Validation of Dissolution Testing with Biorelevant Media: an OrBiTo

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Validation of Dissolution Testing with Biorelevant Media: an OrBiTo study James Mann, Jennifer B. Dressman, Karin Rosenblatt, Lee Ashworth, Uwe muenster, Kerstin Frank, Paul Hutchins, James Williams, Lukas Klumpp, Kristina Wielockx, Philippe Berben, Patrick Augustijns, Rene Holm, Michael Hofmann, Sanjaykumar Patel, Stefania Beato, Krista Ojala, Irena Tomaszewska, Jean-Luc Bruel, and James Butler Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00198 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Molecular Pharmaceutics

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Validation of Dissolution Testing with Biorelevant Media: an OrBiTo study

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James Mann1, Jennifer Dressman2, Karin Rosenblatt3, Lee Ashworth1, Uwe Muenster4, Kerstin Frank5 , Paul Hutchins6, James Williams7, Lukas Klumpp2, Kristina Wielockx8, Philippe Berben9, Patrick Augustijns9, Rene Holm10, Michael Hofmann11, Sanjaykumar Patel12, Stefania Beato13, Krista Ojala14, Irena Tomaszewska15, Jean-Luc Bruel16, James Butler7.

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Pharmaceutical Technology & Development, AstraZeneca (Macclesfield, United Kingdom)

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Institute of Pharmaceutical Technology, Goethe University (Frankfurt am Main, Germany)

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Drug Product Development, AbbVie Deutschland GmbH & Co. KG (Ludwigshafen, Germany)

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Chemical and Pharmaceutical Development, Bayer AG (Wuppertal, Germany)

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Pharmaceutical Development , Boehringer Ingelheim Pharma GmbH (Biberach an der Riß, Germany)

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Drug Product Science and Technology, Bristol-Myers Squibb (Moreton, UK)

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GlaxoSmithKline R&D, Pharmaceutical Development & Supply (Harlow, UK)

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Pharmaceutical Sciences, Janssen Pharmaceutica, Johnson & Johnson (Beerse, Belgium)

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Drug Delivery and Disposition, University of Leuven (Leuven, Belgium)

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Biologics and Pharmaceutical Science, H. Lundbeck A/S (Valby, Denmark). Current Address: Janssen Pharmaceutica, Johnson & Johnson (Beerse, Belgium)

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Department of Biopharmaceutics and Pharmaceutical Technology, University of Mainz (Mainz, Germany)

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Analytical Sciences, MRL, Merck & Co., Inc. (Kenilworth NJ, USA)

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Technical Research and Development, Novartis Pharma AG (Basel, Switzerland)

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Pharmaceutical Sciences, Analytical Development, Orion Pharma (Turku, Finland)

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Analytical Research and Development, Pfizer Ltd. (Sandwich, UK)

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Research & Development, Sanofi-Aventis (Vitry-sur-Seine, France)

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Address correspondence to Dr. James Butler

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GlaxoSmithKline R&D, Pharmaceutical Development & Supply, Harlow, United Kingdom

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Tel +44 1279 63 3216

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E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract

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Background: Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods.

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Purpose: The aim of this study was to determine the inter-laboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2).

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Methods: Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine and a “transfer” (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets.

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Results: The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available media preparation powder were used. Likewise, results were almost identical between the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an inter-laboratory basis. Inter-laboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests.

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Conclusion: Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

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Key Words: Biorelevant; dissolution testing; ring study; OrBiTo; in vivo predictive testing; FaSSIF; transfer model


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1. Introduction

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Compositions of biorelevant media were first introduced in 1998 by Dressman et al.1. These media aim to simulate the composition of the gastrointestinal contents more closely than the media that are commonly used in dissolution testing for quality control purposes. Over the years, a wide range of applications have been established, ranging from solubility studies of new drug candidates2 to determining the ability of probiotics to remain viable in the gastrointestinal tract3.

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One of the main applications of biorelevant media is the dissolution testing of drugs and formulations during pharmaceutical research and development. Many papers have demonstrated the advantages over compendial media in predicting drug and formulation behavior in vivo4,5. For research and development purposes, it is important to be able to compare results for different drugs in order to make rational decisions about the composition and manufacture of formulations of new drug candidates. Additionally, in today’s global working environment, it is critical that results obtained in different laboratories across the world are comparable and can thus be integrated into the development process with confidence.

