A method for CCI testing

Abstract

The present invention provides a method of testing container closure integrity which comprises (i) subjecting a container (10, 30) comprising a transparent wall portion (11, 31) and a drug chamber (17, 37) holding a liquid drug to a test environment (2) comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, (ii) combining at least a portion of the liquid drug with a pH indicator, and (iii) determining a presence or absence of dissolved test gas in the drug chamber (17, 37) from a protonation state of the pH indicator.

Description

[0001] A METHOD FOR CCI TESTING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to container closure integrity testing of liquid holding containers in the pharmaceutical industry.

[0004] BACKGROUND OF THE INVENTION

[0005] Container closure integrity (CCI) testing is an assay that evaluates the adequacy of container closure systems to maintain a sterile barrier against potential contaminants. The industry standard requirement regarding CCI, based on ambitions put forward by the U.S. Food and Drug Administration, is for pharmaceutical companies to be able to detect <20 pm diameter holes in their product containers. Such product containers comprise vials, syringes, and cartridges for use in drug delivery devices.

[0006] CCI testing can be performed in many different ways. The various test options are divided into two major categories, probabilistic and deterministic. Probabilistic methods include Microbial Challenge by Immersion, Bubble Tests, Tracer Gas Tests, and Tracer Liquid Tests, such as Dye Ingress, while deterministic methods include High Voltage Leak Detection (HVLD), Laserbased Gas Headspace Analysis (HSA), Mass Extraction, Pressure Decay, and Vacuum Decay.

[0007] The Dye Ingress test is the most common liquid tracer assay. In this test, the container is submerged in, for example, a blue dye bath and subjected to pressure cycles, after which it is inspected visually or by spectrophotometry to observe any traces of dye inside. Hence, the test is destructive. A further weakness is, as seen when quality assessing the method by testing positive test samples with pre-made holes, that in approximately 5-10% of the cases the containers do not exhibit ingress of the blue dye, and the analysis can therefore not be completed. In general, many of the probabilistic methods suffer from being both slow and laborious. Consequently, there is a move towards using deterministic methods, which are less subject to error, and which provide quantitative results.

[0008] HVLD and HSA are sufficiently fast to function on-line or at-line in a production setup, but these tests have other significant challenges. For instance, on-line HVLD cannot currently measure leaks in container caps and plungers, while HSA requires a headspace inside the container and thus depends on the particular product package. Tuneable Diode Laser Absorption Spectroscopy (TDLAS), one of the most common choices for HSA in CCI testing, is a highly sensitive technique used for the detection and quantification of tracer gases in the headspace of a product container. It can detect very small changes in concentration of gas diffusing into, or out of, the container.

[0009] For CCI applications, TDLAS often measures energy transitions in the near-infrared (NIR) and mid-infrared (MIR) ranges of the electromagnetic spectrum, which is where excitation of discrete combinations of vibrational and rotational modes occur. TDLAS has several advantages for gas analysis, such as high sensitivity, specificity, and speed which allows it to detect gases at very low concentrations, down to the parts-per-bi Ilion range.

[0010] US 2023 / 0088151 (Gasporox AB) discloses a method and system for CCI testing using TDLAS which include obtaining a pressure inside a container by producing, filling and / or sealing the container at hot or cold conditions and transmitting a light signal through a headspace of the container. In case of a leak, gas from the surroundings may enter the container and cause a change to the pressure and / or the concentration of gas inside. Such change, and thereby the integrity of the container, is determined based on the transmitted light signal being detected.

[0011] WO 2016 / 156622 (Gasporox AB) discloses a method for CCI testing which involves subjecting a container to variations in the outside atmosphere to impose a change to the concentration, composition, or pressure of the gas or gases inside the container, in case of a leak, and subjecting the container to optical spectroscopic measurement with the purpose of detecting any variation in the optical signal arising as a consequence of the leak as opposed to the signal where no leak is present.

[0012] In both of these examples, TDLAS is applied to detect changes in the headspace composition as a consequence of a CCI breach. TDLAS employs an infrared laser that can be tuned to a given wavelength where it will excite a gas molecule of interest. Therefore, it can be used to target a very specific gas molecule, avoiding interference from dissimilar molecules.

[0013] WO 2015 / 121064 (Robert Bosch GmbH) discloses a method for CCI testing, where a closed container having a first inner sealed volume filled with a first gaseous substance at a first pressure is arranged in a surrounding second volume, and where a second pressure, greater than the first pressure, is generated in the second volume by introduction of a second gaseous substance. The second gaseous substance has a Raman spectral band that lies in a different wavelength range than the Raman spectral bands of the first gaseous substance. After a predetermined time has passed, the concentration of molecules of the second gaseous substance in the first volume is measured by means of Raman spectroscopy.

[0014] The above prior art documents teach CCI test methods for containers having a headspace. Some containers in the pharmaceutical industry, e.g. many of those that hold an injectable, parenteral drug, are liquid-filled, or substantially liquid-filled, and thus have no, or negligible, headspace. Such containers are thus not eligible for the disclosed methods. Molecules in the liquid phase exhibit different physical and chemical properties compared to those in the gas phase. This is primarily due to differences in the intermolecular forces affecting the molecules. In the gas phase, molecules have weaker intermolecular forces and a high degree of rotational freedom. In contrast, molecules in the liquid phase are affected by stronger intermolecular forces, resulting in a loss of rotational freedom.

[0015] The strong intermolecular interactions from Van Der Waal forces, hydrogen-bonding, and similar effects in the liquid phase leads to significant broadening, and shifts, of spectral lines in TDLAS measurements, for example. These phenomena significantly reduce the spectral resolution, in turn also reducing the sensitivity and specificity of the technique. Additionally, NIR / MIR absorption in aqueous solutions are several orders of magnitude higher than in the gas phase, thus requiring the preparation of optically thin liquid samples for TDLAS to work. Optically thin films are not feasible inside a product container, rendering TDLAS unsuitable for measurements in liquid drugs.

