Application Note

A Hands-On Guide to Ultrafiltration/

Diafiltration Optimization using
Pellicon Cassettes
In ultrafiltration (UF) tangential flow filtration (TFF)
systems, operating parameter selection will have far
reaching impact as the process is scaled to full-scale
manufacturing levels. While there are many factors that
contribute to final system design, several key parameters
should be optimized early in the process development
phase. The goal is to develop a robust process with the
following success criteria: superior product quality,
consistent and high product yield, reproducible process
flux and time, and a cleaning regime that allows extended
membrane reuse.
The following basic experiments should be considered
during development of processing methodology:
Optimization
Impact of transmembrane pressure (TMP) and feed

flow on process flux and retention

Impact of product concentration and buffer

conditions on process flux and retention

Impact of diavolumes on buffer exchange and

contaminant removal
Paper design and full process simulation with chosen

processing parameters
Typically, the first three experiments are performed
sequentially to bracket process performance and obtain
data for analysis. This information is then combined with
actual manufacturing considerations (batch volume,
process time, etc.) to design a process simulation.

The purpose of a process simulation is to duplicate the

entire manufacturing process in a scale-down format, to
confirm sizing, and to assess preliminary product quality
and yield. The intent is to develop an optimized process,
on the bench, that will efficiently scale-up to meet fullscale manufacturing expectations.
Sequence
1. T MP Excursion at Initial Concentration
(Cb initial)

2. C
 oncentration / Volume Reduction
(Cb initial Cb final)

3. T MP Excursion at Final Concentration

(Cb final)

4. D
 iafiltration / Buffer Exchange

5. P roduct Recovery

Purpose
Determine TMP for UF/DF
Determine Feed Flow (QF) for UF/DF
Demonstrate Flux Stability
Confirm Retention of Product
Determine Flux as Function of Concentration
Determine Placement of Diafiltration Step
Determine Flux as Function of Buffer Conditions
Determine TMP for UF/DF
Determine Feed Flow (QF) for UF/DF
Confirm Retention of Product
Determine Diavolume Requirement
Confirm Retention of Product during DF
Crude Assessment of Step Yield
Product Quality Evaluation

Figure 1. Basic Optimization Experiments

Use this step-by-step guide to develop a robust UF/

DF process with Pellicon cassettes (cutoffs of 100 kD
and lower) that will deliver superior product quality,
reproducible results, and high yields.

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

The following are step-by-step protocols for basic optimization experiments.

Set-up and Installation Procedure

Equilibration Procedure

Refer to the Maintenance Procedures for Pellicon and

Pellicon 2 Cassette Filters (P17512) or the Pellicon 3 Filters
Installation and User Guide (AN1065EN00) when performing
actual set-up and installation of Pellicon cassettes.

1. Add 3 L/m2 of the appropriate buffer to the feed tank.

1. Assemble the TFF system as shown in Figure 2.

with 0.1 m2 membrane area, Pellicon 3 with 0.11 m2

membrane area) in the appropriate Pellicon holder.
appropriate cleaning agent (per appropriate
maintenance guide), and flush again.
QR

PR

5. Direct the retentate and permeate to a waste

container.
6. When the feed tank level reaches the minimum level,

PF
Permeate

Feed

QP

Filtration
Module

Feed
Pump

Retentate pressure of 215 psi (0.141.03 bar) to

in total recycle mode1 and recirculate for 10 minutes;

verify that the pH and conductivity in the system have
been equilibrated to the level of the starting buffer.

PP
QF

Feed flow of 5 L/min/m2

4. When half the buffer has been flushed, put the system

Retentate
Retentate
Valve

Feed
Tank

3. Start the feed pump and achieve the following

achieve approximately 30% conversion

3. Flush the system with water, clean with the

Diafiltration
Buffer

2. Direct the retentate and permeate to a waste container.

conditions by partially closing the retentate valve and

adjusting the pump speed:

2. Install the Pellicon cassette(s) (Pellicon 2 Mini

Figure 2. Schematic
of a TFF System

Example: 0.1 m2 membrane area x 3 L/m2 = 0.3 L buffer

open the retentate valve fully and stop the feed pump
to prevent the introduction of air into the system.

