Factor analysis in optimization of formulation of high content uniformity tablets containing low dose active substance

Accepted Manuscript Factor analysis in optimization of formulation of high content uniformity tablets containing low dose active substance

Ivana Lukášová, Jan Muselík, Aleš Franc, Roman Goněc, Filip Mika, David Vetchý PII: DOI: Reference:

S0928-0987(17)30509-2 doi: 10.1016/j.ejps.2017.09.017 PHASCI 4211

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

14 March 2017 1 September 2017 8 September 2017

Please cite this article as: Ivana Lukášová, Jan Muselík, Aleš Franc, Roman Goněc, Filip Mika, David Vetchý , Factor analysis in optimization of formulation of high content uniformity tablets containing low dose active substance, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.09.017

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ACCEPTED MANUSCRIPT FACTOR ANALYSIS IN OPTIMIZATION OF FORMULATION OF HIGH CONTENT UNIFORMITY TABLETS CONTAINING LOW DOSE ACTIVE SUBSTANCE

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IVANA LUKÁŠOVÁ1, JAN MUSELÍK1*, ALEŠ FRANC1, ROMAN GONĚC1, FILIP

Department of Pharmaceutics, Faculty of Pharmacy, University of Veterinary and

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MIKA2, DAVID VETCHÝ1

Pharmaceutical Sciences Brno, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic

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Institute of Scientific Instruments of the CAS, Kralovopolska 147, Brno, Czech Republic

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Corresponding author: e-mail: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Warfarin is intensively discussed drug with narrow therapeutic range. There have been cases of bleeding attributed to varying content or altered quality of the active substance. Factor analysis is useful for finding suitable technological parameters leading to high content

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uniformity of tablets containing low amount of active substance. The composition of tabletting blend and technological procedure were set with respect to factor analysis of

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previously published results. The correctness of set parameters was checked by manufacturing

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and evaluation of tablets containing 1-10 mg of warfarin sodium. The robustness of suggested technology was checked by using „worst case scenario“ and statistical evaluation of European

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Pharmacopoeia (EP) content uniformity limits with respect to Bergum division and process

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capability index (Cpk). To evaluate the quality of active substance and tablets, dissolution method was developed (water; EP apparatus II; 25 rpm), allowing for statistical comparison of dissolution profiles. Obtained results prove the suitability of factor analysis to optimize the

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composition with respect to batches manufactured previously and thus the use of metaanalysis under industrial conditions is feasible.

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Keywords: factor analysis; process optimization; sampling error; content uniformity;

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common blend; worst case; dissolution method.

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ACCEPTED MANUSCRIPT 1. Introduction Warfarin has been used since 1950s to prevent thrombosis and embolism (Tadros & Shakib, 2010). According to EP, the active substance is available either as amorphous or crystalline form, the latter existing as sodium salt clathrate, with 2-propanole trapped in the crystalline

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structure in ratio 2:1 (Gao & Maurin, 2001). Warfarin has low therapeutic index (Benet & Goyan, 1995) and when titrating the dose it is typically increased or decreased by only 5-15%

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of the daily dose (Wittkowsky, 1997). Content uniformity (CU) is an important parameter of

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warfarin tablets (Muselík et al., 2014a), however, its evaluation on purely pharmacopoeial basis cannot be sufficient. Important producers (e.g. DuPont, Taro Pharmaceuticals, and

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Apotex) hence introduced stricter limits: content uniformity within 92.5-107.5% of the

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average and relative standard deviation (RSD) not more than 3% (Sawoniak et al., 2002). In the past, the substitution of original product (Coumadine®) with generic product

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(Panwarfarine®) was identified as a cause of bleeding (Vercaigne & Zhanel, 1998). According

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to literature, this was caused probably by failed content uniformity (Wittkowsky, 1997), however, using amorphous form of the active pharmaceutical ingredient (in Panwarfarine)

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instead of crystalline form (in Coumadine) was also discussed (Jaffer & Bragg, 2003). Although the cause remains unclear (Richton-Hewett et al., 1988) and tablets could not have

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been considered to be therapeutically equivalent (Haines, 2011), quality assurance demands monitoring both content uniformity and the quality of API. The question of generic substitution (Haines, 2011), content uniformity (Arruabarrena et al., 2014), dissolution testing parameters (Rahman et al., 2015a), API quality (Rahman et al., 2015b) and its changes during shelf-life (Fan et al., 2015; Nguyenpho et al., 2015) have been researched intensively. Quality assurance of warfarin tablets thus demands optimal manufacturing parameters leading to required content uniformity as well as corresponding suitable dissolution method.