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With the advent of quality by design (QbD)6,7 the lines between dissolution testing performed for product development and for quality control have begun to blur. If key quality attributes for product performance are optimally measured using dissolution testing in a biorelevant environment, as is often the case for formulations of poorly soluble drugs, then it would be reasonable to utilize biorelevant media for aspects of quality control testing of the product as well as in the development phase. Such an approach has also been embraced by the BioRAM concept8. If biorelevant media are to be used on a regular basis within the quality control paradigm, it is essential that the results obtained are reproducible across laboratories and with different batches of the media.

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The current study was initiated as part of the Oral Biopharmaceutics Tools (OrBiTo)9 project to assess the reliability and robustness of dissolution testing using biorelevant media. The OrBiTo project, a public private collaboration which is funded by the European Union, has the objective of developing methods to assess API and formulation performance which can be used in conjunction with in silico tools to predict in vivo performance after oral administration. The composition of this consortium enabled a large-scale, inter-laboratory assessment of the biorelevant media in both academia and the pharmaceutical industry, with a total of 16 partners from Europe and the USA participating. The principal objective of the study was to determine whether dissolution studies with the biorelevant media can provide results that are sufficiently reproducible to enable their use for cross-laboratory comparison of formulation behavior, including by the regulatory authorities. FaSSGF and FaSSIF were selected as the main media to be investigated following the results of a survey done among consortium members with regard to which version of FaSSIF (fasted state simulating intestinal fluid) is currently most often utilized in pharmaceutical research and development10. Two commercially available drug products, Accolate20 mg (zafirlukast) tablets and Brufen Forte 600 mg (ibuprofen) tablets, were chosen as the test products. Participating consortium partners were Abbvie AG, AstraZeneca, Bayer AG, Boehringer Ingelheim Pharma GmbH, BristolMyers Squibb Pty. Ltd., GlaxoSmithKline, Goethe University, Janssen Pharmaceutica, KU Leuven, Lundbeck A/S, University of Mainz, Merck & Co., Inc., Novartis Pharma AG, Orion Pharma, Pfizer Ltd. and Sanofi-Aventis.



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2. Materials and Methods

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Selection of the test products:

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Sixteen OrBiTo partners participated in this cross-laboratory comparison of dissolution testing with Biorelevant media. It was agreed that the partners would conduct dissolution testing according to protocols that are part of the OrBiTo tool set and are recommended for use on a regular basis in the pharmaceutical industry during drug development. Further, it was agreed that in this initial validation phase, two weakly acidic drugs with different expected dissolution behavior would be studied. According to these considerations, test products for the ring studies (inter-laboratory comparisons) were selected according to the following criteria: More than 10% of the active pharmaceutical (API) must be dissolved within 30 minutes in FaSSIF. It was the intention that one of the APIs should be dissolve rapidly and completely in FaSSIF, while the other API should dissolve slowly and incompletely in this medium, in order to explore a range of behavior. Moreover, it was expected, based on the weak acid criterion, that dissolution of both APIs would be incomplete in the gastric medium and that neither would dissolve completely in the first 10 minutes of exposure to FaSSIF.

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Due to the logistics involved in setting up such a large study, a further criterion was that the drug product should be inexpensive and easy to obtain. Additionally, to ensure that there would be no additional complexity in the protocol beyond the physical set-up, it was required that the APIs chosen should be stable to light, oxygen and relative humidity over the course of the study and that the formulation should not be an “enabling” formulation (e.g. that the drug should not be nanosized or presented as a solid dispersion in the dosage form, that complexing agents like cyclodextrins should not be present in the formulation etc.).