Part 1. TMP Excursion at Initial Concentration

1. Add sufficient volume of product to the feed reservoir

such that final volume or concentration target can be

reached or slightly exceeded (approximately 11.5 L
of final product at final concentration per m2).
Example: if Cinitial = 10 g/L and Cretentate = 80 g/L,
then the concentration factor is 8X. If the minimum
achievable final volume for 0.1 m2 is 0.1 L, calculate the
required initial volume:
Vinitial = Vminimum x VCF = 0.1 L x 8X = 0.8 L
2. Open the retentate valve fully and configure system in

total recycle mode.

conditions by partially closing the retentate valve
and adjusting the pump speed:
Recommended feed flow (QF) rate for the membrane

device, typically 5
cassettes

that stable process flux is achieved2.

5. Record temperatures, pressures, and flows; sample

feed and permeate for product retention3.

6. Increase TMP by 510 psid (0.340.69 bar) by

manipulating the retentate valve while keeping the feed

flow constant. For more open membranes increase by
25 psid (0.140.34 bar). Repeat steps 4 and 5.
7. Repeat step 6 until flux begins to level off4;

typically 46 TMP values are evaluated in total.

8. Open the retentate valve fully and allow system to

3. Start the feed pump and achieve the following

L/min/m2

4. Recirculate the product for 1015 minutes and ensure

for Pellicon 2 and 3

continue in a total recycle.

9. Increase or decrease the feed flow by 23 L/min/m2

and repeat steps 4 through 8. If desired, a third feed

flow rate can be investigated.
10. Plot the data as shown in Figure 3.

Minimal TMP, typically 25 psi (0.140.34 bar) for

more open membranes and 10 psi (0.69 bar) for

tighter membranes.
Tight membranes (1 kD, 5 kD, etc.)
Open membranes (50 kD, 100 kD, etc.)
2

Can use large TMP increases since optimum is typically >30 psi
Can use small TMP increases since optimum is typically <10 psi

200
160
Flux (LMH)

Feed Flux = 5 L/min/m2

Feed P = 20 psid

Optimum Point
TMP = 30 psid
Jf = 150 LMH

Feed Flux = 3 L/min/m

Feed P = 10 psid

120
80

Figure 3.
TMP Excursion at
Two Feed Flows

Optimum Point
TMP = 25 psid
Jf = 86 LMH

40
0
0

10

20

30

40

50

TMP (psid)

[m2]

A
QF [L/min]

Q1 = 5 L/min/m2

Q2 = 3 L/min/m2

Q1/Q2

Volume / Time / 150 LMH

(5 L/min/m2) x Volume / Time / 150 LMH

Volume / Time / 86 LMH

(3 L/min/m2) x Volume / Time / 86 LMH

0.57
0.95

Table 1. Membrane
Area vs. Pump Feed
Rate (Figure 3)

Calculations
The appropriate combination of feed flow rate and
TMP will maximize flux while minimizing the impact of
pumping and shear on the product. The appropriate
combination of these two parameters will also minimize
processing time and/or membrane area. To calculate the
optimum feed flow, compare the required membrane area
with the required pump rate at each of the two feed flow
conditions, as shown in Table 1.
[m2]

Membrane Area
=
Process Volume [L] / (Flux [LMH] x Process Time [h])
In Figure 3:
AreaQ1 = 0.57 x AreaQ2
Pump feed rate [L/min] =
Feed flux [L/min/m2] x Area [m2]
In Figure 3:

Pump feed rateQ1 = 0.95 x Pump feed rateQ2

In this example it is advantageous to run at the higher

feed flow, Q1, since it only requires 57% of the membrane
area used at the lower feed flow rate at almost the
identical pump feed rate.

Note:
Anticipated final volume of over-concentrated product must exceed

minimum working volume of membrane system at selected feed flow rate

(QF); avoid introduction of air and maintain uniform mixing at end of volume
reduction.
Move from least to greatest fouling conditions:
Do not test into pressure-independent regime (past the knee of the flux

vs. TMP curve)4

Avoid exceeding 3040% conversion ratios
Check hysteresis if possible by returning the system to the initial conditions

and taking a final flux measurement; compare initial flux performance to

final flux performance at initial conditions.
Ensure that choice of TMP and feed flow have corresponding retention

values that are acceptable (>0.998) at both initial and final product
concentration and in each buffer5.
There is often very little performance difference versus feed flow rate at low

product concentration. However, at the higher concentrations that will be

investigated in Parts 2 and 3, the benefits of different feed flow rates should
become more pronounced.