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ACCEPTED MANUSCRIPT In commercially produced tablets, the content of API is low (1-10 mg), which has impact on any problems with content uniformity (Carstensen & Dali, 1996). Granulation technology is used widely in the production of warfarin tablets (Nguyenpho et al., 2015) as it improves CU. However, it is time-consuming and increased temperature during drying can have negative impact on stability (Parikh, 2010). In wet granulation, there is a risk of migration of warfarine

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sodium during drying, which deteriorates CU (Chaudry & King, 1972). Because this

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substance is soluble in aqueous wetting agent and there may be residual moisture in the final

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product (Rahman et al., 2015a), a change in quality of active substance and formulation can have impact on dissolution profile (Amann, 1973). Therefore, direct compression remains

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suitable and used technology, requiring the preferred use of crystalline form (Mackin et al., 2002). With the absence of increased temperature or the API dissolving in the wetting agent

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or residual moisture, there is no risk of transition to the amorphous phase (Gao & Maurin, 2001). On the other hand, there is a risk of segregation during blending, blend transfer or

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because of tabletting press vibrations, which has negative impact on CU (Am Ende et al.,

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2007). Thus, an optimization of critical process parameters is essential. The goal was to assess the suitability of factor analysis in the optimization of critical process

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parameters, allowing for the production of tablets of all strengths (1-10 mg) with required CU.

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The choice of qualitative parameters of raw material and process parameters was based on metaanalysis using factor analysis of previously published results of content uniformity of blends and tablets with similar formulation. These papers dealt with the impact of particle size of API and excipients, blending time, addition of lubricant at various phases of blending, and different API concentration on CU of warfarin blends and tablets (Muselík et al., 2014a; Muselík & Franc, 2012; Franc et al., 2013; Muselík et al., 2014b; Franc & Muselík, 2013; Elbl et al., 2016). This analysis based on results of previously manufactured batches can be used in the industry both in the development of dosage form and in optimization of existing 4

ACCEPTED MANUSCRIPT process. The producer is allowed some manoeuvring space in process parameters while having to adhere to declared composition and technology (Commission regulation, 2003; U.S. Food and Drug Administration, 1995). To assess the results of factor analysis, optimized process was used to manufacture tabletting

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blends and compressing them in tablets by direct compression. The technology of common blend for all strengths was used because it is convenient for easier blend CU validation

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(Muselík et al., 2014b), bioequivalence study that can be usually performed on highest

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strength only (U.S. Food and Drug Administration, 2003), and stability tests that are usually run on outlying strengths (U.S. Food and Drug Administration, 2013). Tablets contained

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warfarin sodium clathrate and particular strengths differed in the weight of manufactured

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tablets. To evaluate the robustness of content uniformity, three batches were produced, with two of them representing worst case scenarios, simulating average warfarin content of 96% and 104% of declared content. This is in line with the philosophy of requirements on

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qualification and validation of dosage forms (European Commission, 2015). Content

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uniformity of blends was expected to meet FDA requirements (90-110%; RSD ≤ 5%) and capability index based on EP content uniformity limits (EP 2.9.6: 85-115%). Content

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uniformity of tablets was expected to meet internal limits of commercial producers (Du-Pont,

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Taro Pharmaceuticals, and Apotex) (Sawoniak et al., 2002) and was evaluated by Bergum division based on EP 2.9.40 and capability index based on EP 2.9.6 (85-115%) (Muselík et al., 2014a; Berman et al., 1997). The quality assurance of manufactured tablets also demanded the development of easy and fast dissolution method, allowing for statistical comparison of dissolution profiles using similarity and difference factors.

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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS 2.1. Materials Raw materials used to manufacture tablets, their particle size, and true density are listed in Table 1. All other materials were of analytical grade and were used without further

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purification.

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2.2. Manufacturing of tablets from common blend

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All constituents from Table 1, without magnesium stearate, were sieved through a 250 µm sieve and mixed for 10 minutes. Then, magnesium stearate was added and another 5 minutes

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of mixing followed. Turbula homogenizer (T2C, Switzerland) was used at speed 40 rpm.

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Mass of one batch was 500.0 g. Batch A contained 96% of theoretical API content, batch C contained 104% of theoretical content, and batch B corresponded with theoretical content.

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Tablets were compressed with exocentric tablet press (Korsch EK0, Germany). Theoretical

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weight of tablets was 54.2-542.0 mg, with respect to theoretical content of 1–10 mg of warfarin sodium.

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2.3. Physical testing of blends and tablets True density and particle size of API and excipients are listed in Table 1. True density was

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measured according to EP 2.9.23, using Pycnomatic-ATC helium pycnometer (Porotec, Germany). Particle size was measured by laser diffraction, using HELOS KR (Symaptec, Germany); samples were analysed on dry basis, with compressed air (2 bar) used for deagglomeration. Density and tapped density of tabletting blend were measured according to EP 2.9.15 and EP 2.9.34, using SVM 102 (Erweka, Germany). Flowability was measured according to EP 2.9.16 with Flowability Tester (Medipo-ZT, Czech Republic). Tablet height, diameter, radial hardness, and weight (n=20 tablets) were measured automatically on

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ACCEPTED MANUSCRIPT Pharmatest WHT-1 (Pharmatest, Germany). Weight uniformity was evaluated according to EP 2.9.5 and resistance to crushing of tablets was performed according to EP 2.9.8. Friability was tested according to EP 2.9.7, using TAR 10 (Erweka, Germany). Disintegration was tested according to EP 2.9.1, using ZT 4 (Erweka, Germany).

