Improved Understanding of Cefixime Trihydrate Reactive

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Improved Understanding of Cefixime Trihydrate Reactive Crystallization and Process Scale-up with the Aid of PAT Shuai Yu, Yang Zhang, and Xue Zhong Wang Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00190 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Organic Process Research & Development

Improved Understanding of Cefixime Trihydrate Reactive Crystallization and Process Scale-up with the Aid of PAT Shuai Yu† ‡, Yang Zhang*,†, and Xue Z. Wang*†‡§ †Engineering

Centre for Pharmaceuticals and Advanced Control, Guangdong

Province, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, Guangdong Province, P.R. China, 510640 ‡ Shandong

§School

Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), 19 Keyuan Road, Jinan, Shandong, P.R. China, 250014

of Chemical and Process Engineering, University of Leeds, Leeds LS2 9JT, U.K.

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

AUTHOR INFORMATION Corresponding Authors: Professor Xue Z. Wang Engineering Centre for Pharmaceuticals and Advanced Control School of Chemistry and Chemical Engineering South China University of Technology Guangzhou City

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Guangdong Province P.R. China 510640 Email: [email protected]; ORCID:

Xue Z. Wang: 0000-0001-9515-9492

Tel.: +86 (20) 87114000 or +44 (0) 1133432427 . Dr. Yang Zhang Contact address: ibid Email: [email protected] Tel.: +86 (20) 87114050

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ABSTRACT

Reactive crystallization to make cefixime trihydrate crystals via cefixime disodium reacting with hydrogen chloride was investigated using ATR FTIR and on-line imaging instrument with the aim of developing an improved understanding of the operation and condition optimization and scale-up. The operation involves times of continues introduction of aqueous HCl solution into a cefixime disodium solution in water and also periods at which HCl was stopped for breading of crystals. The concentration profile of the reactant cefixime disodium monitored by ATR FTIR showed almost constant in the first 30-40 minutes since the start of introduction of HCl suggests that cefixime disodium (cefixime2-) reacts with HCl to form cefixime sodium (cefixime-) first prior to the formation of solids. On-line imaging, off-line SEM image analysis, and XRD analysis revealed that spherical amorphous solids were formed first, the solids were then aggregated, followed by phase transition on the surface of aggregates to well-shaped crystals. The causal relationship between the process conditions and the size, impurity content, crystallinity, and whiteness of the cefixime trihydrate crystals was also investigated. It was found that the solution pH value at the time at which introduction of the first portion of HCl solution was stopped to begin breading crystals has major impact on both the size and the whiteness of the crystals. High pH value favors smaller crystals with sharp white appearance. The length of the crystal breeding time was found to be critical to the crystallinity. Explanation of the observed phenomena and an operational envelope was defined. The size distribution, whiteness, impurity content, and crystallinity of cefixime 4


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trihydrate crystals were satisfactory as long as the operation was kept within the defined envelope. The experiments were conducted on a 2L reactor, then successfully scaled-up to 50 L and 4000 L in industry after having considered the scale-up factor in stirrer rotation speed.

Keywords:

Reactive

crystallization;

Cefixime

trihydrate;

Process

analytical

technology; Particle size distribution; Crystallinity

1. INTRODUCTION Cefixime trihydrate, a poorly water-soluble white or slightly yellowish crystalline powder with a molecular formula C16H15N5O7S2·3H2O (CAS No. 125110-14-7), is a third generation oral cephalosporin antibiotic widely used for treatment of infections caused by bacteria such as pneumonia, bronchitis, gonorrhea, and ear, lung, throat, urinary tract infections.1-2 It is often obtained via reactive crystallization by reacting cefixime disodium and hydrogen chloride. Since cefixime trihydrate is not water soluble, it precipitates. The main quality attributes for cefixime trihydrate solids are stability, impurity, purity, particle size distribution, and whiteness. In industrial cefixime trihydrate reactive crystallization, often a mixture of amorphous form (cefixime, C16H15N5O7S2, CAS No.79350-37-1) and crystalline form solids rather than pure crystals, is obtained. Low crystallinity, low purity and high impurity content can lead to bad stability and short shelf life. The wide particle size distribution of cefixime trihydrate crystals can result in bad followability. The whiteness is also found to be 5



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difficult to control, leading to tablets with color difference. In addition, there is often batch to batch variation in product quality in the production of cefixime trihydrate. The above mentioned challenges encountered in industrial production of cefixime trihydrate solids are common to a class of reactive crystallization of pharmaceuticals. In reactive crystallization, the generation rate of supersaturation is very fast and the supersaturation level is high,3 and such critical crystallization parameters including nucleation, crystal growth, agglomeration and attrition are difficult to be obtained.4-6 It is well documented that the quality of reactive crystallization product is difficult to achieve tight control,

7-8

and that it is challenging to obtain pure crystalline form that

has a combination of satisfactory properties including solid state stability, purity, particle size distribution, and filtration and drying characteristics. 5, 9-12 In this study, process analytical technology (PAT) tools13-14 including an on-line imaging

probe,

an

attenuated

total

reflectance-Fourier

transform

infrared

spectrometer (ATR-FTIR), a turbidity probe and an on-line pH meter were applied in order to develop an improved understanding of the cefixime trihydrate reactive crystallization process, and to find

an operational space and scale-up to a 4000L

industrial reactor.