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Two drug products, with contrasting doses, fulfilled the above criteria: ibuprofen (600 mg) and zafirlukast (20 mg). Both drugs are poorly soluble at gastric pH but are more soluble at intestinal pH, are stable and have facile analytical methods. The solubility of Ibuprofen and zafirlukast in FaSSIF is reported to be 1.39 mg/mL and 0.002 mg/mL11 respectively, so ibuprofen is close to its solubility limit when 600 mg is dissolved in 500 mL, and zafirlukast, which is amorphous in this drug product, supersaturates relative to its crystalline solubility during dissolution in FaSSIF. Both products were readily available and could be easily distributed to all partners. Further, both products are simple formulations without “enabling” excipients. It was expected on the basis of solubility data that ibuprofen would dissolve more rapidly in FaSSIF than zafirlukast. The brands used for the study were: ibuprofen tablets (Brufen Forte 600 mg, BGP Products, Maidenhead, UK, Batch 57869PC) and zafirlukast tablets (Accolate 20 mg, AstraZeneca, Luton, UK, Batch LK162). Two types of studies were conducted. The first consisted of single media dissolution studies in fasted state simulating gastric fluid (FaSSGF) and fasted state simulating intestinal fluid (FaSSIF). The second consisted of a two-stage test, representing a simplified form of the transfer model, in which the dosage form was initially subjected to dissolution in FaSSGF12, then FaSSIF was added as a concentrate to bring the final composition of the media to that of FaSSIF. A similar method, but using low volumes of media, had been published previously by Mathias et al.13. The dissolution behavior was characterized both before (i.e. in FaSSGF) and after the addition of the concentrate (i.e. in FaSSIF). The protocols for the two types of studies were as follows.


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A: Single medium biorelevant dissolution in FaSSGF and FaSSIF Media preparation: Media were prepared according to the instructions provided by the manufacturer, (see https://biorelevant.com/site_media/upload/documents/How_to_make_FaSSIF_FeSSIF_and_FaSSGF. pdf) 14 for version 1 of FaSSIF, FeSSIF & FaSSGF Powder (old name: SIF Powder - various batch numbers, refer to Figure 8), all from Biorelevant.com, London, England. The final solution was allowed to equilibrate for 0.5 h (as opposed to the 2 hours recommended by Biorelevant.com) prior to use in order to allow the micelle particle size to stabilize, noting that the resulting solution might appear slightly opalescent. The media were demonstrated to be stable for at least 48 h at room temperature by Kloefer et al.15, consistent with current information on the manufacturer’s website.

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Dissolution method details:

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The details of the protocol for the dissolution method are given below:

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Apparatus: The USP 2 Apparatus (rotating paddle) was implemented at a paddle speed of 75 rpm, at 37°C using a volume of 500 mL for FaSSIF and 250 mL for FaSSGF.

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Sampling time points: 5, 10, 20 and 30 minutes and (if the product proved to be slow dissolving) every subsequent 30 minutes until a plateau was reached. Partners were to add extra early time point(s) if the plateau concentration was reached in 10 minutes or less.

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Sample: Protocol instructions were to mimic the dose of interest in humans. In practice, one unit was added to each dissolution vessel in all experiments. Instructions were to run at least 3 replicates for each type of experiment; most partners ran six replicates.

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Sampling and analysis: Although sampling methodology was allowed to vary among the partners, the typical method used was manual sampling via syringe (5 mL, from which an approximate 0.5 mL analysis sample is taken), followed by HPLC or UPLC analysis using a method appropriate for the drug. It was recommended that the first 2-3 mL passing through the filter and any excess sample in the syringe be returned to the vessel. Further, it was recommended that samples be appropriately diluted with mobile phase and analyzed on the same day, unless stability could be confirmed over a longer period.

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Other sampling and analysis methods (e.g. semi-automated sampling, UV detection without HPLC) were used at the discretion of the partner, provided they were shown to be compatible with the sample properties. Issues to be considered included the possibility of sample precipitation prior to analysis during semi-automated sampling and the potential impact of variable background UV absorbance if using UV detection without HPLC.

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Filtration: Use of a suitable filter was expected, with a check for adsorption of the drug onto the filter. Ideally, % adsorbed was not to exceed 2%. If a fraction larger than 2% was adsorbed, then this was to be clearly stated in the report.

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It was foreseen that the filter would typically be a 0.45 µm 25 mm syringe filter made of PTFE, GHP or glass fiber. It was suggested that a pre-filter (for example, a cannula filter) be used to avoid filter clogging if there was excessive un-dissolved excipient/drug present.



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Each partner purchased appropriate materials (buffers, filters, materials for the analytical work etc.) independently. Partners also purchased the commercially available, ready-made version of FaSSIF powder independently.

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It was additionally noted to partners that when using a 250 mL volume in a standard 1 L dissolution vessel, use of bathless dissolution vessels should be avoided due to heating zone issues. It was also noted that the paddles are very close to the medium surface at this volume and careful sampling is required, and that some variability in results was expected as a consequence.

