Application Note

Automation of biologics formulation stability testing:achieving equivalent results with less manual effort​
Russell Burge, Ph.D., Unchained Labs
Fanny Reynaud, Pharm.D., LFB, Cécile Jaume, Pharm.D., LFB

Application

devel­opment of biopharmaceuticals. The freeslate
system automates many tasks such as formula­tion and analytical sample preparation along withsample processing and stressing. It was designed
to increase productivity while maintaining com­parability to the procedures and analytical tech­niques currently used in formulation laborato­ries.

Formulation development of biopharmaceuti­cals can be a chal­lenging and time consuming
process. To streamline develop­ment activities,
many pharmaceutical companies have imple­
mented laboratory automation for analyticalsample preparation, formulation screening andforced-degradation studies. While automation
can increase lab efficiency and throughput, it
must also provide results comparable to manual
methods. Recently, we performed a study with the
French biopharmaceutical group LFB to compare
manual and automated procedures for protein
drug formulation. Recent updates to regulatory
requirements are increasingly demanding studies
with larger number of samples. In this respect, LFB
was interested in evaluating an automated sys­
tem that could increase both experimental capac­
ity and number of formulations evaluated, as wellas enabling innova­tion in drug development. Here
we present results for seven protein drug formula­tions after stressing and analysis by both manualand automated procedures.

When using the 96‑well plate format, thefreeslate system can screen more than 300
formulations in a single day.

The size and scope of the freeslate BFS can be
tailored to the spe­cific needs of a formulation
workflow and laboratory. The platform used forthis study consisted of two freeslate systems con­nected by a carousel enclosed in a low-particulate
workspace (Figure 1). The freeslate system on theright side has multiple arms and tools that per­form liquid handling and vial and plate transfers.
These arms can access the deck, which includes
heating, cooling and mixing (vortex or magnetic
stir bars) stations as well as proprietary analyti­
cal instruments for vi­sual inspection and viscosity
measurement. The freeslate system on the left
side physically integrates with an incubated plateshaker, dynamic light scattering (DLS) system and
UV/Vis plate reader. The two systems are con­nected by a carousel that provides storage forsamples and consumables (pipetter tips, micro­
plates, vial rack, etc.) and facilitates the movement

Automation designed for formulation development
Automated procedures described in this appli­
cation note were performed on Unchained Labs'
freeslate system configured for biologics formu­
lation. The freeslate system is a comprehensive
automation platform that increases capabilities,
productivity and repeatability in formulation
1

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

laboratory scale (approximately 200 mL) andthen transferred to 2 mL vials and 5 mL tubes. All
formula­tions were subjected to a series of stressconditions in vials or tubes and then tested forevidence of degradation. Molecule 1 formulations
were stressed by stirring at 300 rpm using mag­netic stir bars for 24 hours, rotational agitation at
35 rpm for 24 hours (5 mL tubes only) and heat­ing at 48 °C for 3 hours. Molecule 2 formulations
were stressed by stirring at 500 rpm for 8 hours
using magnetic stir bars, rotational agitation at 35rpm for 8 hours (5 mL tubes only) and heating at
57 °C for 3 hours. Analytical results from UV/Vis(protein content and turbidity), DLS and SEC of
the stressed material were used to determine therelative robustness of each formulation for both
molecules. All processes were performed using
individual laboratory devices and instru­ments
without further automation (Figure 2).

Figure 1: Unchained Labs' freeslate system configured for bio­
logics formulation. The freeslate system on the right prepares
formulations and analytical samples, measures pH and viscosity,and performs visual inspection. The freeslate system on the left
accesses the Wyatt DynaPro® Plate Reader II Dynamic Light
Scattering (DLS) instrument and Molecular Devices SpectraMax®
Microplate Reader. A low-bioburden HEPA enclosure encom­
passes the entire system and work space.

of samples through the system. This unique setof capabilities allows scien­tists to prepare, stressand analyze formulations in a complete and un­interrupted workflow. The freeslate is compatible
with a variety of containers and formats including
ANSI/SLAS micro­plates (e.g., 96-well plates) andserum vials from 2–20 mL.