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2.4. Model blend and its analysis by SEM and EDX Raw materials listed in Table 1 were used to manufacture model blend. 13.7 g of warfarin

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sodium clathrate and 479.5 g of Di-cafos 92-14 were sieved through 250 µm sieve and mixed

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for 10 minutes. Then, 6.8 g of magnesium stearate was added and another 5 minutes of

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mixing followed. Turbula homogenizer (T2C, Switzerland) was used at speed 40 rpm. The ratio of used materials and the manufacturing process were the same as when manufacturing

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tabletting blends. The mixture was placed on a 0.080 µm sieve and vibrated at amplitude 60 (equipment setting) for 1 minute, using AS 200 basic (Retsch & Co., Germany). This

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mechanical stress was applied so as to separate those particles that are not held together by

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physical interaction. Then, a sample for further analysis, weighing approximately 500 mg, was taken by laboratory spoon from the surface from the mixture.

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Extremely high-resolution scanning electron microscope (SEM) with subnanometer resolution down to 1 keV (Magellan 400, FEI, Czech Republic) was used for the analysis. The SEM is

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equipped with through-the-lens and side-attached electron detectors. One of them is fourchannel retractable back-scattered electron detector (BSE) which shows morphology and the material contrast of the powder. The BSE images were taken in standard vacuum conditions at 10-4 Pa at 15 keV. The chemical composition of the model mixture was also proved by energy dispersive spectrometry of X-rays (EDX) at 15 keV (EDAX, Apolo X). The point and area EDX analysis from different places of the mixture was performed. The mapping which shows

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ACCEPTED MANUSCRIPT the phase distribution of elements in an area with several grains of the blend was also performed. 2.5. Sampling and warfarin content measurement Tabletting blend was placed in cylindrical vessel with diameter of 25 cm and levelled to a

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height of about 2 cm by slight horizontal movement. The area was then divided evenly into 10 geometrically equal parts. Approximately 500 mg sample was taken with a small laboratory

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spoon from each one of these parts. For content uniformity of tablets, 10 tablets (batch B) or

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20 tablets (batches A and C) were taken at regular intervals in the course of compressing

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process. Analysis of content by HPLC, including sample treatment, is described in detail in previous paper (Muselík et al., 2014a).

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2.6. Dissolution testing

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For dissolution testing, SOTAX (AT7 Donau Lab, Switzerland) was used. With

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respect to the USP monograph Warfarin Sodium Tablets, water was as dissolution medium. Furthermore, EP phosphate buffer (pH 6.8) was used. The volume of dissolution medium was 900 mL, its temperature was maintained at 37.0±0.5°C. The test was run using EP 2.9.3

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Apparatus II (paddles, 50 rpm) as well as modified method at 25 rpm. 6 tablets containing 1,

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5, or 10 mg of warfarin sodium (batch B) were used for the test. Dissolved amount of active substance was measured by HPLC at timepoints as follows: 5, 10, 20, 30, 60, and 120 minutes.

2.7. Statistical evaluation of results For evaluation of procedure variables (API and filler quantity and particle size, lubrication time) and their interaction with response variables (RSD, Cpk for EP 2.9.6 limits), factor analysis was used, including rotation of factors (Varimax normalized). Prior to

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ACCEPTED MANUSCRIPT modelling, response variables were automatically adjusted by autoscaling. Design evaluation was performed with Statistica 12 (StatSoft, USA). From contents found in samples of tabletting blends, respectively tablets, for each batch, average content, RSD, and Cpk with respect to EP 2.9.6 limits (85-115%) were calculated.

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Moreover, the blends were expected to meet FDA requirements (90-110%; RSD not more than 5%). Tablets were evaluated according to EP 2.9.40, Bergum division and content

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uniformity limits defined by key manufacturers (content within 92.5-107.5% of the average

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and RSD not more than 3%). Dissolution profiles were compared using difference factor (f1)

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and similarity factor (f2). 3. RESULTS AND DISCUSSION

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3.1. Optimization of technological parameters by factor analysis (FA) Based on data in literature (Table 2), the impact of formulation and process parameters

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(particle size distribution of API and filler, content of API, duration of mixing after the

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addition of lubricant) on content uniformity of blend and tablets were evaluated. Data pack of 32 samples with variable RSD and Cpk of tabletting blends, respectively tablets was

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evaluated using factor analysis so as to find optimal technological parameters. Supposedly, content uniformity is improved with decreasing RSD values and increasing Cpk values.

