Method and device for simulating stirring stress on liquid

Abstract

The invention relates to a method for simulating the influence of transport on the quality of a liquid, and to a two-dimensional oscillator suitable for carrying out said method.

Description

Technical Field

[0001] The present invention relates to a method for simulating the effect of transportation on the quality of a liquid, and a two-dimensional oscillator suitable for performing the method. Background Art

[0002] When developing pharmaceutical products, there are many factors to be considered. Among the many factors to be evaluated, product stability and integrity are crucial for ensuring the efficacy and safety of the product. Especially when developing biological and biopharmaceutical products, stability is quite important. The complex molecular composition of these macromolecules makes them extremely vulnerable to conformational and structural changes. External factors such as temperature changes, pH alterations, and surface adsorption may cause protein aggregation, protein particles, and chemical modifications [Das, T.K.; Carroll, J.A. Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products. In Parenteral Medications; Nema, S., Dubois, L., eds.; FL: CRC Press-Taylor & Francis Group, 2019; pp. 219-248; Nowak, C.; K. Cheung, J.; M. Dellatore, S.; Katiyar, A.; Bhat, R.; Sun, J.; Ponniah, G.; Neill, A.; Mason, B.; Beck, A.; Liu, H. Forced Degradation of Recombinant Monoclonal Antibodies: A Practical Guide. MAbs 2017, 9(8), 1217–1230]. Therefore, a large amount of development work is dedicated to finding formulations of biopharmaceuticals that can retain the structure of the biopharmaceutical and thus ensure its efficacy and safety [Das, T.K. et al., 2020; DOI: 10.1016 / j.xphs.2019.09.023; Wang, W. et al., 2012; DOI: 10.1016 / j.ijpharm.2012.04.040].

[0003] Several steps in the production and distribution of biopharmaceuticals are responsible for creating stress conditions that can affect the structure of proteins. One of these steps is the transportation of the drug to the patient [Das, T.K. et al., 2020, DOI: 10.1016 / j.xphs.2019.09.023]. For parenteral administration of biopharmaceuticals, they need to be in liquid formulation, and during transportation, the movement of the liquid causes shear forces or interfacial effects that can degrade / aggregate the protein. Therefore, pharmaceutical companies must test the robustness against vibrations, temperature changes, liquid-gas interfaces, shear stress, and other types of stress that can damage product quality [Nowak, C.; Cheung, K.; Dellatore, M.; Katiyar, A.; Bhat, R.; Sun, J.; Ponniah, G.; Neill, A.; Mason, B.; Beck, A.; Liu, H. Forced Degradation of Recombinant Monoclonal Antibodies: A Practical Guide. MAbs 2017, 9(8), 1217–1230; Koepf, E. et al., DOI: 10.1016 / j.ijpharm.2017.12.043; Maa, Y.F.; Hsu, C.C., 1997, DOI: 10.1002 / (SICI)1097-0290(19970620)54:6<503::AID-BIT1>3.0.CO;2-N]. One way to circumvent this problem is to use lyophilization. Lyophilization is the process of removing water and converting the solution into a solid cake or powder to extend the shelf life and facilitate transportation. However, this process is highly labor-intensive and requires reconstitution, so "ready-to-use" formulations are used M.; Zvonar Pobirk et al., 2020, DOI: 10.1016 / j.ijpharm.2020.119029; Bye, J.W. et al., 2014, DOI: 10.1007 / s10529-013-1445-6]. In addition, the demand for convenient autoinjectors and prefilled syringes has increased, where lyophilized formulations are not an option [Bye, J.W. et al., 2014, DOI: 10.1007 / s10529-013-1445-6; Sassalos, T.M., Paulus, Y.M., 2019, DOI: 10.2147 / OPTH.S169044]. One way to prevent aggregation of liquid-formulated biopharmaceuticals is to add certain inactive ingredients (excipients) to the drug product. Examples of these excipients include: disaccharides and surfactants, while changing the pH and the protein concentration can also prevent protein aggregation [Narhi, L.O. et al., 2022, DOI: 10.1016 / j.xphs.2022.01.011; Das, T.K. et al., 2021, DOI: 10.1016 / j.xphs.2021.09.030].

[0004] The FDA and other health authorities require shipment validation studies, in which the robustness and quality of biopharmaceutical products are assessed after transportation [Nowak, C.; Cheung, K.; Dellatore, M.; Katiyar, A.; Bhat, R.; Sun, J.; Ponniah, G.; Neill, A.; Mason, B.; Beck, A.; Liu, H. Forced Degradation of Recombinant Monoclonal Antibodies: A Practical Guide. MAbs 2017, 9(8), 1217–1230; Narhi, L. O. et al., 2022, DOI: 10.1016 / j.xphs.2022.01.011]. To assess the robustness and quality of products after shipment, pharmaceutical companies need to conduct real-life shipment (RLS) tests, in which the transportation of new drugs is sent through representative distribution routes (usually required by the FDA). To submit new biological drug products to other health authorities (such as the EMA), companies need to conduct a transportation simulation, the so-called ASTM (American Society for Testing and Materials) vibration test (ASTM D4169). There are three transportation methods (rail, truck, and air), and for each method, there are three intensity levels (I, II, III) covered by the ASTM method. Currently, the guidelines recommend a plan in which levels I, II, and III are continuously tested for a specified period of time [ASTM International. ASTM Standard Practice for Performance Testing of Shipping Containers and Systems; 2016; https: / / www.astm.org / d4169-14.html; ASTM International. ASTM D4169 Truck Profile Update Rationale ASTM D4169 Truck Profile Update Rationale; 2016].

[0005] However, studies have shown that the current methods for assessing product quality after transportation stress are less representative and quite expensive P, 2019, DOI: 10.1002 / pts.2434; Németh, Z. et al., 2021, DOI: 10.14513 / actatechjaur.00603]. First, the ASTM D4169 vibration test only covers vertical vibration, even though there are three dimensions of vibration when transporting pharmaceutical products. There is limited knowledge and data regarding the transport stresses that biopharmaceutical products experience in real life and whether adding another dimension changes the stresses to which the pharmaceutical products are exposed.

[0006] In view of such a situation as described above, an object of the present invention is to provide a two-dimensional oscillator in order to more accurately replicate the agitation stress on liquid pharmaceutical formulations during transportation, since two-dimensional vibration is closer to real-life shipping. The devices and methods described herein can be used, in particular, for evaluating liquid formulations during the development process and also to meet the quality requirements as required by health authorities. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 . Different waveforms can be seen between horizontal (X-axis), vertical (Z-axis), and combined (XZ-axis) vibrations. Representative images of 6 ml vials filled with 3.2 ml are shown. The still images were taken from high-speed video footage (200 fps).

[0008] Figure 2. Particle comparison between four different antibodies in a liquid formulation oscillating on 3 different axes (X, Z, and XZ). Each antibody formulation was filled in a 6 ml vial and oscillated for 30 minutes with the ASTM D4169-16 aviation class I curve. For the agitated antibody formulations, N = 3; for the control, N = 1 / 2. A: Visual inspection of the vials, pictures of vials with many particles (left) and unagitated control vials (right) are attached. B: Results of subvisible particle counting obtained by light obscuration. Amount of particles per ml in the size ranges 2 μm to 4 μm, 5 μm to 9 μm, and 10 μm to 24 μm; EP = EP black / white box; SD = Seidnenader.

[0009] Figure 3 . Comparison between 3 different temperatures (5°C, 23°C, and 30°C). The CEA antibody formulation was filled in a 6 ml vial and oscillated for 30 minutes on the XZ axis with the ASTM D4169-16 aviation class I curve. For the agitated antibody formulations, N = 3; for the control, N = 1 / 2. A: Visual inspection of the vials. B: Results of subvisible particle counting obtained by light obscuration. Amount of particles per ml in the size ranges 2 μm to 4 μm, 5 μm to 9 μm, and 10 μm to 24 μm; EP = EP black / white box; SD = Seidnenader.

