Method and arrangement for non-destructive CCI testing

Abstract

The present invention provides a method of, and an arrangement for, testing container closure integrity. The method comprises subjecting a container (10, 30) comprising a transparent wall portion (11, 31) and a drug chamber (17, 37) filled with a liquid drug to a test environment (2) comprising a test gas having at least two atoms in its molecular structure over a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, and, subsequent thereto, obtaining a Raman spectrum of the liquid drug to thereby enable determination of a presence or absence of dissolved test gas in the drug chamber (17, 37).

Description

[0001] METHOD AND ARRANGEMENT FOR NON-DESTRUCTIVE CCI TESTING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to container closure integrity testing of liquid-filled 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. US 4,382,679 (Lee) discloses a method for leak detection based on fluorescent dye ingress.

[0008] 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. 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.

[0009] 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. 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-billion range.

[0010] WO 2016 / 156622 (Gasporox AB) discloses a method for CCI testing by Gas in Scattering Media Absorption Spectroscopy (GASMAS), which is a specialized sub-technique of TDLAS, specifically designed for the detection and measurement of tracer gases in highly scattering media. The GASMAS technique is used for investigating sharp gas spectral signatures, typically 10,000 times sharper than those of the host material, arising from gaseous species trapped in pores or cavities. The described method, reportedly suitable for detecting leaks in containers having only a small volume of gas inside, comprises subjecting the container to variations in outside atmosphere and performing optical measurements on the container. It is cursorily mentioned that the optical measurements may involve a sensor for Raman spectroscopy of the gas inside the container.

[0011] 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.

[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] WO 2016 / 156622, US 2023 / 0088151 , and WO 2015 / 121064 all 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 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 liquid-filled 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] 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 and a setup for reliable CCI testing of a liquid-filled container.

[0020] It is a further object of the invention to provide a method and a setup for reliable CCI testing of a liquid-filled variable volume container forming part of a drug delivery device.

[0021] 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.

[0022] In one aspect, the invention provides a method of testing container closure integrity according to claim 1 .

[0023] Hence, a method is provided which comprises the steps of (i) 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 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, and (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug to thereby enable determination of a presence or absence of dissolved test gas in the drug chamber.

[0024] The test gas has at least two atoms in its molecular structure, as it is then detectable by Raman spectroscopy, and since it 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 gas-liquid 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.

[0025] By obtaining a Raman spectrum of the liquid drug after said period of time has passed, it is possible to determine whether test gas has entered the container through a leak therein, as effects of such entry will be distinguishable as specific Raman peaks. If no such Raman peaks are present in the spectrum, it can be concluded that no test gas has entered the drug chamber and thus that the specific container closure system provides an adequate sterile barrier. Hence, the present invention provides a non-destructive method of CCI testing, as fluid tight test specimens are not negatively affected in any way and therefore suited for patient use afterwards.

[0026] 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).

[0027] 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 the Raman detection. 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.

[0028] 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.

[0029] Obtaining a Raman spectrum of the liquid drug may comprise focusing a laser beam inside the drug chamber, through the transparent wall portion, and measuring Raman scattering from an excitation point of the liquid drug. This may e.g. be done using a confocal Raman instrument, whereby the emitted and the incident light can be handled from the same side of the container, thereby saving space in the measurement setup.

[0030] Alternatively, obtaining a Raman spectrum of the liquid drug may comprise transilluminating the drug chamber with a laser beam, passing through transparent wall portions to an opposite receiver, and measuring Raman scattering from the irradiated portions of the liquid drug.

[0031] The presence or absence of dissolved test gas in the drug chamber may be determined by examining the Raman spectrum for spectral changes in the characteristic spectral bands of the test gas. Such spectral changes would be a direct proof of the presence of test gas molecules in the drug chamber.

[0032] The presence or absence of dissolved test gas in the drug chamber may, alternatively or additionally, be determined by examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug (as a consequence of test gas entering the drug chamber). Such indications may e.g. comprise an indication of a pH shift, a change of concentration of one or more species in the liquid phase, changes in polarity of solutes or solvents, precipitation of a component in the liquid drug, or a co-preci pitation between the absorbed test gas and one or more compounds of the liquid drug.

[0033] The inventors have discovered that chemical interactions in the liquid drug caused by the presence of entered test gas molecules are detectable from the Raman spectrum at lower levels of test gas, and thereby at an earlier stage, than the presence of said test gas molecules themselves.

[0034] Examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug may thus comprise scanning the Raman spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas.

[0035] If the Raman spectrum is scanned only for spectral changes in the characteristic spectral bands of the test gas, there is a risk that a positive test sample falsely passes as intact, because such spectral changes do not present until a significant amount of test gas has been dissolved in the liquid drug. Examining the Raman spectrum outside of the characteristic spectral bands of the test gas enables an even more reliable CCI testing, as said one or more indications of chemical interactions induced in the liquid drug present at a relatively much smaller amount of dissolved test gas. It is accordingly possible to detect smaller leaks and / or to reduce the optimal incubation time for a given test sample.