2. EXPERIMENTAL 2.1.Materials. Cefixime disodium was provided by Guangzhou Baiyunshan Chemical Pharmaceutical Factory. Hydrogen chloride was purchased from Guangzhou 6


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Chemical Reagent Factory, China with analytical reagent grade of 99.7% in mass. Deionized water was obtained by an ultrapure water system (Milli-Q Advantage A10, MERCK MILLIPORE, U.S.).

2.2.Equipment and Crystallization Procedure. The crystallization platform, as depicted in Figure 1, was supplied by Pharmavision (Qingdao) Intelligent Technology Ltd. It consists of a 2L jacketed reactor with a stirrer, a super thermostatic water bath, an ATR-FTIR spectrometer, an on-line imaging probe (2D Vision Probe), an online turbidity meter and an online pH meter (with an accuracy of ±0.002pH).

Figure 1. Schematic diagram of the crystallization system.

Firstly, the crystallizer was filled with cefixime disodium dissolved in water and kept at a fixed temperature for about half an hour. Then aqueous solution of hydrochloric acid (HCl, 4% wt) was prepared and gradually pumped into the crystallizer at a fixed flowrate (about 2 mL/min although it was different from experiment to experiment). After solids were observed at about 60-70 min since the 7



introduction of HCl, HCl solution was continually added in until the pH value reached a fixed value (e.g. about 3.07). Then HCl was stopped, the operation moves into a stage of breading of crystals for a fixed time (e.g. about 1 hr). Then new HCl solution (2% wt) was pumped into the crystallizer until the pH value reached about 2.20. After that, the temperature of the solution was kept at the fixed temperature for about 2h. Then suspension was filtered and crystals dried in a vacuum oven at 40℃ for 10h. The dried crystals were characterized. It is obvious that the above operational procedure is very general, the detailed and quantitative operational steps need to be derived.

2.3. Real time Concentration Measurement Using ATR FTIR. An ATR-FTIR probe was used for online measurement of the concentration of cefixime disodium in water. Calibration experiments were designed to cover the following ranges: 0-120 mg (cefixime disodium)/g (water), and temperature between 15 to 30 ℃. Spectra were collected with a resolution of 8 cm-1, each spectrum was an average of 128 consecutive scans. A partial least-squares (PLS) model for prediction of the concentration of cefixime disodium in water was built using the calibration data. The peaks at 1238 cm-1, 1539 cm-1, 1580 cm-1 and 1765 cm-1 were the feature spectra and used in building the model for prediction of cefixime disodium concentration (Figure 2). It needs further stress that the PLS model does not predict cefixime concentration in water since it does not dissolve in water. Cefixime is a result of 8


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cefixime- reacting with HCl and it exists in solid state. The PLS model predicts the combined concentration of cefixime2- plus cefixime-. There is no noticeable difference in IR absorbance between cefixime2- and cefixime- in water. Please also note that cefixime2- is a result of dissolution of the reactant cefixime disodium in the solvent (i.e. water), while cefixime- is a result of the reaction between cefixime disodium and HCl. Monitoring the concentration of combined cefixime2- plus cefixime- can help the analysis of the reactions and crystallization. The reason to select 1238 cm-1, 1539 cm-1, 1580 cm-1 and 1765 cm-1 as the feature eave numbers can be explained by reference to Figure 2. In Figure 2, the blue curve is the IR spectrum of pure solvent, other curves are the spectra of cefixime solution under different concentrations. Compare the blue spectrum of pure water with spectra of cefixime solutions, one can easily identify the characteristic peaks being 1238 cm-1, 1539 cm-1, 1580 cm-1 and 1765 cm-1, while other peaks are not, for instance, the peak at 1640 cm-1 is clearly due to water, rather than a characteristic peak for dissolved cefixime. There are more advanced approaches for characteristic wave number selection such as using genetic and random fog approaches, as reviewed by the authors recently15. But for this current study, it was found the PLS model built based on the manually selected four characteristic wave numbers gave sufficiently satisfactory result, so more sophisticated feature selection methods were not used.

9



Figure 2 ATR FTIR spectra. In all experiments, the probe was inserted in the same position inside the crystallizer to avoid measurement errors.16 The model R2 of the training set and test set were 0.9999 and 0.9994, respectively.

2.4. On-line Imaging Instrument. The On-line 2D Imaging Probe of Pharmavision (Qingdao) Intelligent Technology Ltd was used for observing crystallization behavior, and measure crystal shape and size. 30 images per minute were recorded in this study. Both photo images and videos were recorded.

