Application Note

High-throughput viscosity measurement of biologics
formulations
Introduction

contamination. The viscosity station measures
samples at user-specified temperatures between 4-40 °C. Scientists can also test multiple shear rates using the viscosity station. The
high-throughput (approximately three minutes
per sample) and low sample volume required forthe viscosity station enables the practical exploration of a variety of formulation conditions
compared to traditional methods.

One of the major challenges in drug development
is the need for rapid formulation of biologic drugcandidates. Developed in conjunction with R&D
departments across the industry, UnchainedLabs 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.

High-throughput viscosity station
The viscosity station is a miniature capillary
viscometer1. A positive displacement tip (PDT)
pipette, integrated on a freeslate system arm,
injects sample through the capillary at a controlled flow rate. The instrument measures thepressure drop across the capillary. For Newtonian liquids, the measured drop in pressure
across the capillary directly relates to the solution viscosity (μ) which is calculated using theequation,

High concentration protein formulations forbiologics can reach viscosities that make themdifficult to manufacture, limit delivery options
or even require costly delivery methods. Consequently, viscosity is becoming an importantconsideration during formulation development.
To this end, viscosity measurements are a bottleneck. To alleviate this challenge, UnchainedLabs developed the automated viscosity station
for the early assessment of formulation viscosity.
The viscosity station uses a temperature-controlled microcapillary for the measurement ofviscosity ranges relevant to biologics formulations (1–20 cP) and can measure viscosities
as high as 100 centipoise (cP). Each viscosity
measurement only requires 100 μL of sample
per measurement. The viscosity station includes
an automated, integrated washing system toeliminate any chance of sample-to-sample cross

μ= πR4ΔP/8LQ
where R is the capillary internal diameter, L isthe capillary length, ΔP is the change in pressure
across the capillary and Q is the flow rate. Twoexperiments were performed to assess the reproducibility of the viscosity station each with twoviscosity standards measured 10 times at a fixedtemperature. The results of each standard areshown in Table 1.

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HIGH-THROUGHPUT VISCOSITY MEASUREMENT OF BIOLOGICS FORMULATIONS

4 °C

40 °C

Viscosity standard

1 cP

99 cP

0.7 cP

36 cP

Average

0.951

91.9

0.679

36.4

Std dev

0.024

0.038

0.038

0.087

%RSD

2.5

0.8

5.6

0.2N10101010Table 1: Reproducibility of the viscosity station over a range of viscosities and temperatures.

Measuring the viscosity of Bovine
Serum Albumin solutions

The standard deviation for each measurement,
repeated in triplicate, was usually less than 3%
and the maximum standard deviation was 7.4%.

Bovine Serum Albumin (BSA) solutions were used
to demonstrate the throughput and range of theviscosity station. For this study, BSA was dissolvedin two different phosphate buffered saline (PBS)
solutions, either containing Tween 80 or containing
Tween 80 plus sucrose. Both known to increase
the viscosity of solutions. The first solution was
10 mM PBS + 0.6% Tween 80; the other solution
was 10 mM PBS + 0.6% Tween 80 + 10% sucrose.
BSA was dissolved in the PBS solutions to prepare
200 mg/mL stock solutions. Samples for analysis
were made by diluting the stock solutions to 20,
50 and 100 mg/mL BSA with their respective PBS
solutions. Measured viscosities of the BSA andstock solutions are presented in Figure 1. Eachmeasurement was made using 100 μL of sample.

Measuring viscosity of Newtonian and non-Newtonian fluids
The viscosity station can measure the viscosity ofNewtonian liquids and non-Newtonian liquids. To
characterize non-Newtonian fluids, measurements
can be made across a range of shear rates (flowrates). The resultant data can be used to calculate
the Weissenberg-Rabinowitsch (WR) correction
factor. The WR correction factor can then be used
to calculate the actual shear rate from the apparent (Newtonian) shear rate.
An example of using the viscosity station tomeasure non-Newtonian solutions is the measurement of Refresh Liquigel® eye rewetting
drops. Refresh Liquigel is a complex non-Newtonian, shear-thinning liquid. It is designed to havea relatively high viscosity at low shear rates inorder to provide good retention on the eye andlow viscosity under shear to enable blinking toproduce a uniform thin layer of the liquid acrossthe eye. Refresh Tears®, on the other hand, is a
Newtonian liquid; the viscosity of this productdoes not vary substantially with shear rate. Theviscosities of Refresh Liquigel and Refresh Tears
were measured using flow rates from approximately 1-15 μL/s, which correspond to shear rates
from approximately 1,000-15,000 s-1. To analyzethe data obtained for the Liquigel product, theWR correction factor was determined and thenapplied to each measurement.