[0036] In particular, examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug may comprise scanning the Raman spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug. The liquid drug may comprise an active pharmaceutical ingredient (API) along with one or more of excipients, buffers, preservatives, solvents, stabilisers, tonicity agents, etc. Each such component of the liquid drug may interact with the test gas in a unique manner that is identifiable at characteristic wavenumbers or wavelengths, or in characteristic ranges of wavenumbers or wavelengths. With knowledge of the composition of the liquid drug, it is thus possible to verify a presence of test gas in the container from spectral changes in the Raman spectrum observed at, respectively in, these characteristic wavenumbers or wavelengths, or characteristic ranges of wavenumbers or wavelengths. The particular characteristic wavenumbers or wavelengths, or characteristic ranges of wavenumbers or wavelengths, may be determined and mapped beforehand through tests. If, and only if, no such spectral changes are observed, it can be concluded that no test gas molecules have entered the drug chamber, and thereby that the container is tight. Examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug may e.g. comprise scanning the Raman spectrum for spectral changes of pH-sensitive molecules in the liquid drug. This will provide for an early detection of a pH shift in the liquid drug associable with an entry of test gas, and thereby with a breach of the CCI.

[0037] For example, in a Raman spectrum obtained for a positive test sample containing semaglutide that has been subjected to a test environment comprising a test gas with pH-lowering capacity, an indication of the pH shift due to protonation of the phosphate buffer ingredient would be observable as spectral changes, in the form of a change of configuration of Raman peaks, in the wavenumber range of 800 cm-1to 1100 cm-1.

[0038] The presence or absence of dissolved test gas in the drug chamber may, alternatively or additionally, be determined by comparing the Raman spectrum with a comparison Raman spectrum obtained from a fluid tight control sample. Such comparison will enable a quick determination of differences between the two spectra.

[0039] Comparing the Raman spectrum with the comparison Raman spectrum may comprise subtracting the Raman spectrum from the comparison Raman spectrum, or vice versa, to provide a difference spectrum, and the presence or absence of dissolved test gas in the drug chamber may thus be determined by examining the difference spectrum for one or more indications of chemical interactions induced in the liquid drug and / or for spectral changes in the characteristic spectral bands of the test gas. The difference spectrum may enhance the visual representation of descriptive Raman peaks and thereby enable a faster determination of the specific spectral changes caused by the entry of test gas into the drug chamber.

[0040] Comparing the Raman spectrum with the comparison Raman spectrum may, alternatively or additionally, comprise normalising the Raman spectrum to provide a normalised Raman spectrum, normalising the comparison Raman spectrum to provide a normalised comparison Raman spectrum, and subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, to thereby provide a normalised difference spectrum. The presence or absence of dissolved test gas in the drug chamber may thus be determined by examining the normalised difference spectrum for one or more indications of chemical interactions induced in the liquid drug and / or for spectral changes in the characteristic spectral bands of the test gas. Normalising the Raman spectra and subsequently producing a normalised difference spectrum is a simple and robust way of eliminating any inter-measurement variations in the absolute Raman intensity, e.g. arising from differences in the positioning of the test samples.

[0041] Hence, examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug may alternatively or additionally comprise scanning the difference spectrum and / or the normalised difference spectrum for the above-mentioned features.

[0042] 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.

[0043] 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.

[0044] 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 is particularly important when it is desired to apply the method close to the production, e.g. on-line or at-line. In some cases, where time perhaps is of less importance, an increased diffusion rate may allow for the use of a less sensitive and cheaper Raman instrument, or for lowering the laser power, or choosing a laser with a higher wavelength.

[0045] The test environment may alternatively be an enclosure for one or more test containers or test devices, operating at atmospheric pressure.

[0046] 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.

[0047] The test environment comprises a test gas having at least two atoms in its molecular structure, i.e. the test environment may contain a single such test gas type or multiple such test gas types.

[0048] 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.

[0049] 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 which contains phenol or meta-cresol as a preservative), a mixture of hydrogen chloride and nitrogen, a mixture of nitrogen dioxide and argon, and a mixture of dichlorodifluoromethane (or another halogenic carbon gas) and nitrogen.

[0050] In principle, all commercial Raman spectrometers, independent of laser frequency, are usable for carrying out the present method, as long as the laser has reasonable transmittance through the transparent wall portion of the container. In practice, at least all lasers above 300 nm are usable, as they will be perfectly transmitted through glass and many common polymers. Lasers in the infrared area, such as 1064 nm lasers, are also applicable, if used in a Fourier Transform Raman spectrometer. Two preferred examples are the 532 nm and 785 nm lasers that are widely commercially available.

