Application Note

High-throughput visible particle, turbidity and color
measurement of biologics formulations
Introduction

light scattering. Visible particles are typicallyassessed using a manual inspection process
to determine the sample’s particle count, color
and turbidity. This results in data that is semiquantitative, varies from user to user and does
not provide for image capture, data retrieval or
future verification, comparison and review. Not tomention the consumption of precious sample thatmany manual methods require.

One of the major challenges in drug development
is the need for rapid formulation of biologic drug
molecules. Developed in conjunction with R&D departments across the industry, Unchained Labs addresses this challenge in both vaccine and protein
formulation with the freeslate system configured
for biologics formulation (Figure 1).
The freeslate system provides the opportunity
to expand drug developers' experimental design
spaces, using less resources and material in lesstime than conventional methods.

To streamline the assessment of visual particles
suspended in solutions, Unchained Labs developed a high-throughput visual inspection station(VIS) that is integrated directly into the freeslate
system (Figure 2).
Through non-destructive imaging, VIS automatically:• Counts the visible particles suspended in solution• Assesses formulation color against known standards• Measures the formulation turbidity using a calibrated scale• Captures images of vial and its contents

Automation where it counts

Figure 1: The freeslate system configured for biologics formulation.

Particle measurement involves vial handling, suspension of particles, image capture and image
analysis. Using a combination of the VIS and Lab
Execution and Analysis (LEA) software, a vial/plategripper transfers a vial to the VIS. Once the vial
is loaded, particles are suspended prior to image
capture by spinning the vial (Figure 3). Acceleration,rotation speed, rotation time and deceleration are
all set by the user and controlled by Unchained Labs'
LEA software.

Streamlining visual measurement through imaging
Assessing the formation and aggregation of bothsub-visible and visible particles under a variety ofstress conditions is a critical step in understanding
drug product stability. Sub-visible particles areassessed using analytical techniques such as
micro-flow imaging, light obscuration and dynamic
1

HIGH-THROUGHPUT VISIBLE PARTICLE, TURBIDITY AND COLOR MEASUREMENTΔtSpin

StopInspect

Figure 3: Mechanism of visible particle detection. Once a vial isloaded into the VIS, it spins the vial to resuspend the particles.
The vial is stopped, and then the system captures a series ofimages.1Particle counting measurement method:
1 Two 3 mL sample vials were prepared. One vial
was filled with approximately 2 mL of water,
the other was filled with the same volume of65% glycerol solution. The second solution hada viscosity of approximately 15 centipoise (cP).
NIST-certified standard particles were added toboth vials. Approximately 55 glass beads with adiameter of 170 μm were placed in the glycerol
solution and 8-9 170 µm diameter polystyrene
beads were added to the vial containing water.

Figure 2: Unchained Labs' visual inspection station (VIS)

Spot the difference
The VIS stops the vial after spinning and captures aseries of images. Successive images are subtracted
to remove “static” features, including vial defects andsmudges. Resultant “difference” images contain onlythose features (i.e., moving particles) that differ fromimage to image. These “difference” images containthe information about particle counts. All images
are stored in the LEA database for later review andre-analysis.

2 Sample vials were loaded into a plate rack,
which was placed on the freeslate system.
3 The freeslate system and automatically loaded
the vials into the VIS.
4 Once loaded, each vial was spun using user-specified motion settings to suspend theparticles.

The particle counting protocol
To mimic protein particles suspended in differentviscosity formulations vials containing 8–9
polystyrene beads in water and approximately
55 glass beads in glycerol were analyzed. The
following protocol was designed and executed toevaluate the particle counting reproducibility ofthe VIS.

5 Once the spinning stopped, multiple images
were acquired at a frame rate of up to 8 fps.
6 A region of interest (ROI), which is the center ofthe vial's image was defined.
7 Successive images were subtracted to remove
static features.

2

HIGH-THROUGHPUT VISIBLE PARTICLE, TURBIDITY AND COLOR MEASUREMENT

8 Resultant difference images were converted tobinary images.

The average particle counts for repeat runs werewithin the expected particle count range. The runto-run count variations are likely due to particle
overlap (especially in the glycerol solution containing over 50 particles) and particles outside of theROI during image acquisition. These results illustrate that the VIS can accurately and reproducibly
count particles suspended in solutions across arange of particle sizes and a range of fluid viscosities that are relevant to drug developers.

9 Analysis was performed to identify and countparticles. Both samples were measured fourtimes. Particle counts for each measurement
are presented in Table 1.

Glass beads in
65% glycerol

Polystyrene
spheres inwater

151

8254

9352

9459

850–60particles

8–9 particles

Run number

Manual count

Assessing formulation color
The VIS can also accurately assess the color ofan unknown sample against a set of standards(Figure 4).
To establish reproducibility, the color of a BY4 sample in a 3 mL vial was assessed 30 times (Table 2).

