Demand Technical background Examples -...

03 May 2013

BioPAT® ViaMass – Real-time, Inline Biomass Measurement for Cell Culture Processes in single-use

03 May 2013

Agenda

• Demand

• Technical background

• Examples

• Outlook & Future Steps

03 May 2013

Biomass Measurement (Capacitance) in reusable systems enables better process control

Cultivation: Insect cells (SF9), 27°C, SF900 media, 180rpm

Correlation online / offline Online measurement

03 May 2013

Single Use: Strong Market Demand for Biomass Measurement

Cell Density 64.0%

Cell Viability 50.0%

Glucose 50.0%

pCO2 40.6%

Flow 32.8%

Most-Wanted Single-Use sensors (Aspen Brooks Report 2011):

03 May 2013

BioPAT ViaMass extends single use process monitoring towards biomass monitoring

Scalable biomass measurement from R&D to production

R&D PD Clinical/Production

Data Information Process

Understanding Demo of

Understanding Process Control

03 May 2013

SU Biomass Measurement – Added Value

Real-time monitoring of viable cell volume for entire batch

Automated process control and process modeling

Automated substrate feed strategies

Harvest point determination

Optimal time of infection for viral production

Reduced risk of contamination due to limited sampling need

Reduced risk of unrepresentative samples (manual handling)

Decreased labor cost for offline analysis

03 May 2013

Agenda

• Demand


• Examples


Status Application Selected Sartorius products Technology

Microwave Resonance

BioPAT®Xgas Fermentation

PAT Toolbox for Enhanced Process Understanding and Process Automation

Ammonia

Refractive Index

Capillar electrophorese

Ammonia Ammonia

Exhaust Gas Analysis

Microwave Resonance

Refractive Index

Microwave Resonance

Refractive Index

Microwave Resonance

Refractive Index

Microwave Resonance

Refractive Index

Microwave Resonance

Refractive Index

Microwave Resonance Microwave Resonance Microwave Resonance Microwave Resonance CIP | Inline Dilution |

Formulation

Fermentation | Cell Culture|

Formulation |Fingerprinting

BioPAT®Reflex

BioPAT®Spectro (NIR | UV-VIS | CCD)

Refractive Index

Spectroscopy

BioPAT®Fundalux Fermentation Turbidity

Fermentation | Cell Culture BioPAT®Trace Glucose / Lactate

Sens

ors

Anal

ytic

al S

yste

ms

Electrochemical sensors SU PH sensor, MU PH sensor, MU redox sensor Mixing | Fermentation | CC

Pressure sensors SU | MU sensor All Unit Operations

Flowmeters SU | MU sensor All Unit Operations

Temperature Pt 100 or Pt 1000, MU sensor All Unit Operations

Conductivity MU sensor, SU under development Mixing Homogeneity

Cell Culture BioPAT®Viamass Biomass

Optical sensors Fermentation | Cell Culture SU Sensors for pH and DO

Sartorius offering

Products available

Products in development

BioPAT®MFCS Bioprocess Chain SCADA

BioPAT®MFCS | DoE Module | BioPAT®MODDE Bioprocess Chain Design of Experiments

Soft

war

e

Auto

mat

ion

BioPAT®DCU Bioprocess Chain Automation Platform

BioPAT®MFCS | MVA Module | BioPAT®SIMCA | -online Bioprocess Chain Multivariate Data Anal.

03 May 2013

03 May 2013

Every cell can be polarized

Cytoplasmic Lipid membrane

impermeable to the free flow of ions

03 May 2013

Influence of electric field is ‘polarising’ viable cells

Electronics controlling the

electric field

+ + + + + +

+ + + + +

+

- - - - - -

- - - - -

- -

+

+ + + + + +

+ + + + +

- - - - - -

- - - - -

-

-

- -

-

-

-

+

+

+

+

+ +

Cell

Polarising ions available in the

cytoplasm and suspending media +

-

Direction of the field

03 May 2013

Dead and ruptured / fragmented cells cannot be polarized

Electronics controlling the

electric field

Direction of the field

+ + -

+ - -

-

+

+

+

- - +

+

-

Cell with badly

ruptured plasma

membrane

Plasma membrane

fragments + -

03 May 2013

In situ four pin electrode probe used for biomass measurement

Dead cells “invisible” to measurement

field

Only live cells polarized

Living biomass is measured

Sensitivity of measurement field approx. 30mm

03 May 2013

Alternative Biomass Measurements do not give similar response

Offline Methods

Trypan blue exclusion

Cells need intact membrane, subjective method

FACS analysis Cells need intact membrane and active enzymes

Offline counter mostly based on Trypan blue exclusion with CCD camera counting

Dry weight / Wet weight not specific, mostly used in microbial fermentation

Online Methods

OD / Turbidity measurement mostly used in microbial fermentation

Offline methods are highly time consuming and do not give direct process feedback