[0010] Figure 4 . Particle comparison between 6 ml and 20 ml vials oscillated at two different vial orientations (horizontal and vertical) with the same vertical fill height (3.2 ml and 6.4 ml respectively). The CEA preparation was filled in 6 ml vials or 20 ml vials and oscillated for 30 minutes on the XZ axis with the ASTM D4169-16 aviation grade I curve. For the agitated antibody preparation, N = 3; for the control, N = 2. A: Visual inspection of the vials. B: Results of subvisible particle counting obtained by light obscuration. Amount of particles per ml in the size ranges 2 μm to 4 μm, 5 μm to 9 μm, and 10 μm to 24 μm; EP = EP black / white box; SD = Seidnenader.

[0011] Figure 5 . Visual inspection of surfactant studies.

[0012] Figure 6. Comparative light obscuration subvisible particle analysis between XZ-axis and Z-axis agitation at different time intervals with different concentrations of PS80. The CEA antibody preparation was filled in 6 ml vials and oscillated for 15, 30, 60, or 120 minutes on the XZ axis (A) or Z axis (B) with the ASTM D4169-16 aviation grade I curve. For all antibody preparations, N = 1. A: Subvisible particle analysis by light obscuration: XZ vibration. B: Subvisible particle analysis by light obscuration: Z vibration.

[0013] Figure 7. Comparative background membrane imaging subvisible particle analysis between XZ-axis and Z-axis agitation at different time intervals with different concentrations of PS80. The CEA antibody preparation was filled in 6 ml vials and oscillated for 15, 30, 60, or 120 minutes on the XZ axis (A) or Z axis (B) with the ASTM D4169-16 aviation grade I curve. For all antibody preparations, N = 1, and three samples were taken from one vial. BMI = Background membrane imaging. A: Subvisible particle analysis by BMI: XZ vibration. B: Subvisible particle analysis by BMI: Z vibration.

[0014] Figure 8 . Surface tension results from CEA at different concentrations of PS80.

[0015] Figure 9 . The 6 mL vials are in a single-package configuration, where the double leaflet (left) is for the vial filled with water, and the single leaflet (right) is for the drug product vial.

[0016] Figure 10. Schematic point mass spring system. A: Spring; B: Body with fixed motion; C: Vibration amplification; D: Rigid transmission; E: Wave damping.

[0017] Figure 11 . Schematic path of wave passing through the packaging. A: Thermal insulation, including cooling; B: Packaging; C: Secondary packaging; D: Pallet; E: Vial; F: Path of wave; G: Surface of vehicle (e.g., airplane or truck).

[0018] Figure 12 . Accelerometer measurement results and positioning of reference sensors (left) MSR165: Recorder with internal battery and sensor (right). A: Probe measurement results; B: Reference measurement results. The amplitude ratio can be calculated by dividing the probe measurement results by the reference measurement results.

[0019] Figure 13 . The accelerometer within its holder is placed inside a shipping box (exemplary).

[0020] Figure 14 . Temperature during transportation.

[0021] Figure 15 . Total vibration energy (Grms) on three axes X, Y, and Z during transportation.

[0022] Figure 16. PSD distribution and density. A: Pallet in refrigerated truck; Z-axis data; B: Pallet in EKR1Active Thermal shipping vehicle (truck); Z-axis data; C: Pallet in EKR1 Active Thermal shipping vehicle (flight); Z-axis data; D: Pallet in refrigerated truck; Z-axis data; E: Pallet in refrigerated truck; Y-axis data; F: Pallet in refrigerated truck; X-axis data; G: Pallet in refrigerated truck; Z-axis data. In the PSD curves of all groups, the assurance levels (levels I, II, and III, as described in Example 2) are indicated by solid lines. For comparison, the relevant ASTM D4169-16 guideline curves are plotted as dashed lines.

[0023] Figure 17 . Impacts recorded during transportation.

[0024] Figure 18Comparison of data from horizontal oscillations during formulation development and real-life shipments. Group A: Sub-visible particles (SvP). Group B: Size-exclusion high-performance liquid chromatography (SE-HMW). In both Groups A and B, labeled: A: Active 2 formulation; B: Active 3 formulation; C: Active 1 formulation; D: Active 1 formulation, containing 0.0.1% surfactant, after 7 days of oscillation at 5 °C; E: Active 1 formulation, containing 0.0.1% surfactant, after 7 days of oscillation at 25 °C; F: Active 1 formulation, without surfactant, after 7 days of oscillation at 5 °C; G: Active 1 formulation, without surfactant, after 7 days of oscillation at 25 °C. Detailed Description

[0025] As used hereinbelow, the terms "having", "including", or "comprising", or any grammatical variants thereof, are used in a non-exclusive manner. Thus, these terms can either refer to a situation where no other features exist in the entity described in this context apart from the features introduced by these terms, or to a situation where one or more other features exist. As an example, the statements "A has B", "A includes B", and "A comprises B" can either refer to a situation where apart from B, no other elements exist in A (i.e., a situation where A consists of B alone and uniquely); or to a situation where apart from B, one or more other elements (such as element C, elements C and D, or even other elements) exist in entity A.

[0026] Furthermore, as used hereinbelow, the terms "specifically", "more specifically", "particularly", "more particularly", or similar terms are used in conjunction with optional features without restricting the possibility of alternatives. Thus, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the present invention can be carried out by using alternative features. Similarly, features introduced by "in one embodiment of the present invention" or similar expressions are intended to be optional features, without any limitation to alternative embodiments of the present invention, without any limitation to the scope of the present invention, and without any limitation to the possibility of combining the features introduced in this way with other optional or non-optional features of the present invention.

[0027] Furthermore, as used herein, transportation refers to the movement of an object from one location to another. Modes of transportation include air, land (rail and road), water, cable, pipeline, and space. In the context of this document, transportation particularly refers to the first three modes. The terms "transportation / transport" and "shipment / shipping" are used interchangeably herein and refer to the same concept.

[0028] In one aspect, the present disclosure provides a method for simulating the impact of transportation on the quality of a liquid, the method comprising the steps of:

[0029] a) Selecting an instruction set that includes: i) one or more PSD curves that include a plurality of frequencies and their corresponding amplitudes on at least two vertical axes (e.g., X and Z), and ii) a schedule that specifies a duration for each of the PSD curves;

[0030] b) Inducing vibrations in the liquid according to the PSD curves; and

[0031] c) Assessing and comparing the quality of the liquid before and after performing step b.

[0032] PSD (power spectral density, or as they are commonly referred to as acceleration spectral density or ASD for vibrations) plots are well known in the art and are used to quantify and compare different vibration environments. The power spectral density (PSD) of a wave (e.g., a vibration) describes the power present in the wave as a function of frequency per unit frequency. The power spectral density is typically expressed in watts per hertz (W / Hz) or g 2 / Hz, where g indicates gravity. As used herein, a PSD curve means a curve that includes information about one or more frequencies and their amplitudes and / or power densities.

[0033] In some specific embodiments, the vibrations on two axes are induced simultaneously, i.e., the liquid is vibrated in two directions during part or the whole process. In some embodiments, the liquid includes a pharmaceutical product. In particular, the pharmaceutical product includes a biological product, such as a protein, an antibody, a nucleic acid, a sugar, or a conjugate, and combinations thereof.

[0034] In some embodiments, the instruction sets for different axes (e.g., X and Z) are different; the differences can be, for example, the PSD curves and / or the duration specified for each frequency that will be applied to induce vibrations in the liquid.

[0035] In some embodiments, the amplitude of the vibration on one axis is proportional (e.g., linearly proportional) to the amplitude of the vibration on another axis; for example, the density of the vibration on one axis can be about 1.5 to 10 times the density of the vibration on another or other axes.

