Method and device for simulation of perturbation stress on liquids - Patents.com

Abstract

The present invention relates to a method for simulating the quality effects of transportation on a liquid, and to a two-dimensional shaker adapted to carry out said method.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for simulating the effects of transport on the quality of a liquid, and to a two-dimensional shaker adapted to carry out said method. [Background technology]

[0002] Background of the Invention When developing a pharmaceutical product, there are many factors that must be considered. Among the many factors to evaluate, product stability and integrity are essential to ensure product efficacy and safety. Stability is of paramount importance, especially when biological and biopharmaceutical products are being developed. The complex molecular makeup of these large molecules makes them highly vulnerable to changes in their conformation and structure. External factors such as temperature changes, pH changes, and surface adsorption can induce protein aggregation, protein particles, and chemical modifications [Das, TK; 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, much development work is dedicated to finding formulations of biopharmaceuticals that can preserve their structure and thereby ensure their efficacy and safety [Das, TK 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 manufacturing and distribution process of biopharmaceuticals can create stress conditions that can affect protein structure. One of these steps is the delivery of the drug to the patient [Das, TK et al, 2020, DOI: 10.1016 / j.xphs.2019.09.023]. To administer biopharmaceuticals parenterally, they must be in liquid formulations. During delivery, the movement of liquids induces shear forces or interfacial effects that can degrade or aggregate proteins. Therefore, pharmaceutical companies must test the robustness against vibration, temperature changes, liquid-air interfaces, shear stress and other types of stress that may harm the quality of their products [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; Koepf, E. et al, DOI:10.1016 / j.ijpharm.2017.12.043; Maa, YF; Hsu, CC, 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 freeze-drying, a process that removes water and converts the solution into a solid cake or powder, extending shelf life and facilitating transportation. However, this process is very labor-intensive and requires reconstitution, so "ready-to-use" formulations are used [Bjelosevic, M.; Zvonar Pobirk et al, 2020, DOI: 10.1016 / j.ijpharm.2020.119029; Bye, J. Wet al, 2014, DOI: 10.1007 / s10529-013-1445-6].Add to this the increasing demand for convenient auto-injectors and pre-filled syringes, and lyophilized formulations are not an option (Bye, JWet et al, 2014, DOI: 10.1007 / s10529-013-1445-6; Sassalos, TM, Paulus, YM, 2019, DOI: 10.2147 / OPTH.S169044). A method to prevent aggregation in liquid-formulated biopharmaceuticals is to add specific inactive ingredients (excipients) to the drug product. Examples of these excipients include disaccharides and surfactants, but altering the pH and protein concentration can also prevent protein aggregation [Narhi, LO et al, 2022, DOI: 10.1016 / j.xphs.2022.01.011; Das, TK et al, 2021, DOI: 10.1016 / j.xphs.2021.09.030].

[0004] The FDA and other health authorities require release validation studies in which the robustness and quality of biologics after shipment are evaluated [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; Narhi, LO et al, 2022, DOI: 10.1016 / j.xphs.2022.01.011]. To assess product robustness and quality after shipment, pharmaceutical companies must conduct real-life release (RLS) studies in which shipments of new drugs are sent through representative distribution channels (typically required by the FDA). To submit new biologics to other health authorities, such as the EMA, companies must conduct a simulation of transportation, known as the American Society for Testing and Materials (ASTM) vibration test (ASTM D4169). There are three modes of transportation (rail, truck, and air), and each mode covered by the ASTM method has three intensity levels (I, II, and III). Currently, guidelines recommend a program in which Levels I, II, and III are tested consecutively 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, research has shown that current methods for evaluating product quality after transport stress are not very representative and are quite expensive [Borocz, P, 2019, DOI: 10.1002 / pts.2434; Nemeth, Z. et al, 2021, DOI: 10.14513 / actatechjaur.00603]. First, the ASTM D4169 vibration test covers only vertical vibration, whereas pharmaceutical products undergo three dimensions of vibration during transport. Knowledge and data about the real-world transport stresses biological products experience and whether adding another dimension alters the stresses to which pharmaceutical products are exposed are limited. Summary of the Invention