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In factor load graph (Fig. 1), the first factor is explained by blend content uniformity and the second factor is explained by content uniformity of tablets. It is clear that blend content uniformity does not correlate with content uniformity of final dosage forms. This can be attributed to sampling error in blend sampling. Blend segregation during sampling is possible (Muselík et al., 2014a; Berman et al., 1997). In factor load graph (Fig. 1), there are correlations, firstly, between particle size of used filler and blend content uniformity represented by Cpk, and secondly, between API particle size and blend content uniformity

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ACCEPTED MANUSCRIPT represented by RSD. These results show that better blend content uniformity was achieved when combining filler with larger particle size (D50=152 μm) and API with smaller particle size (D50=10 μm). This finding corresponds to date in literature; model blend of warfarin sodium with filler (Di-cafos) and magnesium stearate is known to have higher electrostatic charge when using filler with smaller particle size then with larger particle size (Muselík et

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al., 2014a). Electrostatic charge can increase blend sampling error: if it is reduced there is

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positive impact on final uniformity of tabletting blend. Particle size of both API and filler

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does not correlate significantly with content uniformity of tablets (Fig. 1). This means particle size distribution does influence blend sampling error and has no impact on content uniformity

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of tablets.

Factor analysis was also used to evaluate mixing duration after the addition of lubricant

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(“blending” variable). Blending variable correlates partially with blend Cpk; however, there is also some correlation with content uniformity RSD of tablets. Possible explanation suggests

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that homogeneity of a mixture does not increase proportionally to the duration of mixing and that after some time the mixture becomes overblended. Optimal duration of mixing with lubricant is obvious in factor score graph (Fig. 2). The best results were achieved with mixing

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for 5 minutes. On the other hand, blends mixed for shorter time (2 min) or longer time (10 or

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15 min) had deteriorated content uniformity both of blend and tablets. The factor score graph (Fig. 2) suggests that API concentration of 0.5% correlates significantly with content uniformity RSD in tablets, with negative impact on content uniformity. API concentration in range of 2-5.5% does not have significant impact on content uniformity of blend or tablets. The best results were achieved with API concentration of 2, 2.7, or 5%, with duration of mixing after the addition of lubricant 5 minutes, and when using API of small particle size (D50=10 μm) and filler of large particle size (D50=152 μm). Based on factor analysis, raw materials of this particle size distribution and duration of mixing for 5 minutes were chosen 10

ACCEPTED MANUSCRIPT for further experiments. API concentration of 2% was chosen because at this concentration both manufactured batches had suitable content uniformity. This concentration also allows for the manufacture of tablets of all therapeutic strengths (1-10 mg) by common blend technology because in whole range of strengths, the tablets have reasonable dimensions and weight.

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3.2. Manufacturing of tablets from common blend Three batches were manufactured from common blend to check the output of factor

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analysis and to evaluate the robustness of proposed technology. Two of these batches

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represented worst case scenario. Theoretical composition of tablets is shown in Table 1. Batch

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A contained 96% and batch C 104% of theoretical warfarin content, approaching pharmacopoeial limits by „Warfarin Sodium Tablets USP“ that require content within

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95-105% (The United States Pharmacopeia-National Formulary, 2016). The content of warfarin in batch B corresponded with theoretical value: 100%. Physical properties of blends

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- density, tapped density, and flow properties were very similar (see Table 3). Physical

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properties of tablets - radial hardness, friability, disintegration, and weight uniformity - were also similar and met pharmacopoeial limits. Height and diameter of tablets were similar, as

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well. See Table 4. This proves that the manufacturing process of tablets is robust.

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3.3. Validation of content uniformity of blends and tablets The results of evaluation of content uniformity of blends manufactured with respect to the output of factor analysis are shown in Table 5. All blends met EP 2.9.6 and FDA requirements. Cpk≥1 for EP 2.9.6 limits ensures that 99.7% of subsequently manufactured batches with average content within limit (96-104% of theoretical API content) will meet pharmacopoeial limit on content uniformity. Because the results of factor analysis showed the impact of particle size of API and filler (Di-Cafos) on blend content uniformity, model mixture of warfarin sodium, filler, and 11

ACCEPTED MANUSCRIPT magnesium stearate was analysed by scanning electron microscopy and by energy dispersive spectrometry of X-rays. The goal was to elucidate plausible formation of “interactive powder blend” between API and filler. Fig. 3 shows a larger particle of warfarin sodium (black – sodium phase) and filler particles (dark grey – calcium and phosphorus phases). Other warfarin sodium particles (black) are dispersed among filler particles, but they are not

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present on their surface. Obtain results do not confirm the presence of interactive powder

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mixture between API and filler particles. The influence of API and filler particle size on blend

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content uniformity, respectively on the suppression of sampling error when sampling the blend, depends possibly only on changes in electrostatic charge of the blend (Muselík et al.,

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2014a) with absence of interactive powder mixture.

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Tablets containing 1, 2, 2.5, 3, 4, 5, 6, 7.5, and 10 mg of warfarin sodium were manufactured from the blends, with the amount of API in tablet set by the weight of the tablet (Table 4).