[0016] The prior art methods for CCI testing are also not suitable for analysing containers when they form part of a drug delivery device. So-called ‘in-device testing’ and ‘drug-device combination testing’ are further CCI testing requirements for manufacturers of drug delivery devices, the former to show that CCI is not compromised by the container being inserted into, or otherwise coupled with, a drug delivery unit, and the latter to demonstrate CCI when the container and / or the delivery device contains the final product and not just a test substance. Many pharmaceutical products are peptide-based, and since peptides may potentially clog smaller holes, thereby making it impossible to perform proper measurements using known CCI test methods, manufacturers instead customarily test their containers with water inside.

[0017] Finally, TDLAS as well as Raman spectroscopy involve the use of expensive measuring equipment which must be operated by highly specialised personnel to provide meaningful results, rendering these approaches financially burdensome. SUMMARY OF THE INVENTION

[0018] It is an object of the invention to eliminate or reduce at least one drawback of the prior art, or to provide a useful alternative to prior art solutions.

[0019] In particular, it is an object of the invention to provide a method for CCI testing of a container holding a liquid drug, which is reliable and easy to perform.

[0020] It is another object of the invention to provide a method for CCI testing of a container holding a liquid drug, which is both intuitive and comparatively inexpensive.

[0021] It is a further object of the invention to provide a method for CCI testing of such a container, which does not require a certain size headspace be present therein.

[0022] It is a further object of the invention to provide a method for easy and reliable CCI testing of a variable volume container forming part of a drug delivery device.

[0023] In the disclosure of the present invention, aspects and embodiments will be described which will address one or more of the above objects and / or which will address objects apparent from the following text.

[0024] In a first aspect, the invention provides a method of testing container closure integrity according to claim 1.

[0025] Hence, a method is provided which comprises the steps of (i) subjecting a container comprising a transparent wall portion and a drug chamber holding a liquid drug to a test environment comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, in case of a leak in the container, (ii) combining at least a portion of the liquid drug with a pH indicator, and (iii) determining a presence or absence of dissolved test gas in the drug chamber from a protonation state of the pH indicator.

[0026] Since the test gas is in excess concentration, meaning that the concentration of the test gas in the test environment is higher, at least initially, than the concentration of the same gas type in the container (present as free and / or dissolved gas), then if there is a leak anywhere in the container test gas will enter the drug chamber through the leak, following Fick’s laws of diffusion. The entry may either occur as a direct ingress of test gas molecules through an opening in the container, or as a dissolution of test gas molecules in the liquid drug at the gasliquid interface. Depending on which test gas is employed, in many cases the concentration of that particular gas type in the container will, initially, either be zero or close to zero, so the period of time which the container needs to be subjected to the test environment may be relatively short.

[0027] The test gas has pH changing capabilities, in the sense that when the test gas dissolves in the liquid drug it changes the pH thereof, and a presence of dissolved test gas in the drug chamber is thus detectable by combining at least a portion of the liquid drug with a proper pH indicator. The pH indicator is a chemical compound which changes optical (absorption and / or emission) properties depending on its protonation state. If a proper pH indicator has been selected, then when the pH changes the absorption and / or emission properties of the pH- indicator changes as well, and this change, or lack of change, is used to determine the presence, or absence, of dissolved test gas. The protonation state of the pH indicator may be determined by spectroscopy, e.g. using a UV-vis spectrometer to obtain a UV-vis spectrum of the combined pH indicator and liquid drug, or a fluorescence spectrometer to obtain a fluorescence spectrum of the combined pH indicator and liquid drug, or, alternatively, by visual inspection. The present method can accordingly be performed with relatively inexpensive equipment compared to the prior art. The pH change detection approach is furthermore very intuitive, as a simple change in colour signals a presence of test gas inside the container.

[0028] A proper pH indicator may be one with a pKa similar to the pH of the liquid drug, which results in a shift in the optical properties significant enough for detection. In case of UV-vis spectroscopy, non-exhaustive examples of a proper pH indicator are phenol-red and nitrophenol, whereas in case of fluorescence spectroscopy, non-exhaustive examples of a proper pH indicator are BCECF (2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein) and SNARF-1.

[0029] UV-vis spectroscopy may be carried out by placing a sample in a sample holder with a known depth. The sample holder is transparent and absorbs as little light as possible. A light source generates a beam of photons which penetrates the sample, where UV-vis active species will absorb the light. The light will, after having been exposed to the sample, enter a detector system, where a UV-vis spectrum is obtained. The UV-vis spectrum is then used to determine changes in the absorbance of the sample, which, with a proper indicator, will allow for quantification of changes in pH. In some common UV-vis spectrometers, the light source generates a beam of photons with a wavelength ranging from, approximately, 200 nm to 1100 nm. However, other spectrometers of the same type may be able to obtain spectra of higher or lower wavelengths than the above specified. Thus, the term “UV-vis spectrometer” is not to be understood as limiting in respect of explicit wavelength ranges but rather as a convenient term to describe this type of absorption spectrometer. A UV-vis or fluorescence measurement can be carried out in a variety of sample holders which allows for a determination of whether or not test gas has dissolved in the liquid drug either in situ in the container or drug delivery device, or ex situ in e.g. a cuvette or well plate.

[0030] A fluorescence spectrometer measures emitted light from a sample upon exposure to light of specific wavelengths. This process involves excitation, where molecules absorb photons and transition to higher energy states, followed by emission of fluorescent light at longer wavelengths. Simple spectrometers may use single or few excitation lines, often from lasers or LEDs, for analysis. Advanced versions feature a light source, such as xenon lamps or LEDs, and a monochromator for selecting excitation and emission wavelengths. Other advanced versions can utilize ultrashort laser pulses to measure fluorescence lifetime.