[0006] In view of the above, an object of the present invention is to provide a two-dimensional shaker to more accurately replicate the shaking stress on liquid pharmaceutical formulations during transportation, since two-dimensional vibration more closely resembles real-life shipping. The device and method described herein can be used, among other things, for the evaluation of liquid formulations during the development process and to meet quality requirements mandated by health authorities. [Brief explanation of the drawings]

[0007] [Figure 1] Different waveforms are observed between horizontal (X-axis), vertical (Z-axis), and combined (XZ-axis) vibrations. A representative image of a 6 ml vial filled with 3.2 ml is shown. The stills were taken from high-speed video footage (200 fps). [Figure 2A]Comparison of particles among four different antibodies in liquid formulations shaken on three different axes (X, Z, and XZ). Each antibody formulation was filled into a 6 ml vial and shaken for 30 minutes using the ASTM D4169-16 Aviation Level I profile. N=3 for shaken antibody formulations, N=1 / 2 for controls. A: Visual inspection of vials with photographs of a vial containing many particles (left) and a non-shaken control vial (right). B: Subvisible particle count results with light obscuration. Amount of particles per ml in size ranges 2-4 μm, 5-9 μm, and 10-24 μm; EP=EP black / white box; SD=Seidnenader. [Figure 2B] Comparison of particles between four different antibodies in liquid formulations shaken on three different axes (X, Z, and XZ). [Figure 3] Comparison of three different temperatures: 5°C, 23°C, and 30°C. CEA antibody formulations were filled into 6ml vials and shaken in the XZ axis for 30 minutes using the ASTM D4169-16 Aviation Level I profile. N=3 for shaken antibody formulations, N=1 / 2 for the control. A: Visual inspection of vials. B: Subvisible particle count results with light obscuration. Quantity of particles per ml in the size ranges 2-4μm, 5-9μm, and 10-24μm; EP=EP black / white box; SD=Seidnenader. [Figure 4] Comparison of particles in 6ml and 20ml vials with the same vertical fill height (3.2ml and 6.4ml, respectively) shaken in two different vial orientations (horizontal and vertical). CEA formulations were filled in either 6ml or 20ml vials and shaken in the XZ axis for 30 minutes with an ASTM D4169-16 Aviation Level I profile. N=3 for shaken antibody formulation, N=2 for control. A: Visual inspection of vials. B: Subvisible particle count results with light obscuration. Quantity of particles per ml in the size ranges 2-4µm, 5-9µm, and 10-24µm; EP = EP black / white box; SD = Seidnenader. [Figure 5] Study of surfactants by visual inspection. [Figure 6A-1]Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis rocking for different concentrations of PS80 at different time intervals. CEA antibody formulations were filled into 6 ml vials and shaken in XZ-axis (A) or Z-axis (B) for 15, 30, 60, or 120 minutes with ASTM D4169-16 Aviation Level I profile. N=1 for all antibody formulations. A: Subvisible particle analysis with light obscuration: XZ rocking. B: Subvisible particle analysis with light obscuration: Z rocking. [Figure 6A-2] Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 6A-3] Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 6B-1] Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 6B-2] Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 6B-3] Comparison of light-obscured subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 7A-1] Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis shaking for different concentrations of PS80 at different time intervals. CEA antibody formulations were filled into 6 ml vials and shaken in XZ-axis (A) or Z-axis (B) for 15, 30, 60, or 120 minutes with ASTM D4169-16 Aviation Level I profile. N=1 for all antibody formulations, with three samples taken from one vial. BMI=Background membrane imaging. A: Subvisible particle analysis by BMI: XZ shaking. B: Subvisible particle analysis by BMI: Z shaking. [Figure 7A-2] Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 7A-3]Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 7B-1] Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 7B-2] Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 7B-3] Comparison of background membrane imaging subvisible particle analysis between XZ-axis and Z-axis oscillations for different concentrations of PS80 at different time