[0036] In some embodiments, the PSD curve is designed based on measurements of vibrations in one or more real-life shipments (RLSs). The vibrations can be measured, for example, by including sensors in the shipping package during the RLS and recording their frequency and intensity over the shipping period. The intensity in the PSD curve can be derived, for example, by calculating the average of the intensities recorded by several sensors during the RLS. Example 2 provides an exemplary embodiment in this regard. In an embodiment, the PSD curve is as shown in FIGS. 16(A to G). In an embodiment, the PSD curve is within 9 dB (decibels) of the solid or dashed line in the PSD plot of FIGS. 16(A to G), meaning that for a given frequency, the intensity will be the intensity indicated by one of the lines, or up to 9 dB smaller or larger than that intensity (e.g., 1 dB, 2 dB, 3 dB, 4 dB, 5 dB, 6 dB, 7 dB, 8 dB, or 9 dB).

[0037] In some embodiments, the PSD curve is according to the ASTM D4169 standard, such as aviation class I, II, or III, railroad class I, II, or III, or truck class I, II, or III. The ASTM standard (designation: D4169) is published by the American Society for Testing and Materials (ASTM) International and is typically updated every few years (the current ASTM standard was published in 2022 and is thus designated ASTM D4169-22).

[0038] In some embodiments, analytical methods are used for quality assessment of liquids, such as those known to those skilled in the art and applicable to specific liquids, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), analytical ultracentrifugation, visible or sub-visible particle analysis.

[0039] In one aspect, provided herein is a two-dimensional oscillator adapted to perform the steps of the method of the first aspect described above. "Two-dimensional" means that the oscillator has means for simultaneously oscillating an object (such as a liquid container) in two perpendicular directions. Such an oscillator can be designed, for example, by implementing independent linear motors for moving the liquid sample along each axis. In particular, a noise cancellation method is applied to the motor controller to ignore the cross-dependencies between the movements in different directions. This can be achieved, for example, by using sensors on each axis to detect and cancel unwanted vibrations.

[0040] In some embodiments, the oscillator is capable of a frequency sweep mode; the sweep mode includes causing vibrations to start from one extreme of a frequency range (e.g., the lowest frequency) and gradually increasing the frequency towards the other extreme (e.g., the highest frequency). The sweep mode can be performed in parallel mode (i.e., both starting from a low frequency or both starting from a high frequency) or anti-parallel mode (i.e., starting from a low frequency on one axis and starting from a high frequency on the other axis) on two axes. In some embodiments, the vibrations are in the form of a constant sine wave (e.g., without a sweep mode). In some specific embodiments, the frequency range is between 1 Hz and 300 Hz.

[0041] In some embodiments, the oscillator includes a temperature-controlled chamber. This enables the temperature of the liquid to be maintained within an appropriate range during the procedure (e.g., similar to the temperature applied during RLS). For example, a liquid formulation containing a biopharmaceutical is typically maintained at a temperature of 2°C to 8°C. In some embodiments, the temperature in the chamber is maintained at 2°C to 60°C, such as 2°C to 8°C.

[0042] In some embodiments, the oscillator includes a vial holder and / or a holder for a syringe. This enables a liquid sample to be properly placed in various containers. In some embodiments, the holder is adapted to function at different tilt angles.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0044] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description, the drawings, and the claims.

[0045] Example

[0046] Example 1

[0047] In this study, a novel two-dimensional oscillator was used to evaluate the effect of the second dimension on product quality in vibration testing because two-dimensional vibration is closer to real-life shipping. In addition, the measures currently adhered to by pharmaceutical companies regarding protecting drug products from possible additional stresses were also analyzed.

[0048] The vial was vibrated in one or two dimensions, and some external factors (e.g., temperature, surfactant concentration) were varied to assess their effects. It was found that, contrary to one-dimensional vibration, two-dimensional vibration caused splashing in the vial. Two-dimensional vibration also showed more (sub)visible particle formation than one-dimensional vibration. These results raised concerns about the drug stability during shipment, and experiments were carried out using different concentrations of surfactants. The results showed that even the minimum concentration of the tested surfactant (which was 120 times smaller than the concentration commonly used in biopharmaceutical formulations) was sufficient to inhibit (sub)visible particle formation in both one-dimensional and two-dimensional vibrations. This study formed the first basis for the testing of two-dimensional vibration and helped formulation scientists understand how their biopharmaceutical products were agitated during shipment and how best to replicate these conditions internally to test the drug products during development.

[0049] Experimental procedure

[0050] Real-life shipping study

[0051] Real-life shipment studies were carried out in Europe and North America. Accelerometers were placed between the packaged water-filled vials, inside the boxes. The boxes were shipped in five phases (Table 2). After shipment, the accelerometers were analyzed, and power spectral density (PSD) curves and plots were derived from the acceleration measurements.

[0052] Table 2. Real-life shipment plan.

[0053] Truck 1 Truck 2 Air Truck 3 Truck 4 Duration (hours; approximate) 0.5 4 8 10 48

[0054] Monoclonal antibodies and formulations

[0055] Four different monoclonal antibodies (mAb1, mAb2, mAb3, mAb4) formulated in buffer were used in this study, and their different qualities are shown in Table 3. Before shipment, all formulations were filtered through a 0.22-μm pore-size sterile filter (Millex GV) under laminar air flow conditions, all formulations were sealed with bromobutyl polytetrafluoroethylenated and silanized stoppers, and finally all formulations were sealed with aluminum crimp caps. Two vial sizes were tested, 3.2 ml was filled in 6-ml vials, and 6.4 ml was filled in 20-ml vials (Fiolax type I glass, Schott). These fill volumes produced equal fill heights in the vertical orientation, which was beneficial for high-speed video analysis, as found in previous studies. For the surfactant study, different percentages of all polysorbate 80 (0.0005, 0.001, 0.0015, 0.002, 0.003, 0.01, and 0.06) were added to mAb1. The vials were stored at 2 °C to 8 °C.

[0056] Table 3. Antibody overview.

[0057]

[0058] Protein concentration

[0059] For protein concentration measurement, a SoloVPE spectrophotometer (C. Technologies) or a UV / visible light spectrophotometer Lambda35 (Perkin Elmer) was used.

[0060] Vibration study

[0061] The two-dimensional oscillator of the present invention was used to stir the liquid antibody preparation. For the study, the ASTM D4169 guideline 2016 aviation grade IPSD curve was used, and vibration was performed only on the X-axis or Z-axis, or simultaneously on both axes. Parameters such as temperature, vial position, and vibration time could be adjusted with the oscillator. The control was a vial with the same antibody preparation but not stirred.

[0062] High-speed video analysis

[0063] The EoSens mini 1-1 (MIKROTRON) was combined with a Macro 100F2.8 DAT-X PRO (Tokine) camera lens to record high-speed video clips of the vibrating vial at 200 frames per second.

[0064] Visual inspection

[0065] Visual inspection was performed using an EP black / white light box. Magnified visual inspection was performed using a Seidenader V 90-T (Seidenader Maschinenbau GmbH). This method allows visual inspection by the rotation of the drum and includes a 2x magnifying glass.

[0066] Light obscuration

[0067] Use the HIAC 9703+ equipped with an HRLD-150 detector from Beckman Coulter. Analyze the sample using four 200 μl injections. Record the average of the last 3 injections. Use the COUNTCAL 5 μm (3000 particles per ml) counting accuracy standard (catalog number CC05) from Firma Thermo Scientific as the calibration reference. Rinse the equipment with water between each sample analysis until the particle count of ≥2 μm is ≤20, the particle count of ≥5 μm is ≤5, the particle count of ≥10 μm is ≤1, the particle count of ≥25 μm is ≤1, and the particle count of ≥50 μm is 0.

[0068] Background film imaging

[0069] Subvisible particle quantification was also performed using a high-throughput method by analyzing in triplicate with a Horizon instrument (Halo Labs, Burlingame, CA). A 0.4 μm pore polycarbonate background membrane was used, and the sample volume per well was 40 μl, in triplicate. The liquid was removed with a vacuum of 200 mbar. Samples with a membrane coverage higher than 3% were indicated as supersaturated. Use HORIZONVUE software version 3.0.0.121.