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B: Two-stage biorelevant dissolution (to represent gastric to intestinal transfer)

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The two-stage media dissolution method is a simplification of the so-called “transfer” method introduced by Kostewicz et al.12 that is used in several EFPIA companies to screen for precipitation of the drug upon entry into the small intestine. Although no precipitation is expected for the two drugs studied due to their weak acid nature, the test was applied to assess the reproducibility of the method across independent laboratories. The key difference between the two-stage media dissolution method and the transfer model is that instead of using a peristaltic pump to transfer the gastric dissolution medium into the intestinal dissolution medium at a controlled rate, a concentrate of the intestinal medium is quickly (i.e. as a “bolus”) added to the gastric medium.

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Partners were to prepare the media from FaSSIF, FeSSIF & FaSSGF Powder (old name: SIF powder)14, adjusting pH and micelle concentrations as appropriate to give medium 1 (FaSSGF) and 2 (FaSSIF concentrate). More detailed instructions on preparation of the FaSSIF concentrate are given below.

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Two-stage dissolution method details:

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The dissolution method was set up as follows:

Media preparation:

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Apparatus: The USP 2 Apparatus (rotating paddle) was implemented at a paddle speed of 75 rpm, at 37°C using an initial volume of 250 mL for FaSSGF with subsequent addition of 250 mL of the FaSSIF concentrate.

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Initial phase: The dosage form was exposed to 250 mL FaSSGF pH 1.6 (medium 1) for the first 30 min, during which samples were removed after 5, 10, 20 and 30 minutes.

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Media addition: Following the 30-minute sampling time point, 250 mL of FaSSIF concentrate (which has a pH of 7.5 and is designated medium 2) was added to achieve a final volume of 500 mL FaSSIF pH 6.5. Subsequent to the addition of the concentrate, samples were also taken at the following time points: 35, 40, 50, 60 minutes and (if slow dissolving) every subsequent 30 minutes until a plateau was reached. Extra early time point(s) were to be added if the plateau was reached in 10 minutes or less after addition of the FaSSIF concentrate.


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Sampling methodology, filtration, sampling and analysis were to be conducted according to the same methodology as for the single media dissolution experiments. Similarly, any product-specific modifications to the methodology were to be clearly stated in the report.

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It was additionally noted that when using a 250 mL volume, use of bathless dissolution vessels should be avoided due to heating zone issues. It was also noted that the paddles are very close to the medium surface at this volume and careful sampling is required, and that some variability in results was expected as a consequence.

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Preparation of the FaSSIF concentrate

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The following description for the preparation of the FaSSIF concentrate was sent to all participants.

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Step 1. To prepare 5 L of the buffer, dissolve ~4.20 g NaOH (pellets) and 39.54 g sodium dihydrogen phosphate monohydrate (sodium phosphate monobasic monohydrate) in approximately 4.9 L of purified water. Alternatively, 17.19 g sodium dihydrogen phosphate anhydrous can be substituted. Add and dissolve 61.86 g NaCl. Adjust the pH to 7.5 using NaOH (aq), and make up to volume 5 L.

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Analytical Methods

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All partners used an in-house HPLC or UPLC method to analyze samples of ibuprofen and zafirlukast in the studies. Exact details of the methods used are available from the individual partners upon request.

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Typical methods for ibuprofen and zafirlukast are shown in Tables 1 and 2.

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Table 1. Typical conditions for the HPLC analysis of ibuprofen.

Step 2. To prepare 1 L of the FaSSIF concentrate using FaSSIF, FeSSIF & FaSSGF Powder (1L): In a 1 L volumetric flask, dissolve 4.48 g of FaSSIF, FeSSIF & FaSSGF Powder in approximately 500 mL of the phosphate buffer from Step 1. Stir until the FaSSIF, FeSSIF & FaSSGF Powder has dispersed. When a clear solution is obtained, make up to volume (1 L) with the phosphate buffer described in Step 1. The solution will remain clear. This is a 2-X concentrate of FaSSIF; the surfactant concentration and buffer strength are doubled compared to “1X” FaSSIF. The pH of the buffer used to prepare the concentrate is adjusted to pH 7.5 so that after addition of 250 mL medium 2 to 250 ml FaSSGF the final pH will be pH 6.5.