[0031] Fluorescent pH indicators change their fluorescence properties according to their protonation state. These indicators contain chemical groups that transition between protonated and deprotonated states based on the pH of the surrounding environment. In their protonated form, these indicators exhibit one type or level of fluorescence, while in their deprotonated form, they exhibit a different type or level of fluorescence. As the pH changes, the equilibrium between these two states also changes, leading to variations in the fluorescent emission. The alteration in fluorescence intensity and pattern allows for the protonation state of the indicator, and thereby the pH, to be determined.

[0032] There are numerous commercial fluorescent pH indicators available, such as BCECF and SNARF-1 . For instance, when excited at around 490 nm, BCECF will emit a signal peaking at around 515 nm, where an increasing pH value results in an increase in fluorescence. Here, the protonation state of the indicator could be determined based on the intensity of the emission.

[0033] For SNARF-1 , the emission change is more complex. For example, at an excitation of around 490 nm, the spectrum will consist of two peaks at approximately 640 nm and 580 nm, with an isosbestic point at approximately 610 nm. The 640 nm peak will increase with increasing pH, while the 580 nm peak will decrease with increasing pH, and vice versa. Here, the protonation state could be determined based on the intensity of either of the peaks, as well as by a ratiometric approach utilizing both bands.

[0034] Visual inspection may be performed by a human eye, detecting a colour change of the combined pH indicator and liquid drug, and / or by vision technology.

[0035] Hence, the protonation state of the pH indicator may e.g. be determined using image capturing and processing means, such as a camera device.

[0036] Specifically, the protonation state of the pH indicator may be determined using an RGB camera. For example, a sample with phenol red as pH indicator may be incubated in the test environment and subsequently photographed with the RGB camera. The RGB camera filters each pixel through a red filter, a green filter, and a blue filter, and assigns a value based on the intensity of each colour. The pH indicator will change RGB value composition when exposed to the test gas, said change reflecting a quantum of test gas molecules in the drug chamber.

[0037] Alternatively, the protonation state of the pH indicator may be determined using a regular camera with a narrowband light-filter, where the narrowband light-filter is focused on specific wavelengths associated with the colour characteristics of the pH indicator for optimal effect.

[0038] Alternatively, the protonation state of the pH indicator may be determined using a light-source adapted to emit light of a specific wavelength through the combined pH indicator and liquid drug to a camera detector, where the specific wavelength depends on the pH indicator and is selected so as to be absorbed thereby. If the pH indicator changes colour composition, in response to chemical reactions induced in the liquid drug by present test gas molecules, it will change the transmission of the light of the specific wavelength, which will be detected by the camera detector. Any such detection by the camera detector accordingly indicates a CGI breach.

[0039] Combining the at least a portion of the liquid drug with the pH indicator may comprise adding the pH indicator to the liquid drug in the drug chamber, subsequent to step (i). This may, e.g., happen by injection through a sealing component or element of the container. If the container is a syringe, for example, having a slidable sealing plug and a staked needle, then the pH indicator may be injected through the plug. Alternatively, if the container is a cartridge having a slidable sealing plug and a penetrable self-sealing septum, then the pH indicator may be injected through the self-sealing septum. However, the pH indicator may in fact be added to the liquid drug already prior to step (i), e.g. in connection with the filling of the container, thereby eliminating the need for establishing a fluid connection to the drug chamber after sealing of the container.

[0040] Alternatively, combining the at least a portion of the liquid drug with the pH indicator may comprise removing the at least a portion of the liquid drug from the drug chamber and mixing the removed liquid drug with the pH indicator. This will provide for an ex-situ determination of whether test gas exists in dissolved form in the drug chamber. The protonation state of the pH indicator may then be determined from a measurement of the combined pH indicator and liquid drug in e.g. a cuvette or well plate.

[0041] The above-described method for CCI testing is fast, simple, and easy to perform and is furthermore highly sensitive and reliable because the addition of the pH indicator reveals any changes to the pH of the liquid drug as a consequence of test gas entering the drug chamber.

[0042] The period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug is the period of time needed for test gas molecules to induce a detectable chemical change in the liquid drug and may be determined for each drug-container combination and / or drug-device combination by following relevant guidelines from The International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and the United States Pharmacopeia (USP) (e.g. USP 1207.1.4.2) concerning method development and validation of CCI methods to ascertain analytical method parameters such as accuracy (the likelihood of false negatives and / or false positives), specificity (the distinction between positive and negative control samples), and detection limit (the detection limit of the analytical method, i.e. hole size or ingress volume / amount).

[0043] Following these guidelines, the accuracy may initially be determined, using a set of negative control samples and positive control samples, by placing the samples in the test environment under high pressure for many hours, e.g. overnight, thus ensuring that all positive control samples have plenty of test gas dissolved for detection by UV-vis spectroscopy. Following this, an optimal incubation time may be determined, using similar sets of test samples, by iteration, gradually reducing the incubation time until the shortest incubation time which does not entail an introduction of false negatives is reached. The optimal incubation time, which may then correspond to the period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, depends on various parameters such as the specific drug, the type of container, the surrounding pressure in the test environment, the test gas, etc., and may be in the minutes to hours range.

[0044] The above approach may be used to pre-map individual drug-container and drug-device combinations in various test environments, allowing the manufacturer, or other skilled artisan, to easily access information on a recommended period of time in a specific test environment for any of such product combinations, for example by consulting a look-up table. Hence, step (i) essentially comprises, and can thereby alternatively be phrased as, subjecting a container comprising a transparent wall portion and a drug chamber filled with a liquid drug to a test environment comprising a test gas having at least two atoms in its molecular structure over, at least, a predetermined minimum period of time.

