Edited by: Simone Brogi, Università degli Studi di Siena, Italy
Reviewed by: Diego Brancaccio, Università degli Studi di Napoli Federico II, Italy; Ruibing Wang, University of Macau , China
This article was submitted to Medicinal and Pharmaceutical Chemistry, a section of the journal Frontiers in Chemistry
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Herein, we aimed to develop a strategy for evaluating the consistency of pharmaceutically important crystallization processes in real time, focusing on two typical cases of polymorphism. Theoretical analysis using a combination of 13C solid-state nuclear magnetic resonance spectroscopy with other polymorphism analysis techniques identified a number of marker signals, the changes of which revealed the presence of two or more structural orientations (lattices and/or molecular conformations) in both cefazolin sodium pentahydrate (α-CEZ-Na) and cephathiamidine (CETD). The proportions of these forms were shown to be batch-dependent and were defined as critical quality attributes (CQAs) to evaluate process consistency. Subsequently, real-time analysis by chemometrics-assisted near-infrared spectroscopy (NIR) was used to obtain useful information corresponding to CQAs. The pretreated spectra of representative samples were transformed by first derivative and vector normalization methods and used to calculate standard deviations at each wavelength and thus detect significant differences. As a result, vibrational responses of H2O, CH3, and CH2 moieties (at 5,280, 4,431, and 4,339 cm−1, respectively) were shown to be sensitive to the CQAs of α-CEZ-Na, which allowed us to establish a highly accurate discrimination model. Moreover, signals of H2O, CONH, and COOH moieties (at 5,211, 5,284, and 5,369 cm−1, respectively) played the same role in the case of CETD, as confirmed by theoretical results. Thus, we established a technique for the rapid evaluation of crystallization process consistency and deepened our understanding of crystallization behavior by using NIR in combination with polymorphism analysis techniques.
Polymorphism is defined as the ability of a solid substance to exist in various crystal forms (i.e., polymorphs) that exhibit different physical properties such as density, melting point, solubility, and dissolution rate. Therefore, the interconversion of the polymorphic forms of a given drug can strongly affect its bioavailability and therapeutic effect (Aaltonen et al.,
Solid-state substances can be characterized by analytical techniques such as powder X-ray diffraction (PXRD), IR spectroscopy, Raman spectroscopy, and solid-state nuclear magnetic resonance spectroscopy (ssNMR) (Shah et al.,
Since the 1990s, near-infrared spectroscopy (NIR) has steadily evolved to become a popular method of quantifying pharmaceutically relevant materials in the solid state. This technique exhibits numerous advantages that make it well suited for real-time quality monitoring, e.g., it is fast, non-destructive, and does not require sample preparation (Bakeev,
Two drugs were chosen as typical cases to develop and test the above technique, namely cefazolin sodium pentahydrate (α-CEZ-Na), which exhibits a well-defined and relatively fixed solid-state form without isomorphic dehydration, and cephathiamidine (CETD), the structural orientations of which are hard to determine and are strongly affected by manufacturing conditions (Kamat et al.,
The 150 samples of α-CEZ-Na from two processing periods (Period 1: July 2014–January 2015 and September 2015; Period 2: November 2016–March 2017) were provided by Shenzhen Gosun Pharmaceutical Co., Ltd., Guangdong, China (Gosun). To amplify the differences between products, α-CEZ-Na 1–3 (batch no: 1203283, 1203423, 1203403) from Gosun, α-CEZ-Na 4 (batch no: L100200) from Fujisawa Co., Ltd., Kirihara-cho, Fujisawa-shi, Kanagawa, Japan (Fujisawa), and α-CEZ-Na 5 (batch no: 1391) from Kyongbo Phar. Co., Ltd., Silok-ro, Ahsan-si, Choongchungnam-do, South Korea (Kyongbo) were chosen as representative samples for comparison of theoretical characteristics. The quality of every α-CEZ-Na sample complied with the Japanese Pharmacopeia.
The 96 samples of CETD from different batches were provided by Guangzhou Baiyunshan Pharmaceutical Co., Ltd., Guangzhou, China. Among them, CETD 1 (batch no: 1611017), CETD 2 (batch no: 1612010), CETD 3 (batch no: 1703001), CETD 4 (batch no: 1701008), and CETD 5 (batch no: 1704016) were chosen as representative samples to analyze theoretical characteristics. The quality of every CETD sample complied with the Chinese Pharmacopeia.
All ssNMR spectra were recorded on a Bruker AVANCE II WB400 NMR spectrometer using scanning parameters referenced elsewhere (Tian et al.,
Raman mapping was performed using a DXRxi Raman imaging microscope (Thermo Fisher Scientific Inc., Hudson, USA) equipped with a 532 nm excitation laser. The laser power, exposure time, number of scans, and image pixel size equaled 6.0 mW, 0.00286 s, 11, and 5.0 μm, respectively. System control and spectrum acquisition were conducted using the ThermoScientific OMNIC software.