Part 2. Concentration
1. Use the product from Part 1 in the starting buffer.

Based on desired final product concentration factor,

add additional feed volume as needed to ensure
sufficient volume at end of concentration6.
2. Sample feed to confirm product concentration.
3. Put the system in total recycle.

7. Concentrate slightly beyond desired final product

concentration.
8. Repeat the TMP excursion outlined in Part 1 to

determine optimum TMP at the final concentration in

the starting buffer.
9. Diafilter with one diavolume to get product into

4. Start the feed pump and achieve the optimum TMP and

feed flow as determined in Part 1 by partially closing

the retentate valve and adjusting the pump speed.
5. Direct the permeate to a separate container to

concentrate product and reduce volume.

final buffer and dilute with final buffer back to initial

concentration.
10. Repeat the TMP excursion to determine the optimum

TMP at the initial concentration in the final buffer.

11. Repeat Part 2 steps 27 once in final buffer using the

6. Record temperatures, pressures, and flows throughout

the concentration; sample feed and permeate for

product retention7.

optimum TMP as determined above.

12. Plot the data as shown in Figure 4, remembering

to apply a temperature correction in the flux

calculations8.
Starting Buffer
Diafiltration Buffer

Flux (LMH)

Figure 4. Flux vs.

Concentration

0.1

10
Product Concentration [g/L]

100

1000

Calculations
The tradeoff between flux and diafiltration buffer volume
create an optimum bulk concentration at which to
perform diafiltration; this can be calculated using the DF
optimization parameter at each data point:

Plotting the DF optimization parameter as a function of

product concentration yields the optimum concentrations
for diafiltration in both the starting and final buffers, as
shown in Figure 5.

DF Optimization Parameter =
Concentration [g/L] x Flux [LMH]

DF Optimization Parameter

Final Buffer
Copt = 55.0 g/L

Starting Buffer
Copt = 29.0 g/L

1000

Starting Buffer
Diafiltration Buffer

800

Figure 5. DF
Optimization

600
Optimum
Cb for DF

400
200
0
0

20

40

60

80

100

120

Product Concentration [g/L]

There is an alternative approach that may be used
to calculate the optimum concentration at which to
perform diafiltration (Copt). It assumes that the product
is completely retained and that the passage of the
permeating species is constant.
If the flux versus concentration data is plotted as
shown in Figure 4, then the gel concentration, Cg, is the
concentration at which the permeate flux reaches zero
(example: ~80 g/L in the starting buffer, ~110 g/L in the
final buffer). The optimum concentration at which to
perform diafiltration is then calculated as9:
Copt [g/L] = Cg [g/L] / e

In Figure 4:
Starting buffer Copt = 80 / 2.71828 = 29.4 g/L
Final buffer Copt = 110 / 2.71828 = 40.5 g/L

The C g/e method can only be used when the flux vs.
concentration data allows for accurate extrapolation
to zero flux.

Note:
Ensure enough feed material and appropriate system working volume in

order to achieve the final concentration.

Based on the results of the additional TMP excursions performed in Part 2,

the TMPs used for concentration in both the starting and final buffers
should be changed and the concentration should be repeated to obtain more
accurate data.
If the optimum TMP for the dilute solution occurs in the pressure-

independent region (past the knee of the curve) for the concentrated
solution, then the TMP should be decreased to the lowest optimum value.
If the optimum TMP for the dilute solution occurs within the pressure-

dependent region (before the knee of the curve) for the concentrated
solution, then the TMP may be increased to the highest optimum value
to further optimize the flux and reduce the processing time.
Optimum concentration for diafiltration will be different for each buffer;

choose an average or the most conservative.

Restrictions on buffer usage or minimum recirculation volume often

dictate the concentration at which diafiltration occurs.

If the required final concentration is significantly less than the optimum

concentration for diafiltration, over concentration followed by dilution is

a possible option, although rarely chosen. It should only be considered in
cases where diafiltration buffer is limited and the product is stable at the
higher concentrations.

Part 3. TMP Excursion at Final Concentration

1. Use the product from Part 2 at the final concentration in

the final buffer.

Calculations
Reference Part 1.

