Application
Note
SUM111009
Thermo Scientific HyClone Single-Use
Mixer (S.U.M.) Performance Report
Measurement of Particulate Size and Quantity Generated During Operation
Cory Card, Associate Director BioProcess Market Development, Thermo Fisher Scientific
Brett Judd, Validation Engineer, Thermo Fisher Scientific
Tom Smith, Applications Specialist, Thermo Fisher Scientific
April Scott, BMD Applications Manager, Thermo Fisher Scientific
Mohamad Awada, Senior Applications Scientist, Thermo Fisher Scientific
Introduction
Disposables have become increasingly
popular in bioproduction from media
storage bags and filling manifolds to
mixing vessels and bioreactors. The
development of disposable mixing
systems and bioreactors has been
hampered, in part, by inadequate
agitation using only single-use
materials that could be provided
in a sterile, fully integrated format.
Simple designs such as 2-dimensional
pillow bags on rocking platforms and
recirculation loops were early entries
to meet this need. However, this
style of agitation differs significantly
from the industry-standard used in
traditional stainless steel systems.
Other types mixing devices were
later developed, including oscillating
agitators, and magnetically-coupled
impellers. The stirred-tank is the most
commonly utilized style of mixing
vessel for both liquid:liquid and
solid:liquid mixing applications. The
Thermo Scientific HyClone SingleUse Mixer (S.U.M.) was designed
around this industry standard while
providing the unique advantage
of a completely disposable and
portable system. The HyClone®
S.U.M. bioprocess container, or bag,
is supplied in either contained or
open-top formats. The contained
system allows for mixing of sterile
liquid:liquid solutions or dust free
solid:liquid mixing. The solid:liquid
bags contain a 3” tri-clamp port for
powder addition that can be coupled
to another powder dispensing
Thermo Scientific HyClone
Powdertainer II bag. Mixing
applications in downstream processes
require a high level of scrutiny
into factors such as container
integrity, leachables/extractables and
particulate generation. The study
described in this report evaluates
particulate generated during a mixing
operation. Analyses were performed
on samples obtained at regular time
intervals to characterize the size and
quantity of particulates generated.
Materials and Methods
A 50 L S.U.M. stainless steel outer
support container with drive and
controller (SH50212.01) and a
liquid:liquid mixing 50 L S.U.M.
BPC, or BioProcess Container
(SH30767.01) were used in this
evaluation. Since all S.U.M.s and
Thermo Scientific HyClone SingleUse Bioreactors currently utilize
the same seal/bearing assembly,
which is integral to the BPC, and
since the surface to volume ratio of
the 50 L S.U.M. is higher than the
obtained using larger systems, it is
considered to be a “worst case” with
regard to particulate concentration.
Thermo Scientific HyClone Water
for Injection (WFI)(SH30221) was
used to fill the S.U.M. to 50%
nominal volume (25 L). Each trial
was performed at room temperature
(approximately 25° C).
In order to assess particulate
generation over time during a mixing
operation, multiple samples were
obtained. Samples were obtained
by aliquoting a 500 mL sample
from the S.U.M. BPC using a 500
mL polyethylene bottle and cap
(Nalgene, 34240-0650). Initially, the
mixer was operated at 350 RPM for
3 minutes and then sampled for the
first time-point. Thereafter, samples
were collected at 2, 4, 8, 12, 16 and
24 hours with the mixer operating
at 350 RPM. After 24 hours of
mixing, the seal/bearing assembly
was submerged in the WFI water
and agitated again at 350 RPM for
2 hours. A final sample was then
collected.
Second and third trials were
performed identically, with the
exception that a new 50 L S.U.M.
BPC (with integrated seal/bearing
assembly) and fresh WFI water were
used. Additionally, samples were
obtained less frequently, at 0, 12,
24 and 26 (following seal/bearing
assembly submersion) hours in order
to confirm results.
A Multisizer™ 4 (Beckman Coulter)
was used to determine particle
size and quantity. This analyzer
uses Beckman Coulter’s Electrical
Sensing Zone (ESZ) and Digital
Pulse Processing (DPP) to analyze
particle size and quantity. Samples
were prepared for analysis by adding
10 mL of 20% NaCl/H2O filtered
with a 20 nm filter to 200 mL of
each sample in order to make them
as conductive as the electrolyte
(0.9% NaCl/H2O). The analyses were
performed with an aperture setting
of 1 µm to 30 µm using volumetric
control mode and background
subtraction.