[0045] The test environment may be a pressure chamber providing an enclosure for one or more test containers, or test devices. The pressure chamber may be adapted to provide a sufficient pressure for the CCI testing application, which may vary depending on container size and geometry, filling percentage, drug composition, container wall thickness and material, practical requirements for the speed of the CCI test method, etc.

[0046] In some exemplary embodiments of the invention, the pressure chamber is adapted to provide an absolute pressure of at least 2 bar and at most 10 bar. In other exemplary embodiments of the invention, the pressure chamber is adapted to provide up to 15 bar, or even up to just below a limit where the container wall would break due to implosion. In yet other exemplary embodiments of the invention, the pressure chamber is adapted to provide at least 1.1 bar and at most 1.9 bar. In further exemplary embodiments of the invention, the pressure chamber is evacuated using a vacuum pump, and the method is carried out using cycles of vacuum and test gas at an absolute pressure between 200 mbar and 1 .5 bar.

[0047] An overpressure in the test environment increases the rate at which test gas molecules will diffuse into the drug chamber, in the presence of a leak, and the period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug may thus be reduced. This may be particularly important when it is desired to apply the method close to the production, e.g. at-line.

[0048] The test environment may alternatively be an enclosure for one or more test containers or test devices, operating at atmospheric pressure. The liquid drug may e.g. be a peptide-based drug. A compromised container filled with a liquid peptide-based drug presents an insurmountable challenge to many prior art CCI test methods, because a peptide-based drug tends to form protein-clogs when subjected to surrounding air, and a protein-clog may block the open pathway to the drug chamber, rendering ingress of e.g. various tracer liquids impossible. The present invention, however, enables leak detection even in the presence of a clog, because the test gas will be able to penetrate through the clog, or, alternatively or additionally, around a portion of the clog, in case the clog does not completely block the hole.

[0049] The test environment comprises a test gas with pH changing capabilities, i.e. the test environment may contain a single such test gas type or multiple such test gas types.

[0050] In preferred embodiments of the invention, the test environment comprises carbon dioxide (CO2) as the test gas, because CO2has a high solubility in aqueous drug products and is cheap, inert, and non-toxic.

[0051] However, non-limiting examples of alternative suitable test gas types, depending on which liquid drug is in the tested container(s), are ammonia (an alkaline gas that would be suitable for a liquid drug with an acidic pH), a mixture of hydrogen chloride and nitrogen, and a mixture of nitrogen dioxide and argon.

[0052] The container may be a variable volume reservoir, such as e.g. a cartridge type container comprising a self-sealing septum and a displaceable plug or piston, or a syringe comprising a staked needle or means for receiving a needle, said staked needle or means for receiving a needle being sealed by a removable shield, and a displaceable plug or piston. Alternatively, the container may be a constant volume reservoir, such as e.g. a vial or an ampoule, having a penetrable seal.

[0053] Regardless of type, the container is gas-impermeable in a non-compromised state, i.e. neither the transparent wall portion, nor any of the mentioned seals allow any passage of gas to the drug chamber unless flawed by a hole, crack or other unintended opening. In particular embodiments of the invention, the transparent wall portion is non-deformable.

[0054] In some exemplary embodiments of the invention the container is a drug cartridge comprising a cartridge body sealed at one end by a penetrable self-sealing septum and at an opposite end by a slidable plug, and step (ii) is performed by inserting a needle through the penetrable self-sealing septum and injecting the pH indicator into the drug chamber (through the needle). In other exemplary embodiments of the invention the container is a drug cartridge comprising a cartridge body sealed at one end by a penetrable self-sealing septum and at an opposite end by a slidable plug, and step (ii) is performed by inserting a needle through the slidable plug and injecting the pH indicator into the drug chamber.

[0055] In yet other exemplary embodiments of the invention the container is a syringe comprising a syringe barrel carrying an injection needle at one end, sealed by a removable needle shield, and being sealed by a slidable plug at an opposite end, and step (ii) is performed by inserting a needle through the slidable plug and injecting the pH indicator into the drug chamber.

[0056] In particular embodiments of the invention, the drug chamber is filled with the liquid drug.

[0057] In a second aspect, the invention provides a method of testing closure integrity of a container comprising a transparent wall portion and a drug chamber holding a liquid drug, the container forming part of a drug delivery device having a window through which at least a portion of the drug chamber is visible, the method comprising the steps of (i) subjecting the drug delivery device to a test environment comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, in case of a leak in the container, (ii) combining at least a portion of the liquid drug with a pH indicator, and (iii) determining a presence or absence of dissolved test gas in the drug chamber from a protonation state of the pH indicator.

[0058] Also according to this aspect of the invention, which essentially provides a method of in-device testing, as well as a method of drug-device combination testing, step (ii) may be performed before or after step (i), i.e. the pH indicator may be added to the liquid drug either before or after the drug delivery device is subjected to the test environment. If the pH indicator is added before the drug delivery device is subjected to the test environment it may, e.g., happen in connection with the filling of the container, before final assembly of the drug delivery device. If the pH indicator is added after the drug delivery device is subjected to the test environment it may, e.g., happen by injection through a sealing component or element of the container, or by removing the at least a portion of the liquid drug from the drug chamber and mixing it with the pH indicator ex situ, e.g. in a cuvette or well plate.

