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Technical Bulletin

Cell Culture Media Manufactured in the Air


Classifier Mill is Chemically Equivalent and
Performs Comparably to Media Manufactured
in the Continuous Pin Mill and in Ball Mills

SAFC Biosciences can produce cGMP dry powder cell culture enter the center of the disk where they are centrifugally
media (DPM) using any one of three different milling propelled outward and reduced in size by the rotating
methods — the traditional and industry-standard ball milling, intermeshing pins.
as well as two other milling technologies: pin milling and air
classifier impact milling. Both the pin mill system (CM) and The air classifier mill is also considered a high-speed-impact
the air classifier mill system (ACM) are considered continuous mill. Bulk material is fed into the grinding chamber where it
systems wherein the raw materials of a given batch of media is pulverized by impactors fixed to a high-speed rotating disk.
are continually fed into the mill. The milled material is then This product is then drawn by airflow to an independently
blended to assure homogeneity of the final product. Both driven radial flow separator (air classifier wheel), which allows
systems are capable of producing DPM lots of up to particles of the desired size to pass through while returning
4000 kg, assuming the bulk density of the DPM is 0.57 kg/L oversized particles back to the grinding chamber for further
or higher. The maximum lot size is determined by the amount size reduction.
of powder that can physically fit into the blender, and so is
dependent on the bulk density of
each specific DPM formulation. The The following analytical and cell culture data
pin mill system is located in Lenexa, compare three lots of EX-CELL™ CD CHO
KS, with the air classifier mill system Serum-Free Medium produced using each
residing in St. Louis, MO. milling method and demonstrate that,
despite the difference in milling technology,
they are chemically equivalent and show no
The pin mill is a high-speed-impact differences in performance. This media was
mill consisting of a feed inlet, a set chosen because it is complex, yet chemically
of two disks with concentric rings of defined (contains no hydrolysates) and
pins and a product outlet. The raw serum-free.
materials are fed into the mill and

Figure 1. Pin mill chamber in the open


position to allow for viewing of the
disks.

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SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA
Phone
Toll free-USA
+1 913-469-5580
1 800-255-6032
UNITED KINGDOM
Phone
Fax
+44 (0)1264-333311
+44 (0)1264-332412
AUSTRALIA
Phone
Toll free-AUS
+61 (0)3-9362-4500
1 800-200-404
www.safcbiosciences.com
Fax +1 913-469-5584 E-mail info-eu@sial.com Fax +61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com
Analytical Comparison The normal assay variability range for testing components at
A lot of EX-CELL™ CD CHO ball-milled DPM was tested these levels was determined to be 10% for the amino acids
quantitatively for amino acids, vitamins and zinc, a trace and vitamins and 15% for zinc based on preliminary studies
metal, along with a beginning, middle and end sample of lots and information from the testing labs. These data show the
produced in the CM and ACM systems. Figures 2 and 3 show same medium formulation will contain the same amounts of
the results of that comparison. individual components, within the normal assay variability
range, regardless of being produced via ball mill, pin mill or
air classifier mill.

Figure 2

Analytical Comparison
Analytical Comparisonof Ball-Milled, Air Classifier-Milled,
of Ball-Milled, and Pin- and
Air Classifier-Milled
Milled Cell Culture Media
Pin-Milled Cell Culture Media
120
CM Batch
ACM Batch
of Ball-Milled

110
Percent of Ball-Milled

100
Percent

90

80 Ile
a
r

p
Cy al
r

lu

g
s
r
ly
n
p

s
et
e
Th

Se

Ty

Tr
Al

Ph

Hi
Pr

Ar
Le
As
As

Ly
in
V
G

M
st

Media Components
Media components

Figure 2: Amino acid concentration comparison of ball, pin and air classifier mill media.

Figure 3

Analytical
Analytical Comparison
Comparison of Ball-Milled,
of Ball-Milled, Air Classifier-Milled
Air Classifier-Milled, and Pin- and
Pin-Milled Cell Culture
Milled Cell Culture Media Media

120
CM Batch
115 ACM Batch
of Ball-Milled

110
Ball-Milled

105

100
Percentof

95
Percent

90

85

80
nc
e

2
e

in

id
vin

id

B1
in

Ac

Zi
m
ox

fla

ia

in
a

l ic
Th
rid

tin

m
ib

Fo
Py

ta
ico
R

Vi
N

M edia components
Media Components

Figure 3: Vitamin and zinc concentration comparison of ball, pin and air classifier
mill media.