Automated process
The same confidential formulations of Molecule
1 and Molecule 2 were evaluated using the dual
freeslate system. Stress parameters for each mol­ecule were optimized according to the capabilitiesof the robot. Rotational agitation, performed aspart of the manual process, was replaced by vor­
texing. Vortexing speeds were adjusted to achievea similar degree of agitation of the solution to thatobserved in rotational agitation. Stirring intensi­
ties were adjusted to the size of the stir bar in thevials and to the solution volume in order to achieveintended stressing of the sample.

In this particular study, we used this dual system
to perform stressing fol­lowed by analysis to deter­mine the robustness of multiple bio­pharmaceuticalformulations. High-throughput enhanced visualinspection (visible particles, color and tur­bidity),
UV/Vis and DLS analyses were integrated onto
the freeslate deck. Size-exclusion chromatography(SEC) was performed using a UPLC instrumentthat was virtually integrated using Unchained
Labs' software to simplify data interpretation,
analysis and reporting.

The dual freeslate system automated formulationstresses, all analytical sample preparation andanalyses of stressed formulations except SEC,
which was virtually integrated (networked via
a shared database) to the freeslate BFS. Three
confidential formulations for Molecule 1 andfour confidential formulations for Molecule 2
were manually transferred to 2 mL serum vials
inside the low bio­burden freeslate workspace.
All formulations for T0 testing and each stresscondition were prepared in triplicate. After
stressing, the heated and agitated formulations
were analyzed by automated enhanced visualinspection (color, turbidity and visible particle

Case study
Traditional process
Two confidential protein drug products, Molecule
1 and Mol­ecule 2, were provided by LFB for thisstudy. Three confidential formulations of Mole­cule 1 (A–C) and four formulations of Mol­ecule 2(A–C and B') were evaluated by traditional, largely
manual processes. All formulations of Molecule 1
and Molecule 2 were manually prepared at the2

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Manual process

Automated process

Molecule 1
A
BC

Molecule 2
A
B
B´
C

Stirring 300 rpm 24 hours
Agitation 35 rpm 24 hours
Heating 48 °C 3 hours

Stirring 500 rpm 8 hours
Agitation 35 rpm 8 hours
Heating 57 °C 3 hours

UV/VisDLS
SEC

FormulateStressAnalyze

Molecule 1
A
BC

Molecule 2
A
B
B´
C

Stirring 300 rpm 24 hours
Agitation 400 rpm 24 hours
Heating 48 °C 3 hours

Stirring 300 rpm 4 hours
Agitation 400 rpm 4 hours
Heating 57 °C 3 hours

Color
Turbidity
Visible Particle Counting
UV/VisDLS
SEC

Figure 2: Formulation study design. Two protein drug molecules were formulated, stressed and analyzed by a traditional
manual process and an automated process to assess if the automated process provided comparable results.

counting). The stirred formulations were not tested
by enhanced visual inspection due to interferenceby the stir bars. The dual freeslate system
was then used to aliquot and/or dilute protein
formulations into 96-well plates for analysis by
DLS, UV/Vis (A400 and A280) and SEC.

Molecule 2
All Molecule 2 formulations were prepared in 2 mL
serum vials, in triplicate, by manually dispensing
the liquids into vials. Formula­tion B´ was gener­
ated by adding an excipient to formulation B. A
freeslate jr. with solid dispensing capabilities add­
ed 2.2–2.5 mg (target was 2.6 mg) of excipient
into three vials, then 2 mL of formulation B´ wasmanually dispensed into each vial. Vials of formu­
lation B´ were mixed by the vortexer module of thedual freeslate system. The purpose of formulation
B’ was to test automated powder dispensing andalso to investigate the stability of Molecule 2 when
dry excipients were added to the formulation. Notethat formulation B´ is not part of the traditional
formulation assessment, and that in the end B andB´ have the exact same composition.

Molecule 1
The dual freeslate system subjected formulations
A-C of Molecule 1 to stress con­ditions similar to the
traditional processes described above. The exper­
imental procedures for controlling the freeslate
BFS were generated using Unchained Labs'
Library Studio® software. After the scientist set
up the freeslate BFS with starting solutions andconsumables, the experimen­tal design was loaded
and procedures were performed by the system
automatically. Formulations of Molecule 1 in 2 mL
serum vials were stressed by stirring at 300 rpm
with magnetic stir bars for 24 hours, shaking(vortexing) at 400 rpm for 24 hours and heating
at 48 °C for 3 hours. In addition to the study de­scribed above, 250 μL of each Molecule 1 formula­tion was transferred, in triplicate, to a 96-well mi­
crotiter plate and then stored at 48 °C for 3 hours
in an Inheco incubator physically integrated withthe dual freeslate system. Formulations were in­cubated in a 96-well plate to investigate whether
similar results would be obtained between serum
vials and the microtiter plate formats. All stressing
procedures (mag­netic stirring, vortexing, heating
and incubation) were performed in parallel on the
dual freeslate system.