OD Turbidity is a determination of the sum of living and dead cells

03 May 2013

Agenda

• Demand


• Examples


03 May 2013

1. Application – Cultivation of E.coli in single use (STR50)

Comparison of different Methods:

Dry cell weight – DCW

Wet cell weight – WCW

OD 600

Capacitance

03 May 2013

2. Application – Cell Cultures Comparison of offline and online capacitance measurement in 2 cell lines

Cell Line B

0

5

10

15

20

25

0 50 100 150 200 250 300 350 400

Runtime (Decimal Hours)

Cap

acit

an

ce (

pF

/cm

)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

VC

D (

10E

6 c

ell

s/m

L)

Capacitance (pF/cm) Point Capacitance 50-L STR

Cell Line B

0

2

4

6

8

10

12

14

16

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Run Time (Days)

Bio

mas

s C

apac

itan

ce

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

VC

D (

E6

cell

s/m

L)

Capacitance

50L STR VCD

Data provided by:

03 May 2013

Agenda

• Demand


• Examples


BioPAT®ViaMass – Online Measurement of Biomass | Culture control

Implementation of online biomass Measurement in control strategy

Global Trends BioPAT®ViaMass

Measurement of biomass

Determination of cell numbers Compact design for flexible

integration Reduce sampling and labour work

DCU – Local Control Unit

Direct display of capacitance| Cell number

Quick and simple process monitoring

Central data monitoring without delays

BioPAT®MFCS/win + Recipe Module

Recipes for process automation Automatic event-based control of

cultivations to assure batch-to-batch consistency

Fully automated process steps based on cell numbers, for robust processes and reduced labour work

• Single use biomass sensor

• Integrated calibration & cell counting

03 May 2013

03 May 2013

Timelines – Product Availability

Production Transfer + Sensor Qualification + DCU Integration

Cultibag RM Integration

Cultibag STR Integration

RM + STR Launch Q1 / 2014

Milestones:

Application Tests

Customer trials by 09/2013

Customer trials

03 May 2013

Facts for integration

For both systems (RM and STR) the BioPAT®ViaMass will be integrated in bags with the new film (bags with old film will not be considered)

For RM: Only new Rocker can carry bags with biomass sensor!

Any upgrade for old Rocker is not possible!

03 May 2013

Take Home Message

o Number of live cells with intact membranes become polarized and thus measured

o Dead cells with porous membranes are not taken into account

o The resulting capacitance is directly proportional to the total membrane bound volume of the cells

–The number of cells, and the size of the cells

–The total biological volume

o The system is insensitive to cells with leaky membranes, gas bubbles and other debris

o The electric field typically propagates about 30mm from the probe into the liquid

o Be careful when comparing different methods of determining live and dead cells, this is a grey area, and you can get different answers

Appendix

03 May 2013

Online Measurement of Biomass | Viable Cell Volume

03 May 2013

• Real-time monitoring of viable cell volume for entire batch

• allows automated process control and process modeling

automated substrate feed strategies with MFCS

harvest point determination

time of induction / infection

Dead cells “invisible” to RF Field

Only live cells polarized

RF capacitance proportional to viable cell volume

• Reduced risk of contamination due to limited sampling need

• Reduced risk of unrepresentative samples (manual handling)

• Decreased labor cost for offline analysis

• Single use sensor integration for various SU bioreactors

03 May 2013

Measurement details

o System measures capacitance + conductivity between 50 kHz and 20 MHz

o 3 presets , 1 customer setting

o Viable biomass (volume) proportional to capacitance: Calibration = enter proportionality factor

o In future optional: scan of 25 frequencies should be measured

o only one probe table is used for all sensors

03 May 2013

Principle Setup and BioPAT®MFCS integration

USB PC

4-20 mA

MFCS ....

03 May 2013

Integration and retro fit of existing systems

Adaptation cable ODU M12, Modbus

USB PC

4-20 mA ?

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