2.5. Off-line Characterization. Particle size distribution was measured off-line using a Mastersizer 3000 laser particle size analyzer (Malvern Instrument, England). SEM images were acquired on a ZEISS Merlin (Germany). OLYMPUS BX 53 polarization microscope (Japan) was 10


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used to assess the particle size and shape of the cefixime trihydrate. The melting temperature of cefixime trihydrate was determined by differential scanning calorimetry (NETZSCH DSC- STA449 F3, Germany) with a heating rate of 10℃/min under the protection of nitrogen. The impurity content of cefixime trihydrate was analyzed using HPLC (Shimadzu, Japan). The whiteness of cefixime trihydrate was characterized by a WSB-1 whiteness meter (Shanghai Pingxuan Scientific Instrument Co., Ltd., China). The angle of repose of product was measured using a Bettersize Powder Characteristics Tester (BT-1000, China). The sample of cefixime trihydrate was identified by X-ray powder diffraction (XRD), which was carried out using a D8 Advance (Bruker, Germany) instrument. The samples were examined using Cu Kα radiation (λ=1.5418 nm), and the tube voltage and current were set at 40 kV and 30 mA. The data were collected at room temperature from 5° to 60° (2-theta) at a scan speed 1 deg/min under atmospheric pressure. 3. IMPROVED UNDERSTANDING OF THE CRYSTALLIZATION PROCESS

Figure 3. Variation of cefixime concentration during crystallization.

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3.1. Formation of solids. From Figure 3, the concentration profile of the reactant cefixime disodium, the processes taking place in the reactor can be analyzed. In Figure 3, time = 0 is the time HCl solution started to be introduced. Solution concentration seemed remaining almost constant for about 30 minutes before showing a sharp drop, see Section A in Figure 3. The explanation is that in region A of Figure 3, cefixime disodium (cefixime2-) reacts with HCl to form cefixime sodium (cefixime-), as shown in Eq. 1, reaction (a).

(1) Going from region A to region B in Figure 3, where there was a sharp drop in cefixime disodium concentration is a point at which solids began to form which is because sufficient HCl solution was added in and all cefixime2- would have reacted to form cefixime sodium and then cefixime- reacted to solid cefixime, as shown in Eq. 1, reaction (b).

12


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Figure 4. Images at different reaction times of cefixime trihydrate reaction crystallization based on 2D imaging system (a-0 min, b-70 min, c-80 min, d-100 min, e-110 min, f-150 min, g-180 min, h-200min).

Figure 5. SEM images of crystal samples at different times (a-70min, b-85min, c-100min, d-115min, e-130min, f-200min).

While it was considered that solids started to appear at the point going from region A to region B of Figure 3, about 30 to 40 minutes after starting the introduction

13



of HCl, the on-line imaging instrument only detected solids at about 70 min, as shown in Figure 4b. This was because the imaging system can only detect particles larger than 3 µm, an off-line SEM image of a sample taken at almost the same time as Figure 4b is shown in Figure 5a. Figure 4a is an image taken just before HCl was introduced to the cefixime disodium solution. After HCl was introduced, the images remained pretty much the same as Figure 4a until about 70 min as shown in Figure 4b. Figures 4c, d, e, f, g, and h are images taken at 80, 100, 110, 150, 180 and 200 min. 3.2. Aggregation and transformation. During the experiments, the slurry samples were also taken from the reactor every 15 minutes. The samples were filtered and dried before examined using SEM. Figure 5 shows SEM images for samples taken at approximately 70, 85, 100, 115, 130 and 200 min. Figure 5a is for a sample taken at about 70 min, the time the on-line imaging system detected solids for the first time. It can be seen that the particles were of spherical shape with a diameter of about 3-5 µm. The blue lines in Figure 6 corresponding to the crystallization time 90 min prove that the spherical solids were of amorphous form.

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Figure 6. XRD curves of crystals (a: XRD patterns of products with different crystallinity, b: XRD patterns during the time of breeding of crystals).

Analysis of the SEM images in Figures 5b and c, corresponding to times 85 min and 100 min suggests that the spherical solids aggregated together, the size of the aggregates is about 30 µm. The aggregation phenomenon was in some way resembles the phenomenon17-18 seen in spherical crystallization.19-23 The blue lines in Figure 6 corresponding to the crystallization time 90 min prove that the spherical solids and the aggregates were of amorphous form. On-line images taken at similar times, Figures 4c and d corresponding to 80 min and 100 min also showed aggregation. Figures 5d, e, and f, corresponding to 115 min, 130 min and 200 min, showed clearly that on the surface of the aggregates phase transition took place, from amorphous to crystalline form. To verify the observation of phase transition, Figure 15