Table 1: Particle count reproducibility results for small sphericalparticles in solutions.

Match score(%)

Match name199.99

BY4299.99

BY4399.99

BY44100.00

BY45100.00

BY46100.00

BY4..

..

..

15100.00

BY4..

..

..

20100.00

BY4..

..

..

25

100.00

BY4..

..

..

30

100.00BY4Test number

Figure 4: Color assessment of an unknown sample against a setof color standards using LEA software.

Table 2: Repeated color assessment of BY4 sample. All matchscores between test #6 and #30 are the same.

3HIGH-THROUGHPUT VISIBLE PARTICLE, TURBIDITY AND COLOR MEASUREMENT

Normalized luminance = ∆L / L = (Lblank - Lsample) / Lblank

Turbidity measurement method
1 Turbidity standards were placed in 3 mL and
20 mL vials and loaded into the VIS automatically.
2 A blank image (no vial in place) was acquired.
3 The sample vial was loaded into the VIS system.
4 A sample image was acquired.
Figure 5: Blue color standards using LEA software.

5 The normalized luminance was calculated.
6 Steps 2–5 were repeated five times witheach standard.

The capability of the VIS to assess color across a
broader array of standard colors was also evaluated. “Blue” standards were introduced among
the set of color standards and then assessedusing the VIS, which easily achieved a color match(Figure 5).

7 The average normalized luminance values weredetermined for each standard.
8 A calibration curve was generated for eachvial size.

Non-destructive turbidity measurement

The standards used were 1, 2, 5, 10, 20, 40 and
100 Nephelometric Turbidity Units (NTU). The calibration curves are shown in Figure 6.

Turbidity is often a challenging measurement as it
typically requires significant manual involvement
and requires the use of optical cuvettes, which consume samples. To evaluate the accuracy and reproducibility of VIS for turbidity, the system measured
known turbidity standards (from GFS Chemicals).

Reproducibility of turbidity measurements wasevaulated by testing a series of turbidity standards in 3 mL vials (i.e., from 1-100 NTU) seventimes. Measurements were then repeated the nextday (Figure 7). Results show that all measurements were within 5% of the expected values.

Turbid samples scatter light, which causes the average luminance of a sample to decrease compared
to a low turbidity sample. The VIS utilizes a calibration curve obtained from turbidity standards to calculate the turbidity of a sample from the averagemeasured luminance value. Each calibration curve
must be generated using the same type of vial thatwill be used in a particular experiment.

Turbidity calibrations

120
3 mL vial
Linear (3 mL vial)

100

20 mL vial
Linear (20 mL vial)

Turbidity (NTU)80

The VIS employs a normalization method toensure the reproducibility of turbidity measurements. The VIS normalizes luminance by acquiring two images: a “blank” image immediately prior
to loading a sample and a “sample” image oncethe vial is in place. The luminance of this “blank”
image is used to normalize the turbidity measurement using the following formula:60

y=867.8x – 48.26
R2= 0.9999

40

y=465.5x – 26.11
R2= 0.9988

200-20
00.05

0.1

0.15

0.2

0.25

Normalized luminance (ΔL / L)

Figure 6: Turbidity calibrations for 3 mL and 20 mL vials.

40.3

HIGH-THROUGHPUT VISIBLE PARTICLE, TURBIDITY AND COLOR MEASUREMENT• For particle counting, the VIS accurately and reproducibly counted particles suspended in eachsolution across a range of particles and solution
viscosities.

Turbidity reproducibility over two days

0.12

3 mL vial day 10.11

3 mL vial day 20.10• For color assessment, the VIS reliably matchedcolors of samples across a broad range of standards.

ΔL / L0.090.080.07• For turbidity mea­surement, the VIS accurately and reproducibly measured samples from5-100 NTU.

0.06
0.05
0.04

0510152025303540455055Overall, the VIS significantly increased throughput
compared to labor intensive, manual visual inspection methods while maintaining high accuracy andreproducibility. The VIS further enhances the speedof measuring color, turbidity and visible particles
present in liquid protein formulations leading tosignificant gains in productivity in formulation development.

60

Turbidity (NTU)

Figure 7: Turbidity measurement reproducibility data.

Conclusion
Unchained Labs' VIS was evaluated as ahigh-throughput device for three visual inspection
applications—particle counting, color assessment
and turbidity. All measurements were automated,quantitative and non-destructive.

References
1 Challenges and Strategies for Implementing
Automated Visual Inspection for Biopharmaceuticals, Rathore et al., Pharmaceutical Technology, Nov 1, 2009

Unchained Labs
6940 Koll Center Pkwy, Suite 200
Pleasanton, CA 94566
Phone: 1.925.587.9800
Toll-free: 1.800.815.6384
Email: info@unchainedlabs.com© 2016 Unchained Labs. All rights reserved. UnchainedLabs is a registered trademark. All other brands orproduct names mentioned are trademarks owned bytheir respective organizations.

Rev A5