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The results of evaluation of content uniformity of tablets are listed in Table 6. Tablets of all

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strengths produced from common blends (batches A, B, and C) met pharmacopoeial requirements as defined by EP 2.9.6 and EP 2.9.40 (harmonized with USP). Cpk values for

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EP 2.9.6 were not lower than 1.0, thus ensuring that 99.7% of subsequently produced batches with average content within limits will meet pharmacopoeial limit for content uniformity.

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Similarly, meeting of Bergum criteria ensures with 90% assurance, that at least 95.0% of subsequently manufactured batches will meet EP 2.9.40 limits. Only batches B and C containing 1 mg of warfarin sodium failed Bergum division, which corresponds to the fact that these batches also failed internal limits by commercial producers (Du-Pont, Taro Pharmaceuticals, and Apotex) (Sawoniak et al. 2002). 1 mg tablets failed probably because of their low weight as this was the only parameter where there was any difference from other strengths. The weight of sample has impact on the total error of measurement.

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ACCEPTED MANUSCRIPT Certainly, technological parameters chosen with respect to the results of factor analysis lead to tablets that meet pharmacopoeial requirements on content uniformity (EP 2.9.6, EP 2.9.40) on statistically significant level. This is valid also for batches containing deliberately altered amount of API (worst case scenario). Only tablets containing 1 mg of API did not meet the

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strictest requirement – Bergum division, which was probably caused by low tablet weight. 3.4. Development of dissolution method for quality assurance

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For quality assurance of produced tablets, there has to exist a dissolution method that

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is suitable for statistical comparison using f1 and f2 factors. USP, respectively FDA method

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using water as medium is neither discriminatory (Nguyenpho et al., 2015; Ali & Krämer, 1999) nor biorelevant (O´Reilly et al., 1966). Medium with pH 1.2, which is usually used in

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immediate release tablets, is unsuitable because of poor solubility of API in acidic environment (Stella et al., 1984). Similarly, at pH 4.5 the amount of dissolved API is too low

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(tested on 5 mg tablets) and the standard limit for immediate release dosage forms is reached

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only in some cases (Nguyenpho et al., 2015; Quereshi, 2004). In 10 mg tablets, further deceleration of dissolution is expected, as at this pH, the majority of API will be present in

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unionized form (Nguyenpho et al., 2015). Dissolution at pH 6.8 differed not significantly from the USP method (Nguyenpho et al., 2015), although it is offered as alternative method

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(McCormick et al., 1997). There are biorelevant methods: medium with pH 1.2 can be used, followed by switching to pH 7.4 (Wagner et al., 1971) or biphasic method using 1-octanol as second phase (Franc et al., 2016). However, these methods are demanding on equipment and time and not suitable for routine control. Only those dissolution methods that use neutral or slightly alkaline media are able to dissolve higher amounts of warfarin sodium, i.e. strength 5 mg and higher. Nevertheless, API is dissolved so quickly that any statistical comparison of dissolution profiles is out of question.

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ACCEPTED MANUSCRIPT All above mentioned methods use paddles with rotation of at least 50 rpm, which is recommended speed for immediate release dosage forms (U.S. Food and Drug Administration, 1997), although some recent papers suggest that in immediate release dosage forms slower rotation speed can be used of the API is well soluble (Quereshi, 2004).

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Marginal and central strengths (1 mg, 5 mg, and 10 mg) of tablets manufactured by direct compression from common blend were used to find suitable dissolution conditions. Water and

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buffer with pH 6.8 were used as dissolution media. When using USP method, at least 85% of

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API was found to dissolve within 15 minutes. This was valid in all strengths and is in line with previous findings, excluding any statistical evaluation. Similarly, when using buffer with

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pH 6.8 and keeping to the other USP conditions, at least 85% of API was dissolved within

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20 minutes, which is also too fast. With respect to literature, rotation speed was decreased to 25 rpm (Quereshi, 2004).

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At pH 6.8, the decrease of rotation speed to 25 rpm caused all tested strengths to liberate less

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than 75% of API within 120 minutes. Dissolution profiles of particular strengths were similar (Table 7). When using USP method and reducing the speed to 25 rpm, more than 80% of API

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was dissolved within 60 minutes and almost all API was dissolved within 120 minutes, with particular strengths yielding different dissolution profiles (Fig. 4). Because phosphate buffer

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contains sodium ions, lower dissolution rate in the buffer can be caused by common ion effect on the solubility of warfarin sodium (Serajuddin et al., 1987). Altering USP method by reducing rotation speed (water; apparatus II; 25 rpm) resulted in dissolution method that meets requirements on statistical evaluation of dissolution profiles, particularly the calculation of difference and similarity factors. Such comparison of dissolution profiles is essential for the evaluation of stability and shelf-life, for the evaluation of quality after optimization of technological parameters, or for the evaluation of batches with

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ACCEPTED MANUSCRIPT deviations from prescribed procedure during their production (U.S. Food and Drug Administration, 2014). 4. CONCLUSION Thanks to factor analysis, optimal values of critical parameters in the production of

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warfarin sodium tablets were identified. Optimized technology was used to manufacture tablets by direct compression from common blend, ensuring blend content uniformity and

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resulting in the production of tablets of whole therapeutic range (1-10 mg) with content

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uniformity meeting EP requirements. Worst case scenario was used to evaluate the robustness

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of the process. In two out of three batches of common blend, the content of active substance was deliberately altered to 96%, respectively to 104%. Bergum division and process

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capability index were used in statistical evaluation of EP content uniformity limits. Modified pharmacopoeial dissolution method, based on decreasing the rotation from 50 rpm to 25 rpm,

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was found to be suitable for statistical comparison of dissolution profiles of warfarin sodium

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tablets. Optimization of technological parameters with help of factor analysis seems to be a suitable statistical tool that can be put in practice also by commercial manufacturers to

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optimize production following the outputs of produced and released batches.