[0070] Surface tension

[0071] For surface tension analysis, use a Drop Curve Analyzer tensiometer (PAT1M, Sinterface). The analysis of each sample was run at 22 °C for 960 seconds (16 minutes).

[0072] Results

[0073] First, determine whether there are differences in wave formation between one-dimensional and two-dimensional vibrations. Using the strongest ASTM D4169 016 curve (Aviation Grade I), different wave formations and splash patterns can be seen ( Figure 1 ), where the X-axis and Z-axis show no splashing, and the XZ vibration shows splashing and droplet formation.

[0074] XZ simultaneous vibration causes more particle formation than one-dimensional vibration

[0075] To evaluate whether there are different effects on product quality between vibrations on one-dimensional or two-dimensional axes (X, Z, and XZ combinations), vials were placed on a two-dimensional oscillator and oscillated at room temperature for thirty minutes with the ASTM D4169-16 aerospace class I curve. This curve was chosen because it is the most severe vibration curve among the ASTM D4169-16 curves and is most likely to generate particles. Four different antibodies were tested and compared. When observing the results, only the vials subjected to simultaneous XZ vibrations had more than 10 visible particles, which were similar to a particle cloud ( Figure 2A ). In the sub-visible particle range, antibodies mAb1 and mAb4 also had the highest particle amounts for vials subjected to simultaneous XZ vibrations. In the particle range of 2 μm to 4 μm, we could see a difference of at least 1000 particles per ml. Antibodies mAb2 and mAb3 also had the highest particle counts for the 2 μm to 4 μm particle range, but this trend was not supported in the larger particle counts ( Figure 2B ).

[0076] The likelihood of particle formation for formulations vibrated at 5 °C is reduced

[0077] One of the advantages provided by the new prototype two-dimensional oscillator is the ability to regulate the temperature of the vibration chamber. During transportation, biopharmaceuticals are usually stored in a cooling chamber at 2 °C to 8 °C because it is known that higher temperatures accelerate the aggregation pathway [Nowak, C.; K. Cheung, J.; M. Dellatore, S.; Katiyar, A.; Bhat, R.; Sun, J.; Ponniah, G.; Neill, A.; Mason, B.; Beck, A.; Liu, H. Forced Degradation of Recombinant Monoclonal Antibodies: A Practical Guide. MAbs 2017, 9(8), 1217–1230]. Through this experiment, it was aimed to observe whether the intense agitation of XZ-axis vibration is affected by the lower and higher temperatures of the mAb1 formulation. In the vials agitated at 5 °C, their controls, and the 23 °C controls, sub-visible particle formation seemed to be the lowest. The highest sub-visible particles could be seen in the 30 °C control and the vials agitated at 23 °C ( Figure 3 B). Interestingly, the 5 °C control showed more particle formation than its agitated version in visual inspection, which was not reflected in the light obscuration data ( Figure 3 A and 3B). Similarly, the 30 °C control did not show visible particle formation but showed high sub-visible particle formation.

[0078] Differences in particle formation between vial size and orientation

[0079] Previous studies have shown that the orientation of vials affects the shear stress distribution in the vial when oscillated [Bai, G. et al., 2012, DOI: 10.1016 / j.ijpharm.2011.11.044]. Through this experiment, it was aimed to investigate what effects horizontal and vertical orientations have on (sub)visible particle formation and whether different vial sizes would show additional effects on the mAb1 formulation. The vials were re-oscillated at room temperature with a two-dimensional ASTM D4169-16 aviation grade I curve. Almost all vials showed some particles in the visual particle inspection ( Figure 4 A). Only the 6 ml vials stirred horizontally and vertically showed at most one particle in the black box and white box, but showed more than ten particles under Seidnenader analysis. Most notably from the results collected, different vial sizes showed opposite effects in terms of orientation. The 20 ml vials showed more subvisible particles in the horizontal position, while the 6 ml vials showed more subvisible particles in the vertical orientation ( Figure 4 B).

[0080] Polysorbate 80 reduces particle formation in liquid formulations vibrated in one and two dimensions

[0081] To assess whether the drug methods already in use for particle prevention are related to one-dimensional and two-dimensional vibration stresses, mAb1 formulated in buffer was mixed with different concentrations of polysorbate 80 (PS80) (Table 4). These formulations should have roughly the same protein concentration, but due to calculation errors, 0.06% PS80 had a much lower concentration. These different formulations were then stirred on the XZ and Z axes at room temperature for 15, 30, 60, or 120 minutes. Subsequently, the formulations were analyzed for (sub)visible particles using visible particle analysis, light obscuration, and background film imaging. Visible particle analysis showed that there were more than 10 particles in the 0% PS80 formulation oscillated on the XZ axis for 15 and 30 minutes ( Figure 5 ) and in the 0.0005% PS80 formulation oscillated for 60 minutes.

[0082] Table 4. Different concentrations of PS80 and the final CEA protein concentration in the formulations.

[0083] PS 80% (w / v) Protein concentration (mg / ml) 0 72 0.0005 72 0.001 72 0.0015 72 0.002 71 0.003 71 0.01 65 0.06 30

[0084] When observing the light obscuration subvisible particle analysis, the amount of particles in the surfactant-free formulation was higher than that in the formulation containing PS80 ( Figure 6A and 6B)。In the case of surfactant-free formulations, there also appears to be a time-dependent increase in particles, where longer agitation times result in higher particle amounts. Interestingly, in Z-axis agitation, 60 and 120 minutes of oscillation seem to have a much greater effect on particle amount than 15 and 30 minutes of oscillation.

[0085] The results of background film imaging also depict that when surfactant is added to the formulation for XZ-axis agitation, subvisible particles are reduced ( Figure 7A )。However, in Z-axis agitation, there seems to be a higher number of particles in the 0% PS80 formulation agitated for 15 minutes, but there seem to be less significant differences between the remaining formulations ( Figure 7B )。

[0086] Previous studies have shown a correlation between surface tension and aggregation / particle formation. Using a drop curve analysis tensiometer, the surface tension of mAb1 with different concentrations of PS80 as used in the previous experiment was identified. As Figure 8 can be seen, higher concentrations of PS80 result in lower surface tension.

[0087] Discussion

[0088] The purpose of this experiment was to obtain more knowledge about shipping stress and to determine whether current preventive measures during formulation development are sufficient to prevent product quality impacts. The 2016 ASTM D4169 aviation grade I curve was used for this study (since it is the most severe curve) and will surely give some particle amounts that can be compared.

[0089] In this study, the differences between one-dimensional and two-dimensional liquid agitation via vibration were first investigated. The results seem to indicate that simultaneous XZ vibration causes more particle formation than one-dimensional vibration. This is likely due to splashing being seen during high-speed video analysis. Previous studies have investigated that splashing in liquid formulations leads to high shear stress and thus protein aggregation [Koepf, E. et al., DOI: 10.1016 / j.ijpharm.2017.12.043; Maa, Y.F.; Hsu, C.C., 1997, DOI: 10.1002 / (SICI)1097-0290(19970620)54:6<503::AID-BIT1>3.0.CO;2-N; Das, T.K. et al., 2021, DOI: 10.1016 / j.xphs.2021.09.030, Zubiaga, A. et al., 2019, DOI: 10.21152 / 1750-9548.13.1.61]. Internal simulation studies have shown that high local peak shear is expected when droplets impact the liquid, while wave formation results in lower overall shear in the liquid, which is consistent with the observations of particle formation in this study [Hostettler, M.; Brunner, D.; Rosenthal, F.; Clemens, M.; Koepf, E.; Boiger, G.K. Analysis of Falling Droplets into Resting Liquid and Resulting Shear Stresses. In International Conference of Multiphysics, Online; 2020]. As shown in Figure 2, especially particles in the range of 2 μm to 4 μm determined by light obscuration increase when exposed to XZ vibration more than one-dimensional vibration. Even the stable reference molecule mAb2 seems to have more particle formation under XZ vibration.