Results and Discussion
The ESZ method of determining
particle size and quantity allows
one to view the distribution of the
number of particles based upon
their size over a very large range
of sizes. Table 1 lists the sample
identification information, particle
size, quantity and the corresponding
figure containing a histogram for that
sample.
Sample Time
(hours)
Sample Description
Sample ID
1.1
2.1
3.1
1.2
1.3
1.4
1.5
2.2
3.2
1.6
1.7
2.3
3.3
1.8
2.4
3.4
3 minutes post agitation
initiation
0
2
4
8
12
16
24
Submerged seal/bearing
assembly
26
Particle Count
(particle/ml)
1868
2498
1093
3732
5212
8087
10435
7059
7803
12236
16574
11878
12090
15100
12195
12459
Mean Particle
Size (µm)
1.69
1.68
1.61
1.58
1.5
1.44
1.42
1.55
1.38
1.41
1.37
1.47
1.38
1.35
1.41
1.36
Particle Size and
Quantity Distribution
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Table 1. Sampling protocol and results of particle analysis used during a 24-hour mixing evaluation
to determine particle size and quantity.
Sample 1.1
40
Sample 2.1
60
18917A_Sample 1.1_02.#m4
35
18917I_Sample 2.1_01.#m4
50
Number ( per mL)
Number ( per mL)
30
25
20
15
10
30
20
10
5
0
40
1
2
3
4
5
6
7 8
Particle Diameter (µm)
9 10
20
0
30
Figure 1. Sampling protocol and results of particle
analysis used during a 24-hour mixing evaluation to
1
2
3
4
5
6
7 8
Particle Diameter (µm)
9 10
20
30
Figure 2. Histogram of particle size and quantity from
sample 2.1.
determine particle size and quantity.
Sample 3.1
40
80
Number ( per mL)
30
Number ( per mL)
18917B_Sample 1.2_01.#m4
90
35
25
20
15
10
70
60
50
40
30
20
5
0
Sample 1.2
100
18917M_Sample 3.1_01.#m4
10
1
2
3
4
5
6
7 8
Particle Diameter (µm)
9 10
20
0
30
Figure 3. Histogram of particle size and quantity from
sample 3.1.
2
3
4
5
6
7 8
Particle Diameter (µm)
9 10
20
30
Figure 4. Histogram of particle size and quantity from
sample 1.2.
Sample 1.3
120
1
Sample 1.4
200
18917C_Sample 1.3_01.#m4
18917D_Sample 1.4_01.#m4
180
160
80
Number ( per mL)
Number ( per mL)
100
60
40
140
120
100
80
60
40
20
20
0
1
2
3
4
5
6 7 8
Particle Diameter (µm)
9 10
20
Figure 5. Histogram of particle size and quantity from
sample 1.3.
30
0
1
2
3
4
5
6 7 8
Particle Diameter (µm)
9 10
20
Figure 6. Histogram of particle size and quantity from
sample 1.4.
30
Sample 1.5
250
Sample 2.2
180
18917E_Sample 1.5_01.#m4
18917J_Sample 2.2_01.#m4
160
140
Number ( per mL)
Number ( per mL)
200
150
100
120
100
80
60
40
50
20
0
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
0
30
Figure 7. Histogram of particle size and quantity from
sample 1.5.
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
30
Figure 8. Histogram of particle size and quantity from
sample 2.2.
Sample 3.2
200
1
Sample 1.6
300
18917N_Sample 3.2_01.#m4
18917F_Sample 1.6_01.#m4
180
250
140
Number ( per mL)
Number ( per mL)
160
120
100
80
60
40
200
150
100
50
20
0
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
0
30
Figure 9. Histogram of particle size and quantity from
sample 3.2.
Number ( per mL)
Number ( per mL)
300
250
200
150
100
20
30
Sample 2.3
18917K_Sample 2.3_01.#m4
200
150
100
50
50
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
0
30
Figure 11. Histogram of particle size and quantity from
sample 1.7.