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6b plots the XRD patterns of solids at different times. It indeed shows that from 100 min to 110, 120, 130, 150, to 200 min, the crystallinity increased. It is interesting to examine the concentration profile of Figure 3, region C. Although during the period from 80 min to 140 min, there was no introduction of HCl, the concentration of cefixime disodium showed an increase in a short time and then dropped slowly again. The pH value of the reactive solution decreased by about 0.03 in this period. The explanation is that it was due to cefixime reverse to cefixime sodium, therefore, the reverse reaction of Eq. 1-(b) produces hydrogen ions resulting in the pH value dropped and the concentration of cefixime disodium increased. After the small increase of cefixime disodium, it was observed that the concentration decreased and the pH value increased from 3.00 to 3.60. To explore what happened, the SEM images and XRD pattern of sample corresponding to region C in Figure 3 were measured and shown in Figure 5d and Figure 6b (100 min to 150 min), which showed that there brick like crystals formed on the surface of the aggregated particles, and the diffraction peaks of cefixime trihydrate were formed and crystallinity became higher. It can be concluded from the above analysis of experimental results that transformation occurred from cefixime solids to cefixime trihydrate crystals on the surface of agglomerate particles during this period as shown in reactions (c) and (d) in Eq. 1, so H+ icons were expended and pH value increased. The transformation mechanism is considered as “solution-mediated”,4,

24-26

the

change of the solids might be due to that the surface of the solid slightly dissolved first and then new crystals formed on the amorphous particle surface. The solution 16


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mediated phase transition (SMTP) can occur only from a less stable phase to a more stable phase, and the environment including the pH value, reaction temperature, etc. surrounding the solids affects the phase change. In the current study of cefixime reactive crystallization in water solution, cefixime trihydrate solids are more stable than the cefixime solids. SMTP mechanism is more likely based on the information of concentration and SEM images mentioned above. During the transformation period, the crystallinity became higher (Figure 6b). The crystals were confirmed as a hydrate with three molecular H2O according to the experimental TG curve given in Figure 7, in which the mass loss of the final product was about 10-11% in wt% near the temperature of 80℃ to 130℃, and the results also showed that there is no crystal water in the spherical solid form.

Figure 7. TG curves of cefixime and cefixime trihydrate.

17



Figure 8. Particles produced during the secondary adding of HCl solution (a: optical microscope images, 50X, b: SEM image, 8000X).

3.3. Growth. After the crystal breeding period, region C of Figure 3, of approximately one hour when no HCl was added, HCl solution was added again with a fixed adding rate until the pH value reached 2.20 then HCl was stopped. This corresponds to region D in Figure 3. The agglomerated particles were found to grow larger and larger. As indicated in Figures 5 and 8 rod shaped crystals were attached to the surface of the spherical particles. It was considered that aggregation is the leading mechanism for the growth of cefixime trihydrate on the surface of aggregated particles formatted in the solution-mediated step. The crystal growth mechanism was inferred as contact nucleation on the surface of crystals on the spherical particle and growth,25 for the solution concentration has decreased to a lower level for reaction as shown in Eq. 1 reaction (d), the reaction rate decreased and the existing crystal particles on the surface would induce the solid form of new particles to be crystal form while not amorphous form, and crystal growth subsequently. Collision of spherical particle and those between crystal and agitator and vessel also lead to secondary nucleation in this study, which was proved 18


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by some needle crystals obtained and given in Figure 8. It can be found in Figures 4f, g and Figure 9 that the experimental particle size of cefixime trihydrate would grow bigger during the secondary adding of HCl solution compared to region C in Figure 3.

Figure 9. Particle size distributions during the time when the second portion of HCl was added.

In summary, the reactive crystallization of cefixime trihydrate mainly involves three steps. Firstly, spherical solids of amorphous form appeared. Then, the spherical solids aggregate to form larger particles and the amorphous form transformed to crystalline cefixime trihydrate without extra HCl solution being added into the solution at the same time. Finally, new tiny crystalline cefixime trihydrate particles nucleated on the surfaces of crystals on the spherical particles and then growth took place. The procedure is illustrated using Figure 10.

19



Figure 10. Formation, aggregation, transformation and growth of particles.

4. STUDY ON THE CAUSAL RELATIONSHIPS BETWEEN PROCESS VARIABLES AND PRODUCT QUALITY

4.1. Planning of Experiments. In planning the experiments, the objectives for optimization of operational conditions and the process variables to be manipulated or monitored need to be specified. In addition, the interactions between the process variables should be examined. The objective for optimization is to obtain crystalline products with satisfactory properties including stability, impurity content including the total impurity content and the single main impurity content, crystal size distribution which is often defined by the customers, morphology, and whiteness. In addition, batch to batch variation should be minimized. There are other constraints such as that the batch time cannot be too long. In terms of the process variables that are manipulated or monitored, for the original condition for the 4000 L industrial crystallizer, the reaction temperature was 20℃, stirrer speed was 50 r/min, and the HCl feeding rate was 6L/min. Some over 20 initial experiments were conducted in the lab on a 2L crystallizer to repeat the operation in the industrial crystallizer (of course the stirrer speed will be different) or to operate 20