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Acknowledgments

The work is supported by the MEYS CR (LO1212), its infrastructure by MEYS CR and EC (CZ.1.05/2.1.00/01.0017) and by CAS (RVO:68081731). Conflict of interest: none

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ACCEPTED MANUSCRIPT Table 1. Composition of blends. Physical properties of their components. Particle size [µm]a

Density

Component

D10

D50

Content D90

[kg m ] [%]b Warfarin sodium clathrate 1312.8 2.5 10.3 72.4 2 Di-cafos 92-14 2937.9 2.4 152.3 309.8 70 Avicel PH 101 1572.4 14.7 46.3 110.7 25 Ac-Di-Sol 1611.5 12.2 33.1 86.7 2 Magnesium stearate 1085.9 2.6 10.2 23.1 1 a Dx = x% of measured particles smaller than this size [µm]; warfarin sodium clathrate (Pliva, Croatia);

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-3

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Di-cafos – calcium hydrogen phosphate (Budenheim, Germany); magnesium stearate (Peter Greven, Germany);

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Avicel PH101 – microcrystalline cellulose (FMC BioPolymer, Belgium), Ac-Di-Sol – sodium crosscarmellose (FMC BioPolymer, Belgium); bComposition of blends was the same in all batches; in batches A and C, the

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content of API was increased, respectively decreased by 4% of theoretical content.

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ACCEPTED MANUSCRIPT Table 2. Content uniformity of blends and tablets containing particular amount of warfarin sodium, manufactured under particular formulation and process parameters (Muselík et al., 2014a; Muselík & Franc, 2012; Franc et al., 2013; Muselík et al., 2014b; Franc & Muselík, 2013; Elbl et al., 2016). xi blend RSD blend

Batcha

[%]c

xi tbl

RSD tbl

Cpk

blendd

[%]b

[%]c

tbld

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[%]b

Cpk

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2%_W10_D61_2min 106.8 4.59 0.56 105.2 3.70 0.84 2%_W10_D61_2min 111.7 9.77 0.10 102.8 2.16 1.84 2%_W10_D61_5min 103.3 3.41 1.07 100.1 3.20 1.55 2%_W10_D61_5min 102.3 7.18 0.56 103.2 1.62 2.36 2%_W83_D61_5min 103.5 10.57 0.35 100.0 3.03 1.65 2%_W83_D61_5min 104.7 10.59 0.30 100.0 2.92 1.70 2%_W10_D152_5min 100.5 1.37 3.50 102.3 2.52 1.64 2%_W10_D152_5min 99.3 1.76 2.72 99.9 1.67 2.96 2%_W83_D152_2min 99.1 13.61 0.35 99.5 2.49 2.08 2%_W83_D152_2min 103.9 2.38 1.55 105.5 2.91 1.04 2%_W83_D152_5min 100.7 2.62 1.80 100.1 2.54 1.96 2%_W83_D152_5min 105.0 16.38 0.19 103.3 2.42 1.61 0.5%_W10_D152_5min 96.7 6.06 0.66 98.4 2.99 1.52 0.5%_W10_D152_5min 95.3 3.69 0.98 97.4 8.09 0.52 0.5%_W10_D152_10min 99.0 8.33 0.57 96.7 5.76 0.70 0.5%_W10_D152_10min 94.8 6.53 0.52 100.8 8.30 0.63 0.5%_W10_D152_15min 101.1 10.65 0.43 94.4 5.65 0.59 0.5%_W10_D152_15min 100.2 3.43 1.44 95.2 8.24 0.43 5.5%_W10_D152_5min 98.5 1.95 2.34 96.2 1.36 2.85 5.5%_W10_D152_5min 97.2 4.80 0.87 100.8 2.27 2.07 5.5%_W10_D152_10min 100.4 1.48 3.29 98.1 3.42 1.30 5.5%_W10_D152_10min 96.1 4.66 0.83 101.3 2.34 1.93 5.5%_W10_D152_15min 105.5 3.42 0.88 99.2 3.46 1.38 5.5%_W10_D152_15min 109.4 2.66 0.64 95.2 3.53 1.01 2.7%_W10_D152_2min 97.3 3.60 1.17 100.3 2.35 2.08 2.7%_W10_D152_2min 98.1 3.29 1.35 103.0 6.18 0.65 2.7%_W10_D152_5min 95.0 1.64 2.15 96.5 2.75 1.44 2.7%_W10_D152_5min 97.4 2.00 2.17 99.9 2.83 1.76 2.7%_W10_D152_10min 100.1 1.97 2.51 99.0 5.07 0.93 2.7%_W10_D152_10min 101.0 2.54 1.83 100.3 3.14 1.55 2.7%_W10_D152_15min 98.5 2.81 1.63 99.4 5.20 0.93 2.7%_W10_D152_15min 100.0 3.44 1.45 96.9 3.94 1.04 a Batch label: x%_Wy_Dz_mixing time with magnesium stearate in minutes; x – warfarin sodium content in %; y – D50 of warfarin; z – D50 of Di-cafos; D50 – 50% of measured particles are smaller than this size [µm]; b

