ProteinSimple eBook - 16

Simplifying Protein Analysis * Comparability Study of Manual and Automated Particle Characterization with MFI

with Takeda Denmark, we outlined the process for transferring a manual protocol
to an automated format with the Bot1 autosampler and the MFI 5000 series,
and demonstrated comparability of the two modes using a model protein system
composed of 1% BSA at the two sites (ProteinSimple and Takeda Denmark).
No statistically relevant differences were observed between manual and
automated runs at either facility.
Materials and Methods
Sample Preparation and Instrument Operation

Figure 1. Analysis method for manual and automated samples at both
ProteinSimple and Takeda sites. This analysis method was used with the batch
protocol for modified Bot1 Protocol D. All samples were analyzed under the same
conditions, with an analysis volume of 0.77 μL, and Optimize Illumination volume
of 0.22 μL. Particle baseline was established with 0.2 μm-filtered DDI water.

coulter counter cannot do, since they only provide size and count 2,3.
To meet the increasing demand for biopharmaceutical testing of sub-visible
particle characterization, higher throughput and standardization are needed,
and MFI 5000 series easily meets that demand through automated, walk-away
particle analysis with the addition of the Bot1 autosampler. In collaboration
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* 1% BSA sample was prepared, heated at 60 °C overnight (at least 12 hours)
with shaking at 1200 rpm at the ProteinSimple facility. The sample was
then aliquotted and stored at -80 °C. Frozen aliquots were shipped to
the Takeda facility on dry ice and stored at-80 °C until use.
* Flow cell was washed with 1 mL of 10% Triton-X 100 in distilled,
deionized (DDI) water (0.2 micron ﬁltered) and rinsed 4X with
1 mL water before each run.
* A fourth stirring step was added to the protocol to ensure particles did
not settle during the sampling process on the Bot1 (Table 1).
* Manual pipetting steps were performed by turning off the Bot1 autosampler
and manually pipetting sample into the MFI 5200 system via the sample
inlet port. The same analysis method was used for both the automated
and manual protocols (Figure 1).

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