[0090] The novel oscillator is more representative compared to established transport simulations operating under environmental conditions. Our results indicate that temperature has an impact on the formation of (sub)visible particles. Previous studies have shown that accelerated aggregation and denaturation occur above 50 °C [Menzen, T., Friess, W., 2014, DOI: 10.1002 / jps.23827; Vermeer, A.W.P., Norde, W., DOI: 10.1016 / S0006-3495(00)76602-1], however our results seem to suggest that there are also differences between lower temperatures such as 5 °C and 23 °C. This is not entirely surprising as temperature can change the viscosity of the liquid and this subsequently affects the shear stress experienced by the liquid [Woldeyes, M.A. et al., 2020, DOI: 10.1021 / acs.molpharmaceut.0c00552; Iqbal, M.J.; Chaudhry, M.A., 2009, DOI: 10.1016 / j.jct.2008.09.016]. Finally, there are also reports that room temperature is considered the worst-case scenario as companies are used to shipping their pharmaceutical products in temperature-controlled chambers [Ammann, C., 2011, DOI: 10.1208 / s12249-011-9684-0; Fleischman, M.L. et al., 2017, DOI: 10.1016 / j.xphs.2016.11.021].

[0091] For the horizontal and vertical vial orientation experiments, the visible particle inspection and subvisible particle analysis were not entirely consistent. This may be due to the presence of more subvisible / smaller-sized particles in the agitated vials and more visible particles in the controls. The fact that more subvisible particles were seen in the 6 ml vials in the vertical orientation and in the 20 ml vials in the horizontal orientation can be explained by the difference between the gas-liquid interface stress and the shear stress [Koepf, E. et al., DOI: 10.1016 / j.ijpharm.2017.12.043; Maa, Y.F.; Hsu, C.C., 1997, DOI: 10.1002 / (SICI)1097-0290(19970620)54:6<503::AID-BIT1>3.0.CO;2-N; Narhi, L.O. et al., 2022, DOI: 10.1016 / j.xphs.2022.01.011]. In the horizontal position, the formulation in the 20 ml vial had a larger air surface and splashed onto the neck of the vial, which is the situation where Bai et al. found the maximum shear stress in the vial [Bai, G. et al., 2012, DOI: 10.1016 / j.ijpharm.2011.11.044]. On the other hand, the 6 ml vial in the horizontal orientation did not splash onto the neck of the vial but onto the middle of the stopper. In the vertical position, the 20 ml vial did not really show any splashing, and the 6 ml vial (as Figure 1 seen) did. Due to the complexity of the interaction between the fill volume, fill height, and the influence of the vial geometry that depends on the vial orientation, it is not straightforward to find a simple head-to-head comparison for this research question. As in the preliminary image analysis study, the fill volume chosen was ideal for comparing vertical vibrations. Setting a range of fill parameters in a follow-up study will allow for a more detailed investigation. Additionally, when in contact with the product solution, the silicone oil leached from the stopper may result in higher subvisible particles, as was the case for the 6 ml vial compared to the 20 ml vial [Grapentin, C. et al., 2020, DOI: 10.1016 / j.xphs.2020.03.010; Pavanetto, F. et al., 1991, 10.1016 / 0378-5173(91)90234-F; Sendo, T. et al., 1995, DOI: 10.1002 / jps.2600841218]. Fourier transform infrared spectroscopy (FT-IR) analysis will give information about the composition of the particles and can help us understand their origin.

[0092] To prevent protein aggregation, formulation developers add excipients, such as surfactants and sugars, to drug product formulations. PS80 was used in this study because it is said to provide higher protection against aggregation detected by agitation [Singh, S.M. et al., 2017, DOI: 10.1016 / j.xphs.2017.08.011]. The results showed that the amount of (sub)visible particles in the surfactant-containing formulations was much lower than that in the formulations without PS80. In the BMI results, there also seemed to be a correlation between the amount of surfactant and the amount of particles. This was expected because the surface tension measurement results showed a lower surface tension at higher PS80 concentrations ( Figure 8 ), and this was consistent with previous investigations [Das, T.K. et al., 2021, DOI: 10.1016 / j.xphs.2021.09.030; Kannan, A. et al., 2021, DOI: 10.1016 / j.xphs.2020.10.036]. However, due to calculation errors, the protein concentration of mAb1 was almost half of the target concentration (72 mg / ml) in the 0.06% PS80 formulation, and the light obscuration measurement results did not follow the same trend.

[0093] It should be noted that light obscuration is a noisy method, and there can be significant differences between samples processed in the same way. For this reason, product quality analysis is not limited to subvisible analysis and visual inspection, but also to other stability indicating methods, such as size exclusion HPLC. Since light obscuration produced different results, only particles with a size up to 10 μm were shown in the light obscuration results. Background membrane imaging is also a method used to measure subvisible particles and has some advantages over light obscuration, but also has disadvantages [Vargas, S.K. et al., 2020, DOI: 10.1016 / j.ijpharm.2020.119072]. Therefore, it was used together with light obscuration in this study.

[0094] Although this study showed interesting results, such as an increase in (sub)visible particles in the liquid formulated biopharmaceuticals agitated two-dimensionally, it was also well seen that the addition of surfactants significantly reduced the formation of (sub)visible particles, and thus, the prevention measures taken by formulation developers against aggregation and particle formation were effective. The use of a two-dimensional shaker as disclosed herein may be very useful for: enabling formulation scientists and pharmaceutical companies to understand how their biopharmaceutical products are agitated during transportation and how to best replicate these conditions internally to test drug products during development.

[0095] Example 2

[0096] Range

[0097] Product Quality Impact (PQI) studies are conducted on large molecule products such as antibody drugs to evaluate the impact of commercial shipping conditions on the quality attributes of drug products via representative shipping routes. The main objective of this study is to record and analyze the vibrations and impacts that occur during real-life shipments and how they propagate through the entire stack of products on a pallet. These shipping stresses will be recorded by accelerometers along a representative supply chain for EU / US distribution.

[0098] This study provides a better understanding of the long-term vibrations within the package and their orientation. From simulations and experiments, it is well known that horizontal and vertical vibrations do have different effects on the movement of fluids and thus different contributions to the stress on the drug (Zubiaga et al., 2019).

[0099] Three different investigational drugs (Active 1, Active 2, and Active 3) are shipped together with accelerometers and analyzed for the potential quality impact of the shipment on the drug product (DP). This will allow a direct correlation between the recorded agitation stress profiles and the corresponding product quality impacts. The aim is to compare existing model systems with respect to product quality impact and develop novel laboratory-based methods to replace real-life shipment studies.

[0100] The knowledge collected allows testing of products in the laboratory under more realistic (i.e., representative) shipping conditions. Such laboratory-based model systems will help save the cost, resources, and environmental footprint of real-life shipment studies. Further in-depth understanding of the damping and resonance effects of real shipping configurations (such as stacks of boxes) will enable those skilled in the art to account for these effects by adjusting the resulting model system conditions and parameters. In addition, a data base of the agitation of investigational drug product vials within their secondary packaging is also collected.

[0101] Drug product selection

[0102] This study assesses the impact of commercial shipping conditions on the quality attributes of drug products. In addition to water-filled vials used to fill the boxes and place the accelerometers, three different drugs will be used for this shipping study (Table 5).

[0103] Table 5. Drug product information. The formulation contains an antibody at a concentration range of 25 mg / ml to 180 mg / ml, a suitable buffer in the range of 10 mM to 200 mM as used in the art for parenteral formulations of antibodies, and a pH of 5.5 to 6.0.

