Physicochemical stability of Cabazitaxel Zentiva ® solution in vials after opening and diluted solutions in three infusion bags

Objectives: To the best of our knowledge, few studies have been published on the stability of cabazitaxel in infusion bags. Stabilis ® database has selected a study demonstrating the stability of this molecule at 0.15 mg/mL for 28 days at 4 ° C and 25 ° C in polyole ﬁ n bags. The aim of this work was to study the physicochemical stability of Cabazitaxel Zentiva ® solutions in vials after “ opening ” with a vented Chemo-Clave ® Spike, at 25 ° C, protected from light and in solutions diluted at 0.1 and 0.26 mg/mL in 0.9 % sodium chloride (0.9 % NaCl) or dextrose 5 % (D5W) in 3 types of infusion bags (Easy ﬂ ex ® and Via ﬂ o ® at 25 ° C, Free ﬂ ex ® between 2 and 8 ° C, protected from light). Methods: The chemical stability was analyzed after preparation and then after 14 and 28 days of storage by high performance liquid chromatography (HPLC), coupled to a diode array detector, at the analysis wavelength of 232 nm. The method has been validated according to ICH Q2 (R1) standards. For the study in infusion bags, three preparations were realised for each condition. At each time of analysis, for each bag, a sample wasprepared and analyzed by HPLC. Two vials after “ openings ” were kept at 25 ° C and three samples per vial were prepared and analyzed at the three analysis times (D0, D14 and D28). Physical stability was assessed by visual examination (change in colour, appearance of precipitate, gas formation). The pH of the solutions prepared in infusion bags was evaluated at each analysis time. Results: Cabazitaxelsolutionsat0.1and0.26 mg/mLdilutedin 0.9 %NaClorD5WinEasy ﬂ ex ® (polyole ﬁ n),Via ﬂ o ® (multilayer high density polyethylene, polyamide, polypropylene) bags retained more than 95 % of the concentration after 28 days at 25 ° C. In the Free ﬂ ex ® bag (polypropylene multilayers), cab-azitaxelsolutionsat0.1and0.26 mg/mLdilutedin0.9 %NaClor D5W retained more than 95 % of the initial concentration be-tween 2 and 8 ° C for 28 days. In vials with a Spike, cabazitaxel solutions at 20 mg/mL retained more than 95 % of the initial concentrationfor28daysat25 ° C.Foralltheconditionsstudied, no visual modi ﬁ cation was observed. The pH of solutions in bags were constant during the stability study. Conclusions: Cabazitaxel Zentiva ® diluted at 0.1 and 0.26 mg/mL in 0.9 % NaCl or D5W was stable for 28 days at 25 ° C and between 2 and 8 ° C. These stability data allow preparations to be made in advance. The remainder of the cabazitaxel vial ﬁ tted with a Spike was stable for 28 days at 25 ° C, allowing the remainder of the vial to be used over several days.


