Technical Bulletin

Cell Culture Media Manufactured in the

Continuous Pin Mill is Chemically Equivalent
and Performs Comparably to Media
Manufactured in Ball Mills
In 2005 SAFC Biosciences opened a fully validated, state-of- Homogeneity
the-art, cGMP-compliant continuous pin mill facility for The pin-mill manufacturing process was fully validated to
manufacturing dry powder cell culture media (DPM) in show that it uniformly blends components to achieve a
Lenexa, Kan. The facility employs two different sized homogenous and quality product. Testing proved that any
continuous pin mill systems, each consisting of a stainless given component was present at the same concentration at
steel pin mill (Hosokawa) combined with three-dimensional any given location throughout a batch of media. Multiple
conical blenders to produce pharmaceutical-quality DPM in samples (approximately 10 - 40 depending on the lot size)
lots sizes of 10 to 4000 kg. The large-scale continuous pin mill throughout the lot were pulled, hydrated and sent for
system is referred to as the CM and the smaller-scale quantitative analysis of amino acids, vitamins and zinc. The
continuous pin mill system as the SSPM. media contained a very low level — approximately 20 ppm
— of the trace metal zinc.
Pin mills are a type of high-speed-impact mills consisting of a
feed inlet, a set of two disks with concentric rings of pins and
a product outlet. The raw materials are fed into the mill and Percent relative standard deviation (% RSD) was chosen as
enter the center of the disk where the metric to evaluate homogeneity. The
they are centrifugally propelled % RSD is the standard deviation divided by
outward and reduced in size by the the mean and multiplied by 100 in order to
rotating intermeshing pins. The raw be expressed as a percentage. It is used to
materials are mixed in a blender indicate the variation between samples in a
both before entering and after given population. The smaller the % RSD, the
leaving the pin mill chamber to less variation and, in this case, the more
ensure a consistent feed into the homogenous the product. There will be an
mill and a homogenous final inherent amount of variability due to sample
product after being milled. preparation and the analytical method itself,
which will be larger for components present
As part of the validation and in smaller quantities.
qualification of the continuous pin Figure 1. Pin mill chamber in the open
mill, it was necessary to position to allow for viewing of the Based on preliminary work and information
demonstrate that the DPM disks. from the testing laboratories regarding the
produced was both homogenous assay variability, an acceptance criterion of
and comparable to the tradition and industry-standard ball- < 10% RSD for the amino acids and vitamins and < 15% RSD
milled DPM. This was done by chemical analysis and cell for the trace metal zinc was chosen to indicate a
growth studies comparing different lots sizes of pin-milled homogeneous product.
DPM against ball-milled control DPM.

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
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
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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
Figures 2 and 3 show homogeneity results obtained as part of (EX-CELL™ CD CHO Serum-Free Medium) at 300 and 10 kg
the validation for a classical medium (DMEM/F12 at 2400 kg batch sizes. All of the components met their criteria for being
batch size), a hydrolysate-containing serum-free medium homogeneously distributed throughout the pin mill media
(EX-CELL™ 302 Serum-Free Medium for CHO Cells at 800 kg regardless of batch size or formulation.
batch size) and a chemically defined serum-free medium

Figure 2

10,
10, 300, 800, and 2 4300,
0 0 k g800 and
B a tc h e s2400
o f C kg
o m Batches
p le te C e llof
C u ltu re
Complete M e Cell
d ia Culture Media
120
2400 kg Batch 300 kg Batch
800 kg Batch 10 kg Batch

110
t (%
A m o u n(%)

100
Amount

90

80 C y s tin e

M et
Val

H is
Thr

Ser

Phe
T yr
Asp

Asn

G ly

Leu

L ys
G lu

G ln

Arg
P ro

T rp
A la

M e d ia C o m p o n e ts Ile
Media components
Figure 2: Amino acid homogeneity of pin mill media.

Note: Gln was not in the media formulations for the 300, 800 and 10 kg batches.

Figure 3

10, 300, 800, and10, 300,

2400 800
kg B andof2400
atches kg Batches
C om plete of Media
C ell C ulture
Complete Cell Culture Media
120
2400 kg Batch 300 kg Batch
115 800 kg Batch 10 kg Batch

110
Amount (%)

105

100

95

90

85

80
e

12
e

d
n

in

c
id
in

ci
vi

in
am

B
am
ox

A
la

Z
in
of

hi

ic
id

in

m
ol
ib

T
yr

ot

it a
R

F
P

ic

V
N

M e d ia Co mp o n e n ts
Media components

Figure 3: Vitamin and zinc homogeneity of pin mill media.

www.safcbiosciences.com
Analytical Comparison sample of pin-milled media made in both the CM and SSPM.
An analytical comparison of pin-milled and ball-milled media Figures 4 and 5 show the results of that comparison.
was performed using EX-CELL™ CD CHO DPM. This medium
was chosen because it is complex, yet chemically defined These data show the same formulation will contain equal
(contains no hydrolysates) and serum-free. One lot of ball- amounts of individual components, within the normal assay
milled DPM was tested quantitatively for amino acids, variability range, regardless of being produced via ball mill,
vitamins and zinc, along with a beginning, middle and end large-scale (CM) or small-scale pin mill (SSPM).