intervals. [Figure 8] Surface tension results obtained from CEA with different concentrations of PS80. [Figure 9] 6 mL vials in single packet packaging configuration with double leaflets for the water-filled vials (left) and a single leaflet for the drug product vial (right). [Figure 10] Schematic mass-spring system. A: spring; B: fixed moving body; C: vibration amplification; D: stiffness transmission; E: wave damping. [Figure 11] Schematic path of a wave through packaging. A: Thermal insulation including cooling; B: Packaging; C: Secondary packaging; D: Pallet; E: Vial; F: Wave path; G: Vehicle surface (e.g., airplane or truck). [Figure 12] Accelerometer readings and reference sensor position (left); MSR165: logger with internal battery and sensor (right). A: Probe reading; B: Reference reading. The amplitude ratio can be calculated by dividing the probe reading by the reference reading. [Figure 13] The accelerometer inside the holder is placed inside the shipping box (exemplary). [Figure 14] Temperature during transport. [Figure 15] Total vibration energy (Grms) during transportation on three axes: X-axis, Y-axis, and Z-axis. [Figure 16A] PSD distribution and density. A: Refrigerated truck pallet; Z-axis data; B: EKR1 active thermal shipper (truck) pallet; Z-axis data; C: EKR1 active thermal shipper (flight) pallet; Z-axis data; D: Refrigerated truck pallet; Z-axis data; E: Refrigerated truck pallet; Y-axis data; F: Refrigerated truck pallet; X-axis data; G: Refrigerated truck pallet; Z-axis data. For all panels, the PSD profiles indicate the assurance levels (Levels I, II, and III, as described in Example 2) with solid lines. For comparison, the relevant ASTM D4169-16 guideline profiles are plotted with dotted lines. [Figure 16B] PSD distribution and density. [Figure 16C] PSD distribution and density. [Figure 16D] PSD distribution and density. [Figure 16E] PSD distribution and density. [Figure 16F] PSD distribution and density. [Figure 16G] PSD distribution and density. [Figure 17] Shock recorded during transport. [Figure 18A] Comparison of data from horizontal shaking during formulation development with actual shipping data. Panel A: Subvisible Particles (SvP). Panel B: Size Exclusion High Performance Liquid Chromatography (SE-HMW). In both Panels A and B, labels are as follows: A: Active 2 formulation; B: Active 3 formulation; C: Active 1 formulation; D: Active 1 formulation with 0.01% surfactant after 7 days of shaking at 5°C; E: Active 1 formulation with 0.01% surfactant after 7 days of shaking at 25°C; F: Active 1 formulation without surfactant after 7 days of shaking at 5°C; G: Active 1 formulation without surfactant after 7 days of shaking at 25°C. [Figure 18B] Comparison of data from horizontal shaking during formulation development with data from real-world shipping. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description of the Invention When used below, the terms "having," "comprising," or "including," or any grammatical variants thereof, are used in a non-exclusive manner. Thus, these terms can refer both to a situation in which no further features are present in the entity described in this context other than the features introduced by these terms, and to a situation in which one or more further features are present. As an example, the expressions "A has B," "A comprises B," and "A includes B" can both refer to a situation in which no other elements are present in A other than B (i.e., a situation in which A consists solely and exclusively of B), and to a situation in which one or more further elements are present in entity A other than B, such as element C, elements C and D, or further elements.

[0009] Furthermore, when used hereinafter, the terms "particularly," "more particularly," "particularly," "more particularly," or similar terms are used in conjunction with optional features without limiting the possibility of substitution. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The present invention can be implemented by using alternative features, as recognized by those skilled in the art. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be optional features without any limitation regarding alternative embodiments of the invention, without any limitation regarding the technical scope of the invention, and without any limitation regarding the possibility of combining the feature introduced in this way with other optional or non-optional features of the invention.