One set of the Molecule 2 formulations (A–C and B´)remained unstressed, T0, while the remaining vials
were exposed to the following stress conditions:stirring at 300 rpm for 4 hours us­ing magnetic
stir bars, shaking at 400 rpm for 4 hours and heatstress at 57 °C for 3 hours. Stressing procedures
were performed in parallel on the freeslate BFS.

Results
Traditional process
The rank orders for the stability of these formu­
lations were estab­lished using data derived frompreviously-conducted manual experi­ments includ­ing analysis by DLS, UV/Vis (A400 and A280) and3

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

SEC. For Molecule 1, formulation B was the best
performing followed by C and A (B > C > A). For Mol­ecule 2, formulation C was the most stable followed
by B (includes B´), and formulation A was the least
stable (C > B > A). These results provided the bench­
mark for comparison with the automated process.

ness of protein formulations. Stirring stress was so
damaging to the protein that monomer peaks had
average intensities between 0% and 4% in every
formulation (Table 2). Heating in vials showedsmall but meaningful differences in the stabilitiesof all formula­tions (Table 3). Shaking stress was
not optimized and did not permit clear discrimina­tion of the robustness of the formulations(Table 4). Heating in vial or in microplate showedsome varia­tions that may be explained by thedifference in heat transfer performance (Tables 3
and 5). Specifically, heat transfer was far more
efficient in the 96-well plates when in an Inheco
incubator compared to heating vials in a vial rack
made of aluminum.

Automated process: Molecule 1
DLS
Results from DLS measurements (summarized inTables 1–5) provided insights into the compara­tive performance for each formulation of Mole­cule 1. Representative regularization graphs for all
formulations of Molecule 1 after stirring stress areshown in Figures 3–5. Peaks with radii between
3–9 nm were assigned as monomers, and all larg­er peaks were consid­ered aggregates. Intensities
of monomer peaks were used to qualitatively as­
sess the amount of monomer in each formula­tion.
Tables 1–5 summarize DLS results for all formula­tions and conditions studied for Molecule 1. De­
creases in percent intensity of monomer provided
evidence that protein aggregation had occurred,
and these data were used to assess the robust­

Average monomer radii and percent intensity re­sults indicated formulation B was the most robust
in all stress conditions except heat stressing in
a 96-well plate using the Inheco incubator. For­
mulation C was the second most robust formula­tion in all condi­tions except for at T0 and heating in
the 96-well plate, where this formulation had ef­
fectively the same amount of monomer as formu­
lation B. Formulation A had the lowest monomer

A

B

100

100

90

90

Formulation A T0 1

80

Formulation A T0 3

60
50
40

60
50
40

30

30

20

20

10

10

0
0.01

0.10

1.00

10.00

100.001.0 E+3

Formulation A stirring 2

70% Intensity% Intensity

70

Formulation A stirring 1

80

Formulation A T0 2

00.01

1.0 E+4

Radius (nm)

0.10

1.00

10.00

100.001.0 E+3

1.0 E+4

Radius (nm)Figure 3: (A) Regularization chart of DLS results for Molecule 1 formulation A at T0 with is shown. (B) Regularization graph ispresented for Molecule 1 formulation A after stirring stress. One replicate was not stirred as intended and was removed from theanalysis.

4

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

A

B
100

100

90

90

Formulation B T0 1

80

Formulation B T0 3

60
50
40

50
40
30

20

20

10

10

0.10

1.00

10.00

100.001.0 E+3

Formulation B stirring 3

60

30

0
0.01

Formulation B stirring 2

70% Intensity% Intensity

70

Formulation B stirring 1

80

Formulation B T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

1.0 E+4

Radius (nm)Radius (nm)Figure 4: (A) Regularization chart of DLS results for Molecule 1 formulation B at T0 is presented. (B) Regularization graph is shownfor Molecule 1 formulation B after stirring stress.

