APPLICATION NOTE

HyPerforma Single-Use Mixer

HyPerforma Single-Use Mixer(S.U.M.) performance evaluation
Measurement of particulate size andquantity generated during operation
Introduction
Disposables have become increasingly popular in
bioproduction, from media storage bags and filling
manifolds to mixing vessels and bioreactors. Simple
designs such as 2-dimensional “pillow” bags on rocking
platforms and recirculation loops were early attempts tomeet this need. However, this style of agitation is similar
to the industry standard used in traditional stainlesssteel systems. Other types of mixing devices were later
developed, including oscillating agitators and magneticallycoupled impellers.
The stirred tank is the most commonly utilized style ofmixing vessel for both liquid–liquid and powder–liquid
mixing applications. The Thermo Scientific™ HyPerforma™
Single-Use Mixer (S.U.M.) was designed to meet thisindustry standard but also to provide the unique advantage
of a completely disposable and portable system. The
S.U.M. BioProcess Container (BPC) is supplied ineither contained or open-top formats. The contained
system allows for sterile liquid–liquid mixing or dust-freepowder–liquid mixing. The powder–liquid BPCs containa 3-inch tri-clamp port for powder addition that can becoupled to another powder-dispensing Thermo Scientific™
Powdertainer™ II BPC. Mixing applications in downstream

processes require a high level of scrutiny into factors
such as container integrity, leachables, extractables,and generation of particulates. This study evaluatedparticulates generated during a mixing operation.
Analyses were performed on samples obtained at regulartime intervals to characterize the size and quantity ofparticulates generated.

Materials and methods
A Thermo Scientific™ 50 L S.U.M. stainless steel outer
support container with drive and controller and a
Thermo Scientific™ liquid–liquid mixing 50 L S.U.M. BPC(Cat. No. SH30767.01) were used in this evaluation. Since
all S.U.M.s and single-use bioreactors (S.U.B.s) currently
utilize the same bearing-and-seal assembly, which is
integral to the BPC, and since the surface-to-volume ratioof the 50 L S.U.M. is higher than that of larger systems,
this setup is considered to be a worst-case scenario withregard to particulate concentration. Water for injection(WFI) was used to fill the S.U.M. to 50% nominal volume(25 L). Each trial was performed at room temperature(approximately 25°C).
To assess particulate generation over time during a mixing
operation, multiple samples were obtained. Samples
were obtained by aliquoting a 500 mL sample from theS.U.M. BPC using a 500 mL Thermo Scientific™ Nalgene™
polyethylene bottle and cap (Cat. No. 34240-0650). Initially,
the mixer was operated at 350 rpm for 3 minutes andthen sampled for the first time point. Thereafter, samples
were collected at 2, 4, 8, 12, 16, and 24 hours, with themixer operating at 350 rpm. After 24 hours of mixing, thebearing-and-seal assembly was submerged in WFI andagitated again at 350 rpm for 2 hours. A final sample wasthen collected. For confirmation, second and third trials
were performed similarly, with a new 50 L S.U.M. BPC (withintegrated bearing-and-seal assembly) and fresh WFI, but
samples were obtained less frequently at 0, 12, 24, and26 (following bearing-and-seal assembly submersion) hours.

A Multisizer™ 4 counter (Beckman Coulter) was used todetermine particle size and quantity. This analyzer uses
electrical sensing zone (ESZ) and digital pulse processing(DPP) methods to analyze particle size and quantity.
Samples were prepared for analysis by adding 10 mL of
20% NaCl in H2O (filtered with a 20 nm filter) to 200 mLof each sample, to make the samples as conductive as
the electrolyte (0.9% NaCl in H2O). The analyses wereperformed with an aperture setting of 1 µm to 30 µm usingvolumetric control mode and background subtraction.

Results and discussion
The ESZ method allows determination of the distribution of the number ofparticles, based on their size, over a very large range of sizes. Table 1 lists thesample identification information, particle size, and quantity for that sample.

Table 1. Sampling protocol used during a 24-hour mixing evaluation to determine particle size and quantity,and results of particle analysis.
Sample ID

Particle count(particles/mL)

Mean particle
size (µm)

Count of>10 µm particles

Count of>25 µm particles1.1

1,868

1.69

10

2.1

2,498

1.68

8

0

3.1

1,093

1.61

302

1.2

3,732

1.58

304

1.3

5,212

1.50

008

1.4

8,087

1.44

10

1.5

10,435

1.42

0

0

2.2

7,059

1.55

5

0

3.2

7,803

1.38

00

1.6

12,236

1.41

0

0

1.7

16,574

1.37

00

2.3

11,878

1.47

00

3.3

12,090

1.38

00

1.8

15,100

1.35

0

0

2.4

12,195

1.41

0

0

3.4

12,4591.36

00

Sample time(hours)

0Sampledescription

3 minutes after
initiation of agitation

12162426

Submerged
bearing-and-seal
assemblyIt is apparent from all three trials that the mean particle size
decreased with increased mixing time. The samples taken
at 3 minutes after initiation of agitation 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 insize is most likely due to particles breaking apart throughagitation. The total average number of particles 1–30 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 idea that the particles werebreaking apart.
United States Pharmacopeia (USP) Chapter 788,Particulate Matter in Injections, requires that injectable
solutions of <100 mL per injection (small-volumeinjections) do not exceed a maximum particle countof 6,000 particles/container of ≥10 µm in size and600 particles/container of ≥25 µm in size. For injectable
solutions of >100 mL (large-volume injections), USP
788 requires not exceeding a maximum particle countof 25 particles/mL of ≥10 µm in size and 3 particles/mLof ≥25 µm in size as determined by light obscuration.
Results from this evaluation show the total average
number of particles ≥10 µm in size to be 3.3 particles/mLand particles ≥25 µm in size to be 0 (none found). The
S.U.M. mixing system, even when evaluated at a highsurface-to-volume ratio with the bearing-and-seal assemblyrotating at maximum speed for 24 hours, meets USP
788 requirements for particle count.

The methods stated for use in USP 788, however, arelight obscuration or microscopic particle counting. In
this evaluation, we utilized the ESZ method to determine
the distribution of particle sizes within a greater range
than required by USP 788. These results also meet therequirements under USP 788 for microscopic particle
count (small-volume injections: 3,000 particles/containerof ≥10 µm in size and 300 particles/container of ≥25 µm insize; large-volume injections: 12 particles/mL of ≥10 µm insize and 2 particles/mL of ≥25 µm in size).
Summary
Based upon the ESZ method for particle and size
determination, the HyPerforma S.U.M. meets the particle
size and quantity requirements of USP 788. The evaluation
included a worst-case scenario of a high surface-to-volume
ratio, maximum rotational speed (350 rpm) of the
bearing-and-seal assembly (including impeller), and a
24-hour mixing time with an additional 2 hours of mixing
at maximum rotational speed while the bearing-and-seal
assembly was submerged. Multiple samples wereobtained during three trials in order to assess particle
size and quantity. In each of the three trials, the highest
counts of particles of ≥10 µm in size were found in the firstsamples taken. These samples contained an average of3.3 particles of ≥10 µm. No particles of ≥25 µm were foundin any samples collected throughout the evaluation.

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