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the process surround the industrial condition. The experiments revealed that batch to batch variation exists and is difficult to be avoided. These initial experiments led us to find a new variable to monitor, which is the difference of two pH values, the pH at the point solids are seen to form, and the pH at which breading of crystals begins. Throughout the rest of the article, △pH is used to represent the difference of the two pH values. It was found that by controlling △pH, batch to batch variation can be minimized. More detailed discussion about this new variable, △pH, will be given below in Section 4.2. So in summary, the process variables that are manipulated or monitored are reaction temperature, stirrer speed, HCI feeding rate, length of breading time of crystals, and △pH. A logical thinking could be that as ATR FTIR was used to measure concentration why supersaturation was not used as the basis to address batch to batch variation? This is a reactive crystallization process, the crystals are cefixime crystals, since cefixime does not dissolve in water, once produced via reaction, it immediately forms solid crystals; so in this case if there was such a concept as supersaturation for cefixime, then its supersaturation would be close to +∞. So we cannot make use of supersaturation concept here to explain the crystallization process in the same way as for cooling or anti-solvent crystallization. Then what concentration is measured by ATR FTIR? The concentration measured by ATR FTIR is the combined concentration of cefixime2- plus cefixime-. Cefixime2- is a result of dissolution of the reactant cefixime disodium in the solvent (i.e. water), while cefixime- is a result of the reaction between cefixime disodium and HCl. There is no noticeable difference in IR 21



absorbance between cefixime2- and cefixime- in water. This analysis naturally leads to a consideration that is can the combined concentration of combined cefixime2- plus cefixime- be used to achieve consistent quality control? This was analyzed but a way was not yet found, partly due to the complexity such as the fact that the reaction is reversible. The potential interactions between the process variables were also examined and the conclusion is discussed here. The impact of the process variables on the product quality, as well the potential interactions between the process variables were examined only in the feasible ranges of values for the variables (the detailed ranges for each variable will be discussed in the Result and Discussion section below). It was found that the within this narrow space, the reaction temperature and △pH are the main variables that affect crystal size, while the feeding rate and the stirrer speed mainly affects impurity content. Considering there might be an interaction between the ΔpH value and the reaction temperature on the particle size, an interactive impact pretesting with the reaction temperature of 20℃, the ΔpH value of 0.05 with other parameter conditions settled the same as Exp.4 in Table 1 was designed. The result is shown in Figure 11.The x-axis is the reaction temperature, the y-axis is the mean particle size (d50), the red line corresponding the ΔpH value of 0.03, and the blue line corresponding the ΔpH value of 0.05. The two lines are almost in parallel which is indication that the interaction between the ΔpH and the temperature is small27. In the Section 4.3 below, the potential interaction between the feeding rate and stirrer

22


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speed was also discussed, and the conclusion was that there is no need to study the interactions between the feeding rate and the stirrer speed.

Figure 11 Interaction between the ΔpH value and the reaction temperature.

Table. 1 The experiments Feeding Experiment

Reaction

Stirring rate

△pH No.

Length of

temperature (℃)

speed (r/min)

breeding time (h) (mL/min)

(0.01, 0.02, 0.03, 0.05, Exps 1-5

25

250

2.0

1

250

2.0

1

2.0

1

0.10) 10, 15 20, 25, Exps 6-10

0.03 30

20, 100, 150, Exps 11-16

0.03

25

200, 250, 300

23



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0.5, 1.0, Exps 17-22

0.03

25

250

2.0, 3.0,

1

5.0, 8.0 0, 0.5, 1.0, 1.5, Exps 23-27

0.03

25

250

2.0 2.0

4.2. Factors Affecting Particle Size Distribution.

4.2.1. Effect of ΔpH. ∆pH = 𝑝𝐻1 ― 𝑝𝐻2

(2)

where 𝑝𝐻1 represents the pH value corresponding to the emergence of spherical solids seen by the online imaging probe (Figure 4b), 𝑝𝐻2 is the solution pH value at the start for the first time interval of crystal breeding. Five different ΔpH values 0.01, 0.02, 0.03, 0.05 and 0.10 were investigated with other parameters unchanged (Exps 1-5), as shown in Table 2 and Figure 9. The results indicated that large product particles were obtained when constant ΔpH value for every run was changed from 0.10 to 0.01. During the addition of HCl solution, the spherical solid particles were observed appearing continuously from solution from Figure 5, the total number of formed particles increase more when the ΔpH value increases, and then the spherical solid particle will agglomerate growth until aggregates with size of about 30μm were formed in solution after stopping adding the HCl solution shown in Figure 3b and c. According to the law of conversation of mass, the more aggregated particle

24


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formatted in this period, the particle size of the final product would be smaller as shown in Figure 9d.