average content of 10 samples from a particular batch; crelative standard deviation of 10 samples from a

particular batch; dprocess capability indexes for EP 2.9.6 limits.

17

ACCEPTED MANUSCRIPT

Table 3. Evaluation of blends. α

Fwa

[°]

[g/s]

1.18

28.0

2.51 ± 0.14

12.67

1.15

29.0

2.46 ± 0.18

18.06

1.22

30.0

2.57 ± 0.11

DB

DT

CI

[g/cm3]

[g/cm3]

[%]

A

0.62

0.73

15.06

B

0.62

0.71

C

0.59

0.72

HR

PT

Batch

RI

DB, bulk density; DT, tapped density; CI, compressibility index; HR, Hausner ratio; α, angle of repose; F w, flow

AC

CE

PT E

D

MA

NU

SC

rate; a Values expressed as mean ± standard deviation (n=3).

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ACCEPTED MANUSCRIPT Table 4. Evaluation of tablets. Rh

Fr

Ds

He

We

Di

[N]

[%]

[s]

[mm]

[mg]

[mm]

Batch

CE

PT E

D

MA

NU

SC

RI

PT

1_A 36.7 ± 2.6 0.67 32 2.83 ± 0.05 54.2 ± 0.7 5.05 ± 0.01 1_B 35.1 ± 5.0 0.64 35 2.91 ± 0.04 53.9 ± 1.1 5.01 ± 0.07 1_C 32.2 ± 4.8 0.61 30 2.79 ± 0.06 53.8 ± 0.8 5.01 ± 0.01 2_A 45.8 ± 1.9 0.55 38 3.49 ± 0.03 108.3 ± 1.6 5.50 ± 0.00 2_B 50.5 ± 12.5 0.47 44 3.58 ± 0.06 108.4 ± 0.9 5.51 ± 0.00 2_C 75.2 ± 9.9 0.36 54 3.37 ± 0.04 108.2 ± 0.7 5.50 ± 0.01 2.5_A 80.9 ± 5.4 0.36 64 4.03 ± 0.03 133.5 ± 2.2 5.50 ± 0.00 2.5_B 70.2 ± 8.3 0.48 56 4.21 ± 0.06 135.2 ± 1.1 5.51 ± 0.00 2.5_C 64.1 ± 9.3 0.55 48 4.16 ± 0.04 135.4 ± 0.9 5.50 ± 0.00 3_A 61.8 ± 18.5 0.51 55 2.86 ± 0.08 160.1 ± 5.3 6.99 ± 0.01 3_B 52.2 ± 6.3 0.57 60 2.79 ± 0.07 162.5 ± 1.3 7.01 ± 0.00 3_C 33.1 ± 9.2 0.69 35 2.89 ± 0.09 163.1 ± 1.5 7.03 ± 0.11 4_A 93.2 ± 4.1 0.23 75 3.38 ± 0.03 213.7 ± 4.8 7.00 ± 0.01 4_B 74.2 ± 8.7 0.37 62 3.47 ± 0.05 215.9 ± 1.7 7.01 ± 0.00 4_C 64.1 ± 9.7 0.42 50 3.56 ± 0.04 216.3 ± 1.1 7.00 ± 0.00 5_A 44.3 ± 16.6 0.59 38 2.21 ± 0.02 268.6 ± 5.6 10.02 ± 0.01 5_B 50.3 ± 8.1 0.60 45 2.29 ± 0.07 271.5 ± 1.9 10.04 ± 0.00 5_C 45.7 ± 11.4 0.52 41 2.31 ± 0.03 270.1 ± 2.1 10.02 ± 0.01 6_A 56.2 ± 2.7 0.55 45 2.75 ± 0.04 320.4 ± 7.9 10.02 ± 0.00 6_B 60.1 ± 17.1 0.53 50 2.71 ± 0.09 325.5 ± 1.5 10.04 ± 0.00 6_C 57.2 ± 14.2 0.58 48 2.67 ± 0.11 324.3 ± 2.8 10.02 ± 0.00 7.5_A 64.1 ± 8.5 0.41 58 3.43 ± 0.02 400.3 ± 5.1 10.03 ± 0.01 7.5_B 71.2 ± 15.6 0.45 65 3.33 ± 0.08 407.2 ± 2.2 10.04 ± 0.00 7.5_C 76.5 ± 31.1 0.36 60 3.26 ± 0.08 403.6 ± 1.8 10.02 ± 0.00 10_A 43.7 ± 21.1 0.64 40 2.76 ± 0.04 541.9 ± 4.8 13.08 ± 0.01 10_B 55.5 ± 10.0 0.57 60 2.85 ± 0.06 543.2 ± 0.9 13.09 ± 0.01 10_C 60.7 ± 8.7 0.52 55 2.87 ± 0.07 536.8 ± 3.1 13.10 ± 0.02 a Batch label: API content_batch. Rh – Radial hardness; Fr – friability; Ds – disintegration time; He – height;