[0059] It is implicit that any of the features and details mentioned in connection with the abovedescribed first aspect and embodiments of the invention are applicable to this second aspect of the invention. The drug delivery device may comprise a housing accommodating a dose expelling unit. The container may be a variable volume reservoir, e.g. comprising an outlet end portion, an operator end portion, and a cylindrical wall extending therebetween, such as e.g. a drug cartridge comprising a cylindrical wall with a necked down outlet end portion, sealed by a penetrable septum and a slidable sealing plug, or a syringe comprising a syringe barrel, a staked needle sealed by a removable needle shield, or means for receiving a needle sealed by a removable port shield, and a slidable sealing plug. The variable volume reservoir may be embedded in, or attached to, the housing, in a way as to enable the dose expelling unit to reduce the volume of the drug chamber therein.

[0060] If the container is a syringe, for example, then the pH indicator may be injected through the plug. Alternatively, if the container is a cartridge, then the pH indicator may be injected through the self-sealing septum. In conventional pen-type injection devices, e.g. as known within diabetes care, where the container is a cartridge and the cartridge is accommodated in a dedicated cartridge holder, the pH indicator may be added via a needle inserted into an end opening of the cartridge holder and through the self-sealing septum.

[0061] The drug delivery device may further comprise a container holding member, such as e.g. the aforementioned cartridge holder, for accommodating and / or retaining the container. The container holding member may form part of the housing or may be permanently or removably secured to the housing, to respectively prevent or allow an exchange of the container. The window may be arranged in the container holding member.

[0062] In a third aspect, the invention provides a method of testing container closure integrity comprising (I) subjecting a container comprising a transparent wall portion and a drug chamber holding a liquid drug comprising a constituent serving as an intrinsic pH indicator to a test environment comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and (II) determining a presence or absence of dissolved test gas in the drug chamber from a protonation state of the intrinsic pH indicator.

[0063] A liquid drug comprising a constituent serving as an intrinsic pH indicator is a liquid drug which comprises at least one type of molecule suitable to act as a pH indicator. In that case the liquid drug itself is capable of reflecting a change in pH, and no external pH indicator is thus needed to determine the presence or absence of dissolved test gas in the drug chamber. The method according to any of the above aspects of the invention may further comprise determining an amount of test gas which has entered the drug chamber from the protonation state of the pH indicator, or of the intrinsic pH indicator, using a reference.

[0064] The reference may be a reference curve, a reference colour scale, a look-up table, or some other set of reference data which correlates known amounts of dissolved test gas with the protonation state of the pH indicator, or of the intrinsic pH indicator, and which therefore provides for an easy determination of how much test gas has entered the drug chamber once the protonation state of the pH indicator, or of the intrinsic pH indicator, has been determined.

[0065] With a reliable determination of the amount of test gas which has entered the drug chamber, it is possible to provide an estimation of the size of the leak geometry in the container based on known process parameters such as the pressure in the test environment and the incubation time for the specific container.

[0066] Certain containers are practically impossible to fill completely with a liquid substance, using certain filling methods. Hence, in the present context, terms such as “filled with the liquid drug” and “a liquid-filled container” should be understood as, respectively, “at least substantially filled with the liquid drug” and “an at least substantially liquid-filled container”. In other words, a drug chamber “filled with a liquid drug” may, in the present context, either contain the liquid drug and nothing else, or the liquid drug and a miniscule volume of gas. In any case, importantly, a drug chamber “filled with a liquid drug” does not have sufficient headspace to allow for spectroscopic headspace analysis (such analysis would require a free optical path for the laser to pass through the container walls and the headspace without meeting any part of the liquid and without being deflected by angled or curved container surfaces).

[0067] For the avoidance of any doubt, in the present context the term “injection device” designates an apparatus suitable for injecting fluid media into the body of a subject, e.g. with the aid of an attachable needle device, and the term “drug” designates a medium which is used in the treatment, prevention or diagnosis of a condition, i.e. including a medium having a therapeutic or metabolic effect in the body. Further, the terms "distal" and "proximal" denote positions at, or directions along, a drug delivery device, a drug reservoir, or a needle unit, where "distal" refers to the drug outlet end and "proximal" refers to the end opposite the drug outlet end.

[0068] In the present specification, reference to a certain aspect or a certain embodiment (e.g. "an aspect", "a first aspect", "one embodiment", "an exemplary embodiment", or the like) signifies that a particular feature, structure, or characteristic described in connection with the respective aspect or embodiment is included in, or inherent of, at least that one aspect or embodiment of the invention, but not necessarily in / of all aspects or embodiments of the invention. It is emphasized, however, that any combination of the various features, structures and / or characteristics described in relation to the invention is encompassed by the invention unless expressly stated herein or clearly contradicted by context.

[0069] The use of any and all examples, or exemplary language (e.g., such as, etc.), in the text is intended to merely illuminate the invention and does not pose a limitation on the scope of the same, unless otherwise claimed. Further, no language or wording in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In the following the invention will be further described with references to the drawings, wherein

[0072] Fig. 1 is a schematic representation of an incubation arrangement for CCI testing according to an embodiment of the present invention,

[0073] Fig. 2 is a side view of a prefilled syringe containing a liquid drug and illustrates a test gas being absorbed in the liquid drug through a hole in the syringe wall,

[0074] Fig. 3 is a perspective view of a cartridge-based injection device suitable for undergoing CCI testing according to an embodiment of the invention,

[0075] Fig. 4 is a longitudinal section view of the cartridge assembly forming part of the injection device of Fig. 3,

[0076] Fig. 5 shows UV-vis spectra of phenol-red at pH ranging from 6 to 8,

[0077] Fig. 6 presents the pH of a phenol-red solution, determined by UV-vis, as a function of the pH determined by a pH electrode,

[0078] Fig. 7 illustrates the absorption spectrum of nitrophenol as obtained in one exemplary embodiment of the invention, Fig. 8 illustrates the correlation between the pH of nitrophenol as predicted by the UV-vis spectra and the pH as determined by a pH electrode, and

[0079] Fig. 9 illustrates the correlation between injected C02and an acidification of the sample.