[0104]

[0105] Rationale for drug product selection

[0106] These formulations have different sensitivities to changes in product quality under agitation stress. For all products, the fill volume and vial configuration are kept the same to rule out the headspace-to-fill volume ratio and surface-to-fill volume ratio as potential influencing factors. It is known that ACTIVE 1 and ACTIVE 2 are sensitive to surface-related aggregation. In contrast, Active 3 is assumed to be more stable upon oscillation. The formulations of ACTIVE 1 and ACTIVE 2 as described herein contain only minimal amounts of surfactant. Therefore, these formulations are more likely to exhibit relevant quality changes upon agitation.

[0107] The purpose of shipping these pharmaceutical product formulations is to assess the potential impact of agitation stress on the DP quality during a representative shipment. The study results will be interpreted by comparing the stress of the shipped samples and the control samples. Therefore, the materials should be subdivided into shipped samples and non-shipped control samples. The control samples will be stored in a controlled environment at 2°C to 8°C under the recommended storage conditions. To ensure that there are no visible particles, all samples and controls will be visually inspected before shipping. The DP will be analyzed after return. Vials filled with water are used as fillers in the shipping box, which achieves the true positioning of the accelerometer and the DP. The vials filled with water will not be analyzed.

[0108] Packaging configuration

[0109] Shipping case (tertiary packaging)

[0110] All vials and accelerometers are placed in a shipping box with a representative configuration. One shipping box contains 105 vials in secondary packaging.

[0111] Twenty vials of each laboratory-scale produced DP, i.e., a total of 60 samples, are included in the shipment. The DP vials will be distributed in the boxes containing accelerometers. The remaining space in each of these shipping boxes containing DP samples will be filled with vials filled with water. All other shipping boxes are specifically packed with vials filled with water.

[0112] Pallet configuration

[0113] A total of 24 shipping containers are placed on a pallet, with 3 layers, 8 secondary packaging boxes per layer (corresponding to the maximum allowable height of the pallet within a single container). Thus, the pallet will contain a total of 2,510 vials in secondary packaging (24 boxes à 105 vials -> 2,520 vials; minus 10 recorders -> 2,510). Of these, 2,450 are vials filled with water, and 60 are DP vials. The entire pallet is transferred in a hot shipping system (such as RKN e1 or Skycell 1500C) for air transportation. The pallet is placed at the bottom of the aircraft.

[0114] Agitation study Background Art

[0116] The wave changes as it travels through the medium, and an accelerometer is used to measure the vibration intensity at different positions within the pallet. A good example of practice is a weighing platform, where a large marble block is located on an elastomer. This system produces a very low resonance frequency. For such a system, any wave below this resonance frequency undergoes "rigid transmission", i.e., the wave does not change as it travels through the elastomer. If the excitation is at the resonance frequency, the vibration amplitude increases as it travels through the system (unless the system is above critical damping). The weighing platform system is designed such that all vibration frequencies are higher than the resonance frequency. In this case, the vibration intensity decreases significantly, and the measurement is to some extent isolated from external vibrations. The system can be considered a "mass - spring system", which, as Figure 10 shown, has its three colored regions (rigid transmission, vibration amplification, wave damping).

[0117] Similarly, a shipping container can be regarded as a mass - spring system; however, it essentially has an infinite number of degrees of freedom. Since the resonance frequency is likely to be below or within our range, it is expected that the packaging will affect the vibration intensity at the vials ( Figure 11 ).

[0118] When considering vibrations during transportation, there are generally two aspects, namely: impact and long - term vibration intensity. For many applications, impacts are more important because they impose high stresses on the structure, which can lead to the failure of the structure. On the other hand, general vibrations produce long - term loads, resulting in fatigue.

[0119] When considering fluids, impacts may be less important as there is no structure to be damaged. Impacts may cause brief intense movements, but over time, have little effect on the drug. On the other hand, long-term vibrations may potentially cause continuous movement and frequent splashing, and thus stress the drug, which may lead to its degradation. It is well known that horizontal and vertical vibrations do have different effects on the movement of fluids and thus different contributions to the stress on the drug (Zubiaga et al., 2019). Therefore, it is very important to characterize the stress input and propagation within our shipping system.

[0120] Agitation measurement

[0121] Two different objectives should be accomplished for the agitation measurements. First, obtain a complete time series of 3-axis acceleration data at a resolution of 1600 Hz throughout the shipping process. Additionally, the second objective is to understand the effect of the packaging on the vibrations up to the level of the secondary packaging (in which each vial is packed).

[0122] The accelerometer mounted on the lowest layer serves as a reference sensor, which measures the excitation vibrations of the pallet with sufficient accuracy for the purposes of this investigation. Laboratory studies have shown that the measurements regarding the lowest layer within the packaging configuration are comparable to the vibrations of the pallet located below. Due to the limited battery life and storage capacity of each recorder, five reference accelerometers will be activated continuously to cover a maximum span of 15 days.

[0123] To achieve the second objective, for example, to quantify the variation in vibration intensity, accelerometers are placed at different locations within the pallet. The reference measurements will be compared with the measurements of the remaining probe sensors placed at five different locations within the pallet. The reason for making multiple measurements is that the wave paths are significantly different for the secondary packaging closer to the bottom compared to that closer to the top. Comparing the data from the reference with the data from the probe sensors helps to understand and determine the vibration transfer from the shipping container through the pallet to the secondary packaging. This vibration transfer is characterized by the frequency-dependent vibration intensity ratio, which is the amplitude ratio of the probe measurement to the reference measurement.

[0124] The probe sensors record at intervals of 2 minutes of recording and 8 minutes of pause to cover the same span. Since the spectra are compared, this is sufficient to characterize the transmission of the vibrations.

[0125] Figure 12The experimental setup is conceptualized where the reference measurements are made on the lowest layer (black) inside the shipping box and the remaining probe sensors at different locations. The accelerometers (sensors, recorders, and batteries inside the device) are shown on the right side of the figure. Importantly, the pallet and the box should be handled as representative as possible while still remaining within the recording period of the accelerometers. Any special handling may lead to misinterpretation of the data obtained.

[0126] Accelerometer and recorder holder

[0127] A total of 10 MSR data loggers are used to record the agitation stress during the entire real - life shipping process. The accelerometers will be located in custom - made logger holders that have the same external dimensions as the secondary carton packaging of a 6 - ml vial. The holder is milled from solid aluminum and the accelerometers are fixed to the holder by three M3 machine screws. The accelerometer holder is designed such that it allows for a “rigid” transition within the target frequency spectrum. This means there are no resonance frequencies below 500 Hz. This ensures that we only measure external vibrations and the holder itself does not affect the measurement.

[0128] Drug product sample and sensor location

[0129] Based on the basic principle of positioning as explained above, the reference accelerometers are placed as close as possible to the center of the pallet, not in the corners of the box or in direct contact with each other. The probe accelerometers are distributed to cover the entire pallet and the edges. DP vials are added to the same box as the accelerometers, firstly to make a direct measurement of the vibrations inside the box and additionally to account for the packaging procedures and the operational aspects of accelerometer activation. This results in the following distribution of accelerometers and DP vials:

[0130] Boxes 8, 10, 13, 23, and 24 contain three vials of each DP and one probe accelerometer; box 7 contains five vials of each DP and two reference accelerometers; boxes 2, 3, and 6 contain one reference accelerometer.

[0131] The holder with the accelerometer contained therein is placed in the same way as the ordinary secondary packaging with vials. The exact positioning of each sensor within the pallet is defined ( Figure 13 and 14 ). Figure 13 An exemplary picture of the accelerometer in the holder inside the shipping box packed with vials in secondary packaging is shown.

[0132] Real-life shipping study procedure

[0133] All vials (including the accelerometers placed at dedicated locations) are subjected to real - life shipping conditions. Before starting the shipping study, all DP samples must be stored at 2 °C to 8 °C.

[0134] The unshipped control samples are stored at 2°C to 8°C for the duration of the shipping procedure. After the samples are returned, they are stored at 2°C to 8°C until analysis, and aliquots can be frozen at –70°C ± 20°C for future analysis.