Parameter/Method HPLC Column Mobile Phase pH Mobile Phase Flow rate Wavelength Injection Volume Analysis Time

Hitachi LaChrom Elite® Kinetex® 2.6 µm XB-C18 100 Å Acetonitrile/Water/Triethylamine/Phosphoric acid (600/400/2.65/1.425) 3 1.5 mL/min 254 nm 10 µl 3 min

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With this method the retention time of ibuprofen was ~1.7 minutes and the correlation coefficient (r2) of the calibration curve was > 0.999.

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Table 2. Typical conditions for the HPLC analysis of zafirlukast

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Parameter/Method HPLC Column Mobile Phase pH Mobile Phase Flowrate Wavelength Injection Volume Analysis Time

Hitachi LaChrom Elite® Spherisorb ODS-2 C18 5µm, 50x4.6mm, Waters Acetonitrile/Water/Phosphoric acid (600/400/3) 3 1.5 mL/min 224 nm 20 µl 3.25 min

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The retention time of zafirlukast under these conditions was ~2.4 minutes and the correlation coefficient (r2) of the calibration curve was > 0.999.

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Data Analysis and Statistical Methods

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Data are shown as the mean profiles with the error bars representing the highest and lowest % release at each time point for each partner, noting that for many experiments, the error bars were smaller than the symbols. For each product/dissolution experiment combination, the overall mean profile and the 95% confidence interval (CI) around the overall mean was calculated.

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Additionally, the overall mean profile and a 10% absolute deviation around the mean profile are presented and compared with the individual data for each product/dissolution experiment combination. This approach is in line with the FDA requirement for an f2 > 50 for the comparison of mean profiles. The f2 statistic is a measure of the overall similarity of the profile, requiring a deviation of less than 10% between the mean profiles for acceptance of their similarity16. By comparing the data at each time point, the probability of determining a greater than 10% difference is actually higher than when comparing the mean profile, thus this test is a stricter measure of similarity than the f2 statistic.

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Additionally, the f2 statistic was calculated for the comparison of the results obtained with the commercially available product and FaSSIF manufactured in house by one of the partners, as well as to determine the effect of preheating the media or other deviations from the protocol.

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Variations from the protocols

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As the protocols provided some flexibility in the experimental design, there were several instances in which variations on the protocol occurred or that a partner chose to explore some of the parameters that were not explicitly specified in the protocol. Since the partners independently sourced the commercial biorelevant media powders, several different batches were used for the studies. This enabled an assessment of how reproducible the powders are on a batch-to-batch basis. Further, one of the partners prepared biorelevant media from the individual ingredients as well as using the commercial powders. In this case the in-house


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preparation method followed the method described by Kloefer et al.15, a process that took up to 5 hours in total, and used pre-dispersion of bile salt and lecithin in a small volume of water at 37°C until clear, followed by cooling to room temperature, addition of the buffer, dilution to volume and pH adjustment.

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Although the protocol intended that the media be pre-heated, one partner ran the studies without pre-heating the media and was asked to repeat the studies using pre-heated media. The data obtained were compared to determine to what extent variations in this step of the protocol influence the results. Finally, one partner ran the studies in a semi-automated set-up and was then asked to repeat the work using manual sampling. This partner also switched from not pre-heating the media to preheating the media. Again, the results were compared to determine whether the degree of sampling automation had any effect on the results.

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3. Results

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A. Single media studies with ibuprofen Since ibuprofen is a weak acid and is poorly soluble at low pH, with a solubility of 38µg/ml at pH 1 and 37°C17, the dissolution results in FaSSGF were consistently very low (less than 5% of label strength) and are therefore not shown separately. To gain an impression of the behavior of ibuprofen in FaSSGF, the reader is directed instead to Figure 3, in which the first 30 minutes of the two-stage test depict the release in FaSSGF. In FaSSIF, where the pH of 6.5 is considerably higher than the pKa of ibuprofen, which is 4.217, dissolution is almost complete (in most cases over 90%) within 30 minutes. The mean profiles from each individual partner are shown in Figure 1. In Figure 1 Panel A these are accompanied by the 95% confidence interval around the overall mean. The same individual mean profiles, attended by a deviation of +/- 10% (on an absolute basis) from the overall mean profile, are shown for comparison in Figure 1 Panel B.