A

B
100

100

90

90

Formulation C T0 1

80

Formulation C T0 3

60
50
40

50
40
30

20

20

10

10

0.10

1.00

10.00

100.001.0 E+3

Formulation C stirring 3

60

30

0
0.01

Formulation C stirring 2

70% Intensity% Intensity

70

Formulation C stirring 1

80

Formulation C T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

1.0 E+4

Radius (nm)Radius (nm)Figure 5: (A) Regularization chart of DLS results for Molecule 1 formulation C at T0 is presented. (B) Regularization graph is shownfor Molecule 1 formulation C after stirring stress.

5

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Molecule 1 T0

Formulation

A

B

C

Averagemonomerradius (nm)Preparation

Monomerradius (nm)Monomerintensity (%)

1

3.6

2

3.3

3

3.4

92.7

1

4.2

96.0

2

4.1

3

4.2

96.9

1

4.0

96.6

2

4.2

3

4.0

Averagemonomerintensity (%)

91.6
3.4

4.1

4.1

91.6

94.2

97.5

92.0

95.7

96.7

96.0

Table 1: Summary of DLS results for Molecule 1 at T0.

Molecule 1 stirring

Formulation

A

B

C

Averagemonomerradius (nm)Preparation

Monomerradius (nm)Monomerintensity (%)

1

NA

2

NA

3

NA

NA

1

4.0

0.3

2

5.2

3

7.2

9.1

1

8.5

2.2

2

7.4

3

5.7

Averagemonomerintensity (%)

NA
NA

5.5

7.2

NA

1.3

2.5

NA

3.6

2.2

1.9

Table 2: Summary of DLS results for Molecule 1 after stirring stress. NA: not applicable; no monomer detected.

6

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Molecule 1 heat stress in vial

Formulation

A

B

C

Averagemonomerradius (nm)Preparation

Monomerradius (nm)Monomerintensity (%)

1

3.9

2

3.8

3

4.6

97.4

1

6.1

97.3

2

8.6

3

8.4

99.3

1

4.3

96.8

2

4.4

3

4.6

Averagemonomerintensity (%)

94.6
4.1

7.7

4.5

89.1

99.2

95.4

93.7

98.6

96.2

1.9

Table 3: Summary of DLS results for Molecule 1 after heat stressing in vials.

Molecule 1 shaking

Formulation

A

B

C

Averagemonomerradius (nm)Preparation

Monomerradius (nm)Monomerintensity (%)

1

4.3

2

4.7

3

4.4

92.9

1

9.1

100.0

2

7.3

3

9.5

98.4

1

8.6

99.3

2

8.2

3

5.9

Averagemonomerintensity (%)

91.9
4.5

8.6

7.6

96.0

96.3

100.0
84.6

Table 4: Summary of DLS results for Molecule 1 after shaking stress in vials.

7

93.6

98.2

94.6

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Molecule 1 heat 96-well plate

Formulation

Averagemonomerradius (nm)Preparation

Monomerradius (nm)

1

2.7*

2

8.5

3

5.5

96.7

1

5.1

76.9

2

6.9

3

7.5

94.4

1

5.6

97.0

2

6.6

3

5.5

A

B

C

Monomerintensity (%)

Averagemonomerintensity (%)

0.4*
7.0

6.5

5.9

96.3

84.3

100.0

96.5

85.2

96.8

93.3

Table 5: Summary of DLS results for Molecule 1 after heat stressing in a 96-well plate using Inheco incubator.
*: Outlier data were omitted from averaged results.

content in all formulations and conditions, except
heating in 96-well plates. However, after heatingin the 96‑well plate, formulation A showed a small
increase in main peak radius compared to theother for­mulations. Taken together, the DLS resultsfor Molecule 1 suggest that formulation B was themost robust followed by formulations C and thenA (B > C > A).

Automated enhanced visual inspection
Results from automated enhanced visual inspec­tion including color, turbidity and visible particle
counting indicated no sig­nificant changes acrossthe formulations stressed by heat and agitation
for Molecule 1 (data not shown). Automated visualinspection was not performed on stirring stresssamples due to the presence of stir bars in thevials.