[0051] The container may be a variable volume reservoir, such as e.g. a cartridge type container comprising a self-sealing septum and a displaceable piston, or a syringe comprising a staked needle, or means for receiving a needle, and a displaceable piston. Alternatively, the container may be a constant volume reservoir such as e.g. a vial.

[0052] In another aspect, the invention provides a method of testing container closure integrity of a container comprising a transparent wall portion and a drug chamber filled with 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 having at least two atoms in its molecular structure over a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug to thereby enable determination of a presence or absence of dissolved test gas in the drug chamber.

[0053] 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, the Raman spectrum may be obtained by focusing a laser beam inside the drug chamber, through the window and the transparent wall portion, and measuring Raman scattering from an excitation point of the liquid drug. The drug delivery device may comprise a second window radially opposite the window, in which case the Raman spectrum may be obtained as mentioned above, or, alternatively, by transilluminating the drug chamber with a laser beam, passing through the windows and transparent wall portions, and measuring Raman scattering from the irradiated portions of the liquid drug.

[0054] It is implicit that any of the details mentioned in connection with the above-described aspect and embodiments of the invention are applicable to this other aspect of the invention.

[0055] In exemplary embodiments of the invention, only a portion of the drug chamber is visible through the window, or windows.

[0056] The drug delivery device may comprise a housing accommodating a dose expelling unit. The container may be a variable volume reservoir 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, or means for receiving a needle, 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.

[0057] The drug delivery device may further comprise a container holding member 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, and the second window, if present, may be arranged in the container holding member. In a further aspect, the invention provides an arrangement, or system, adapted to test container closure integrity of a container comprising a transparent wall portion and a drug chamber filled with a liquid drug, the arrangement, or system, comprising a test environment comprising a) a test gas having at least two atoms in its molecular structure, the test environment being adapted to surround the container for a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and b) a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber to a laser beam, through the transparent wall portion, and to measure the resulting Raman scattering from the liquid drug.

[0058] The arrangement, or system, according to this particular aspect of the invention is applicable to carry out the methods according to the previously described aspects and embodiments of the invention.

[0059] In a further aspect, the invention provides an arrangement, or system, adapted to test container closure integrity of a drug delivery device comprising a container having a transparent wall portion and a drug chamber filled with a liquid drug, and a container holding member accommodating the container such that at least a portion of the drug chamber is visible through a window of the container holding member, the arrangement, or system, comprising A) a test environment comprising a test gas having at least two atoms in its molecular structure, the test environment being adapted to surround the drug delivery device for a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and B) a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber to a laser beam, through the window and the transparent wall portion, and to measure the resulting Raman scattering from the liquid drug.

[0060] The arrangement, or system, according to this particular aspect of the invention is applicable to carry out the method according to the previously described aspect and embodiments of the invention that concern in-device testing and drug-device combination testing.

[0061] Further exemplary embodiments of the invention are listed in the following.

[0062] Embodiment 1 : A method of testing container closure integrity, comprising: (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug to thereby enable determination of a presence or absence of dissolved test gas in the drug chamber.

[0063] Embodiment 2: A method according to Embodiment 1 , wherein obtaining a Raman spectrum of the liquid drug comprises focusing a laser beam inside the drug chamber, through the transparent wall portion, and measuring Raman scattering from an excitation point of the liquid drug.

[0064] Embodiment 3: A method according to Embodiment 1 or 2, wherein the presence or absence of dissolved test gas in the drug chamber is determined by comparing the obtained Raman spectrum with a comparison Raman spectrum obtained from a fluid tight control sample.

[0065] Embodiment 4: A method according to Embodiment 3, wherein comparing the obtained Raman spectrum with the comparison Raman spectrum comprises subtracting the comparison Raman spectrum from the obtained Raman spectrum.

[0066] Embodiment 5: A method according to Embodiment 3, wherein comparing the obtained Raman spectrum with the comparison Raman spectrum comprises normalising the obtained Raman spectrum to provide a normalised obtained Raman spectrum, normalising the comparison Raman spectrum to provide a normalised comparison Raman spectrum, and subtracting the normalised comparison Raman spectrum from the normalised obtained Raman spectrum.

[0067] Embodiment 6: A method according to any of Embodiment 1-5, wherein the presence or absence of dissolved test gas in the drug chamber is determined by examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug.

[0068] Embodiment 7: A method according to Embodiment 6, wherein the one or more indications of chemical interactions comprise an indication of a pH shift.

[0069] Embodiment 8: A method according to Embodiment 6, wherein the one or more indications of chemical interactions comprise precipitation of a component in the liquid drug.

[0070] Embodiment 9: A method according to any of Embodiments 1-8, wherein the test environment is a pressure chamber. Embodiment 10: A method according to any of Embodiments 1-9, wherein the liquid drug is a peptide-based drug.

