Shear Stress Sensor for Peristaltic Pump Head as an in-line PAT Tool for

Protein Solutions Ajit S. Narang1, Vadim Stepaniuk2, Valery Sheverev2,

Mary E. Krause1, and Smeet Deshmukh1

1Bristol-Myers Squibb, Co., New Brunswick, NJ 2Lenterra, Inc., Newark, NJ

AAPS-NBCMay 2016

M1036

AbstractPurpose: Shear stress during processing can impact protein stability. Closed loop pumping of protein solutions, especially during tangential flow filtration, can impact the stability of sensitive proteins. Although shear stress rates have been modeled in tubular flow systems, direct, experimental measurement of the rate of shear stress on the protein in a peristaltic pump head has beenchallenging. In this study, we demonstrate the application of wall shear stress (WSS) sensor in quantitating shear experienced by viscous aqueous solutions in the peristaltic pump head.

Methods: An F-series Lenterra’s RealShear™ stress sensor was adapted to measure wall shear stress on the wall of plastic tube within and outside Flexicon PF6 peristaltic filling machine (Figure 1). Water (1cP viscosity) and two PEG (20,000 molecular weight) water solutions (6 w% and 10 w%) with viscosities of 7 cP and 13 cP, respectively, were used in tests. Also, some tests were carried out without any fluid, effectively pumping air. Flexicon PF6 peristaltic filling machine motor speed was in the range from 30 RPM to 250 RPM. WSS sensor response was recorded during pumping of three different fluids at a range of RPMs. The WSS measurement rate was held at 500 Samples/s throughout the tests. Flow rates were determined by measuring the weight of the fluid accumulated in a beaker in a set time interval. Pulse amplitude data was analyzed for various RPM for different fluids, both for the setup where the sensor was inside and outside the pump.

Results: All raw data plots showed periodic structure with pulses corresponding to successive roller occurrences. The tests demonstrated that the flow properties inside the peristaltic pump are of complex nature. The measured values of WSS are much greater than those estimated from the fully developed flow model. It was shown that these high values of WSS could not be explained by probe vibration and movement experienced during the tests. The temporal dependence of WSS was found to be different for fluids with different viscosities.

Conclusions: Measured magnitudes of the generated WSS pulses (several hundreds Pa) were significantly higher than the expected wall shear stress in a circular cross-section in fully developed flow (a fraction of Pa for realized flow rates). The shape of the pulse varied for different fluids: for lower viscosity fluids the pulse had a shape of two partly overlapping peaks with earlier peak being higher than the later one, and for higher viscosity fluids the pulse shape showed a single peak with relatively slowly rising front edge and a sharp back edge. In addition, reversal of the flow direction was observed in the pipe that is being compressed by theroller. Understanding of the flow patterns and the wall shear forces inside the plastic tubing in the pump can help understand the stresses experienced by protein solutions during processing.

2

Purpose: Shear stress during processing can impact protein stability

http://www.emdmillipore.com/US/en/product/Amicon-Ultra-0.5%C2%A0mL-Centrifugal-Filters-for-DNA-and-Protein-Purification-and-Concentration,MM_NF-C82301http://www.pall.com/main/laboratory/literature-library-details.page?id=34212

Normal flow filtration (NFF) Tangential Flow Filtration (TFF)

33

Effect of process shear on aggregation and viscosity after accelerated storage

0 10 20 30 40 500

10

20

30

40

storage temp (οC)

Visc

osity

(cP

)

0 10 20 30 40 500

5

10

15TFFNFF

storage temperature (οC)

%H

MW

S

Processing lead to increased aggregation and higher viscosity(TFF > NFF)

Protein was concentrated to 140 mg/L using an NFF process and a TFF process, then placed on station for 3 month

Viscosity Aggregation

4

F i L t R lSh ™ ll h t F-series Lenterra RealShear™ wall shear stress sensorProbe:

Sensor directly measures wall shear stress induced by fluids on construction walls in mixers, pipes, turbines, pumps, extruders and other devices

Probe is mounted flush with an inner surface of the flow channel wall to provide in line measurement with noprovide in-line measurement with no disruption of process flow

Measures both wall shear and temperaturep

Detection principle: Two optical strain gages, Fiber Bragg Gratings (FBG), are affixed to the

opposite sides of the cantileveropposite sides of the cantilever The FBG assembly detects a minute deflection of the tip of the pin (< 10

nm) and which is related to equivalent force on the tip Directional measurement – measures wall shear stress projection on the