SEC
Molecule 1 formulations were also evaluated by
SEC. The monomer and aggregate peak area %are shown in Figures 6–7. The low molecule weightpeak data is not shown. Monomer peak area
percentages for Molecule 1 (Figure 6) show that
formulation B best stabilized the protein across all
stress conditions. Formulation B also had the least
aggregation followed by formulations C and thenA (Figure 7) (B > C > A).

Combined analysis and assessment for Molecule 1
Results from DLS, SEC and absorbance at 400 nm
indicated that the most robust formulation for
Molecule 1 was B followed by formulation C, and fi­
nally the least stable formulation was A (B > C > A).

Automated process: Molecule 2
DLS
Results from DLS (Tables 6–9) provided an un­derstanding of the comparative robustness for
all formulations of Molecule 2. Representative
regularization graphs with peak percent intensity
and radii results for all formulations of Molecule 2
after heat stressing are shown in Figures 8–11.

UV/Vis (A400 and A280)
All formulations and conditions for Molecule 1
showed no significant changes in protein content
or turbidity as measured by absorbance at 280
nm and 400 nm, respectively (data not shown).

8

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

40
A

90

B

C

Aggregate peak area percentage (%)

Monomer peak area percentage (%)

100

80
70
60
50
40
30
20
10
0

t0

Stirring

Heat

ShakingInhecoheatedincubation

35
30

B
C

25
20
15
10
5
0

AverageA

t0

Stirring

Heat

ShakingInhecoheatedincubation

Average

Figure 6: Automated Process Molecule 1 SEC results. SEC
results for aggregate peak percentages for all Molecule 1 for­mulations and conditions.

Figure 7: Automated Process Molecule 1 SEC results for aggre­
gate peak percentages for all Molecule 1 formulations.

Peaks with radii of 2-15 nm were considered asmonomers. Intensities of the monomer peaks wereused to qualitatively assess the amount of mono­
mer in the formulations.Tables 6–9 summarize
the various formulations and conditions studied
for Molecule 2. Decreases in monomer percent
intensity provided evidence that protein aggrega­tion had occurred, and these data were used toassess the robustness of protein formulations. As
expected, at T0 all formulations had similar aver­age monomer radii and percent intensity results(Table 6). After stirring, formulation C was themost robust, when monomer intensities were av­
eraged for formulations B and B’ (13.2% aver­age
monomer intensity) (Table 7). Formulation A wasthe least stable after stirring stress. Heat stressingin vials clearly showed that formulation A was theleast robust and that formulations B and C hadvery similar stabilities (Table 8). Shaking stress did
little to help determine the relative robustness ofthe formulations (Table 9). Taken together the DLS
results for Molecule 1 suggest that formula­tion
C was the most robust, followed by formulations
B /B' and then A (C > (B ≈ B´) > A).

SEC
Significant changes in the area percentages forboth the main peak and aggregate peak values
in formulation A suggest that this for­mulation
was comparatively the least robust (Figure 12).
According to the SEC results, formulation C was
the most stable, and formula­tions B and B´ were
nearly equal in robustness (C > (B ≈ B´) > A).
UV/Vis (A400 and A280)
Absorbance values at 400 nm added to theunderstanding of comparative performances foreach formulation (Figure 13). Average A400 in­
tensity values were essentially unchanged after
heating and shaking stresses; however, stirring
stress led to significant increases in A400 values forformulations A and B. Increased A400 values sug­gested formation of particles and/or aggregatesin formulations A and B and suggest that thesefor­mulations are less stable than C and B´. Overall,
the A400 data indicate that formulation B´ is themost stable, followed by C, B and A (B´ > C > B > A).

9

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

A

B

100

100
90

90

Formulation A T0 1

80

Formulation A T0 3

70

% Intensity% Intensity

50
40

50
40
30

20

20

10

10

1.00

10.00

100.001.0 E+3

Formulation A heat stress 3

60

30

0.10

Formulation A heat stress 2

70

60

0
0.01

Formulation A heat stress 1

80

Formulation A T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

Radius (nm)Radius (nm)Figure 8: (A) Regularization chart of DLS results for Molecule 2 formulation B at T0 is presented. (B) Regularization plot is shown for
Molecule 2 formulation A subjected to heat stress in vials.

