Rapid, Automated Development of a Pharmaceutical Small

Molecule Separation Using A Two Phase Method Screening and

Optimization Approach

Introduction
This work, conducted in collaboration with Pfizer Inc., Ann Arbor, describes the use of Fusion
AE for Galaxie in a two-phase rapid screening and optimization experiment designed to
optimize the HPLC separation of a complex mixture of small organic molecules typically found
in pharmaceutical products.
Phase 1 - Rapid Screening - consisted of a rapid screen utilizing a novel Trend Response
data analysis algorithm designed to to identify the correct column, mobile phase and
approximate gradient conditions needed to separate a complex mixture of two API’s and
several known impurities.
Phase 2 – Optimization - comprised a method optimization experiment that identified the run
conditions that gave the best results in terms of resolution and assay time using the column and
mobile phase identified from the Phase 1 screen. Overall method robustness was also
determined using a novel robustness calculation. The results of this work are presented below.

Materials and Methods

Instrument – 1100 HPLC with Diode Array detector (Agilent Inc.). Model 500 Column Valve
Module (Varian Inc.)
Columns – 150 x 4.6 mm Gemini C18, Synergi Fusion RP, Luna C18, (Phenomenex Inc.),
Pursuit DiPhenyl (Varian Inc.), Sunfire C18 (Waters Inc.)
Buffer System – Aqueous: Ammonium Acetate Buffer (pH5), Potassium Phosphate (pH 2.5 &
7). Organic: Acetonitrile
System Parameters Included as Experiment variables – Flow Rate, Gradient Slope,
Gradient Time, pH and Column Type

Rapid Method Development Platform – Instrument control, chromatogram generation, peak

processing: Varian Galaxie chromatography data system (CDS), (Varian Inc.) Statistical
experimental design, data analysis, modeling, optimization: Fusion AE for Galaxie (S-Matrix
Corp. CA.)

Experimental Method – Study factors for the Phase 1 Rapid Screening experiment were
varied according to a model-robust screening design generated by Fusion AE, which
constructed the 38 run design as a set of ready-to-run methods and the corresponding
sequence in the CDS. The experiment was run overnight on the HPLC under Galaxie CDS
control. Peak results were imported from the CDS into Fusion AE, using three trend response
variables viz. Total number of peaks and total number of resolved peaks (R>1.5 & R>2.0), for
automated data analysis.
Figure 1. Phase 1 – Rapid Screening. Experimental parameters (study variables)
including, Gradient Slope, Gradient Time Mobile Phase pH and Column Type were entered
into a standardized template.

Experiment Range or Level

Variable Setting
Gradient Time 15.0 — 40.0
(min)
pH 2.5, 5.0, 6.5
Column Type Gemini C18
Synergi Fusion RP
Luna C18
Pursuit DiPhenyl
Sunfire C18

Gradient Slope (% 5.0 — 95.0

Organic)
Organic Solvent Acetonitrile
Type

Figure 2. Completing the Fusion experimental design template involved setting the
upper and lower bound values for the gradient time, target pH range, the specific
columns to be screened, and the desired organic solvent type and percentage
Figure 3. A Model-robust Screening type of
design was used to efficiently screen large ranges
of the variables and quantify their effects on
method performance.

Figure 4. Software generated a statistical experimental design. The

variables included process and mixture types. Fusion AE therefore selected a
mixture process algorithm design.
Figure 5. A Galaxie sequence, comprising 38 individual automatically generated methods
based on the experimental design parameters was built by the Fusion AE software within
the Galaxie CDS. This sequence was started by user and all lines were run in “inject and
forget” mode.

Figure 6. Peak results data were automatically imported from the

Galaxie CDS using file-less data transfer.
Experimental Method cont..– Study factors for the Phase 2 Optimization experiment
were varied according to a model-robust optimization design generated by Fusion AE, which
constructed a 14 run design as a set of ready-to-run methods and the corresponding
sequence in the CDS. The experiment was run overnight on the HPLC under Galaxie CDS
control. Peak results were imported from the CDS into Fusion AE, using a file-less exchange
module, for automated analysis. Optimization solution searches were conducted with the
Fusion AE numerical and graphical optimizers using the following goals:

USP Resolution >= 2.5 Cp Resolution Robustness >=1.25

Results and Discussion

Table 1 Phase 1 – Rapid Screening experiment design generated from the template
along with the Trend Response results computed directly from the chromatogram data.
Table 2. Regression analysis results for the Total Peaks trend response.
Coefficient Coefficie Lower 95% Upper 95%
Coefficient Standard nt t Confidence Confidence
Parameter Name Value Error Statistic P-Value F-Ratio Limit Limit

Constant 1,415.59 44.80 --- --- --- 1,324.10 1,507.08

Gradient Δt -662.76 121.51 -5.4545 <+/- 0.0001 29.7514 -910.91 -414.61

Column 4 -462.68 105.27 -4.3952 0.0001 19.3178 -677.67 -247.69

(Grad. Δt)*pH -113.54 55.15 -2.0589 0.0483 4.2389 -226.16 -0.92

(Grad. Δt)*Column 2 442.20 168.63 2.6223 0.0136 6.8762 97.81 786.60

(Grad. Δt)*Column 3 901.09 170.96 5.2707 <+/- 0.0001 27.7807 551.94 1,250.24

(Grad. Δt)*Column 4 781.87 164.97 4.7396 <+/- 0.0001 22.4635 444.96 1,118.77

(Grad. Δt)*Column 5 1,075.53 163.78 6.5667 <+/- 0.0001 43.1221 741.04 1,410.02

Table 2 contains two important results. Firstly, all equation (study parameter effect) terms
were found to be statistically significant. This can be seen from the significance test
values associated with each term in the table (P-Value less than 0.0500, F-Ratio value >
4.0000, zero outside the 95% confidence interval). Secondly, all study parameters are
represented in the equation in a form related to the nature of their effects (nonlinear,
interaction, etc). In fact, as expected, a ranking of the effect coefficients identifies the
largest effect as due to changing columns.

Table 3. Regression analysis results for the Resolved Peaks (> 1.50) trend response.
Coeffici Coefficient Coefficie Lower 95% Upper 95%
ent Standard nt t Confidence Confidence
Parameter Name Value Error Statistic P-Value F-Ratio Limit Limit

Constant 9.13 0.12 --- --- --- 8.88 9.37

Gradient Δt -2.01 0.32 -6.2877 <+/- 39.5346 -2.66 -1.36

0.0001

Column 4 -1.26 0.28 -4.5450 0.0001 20.6568 -1.82 -0.69

(Grad. Δt)*pH -0.38 0.15 -2.6404 0.0130 6.9715 -0.68 -0.09

(Grad. Δt)*Column 2 1.90 0.44 4.2800 0.0002 18.3185 0.99 2.80

(Grad. Δt)*Column 3 3.53 0.45 7.8523 <+/- 61.6582 2.61 4.45

0.0001

(Grad. Δt)*Column 4 2.56 0.43 5.8987 <+/- 34.7952 1.67 3.45

0.0001

(Grad. Δt)*Column 5 3.51 0.43 8.1422 <+/- 66.2962 2.63 4.39

0.0001
Table 3 shows that all the equation (study parameter effect) terms for the resolution
trend responses are statistically significant, and all the study parameters are
represented in the equation in a form related to the nature of their effects (nonlinear,
interaction, etc). These results show that a predictive equation has been developed for
Fusion AE which accurately and quantitatively relates the study parameter effects to a
second key aspect of compound separation – the separation of each compound from all
other compounds to the extent required.

Once the software derived the equations from the Trend Response data sets, these
equations were linked to a numerical algorithm that identified the study parameter settings
that maximized both responses. In this study the Fusion AE automated optimization
analysis immediately identified the column type, pH, and gradient conditions that should be
used in the second phase of the method development workflow. These results are
presented in Table 4 below.

Table 4. Phase 1 – Rapid screening experiment Automated Optimizer results.