[0010] 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 refers specifically to the first three modes. The terms "transportation," "transport," "shipment," and "shipping" are used interchangeably herein and refer to the same concept.

[0011] In one aspect, provided herein is a method for simulating the quality impact of transportation on a liquid, comprising: a) selecting a set of instructions including: i) one or more PSD profiles including a plurality of frequencies and their corresponding amplitudes in at least two perpendicular axes (e.g., X and Z); and ii) a timetable specifying a duration for each of the PSD profile(s); b) inducing vibrations in the liquid according to said PSD profile(s); c) evaluating and comparing the quality of the liquid before and after performing step b; A method is provided, comprising:

[0012] PSD (Power Spectral Density, or as it is often called, Acceleration Spectral Density for Vibration, or ASD) 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., vibration) describes the power present in a wave as a function of frequency per unit frequency. Power Spectral Density is commonly expressed in watts per hertz (W / Hz) or g 2 / Hz, where g represents g-force. As used herein, a PSD profile refers to information that includes one or more frequencies along with their amplitude and / or power density.

[0013] In some specific embodiments, vibrations in two axes are induced simultaneously, i.e., the liquid is vibrated in both directions either for part or the entire process. In some embodiments, the liquid comprises a pharmaceutical. In particular, the pharmaceutical comprises a biological product, such as a protein, an antibody, a nucleic acid, a sugar, or conjugates and combinations thereof.

[0014] In some embodiments, the sets of commands for different axes (e.g., X and Z) will be different. The differences may be, for example, in the PSD profile and / or the period specified for each of the frequencies applied to induce vibrations in the liquid.

[0015] In some embodiments, the amplitude of vibration in one axis is proportional (e.g., linearly proportional) to the amplitude of vibration in the other axis, e.g., the vibration density in one axis can be about 1.5 to 10 times the vibration density in the other axis.

[0016] In some embodiments, a PSD profile is designed based on measurements of vibrations during one or more real-world shipments (RLS). The vibrations can be measured, for example, by including sensors in packages shipped in the RLS and recording their frequency and density over the shipping period. The density in the PSD profile can be estimated, for example, by calculating the average of the densities recorded by several sensors during the RLS. Example 2 demonstrates an exemplary embodiment in this regard. In an embodiment, the PSD profile is similar to that shown in Figures 16(A-G). In an embodiment, the PSD profile is within 9 dB (decibels) of the solid or dotted lines of the PSD plots in Figures 16(A-G), meaning that for a given frequency, the intensity will be as indicated by one of the lines or be up to 9 dB less or more (e.g., 1 dB, 2 dB, 3 dB, 4 dB, 5 dB, 6 dB, 7 dB, 8 dB, or 9 dB) than that intensity.

[0017] In some embodiments, the PSD profile complies with the ASTM D4169 standard, e.g., aviation level I, II, or III, railroad level I, II, or III, or truck level I, II, or III. The ASTM standard (designated 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 therefore designated ASTM D4169-22).

[0018] In some embodiments, the quality assessment as a liquid is performed using analytical methods, such as analytical methods known to those skilled in the art and applicable to the particular liquid, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), analytical ultracentrifugation, visible or subvisible particle analysis.

[0019] In an embodiment, provided herein is a two-dimensional shaker adapted to perform the steps of the method of the first embodiment described above. By "two-dimensional," we mean that the shaker has a means that allows it to shake an object (e.g., a liquid container) in two perpendicular directions simultaneously. Such a shaker can be devised, for example, by implementing independent linear motors for moving the liquid sample along each axis. In particular, noise cancellation methods are applied to the motor controllers to eliminate interdependencies between movements in different directions. This is achieved, for example, by using sensors on each axis to detect and counteract unwanted vibrations.