Introduction
Cabazitaxel (Figure 1) is a compound belong to the class of taxanes, a second-generation semisynthetic taxane.
Cabazitaxel is an antineoplastic agent that acts by disrupting the microtubular network in cells.Cabazitaxel binds to tubulin and promotes the assembly of tubulin into microtubules while simultaneously inhibiting their disassembly.This leads to the stabilisation of microtubules, which results in the inhibition of mitotic and interphase cellular functions.Cabazitaxel in combination with prednisone or prednisolone is indicated for the treatment of adult patients with metastatic castration resistant prostate cancer previously treated with a docetaxel-containing regimen.The recommended dose of cabazitaxel is 25 mg/m 2 administered as a 1-hour intravenous infusion every 3 weeks in combination with oral prednisone or prednisolone 10 mg administered daily throughout treatment [1].
The product was initially marketed under the name of Jevtana ® in the form of a concentrated vial accompanied by a solvent.The concentrate vial contained polysorbate 80 and citric acid as excipients and 60 mg of cabazitaxel.The solvent vial contained ethanol 96 % and water for injection.Each vial was overfilled so that after dilution with the entire content of the accompanying solvent, the solution contained 10 mg/mL cabazitaxel.For the reconstituted vial, the manufacturer indicated a 1 h stability at ambient temperature and a 24 h stability between 2 °C and 8 °C.After dilution in infusion bags at a concentration between 0.1 and 0.26 mg/mL, an 8 h stability at ambient temperature and a 48 h stability under refrigeration were indicated [2].
Then, six generics appeared on the market in France, one with the same formula [3], and another in a one-vial concentrate at 10 mg/mL with polysorbate 80, ethanol anhydrous, macrogol and citric acid as excipient.Infusion stability was extended to 48 h at a temperature below 25 °C and 14 days under refrigeration [4].Two other generics were marketed as a one-vial at 20 mg/mL with the same excipient but without macrogol.The Accord ® generic indicated the same stability as Jevtana ® [5].The Zentiva ® generic indicated a 4 week stability between 2 °C and 8 °C after opening of the vial and for the infusion, a 48 h stability at 25 °C and a 14-day stability between 2 °C and 8 °C [1].All stability data was demonstrated in polyolefin containers as the presence of polysorbate 80 contraindicated the use of PVC containers due to the release of phthalates into the solution and an in-line filter of 0.22 µm is recommended during administration because the infusion solution is supersaturated and may crystallize overtime.
Few stability studies have been published concerning cabazitaxel infusion stability.Spindeldreier et al. studied the stability in Freeflex ® infusion bags diluted in 0.9 % sodium chloride (0.9 % NaCl) and 5 % dextrose (D5W) after storage at 25 °C and refrigerated.They concluded there was of a 28 day stability for the 0.1 and 0.26 mg/mL solutions, however great variability occurred in the results and particles were observed in some cabazitaxel infusions after 21 days of storage [6].Lazzarini et al. studied the stability at 0.15 mg/mL in 0.9 % NaCl in Viaflo ® infusion bags and demonstrated a 4-week stability at ambient and refrigerated temperatures [7].Both studies were performed using Jevtana ® .No publication is available on the stability of the concentrated solution partially used with a Spike system inserted in the vial.
The objectives of this study were to evaluate the stability of Cabazitaxel Zentiva ® under several storage conditions: the stability of the partially-used vials with a Spike device (ICU Medical) inserted into the vial, the stability of the solutions at 0.1 and 0.26 mg/mL in 0.9 % NaCl and D5W at 25 °C and between 2 °C and 8 °C in three types of infusion bags: Easyflex ® made of ethylene/propylene copolymer, Freeflex ® film containing polypropylene and thermoplastic elastomers and Viaflo ® made from a multilayer sheeting (PL-2442) composed of polypropylene (PP), polyamide (PA) and polyethylene (PE) [8][9][10].

Preparation of test solutions
All manipulations were performed under a biological safety cabinet.
Stability in infusions bags: Stability study in three types of infusion bags (Easyflex ® , Viaflo ® , Freeflex ® ) at 0.1 mg/mL (10 mg/100 mL) solutions in 0.9 % NaCl and D5W: the bags are usually overfilled compared with the theoretical volume of 100 mL.The overfilling depends on the batch used.The overfilling of each batch was measured in our laboratory by emptying three bags into a graduated cylinder and by calculating the average value.For each solution, the overfilling volume and 0.5 mL (corresponding to the volume of cabazitaxel to be added) were removed and 0.5 mL of cabazitaxel 20 mg/mL was injected in the infusion bag.
Stability study in three types of infusion bags (Easyflex ® , Viaflo ® , Freeflex ® ) at 0.26 mg/mL in 0.9 % or D5W (26 mg/100 mL): the overfilling volume of infusion bags and 1.3 mL of solvent were removed, then 1.3 mL of cabazitaxel 20 mg/mL was injected into the infusion bag.The solutions were homogenized by stirring.
Stability in partially-used vials: The impact of using a ChemoClave ® vented vial Spike: to secure the preparation of cabazitaxel infusions in daily practice, the use of ChemoClave ® Spike has been investigated to pick up the cabazitaxel solution in vials.Two vials of cabazitaxel with two different batches were analysed and stored at 25 °C (CL2105, CL210102).The physico-chemical stability was analysed after addition of a Spike, after 14 and 28 days.