2. Repeat steps 210 of Part 1.

Note:
Reference Part 1 and Part 2 notes.

Part 4. Diafiltration
1. Use the product from Part 3 at the optimum

Use 35 diavolumes as an initial estimate

concentration for diafiltration; dilute as needed using

the final buffer.
2. Configure the system for constant volume diafiltration.
3. Start the feed pump and achieve the optimum TMP and

feed flow as determined in Part 1 and Part 3.

4. Diafilter the product with the chosen number of

diavolumes:
Choose the number of diavolumes based on the

for upstream UF/DF steps, or

Use 712 diavolumes as an initial estimate for

final formulation UF/DF steps

5. Record temperatures, pressures, and flows at every

diavolume; sample feed and permeate for both product

retention, and retention and concentration of the
contaminant of interest.
6. Plot the data as shown in Figure 6.

product purity specifications (if known, see calculation

below) and add a safety factor of 2 diavolumes, or
100
Contaminant Retained
[% of original]

Figure 6. contaminant
removal vs. Diavolumes

10
1
R = 0.4

0.1

R = 0.2

0.01

0.001

R=0
0

Diavolumes

10

15

Calculations
The percentage of the original contaminant in the retentate
at each diavolume can be calculated from the retention
values using the following:

Remaining Contaminant [%] = 100 x e (Retention1) x N

diafiltration steps, such as final formulation. For upstream

steps, add 12 diavolumes. If the goal of diafiltration is not
to wash out a contaminant but rather to reach a target pH
or conductivity, then the measurement of that quality can
be plotted against the number of diavolumes instead.

where N is the number of diavolumes.

However, since contaminant concentration is being directly
measured in each feed sample throughout diafiltration, plot
these concentrations as a percentage of the original and
use the above equation to plot several lines of theoretical
retention, as shown in Figure 6. This plot will help demonstrate
the contaminant removal at various retentions.
Select the whole number of diavolumes based on the
acceptable contaminant levels for the product; always
add 23 diavolumes as a 10-fold safety factor for critical
6

Note:
If it appears necessary to diafilter past ~14

diavolumes, any dead-legs or poor mixing areas in

the system will increase the apparent retention of the
contaminant and make further removal difficult.
Ensure that choice of TMP and feed flow have

corresponding product retention values that are

acceptable (>0.998) throughout diafiltration.

Part 5. Product Recovery

There are various methods for product recovery at
large-scale10. However, at small-scale, sufficient product
recovery can be achieved by manually tilting the system
and breaking the piping at low-points to drain the product.
Samples of the final retentate should then be analyzed for
product concentration and quality.
1. After the product has been drained from the system, add

one system volume of diafiltration buffer to the

feed tank.

2. Recirculate at the selected feed flow rate with

the retentate valve fully open for 10 minutes.

3. Recover the buffer in a separate container using the

same methods that were used to recover the product.

Samples of this buffer rinse should be analyzed for
product concentration.
4. After the product is recovered, the system should be

cleaned with the appropriate solutions11.

Calculations
Ideally, the total product mass recovered in the retentate,
permeate, and buffer flush as well as unrecoverable
holdup volume should equal the total mass of product in
the feed. If the total product mass recovered is less than
the initial product mass, it is typically due to adsorption
and/or solubility losses during processing12. However, it is
important to perform a mass balance and calculate total
yield to ensure optimum process parameters.

The theoretical yield can also be calculated based on the

membrane retention and compared to the actual yield.

Theoretical Yield [%] = 100 x e (Retention1)(N + lnX)

where N = number of diavolumes and X = concentration

factor.

Actual Yield [%] =

100 x (Vretentate [L] x Cretentate [g/L]) / (Vinitial [L] x Cinitial [g/L])

Mass Balance [%] =

100 x {(Vretentate [L] x Cretentate [g/L]) + (Vpermeate [L] x Cpermeate [g/L]) + (Vrinse [L] x Crinse [g/L])} / (Vinitial [L] x Cinitial [g/L])

Note:
All calculations are estimates; during these optimization steps,

the product has undergone more processing than normal.

Product degradation and yield may be slightly affected. For a
true indication of processing on product quality, perform the
entire optimized process using fresh feed and new membranes.
Product can be very viscous when recovered and may affect

assays; perform serial dilutions for more accurate assay results.