[0020] In some embodiments, the shaker can perform a frequency sweep, which involves inducing vibrations starting at one extreme of the frequency range (e.g., the lowest frequency) and gradually increasing the frequency toward the other extreme (e.g., the highest frequency). The sweep can be performed on two axes in a parallel fashion (i.e., both starting at a low frequency or both starting at a high frequency) or an anti-parallel fashion (i.e., starting at a low frequency on one axis and a high frequency on the other). In some embodiments, the vibration is in the form of a constant sine wave (e.g., no sweep). In some specific embodiments, the frequency ranges from 1 to 300 Hz.

[0021] In some embodiments, the shaker includes a temperature-controlled chamber, which allows the temperature of the liquid to be maintained within an appropriate range during the procedure, e.g., a temperature similar to that applied during RLS. For example, liquid formulations containing biopharmaceuticals are typically kept at a temperature of 2-8°C. In some embodiments, the temperature within the chamber is maintained at 2-60°C, e.g., 2-8°C.

[0022] In some embodiments, the shaker includes a vial holder(s) for syringes, allowing liquid samples to be placed in a variety of containers as needed. In some embodiments, the holder is adapted to function at various tilt angles.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to implement the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0024] The 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 and drawings, and from the claims. [Example]

[0025] Example 1 In this study, a new 2D shaker was used to evaluate the impact of 2D in vibration testing on product quality, as 2D vibration is closer to real-life shipping. Furthermore, the current practices followed by pharmaceutical companies regarding protecting formulations from possible extra stress were analyzed.

[0026] Vials were shaken in either one or two dimensions, and several external factors (e.g., temperature, surfactant concentration) were varied to evaluate their effects. Two-dimensional vibration, in contrast to one-dimensional vibration, was found to induce droplets within the vial. Two-dimensional vibration also showed the formation of more sub-visible particles than one-dimensional vibration. These results raised concerns about drug stability during shipping, and experiments using different concentrations of surfactant were conducted. Results showed that even the smallest concentration of surfactant tested, 120 times smaller than concentrations commonly used in biopharmaceutical formulations, was sufficient to inhibit the formation of sub-visible particles in one and two-dimensional vibration. This study formed the initial basis for testing two-dimensional vibration and helped formulation scientists understand how biopharmaceuticals are shaken during shipping and how these conditions can best be replicated in-house for testing formulations during development.

[0027] Experimental procedure Real-world shipping research A real-world shipping study was conducted in Europe and North America. Accelerometers were placed inside boxes between packaged, water-filled vials. These boxes were shipped in five stages (Table 2). After shipping, the accelerometers were analyzed, and power spectral density (PSD) profiles and plots were derived from the acceleration measurements. [Table 1]

[0028] Monoclonal Antibodies and Formulations Four different monoclonal antibodies (mAb1, mAb2, mAb3, and mAb4) formulated in buffer were used in this study, and their different properties are shown in Table 3. All formulations were filtered under laminar airflow conditions through 0.22-micrometer pore size (Millex Sterivex® GV) sterile filters before shipping, closed with bromobutyl Teflonized and siliconized stoppers, and finally sealed with aluminum crimp caps. Two vial sizes were tested: 3.2 ml was filled into 6 ml vials and 6.4 ml into 20 ml vials (Fiolax Type I glass, Schott). These fill volumes resulted in equal vertical fill heights, which, as found in previous studies, is beneficial for high-speed video analysis. For surfactant studies, different percentages of total oleate PS80 (0.0005, 0.001, 0.0015, 0.002, 0.003, 0.01, and 0.06) were added to mAb1. Vials were stored at 2-8 °C. [Table 2]

[0029] Protein concentration For the measurement of protein concentration, either a SoloVPE spectrophotometer (C.Technologies) or a UV / Vis-photometer Lambda35 (Perkin Elmer) was used.