[0135] The shipping plan is designed to represent typical shipments of the drug product. A qualified thermal shipping system is used to avoid temperature-related effects on the product. The product temperature needs to be maintained at 2°C to 8°C before shipping, throughout the shipping route, and after shipping until analysis. Each branch of the proposed route covers the following:

[0136] - Branch 1: By truck; duration: approximately 45 minutes.

[0137] - Branch 2: By truck; duration: approximately 5 hours.

[0138] - Branch 3: By flight; duration: approximately 9 hours.

[0139] - Branch 4: By truck; duration: approximately 11 hours.

[0140] - Branch 5: By truck; duration: approximately 57 hours.

[0141] Product quality assessment and acceptance criteria

[0142] The assessment of the quality of the DP after shipping and the corresponding control are based on quantitative tests and qualitative comparisons.

[0143] Table 6. Sampling test information. X = analysis, (X) = optional reading, NT = not tested.

[0144]

[0145]

[0146] Since the DP formulation and configuration used in the study are specifically designed for this study, acceptance criteria are not given. Therefore, the results are evaluated according to the acceptance criteria (within the determination accuracy) with no significant changes for each test method. The results of the shipped samples are directly compared with the unshipped vials used as controls.

[0147] Regarding the following, the chromatographic (SE-HPLC and IE-HPLC) curves of the unshipped controls and the shipped samples are compared and evaluated: a) the absence of new peaks and b) the overall curve comparability (within the determination accuracy). In addition, the results of the shipped samples are directly compared with the unshipped vials used as controls to assess only the impact of shipping on the product quality.

[0148] Results

[0149] Monitor the temperature during transportation. After the initial cooling of the sensor and the package, the temperature remains stable within the expected range( Figure 14 ).

[0150] Vibration monitoring

[0151] Monitor vibrations to understand real-world stress conditions. Measure the overall vibration input to the pallet and additionally determine the distribution of vibrations within the stack of packages throughout the package.

[0152] Long-term vibration

[0153] Record vibrations by measuring 3-axis acceleration at 1600 Hz, so there are 1600 data points per second. To cover transportation, five reference recorders at the bottom of the pallet record continuously.

[0154] Total vibration energy

[0155] Grms (root mean square of gravity), defined as the area under the PSD curve, measures the total energy input through random vibration.

[0156] The time series shows that vibrations occur during branches (1 to 5) of transportation. Compared to flights (branch 3), vibrations are stronger during truck transportation (branches 4 and 5). Additionally, recorders at different heights within the pallet show that the total energy is highest at the top (M5) and has a decreasing order down to the reference recorder (R) at the bottom of the pallet( Figure 15 ). This indicates that vibrations are amplified through the stack of packages.

[0157] The overall vibration intensity is lower than that recommended by typical vibration test guidelines such as ASTM D4169-16.

[0158] Spectral density of vibration

[0159] To analyze the power spectral density (PSD) of vibrations, combine, perform a fast Fourier transform (FFT) on 2-minute intervals. The PSD curve describes the random vibration spectral density, e.g., at which frequencies what energy levels occur during that 2-minute interval. Plot the PSD curves of all combined 2-minute intervals as density. The density shows the distribution of the intervals. Based on the density, quantiles and corresponding PSD curves can be derived in the same way as for the ASTM D4169 guidelines, e.g., the 10% or 5% most severe vibrations.

[0160] The PSD distribution is plotted, and the color gradient shows the quantiles or frequencies of such PSD curves to appear. The darkest areas are the most frequent during shipping, such as average vibrations. Yellow is less likely to occur, such as very low or high vibrations. Similar to the ASTM D4169-16 guidelines, the corresponding assurance levels are plotted in solid black.

[0161] Assurance level:

[0162] Level I: 99% of the vibrations that occur exceed this line

[0163] Level II: 95% of the vibrations that occur exceed this line

[0164] Level III: 90% of the vibrations that occur exceed this line

[0165] For comparison, the relevant ASTM D4169-16 guideline curve is plotted as a blue dashed line.

[0166] The vibrations between branches are significantly different. Air transportation has the lowest vibration intensity, and the solid black PSD assurance level is significantly lower than the blue dashed line. Therefore, smaller stresses are detected during transportation than those typically tested during laboratory-based ASTM D4169-16 tests. The reasons for this may be smooth flights, vibration absorption by the thermal shipping system, or overall improved aviation technology and large aircraft.

[0167] During truck transportation, the overall intensity is stronger than during air transportation. Compared with the general test guidelines, some frequencies between 8 Hz and 20 Hz and below 3 Hz are stronger. Generally speaking, for most frequencies, the measured vibrations are within or below expectations. The results are presented in Table 16.

[0168] Transport legs and modes

[0169] To compare different branches of transportation, the data of the reference recorder are compared. Similar amplification patterns are found for all transportation routes. Frequencies between 10 Hz and 60 Hz are amplified in the entire stack of packages, while frequencies above 200 Hz are attenuated. The results are consistent with laboratory-scale tests that observed the same effect.

[0170] Impact

[0171] Most impacts are recorded during the loading and writing of the goods and during truck transportation (branches 1, 2, 4, and 5) ( Figure 17 ).

[0172] Product quality

[0173] No impact on product stability was detected. The stress conditions did not result in measurable degradation of the selected model drug. Subsequently, no impact of the position of the drug product container within the pallet was detected either.

[0174] No impact of the stress conditions on the following quality attributes was found: size variants, charge variants, color, turbidity, visible particles, sub-visible particles.

[0175] Discussion

[0176] Vibration

[0177] The comprehensive vibration data set shows real-life stress conditions during transportation. Novel data on lateral vibration was collected and this data can be used for testing vibrations on multiple axes. The overall energy input was lower than the current laboratory test procedures. An impact of the position within the pallet was observed. The PSD frequency between 10 Hz and 60 Hz was highest on top of the pallet and higher than the frequency according to the current laboratory test guidelines. Therefore, the worst-case curve of the highest PSD on the combined pallet can be used as the future worst-case test curve.

[0178] Impact on product quality

[0179] No significant decrease or impact on the quality attributes was detected. Neither the stable reference nor any less stable formulations showed any decrease. Even with the addition of a small amount of surfactant (0.01% w / v), PS20 clearly protected the protein from aggregation (Figure 20). Therefore, the vibration intensity and stress conditions were lower than those typically tested during the development of the drug product. It is assumed that the transportation intensity was lower than expected and that the drug product was stable enough to withstand these conditions.

[0180] During development, data on SvP formation and aggregate formation was observed using an oscillating model system. Therefore, the intensity of real-life transportation was less.

[0181] Example

[0182] Further specific embodiments of the present invention are listed below.

[0183] 1. In an embodiment, a method for simulating the quality impact of transportation on a liquid is disclosed, the method comprising the steps of:

[0184] a) Selecting a set of instructions, the set of instructions comprising: i) one or more PSD curves, which include a plurality of frequencies and their corresponding amplitudes on at least two vertical axes (e.g., X and Z), and ii) a schedule, which specifies the duration for each of the PSD curves;

[0185] b) inducing vibrations in the liquid according to the PSD curve; and

[0186] c) assessing and comparing the quality of the liquid before and after performing step b).

[0187] 2. In an embodiment, a method according to embodiment 1 is disclosed, wherein the vibrations on two axes are induced simultaneously.

[0188] 3. In an embodiment, a method according to embodiment 1 or 2 is disclosed, wherein the liquid comprises a pharmaceutical product.

[0189] 4. In an embodiment, a method according to any one of embodiments 1 to 3 is disclosed, wherein the liquid comprises a biological product.

[0190] 5. In an embodiment, a method according to embodiment 4 is disclosed, wherein the biological product is a protein (such as an antibody), nucleic acid, sugar or conjugate and combinations thereof.

[0191] 6. In an embodiment, a method according to any one of the foregoing embodiments is disclosed, wherein the instruction set as in embodiment 1a is different for different axes.