Table 2. Whiteness, Size Distribution and Angle of Repose of Final Products under Different ΔpH Values ΔpH

Whiteness

Particle size (d50, μm)

Angle of repose (°)

0.01

60

90

30

0.02

66

75

35

0.03

70

65

40

0.05

73

50

50

0.10

80

40

55

As discussed in Section 3.2, pH value also increased during the transformation process from amorphous state to hydrate crystalline form, and it was found in Figure 12 that the transformation rate can be affected by the ΔpH value. Such as ΔpH=0.10, the starting time of transformation is about 10min after stopping adding the HCl solution, the transformation rate was faster than the other four ΔpH values.

25



Figure 12. Change of absolute pH value during the first time interval of crystal breeding. It was found that large particle size resulted in weaker whiteness and the better flowability of the product, as shown in Table 2. Therefore, for comprehensive consideration of improved whiteness and flowability of the final particle, 0.03 was selected as the best ΔpH value for reactive crystallization of cefixime trihydrate.

4.2.2. Effect of temperature. The reaction temperature was found to be another important factor that influences the particle size distribution of cefixime trihydrate product. The experimental result of different reactant temperatures on particle size and crystal shape are shown in Table 3 and Figure 13, which indicated that the particle size increased when increasing the reactant temperature over the temperature range from 10℃ to 30℃ with other parameters unchanged (Exps 6-10). The result is in agreement with that of Seyssiecq et al. (1998)28, Yamada (1980)29, and Sakamoto et al. (1976)30 who also investigated the effect of temperature on crystal size distribution. Their analysis was that at low supersaturation the size 26


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change was mainly due to surface growth, while at high supersaturation it was mainly due to aggregation of particles. Although our process is reactive crystallization while their work was conducted for cooling crystallization, the mechanism is similar. According Chen et al31, in reactive crystallization where the product crystal does not dissolve in the solvent, the crystal size increase was due to a combination of aggregation and growth, with the aggregation dominates in most operational conditions. At high temperature, the reaction rate is high, so the generation rate is high for cefixime, leading to high possibility of aggregation of particles, causing crystal size to increase. This is similar to cooing crystallization when the supersaturation is high. The temperature also affected the morphology of the crystals. Figure 13 shows the SEM images of crystals obtained different temperatures. While the shape looks different, XRD analysis confirmed that they are of the same polymorph. The morphology varies under different temperatures is understandable, just as the case of temperature influence on size. However, we did not perform on detailed study here on cefixime. Detailed investigations on crystal morphology has been reviewed in literature32-33.

Table 3. Size Distribution of Final Products under Different Reaction Temperature Reaction temperature (℃)

Particle size (d50, μm)

Whiteness

Angle of repose (°)

10

45

82

65

15

57

78

60

27



Page 28 of 43

20

61

72

45

25

65

70

40

30

75

38

35

Figure 13. SEM images of final products under different reaction temperature (a-10 ℃, b-15 ℃, c-20 ℃, d-25 ℃, e-30 ℃).

4.3. Factors Affecting the Impurity Content. There are more than six impurities that were known according to HPLC analysis, their names have been added into the revised

manuscript.

These

are

cefixime

methyl

ester,

cefixime-13C-15N2,

cefixime-13C3-15N2, cefixime 7-epimer, (E)-cefixime, and 3-desethenyl-3-methyl cefixime.. The main impurity represents the impurity with the largest composition, and the total impurity content is the sum of all the impurity contents.

4.3.1. Effect of feeding rate. The feeding rate was found to have noticeable effect on the impurity content in cefixime trihydrate products. In Table 4, the feeding rate was

28


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changed in the range between 0.5 mL/min (HCl solution) to 8.0 mL/min (HCl solution). The HPLC data (relative area, Table 4) showed that feeding rate of 8.0 mL/min generated crystals of the highest impurity content. There is no obvious difference among the extra feeding rates of 0.5 mL/min, 1.0 mL/min, and 2.0 mL/min. The feeding rate of HCl solution has a direct impact on the reaction rate for the reaction process, and the reaction rate and diffusion rate decide the deposit process. The analysis is that rapid feeding rate could result in the fast formation of solid particles while impurities were not given sufficient time to be removed due to slowly mass transfer rate. Considering the impurity content and production time, the best feeding rate for acid was selected as 2.0 mL/min.

Table 4. Impurity Content of Product under Different HCl Solution Feeding Rates Feeding rate

The main impurity content

Total impurity content (0 day, %; less

Total impurity content

(mL/min)

(0 day, %)

than 0.1% was ignored)

(0 day, %)

0.5

0.091

0.091

0.145

1.0

0.091

0.091

0.151

2.0

0.095

0.095

0.162

3.0

0.125

0.125

0.200

5.0

0.142

0.162

0.233

8.0

0.230

0.381

0.455

HPLC measurement was according to Chinese pharmacopeia 2015 edition.