[0030] Vibration Test The two-dimensional shaker of the present invention was used to agitate liquid antibody formulations. In the described study, an ASTM D4169 Guideline 2016 Aviation Level I PSD profile was used, with vibration in either the X-axis or Z-axis only, or both axes simultaneously. Parameters such as temperature, vial position, and vibration time were adjustable on the shaker. Controls were vials with the same antibody formulation but without agitation.

[0031] High-Speed ​​Video Analysis High-speed video footage of the shaking vial was recorded at 200 frames per second using a MotionBLITZ® EoSens mini 1-1 (MIKROTRON) combined with a Macro 100 F2.8 D AT-X PRO (Tokine) camera lens.

[0032] Visual inspection Visual inspection was performed using an EP black / white light box. Magnified visual inspection was performed using a Seidenader V90-T (Seidenader Maschinenbau GmbH). This method includes visual inspection through roller rotation and a 2x magnification lens.

[0033] light shielding A HIAC 9703+ equipped with a Beckman Coulter HRLD-150 detector was used. Samples were analyzed using four 200-microliter injections. The average of the last three injections was recorded. A COUNT CAL 5 μm (3000 particles per ml) Count Precision Standard from Firma Thermo Scientific (catalog number CC05) was used as the calibration standard. Between each sample analysis, the instrument was rinsed with water until the following particle counts were obtained: 20 particles or greater than 2 μm, 5 particles or greater than 5 μm, 1 particle or greater than 10 μm, 1 particle or greater than 25 μm, and 0 particles or greater than 50 μm.

[0034] Background membrane imaging Subvisible particle quantification was also performed using a high-throughput method by analyzing triplicate measurements with a Horizon instrument (Halo Labs, Burlingame, CA). A 0.4 μm pore size polycarbonate background membrane plate was used, with a sample volume of 40 μl per well in triplicate. Liquid was removed by vacuum at 200 mbar. Samples exceeding 3% membrane coverage were indicated as supersaturated. Horizon VUE software version 3.0.0.121 was used.

[0035] surface tension A drop profile analysis tensiometer (PAT1M, Sinterface) was used for surface tension analysis. The analysis was performed at 22°C for 960 seconds (16 minutes) per sample.

[0036] result First, we determined whether there were differences in waveforms between 1D and 2D vibrations. Using the strongest ASTM D4169016 profile (Aviation Level I), we could see different waveforms and splashing patterns (Figure 1). X-axis and Z-axis vibrations showed no splashing, while XZ vibrations showed splashing and droplet formation.

[0037] Simultaneous XZ oscillation induces more particle formation than one-dimensional oscillation To assess whether product quality was affected differently during vibration in one or two axes (X, Z, and a combination of XZ), vials were placed in a two-dimensional shaker and shaken at room temperature for 30 minutes using the ASTM D4169-16 Aviation Level I profile. This profile was chosen because it is the most severe vibration profile of the ASTM D4169-16 profiles and was most likely to generate particles. Four different antibodies were tested and compared. Observing the results, only vials exposed to simultaneous XZ vibration had more than 10 visible particles, resembling a particle cloud (Figure 2A). In the subvisible particle range, antibodies mAb1 and mAb4 also had the highest particle amounts for vials exposed to simultaneous XZ vibration. In the 2-4 μm particle range, a difference of at least 1,000 particles per ml is observed. Antibodies mAb2 and mAb3 also had the highest particle counts in the 2-4 μm particle range, but this trend did not persist at higher particle counts (Figure 2B).