Figure 4

AnalyticalofComparison
Analytical Comparison ofPin-Milled
Ball-Milled and Ball-Milled
Celland
Culture
Pin-Milled Cell Culture Media
Media

120
CM Batch
ball-milled

SSPM Batch

110
Ball-Milled

100
Percent ofof
Percent

90

80
I le

u
r

p
r

lu

Cy al

g
s
ly

r
n
p

s
et
e
Th

Se

Ty

Ph

Tr
Pr

Al

Ar
Hi
As
As

Le

Ly
in
G

M
st

Mediacomponents
Media Components

Figure 4: Amino acid concentration comparison of ball mill and pin mill media.

Figure 5

Analytical
Analytical Comparison Comparison
of Ball-Milled of Ball-Milled
and Pin-Milled and Media
Cell Culture
Pin-Milled Cell Culture Media
120
CM Batch
115 SSPM 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

Media
M edia components
Components

Figure 5: Vitamin and zinc concentration comparison of ball mill and pin mill media.

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Biological Comparison culture longevity and antibody production in EX-CELL™ CD
CHO media produced via ball milling and pin milling. Figures
A cell culture comparison of ball-milled and pin-milled media
6, 7 and 8 demonstrate that there are no differences in
was performed with the CHO cell line B13-24, a derivative of
growth, viability or IgG production between the media.
the DX-B11 line which secretes humanized immunoglobulin
IgG. The studies evaluated cell growth over multiple passages,

Figure 6

Cell
C omparison of Growth
Grow th ofComparison
D X-B 11 C ellsof
in Ball-Milled and
B all-milled and Pin-
Pin-Milled Cell Culture
milled EX-C E LL C D C H O MediaMedia
3.50E + 06
100
3.00E + 06
Celldensity/mL

cells
Density/m L

2.50E + 06 80

V iable cells
2.00E + 06 60

viable
1.50E + 06
40

%%
Cell

1.00E + 06
20
5.00E + 05

0.00E + 00 0
P ass 1 P ass 2 P ass 3 P ass 4 P ass 5

Passage
P a ssa ge

B all-M illed CM SSPM

B all-m illed V iability CM V iability S S P M V iability

Figure 6: Comparison of growth of DX-B11 cells in ball-milled and pin-milled

EX-CELLTM CD CHO Serum-Free Medium.

Figure 7

Longevity
Comparison Comparison
of Longevity of Cells
of DX-B11 Cells in Ball-Milled
in Ball-Milled and
and Pin-Milled
Pin-Milled
EX-CELL CD Cell
CHOCulture
Media Media
4.00E+06
100
3.50E+06
Celldensity/mL

cells
3.00E+06 80
Density/m L

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
Pa ssa ge

Ball-Milled CM SSPM

Ball-Milled Viability CM Viability SSPM Viability

Figure 7: Comparison of longevity of DX-B11 cells in ball-milled and pin-milled

EX-CELLTM CD CHO Serum-Free Medium.

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 Figure 8

Production
C o m p ariso n o f Gro w Comparison
th o f D X -B 11 of Cells
C ells in Bin Ball-Milled
all-m ille d an d Pand
in -
Pin-Milled Cell Culture Media
m ille d E X -C E L L C D C H O M ed ia
3.50E + 06
100
3.00E + 06
density/mL

V iab lecells
Den sity/m 2.50E + 06 80

cell
2.00E + 06 60

viable
1.50E + 06
40
Cell

%%
1.00E + 06
Cell

20
5.00E + 05

0.00E + 00 0
P as s 1 P as s 2 P as s 3 P as s 4 P as s 5

Passage
P a ssa ge

B all-M illed CM SSPM

B all-m illed V iability CM V iability S S P M V iability

Figure 8: Comparison of growth of DX-B11 cells in ball-milled and pin-milled

EX-CELLTM CD CHO Serum-Free Medium.

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Particle Size Comparison differences in particle size distribution do exist, does not affect
other finished product testing such as solubility, pH or
The particle size distribution of EX-CELL™ CD CHO media
osmolality, and does not result in any difference in the
produced in the CM, SSPM and in a ball mill was compared
performance of the media on cells.
for informational purposes and is shown in Figure 9.

The "S"-shaped part of the curve represents the percentage of

particles that are under a certain size. For example, 90% of
the particles are < 71.6 µm, < 79.7 µm and < 96.1µm for the
SSPM, CM and ball-milled media, respectively. Whatever

Figure 9

SSPM
(Mean value = 35.5 µm)

CM
(Mean value = 33.4µm)

Ball-Milled
(Mean value = 41.1µm)

Figure 9: Particle size distribution curves of ball mill and pin mill media.

Summary For more information about this subject or other SAFC

Through both analytical and cell-based studies it is demon- Biosciences’ products and services, please call our
strated that continuous pin milling is an effective and scaleable Technical Services department or e-mail us at
technique that can produce quality, homogenous cell culture technicalservices@sial.com.
media which performs equivalently to ball-milled media. Pin
milling can provide an alternative manufacturing site and
process for customers considering risk management and
contingency plans pertaining to their raw materials supply
chain.

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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 T093

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