Chemical stability
HPLC assays: Cabazitaxel solutions were analysed by a stabilityindicating reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode array detection, adapted from Lazzarini et al. [7] The HPLC system consisted of an ELITE LaChrom VWR/Hitachi plus autosampler, a VWR photodiode array (PDA) detector L-2455 and a VWR L-2130 HPLC-pump.Data were acquired and integrated by using EZChrom Elite software (VWR, Agilent).The column used was a LiChrospher ® 100 RP-18 (5 µm) 12.5 cm and 5 µm particle size.The mobile phase was in isocratic mode constituted of 42 % of water for chromatography, 32 % of methanol and 26 % of acetonitrile.The flow rate was set at 1.2 mL/min, with an injection volume of 25 µL.The temperatures of the column oven and of the injector were set at 40 °C and 10 °C respectively.The detection of cabazitaxel was performed at 232 nm.The photodiode array detector evaluated the UV spectrum of the chromatographic column effluent every 0.4 s.The wavelengths of the analysis spectrum were between 200 and 400 nm.
Validation of the analytical method: The analytical method was validated as recommended by the International Conference on Harmonisation (ICH) Q2 (R1) [11].The calibration curve was constructed from plots of peak area vs. concentration.The linearity of the method was evaluated with five concentrations (50, 100, 200, 300, 400 μg/mL).One calibration curve was performed daily for three days.For the calibration curves, the homogeneity of variances was evaluated with a Cochran test whose significance level was set at p<0.05.An analysis of variation (ANOVA) of the linear regression data was performed to assess the significance (p<0.05) of the proposed method.The intra-day precision was evaluated using three determinations for each concentration at 100, 200 and 300 μg/mL.For interday precision, three determinations for each concentration at 100, 200 and 300 μg/mL of cabazitaxel were assayed daily on three different days.
The evaluation of the stability in the autosampler was realized with the concentration at 200 μg/mL by injecting every hour during 15 h.The specificity of our method was evaluated with the analysis of excipient solutions of Cabazitaxel Zentiva ® by HPLC: polysorbate 80 (Acros Organics, batch A0389530), ethanol 95 diluted with ultrapure water (1/4) (Cooper, batch: 2007024B) and 5-hydroxymethylfurfural (5-HMF) solution at 0.1 mg/mL (Acros organics, batch: A0406690), the main degradation product of dextrose.
The stability-indicating capability was evaluated by analysing forced degraded cabazitaxel solution.The aim of this forced degradation was to obtain degradation products with retention times different from those of cabazitaxel.The objective was to obtain a degradation close to 20 % of our molecule of interest [12].Conditions for the degradation of cabazitaxel were as follow: -Acidic conditions: one mL of an 800 μg/mL cabazitaxel solution was diluted with 1 mL HCl 1.69 M, stored at 20-25 °C for 10 min, neutralized by 1 mL of NaOH 1.69 M and diluted with 1 mL of mobile phase to obtain a theoretical concentration of 200 μg/mL.-Alkaline degradation: one mL of an 800 μg/mL cabazitaxel solution was diluted with 1 mL NaOH 0.05 M, stored at 20-25 °C for 1 min, neutralized by 1 mL of HCl 0.05 M and diluted with 1 mL of mobile phase to obtain a theoretical concentration of 200 μg/mL.-Heat degradation: an 200 μg/mL cabazitaxel solution was exposed to a temperature of 80 °C during 24 h.The solution was analysed by HPLC, directly without dilution.-Oxidative degradation: one mL of an 800 μg/mL cabazitaxel solution was diluted with 3 mL 6 % H 2 O 2 and analysed by HPLC.-Photolytic degradation: a 200 μg/mL cabazitaxel solution was exposed to a UV source of 254 nm for 24 h.The solution was analysed by HPLC, directly without dilution.
Sample dilution for analysis by RP-HPLC: For each bag prepared, one sample was analysed by HPLC without dilution: 0.1 mg/mL and 0.26 mg/mL are concentrations included in the standard range.For vials, samples were prepared in triplicate for each vial and diluted to obtain a concentration for HPLC analysis at 200 μg/mL.This process was repeated at each time of the analysis.Total run time was set at 30 min.Chemical stability was defined as not less than 90 % of the initial cabazitaxel concentration [13].The evaluation of additional peaks has been carried out.
pH measurements: pH measurements were performed using CRISON PH25 pH meter.Analysis was carried out for each infusion bag at each time of analysis.pH values were considered to be acceptable if they did not vary by more than 1 pH unit from the initial measurement [13].pH measurements were not performed on partially-used vials due to insufficient volume.