[0038] Potential for reduced particle formation for formulations shaken at 5°C One advantage offered by the new prototype two-dimensional shaker is the ability to adjust the temperature of the shaking chamber. During transport, biopharmaceuticals are typically kept in a cooling cell at 2–8 °C, as higher temperatures are known to accelerate aggregation pathways [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]. In this experiment, we aimed to determine whether the harsh XZ-axis shaking would affect low and high temperatures for mAb1 formulations. Subvisible particle formation appeared to be lowest in the 5 °C shaken vial, its control, and the 23 °C control. The highest subvisible particles can be seen in the 30°C control and 23°C shaken vials (Figure 3B). Interestingly, the 5°C control showed more particle formation by visual inspection than its shaken version, which is not reflected in the light obscuration data (Figures 3A and 3B). Similarly, the 30°C control showed no visible particle formation but high subvisible particle formation.

[0039] Differences in particle formation between vial sizes and orientations Previous research has shown that the orientation of a vial when shaken affects the shear stress distribution in the vial [Bai, G. et al., 2012, DOI: 10.1016 / j.ijpharm.2011.11.044]. In this experiment, we aimed to investigate the impact of horizontal versus vertical orientation on (sub)visible particle formation and whether different vial sizes would have additional effects on mAb1 formulations. Vials were again shaken at room temperature in a two-dimensional ASTM D4169-16 Aviation Level I profile. Nearly all vials exhibited several particles upon visual particle inspection (Figure 4A). Only the 6 ml vials shaken horizontally and vertically exhibited a maximum of one particle in the black-white box, but more than 10 particles under Seidnenader analysis. From the collected results, it was striking that different vial sizes exhibited opposing effects with respect to orientation. The 20 ml vial shows more subvisible particles in the horizontal position, while the 6 ml vial shows more subvisible particles in the vertical position (Figure 4B).

[0040] Polysorbate 80 reduces particle formation in one- and two-dimensional vibratory liquid formulations To evaluate whether previously used pharmaceutical approaches to particle prevention are relevant to 1D and 2D vibrational stress, mAb1 formulated in buffer was mixed with different concentrations of polysorbate 80 (PS80) (Table 4). The formulations were assumed to have approximately the same protein concentration, but due to calculation errors, the 0.06% PS80 formulation had a much lower concentration. These different formulations were then agitated in the XZ and Z axes for 15, 30, 60, or 120 minutes at room temperature. The formulations were then analyzed for (subvisible) visible particles using visible particle analysis, light obscuration, and background film imaging. Visible particle analysis showed more than 10 particles in the 0% PS80 formulation shaken for 15 and 30 minutes in the XZ axis (Figure 5) and in the 0.0005% PS80 formulation shaken for 60 minutes. [Table 3]

[0041] When considering light-obscured subvisible particle analysis, the amount of particles in the surfactant-free formulation is higher than in the formulation with PS80 (Figures 6A and 6B). Also, when surfactant-free formulations are involved, there appears to be a time-dependent increase in particles, with longer shaking times resulting in higher particle amounts. Interestingly, for Z-axis rocking, 60 and 120 minutes of shaking appear to have a much greater impact on particle amount than 15 and 30 minutes.

[0042] The background membrane imaging results also depict a decrease in subvisible particles when surfactant is added to the formulations for XZ rocking (Figure 7A). However, for Z rocking, the number of particles in the 0% PS80 formulation rocked for 15 minutes appears to be higher, but there appears to be less significant difference between them and the remaining formulations (Figure 7B).

[0043] Previous studies have shown a correlation between surface tension and aggregation / particle formation. Using a droplet profile analysis tensiometer, we determined the surface tension of mAb1 with different concentrations of PS80 used in previous experiments. As shown in Figure 8, higher concentrations of PS80 conferred lower surface tension.

[0044] Consideration The purpose of this study was to gain more knowledge about transportation stresses and determine whether current precautions during formulation development are sufficient to prevent impacts to product quality. The 2016 ASTM D4169 Aviation Level I profile was used for this test because it is the most rigorous profile and ensures several particle loads that can be compared.