Parameter Name Optimizer Result Level Setting

Gradient Time 40.0

pH 2.5

Column Column 3

At this point is is important to remember that in practice, the Trend Response approach will
not always yield the optimum HPLC method (instrument parameter settings) in a single
experiment, and indeed it is not meant to. The Trend Response approach is part of a
phased workflow in which the trend responses enable the experimenter to identify the best
settings of parameters such as Column Type and pH; parameters that normally have the
greatest effect on separation and therefore cause the most inherent data loss. Once these
settings have been identified, these parameters can then held constant in a second
experiment to designed optimize the HPLC instrument method.
Table 5. Experiment design generated from the modified template along with the
Resolution response results imported directly from the CDS for the Phase 2 Optimization
experiment chromatograms. Resolution results were imported for four critical peak pairs (1-
2, 2-3, 5-6, and 9-10), as the compounds corresponding to the other sample peaks were
well resolved in all experiment chromatograms.

Figure 7. A trellis of four resolution response surface graphs illustrating the changes in
resolution of four critical peak pairs (1-2, 2-3, 5-6, and 9-10) as a function of changing the
pump flow rate (X axis) and the final percent organic (Y axis).
 Figure 8. Response Overlay graph showing multiple response goals from the
Phase 2 Optimization experiment overlaid on one graph. Resolution goals
(Maximize, all Lower Bounds = 2.5) for all four critical peak pairs in the DOE-based
are displayed.

Figure 9 Response Overlay graph for Phase 2 Optimization experiment with additional
overlays of method Robustness Cp goals (Maximize, all Lower Bounds = 1.25) defined for
all peak pairs having predicted mean resolution values below 4.00. responses. The
unshaded region in this final overlay graph represents the level setting
combinations of the study factors that exceed the defined goals for both mean
performance and robustness.
 Figure 10. Chromatogram obtained by injecting a test sample on the HPLC set at the
optimum method parameter settings identified in the Phase 1 and 2 Fusion AE
experiments. The final method conditions are defined below. It is noteworthy that the
total experimental work required to obtain this final method consisted of two multi-
factor statistically designed experiments, both of which were carried out overnight in
fully automated inject-and- forget (walk-away) mode.

Phase 1 – Column/Solvent Screening

Column Type – Column 3
pH – 2.5
Gradient Time – 40 minutes

Phase 2 – Method Optimization

Pump Flow Rate – 0.67 mL/min
Final % Organic – 70 %

Conclusions

Chromatographic analytical method development work normally begins with selection

of the analytical column, the pH, and the organic solvent type. A major risk of using a
trial-and-error based one-factor-at-a-time (OFAT) approach is that it provides no ability
to visualize or understand the interaction effects usually present among these key
instrument parameters. In addition, this approach often results in significant inherent
data loss in key chromatographic performance indicators such as compound resolution
due to the large amount of peak exchange and compound co-elution common in these
experiment data sets.
This inherent loss makes it difficult or impossible to quantitatively analyze and model these
data sets, reducing the analysis to a pick-the-winner strategy based solely on visual
inspection of the chromatograms. The 2 phased Quality by Design based approach
described here, uses statistical experimental design coupled with automatically computed
Trend Responses™. This new practice successfully overcomes these problems to provide a
rigorous and quantitative methodology for column/solvent screening without the need for
difficult and laborious peak tracking implemented in a fully automated HPLC experimentation
platform.

The Phase 1 – Rapid Screening experiment identified the correct analytical column, pH, and
organic solvent type. Once these instrument parameters had been identified, the Phase 2 -
Optimization experiment involved manipulating the remaining important instrument
parameters to obtain a method that met all the performance goals including overall method
robustness. The novel Quality-by-Design based methodology used for the Phase 2
experiment combined Design of Experiments methodology with a Monte Carlo simulation to
successfully integrate quantitative robustness metrics into the method optimization process
resulting a the development of an analytical HPLC method capable of separating four critical
peak pairs simultaneously.

Authors

Patrick H. Lukulay, Ph.D., Manager, Drug Quality Control and Training

USP Drug Quality and Information
U.S. Pharmacopeia, Rockville, MD

Richard Verseput, President,

S-Matrix Corporation. Eureka, CA

Acknowlegements
The authors are grateful to Dr. Graham Shelver, Varian, Inc. for providing hardware,
software, and expertise in support of the live experimental work conducted to prove out
the Quality-by-Design approach to method development presented in this paper. The
authors also want to thank Dr. Raymond Lau, Wyeth Consumer Healthcare, and Dr.
Gary Guo and Mr. Robert Munger, Amgen, Inc. for the experimental work done in their
labs which supported refinement of the phase 1 and phase 2 rapid development
experiment templates.