Cell Density

Applications eBook
Publication Overview
Hamilton's on-line cell density sensors have been used by a variety
of customers for applications ranging from feed control to continuous
processing. This eBook provides a summary of relevant data and
references to published research that employed those sensors.

Introduction
Discover On-Line Cell Density............................................... 3
About Incyte.......................................................................... 4
About Dencytee..................................................................... 5

By Application
Monitoring Viable and Total Cells at the Same Time.............. 6
Feed Control.......................................................................... 7
Continuous Processing.......................................................... 8
Cells on Microcarriers............................................................ 9
Correlation of On-Line to Off-Line Measurements................10

By Cell Line / Strain

Mammalian Cells (CHO)......................................................... 6
Mammalian cells (BHK).......................................................... 9
Insect cells (SF9)...................................................................10
Yeast (Orgatae).....................................................................11
Bacteria (E. coli)....................................................................12
Filamentous Fungi (Absidia)..................................................13

2
Discover On-Line
Cell Density
Innovative Methods that
Improve and Streamline
Cell Density Monitoring
Traditional cell density measurement and monitoring is time- and labor-
intensive. It also lacks real-time, continuous data and makes assumptions
based off of off-line sampling and manual cell counting.

Hamilton Process Analytics has revolutionized and modernized this

process. Rather than counting the cells directly, Hamilton sensors
use the principles of permittivity measurement (capacitance per area)
and light absorbance which correlate to the cell count in solution at
the production line. It’s an improvement that saves time and allows
for advanced process control.

The following pages detail real applications of these sensors and

measurement principles to show the benefits and opportunities
offered by this shift in technology.

3
About Incyte
Real-Time, On-Line Viable Cell Density
Measurement by Permittivity
Incyte sensors measure permittivity in the on-line process to
give a continuous, in-situ reading of viable cells in solution.
This eliminates time-intensive manual cell counting.

Incyte Highlights:
■■ Not influenced by changes in the media, microcarriers, dead cells, or debris
■■ Can increase yield and lower production costs
■■ Can detect changes in cell physiology with frequency scanning
■■ Offers precise data to control harvesting in continuous culture
■■ Offers early detection of process deviations
■■ Available in optimized versions for multiple conductivity ranges

A LT E R N AT I N G
ELECTRIC
FIELDS P L AT I N U M
ELECTRODES

DEAD CELLS
VIABLE CELLS H AV E DA M AG E D
POL ARIZE MEMBR ANES AND
DO NOT POL ARIZE

4
About Dencytee
Real-Time, On-Line Total Cell Density
Measurement by Optical Density
Dencytee sensors determine optical density by measuring how cells in solution
absorb and scatter light. These on-line, continuous readings eliminate the need
for a technician to manually sample and count cells off the production line.

Dencytee Highlights:
■■ Simple on-line measurement of cell growth
■■ Reliable values during the growth phase
■■ Early detection of process deviations

LIGHT SOURCE INCREASES

I N T E N S I T Y T O C O M P E N S AT E
FOR THE TURBIDIT Y OF
T H E C U LT U R E B R O T H

5 mm
LIGHT WINDOW
DE TECTOR

5
 Viable and Total Cells
Mammalian Cells (CHO)
Obtaining relevant on-line data about viable and total cell density.

■■ Incyte correlates well with viable cell counts throughout process

■■ Incyte and Dencytee together can be used to estimate viability

165 165
Viable Cell Count [105 cells/mL]

Viable Cell Count [105 cells/mL]

110 110

55 55

0 0

0 65 130 195 260

Time [h]

■■ Viable Cell Counts ■■ Dencytee Measurement ■■ Incyte Measurement

Return to Index
Kandra, Kulwant, et al. On-Line Monitoring of CHO Cell Culture. Hamilton Company, 2014.
6
Optimize the Process
Mammalian Cells (CHO)
Monitoring permittivity provides the possibility to control feeding and enable
optimal nutrition supply.

In this study, alanine is an indicator for the protein of interest. As glutamine

nears depletion, the metabolism of the cells change and alanine is similarly
depleted. At that point there is no productivity.

■■ Drop in permittivity, as measured by Incyte, triggered glucose and

glutamine feeding
■■ Expanded alanine production phase of the protein of interest by three
days as compared with a non feed-controlled process (greatly reducing
workload of new batch startup)

11 7

10
6
9

8 5
Permitivity [pF/cm]

Biovolume [Litre]
7
4
6

5
3
4

3 2

2
1
1

0 0
0 50 100 150 200 250

Time [h]

Glutamine [mmol/L] Alanine [mmol/L] Glucose [g/L] Incyte Measurement

Return to Index
Roche (2005) CHO Biomass nutrients limit. Poster at ESACT19
7
 Continuous
Processing
Mammalian Cells (CHO)
Viable Cell Density monitoring correlates with the off-line Cedex counts.

■■ Incyte and Dencytee correlate throughout continuous (perfusion) process

■■ No sensor adjustment needed when harvesting

140

6.30
120

100 5.30
VCD [10^5 Cells/mL]

80

TCD [OD880 ]
4.30

60

3.30

40

2.30
20

0 1.30

0 50 100 150 200 250 300

Time [h]

■■ Off-Line ■■ Incyte Measurement ■■ Dencytee Measurement

Return to Index
Real-time Cell Density Measurement for PAT Applications,
Dr. Heino Büntemeyer, Anica Schmidt, Zellkulturtechnik, Technische Fakultät, Universität Bielefeld. 8
 Cells on Microcarriers
Mammalian Cells (BHK)
The correlation of permittivity to viable cell count is not influenced
by the presence of microcarriers.