29



4.3.2. Effect of stirrer speed. The effect of stirrer speed on the impurity content of cefixime trihydrate product was also investigated. The result is shown in Table 5. The result indicated that if all other conditions were the same when the agitation rate was increased, both the maximum single impurity content and total impurity content were reduced effectively. The worst mixing speed was below 150r/min. It was found that there was no obvious difference in product impurity content when the stirrer speed was above 250r/min. In H. Charmolue and R. Rousseau (AIChE J)34 and Jeﬀrey C. Givand, Amyn S. Teja, Ronald W. Rousseau (J Crystal Growth)35 investigated in a cooling crystallization the impact of stirrer speed on the impurity content of methanol in L-serine crystals. They found that the impurity content firstly became lower with the stirrer speed increased from 500rpm to 1000rpm, and then the impurity content would become higher with the stirrer speed increased from 1000rpm to 1500rpm. Their explanation was that a low stirrer speed enhanced the entrapped impurity because of the poor solid dispersion. And in the work of Norshafika Yahya’s team published in Chemical Engineering Transactions36, it was found that high shear force will bring away the impurity which is entrapped between the dendritic structure of ice layer during the crystallization process. In our research, although we didn’t find an inflection point between the stirrer speed and the impurity content. It was found that the impurity content decreased with the increase of stirrer speed was found. The possible explanation is similar in what was discussed in the literature that the higher stirrer speed enhanced the shear force and provided better mixing and so decreased the entrapped impurity. 30


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Table 5. Impurity Content of Products under Different Stirrer Speed Stirring rate (r/min)

The main impurity content (0 day, %)

Total impurity content (0 day, %)

50

0.152

0.152

100

0.130

0.130

150

0.115

0.115

200

0.105

0.105

250

0.095

0.095

300

0.093

0.093

When the feeding rate changes from 0.5 mL/min to 8.0 mL/min, the total impurity content would increase from 0.091% to 0.381%. But with the change of the stirrer speed ranging from 50rpm to 300rpm the total impurity content only changes from 0.152% to 0.093%. Although it seems that the feeding rate has an obvious effect on the impurity content within the range of experimental conditions, there might be an interaction between the HCl feeding rate and the stirrer speed on the impurity content. But according to the experimental data, the impurity content would not change significantly when the HCl feeding rate was under 2.0 mL/min. Although it is the slower the better for the HCl feeding rate, considering that the production time should not be too long in industrial operation, the HCl feeding rate was set at 2.0 mL/min and not be changed in other experiments. As for the stirrer speed, there is no obvious change of the impurity content when the stirrer speed larger than 250rpm, so 31



the stirrer speed was also fixed. And the investigation of the interaction between the HCl feeding rate and the stirrer speed was not very necessary.

4.4. Factors Affecting Crystallinity. The crystallinity is positively associated with the stability,37 high crystallinity organic drug often results in long shelf time.21 Process conditions on the crystallinity of cefixime product prepared by reactive crystallization were investigated in this study. The crystallinity can be drawn from peaks heights of X-ray powder diffraction patterns, the diffraction peaks at values of 2θ at 9.0°, 15.0°, 18.5°, and 23.1° were the main peaks of cefixime trihydrate (the black solid line in Figure 6a). It is generally accepted that high peaks correspond to higher crystallinity9, 35, In the current study, it was observed that for a given powder sample, the characteristic peaks were either all high, or all low. In other words, when comparing the crystallinity of different samples, one could compare the peak heights of different samples at a particular peak, e.g. 9.0°. The reason to choose 9.0°(2θ) as shown in Figure 6(b) is that it is the highest peak, allowing the differences between the seven samples to be clearly distinguished. The reason not to plot all the characteristic peaks together for the seven samples, is that it will make the curves overlapped and could not see clearly the differences between the seven samples. Figure 6b shows the variation of XRD patterns of particles under different crystal breeding time. It can be seen from Figure 6b that the peak height in 9° increased from 1000 (start point of first time interval of crystal breeding) to 5100 (1h later), but 32


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doesn’t change obviously later, which is indication that the products change gradually from amorphous to crystalline state during the agglomeration and growth process, which in coincidence with the transformation process described in Figure 5.

Table 6. Impurity Content of Products under Different Crystallinity The main impurity


The main impurity content


content (0 day,

(0 day, %)

(10 day, %)

(10 day, %)

Sample No.