[0071] Embodiment 11 : A method according to any of Embodiments 1-10, wherein the container forms part of a drug delivery device having a window through which at least a portion of the drug chamber is visible.

[0072] Embodiment 12: A method according to Embodiment 11 , wherein the container is a variable volume reservoir comprising an outlet end portion, an operator end portion, and a cylindrical wall extending therebetween, and wherein the operator end portion is attached to a receiving end of a drug delivery unit comprising dose expelling means.

[0073] Embodiment 13: A method according to Embodiment 11 , wherein the container is a variable volume reservoir embedded in a reservoir holding member of the drug delivery device, the reservoir holding member comprising the window through which at least a portion of the drug chamber is visible.

[0074] Embodiment 14: An arrangement adapted to test container closure integrity of a container comprising a transparent wall portion and a drug chamber filled with a liquid drug, the arrangement comprising: a test environment comprising a test gas having at least two atoms in its molecular structure, the test environment being adapted to surround the container for a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber to a laser beam, through the transparent wall portion, and to measure the resulting Raman scattering from the liquid drug.

[0075] Embodiment 15: An arrangement adapted to test container closure integrity of a drug delivery device comprising a container having a transparent wall portion and a drug chamber filled with a liquid drug, and a container holding member accommodating the container such that at least a portion of the drug chamber is visible through a window of the container holding member, the arrangement comprising: a test environment comprising a test gas having at least two atoms in its molecular structure, the test environment being adapted to surround the drug delivery device for a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber to a laser beam, through the window and the transparent wall portion, and to measure the resulting Raman scattering from the liquid drug.

[0076] Embodiment 16: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, and (iii) examining the Raman spectrum for spectral changes in the characteristic spectral bands of the test gas.

[0077] Embodiment 17: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, and (iii) examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug.

[0078] Embodiment 18: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, and (iii) examining the Raman spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas.

[0079] Embodiment 19: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, and (iii) examining the Raman spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug. Embodiment 20: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, and (iii) examining the Raman spectrum for spectral changes of pH-sensitive molecules in the liquid drug.

[0080] Embodiment 21 : A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, thereby providing a difference spectrum, and (iv) examining the difference spectrum for one or more indications of chemical interactions induced in the liquid drug.

[0081] Embodiment 22: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, thereby providing a difference spectrum, and (iv) examining the difference spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas.

[0082] Embodiment 23: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, thereby providing a difference spectrum, and (iv) examining the difference spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug.

[0083] Embodiment 24: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, thereby providing a difference spectrum, and (iv) examining the difference spectrum for spectral changes of pH-sensitive molecules in the liquid drug.

[0084] Embodiment 25: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) normalising the Raman spectrum to provide a normalised Raman spectrum, (iv) normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, (v) subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, thereby obtaining a normalised difference spectrum, and (vi) examining the normalised difference spectrum for one or more indications of chemical interactions induced in the liquid drug.

[0085] Embodiment 26: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) normalising the Raman spectrum to provide a normalised Raman spectrum, (iv) normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, (v) subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, thereby obtaining a normalised difference spectrum, and (vi) examining the normalised difference spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas.

[0086] Embodiment 27: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) normalising the Raman spectrum to provide a normalised Raman spectrum, (iv) normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, (v) subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, thereby obtaining a normalised difference spectrum, and (vi) examining the normalised difference spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug.

[0087] Embodiment 28: A method of testing container closure integrity, comprising (i) 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 a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, (ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug, (iii) normalising the Raman spectrum to provide a normalised Raman spectrum, (iv) normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, (v) subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, thereby obtaining a normalised difference spectrum, and (vi) examining the normalised difference spectrum for spectral changes of pH-sensitive molecules in the liquid drug.

[0088] Embodiment 29: A method of testing container closure integrity of a drug delivery device comprising a) a container comprising a transparent wall portion and a drug chamber filled with a liquid drug, and b) a window through which at least a portion of the drug chamber is visible, the method comprising (i) subjecting the drug delivery device to a test environment comprising a test gas having at least two atoms in its molecular structure over a period of time sufficient to allow dissolution of any test gas from the test environment in the liquid drug, and (ii) carrying out the subsequent steps specified in any one of Embodiments 16-28. Certain containers are practically impossible to fill completely with a liquid substance, using certain filling methods. Hence, in the present context, the terms “filled with a liquid drug” and “a liquid-filled container” should be understood as, respectively, “at least substantially filled with a 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).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] BRIEF DESCRIPTION OF THE DRAWINGS

[0093] In the following the invention will be further described with references to the drawings, wherein Fig. 1 is a schematic representation of an incubation arrangement for CCI testing according to an embodiment of the present invention,

[0094] Fig. 2 is a schematic representation of a prefilled syringe in a Raman spectrometer,

[0095] Fig. 3 is a perspective view of a cartridge-based injection device suitable for undergoing CCI testing according to an embodiment of the invention,