9BSA in PBS + 0.6% Tween 80

8BSA in PBS + 0.6% Tween 80 + 10% sucrose

Average viscosity (cP)

7654
3
2
1
0050

100

150

200

250

BSA concentration (mg/mL)

Figure 1: BSA solution viscosity versus concentration results
collected using the automated viscosity station.

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HIGH-THROUGHPUT VISCOSITY MEASUREMENT OF BIOLOGICS FORMULATIONS

formulations (Figure 4) increased significantly duringstorage. The viscosities of many 210 mg/mL formulations stored at 40 °C approached or exceeded theinstrument measurement range (100 cP). By using
the viscosity station for this stability study, we wereable to rapidly assess the effect formulation, antibody
concentration and storage conditions had on theviscosity of each antibody solution. The integration of
the viscosity station into the freeslate systems enables the unattended high-throughput measurementof viscosity in protein solutions and biotherapeuticformulations.

The actual (corrected) shear rate was used in theviscosity calculation. As expected, the viscosityof the Refresh Tears (Figure 2) did not change
appreciably across this range of shear rates.
Conversely, the viscosity of the Refresh Liquigel
dropped by more than a factor of two across theapplied range of shear rates. Using as little as
100 μL of sample, the shear rates of solutions were
accurately measured with the viscosity station.

25Liquigel
Refresh Tears

3.0

15

2.5

10

Average viscosity (cP)Viscosity (cP)

20

5002

468

1012141618

Shear rate (1,000/s)

Figure 2: Viscosity measurement of Newtonian and non-Newtonian solutions.

t0

T4C 45d

T40C 45d

T25C 45d

T4C 90d

T40C 90d

T25C 90d

2.01.51.00.50F13F14F15F16F17F18F19F20F21F22F23F24Formulation code

High-throughput viscosity measurement of monoclonal antibody
formulations

Figure 3: Viscosity measurements during stability testing of 12mAb formulations at 50 mg/mL (F13–F22 and F24) or 10 mg/mL(F23).

A set of 24 different monoclonal antibody (mAb)
formulations were evaluated in a stability study. ThemAb concentration in 12 of the formulations (F01–F12) was 210 mg/mL. The mAb concentrations of theremaining formulations was 50 mg/mL (F13–F22 andF24) or 10 mg/mL (F23). In addition to mAb concentration, the selected formulations also vary widely in
pH buffer species, excipient types and excipient levels.
The initial viscosity of each formulation was measured(t0), and six other sets of formulations were stored at
4 °C, 25 °C or 40 °C for 45 and 90 days. At the end ofeach storage period, the viscosity of each formulation
was measured. The viscosity station detected viscosity differences resulting from differences in formulation composition including mAb concentration andexcipients. Viscosities of 50 mg/mL formulations (Figure 3) did not change dramatically during the stabilitystudy. In some cases, the viscosities of the 210 mg/mL

10090t0

T4C 45d

T40C 45d

T25C 45d

T4C 90d

T40C 90d

T25C 90d

Average viscosity (cP)

80
7060
50
40
30
20
100

F01

F02

F03

F04

F05

F06

F07

F08

F09

F10

F11F12Formulation code

Figure 4: Viscosity measurement of twelve 210 mg/mL mAb formulations during stability testing. Viscosities >100 cP are beyondthe performance specifications of the viscosity station and arereported as 100 cP.

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HIGH-THROUGHPUT VISCOSITY MEASUREMENT OF BIOLOGICS FORMULATIONS

Conclusion

The freeslate systems configured for biologics formulation enable the automated characterizationof an array of candidate formulations. And, whenused in conjunction with Lab Execution and Analysis (LEA) software from Unchained Labs, datafrom off-line analytics (e.g., SEC, cSDS and cIEF),
can be associated with a particular formulation forreporting. The combination of the viscosity stationon the freeslate system combined with the associated LEA software provide a comprehensive andpowerful system for formulation screening androbustness assays of biotherapeutics.

Biologics formulations are often highconcentration (50–150 mg/mL), and the viscosities
of such solutions can pose challenges to drug
developers. An integral solution to this challenge
is the viscosity station on the freeslate system,
which was designed specifically to target theacceleration of formulation development with thefollowing benefits:• High-throughput viscosity measurements using
100 μL of sample• Measurement of biologics formulations with viscosities ranging from 1-100 cP• Viscosity measurements and automatic cleaning
in less than three minutes• Automated cleaning of the capillary between
measurements• Viscosity measurement over a wide temperaturerange• Viscosity measurement of Newtonian andnon-Newtonian solutions

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.

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