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Ave. % ibuprofen released

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95% CI

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B

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Figure 1. Individual mean results for dissolution of ibuprofen from Brufen Forte 600 mg tablets in FaSSIF: Panel A with the 95% confidence interval for the data shown in grey and Panel B with an absolute deviation of 10% from the overall mean concentration profile shown in grey.

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For fourteen of the sixteen partners, results were very tight, with deviations of only a few percent over first sixty minutes of release, at which time release was 90% or more. One partner recorded consistently lower results, but no technical explanation could be identified for the low results in that case. On the other hand, one partner recorded substantially higher results at the 5-minute sample time. This was attributed to the use of semi-automated sampling in conjunction with a 45 µm cannula filter. The coarse filter enabled some undissolved ibuprofen to pass through the filter and contribute to the measured concentration.

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Figure 1 Panel B shows the mean profile obtained by pooling data from all partners, along with a 10% deviation on an absolute basis from the mean % release at each time point, along with the individual mean dissolution profile from each partner. The graph illustrates that all but one profile from a total of 16 profiles obtained were able to meet the acceptance criteria set by the FDA (f2>50, which corresponds to an overall absolute deviation of 10% or less between the mean profiles).

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B. Single media studies with zafirlukast

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Zafirlukast, like ibuprofen, is a weak acid and is also poorly soluble at low pH. As for ibuprofen, results are not shown for release in FaSSGF, since the percent released did not exceed 5%. However, the behavior in FaSSGF is reflected in the first medium of the two-stage studies (see section D).

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In FaSSIF, where the pH of 6.5 is higher than the pKa of zafirlukast, which according to the AstraZeneca safety sheet for zafirlukast is 5.5 18, dissolution is somewhat slower than for the ibuprofen product, with an overall mean release of 90% achieved after 90 minutes or longer. The profiles from all partners are shown in Figure 2, together with the 95% confidence interval in Panel A of Figure 2. The same profiles attended by an absolute deviation of +/- 10% from the overall mean profile are shown for comparison in Figure 2 Panel B.

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Ave. % zafirlukast released

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95% CI


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Figure 2. Individual mean results for dissolution of zafirlukast from Accolate 20 mg tablets in FaSSIF: Panel A with the 95% confidence interval for the data shown in grey and Panel B with an absolute deviation of 10% from the overall mean concentration profile shown in grey

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Fifteen partners participated in the zafirlukast studies in FaSSIF, recording highly consistent results (Figure 2, Panel A). The form of the curves was also highly consistent among the partners.

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Panel B in Figure 2b shows the same profiles, along with a 10% deviation on an absolute basis from the mean % release at each time point obtained by pooling data from all partners. The graph illustrates that all 16 profiles were able to meet the acceptance criteria set by the US-FDA (f2>50, which corresponds to an absolute deviation of 10% or less between the mean dissolution profiles).

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C. Two-stage studies with ibuprofen Results for the two-stage studies with Brufen Forte 600 mg tablets are shown in Figure 3.



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Figure 3. Individual mean results for dissolution of ibuprofen from Brufen Forte 600 mg tablets in the two-stage dissolution test: in Panel A these are shown with the 95% confidence interval for the data shown in grey and in Panel B together with an absolute deviation of 10% from the overall mean concentration shown in grey.

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As expected from the low solubility of ibuprofen in acidic conditions, release was slow and after 30 minutes had reached less than 5% in the FaSSGF medium. Upon addition of the FaSSIF concentrate, the release rate became much faster and the %released reached a plateau value within 60 minutes of exposure to FaSSIF in most cases (some partners truncated the experiment after 30 minutes of exposure to FaSSIF, so it is not possible to say for sure if the release had achieved a plateau in these cases). One partner achieved a % release of 120%. The high value was attributed to the filtration method used by that partner. As the same filter was used throughout both stages of the experiment, the filter efficiency may have been compromised by excessive pressure on the filter, and/or solid particles remaining on the filter from the FaSSGF samples may have re-dissolved upon exposure to the FaSSIF medium. One partner had a substantially lower profile than the others. This partner had experienced some initial analytical problems and it is not clear whether they were fully resolved at the time the profile was generated.

