Implementation of QbD Approach to the Development of Chromatographic Methods for the Determination of Complete Impurity Profile of Substance on the Preclinical and Clinical Step of Drug Discovery Studies

Abstract

The purpose of this work was to demonstrate the use of the AQbD with the DOE approach to the methodical step-by-step development of a UHPLC method for the quantitative determination of the impurity profile of new CPL409116 substance (JAK/ROCK inhibitor) on the preclinical and clinical step of drug discovery studies. The critical method parameters (CMPs) have been tested extensively: the kind of stationary phase (8 different columns), pH of the aqueous mobile phase (2.6, 3.2, 4.0, 6.8), and start (20-25%) and stop (85-90%) percentage of organic mobile phase (ACN). The critical method attributes (CMAs) are the resolution between the peaks (≥2.0) and peak symmetry of analytes (≥0.8 and ≤1.8). In the screening step, the effects of different levels of CMPs on the CMAs were evaluated based on a full fractional design 2². The robustness tests were established from the knowledge space of the screening step and performed by application fractional factorial design 2^(4-1). Method operable design region (MODR) was generated. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations. In relation to literature such a complete AQbD approach including screening, optimization, and validation steps for the development of a new method for the quantitative determination of the full profile of nine impurities of an innovative pharmaceutical substance with the structure-based pre-development pointed out the novelty of our work. The final working conditions were as follows: column Zorbax Eclipse Plus C18, aqueous mobile phase 10 mM ± 1 mM aqueous solution of HCOOH, pH 2.6, 20% ± 1% of ACN at the start and 85% ± 1% of ACN at the end of the gradient, and column temperature 30 °C ± 2 °C. The method was validated in compliance with ICH guideline Q2(R1). The optimized method is specified, linear, precise, and robust. LOQ is on the reporting threshold level of 0.05% and LOD at 0.02% for all impurities.

Keywords: Analytical Quality by Design (AQbD); CHI logD; CPL409116; JAK/ROCK inhibitor; analytical method development; design of experiment (DOE); method operable design region (MODR); pharmaceutical impurity profiling.

Figure 1

AQbD methodology workflow on the base of Tome at al. [27].

Figure 2

Chemical structure of CPL409116.

Figure 3

Ishikawa diagram for critical method parameters (CMP).

Figure 4

Screening experiment results showing the interaction effect of columns, % of organic solvent at the start and end gradient, and pH with CMAs as a desirability plot of resolutions (R_S). C1—ZORBAX Eclipse Plus C18 (2.1 × 50 mm, 1.8 µm, Agilent Technologies), C2—Kinetex EVO C18 (2.1 × 50 mm, 1.7 µm, Phenomenex), C3—ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 µm Waters), C4—InfinityLab Poroshell 120 Phenyl Hexyl (2.1 × 50 mm, 1.9 µm, Agilent Technologies), C5—Kinetex Biphenyl (2.1 × 50 mm, 1.7 µm, Phenomenex), C6—Kinetex PFP (2.1 × 50 mm, 1.7 µm, Phenomenex), C7—ACQUITY UPLC CSH C18 (2.1 × 50 mm, 1.7 µm, Waters), and C8—ACQUITY UPLC CSH Fluoro-Phenyl (2.1 × 100 mm, 1.7 µm, Waters).

Figure 5

Screening experiment results showing the interaction effect of columns, % of organic solvent at the start and end gradient, and pH with CMAs as a desirability plot of symmetry factors (A_S). C1—ZORBAX Eclipse Plus C18 (2.1 × 50 mm, 1.8 µm, Agilent Technologies), C2—Kinetex EVO C18 (2.1 × 50 mm, 1.7 µm, Phenomenex), C3—ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 µm Waters), C4—InfinityLab Poroshell 120 Phenyl Hexyl (2.1 × 50 mm, 1.9 µm, Agilent Technologies), C5—Kinetex Biphenyl (2.1 × 50 mm, 1.7 µm, Phenomenex), C6—Kinetex PFP (2.1 × 50 mm, 1.7 µm, Phenomenex), C7—ACQUITY UPLC CSH C18 (2.1 × 50 mm, 1.7 µm, Waters), and C8—ACQUITY UPLC CSH Fluoro-Phenyl (2.1 × 100 mm, 1.7 µm, Waters).

Figure 6

An example of chromatograms from screening experiments on the eight tested columns: C1—ZORBAX Eclipse Plus C18 (2.1 × 50 mm, 1.8 µm, Agilent Technologies), C2—Kinetex EVO C18 (2.1 × 50 mm, 1.7 µm, Phenomenex), C3—ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 µm Waters), C4—InfinityLab Poroshell 120 Phenyl Hexyl (2.1 × 50 mm, 1.9 µm, Agilent Technologies), C5—Kinetex Biphenyl (2.1 × 50 mm, 1.7 µm, Phenomenex), C6—Kinetex PFP (2.1 × 50 mm, 1.7 µm, Phenomenex), C7—ACQUITY UPLC CSH C18 (2.1 × 50 mm, 1.7 µm, Waters), and C8—ACQUITY UPLC CSH Fluoro-Phenyl (2.1 × 100 mm, 1.7 µm, Waters).

Figure 7

Chromatogram of JAK01 substance solution spiked with impurities at 0.15% level on the Kinetex EVO C18 (2.1 × 50 mm, 1.7 µm, Phenomenex) column.

Figure 8

The Pareto chart for Rs between JAK01 and JAK07 and As for JAK01 for robustness tests.

Figure 9

Overlay chromatograms JAK01 test solution (A), and JAK01 test solution spiked with impurities at 0.15% level at 230 nm (B), and at 297 nm (C), on the ZORBAX Eclipse Plus C18 (2.1 × 50 mm, 1.8 µm, Agilent Technologies, Waldbronn, Germany) column.