[0192] 7. In an embodiment, a method according to any one of the foregoing embodiments is disclosed, wherein the amplitude of the vibration on the first axis is proportional to the amplitude of the vibration on the second axis.

[0193] 8. In an embodiment, a method according to any one of the foregoing embodiments is disclosed, wherein the PSD curve is designed based on measurements of vibrations in one or more real-life shipments.

[0194] 9. In an embodiment, a method according to any one of embodiments 1 to 8 is disclosed, wherein the frequency range in the PSD curve is between 1 Hz and 300 Hz.

[0195] 10. In an embodiment, a method according to any one of embodiments 1 to 8 is disclosed, wherein the frequency range in the PSD curve is between 15 Hz and 80 Hz.

[0196] 11. In an embodiment, a method according to any one of embodiments 1 to 7 is disclosed, wherein the PSD curve is as represented in FIGS. 16(A to G).

[0197] 12. In an embodiment, a method according to any one of embodiments 1 to 7 is disclosed, wherein the PSD curve is according to the ASTM D4169 standard, such as aviation class I, II or III, railway class I, II or III, or truck class I, II or III.

[0198] 13. In an embodiment, a method according to any one of the foregoing embodiments is disclosed, wherein an analytical method, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), analytical ultracentrifugation, visible or sub-visible particle analysis, is used to perform the quality assessment according to Example 1c.

[0199] 14. In an embodiment, a two-dimensional oscillator suitable for performing the steps of the method according to any one of the foregoing embodiments is disclosed.

[0200] 15. In an embodiment, a two-dimensional oscillator according to Example 14 is disclosed, wherein the oscillator includes a temperature control chamber.

[0201] 16. In an embodiment, a two-dimensional oscillator according to Example 15 is disclosed, wherein the chamber temperature is maintained at 2°C to 60°C, such as 2°C to 8°C.

[0202] 17. In an embodiment, a two-dimensional oscillator according to any one of Examples 14 to 16 is disclosed, wherein the oscillator includes a vial holder and / or a holder for a syringe.

[0203] 18. In an embodiment, a two-dimensional oscillator according to Example 17 is disclosed, wherein the holder is adapted to act at different tilt angles.

[0204] 19. In an embodiment, a device for simultaneously inducing vibrations in at least two perpendicular directions (e.g., the X and Z axes) in a liquid is disclosed.

[0205] 20. In an embodiment, a device according to Example 19 is disclosed, wherein the device includes at least one individually controlled linear motor for each axis.

[0206] 21. In an embodiment, a device according to Example 19 or 20 is disclosed, wherein the device induces vibrations based on an instruction set received by the device, the instruction set including: i) one or more PSD curves, which include a plurality of frequencies and their corresponding amplitudes on at least two perpendicular axes (e.g., X and Z), and ii) a schedule, which specifies the duration for each of the PSD curves.

[0207] 22. In an embodiment, a device according to any one of Examples 19 to 21 is disclosed, wherein the liquid includes a pharmaceutical product.

[0208] 23. In an embodiment, a device according to any one of Examples 19 to 22 is disclosed, wherein the liquid includes a biological product.

[0209] 24. In an embodiment, there is disclosed an apparatus according to embodiment 23, wherein the biological product is a protein (such as an antibody), nucleic acid, sugar, or conjugate, and combinations thereof.

[0210] 25. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 24, wherein the instruction set as in embodiment 21a is different for different axes.

[0211] 26. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 25, wherein the PSD curve is designed based on measurements of vibrations in one or more real-life shipments.

[0212] 27. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 26, wherein the frequency range in the PSD curve is between 1 Hz and 300 Hz.

[0213] 28. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 26, wherein the frequency range in the PSD curve is between 15 Hz and 80 Hz.

[0214] 29. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 25, wherein the PSD curve, i.e., the frequency and its corresponding amplitude, is represented by a solid or dashed line as in FIGS. 16(A to G) or is within a 9-dB range in its amplitude.

[0215] 30. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 25, wherein the PSD curve is according to the ASTM D4169 standard, such as aviation class I, II, or III, railway class I, II, or III, or truck class I, II, or III.

[0216] 31. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 30, wherein the apparatus includes a temperature-controlled chamber.

[0217] 32. In an embodiment, there is disclosed an apparatus according to embodiment 31, wherein the chamber temperature is maintained between 2°C and 60°C, such as between 2°C and 8°C.

[0218] 33. In an embodiment, there is disclosed an apparatus according to any one of embodiments 19 to 32, wherein the apparatus includes a vial holder and / or a holder for a syringe.

[0219] 34. In an embodiment, there is disclosed an apparatus according to embodiment 33, wherein the holder is adapted to act at different tilt angles.

[0220] 35. In an embodiment, there is disclosed a device according to any one of embodiments 19 to 34, which is used for the method according to any one of embodiments 1 to 13.

Claims

1. A method for simulating the impact of transportation on the quality of a liquid, the method comprising the following steps: a) Selecting an instruction set, the instruction set including: i) one or more PSD (Power Spectral Density) curves, which include a plurality of frequencies and their corresponding amplitudes on at least two vertical axes (e.g., X and Z), and ii) a schedule that specifies the duration for each of the PSD curves; b) Inducing vibrations in the liquid according to the PSD curves; and c) Evaluating and comparing the quality of the liquid before and after performing step b.

2. The method according to claim 1, wherein the vibrations on the two axes are induced simultaneously.

3. The method according to claim 1 or 2, wherein the liquid includes a pharmaceutical product.

4. The method according to any one of claims 1 to 3, wherein the liquid includes a biological product, such as a protein (e.g., an antibody), a nucleic acid, a sugar, or a conjugate, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the instruction set as in claim 1a is different for different axes and / or wherein the amplitude of the vibration on the first axis is proportional to the amplitude of the vibration on the second axis.

6. The method according to any one of claims 1 to 5, wherein the PSD curve is designed based on measurements of vibrations in one or more real-life shipments.

7. The method according to any one of claims 1 to 6, wherein the frequency range in the PSD curve is between 1 Hz and 300 Hz or between 15 Hz and 80 Hz.

8. The method according to any one of claims 1 to 5, wherein the PSD curve, i.e., the frequency and its corresponding amplitude, is represented by the solid or dashed lines in FIGS. 16(A to G) or is within a 9-dB range in its amplitude.

9. The method according to any one of claims 1 to 5, wherein the PSD curve is according to the ASTM D4169 standard, such as aviation class I, II, or III, railway class I, II, or III, or truck class I, II, or III.

10. The method according to any one of claims 1 to 9, wherein an analytical method, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), analytical ultracentrifugation, visible or subvisible particle analysis, is used for the quality evaluation as in claim 1c.

11. A two-dimensional oscillator adapted to perform the method steps as in claim 1b or any one of claims 2 to 9 dependent thereon.

12. A device for inducing vibrations in a liquid simultaneously in at least two vertical directions (e.g., X and Z axes), optionally wherein the device induces vibrations based on an instruction set received by the device, the instruction set including: i) one or more PSD curves, which include a plurality of frequencies and their corresponding amplitudes on at least two vertical axes (e.g., X and Z), and ii) a schedule that specifies the duration for each of the PSD curves.

13. The device according to claim 12, wherein the PSD curve is designed based on measurements of vibrations in one or more real-life shipments (e.g., as described in claim 8), and / or the frequency range in the PSD curve is between 1 Hz and 300 Hz (e.g., 15 Hz to 80 Hz).

14. The two-dimensional oscillator according to claim 11, or the device according to claim 12 or 13, further comprising a temperature control chamber, optionally wherein the chamber temperature is maintained between 2 °C and 60 °C (e.g., 2 °C to 8 °C).

15. The two-dimensional oscillator according to claim 11 or 14, or the device according to any one of claims 12 to 14, further comprising a vial holder and / or a holder for a syringe, optionally wherein the holder is adapted to act at different tilt angles.

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