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
30
Figure 12. Histogram of particle size and quantity from
sample 2.3.
Sample 3.3
350
Sample 1.8
400
18917O_Sample 3.3_01.#m4
18917H_Sample 1.8_01.#m4
350
300
300
Number ( per mL)
250
Number ( per mL)
4
5
6 7 8 9 10
Particle Diameter (µm)
250
350
200
150
100
50
0
3
300
18917G_Sample 1.7_01.#m4
400
0
2
Figure 10. Histogram of particle size and quantity from
sample 1.6.
Sample 1.7
450
1
250
200
150
100
50
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
Figure 13. Histogram of particle size and quantity from
sample 3.3.
30
0
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
Figure 14. Histogram of particle size and quantity from
sample 1.8.
30
Sample 2.4
300
300
250
250
200
150
100
50
0
In addition to these
Sample 3.4
350
18917L_Sample 2.4_01.#m4
Number ( per mL)
Number ( per mL)
350
18917P_Sample 3.4_01.#m4
offices, Thermo Fisher
Scientific maintains a
network of representative
organizations throughout
the world.
200
150
100
50
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
Figure 15. Histogram of particle size and quantity from
sample 2.4.
It is apparent from all three trials
that the particle size decreased with
increased mixing time. The samples
taken at 3 minutes post agitation
initiation had a total average size of
1.66 µm, whereas the samples taken
at 26 hours had a total average size
of 1.37 µm. This reduction in size is
most likely due to particles breaking
apart through agitation. The total
average number of particles from
1 to 30 µm in size from the first
samples taken in the three trials
was 1,819, while the total average
from the final samples was 13,251,
supporting the fact that the particles
may have been breaking apart.
United States Pharmacopeia (USP)
Chapter 788, Particulate Matter in
Injections, requires that injectable
solutions of 100 mL or less per
injection (small volume injections)
have a maximum particle count of
6000 particles/container greater
than or equal to 10 µm in size and
600 particles/container greater
than or equal to 25 µm in size. For
large volume injections (injectable
solutions of greater than 100 mL),
USP 788 requires a maximum
particle count of 25 particles/mL
30
0
1
2
3
4
5
6 7 8 9 10
Particle Diameter (µm)
20
Figure 16. Histogram of particle size and quantity from
sample 3.4.
greater than or equal to 10 µm
in size and 3 particles/mL greater
than or equal to 25 µm in size as
determined by light obscuration.
Results from this evaluation show
the total average of particles greater
than or equal to 10 µm in size to
be 3.3 particles/mL and particles
greater than or equal to 25 µm
in size to be 0 (none found). The
S.U.M. mixing system, even when
evaluated at a high surface area per
volume ratio with the seal/bearing
assembly rotating at maximum
speed for 24 hours, meets the USP
788 requirements in particle count.
The methods stated for use in USP
788, however, are light obscuration
or microscopic particle count. In
this evaluation, we utilized the ESZ
method in order to determine a
distribution of particle sizes within
a greater range than required by
USP 788. These results also meet
the requirements under USP 788 for
microscopic method particle count
(small volume injections: 3000
particles/container greater than
or equal to 10 µm in size and 300
particles/container greater than or
equal to 25 µm in size; large volume
injections: 12 particles/mL greater
than or equal to 10 µm in size and 2
particles/mL greater than or equal to
25 µm in size).
Summary
Based upon the ESZ method for
particle enumeration and size
determination, the S.U.M. meets the
particle size/quantity requirements
of USP 788, Particulate Matter in
Injections. The evaluation included
a “worst-case” scenario of a high
surface area to volume ratio,
maximum rotational speed (350
RPM) of the seal/bearing assembly
(including impeller) and a 24 hour
mixing time with an additional 2
hours mixing at maximum rotation
speed while the seal/bearing
assembly was submerged. Multiple
samples were obtained during three
trials in order to assess particle size
and quantity. The highest particle
counts observed in each of the
three trials of size greater than or
equal to 10 µm were found in the
first samples taken. These samples
contained an average of 3.3 particles
of 10 µm or larger. No particles of
25 µm or larger were found in any
samples collected throughout the
evaluation.
30
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