A

B

100

100

90

90

Formulation B T0 1

80

Formulation B T0 3

60
50
40

50
40
30

20

20

10

10

0.10

1.00

10.00

100.001.0 E+3

Formulation B heat stress 3

60

30

0
0.01

Formulation B heat stress 2

70% Intensity% Intensity

70

Formulation B heat stress 1

80

Formulation B T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

Radius (nm)Radius (nm)Figure 9: (A) Regularization graph for Molecule 2 formulation B at T0 is presented. (B) Regularization graph is shown for Molecule 2
formulation B subjected to heat stress in vials.

10

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

A

B
100

100

90

90

Formulation C T0 1

80

Formulation C T0 3

60
50
40

50
40
30

20

20

10

10

0.10

1.00

10.00

100.001.0 E+3

Formulation C heat stress 3

60

30

0
0.01

Formulation C heat stress 2

70% Intensity% Intensity

70

Formulation C heat stress 1

80

Formulation C T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

Radius (nm)Radius (nm)Figure 10: (A) Regularization graph for Molecule 2 formulation C at T0 is presented. (B) Regularization graph is shown for Molecule 2
formulation C subjected to heat stress in vials.

A

B
100

100

90

90

Formulation B’ T0 1

80

Formulation B’ T0 3

60
50
40

50
40
30

20

20

10

10

0.10

1.00

10.00

100.001.0 E+3

Formulation B’ heat stress 3

60

30

0
0.01

Formulation B’ heat stress 2

70% Intensity% Intensity

70

Formulation B’ heat stress 1

80

Formulation B’ T0 2

00.01

1.0 E+4

0.10

1.00

10.00

100.001.0 E+3

Radius (nm)Radius (nm)Figure 11: (A) Regularization graph for Molecule 2 formulation B´ at T0 is presented. (B) Regularization graph is shown for Molecule 2
formulation B´ subjected to heat stress in vials.

11

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Molecule 2 T0

Formulation

A

B

C

B´

Averagemonomerradius (nm)Prep

Monomerradius (nm)Monomerintensity (%)

1

5.8

2

5.7

3

5.8

96.5

1

6.1

96.1

2

7.4

3

6.8

98.6

1

8.2

94.8

2

7.3

3

7.1

88.9

1

8.2

96.6

2

7.2

3

7.2

Averagemonomerintensity (%)

96.5
5.7

6.7

7.5

7.5

96.8

97.3

94.7

95.3

96.6

97.3

92.8

95.8

95.4

Table 6: Summary of DLS results for Molecule 2 at T0.

Molecule 2 stirring

Formulation

A

B

C

B´

Averagemonomerradius (nm)Prep

Monomerradius (nm)Monomerintensity (%)

1

4.7

2

5.3*

3

5.8*

68.8*

1

NA*

NA*

2

6.9

3

7.0

9.2

1

8.6*

67.3*

2

NA*

3

8.2

15.1

1

NA*

NA*

2

9.6*

3

10.2

Averagemonomerintensity (%)

10.3
4.7

6.9

8.2

10.2

71.8*

5.4

NA*

80.2*
19.0

Table 7: Summary of DLS results for Molecule 2 after stirring stress. NA: data not acquired.
*: samples not stirred as intended; DLS data were removed from averaged results.

12

10.3

7.3

15.1

19.0

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Molecule 2 heat stress in vials

Formulation

A

B

C

B´

Averagemonomerradius (nm)Prep

Monomerradius (nm)Monomerintensity (%)

1

6.3

2

6.5

3

6.3

21.5

1

1.6

NA

2

1.6

3

1.6

51.7

1

1.7

57.7

2

1.8

3

1.8

56.3

1

1.7

52.0

2

1.7

3

1.7

Averagemonomerintensity (%)

25.4
6.4

1.6

1.8

1.7

24.7

51.5

57.5

52.6

23.9

51.3

57.2

52.7

53.4

Table 8: Summary of DLS results for Molecule 2 after heat stressing in vials.