Actual yield and mass balance percentages should be close

to 100% and/or theoretical yield. If significant losses occur,

process parameters (including membrane type) may have to
be changed and then re-optimized.
In a robust process, adsorption and solubility losses should

be very low.

Paper Design and Process Simulation

The optimization parameters obtained from the previous
experiments can be combined to design a full process
simulation: concentration, diafiltration, (concentration,)
and recovery. If time permits, a process simulation should
be run immediately following the optimization work, and
should employ the following:
New set of cassettes; same membrane type,

same cassette path length

After performing the process simulation, the system

should be cleaned with the appropriate solution according
to EMD Millipore recommendations11. If possible, the
process should be rerun using the cleaned membranes to
determine the effectiveness of the cleaning cycle and the
consistency of membrane performance from run-to-run.
If the cleaning cycle does not prove effective, the cleaning
parameters or cleaning solutions will need to be changed
and the cleaning cycle will have to be tested again.

Fresh feedstock
Fresh buffer(s)
Optimized process parameters
See detailed process simulation calculations below.

Calculations
The membrane area can be optimized to allow the
entire process (both concentration and diafiltration) to
be completed in the specified timeframe (34 hours is
recommended). The average flux for each concentration
and diafiltration step can be estimated from the
optimization data and combined with the desired volumes
to be processed. The approximate required membrane
area can then be calculated for both manufacturing scale
and scale-down runs.
Assume an example process scenario (this would have
been determined by optimization data, DF parameter, etc.):
2.9X Concentration:

10 g/L to 29 g/L; flux decreases from 150 LMH to 80 LMH

7X Diafiltration:

29 g/L; flux increases from 80 LMH to 85 LMH

3.4X Concentration:

29 g/L to 100 g/L; flux decreases from 85 LMH to 20 LMH

Desired process time is 4 hours

Manufacturing scale volumes as determined by the

customer:
Feed volume = 5000 L
Retentate volume at end of 2.9X concentration =

5000 L/2.9 = 1724 L

Permeate volume removed during 2.9X concentration =

5000 L1724 L = 3276 L

7X Diafiltration buffer volume = 7 x 1724 L = 12,068 L
Retentate volume at end of 3.4X Concentration =

1724 L/3.4 = 507 L

Permeate volume removed during 3.4X concentration =

1724 L507 L = 1217 L

Average process flux for concentration step13:

Javg = Jfinal + 0.33 (JinitialJfinal) = Jinitial x 0.33 + Jfinal x 0.67
For 2.9X concentration:
Javg = 150 LMH x 0.33 + 80 LMH x 0.67 = 103 LMH
For 3.4X concentration:
Javg = 85 LMH x 0.33 + 20 LMH x 0.67 = 41 LMH
Average process flux for diafiltration step:
For diafiltration the average flux can be estimated as the
initial and final process flux during the diafiltration step.
Required area:
Area = [(Permeate volume/Average flux) Concentration + (Permeate volume/Average flux) Diafiltration + ] / Time
In this example:
Area = [(3,276 L/103 LMH) + (12,068 L/83 LMH) + (1,217 L/41 LMH)] / 4 hours = 51.6 m2
Add 20% safety factor:
Area = 62 m2
To perform a scale-down process simulation, the same
volume to area ratio is used and scaled based on either the
feed volume that can be used for the simulation or the area
of the desired filtration device. For example, if the process
is to be performed on one Pellicon 2 Mini cassette (with
an area of 0.1 m2), then the required feed volume will be:

Scale-down feed volume = 0.1 m2 x (5000 L/62 m2) = 8 L

Instead, if there is a specific volume of feedstock to process

(example: 25 L), then the required membrane area will be:

Scale-down membrane area = 25 L x (62 m2/5000 L) = 0.3 m2

The process parameters, including Pellicon device type,

should be consistent between scales, allowing the process
to be completed in a similar timeframe with similar fluxes,
pressures and loadings. The concentration factors, number
of diavolumes and feed quality should be kept consistent
at all scales to ensure robust scalability. However, to
demonstrate process robustness and repeatability, the
process should be tested at pilot scale before proceeding to
manufacturing.