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Considering the added complexity of the two-stage dissolution experiment, and given that many partners had not run this type of experiment before, the deviation from the overall mean profile was low for the majority of the profiles. One EFPIA partner with prior experience in two-stage testing confirmed that variability is usually somewhat higher in this kind of test than in single medium studies, although in their experience the results continued to be sufficiently reliable. This partner opined that the higher variability could be due to some variation in the rate of increase in micellar size, which occurs when FaSSIF concentrate is added to FaSSGF.

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D. Two-stage studies with zafirlukast Results for the two-stage studies with Accolate 20 mg tablets are shown in Figure 4.

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Figure 4. Individual mean results for dissolution of zafirlukast from Accolate 20 mg tablets in the two-stage dissolution test: a) With the 95% confidence interval for the data shown in grey (Panel A) and b) with an absolute deviation of 10% from the overall mean concentration shown in grey (Panel B).

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Results for the two-stage test with zafirlukast were generally more reproducible than for the twostage test for ibuprofen. This might be attributable to the “learning curve” effect – almost all of the partners ran the ibuprofen two-stage tests before the zafirlukast tests and many had not had previous experience with the two-stage test protocol. However, an alternative explanation would be that the dissolution of the ibuprofen tablets is intrinsically more variable than that of the zafirlukast tablets, consistent with the single medium results. One partner observed higher than expected concentrations of zafirlukast in FaSSGF. Review of the test conditions revealed that the high concentrations were attributable to use of a 10 µm cannula filter, which allowed some fine zafirlukast particles to slip through and then be dissolved in the filtrate when acetonitrile was added to the sample (to ensure that any dissolved zafirlukast was held in solution prior to analysis). On the other hand, another partner reported substantially lower results in the acceptor medium (FaSSIF). This partner also reported lower values for the ibuprofen two-stage test than the other partners, but no obvious technical reason for the lower results could be identified.

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Similarly, as shown in Panel B of Figure 4, all but two of the results for the zafirlukast two-stage test fell within the FDA corridor of +/- 10% from the overall mean % dissolved, indicating excellent reproducibility.

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In-house manufacturing vs. commercially available biorelevant media

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Figure 5 shows the results of dissolution testing of Accolate 20 mg tablets in FaSSIFv1 prepared from the commercially available powder versus in-house preparation from the individual ingredients.



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Figure 5. Release of zafirlukast from Accolate 20 mg tablets in FaSSIF prepared in-house (triangles) and from the commercially available powder (circles). Error bars represent the maximum and minimum % released from the individual tablets, in some cases these lie within the symbol.

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The results indicate that there was less than 5% difference in results between the commercial powder form of FaSSIF and preparing the medium in-house from the individual ingredients (f2= 72). Further, for each experiment the variation in the results from tablet to tablet was extremely low. Although only data for Accolate tablets are shown above, profiles for Brufen tablets in the two media preparations also very closely matched each other.

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Effect of technical adjustments to the procedure

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The protocol specified that the FaSSIF concentrate medium was to be pre-heated to 37°C before starting the dissolution test, but left the choice of sampling method (manual or semi-automated) open. One partner explored the effect of pre-heating the dissolution medium prior to starting the test, for the dissolution of both ibuprofen and zafirlukast. A second partner also ran studies both with and without pre-heating, however, in this case, a semi-automated sampling method was also introduced upon repeating the test with pre-heated medium. The purpose was to explore to what extent transferring the method to different types of dissolution equipment would influence the results. The results from these studies are shown in Figure 6.


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Figure 6. Results of trials in which various factors deviated from the study protocol. Panels A and C represent the effect of preheating the medium for ibuprofen (Panel A) and for zafirlukast (Panel C). Triangles represent preheated medium; circles represent the experiment in which the medium was not preheated; both were conducted manually. Panels B and D represent the effect of switching from manual to semi-automated equipment and from no preheating to preheating of the dissolution medium for ibuprofen (Panel B) and for zafirlukast (Panel D). Triangles represent semi-automated experiments with preheated media, circles represent manual experiments in which the medium was not preheated.

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Panels A and C represent the effect of preheating/not preheating the medium. For ibuprofen the f2 factor was 93 (Panel A) and for zafirlukast the f2 factor was 78 (Panel C), indicating only small differences between the dissolution profiles (

Validation of Dissolution Testing with Biorelevant Media: an OrBiTo

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