Samples were directly scanned in vials using a Fourier transform NIR integrating sphere (MPA, Bruker, Switzerland). The scan wavelength range and resolution were set to 12,000–4,000 cm−1 and 8 cm−1, respectively. All spectra were obtained by averaging the results of 32 scans, and six spectra of the same sample were averaged to a representative mean spectrum.
Raman mapping produced microscopic images as three-dimensional data sets that comprised two-dimensional data matrices containing information on the number of pixels and the number of data points in each spectrum. Before the application of any imaging algorithm, each individual spectrum (3,400–50 cm−1) was compared with all other spectra within a given data set. The largest variations were observed for the relative intensities at 1,644 and 1,658 cm−1, and the ratio of these intensities (
Chemometrics is widely used to transform original NIR spectra and establish qualitative or quantitative models. Herein, first derivative (1st Der) transformations using the Savitzky-Golay (SG) convolution filter (17, 3) and vector normalization (Chu,
Our previous study (Tian et al.,
Distinct differences were observed for the shape of peak C19 (i.e., for the intensity of the shoulder peak of C19) and the chemical shifts of peaks C14 and C9 (Figures
Standard deviations (SDs) of pretreated spectra were calculated to reveal the dispersion of response values at a given wavelength (Figure
NIR spectra of α-CEZ-Na 1–5 produced by different vendors (Gosun, Fujisawa, and Kyongbo).
CEZ-Na contained only one methyl group (C17), the vibration of which was probably described by the absorption band at 4,405 cm−1. The above methyl group was connected to C14 and was therefore expected to feature a vibration performance dependent on the variation of C14 electron cloud density, which was likely to happen according to the results of ssNMR analysis. Figure
The higher proportion of conformation 2 in Kyongbo samples with lower NIR intensities (Figure
MLR-DA results for all α-CEZ-Na products obtained in Periods 1 and 2.
In the case of CETD, the Raman spectra of specified positions were scanned to reflect the differences of solid-state forms (Figure
Raman spectra (1,550–1,830 cm−1) of five CETD crystals marked in optical images by “+”.
The 13C NMR data obtained in dimethyl sulfoxide-
NMR data (δ) for CETD.
C2 | 115.8 | 114.4; 106.9 |
C3 | 131.9 | 132.8; 131.3 |
C4 | 25.3 | 24.2; 23.3 |
C6 | 56.8 | 58.4 |
C7 | 58.9 | 53.9; 53.3 |
C8 | 162.8 | 158.7 |
C9 | 163.6 | 162.8; 161.5 |
C10 | 63.8 | 69.0; 63.1 |
C12 | 170.4 | 170.1 |
C13 | 20.7 | 19.3a |
C15 | 170.1 | 169.1 |
C16 | 34.0 | 32.8; 29.8 |
C18 | 170.1 | 167.5; 165.7 |
C20 | 45.5 | 44.1; 43.4 |
C21(C21′) | 22.9 | 19.3a |
C23 | 49.3 | 48.1 |
C24(C24') | 22.9 | 19.3a |
Figure
All NIR spectra of CETD were transformed by SG(17, 3) 1st Der and vector normalization, and SDs at each wavelength were calculated for the intensities of these pretreated spectra (Figure
NIR spectra of CETD 1–5 obtained from different batches.
All samples were classified according to the intensities of peaks at 5,211, 5,284, and 5,369 cm−1 in pretreated spectra using HCA. Based on the results in Figure
Results of HCA (Ward's method) performed using intensities of peaks at 5,211, 5,284, and 5,369 cm−1 in pretreated NIR spectra of all samples. Among these results, CETD 1–5 were emphasized by their own microscope Raman images, which mapping by the ratio of intensities at 1,644 and 1,658 cm−1 (
ssNMR and Raman microscopy allowed for effective characterization of the mixed solid-state forms of investigated drugs, and changes of peak shape and/or peak intensity were shown to be sensitive to those of polymorph proportions. These variations could be defined as CQAs for each drug to reveal the consistency of the corresponding crystallization processes. Considering the requirement of real-time detection, NIR was identified as a practical technology to replace theoretical analysis methods despite requiring the assistance of chemometrics. First derivative and vector normalization were proposed as methods of transforming NIR spectra to achieve baseline correction and high resolution, and SD spectra of representative samples allowed one to easily find variables corresponding to CQAs. Therefore, these characteristic features could be used to establish a model for the rapid on-line evaluation of API crystallization degree or the consistency of mixed polymorphs. Most importantly, this strategy (Chart
Strategy to control of critical quality attributes of polymorphic drugs in crystallization process.
However, revealing the reason of process variation scientifically remains a challenge even for modern analytical techniques, necessitating the use of supporting methods such as terahertz, IR and thermogravimetric analysis in certain cases. In addition, computational chemistry methods are required for clarifying certain structural aspects in detail. For seeking right protective measures of process consistency, NIR analysis can be assisted by other chemometric algorithms as long as they benefit the extraction of key information corresponding to CQAs.
S-YQ: design of experiments, Raman and NIR data acquisition, analysis of experimental data; YT: SSNMR data acquisition; W-BZ: PXRD data acquisition; C-QH: design of experiments, analysis of experimental data.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.