Physical stability
Physical stability was defined as the absence of colour change, particulates or gas formation.The samples were visually inspected against a white/black background with unaided eye at each analysis time by two technicians [13].
A subvisual examination was carried out by performing a particulate counting test to detect microparticles invisble to the naked eye.It was assessed by PAMAS Particle counter.The method consists in intercepting of a light ray, allowing the automatic determination of the size of the particles and their number by size.As recommended by the European Pharmacopoeia [14], preparations of a volume of less or equal to 100 mL comply with the particulate contamination test if the number of particles measured does not exceed 6,000 particles with a size ≥10 µm per container and 600 particles with a size ≥25 µm per container.These tests were performed on each cabazitaxel solutions in infusion bags, at each times of analysis.Due to volume limitation in the vials, these sub-visual tests were not performed on cabazitaxel vials.

Validations of the analytical method
The retention time of cabazitaxel was near to 9.5 min (Figure 2).The UV spectrum of cabazitaxel is shown in Figure 3.
A set of forced degradations performed is shown in Figure 4 and the mass balance is presented in Table 1.We were able to degrade cabazitaxel between 6 % with photolytic degradation and up to 16 % with acidic degradation.None of the degradation products interferes with the cabazitaxel peak.Forced degradation under thermal and oxidative conditions did not allow the degradation of cabazitaxel.Stability-indicating capacity was proved by using various stressed conditions.

HPLC assay of cabazitaxel
The remaining percentage of the initial concentration of cabazitaxel in different types of infusion bags and in vials is shown in Tables 2 and 3 respectively.Regardless of containers and storage temperatures (Easyflex ® and Viaflo ® at 25 °C, Freeflex ® at 2-8 °C), solvent (0.9 % NaCl or D5W) and concentrations studied (0.1 or 0.26 mg/mL), solutions retain more than 95 % of the initial concentration up to 28 days.On some chromatograms obtained for solutions tested, additional peaks were observed but represented a small percentage compared to the cabazitaxel peak.Some are random (for example, appearance from D0 and absent on the other days of analysis), some peaks were present from D0, as well as at D14 and D28, but minor variations have been observed over time and some peaks also have a peak area below the exclusion limit and were therefore not considered.
Regarding pH measurements, minor changes in pH for all the conditions studied, during the stability study: maximum variation of 0.39 units pH on D14 and 0.24 pH unit on D28.20 mg/mL cabazitaxel solutions in glass vials, stored at 25 °C, retained more than 95 % of the initial concentration after 28 days.

Physical stability of solutions
For each condition tested, infusion bags and vials were clear and colourless at D0, D14 and D28 for all the analysis conditions.
For the subvisual examination, the results were within the acceptance criteria of European Pharmacopeia for solutions prepared in Easyflex ® infusion bags at T0 and were outside of these criteria for solutions in Freeflex ® and Viaflo ® at each times of analysis and for solutions in Easyflex ® at D14 and D28.Morever, great and incoherent variations were observed for the number of particles detected mainly over 10 µm.