[0096] Fig. 4 is a schematic representation of possible locations of CCI testing,

[0097] Fig. 5 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,

[0098] Fig. 6 shows a Bjerrum diagram of the dissociation of carbonate,

[0099] Fig. 7 shows normalised Raman spectra of positive and negative control samples containing a liquid formulation of semaglutide after incubation in pressurised C02(top), and the resulting difference spectrum (bottom),

[0100] Fig. 8 shows Raman spectra of positive and negative control samples with a placebo drug formulation after incubation in C02atmosphere,

[0101] Fig. 9 shows Raman spectra of positive and negative control samples of a single dose cagrilintide formulation after incubation in C02,

[0102] Figs. 10 and 11 present Raman spectra of phenol in water at acidic and alkaline pH levels,

[0103] Fig. 12 shows Raman difference spectra for positive and negative control samples containing multidose formulations of, respectively, semaglutide and liraglutide after incubation in C02,

[0104] Fig. 13 shows normalised Raman spectra of positive and negative control samples containing multidose long-acting insulin icodec after incubation in C02(top), and the resulting difference spectrum (bottom), and

[0105] Fig. 14 shows the development of spectral properties from CO2-induced precipitation in a glp- 1 analogue. In the figures like structures are mainly identified by like reference numerals.

[0106] DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0107] 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.

[0108] 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.

[0109] One of the plurality of syringes 10 can be seen in Fig. 2. The shown syringe 10, which is representative of all the syringes 10 tested in the pressure chamber 2, comprises a transparent cylindrical syringe barrel 11 with a necked down outlet portion 13, and a staked injection needle 15 sealed off by a rigid needle shield 14. A slidable sealing plug 19 is arranged in the syringe barrel 11 axially opposite the injection needle 15, and the chamber 17 resultantly defined by the syringe barrel 11 and the plug 19 is filled with a liquid drug (not visible).

[0110] 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. In the present case, a test gas pressure of 6 bar is applied in the interior 6, enabling a short and efficient incubation. 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. The overpressure created in the interior 6 promotes this process.

[0111] The dissolution of test gas molecules in the liquid drug causes a change in the chemical species that are present, and / or a change in the equilibrium between chemical species. The chemical effects thus induced in the liquid drug could, for example, be a change in pH, a change in the concentration of one or more species in the liquid phase, precipitations of a component in the liquid drug, or a co-precipitation between the absorbed test gas and one or more compounds of the liquid drug. All such effects are liquid phase specific and, as realised by the inventors of the present method, non-destructively detectable by use of Raman spectroscopy.

[0112] 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.

[0113] An exemplary period of time, realisable by the 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 placed in a Raman spectrometer (not shown). Fig. 2 shows the Raman measurement setup schematically, where one of the syringes 10 is placed on a carrier plate 51 , and a laser beam L is directed through a confocal lens 55 to a focus point P in the chamber 17. Raman scattered light S from the excited molecules in the liquid drug is collected by the confocal lens 55 and analysed in the Raman spectrometer for evidence of ingressed test gas.

[0114] 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 non- releasably attached to the housing 21 . The cartridge holder 22 retains a drug cartridge 30 with a transparent body 31 , e.g. of glass, which is sealed proximally by a slidable piston (not visible) and distally by a penetrable self-sealing septum (not visible). The drug cartridge 30 with the piston and the self-sealing septum defines 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.

[0115] 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 will be penetrated by a rear end of an injection needle (not shown), 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 is pushed distally in the drug cartridge 30 and the set dose of liquid drug is expelled through the injection needle.

[0116] The autoinjector 20 is an example of an injection device which can be CCI tested, non- destructively, 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, followed by an individual, or a batch, analysis in a Raman spectrometer it is possible to determine, if the devices have leakages that enable ingress 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, inducing chemical interactions that are detectable using Raman spectroscopy.

[0117] Hence, by the present method it is possible to perform in-device testing as well as drug-device combination testing of the container closure integrity without having to destroy the test sample. Consequently, the CCI testing can in principle be performed in-line, on-line, or at-line in a production line (in case of stand-alone container testing), or in an assembly line (in case of indevice testing), as opposed to the previous off-line testing.

[0118] As illustrated in Fig. 4, in-line testing 101 is performed in a production line 100, or an assembly line, at production speed. This allows for testing of 100% of a batch instead of only selected samples. In an on-line testing 200 test samples are taken out of the production line, or the assembly line, run through a fully or partly automated test setup, capable of providing results quickly, and thereafter returned to the batch. At-line testing 300 designates a process where test samples are taken out of the production line, or the assembly line, 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, in-line testing 101 , on-line testing 200, and at-line testing 300 are all superior to off-line testing 400, where test samples are transported to a Quality Control laboratory for CCI testing by trained laboratory technicians.

[0119] Some examples further illustrating the invention are summarised in the following.

[0120] Example 1 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.