%) S1

0.102

0.102

1.120

3.830

S3

0.105

0.105

0.895

2.360

S5

0.105

0.105

0.480

2.010

S7

0.104

0.104

0.325

1.520

The accelerated stability test was settled as 10 days at the temperature of 60℃, and the content of impurities was used to evaluate the crystallinity. The results in Table 6 showed a significant negative correlation between impurity content and crystallinity. The named of samples from S1 to S7 were corresponding to the samples collected under different first time interval of crystal breeding shown in 33



Figure 6b. Higher crystallinity often along with good stability as mentioned above. The total impurity content of different crystallinity samples before and after the accelerated stability test was shown in Table 5 (the composition of an impurity was written as zero in the table is it is less than 0.1%). And there is no obvious relation between the crystallinity and the impurity content before the accelerated stability test. It was also found that the crystallinity further improved during the growth step in Region D in Figure 3 for the peak height of the XRD pattern of the final product in 9° was further improved. According to the growth mode mentioned above, the nucleation and crystal growth will occur on the surface of the formed aggregated solid particle once transformation process is finished, while not amorphous solid state during this period. Therefore, no detailed experiment about the second time of crystal breeding was investigated in this study.38 In addition, the crystal breeding time was not taken into consideration in the original operational procedure, which leads to the crystal transformation incomplete and poor crystallinity and stability. For the different △pH values mentioned above, one hour was found to be sufficient for the amorphous to transform to crystal form except for the △pH value of 0.01 (Figure 6b 90 min-100 min and Figure 12). The result of the experiments indicates △pH and the reaction temperature are the main factors that influence the particle size, but they showed little impact on other indicators (Exps 1-5 and Exps 6-10). The stirrer speed and the HCl feeding rate would influence the impurity content but almost have no effect on other indictors as well (Exps 11-16 and Exps 17-22). And the length of the breeding time would 34


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influence the crystallinity (Exps 23-27). The quality of the indicators changes as monotonous with the change of the variable level, it is difficult to find an optimal point to proceed further investigation. Another challenge, towards a multiple objective optimization process there is a relationship between some quality indicators. Such as the increase of the △pH value would lead the decrease of the particle size, but the whiteness would increase and the followability would decrease. But the particle size, whiteness, and flowability are all important quality indicators for the cefixime trihydrate medicine. Moreover, the optimized △pH value obtained from different target indicators were also different. Therefore, it is necessary to choose the optimized variable value that takes into account of the above indicators. In planning the experiments, the whole experiments were not selected, the reason is that the impurity content was monotone decreasing with the increasing stirrer speed and decrease of the HCl feeding rate, but the impurity content value would not change significantly when the stirrer speed reached 250r/min and the HCl feeding rate slower than 2.0 mL/min.

4.5 Scale-Up. All the above experiments were conducted in the 2 L crystallizer, and the optimized conditions were firstly scaled up to a 50 L pilot crystallizer and then an industrial 4000 L reactor. The optimized laboratory scale crystallization: reaction temperature was 25℃, feeding rate was 2.0 mL/min, ΔpH = 0.03, the first time interval of crystal breeding was 1h, and the stirring speed was 250 r/min.

35



In scale-up the operational envelope to the 4000 L industrial reactor, the operational condition was as follows: (1) the reaction temperature was 25℃, the same as in laboratory scale experiments; (2) the feeding rate of HCl solution was 4L/min, scaled-up based on the difference in throughputs between the lab and industrial sized reactor; (3) ΔpH was 0.03, the same as in the lab; (4) the first breeding time of crystals was 1h, the same as in lab scale; (5) the stirrer speed was set at 70 rpm, different from in the lab experiments. The stirrer speed in the 4000 L reactor was decided based on computational fluid dynamics (CFD) simulation. The contours of volume fraction and magnitude of velocity at four different stirrer speeds, i.e. 50, 60, 70 and 80 rpm were simulated. It indicated that the mixing conditions improved as increasing the stirrer speed from 50 rpm to 70 rpm, but there was no significant improvement in mixing as increasing the speed to 80 rpm. Consequently, 70 rpm was used in the 4000 L crystallizer. The experimental XRD pattern and particle size distribution were shown in Figures 14 and 15. No significant difference for crystallinity and particle size distribution between the laboratory, pilot plant scale, and industrial scale. With the optimized conditions, the final industrial product showed high crystallinity, whiteness large than 70, the angle of repose less than 40°, and the mean particle size was about 63μm with a narrow distribution (variable coefficient, C.V. less than 43.5%, C.V. =

100(𝐷90 ― 𝐷10) 2𝐷50

%). Detailed comparison of the quality of the product between the

original process and the optimized process is shown in Table 7. The particle could be controlled about 65μm with a narrow distribution in the optimized process. And what 36


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is more, the impurity content could be less than 0.20% in both laboratory and industrial scales under the optimized conditions.

Figure 14. XRD curves of final products under different process scale.

Figure 15. Particle size distribution of final products under different process scale (△pH=0.03).

37



Page 38 of 43

Table 7. Quality of the Product Produced with the Original Process and the Optimized Process.

Particle Process name

Total impurity

Total impurity

content (0 day,

content (10 day,

%)

%)

Angle of repose C.V.(%)

Whiteness

size (d50, μm)

(°)

Laboratory scale 40-100

>40

60-70

35-50

0.10-0.12

>0.70

60-70

70

Improved Understanding of Cefixime Trihydrate Reactive

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