[0080] In the figures like structures are mainly identified by like reference numerals.

[0081] DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0082] When / lf relative expressions, such as "upper" and "lower", "left" and "right", "horizontal" and "vertical", "clockwise" and "counter-clockwise", etc., are used in the following, these refer to the appended figures and not necessarily to an actual situation of use. The shown figures are schematic representations for which reason the configuration of the different structures as well as their relative dimensions are intended to serve illustrative purposes only.

[0083] Fig. 1 is a schematic representation of an incubation arrangement 1 for CCI testing according to an embodiment of the present invention. The incubation arrangement 1 comprises a test environment provided by a pressure chamber 2 having an interior 6 with sufficient space to accommodate a plurality of syringes 10. The pressure chamber 2 is fluidly connected to a test gas container 3 with a reduction valve 4 operable to control supply of test gas to the interior 6. A manometer 5 indicates the current pressure of the test gas in the pressure chamber 2.

[0084] One of the plurality of syringes 10 can be seen in Fig. 2. The shown syringe 10 comprises a transparent cylindrical syringe barrel 11 with a necked down outlet portion 13, and a staked injection needle 15 sealed off by a needle shield 14. A slidable sealing plug 19 is arranged in the syringe barrel 11 axially opposite the injection needle 15, and a chamber 17 resultantly defined by the syringe barrel 11 and the plug 19 is filled with a liquid drug (not visible).

[0085] The test gas is supplied to the pressure chamber in a quantity which ensures that it is present in excess concentration, i.e. which ensures that the concentration of test gas in the pressure chamber 2 exceeds that within each individual syringe 10. The test gas will interact with the surface areas of the syringes 10, and if any of the syringes exhibit leakage, e.g. in the form of one or more holes, cracks, or other openings in the syringe barrel 11 and / or the plug 19, test gas molecules will, over time, due to the excess concentration in the surroundings, diffuse into the chamber 17 and dissolve in the liquid drug, as indicated in the enhanced section of Fig. 2, which reveals a crack 16 in the syringe barrel 11. The time it takes for test gas molecules to diffuse through an opening in the syringe 10 and dissolve in the liquid drug depends to a great extent on the pressure in the interior 6. Basically, the higher the pressure, the faster the process. It is noted, however, that an overpressure is not needed to occasion a migration of test gas molecules into the chamber 17 of a leaking syringe 10 if the test gas surrounding the syringe 10 is in excess concentration. The pressure in the test setup is thus a parameter that can be varied to control the period of time which the syringes 10 need to be incubated in the test environment for the migration and dissolution to occur.

[0086] An exemplary period of time, realisable by a test gas pressure of 6 bar, is 10 minutes. Hence, in this case, after 10 minutes the syringes 10 are removed from the pressure chamber 2 and a pH indicator is injected into each chamber 17 through each plug 19. A dissolution of test gas molecules in the liquid drug will cause a change to the pH thereof, which is detectable by UV- vis spectroscopy, potentially also by visual inspection. It can thus quickly and easily be verified if any test gas has entered the drug chamber 17.

[0087] Fig. 3 is a perspective view of a pen-type autoinjector 20 comprising a housing 21 , which accommodates a spring-driven dose expelling unit (not visible), and a cartridge holder 22 which is non-releasably attached to the housing 21 and which retains a drug cartridge 30. Fig. 4 is a longitudinal section view of the cartridge holder 22 with the drug cartridge 30. The drug cartridge 30 comprises a transparent body 31 , e.g. of glass, which is mainly cylindrical and has a necked down region 33 forming an outlet end portion. The body 31 is sealed proximally by a slidable piston 39 and distally by a penetrable self-sealing septum 34. The body 31 , the piston 39 and the self-sealing septum 34 together define a chamber 37 which is filled with a liquid drug (not visible). A portion of the chamber 37 is visible through a window 25 in the cartridge holder 22.

[0088] The autoinjector 20 is operated by a user firstly attaching an injection needle assembly (not shown) to a needle mount 23 formed on a distal end portion of the cartridge holder 22, whereby the self-sealing septum 34 will be penetrated by a rear end of an injection needle (not shown) entering through a distal opening 24 in the cartridge holder 22, and then setting a dose to be injected by turning a dose dial 26. The dose dial 26 is operatively coupled with a scale drum 28, which is visible through a housing window 29. The scale drum 28 is provided with a plurality of dose indicating numbers to indicate a selected dose. Once a desired dose has been set and a front end of the injection needle has been inserted into an injection site the user depresses a dose button 27, thereby causing the release of a strained injection spring in the dose expelling unit. The injection spring is operatively coupled with a piston rod (not visible) such that the released energy resultantly moves the piston rod distally relative to the housing 21 , whereby the piston 39 is pushed distally in the drug cartridge 30 and the set dose of liquid drug is expelled through the injection needle.

[0089] The autoinjector 20 is an example of an injection device which can be CCI tested in accordance with the present invention. By subjecting e.g. cartridge based or syringe based injection devices, either individually or in batches, to a test environment such as the one described above, and adding a pH indicator to the liquid drug, it is possible to determine, by vision or spectroscopy, if the devices have leakages that enable entry of test gas. In case of the autoinjector 20, as with the syringes 10, if any leakage exists in the drug cartridge 30 the test gas will enter the chamber 37 and dissolve in the liquid drug, thereby causing a change to the pH thereof and to the UV-vis spectrum of the pH indicator. The pH indicator may be added to the liquid drug by injection into the chamber 37 through a dedicated needle (not shown) penetrating the self-sealing septum 34. However, alternatively, a portion of the liquid drug may be expelled or extracted from the chamber 37, through a needle (not shown) penetrating the self-sealing septum 34, and mixed with the pH indicator in e.g. a cuvette or well plate to thereby conduct an ex situ evaluation of the liquid drug.

