity
% Recovery
% RecoveryBefore processing After processing
1B
3B
Before processing After processing C
Test 2
Test 1
Test 2
Test 1
PROCESS MAP: 1-2L VOLUME, 1-40E6/ ML T-CELLS
Viable cell recovery vs volume and cell density Typical T-cell expansion post-ACW
A B
CONCLUSIONS
Experimental setup of the acoustic fluidized bed. Depicted is the stand (s), the column (c) and the acoustic section (a). The inset shows the column section filled with 50mm affinity beads
CONCLUSIONS
S
C
A
Region Parameters45ml/min, 5mL product45ml/min, 9mL product45ml/min, 2 draws, 12mL product45ml/min, 30mL product45ml/min, 100mL product45ml/min, 120mL product60ml/min, 5mL product60ml/min, 9mL product60ml/min, 12mL product60ml/min, 100mL product60ml/min, 100mL product60ml/min, 120mL productEstimated - 45ml/min, 30mL productEstimated - 60ml/min, 30mL productEstimated - 60ml/min, 100mL productNot yet defined