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Biological Comparison antibody production in EX-CELL™ CD CHO. Typically, for cell
Another comparison of ball-milled, pin-milled and air studies, differences of up to 20% between groups is
classifier-milled media was performed using cell culture considered acceptable variability. It is illustrated in Figures 4,
studies with the CHO (Chinese Hamster Ovary) cell line 5 and 6 that there are no significant differences in growth,
B13-24, a derivative of the DX-B11 line which secretes viability or IgG production between the media.
humanized immunoglobulin IgG. The studies evaluated cell
growth over multiple passages, culture longevity and

Figure 4

Comparison of Growth of DX-B11 Cells in Ball-Milled,


Comparison ofAir Classifier-Milled
Growth of DX-B11 Cellsand Pin-Milled
in Ball-milled, Air Classifier-
EX-CELL
Milled
TM
CD CHO
and Pin-milled Serum-Free
EX-CELL CD CHOMedium
Media
3.50E+06
100
3.00E+06
Celldensity/mL

cells
2.50E+06 80
Density/mL

Viable cells
2.00E+06 60

%%viable
1.50E+06
40
1.00E+06
Cell

20
5.00E+05

0.00E+00 0
Pass 1 Pass 2 Pass 3 Pass 4 Pass 5
Passage
Passage

Ball-Milled ACM CM
Ball-Milled Viability ACM Viability CM Viability

Figure 4: Cell growth comparison of ball, pin and air classifier mill media.

Figure 5

Comparison of Longevity of DX-B11 Cells in Ball-Milled,


Air Classifier-Milled
Comparison of Longevity of DX-B11 Cells and Pin-Milled
in Ball-Milled, Air Classifier-
EX-CELL
Milled,
TM
CD CHO
and Pin-Milled Serum-Free
EX-CELL CD CHO MediaMedium
4.00E+06
100
3.50E+06
Celldensity/mL

cells

3.00E+06 80
Density/mL

Viable cells

2.50E+06
60
%%viable

2.00E+06
1.50E+06 40
Cell

1.00E+06
20
5.00E+05

0.00E+00 0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9

Passage
Passage

Ball-Milled ACM CM
Ball-Milled Viability ACM Viability CM Viability

Figure 5: Cell longevity comparison of ball, pin and air classifier mill media.

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Figure 6
Comparison of Human IgG Production of DX-B11 Cells in
Comparison of HumanAir
Ball-Milled, IgGClassifier-Milled
Production of DX-B11
and Cells in Ball-Milled,
Pin-Milled
Air Classifier-Milled,
EX-CELLTM and CD Pin-Milled EX-CELL Medium
CHO Serum-Free CD CHO Media
800

700

600

ng/mL 500

400

300

200

100

0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9
Passage
Passage

Ball-Milled ACM CM

Figure 6: Production comparison of cells in ball, pin and air classifier mill media.

No significant differences are observed in the performance of


the media regardless of being produced via ball milling, pin
milling or air classifier milling.

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Figure 7

CM
(Mean value = 33.4µm)

Ball Mill
(Mean value = 41.1 µm)

ACM
(Mean value = 48.2µm)

Figure 7: Particle size distribution curves of ball, pin and air classifier mill media.

Particle Size Comparison Summary


The particle size distribution of EX-CELL™ CD CHO media Both pin milling and air classifier milling can provide an
produced in both the CM and ACM as well as in a ball mill alternative manufacturing process for customers considering
was compared for informational purposes and is shown in risk management and contingency plans pertaining to their
Figure 7. raw materials supply chain. Through both analytical and cell-
based studies, it is demonstrated that continuous pin milling
The "S"-shaped part of the curve represents the percentage of and air classifier milling produce quality media that is
particles that are under a certain size. For example, 90% of equivalent to ball-milled media.
the particles are < 94.8 µm, < 79.7 µm and < 96.1µm for the
ACM, CM and ball-milled media, respectively. Although the For more information about this subject or other SAFC
curve shape for the differently milled media are not identical, Biosciences’ products and services, please call our
the mean particle size values of all three media are relatively Technical Services department or e-mail us at
similar. Any differences in particle size distribution that may technicalservices@sial.com.
exist did not affect other finished product testing such as
solubility, pH or osmolality, and did not result in any
difference in the performance of the medium with cells.

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Warranty, Limitation of Remedies
SAFC Biosciences warrants to the purchaser for a period of one year from date of delivery that this product conforms to
its specifications. Other terms and conditions of this warranty are contained in SAFC Biosciences’ written warranty, a
copy of which is available upon request. ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING THE IMPLIED
WARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, ARE EXCLUDED. In no case will SAFC
Biosciences be liable for any special, incidental, or consequential damages arising out of this product or the use of this
product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort,
or any other legal theory. SAFC Biosciences expressly disclaims any warranty against claims by any third party by way of
infringement or the like. THIS PRODUCT IS INTENDED FOR PURPOSES DESCRIBED ONLY AND IS NOT INTENDED FOR
ANY HUMAN OR THERAPEUTIC USE.
Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.

EX-CELL™ is a trademark of SAFC Biosciences, Inc.

© 2007 SAFC Biosciences, Inc.

Issued May 2007 T094

United States Europe Asia Pacific


SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
www.safcbiosciences.com USA
Phone +1 913-469-5580
UNITED KINGDOM
Phone +44 (0)1264-333311
AUSTRALIA
Phone +61 (0)3-9362-4500
Toll free-USA 1 800-255-6032 Fax +44 (0)1264-332412 Toll free-AUS 1 800-200-404
Fax +1 913-469-5584 E-mail info-eu@sial.com Fax +61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com