Reversed phase HPLC
Cabazitaxel was analysed by a stability-indicating reversed phase HPLC, a method adapted from Lazzarini et al. [7].The validation of the analytical method was complete: the stability indicating capacity of the method has been proved with forced degradation of cabazitaxel in extreme conditions (acidic, basic and photolytic conditions).The percentage of degradation was near to 10-20 % in accordance with the recommendations of Bardin et al. [12].The extreme stress conditions tested allowed good separation and detection of the degradation products of cabazitaxel peak.About the specificity of the method, one excipient (citric acid) was not analysed by HPLC system due to a supply problem.

Chemical and physical stability of solutions
Cabazitaxel solutions at 0.1 and 0.26 mg/mL diluted in 0.9 % NaCl or D5W in Easyflex ® bags (polyolefin) or Viaflo ® (multilayer high density polyethylene, polyamide, polypropylene) were stable physically and chemically for 28 days at 25 °C.Cabazitaxel solutions at 0.1 and 0.26 mg/mL diluted in 0.9 % NaCl or D5W in Freeflex ® bags (polypropylene multilayers) are physically and chemically stable for 28 days at 2-8 °C.A Cabazitaxel 20 mg/mL vial with Spike was physically and chemically stable for 28 days at 25 °C.The use of a Spike device has been demonstrated and did not lead to a decrease in the concentration of cabazitaxel.The use of this device allows to shorten the preparation time of cabazitaxel and to secure the reconstitution of vials.For all conditions, solutions retained above 95 % of the initial concentration and no visual change was observed.The pH of the solutions in the infusion bag was constant over time over the period analyzed.The additional peaks are in small proportions and minor variations were observed over time.
The concentration measured at D0 for bag No. 3 Easyflex ® 0.1 mg/mL-D5W was 0.115 mg/mL (*).On D14 and D28, the measured concentration was 0.1038 and 0.1029 mg/mL respectively.This higher value at D0 may be due to a homogenization problem.These two values are not considered for the final conclusion on chemical stability.In another condition (bags No. 1 and 2 -Freeflex ® -0.1 mg/mL -0.9 % NaCl), the remaining percentage of the initial concentration of cabazitaxel was 105.27 (No. 1) and 103.47 % (No. 2) at D14 and 99.83 and 96.16 % respectively at D28 (**).The purity of the cabazitaxel peak was checked and was close to 1.An analytical or instrumentation problem may explain this high value at D14.Additional peaks observed during the stability study were already observed during the forced degradation and are minor in comparison of the peak of interest.
Physical stability was assessed by performing a particulate contamination test (Particle counter; PAMAS SVSS) at each time of analysis.Some of these analyses are compliant and others not compliant for the same preparation conditions.For example, at D0, in the same condition of preparation, the results were compliant for Easyflex ® bag and noncompliant for Freeflex ® and Viaflo ® bags.These solutions prepared in accordance with the recommendations of the SmPC of Cabazitaxel Zentiv should be in accordance with European Pharmacopeia.The results obtained were incoherent, probably due to the complexity of the excipients composition in the formulation of the product, which alters the analyses.
These products were chosen in relation due to their composition close to that of cabazitaxel Zentiva.Docetaxel contains notably polysorbate 80 as excipient [15] and ciclosporin contains polyoxyl 35 castor oil [16].Results were consistent with analysis times for polysorbate 80 and cabazitaxel powder diluted with only ethanol.The results were non-compliant from T0 for the docetaxel and ciclosporin solutions prepared in accordance with their respective SmPC.In view of these results, we believe that particle counting for these products with a complex formulation is probably not well adapted.
Cabazitaxel 20 mg/mL vials with Spike were physically and chemically stable for 28 days at 25 °C.

Table  :
Mass balance of cabazitaxel solutions after various stressed degradations (expressed as peak areas).

Table  :
Chemical stability of cabazitaxel at . and . mg/mL in three infusion bags in . % sodium chloride (. % NaCl) or in dextrose  % in water (DW).

Table  :
Chemical stability of cabazitaxel at  mg/mL in vials.