5

p jplane formed by FBGs

Setup of stress sensor with peristaltic pump

Cross-section of plastic tubing t-junction and RealShear™ sensor

Sensing surfaceof the sensor

Sensor

Plastic tubing t-junction

Optical fibers connected to optical interrogator

Hermeticallysealed

Movable part of tube bridge

Rollers

T-junction tubing

Wall shear stress sensor

An F-series Lenterra’s RealShear™ stress sensor was adapted to measure wall shear stress (WSS) on the wall of plastic tube within and outside Flexicon PF6 peristaltic

filling machine

6

Sensor calibration: vibration evaluation

Time, s

4.2 4.4 4.6 4.8 5 5.2 5.4 5.6

Sen

sor

resp

onse

, Pa

-100

-80

-60

-40

-20

0

20

40

60

80

100Sensor response vs. time

Air: Movable part of the bridge was pushed up at ~4.3s and released at ~4.6s

Oscillation frequency: 76±1 Hz

Water: Movable part of the bridge was pushed up at ~12.15s and released at ~12.65s

Free mechanical oscillations of the probe floating element

Time, s

12 12.2 12.4 12.6 12.8 13 13.2 13.4

Se

nso

r re

spo

nse

, P

a

-100

-80

-60

-40

-20

0

20

40

60

80

100Sensor response vs. time

Oscillation frequency: 77±1 Hz.

While the sensor oscillation might generate some false signal in the sensor output, the signal shapes and levels observed in tests with PF6 are different

from calibration testing; cannot be explained by sensor shaking alone.

• In the flow path, complex probe motion is observed, with vertical andlateral displacements.

7

Method development: Studies with PEG and water

3 solutions tested at 30, 50, 75, 100, 150, 200,

and 250 rpm:Water

6% PEG (7 cP) 10% PEG (13 cP)

0

5

10

15

20

25

30

35

0 50 100 150 200 250 300

Flow

rate

, ml/s

Pump RPMWater PEG 7cP PEG 13 cP

Flow rate was measured for three different solution at different pump speeds

8

Calculation of force pulse magnitude (FPM)

• FPM - difference between the raw signal at a maximum value and the preceding minimum.• Characterizes passing of one roller under the probe pin.

FPM1 FPM2FPM3 FPM4

Blade 2 Blade 3Blade 1 Blade 4

FPM1 FPM2FPM3 FPM4

Blade 1 Blade 2 Blade 3 Blade 4

9

Pulse shape: sensor in tubing downstream of the pump

Time, s

19.8 19.85 19.9 19.95 20 20.05 20.1 20.15 20.2

WSS

, Pa

-50

-40

-30

-20

-10

0

10

20

30

40WSS vs. time

WaterTime, s

20.1 20.15 20.2 20.25 20.3 20.35 20.4 20.45 20.5

WSS

, Pa

-50

-40

-30

-20

-10

0

10

20

30

40WSS vs. time

PEG 7 cP

Time, s

19.9 19.95 20 20.05 20.1 20.15 20.2 20.25 20.3

WSS

, Pa

-50

-40

-30

-20

-10

0

10

20

30

40WSS vs. time

PEG 13 cP

Sensor was placed in tubing downstream of the pump and WSS was monitored

Periodic structure - pulses correspond to successive roller occurrences.

Pulse shape varies for fluids with different viscosities

10

Pulse shape: sensor inside the pump

Time, s

20.1 20.15 20.2 20.25 20.3 20.35 20.4 20.45 20.5

WSS

, Pa

-350

-300

-250

-200

-150

-100

-50

0

50

100

150

200WSS vs. time

PEG 7cP

Time, s

20 20.05 20.1 20.15 20.2 20.25 20.3 20.35 20.4

WSS

, Pa

-350

-300

-250

-200

-150

-100

-50

0

50

100

150

200WSS vs. time

PEG 13 cP

Time, s

20 20.05 20.1 20.15 20.2 20.25 20.3 20.35 20.4

WSS

, Pa

-350

-300

-250

-200

-150

-100

-50

0

50

100

150

200WSS vs. time

Water

High frequency oscillations observed in water are due to resonant oscillations of

the sensor’s floating element.

Pulse shape varies for fluids with different viscosities

Sensor was placed in the peristaltic pump and WSS was monitored

11

Impact of flow rate and viscosity on magnitude of WSS within the pump

• Magnitudes of WSS minimum and maximum are higher for fluids with lower viscosity (water)

• In general, minimal systematic change with flow rate.

050

100150200250300350

0 5 10 15 20 25 30 35

Min

(abs

val

ue),

Pa

Flow rate (mL/s)

Minimum (plotted as absolute value)

Water PEG 7cP PEG 13cP

0

50

100

150

200

250

300

350

Max

, Pa

Flow rate (mL/s)

Maximum

Water PEG 7cP PEG 13cP

• With the sensor in the peristaltic pump head, fluid was pumped for 1 minute with monitoring (triplicate for each fluid/flow rate)

• Data presented are averages of 10 roller occurrences from each of 3 pumping events.

12

Impact of flow rate and viscosity on FPM within the pumpFP

M -f

orce

pul

se m

agni

tude

FPM is higher for fluids with lower viscosity (water).

