development of novel chemically defined media for cho cell applications

Development of novel chemically-defined media for CHO cell applications

Dr. Dipl-Ing. Jörg von Hagen

Head of Process Development & Launch

Management Merck Millipore, Darmstadt, Germany

Cell Culture World Congress, Munich 2013

2

Presentation Outline

CHO Cell Media & Feed – Performance evaluation 1

2 Lot to Lot Consistency

Platform Consistency 3

2

5 Powder handling improvements

Solubility Toolbox for high performing media 6

Perspectives 7

4 Media Performance Prediction

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0 3 4 5 6 7 8 9 10 11 12 13 14

Elapsed Time (Day)

Cell

Via

bil

ity (

% V

iab

le C

ell

s)

0

0.5

1

1.5

2

2.5

3

3.5

IgG

Tit

er

(g/L

)

Competitor A Competitor B Cellvento CHO-200


A Comparison of IgG Production in CVC-200 and Two Competitor Media and Feed Supplements.

IgG Production in Cellvento CHO-200 was 2-3 fold higher than two competitor

products over a 14 day fed batch culture.

4

Cell Growth (VCD) - Competitive Evaluation in Fed Batch

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14.00

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Elapsed Time (Day)

VC

D (

10e6 c

/ml)


Cell growth in Cellvento CHO-200 achieved above 1x107 cells /mL in a 14 day

fed batch culture.

A Comparison of Cell Growth in CVC-200 and Two

Competitor Media and Feed Supplements

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5



Perspectives 7


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Elapsed Time (Days)

Inte

gra

l V

iab

le C

ell

Den

sit

y

(10e6 c

ell

day/m

l)

Lot 006_1

Lot 006_2

Lot 006_3

Comparison of Average Cell Growth in Batch Culture

Cell growth in Cellvento CHO-200 was consistent in batch culture across three

lots of media

7

Comparison of Cell Growth in Fed Batch Culture

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Via

ble

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ll D

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e6

c/m

l)

Elapsed Time (Day)

Three Lots of Feed tested

Feed Lot 007_1

Feed Lot 007_2

Feed Lot 007_3

Cell growth in Cellvento CHO-200 was consistent in fed batch culture across

three lots of feed

8

Comparison of Volumetric Titers Across Three Lots of Feed

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1.0

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1.8

2.0

7 10

Sample Day

IgG

(g

/L) Feed Lot 007_1

Feed Lot 007_2

Feed Lot 007_3

Comparison of IgG Production in Fed Batch Culture

IgG production in Cellvento CHO-200 was consistent in fed batch culture

across three lots of feed

PSD Media

9

10

PSD Feed

Presentation title in footer | 00 Month 0000 11

0

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120

His Asn Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Met Val Ile Leu Phe

Am

ino

aci

d c

on

cen

trat

ion

(in

% o

f th

e

theo

reti

cal v

alu

e)

Cellvento CHO 200 media

Batch 1

Batch 2

Batch 3

Reproducibility amino acid content

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12



Perspectives 7


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0 2 4 6 8 10 12 14

Via

ble

Ce

ll D

en

sit

y (

10

e6

c/m

l)

Elapsed Time (Day)

3L Cellready Bioreactor

50 ml Spin Tubes

A Comparison of Cell Growth in Spin Tubes and 3L Benchtop Bioreactors

Similar growth performance was achieved with Cellvento CHO-200 in spin tubes

and benchtop bioreactors over a 14 day fed batch culture.

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1

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1,8

7 10

IgG

(g

/L)

Sample Day

50 ml Spin Tubes

3L Cellready Bioreactor

A Comparison of IgG Production in Spin Tubes and 3L Benchtop Bioreactors

IgG titers averaged 1.5 g/L after 10 days of fed bath culture using

Cellvento CHO-200 in spin tubes and benchtop bioreactors.

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Perspectives 7


NIR - Near Infrared Spectroscopy

Measurement conditions

Integrating sphere, PbS-detector, diffuse reflexion

Instrument: BrukerVector 22/N

Number of scans: 16, Resolution: 8 cm-1, Measurement Range: 12000 – 4000 cm-1

PCA-Possibility for clustering good and poor performing media batches

A fingerprint method for media performance prediction

low performance

medium

performance

high performance

EMD Millipore

Supplier C-1

Supplier C-2

Supplier C-3

Supplier B

Supplier A

Supplier S

Principle component analysis (PCA) Correlation of cellular performance and NIR analysis

PCA of DMEM-F12. Analysis by NIR-Spectroscopy (Bruker, Vektor MPA)

Quality Testing of Media …

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20

25

0 days 3 days 5 days 7 days 10 days 12 days 14 days 17 days

cu

mu

lati

ve P

DL Mill Tech 1-1

Mill Tech 1-2

Mill Tech 1-3

Mill Tech 2-1

Mill Tech 2-2

Mill Tech 2-3

Impact of the milling technology on CHO-S cells grown in chemically defined media.

No significant differences are observed in the basic cell culture assay analyzing the

cumulative population doubling levels (PDL) using different produced mammalian

cell culture media.

…depends on the appropriate test system

Sensitive Cell Culture Assays

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10

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30

40

50

60

70

Supplier A Mill Tech 1-1 Mill Tech 1-2 Mill Tech 1-3 Mill Tech 2-1 Mill Tech 2-2 Mill Tech 2-3

pla

tin

g e

ffic

acy (

%)

Impact of the milling technology on CHO-S plating efficacy in chemically defined media.

CHO-S cells were diluted in the corresponding media and analyzed after 2 weeks on

plating efficacy.

… underline differences in cell culture media performance

Impact of Processing Technologies

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110

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140

Supplier A Mill Tech 2

Tit

er

( %

to

in

tern

al

refe

ren

ce)

The media were tested in the production of a monoclonal antibody.