[0090] Hence, by the present method it is possible to perform in-device testing as well as drug-device combination testing with the final drug product. Consequently, the CCI testing can in principle be performed at-line, as opposed to the previous off-line testing. At-line testing designates a process where test samples are taken out of the production line (in case of stand-alone container testing), or the assembly line (in case of in-device testing), and CCI testing is carried out by operators on-location, i.e. without the samples leaving the facility. In terms of both speed and cost, at-line testing is superior to off-line testing, where test samples are transported to a Quality Control laboratory for CCI testing by trained laboratory technicians.

[0091] Some examples further illustrating the invention are summarised in the following.

[0092] Example 1

[0093] In one example C02was used as the test gas. The drug was an API formulated in water buffered with phosphate buffer at pH 7.4. To the drug 2 mg / 50 mL of an indicator of phenol red was added before filling of the container. The positive control samples were prefilled glass syringes, with 10 pm laser drilled holes. Five positive control samples were placed in a pressure chamber and the pressure was raised to 5 bar, comprising 1 bar air and 4 bar C02, and the samples were incubated for 10 minutes. All samples were subsequently spectroscopically analysed.

[0094] If the C02gets in contact with the drug through a hole in the container, diffusion through the gas-liquid interface and into the drug follows Fick’s laws of diffusion. When C02is absorbed and dissolved in the drug, several chemical reactions occur which change the chemical composition of the drug. This illustrated in Fig. 2, showing an exemplary syringe 10 of the above-mentioned type with a crack 16 in the syringe barrel 11.

[0095] The first and primary reaction is between C02and H20 to form carbonate:

[0096] CO2(aq. ) + H20 -> H2CO3

[0097] Followed by a dissociation of the proton:

[0098] H2CO3H++ HCO3

[0099] Due to the presence of buffering species in the drug this equation becomes more complicated, here the protons are donated by carbonate. The increase of carbonate in the drug is shifted towards the left:

[0100] H2P0; H++ HP0 “

[0101] A similar effect can be observed on phenol-phenolate equilibrium:

[0102] C6H50H C6H50- + H+

[0103] Every molecule in the drug which is part of a protonated equilibrium will change as a function of an increase in the carbonate concentration and the resulting acidification.

[0104] A significant change in the pH can be visibly determined by the naked eye, while smaller changes can easily be determined using a UV-vis spectrometer. The pH of solutions containing pH indicators can be calculated from their UV-vis spectra using Lambert Beer’s law and the Henderson-Hasselbalch equation. Lambert Beer’s law correlates the absorption of a compound at a given wavenumber with its concentration:

[0105] AbsA= £A■ I ■ [ ] where A is the light wavelength, sAis the molar attenuation coefficient for the compound A at the wavelength A, and I is the pathlength. The Henderson-Hasselbalch equation makes a correlation between the pH of a solution, and the pKa and concentrations of the acid and conjugated base of the solution: where [HA] and [A-] represent the concentrations of the acid and base pairs of the indicator.

[0106] Combining the above two equations and refactoring, the pH can be expressed in terms of the absorbance of the acid and the conjugated base of the indicator: where the term log10(£HA, 2) is the logarithm of the ratio of the extinction coefficients of the \£A~, 1 / acid and its conjugated base and can be calculated through a calibration experiment.

[0107] To demonstrate this, a titration experiment with the pH indicator phenol red was prepared in the pH range between 6.10 and 7.81. At each titration step, 1 mL of the solution was transferred to a prefilled syringe and the UV-vis spectrum was recorded. Fig. 5 shows the changes in the UV-vis spectrum of phenol red as the pH increases from 6 (light grey) to 8 (black), while Fig. 6 shows the comparison between a reference pH determined by a pH electrode and the predicted pH calculated from the UV-vis spectra using the above equation and a log10(£HA 2) of 0.23. This experiment demonstrates that UV-vis spectra can be used to calculate the pH of solutions containing pH indicators.

[0108] Example 2 For peptide-based drugs, which, when they get in contact with air, might form protein-clogs that block the holes through which the test gas is to ingress, precautions need to be put in place. The challenge with protein-clogging is insurmountable for many of the alternative methods, i.e., dye ingress, vacuum and pressure decay, and for samples that have no headspace available for the gas headspace analysis. A protein-clog, however, is manageable with gas ingress according to the present invention because the high mobility of the test gas molecules means that any clog must be gas tight, and must remain gas tight even if exposed to high pressure, to prevent passage, as opposed to the dye ingress method, where a partial clogging is enough to prevent the ingress of the dye. Also, the option for multiple pressure cycles, as well as the possibility of multiple different test gases, allows for chemical absorption to overcome protein clogging.

[0109] To demonstrate this an experiment was prepared with four negative control prefilled syringes and 20 prefilled syringes with 20 pm laser drilled holes. All control samples contained a peptide-based drug with a pH of 7.4. The 24 samples were placed in the pressure chamber, and 4 bar of C02were then added to the atmosphere. After 10 minutes of incubation the chamber was vented to ambient, and the samples were analysed by UV-vis spectroscopy. This process was repeated twice.

[0110] The dissolution of C02in each sample, after each incubation, was followed through pH changes in the following way: The spectra of all experiments were subjected to a multivariate PLS calibration model that calculated the pH value from each spectrum. Therefore, the dissolution of C02in each positive control sample, after each incubation, could be monitored and quantified through the change in pH determined by UV-vis spectroscopy and multivariate analysis.

[0111] The below table shows the number of samples in different pH intervals after incubation cycles in the C02-containing environment. This example demonstrates how repeating cycles, changing between an incubation under a pressure of C02and ambient pressure, progressively dissolves more C02in the peptide drug containing positive control samples, until all samples have a significant amount of C02dissolved in the drug phase. The example demonstrates how challenging samples can be handled by repeating the incubation cycles.