[0121] The positive control samples were prefilled glass syringes, with 10 pm laser drilled holes. Five positive control samples were places 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 analysed with a confocal Raman instrument, using a 40 mW, 532 nm laser. Acquisition time per scan was 1 second, and each final spectrum was made by average of 360 individual scans.

[0122] 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 the C02is absorbed and dissolved in the drug, several chemical reactions occur which change the chemical composition of the drug. This illustrated in Fig. 5, which shows an exemplary syringe 10 of the above-mentioned type with a crack 16 in the syringe barrel 11 .

[0123] The first and primary reaction is between C02and H20 to form carbonate:

[0124] CO2(aq. ) + H20 -> H2C03

[0125] Followed by a dissociation of the proton:

[0126] H2C03H++ HCO3

[0127] The relative equilibria of the different protonated states as a function of pH can be determined based on the pKa of carbonate, using a Bjerrum diagram as shown in Fig. 6.

[0128] 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:

[0129] H2P0; H++ HP0 “

[0130] A similar effect can be observed on phenol-phenolate equilibrium:

[0131] C6H50H C6H50- + H+ 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.

[0132] At a first glance the spectra of positive and negative control samples looked alike. However, when taking the difference between a positive and a negative spectrum there was very clear signs of the incubation in the resulting spectrum. Fig. 7 presents both the raw spectra (top diagram) and the difference spectrum (bottom diagram). It is clear that whereas it is practically impossible to distinguish the positive control sample spectrum from the negative control sample spectrum in the top diagram with the bare eye, in the difference spectrum all the interactions caused by C02are evident: disappearance of HPO^- bands (‘A’), appearance of H2PC>4 bands (‘B’), and appearance of bicarbonate bands (‘C’) and dissolved C02molecules CD’).

[0133] Example 2

[0134] In another example the drug was filled into 3mL glass cartridges. In this case the drug was a placebo formulation, hence contained no active API but only an aqueous solution of a phosphate buffer and sodium chloride as tonicity agent. One positive control with a 10-micron laser drilled hole and one negative control was placed in a small glass chamber. The chamber was flushed and pressurised with C02at 2 bar. After incubation for one hour the control samples were analysed using Raman spectroscopy for around six minutes each.

[0135] In products with phosphate buffer, the C02will react with the buffer system and form HCO3 through converting the HPO^- into H2PC>4. Although the HCO3 ion has a very weak Raman spectrum the two ions of the phosphate buffer system have very intense and very distinct Raman spectra (1078 cm-1and 875 cm-1for H2P04 , and 990 cm-1for HPO^-).

[0136] When comparing a Raman spectrum of a positive control sample’s reaction with a negative control reference (difference spectrum), a very clear difference in the absorbance changes of the three buffer peaks is observed, as shown in Fig. 8, where the spectra in the insert are baseline corrected for visual clarity.

[0137] This example illustrates that the invention can be used for CCI testing in a buffered drug solution through spectroscopic identification of the changes in the buffer species of the positive control samples due to the acidic reaction of dissolved C02, after the positive and negative control samples have been incubated in C02and the difference spectra have been obtained. Example 3

[0138] In this example, 20 positive control samples (prefilled syringes) with 20 pm laser drilled holes, where 10 was stored at 5 °C and 10 were stored at room temperature (RT), and two negative control samples were investigated. After four weeks the samples were all placed in the pressure chamber, incubated at 5 bar (4 bar CO2 + 1 bar air) for 30 minutes, and subsequently analysed by Raman spectroscopy.

[0139] Table 1 shows the fraction of successfully incubated samples per incubation cycle. The 10 samples stored at 5 °C were all easily identified as positives after incubation, while seven out of 10 of the samples stored at RT had resisted incubation, presumably due to the formation of protein clogs.

[0140] Table 1.

[0141] Following Raman analysis, the false negative samples stored at RT were stored for another 30 days and then re-incubated and re-analysed. It was found that five out of seven of the previously false negatives had successfully been lowered in pH.

[0142] These incubation- and analysis-cycles were repeated until all samples had been successfully incubated with the test gas and a pH change had been determined with Raman spectroscopy. The results demonstrate that the method, by repeating the incubation cycle, can handle difficult, aged peptide-based drugs. This is significant because peptide-based drugs tend to form clogs that prevent other CCI test methods from detecting leaks.

[0143] Example 4

[0144] In the following example the method for CCI analysis was used on an amylin analogue drug formulation, in prefilled syringes with a 20 pm laser drilled hole, buffered by glutamic acid at pH 4.0. Glutamic acid has two carboxylic groups (pKaat around 2.16 and 4.15) and one amino group (pKaat around 9.58). Negative controls were incubated in 4 bar C02for 30 minutes, together with positive controls. After incubation, Raman spectra of the samples were obtained and analysed. The positive samples could be identified from a strong Raman peak at 1385 cm-1due to dissolved C02(aq.), as shown in Fig. 9. This example illustrates that the invention can be used for CCI testing in an acidic drug, by identifying molecular dissolved C02in the positive control sample, after incubation in C02.