FPM decreases (slight) with increasing flow rate.

050

100150200250300350400450

0 5 10 15 20 25 30 35

FPM

, Pa

Flow rate (mL/s)

Water PEG 7cP PEG 13cP

13

Impact of flow rate and viscosity on magnitude of WSS in tubing

Higher WSS with lower viscosity

Higher WSS with increasing flow rate

020406080

100120

0 10 20 30 40

Min

, Pa

(abs

olut

e va

lues

)

Flow rate (mL/s)

Minimum (plotted as absolute value)

PEG 13cP PEG 7cP Water

020406080

100120

0 10 20 30 40M

ax, P

aFlow rate (mL/s)

Maximum

PEG 13cP PEG 7cP Water

• With the sensor in tubing downstream of the pump, fluid was pumped for 1 minute with monitoring (triplicate for each fluid/flow rate)

• Data presented are averages of 10 roller occurrences from each of 3 pumping events.

14

Impact of flow rate and viscosity on FPM in tubing

FPM increases with increasing flow rate

FPM is higher for fluids with lower viscosities

0

50

100

150

200

250

0 5 10 15 20 25 30 35

FPM

, Pa

Flow rate (mL/s)

PEG 13cP PEG 7cP Water

15

Summary of water/PEG studies: Fluid viscosity and flow rate impact shear

• Flow properties inside the peristaltic pump are of complex nature.

• The roller generates a negative pulse in all liquids and in air. • Floating element of the sensor moves against the flow direction in the pipe.• The observed reversal of the flow direction in the pipe that is being

compressed by the roller is not immediately obvious.

• The measured values of WSS are much greater than those estimated from the fully developed flow model.

• In general, WSS is higher for fluids with lower viscosities (water). Further understanding of FPM trends is needed

• WSS dependence on flow rate is different in the pump head (slight decrease with increased flow rate) and in the tubing downstream of the pump head

16

Application to protein system

• Protein was tested in different formulations with different viscosities

• pH 4.0• pH 4.0 + 60% sucrose (for high viscosity)

• Tests were only performed with the probe inside the peristaltic pump

• Measurements were recorded for one minute (in triplicate) at each flow rate

• Maximum, minimum, and FPM values were found for each roller

17

Pulse shape changes with protein formulation

Time, s

14.7 14.75 14.8 14.85 14.9 14.95 15 15.05 15.1

WS

S, P

a

-100

-50

0

50

100

150WSS vs. time

pH 4 sucroseTime, s

14.7 14.75 14.8 14.85 14.9 14.95 15 15.05 15.1

WS

S, P

a

-100

-50

0

50

100

150WSS vs. time

pH 4

• Pulse shape is different for sample with and without sucrose (effect of viscosity)

• Pulse shape is also modified in the presence of protein • pH 4 protein formulation has different pulse shape than water

(similar viscosity)

• Viscosity may not be the only factor impacting shear 18

Impact of protein on how fluid viscosity and flow rate effect shear

0.050.0

100.0150.0200.0250.0300.0350.0

0 10 20 30 40

Min

WS

S, P

a

Flow rate (mL/s)

Minimum (plotted as absolute values)

pH 4 sucrose pH 4

0.050.0

100.0150.0200.0250.0300.0350.0

0 10 20 30 40

Max

WS

S, P

aFlow rate (mL/s)

Maximum

pH 4 sucrose pH 4

• Magnitude of maximum/minimum WSS increases with increasing flow rate.

• Magnitude is higher with higher viscosity.

Data presented are averages of 10 roller occurrences from each of 3 pumping events.

19

Impact of protein on how fluid viscosity and flow rate effect shear

0.0

100.0

200.0

300.0

400.0

500.0

600.0

0 5 10 15 20 25 30 35

FPM

, Pa

pH 4 sucrose pH 4.0

For protein solutions, FPM increases with both increasing flow rate and with higher viscosity

20

Impact of shear on protein: degradation experiments

0

50

100

150

200

250

300

350

400

0:00 1:12 2:24 3:36 4:48 6:00

FPM

, Pa

Elapsed time, h:min

Sucrose pH4

pH4

Continuous pumping (6 mL/s) and monitoring every 15 min over 6 hours

See Poster M1037

• Shear impacts protein conformation (see poster M1307)

• Sucrose has a protective effect against shear stress21

Conclusions: Viscosity may not be the only factor impacting shear

• Fluid viscosity and flow rate impact shear differently in the presence and absence of protein

• Further understanding of FPM trends observed in the absence of protein is needed

• Shear impacts protein conformation

In the absence of protein: • FPM decreases with increasing flow

rate

• FPM is lower with higher viscosity

In the presence of protein:• FPM increases with increasing flow

rate.

• FPM is higher with higher viscosity.

For solutions with similar viscosity:

22