■■ Incyte helps avoid misinformed decisions caused by outliers

(marked with the red circle)
■■ Microcarriers have no impact on permittivity measurements

140 7.0E+06
This data point is an
outlier that can be seen
as compared to the
120 Incyte measurement. 6.0E+06
Without Incyte, an
incorrect process
estimation would
likely happen.

Viable Cell Count [cells/mL]

100 5.0E+06
Permittivity [pF/cm]

80 4.0E+06

60 3.0E+06

40 FEED 2.0E+06

20 1.0E+06

0 0.0E+06

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00

Time [h]

■■ Viable Cell Count ■■ Incyte Measurement

Return to Index
On-ine Viable Cell Density Measurement on Microcarriers;
Marlene Frank, Ruma Lüthi, Iris Poggendorf, Caspar Demuth, Waedenswil 9
 Biovolume
to Permittivity
Insect Cells (SF9)
Permittivity shows a high correlation to off-line measurements of biovolume.

■■ Incyte correlates with biovolume throughout process

SF9 Insect Cell Growth Linear Relationship

and Infection Monitoring Permittivity - Biovolume
4.5 10 10 10 10 10
9 9 9 9 9
INFECTION

4
y =y1.9788x
= 1.9788x
Biovolume [µm3/mL]
Biovolume [µm3/mL]

Biovolume [µm3/mL]
Biovolume [µm3/mL]
Biovolume [µm3/mL]

8 8 8 8 8
Permitivity [pF/cm]

3.5
7 7 7 7 7
3
6 6 6 6 6
2.5
5 5 5 5 5
2
4 4 4 4 4
1.5 3 3 3 3
3
1 2 2 2 2 2
0.5 1 1 1 1 1
0 0 0 0 0 0
0 20 40 60 80 100 120 140 160 0 0 1 1 2 2 3 3 4 4 5 5
Culture Time [h] Permitivity
Permitivity
[pF/cm]
[pF/cm]

■■ Biovolume ■■ Incyte Measurement ■■ Biovolume ■■ Incyte Measurement

Esteban, G. (2006) On-Line Viable Cell Density and Physiological States Monitoring by Dielectric
Spectroscopy sf9 Growth and Infection Process. Poster at Cell Culture Engineering X. Return to Index
Related work: On-Line Monitoring of Infected Sf-9 Insect Cell Cultures by Scanning Permittivity
Measurements and Comparison with Off-Line Biovolume Measurements.
Sven Ansorge. Cytotechnology (2007) 55:115–124 10
 Concentration
to Permittivity
Yeast (Orgatae)
Viable cell density monitoring correlates with the off-line measurement.

■■ Good correlation with dry weight throughout process

■■ On-line monitoring provides data 24 hours/day and 7 days/week, as compared
with off-line sampling that is only done when technicians are on duty

Propagation of Ogateae Yeast in Bench-top Bioreactor

2.50
12

2.00 10

Cell Concentration [g/L]

Permitivity [pF/cm]

8
1.50

6
1.00
4

0.50
2

0.00 0
0 50 100 150 200 250

Time [h]

■■ Off-Line Cell Count ■■ Incyte Measurement

Return to Index
Xylitol Fermentation Experiments with Different Yeast Strains,
Csaba Feher, Zsolt Barta, Budapest University of Technology and Economics 11
Dry Weight
to Permittivity
Bacteria (E. coli)
Viable cell density monitoring correlates with the dry weight measurement.

■■ Incyte correlates to dry cell weight throughout feed and

differs after induction, when production phase starts
■■ IPTG Induction starts the production phase and leads
to increased bacterial size and permittivity

140 140
FEED STARTS

IPTG INDUCTION
120 120

100 100

80 80
Cx [g/L]

Cx [g/L]
60 60

40 40

20 20

0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time [h]

■■ Cell Dry Weight ■■ Incyte Measurement

Return to Index
Bioprocess Biosyst Eng 2012_Horta et al_iIntensification of high cell-density cultivations of E. coli
12
 Dry Weight
to Permittivity
Filamentous Fungi (Absidia)
Viable cell density monitoring correlates with the off-line method.

■■ Filamentous fungi show a high heterogeneity during cultivation,

therefore reliable off-line sampling is very difficult
■■ Incyte can be used for on-line measurement of viable
microorganisms – no sampling needed

25 12
Permittivity [pF/cm], Conductivity [mS/cm]

10
20

Dry Weight [g/L]

15

10
4

5
2

0 0
0 10 20 30 40 50

Time [h]

■■ Cell Dry Weight ■■ Incyte Measurement

Return to Index
INRA - French Agronomical Research Institute
13
 Talk to a Hamilton
Expert About Your
Cell Density Application
Now that you’ve seen Incyte and Dencytee in action,
contact a Hamilton sales representative about how to
apply this technology to your application and process.

sensors@hamiltoncompany.com
US: 800-648-5950
Europe: +41-58-610-10-10

©2018 Hamilton Company. All rights reserved.

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Lit. No. L30019 — 01/2018

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