Titer was measured by Surface Plasmon Resonance Spectroscopy.

Red line indicates the minimum achievable titer according to the specifications.

Impact of the milling technology on mAb titer

Milling Process Impact on final NBE Attribute

0

5

10

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25

30

35

Supplier A Mill Tech 2

Cri

tical

Pep

tid

es (

%)

Final molecules after capture were analyzed by HPLC to determine the amount (%) of

critical peptides that are known to be correlated with lower half life of the drug substance

in patient serum samples. The amounts are directly linked with the raw material quality

and the production process.

Red line indicates the max. value given by the authorities. Dotted red line represents the internal specification limit.

Range between dotted blue lines show the target space for good media performance.

Impact of the milling technology on critical antibody attributes

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Perspectives 7


Humidity and impact on powder flowability

After opening the package the first time the CDM II powder is clumpy. The DMEM-

F12 and CDM I media are opened several times and are free flowing powders.

CDM I

DMEM-F12

CDM II

CDM II: particle size distribution

CDM II particle size analysis is

technically not feasible by air-jet

sieving. After 3 min all material

gets like chewing gum, whereas

for CDM I and DMEM-F12

particle size distribution analysis

went without clumping.

CDM composition comparison

The CDM I and II composition have the following critical hygroscopic

ingredients:

– Calcium Chloride 2H2O

– Choline Chloride

– Ferric Nitrate 9 H2O

– Ferrous Sulfate 7 H2O

– Sodium phosphate, monobasic, monohydrate

the above listed ingredients are present with a concentration in sum of 1.5% in

the CDM I and 9.7% in the CDM II formulation.

6.3-fold enrichment of hygroscopic compounds resulted in a final H2O titer

of 4.05% measured by Karl Fischer Titration in the CDM II powder

(compared to 1.05 to 1.2 in DMEM-F12 and CDM I.)

Humidity effects solubility of DMEM F12

6 h / 28 °C / 90 % rH. 2 – 8 °C 4 fold accelerated (5min real)

17° increase in avalanche angle after DMEM F12 exposure to 90% rH

DMEM F12 before and after flowability measurement (3h/25°C at 90% rH)

DMEM-F12 compacted

uncompressed 20KN 30KN 40KN 50KN 60KN

R = Rosa = pinkish

DMEM-F12 compacted

uncompressed 20KN 30KN

40KN 50KN 60KN 1-3 mm particle sizes

1 mm 1 mm 1 mm

1 mm 1 mm 1 mm

Solubility of DMEM F12 powder vs. granules

Filterability of Powder vs. Granules of DMEM F12

Medium Compaction

(kN)

Filter

Family

Filter

Name

Filter Pore

Size

(µm)

Vmax

(L/m2)

Amin

(m2)

DMEM

F-12

0

0

Express SHR 0,1 9 305 0,28

Express SHR-2L 0.5/0.1 20 555 0,29

30

30

Express SHR 0.1 11 221 0,29

Express SHR-2L 0.5/0.1 37 911 0,31

60 Express SHR 0.1 10 596 0,30

60 Express SHR-2L 0.5/0.1 44 219 0,31

The Amin represents the minimal surface of membrane needed to process 500L

within the defined time.

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Perspectives 7


Tyrosine solubility in feed with >110 g/L amino acid & vitamin load

1 1 50 70

g/L @ pH 7.0 +/- 0.2

L-Tyr L-Tyr 2Na mod L-Tyr I mod L-Tyr II

CHO-S growth profile comparing L-Tyrosine Disodium salt vs. modified Tyrosine

time (days)

VC

D (

x1

05 v

c/m

L)

Medium 0 1 4 5 6 7 8

1 1 29,6333333 753,033333 1264,53333 1541,13333 988,936665 333,58 0

2 1 33,2 681,6 1218,13333 1470,4 1108,24002 548,75

3 1 11,5333333 61,0666667 73,0666667 64,8999992 46,4400007 63,1033333 1,92

4 1 24,15 528,6 1318,4 1409,82503 1393,7 1324,422 2,07

5 1 24,6666667 462,9 1197,26667 1441,11333 1578,69001 1502,80333 2,49

6 1 19 85,2666667 157,4 266,206664 375,935004 415,776667 0,29546573

7 1 16,4666667 116,266667 135,9 117,206667 78,376667 71,34

18,33

0,31

0

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1 3 5 7 9 11

time

Leb

en

sze

llzah

l [vc

/ml]

x1

0^

5

4,5mM 2Na-Tyr

4,5mM 2Na-P-Tyr

9mM 2Na-P-Tyr

4.5 mM L-Tyr 2Na

4.5 mM mod L-Tyr II

9.0 mM mod L-Tyr II

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Leb

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llzah

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/ml]

x1

0^

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4,5mM 2Na-Tyr

4,5mM 2Na-P-Tyr

9mM 2Na-P-Tyr

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4,5mM 2Na-Tyr

4,5mM 2Na-P-Tyr

9mM 2Na-P-Tyr

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Leb

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x1

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4,5mM 2Na-Tyr

4,5mM 2Na-P-Tyr

9mM 2Na-P-Tyr

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Perspectives 7


Perspectives in media preparation • The raw material quality is key for optimal

media performance on cellular level

• Raw material selection

• Raw material pre-processing

• Novel raw material development

• Supply chain

• The production process is directly linked with

batch to batch consistency and cellular

performance

• Milling process

• Mixing process

• Packaging

• The combination of raw materials and the

production process is mandatory for excellent dry

powder media performance

Raw material quality

Production process

Cellular performance

development of novel chemically defined media for cho cell applications

Health & Medicine