[0145] Example 5

[0146] 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.

[0147] 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 Raman spectroscopy. This process was repeated twice.

[0148] 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 Raman spectroscopy and multivariate analysis. Table 2 shows the number of samples in different pH intervals after incubation cycles in the C02-containing environment.

[0149] Table 2.

[0150] 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, as well as how Raman spectroscopy combined with multivariate analysis is used in the CCI testing. In a practical application of this example, Raman spectroscopy and multivariate analysis might be used only after the last incubation cycle.

[0151] Example 6

[0152] A multidose drug formulation for injection contains a preservative to prevent bacterial growth while used by the patient. Often the preservative could be a smaller aromatic molecule like phenol, or a meta-cresol, or similar molecules. Phenol and meta-cresol are both very weak acids with a pKaat around 10, meaning that they can deprotonate to make the anion form at more alkaline pH values. In the following phenol was considered as a non-limiting example for an aromatic preservative.

[0153] Phenol and phenolate both have extremely strong Raman spectral characteristics with an extremely intense ring-breathing peak located at around 1000 cm’1. However, the phenol and phenolate anion do have slightly different Raman spectra, i.e., the strong ring breathing is located at 1000 cm'1for phenol but at 990 cm'1for phenolate, cf. Figs. 10 and 11. A similar shift is observed for the other Raman bands of the two related molecules, as shown in Fig. 11. The majority of multidose peptide drugs are formulated around neutral pH values, in which the dominant form of phenol is in its native protonated form. However, despite the phenol form being dominant there still exists a small fraction of phenolate, approximately 0.3 %.

[0154] Due to the strong intensity of the phenol and phenolate bands in Raman spectroscopy, even a slight decrease in pH can be detected through the shift in Raman spectra indicating the last fraction of phenolate being converted into phenol. In the following it is demonstrated on various multidose drug products how this can be exploited in the present invention.

[0155] In one experiment, positive and negative control samples for CCI testing were prepared with two different multidose GLP-1 drugs for treatment of type-2 diabetes. In the first series the subject was a multidose formulation of the GLP-1 analogue semaglutide, and in the second series it was a multidose formulation of the GLP-1 analogue liraglutide. Both series of samples were incubated for 30 minutes in 5 bar (1 bar atmospheric air and 4 bar C02), after which the Raman spectra of all samples were recorded using a 532 nm dispersive Raman instrument.

[0156] In Fig. 12 the difference spectra (positive minus negative control) are shown, for semaglutide (top) and liraglutide (bottom). In the difference spectra several features reveal a conversion from phenolate to phenol, indicating a small pH shift induced by the dissolved C02in the positive control samples. Most notable is the shift around the intense ring- breathing mode at 1000 cm-1, indicated by ‘A’ in the two figures. Weaker but significant indications show the appearance of dihydrogen phosphate (‘B’), as well as a minor amount of molecular C02(‘C’).

[0157] In another experiment, positive and negative control samples of a multidose formulation of the long-acting insulin icodec were incubated in C02. After incubation, the Raman spectra of the positive and negative control samples were recorded and normalized (Fig. 13, top), and then the difference spectrum of the normalized spectra was produced (Fig. 13, bottom). The dissolution and accompanying drop in pH was detected by shifts in the phenol peak indicating full conversion into phenol form (‘A’), while (‘B’) indicates the bicarbonate formation. The lack of a buffer agent in the formulation results in a strong signal due to molecular C02(‘C’).

[0158] The current examples illustrate how excipients like aromatic preservatives play an underlying role in some applications of this invention, where their changes in the Raman spectrum is a strong marker for the dissolution of the test gas during CCI testing.

[0159] Example 7 In this example a prefilled syringe containing a glp-1 drug formulation buffered with phosphate was used. The prefilled syringe was incubated overnight, and the successful incubation was immediately observable as a milky white precipitation and was confirmed with Raman as a general loss of signal intensity. Fig. 14 shows the development of the spectral properties from CO2-induced precipitation. When pH drops, as more C02is added to the sample, the general intensity of the spectrum decreases, particularly the water peaks located above 3000 cm-1, at 1600 cm-1, and below 500 cm-1.

[0160] In a similar example, a positive control sample containing a weakly buffered multidose insulin formulation incubated in C02will, on top of the phenol shift and the detection of a C02(aq.) peak, also produce a milky precipitation of the insulin as the histidine gets protonated at pH<5. This results in a destabilisation of the zinc hexamer complex, as well as the insulin losing its stabilizing charge, and will cause instant precipitation, detectable by Raman as a strong loss of signal due to Mie scattering.