Definitions
Apparent Sieving (Sapp)
The fraction of a particular protein that passes through
the membrane to the permeate stream based on the
measurable protein concentrations in the feed and permeate streams. A sieving coefficient can be calculated for
each protein in a feedstock.
Sapp [] = (Concentration in permeate, CP) /
(Concentration in feed, Cb)
Concentration Factor (CF)
The amount that the product has been concentrated in
the feed stream. This depends on both the volume concentration factor and the retention. As with the VCF, for a
Fed-Batch concentration process, calculate CF based only
on the volume of feedstock added to the TFF application.
CF [] = Final product concentration /
initial product concentration = VCF(Rapp)
Conversion Ratio (CR)
The fraction of the feed side flow that passes through the
membrane to the permeate.
CR [] = QP /QF
Diavolume (DV or N)
A measure of the extent of washing that has been performed during a diafiltration step. It is based on the volume of diafiltration buffer introduced into the unit
operation compared to the retentate volume. If a constant-volume diafiltration is being performed, where the
retentate volume is held constant and diafiltration buffer
enters at the same rate that permeate leaves, a diavolume
is calculated as:
DV or N [] = Total buffer volume introduced
to the operation during
diafiltration/retentate volume
Intrinsic Sieving (Si)
The fraction of a particular protein that passes through
the membrane to the permeate stream. However, it is
based on the protein concentration at the membrane surface. Although it cannot be directly measured, it provides
a better understanding of the membranes inherent separation characteristics.
Si [] = (Concentration in permeate, CP) /
(Concentration at membrane wall, Cw)

10

Mass Flux (Jm )

The mass flow of protein through the membrane normalized for the area of membrane (m2) through which it is
passing.
Jm [g m2 h1] = QP x CP / area
Permeate Flux (Jf )
The permeate flow rate normalized for the area of membrane (m2) through which it is passing.
Pressure Drop (P)
The difference in pressure along the feed channel of the
membrane from inlet to outlet.
P [bar] = PFPR
Retention (R)
The fraction of a particular protein that is retained by the
membrane. It can also be calculated as either apparent or
intrinsic retention. Retention is often also called rejection.
Rapp [] = 1Sapp or Ri = 1Si
Transmembrane Pressure (TMP)
The average applied pressure from the feed to the permeate side of the membrane.
TMP [bar] = [(PF + PR)/2] - PP
Volume Concentration Factor (VCF or X)
The amount that the feed stream has been reduced in
volume from the initial volume. For instance, if 20 L of
feedstock are processed by ultrafiltration until 18 L have
passed through to the permeate and 2 L are left in the
retentate, a ten-fold concentration has been performed
so the Volume Concentration Factor is 10. In a Fed-Batch
concentration process, where the bulk feedstock is held in
an external tank and added to the TFF operation continuously as permeate is removed, VCF should be calculated
based only on the volume that has been added to the TFF
operation.
VCF or X [] = Total starting feed volume added to
the operation / current retentate volume

References/Footnotes
1. Total recycle means retentate and permeate lines return to feed vessel
2. If process flux is unstable, it may be necessary to allow additional

time or investigate other membrane options

3. Retention samples are not required at every data point; sampling at

lowest and highest TMP is typical

4. The point at which the flux levels off is defined as the point around

which the slope of the flux vs. TMP curve decreases to 50% of the
previous slope. This point is also referred to as the knee of the curve.
5. These other conditions are described in more detail in Parts 2 and 3.
6. Example: 10X concentration with a final volume of 300 mL requires

(300 mL x 10) = 3 L of feed

7. Retention samples are not required at every data point; initial and

final concentration are typical. Typical data recording interval is

approximately every 1015 minutes.
8. See Guide: Maintenance Procedures for Pellicon and Pellicon 2

Cassette Filters (P17512) or Pellicon 3 Filters Installation and User

Guide (AN1065EN00)
9. Ng P, Lundblad J, and Mitra G, Optimization of Solute Separation by

Diafiltration, Separation Science, 11(5): 499-502, 1976.

10. See Technical Brief: Protein Concentration and Diafiltration by

Tangential Flow Filtration (TB032)

11. See Guide: Maintenance Procedures for Pellicon and Pellicon 2

Cassette Filters (P17512) or Pellicon 3 Filters Installation and User

Guide (AN1065EN00)
12. See Technical Note: Increase Product Yield in Your UF/DF Processes

(AN1026EN00)
13. Average flux can also be calculated for each step by dividing the total

process volume by the total process time

11

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