[0112] Example 3

[0113] In this example the quantifiability of the method is demonstrated. A solution containing a known concentration of a pH indicator and of a peptide-based drug was prepared. The pH indicator in this example was nitrophenol, with an absorption peak at around 400 nm at a pH of 7.5 which shifts into an absorption peak at around 330 nm when the pH drops. The known pH indicator concentration was previously estimated by a calibration curve with different concentrations and their respective absorbances. Interpolation was used to determine the pH indicator concentration of the solution at an absorbance of 0.9. Using the appropriate calculations, the required pH indicator amount was determined and added to a 1 mL pre-filled syringe, with a 20 pm hole in its outer structure. Two samples were prepared, and their initial absorbance was measured. Note that the following procedure is repeated per each syringe.

[0114] The samples were incubated in a pressure chamber for 30 minutes at a constant pressure of 5 bar (4 bars of C02and 1 bar atmosphere). Post incubation, the chamber was vented at a rate of 1 bar per minute to prevent a high-pressure difference that could cause the plunger to move. The absorbance was then measured, and the samples were evaluated visually, where an apparent colour change, going from yellow (400 nm) to colourless (330 nm), was observed.

[0115] Figure 7 illustrates the absorption spectrum of nitrophenol with an observable isosbestic point at 350 nm. The spectrum changes as a function of changes in pH, as shown in the range 6.5 to 7.5.

[0116] Figure 8 illustrates the correlation between the pH as predicted by the UV-vis spectra and the pH as determined by a pH electrode. The linearity of the correlation is an attribute of a proper pH indicator.

[0117] A small amount of C02was added to the sample, via a Hamilton injection syringe, through the plunger. The induced pH change correlates with the amount of C02injected, as seen in Fig. 9. The linearity of the correlation between the amount of C02and the change in pH is a sign that the method is quantifiable.

[0118] The example concludes that the method is quantifiable because it can correlate changes in pH to an amount of absorbed C02. Since the amount of C02can be quantified and since various parameters related to the incubation, such as time and pressure, can be controlled, the size of the hole is determinable by a mathematical approach.

Claims

CLAIMS1. A method of testing container closure integrity, comprising:(i) subjecting a container (10, 30) comprising a transparent wall portion (11 , 31) and a drug chamber (17, 37) holding a liquid drug to a test environment (2) comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug,(ii) combining at least a portion of the liquid drug with a pH indicator, and(iii) determining a presence or absence of dissolved test gas in the drug chamber (17, 37) from a protonation state of the pH indicator.

2. A method according to claim 1 , wherein the protonation state of the pH indicator is determined from an obtained UV-vis spectrum of the combined pH indicator and liquid drug, or from an obtained fluorescence spectrum of the combined pH indicator and liquid drug.

3. A method according to claim 1 , wherein the protonation state of the pH indicator is determined by visual inspection.

4. A method according to claim 2 or 3, further comprising:(iv) determining an amount of test gas which has entered the drug chamber (17, 37) from the protonation state of the pH indicator using a reference.

5. A method according to any of claims 1 - 4, wherein the liquid drug is a peptide-based drug.

6. A method according to any of claims 1 - 5, wherein step (ii) comprises adding the pH indicator to the liquid drug in the drug chamber (17, 37).

7. A method according to any of claims 1 - 5, wherein step (ii) comprises adding the pH indicator to the liquid drug prior to step (i).

8. A method according to any of claims 1 - 5, wherein step (ii) comprises removing the at least a portion of the liquid drug from the drug chamber (17, 37) and mixing it with the pH indicator.

9. A method according to claim 8, wherein the protonation state of the pH indicator is determined from a measurement of the combined pH indicator and liquid drug, carried out in a cuvette or in a well plate.

10. A method according to claim 6, wherein the container is a drug cartridge (30) comprising a cartridge body (31) sealed at one end by a penetrable self-sealing septum (34) and at an opposite end by a slidable plug (39), and wherein step (ii) is performed by inserting a needle through the penetrable self-sealing septum (34), or through the slidable plug (39), and injecting the pH indicator into the drug chamber (37).11 . A method according to claim 6, wherein the container is a syringe (10) comprising a syringe barrel (11) carrying an injection needle (15) at one end and being sealed by a slidable plug (19) at an opposite end, and wherein step (ii) is performed by inserting a needle through the slidable plug (19) and injecting the pH indicator into the drug chamber (17).

12. A method according to any of claims 1 - 9, wherein the container (10, 30) forms part of a drug delivery device having a window through which at least a portion of the drug chamber (17, 37) is visible.

13. A method according to claim 12, wherein the container (30) is a variable volume reservoir embedded in a reservoir holding member (22) of the drug delivery device (20), the reservoir holding member (22) comprising the window (25) through which at least a portion of the drug chamber (37) is visible.

14. A method according to claim 13, wherein the variable volume reservoir is a drug cartridge (30) comprising a cartridge body (31) sealed at one end by a penetrable self-sealing septum (34) and at an opposite end by a slidable plug (39), and wherein step (ii) is performed by inserting a needle into an end opening (24) of the reservoir holding member (22) and through the penetrable self-sealing septum (34) and injecting the pH indicator into the drug chamber (37).

15. A method of testing container closure integrity, comprising:(i) subjecting a container (10, 30) comprising a transparent wall portion (11 , 31) and a drug chamber (17, 37) holding a liquid drug comprising a constituent serving as anintrinsic pH indicator to a test environment (2) comprising a test gas with pH changing capabilities over a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, and(ii) determining a presence or absence of dissolved test gas in the drug chamber (17, 37) from a protonation state of the intrinsic pH indicator.

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