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) filled with a liquid drug to a test environment (2) comprising a test gas having at least two atoms in its molecular structure over a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, and(ii) subsequent to (i), obtaining a Raman spectrum of the liquid drug to thereby enable determination of a presence or absence of dissolved test gas in the drug chamber (17, 37).

2. A method according to claim 1 , wherein obtaining a Raman spectrum of the liquid drug comprises focusing a laser beam (L) inside the drug chamber (17, 37), through the transparent wall portion (11 , 31), and measuring Raman scattering (S) from an excitation point (P) of the liquid drug.

3. A method according to claim 1 or 2, wherein the presence or absence of dissolved test gas in the drug chamber (17, 37) is determined by examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug.

4. A method according to claim 3, wherein examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug comprises at least one of:- scanning the Raman spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas,- subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, to thereby provide a difference spectrum, and scanning the difference spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas, or- normalising the Raman spectrum to provide a normalised Raman spectrum, normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, subtracting the normalisedRaman spectrum from the normalised comparison Raman spectrum, or vice versa, to thereby provide a normalised difference spectrum, and scanning the normalised difference spectrum for spectral changes at wavenumbers or wavelengths that are distinct from the characteristic spectral bands of the test gas.

5. A method according to claim 3, wherein examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug comprises at least one of:- scanning the Raman spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug,- subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, to thereby provide a difference spectrum, and scanning the difference spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug, or- normalising the Raman spectrum to provide a normalised Raman spectrum, normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, to thereby provide a normalised difference spectrum, and scanning the normalised difference spectrum for spectral changes at predetermined wavenumbers or wavelengths, or in predetermined ranges of wavenumbers or wavelengths, which predetermined wavenumbers or wavelengths, or ranges of wavenumbers or wavelengths, depend on the liquid drug.

6. A method according to claim 3, wherein examining the Raman spectrum for one or more indications of chemical interactions induced in the liquid drug comprises at least one of: scanning the Raman spectrum for spectral changes of pH-sensitive molecules in the liquid drug,- subtracting the Raman spectrum from a comparison Raman spectrum obtained from a fluid tight control sample, or vice versa, to thereby provide a difference spectrum, and scanning the difference spectrum for spectral changes of pH-sensitive molecules in the liquid drug, or- normalising the Raman spectrum to provide a normalised Raman spectrum, normalising a comparison Raman spectrum obtained from a fluid tight control sample to provide a normalised comparison Raman spectrum, subtracting the normalised Raman spectrum from the normalised comparison Raman spectrum, or vice versa, to thereby provide a normalised difference spectrum, and scanning the normalised difference spectrum for spectral changes of pH-sensitive molecules in the liquid drug.

7. A method according to claim 3, wherein the one or more indications of chemical interactions comprise an indication of a pH shift, a change of concentration of one or more species in the liquid phase, changes in polarity of solutes or solvents, and / or precipitation of a component in the liquid drug.

8. A method according to claim 1 or 2, wherein the presence or absence of dissolved test gas in the drug chamber (17, 37) is determined by examining the Raman spectrum for spectral changes in the characteristic spectral bands of the test gas.

9. A method according to any of the preceding claims, wherein the test environment (2) is a pressure chamber.

10. A method according to any of the preceding claims, wherein the liquid drug is a peptide- based drug.

11. A method according to any of the preceding claims, 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.

12. A method according to claim 11 , wherein the container (30) is a variable volume reservoir comprising an outlet end portion, an operator end portion, and a cylindrical wall extending therebetween, and wherein the operator end portion is attached to a receiving end of a drug delivery unit comprising dose expelling means.

13. A method according to claim 11 , 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. An arrangement adapted to test container closure integrity of a container (10, 30) comprising a transparent wall portion (11 , 31) and a drug chamber (17, 37) filled with a liquid drug, the arrangement comprising:- a test environment (2) comprising a test gas having at least two atoms in its molecular structure, the test environment (2) being adapted to surround the container (10, 30) for a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, and- a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber (17, 37) to a laser beam, through the transparent wall portion (11 , 31), and to measure the resulting Raman scattering from the liquid drug.

15. An arrangement adapted to test container closure integrity of a drug delivery device (20) comprising a container (30) having a transparent wall portion (31) and a drug chamber (37) filled with a liquid drug, and a container holding member (22) accommodating the container (30) such that at least a portion of the drug chamber (37) is visible through a window (25) of the container holding member (22), the arrangement comprising:- a test environment (2) comprising a test gas having at least two atoms in its molecular structure, the test environment (2) being adapted to surround the drug delivery device (20) for a period of time sufficient to allow dissolution of any test gas from the test environment (2) in the liquid drug, and- a Raman spectrometer operable to obtain a Raman spectrum of the liquid drug, the Raman spectrometer being adapted to expose the drug chamber (37) to a laser beam, through the window (25) and the transparent wall portion (31), and to measure the resulting Raman scattering from the liquid drug.

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