Molecule 2 shaking

Formulation

A

B

C

B´

Averagemonomerradius (nm)Prep

Monomerradius (nm)Monomerintensity (%)

1

5.4

2

5.3

3

5.4

95.7

1

7.2

94.0

2

7.7

3

6.4

91.4

1

8.3

95.2

2

7.1

3

7.9

93.5

1

9.1

96.4

2

9.2

3

9.9

Averagemonomerintensity (%)

95.5
5.4

7.1

7.8

9.4

100.0

95.2

93.3

95.3
94.8

Table 9: Summary of DLS results for Molecule 2 after shaking stress in vials.

13

97.1

93.5

94.0

95.5

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

6
A

C

A

C

B

B´

B

B´

5

20

Absorbance at 400 nm (AU)

Aggregate peak area percentage (%)

25

15

20

5

0

t0

Stirring

Heat

Shaking

4

3

2

1

0

Average

Figure 12: Automated process Molecule 2 SEC results. SEC
results for aggregate peak percentages for all Molecule 2 for­mulations and conditions.

t0

Stirring

Least stable

Shaking

Average

Figure 13: Automated process Molecule 2 turbidity result. Turbid­ity (A400) values for all formulations and conditions for Molecule 2.
Absorbances greater than 6 absorbance units (AU) are notshown.

Molecule 1

Most stable

Heat

Molecule 2

Traditional
process

Automated
process

Traditional
process

Automated
process

B

B

C

C

C

C

B, B´

B, B´

A

A

A

A

Table 10: Summary of rank order comparison of formulation stability evaluated by both the traditional process and an automatedprocess. The automated process provides the same results as the traditional manual process.

Conclusion

Automated enhanced visual inspection
No significant changes in enhanced visual inspec­tion (turbidity, color and particles) were observedin all Molecule 2 formulations (A–C, B´) stressed byheat and agitation (data not shown). Automatedvisual inspection was not performed on stirring
stress samples due to the presence of stir bars in
the vials.

Rank order of formulation stability
Table 10 shows the rank order of stability for all
formulations for both manual and automated pro­
cesses. The results demonstrate a high-through­put workflow can provide the same formulation
rank orders as the manual workflow and proce­dures performed at LFB. For both automated andtra­ditional processes, the rank order of Molecule
1 formulations from most to least stable was B, C
and A. Results from the Molecule 2 study show arank order from most to least stable: C, B≈B´, A .
This study demonstrates that the freeslate plat­form and automated high-throughput procedures
can generate comparable results to traditional
manual methods.

Combined analysis and assessment
Results from A400, DLS and SEC all indicated that
formulation A was the least robust. Taken together,
the other analytical results suggested that the rankorder of the formulations was as follows from mostto least robust: C, B≈B´ and then A (C > (B ≈ B´) > A).

14

AUTOMATION OF BIOLOGICS FORMULATION STABILITY TESTING

Comparability of formulation stability in
96-well plates to previous results

by eliminating manual, tedious steps such as
for­mulation preparation, sample stressing andsample preparation for SEC and DLS while simpli­
fying data analysis and compilation. Automation
also provides an opportunity for scientists to focus
on higher value activities including experimental
design, evalua­tion of new and novel formulations,interpreting results and more extensive biophysi­
cal characterization.

In this study, automation was used to prepare
and stress for­mulations in both a final container/
closure system and 96-well microplates. Testing
formulation robustness in 96-well plates has tre­
mendous advantages by reducing material con­sumption while increasing the number of formu­
lations that can be screened in parallel. The SEC
results from heat stressing Molecule 1 in a 96well microtiter plate show comparability to finalcontainer/closures and traditional results (Fig‑ures 6–7). These results support the justificationof using the microtiter plate in forced degradation
studies of some protein formulations. Us­ing a 96well plate format on the freeslate system enables
scientists to screen more than 300 formulations ina single day.

This study proved to LFB that the automated sys­
tem could provide comparable results while en­abling time and material savings. The majority oftime savings were realized from sample prepara­tion, such as dilutions and also in regards to datacompilation. LFB also expects to see improve­
ment in reproducibility with an automated robotic
system when compared to the manual pro­cess,specifically in regards to the visual inspection
analysis and sample preparation. With a simple
and logical design space, LFB scientists found the
LEA software and specifically, Library Studio, easy
to use. While system handling requires expertise, it
shows tremendous promise for future use at LFB
and other biopharmaceutical companies.

Benefits of automation
Fully integrated automation, including both hard­ware and data integration for formulation stud­ies can increase lab productivity and efficiency

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Rev B

15