AC

We – weight; Di – diameter. Radial hardness, height, and weight expressed as mean ± standard deviation (n=20).

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ACCEPTED MANUSCRIPT

Table 5. Content uniformity of blends. Batch

xi a

Cpkb

[%]

PT

[%]

RSD

AC

CE

PT E

D

MA

NU

SC

RI

A 96.2 2.76 1.40 B 98.6 3.61 1.27 C 106.8 1.82 1.40 a Average content of 20 (Batch A and C) or 10 (Batch B) samples; bprocess capability index for EP 2.9.6 limits.

20

ACCEPTED MANUSCRIPT Table 6. Content uniformity of tablets.

Batcha

xib

Cpk

Bergum

DuPont

(2.9.6)

divisionc

specificationc

RSD

[%]

AC

(–) failed.

CE

PT E

D

MA

NU

SC

RI

PT

1_A 98.4 2.88 1.57 + + 1_B 100.9 4.68 1.00 1_C 104.3 3.28 1.04 2_A 98.2 2.37 1.89 + + 2_B 101.3 1.67 2.71 + + 2_C 102.5 2.31 1.76 + + 2.5_A 96.0 2.14 1.89 + + 2.5_B 97.4 1.59 2.66 + + 2.5_C 103.0 1.97 1.96 + + 3_A 96.5 1.97 2.02 + + 3_B 99.1 2.80 1.70 + + 3_C 103.5 1.23 3.02 + + 4_A 96.9 1.21 3.39 + + 4_B 98.7 2.30 2.01 + + 4_C 102.1 1.37 3.08 + + 5_A 95.7 1.41 2.62 + + 5_B 101.4 2.63 1.69 + + 5_C 103.7 2.50 1.45 + + 6_A 95.5 1.26 2.91 + + 6_B 97.5 2.63 1.62 + + 6_C 102.2 1.92 2.18 + + 7.5_A 95.0 1.34 2.63 + + 7.5_B 98.8 1.97 2.35 + + 7.5_C 103.4 2.69 1.40 + + 10_A 98.2 1.54 2.91 + + 10_B 98.5 1.27 3.60 + + 10_C 105.4 1.93 1.58 + + a Batch label: API content_batch; baverage content of 20 (Batch A and C) or 10 tablets (Batch B); c(+) passed;

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ACCEPTED MANUSCRIPT

Table 7. Difference factor and similarity factor. Modified USP dissolution method (apparatus II; 25 rpm).

Sample

Reference

f2

f1

1_B 5_B 10_B 1_B 5_B 10_B

5_B 10_B 1_B 5_B 10_B 1_B

26.5 42.8 19.3 58.2 90.9 61.9

23.6 17.3 36.7 13.5 2.3 11.5

PT

Dissolution

SC

NU MA D PT E CE AC

water water water pH 6.8 pH 6.8 pH 6.8

RI

medium

22

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 1. Factor loads.

23

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Fig. 2. Factor score.

24

NU

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ACCEPTED MANUSCRIPT

Fig. 3. Model mixture containing warfarin sodium: BSE high-resolution SEM image (A) and

MA

EDX phase map image (B):( ) – sodium phase (corresponding to warfarin sodium); ( ) – calcium and phosphorus phase (corresponding to Di-cafos filler);

AC

CE

PT E

D

(corresponding to carbon table).

– carbon phase

25

D

MA

NU

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ACCEPTED MANUSCRIPT

PT E

Fig. 4. Dissolution profiles of warfarin sodium tablets: A – water, 50 rpm; B – phosphate buffer pH 6.8, 50 rpm; C – water, 25 rpm; D – phosphate buffer pH 6.8, 25 rpm; ( ) 1_B;

AC

CE

( ) 5_B; ( )10_B; (mean ± standard deviation; n=6).

26

ACCEPTED MANUSCRIPT REFERENCES Ali, S.L., Krämer, J., 1999. Pharmaceutical Quality of Warfarin Sodium tablets, A multinational postmarket comparative study. Die Pharmaceutische Industrie 61, 363-368. Am Ende, M.T., et al., 2007. Improving the Content Uniformity of a Low-Dose Tablet

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Graphical abstract

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