[0045] In this study, we first investigated the difference between one-dimensional and two-dimensional liquid fluctuations due to vibration. The results seem to indicate that simultaneous XZ vibrations induce more particle formation than one-dimensional vibrations. This is most likely due to the splashing observed during high-speed video analysis. Previous studies have investigated that droplets in liquid formulations cause high shear stress and therefore protein aggregation [Koepf, E. et al, DOI: 10.1016 / j.ijpharm.2017.12.043; Maa, YF; Hsu, CC, 1997, DOI: 10.1002 / (SICI)1097-0290(19970620)54:6<503::AID-BIT1>3.0.CO;2-N; Das, TK et al, 2021, DOI: 10.1016 / j.xphs.2021.09.030, Zubiaga, A. et al, 2019, DOI: 10.21152 / 1750-9548.13.1.61]. Our internal simulation studies showed that high local peak shear is expected when droplets impact a liquid, whereas wave formation results in a decrease in overall shear in the liquid. This is consistent with the particle formation observations 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]. Figure 2 shows that particles, particularly those determined by optical obscuration to be in the 2-4 μm range, increase when exposed to XZ oscillations beyond one-dimensional oscillations. Even the stable reference molecule, mAb2, appears to have more particle formation under XZ oscillations.

[0046] The novel shaker is more representative compared to established transport simulations operating under ambient conditions. Our results indicate that temperature influences 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, AWP, Norde, W., DOI:10.1016 / S0006-3495(00)76602-1], but our results seem to indicate differences even at lower temperatures, such as 5°C and 23°C. This is not entirely surprising, as temperature can change the viscosity of a liquid, which in turn affects the shear stress it experiences [Woldeyes, MA et al, 2020, DOI: 10.1021 / acs.molpharmaceut.0c00552; Iqbal, MJ; Chaudhry, MA, 2009, DOI: 10.1016 / j.jct.2008.09.016]. Finally, there are reports where room temperature has been considered a worst-case scenario, as companies utilize temperature-controlled chambers to ship formulations [Ammann, C., 2011, DOI: 10.1208 / s12249-011-9684-0; Fleischman, ML et al, 2017, DOI: 10.1016 / j.xphs.2016.11.021].

[0047] For horizontal vs. vertical vial orientation experiments, the visible particle examination does not completely match the subvisible particle analysis. This may be due to the presence of more subvisible / smaller sized particles in the shaken vial and more visible particles in the control. The fact that more subvisible particles were observed in the vertically oriented 6 ml vial than in the horizontally oriented 20 ml vial can be explained by differences in air-liquid interfacial stress and shear stress [Koepf, E. et al, DOI: 10.1016 / j.ijpharm.2017.12.043; Maa, YF; Hsu, CC, 1997, DOI: 10.1002 / (SICI)1097-0290(19970620)54:6<503::AID-BIT1>3.0.CO;2-N; Narhi, LO 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 against the bottleneck, which is where Bai et al. found the most shear stress occurred in the vial [Bai, G. et al, 2012, DOI:10.1016 / j.ijpharm.2011.11.044]. On the other hand, the horizontally oriented 6 ml vial splashed toward the center of the stopper, rather than against the bottleneck. In the vertical position, as seen in Figure 1, the 20 ml vial practically did not exhibit any splashing, while the 6 ml vial did. Finding a simple direct comparison for this research question is not straightforward due to the complex interactions between fill volume, fill height, and the effects of vial shape, which depend on vial orientation. The selected fill volume is ideal for comparison with vertical vibration, as well as preliminary image analysis studies. Setting a range of filling parameters in follow-up studies will allow for more detailed investigation.Additionally, silicone oil leaching from the stoppers can result in higher subvisible particles upon contact with the product solution, as did 6 ml vials compared to 20 ml vials [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 can provide information about the composition of particles and help understand their origin.

[0048] To prevent protein aggregation, formulation developers add excipients such as surfactants and sugars to pharmaceutical formulations. In this study, PS80 was used because it is said to provide greater protection against aggregation caused by rocking [Singh, SM et al, 2017, DOI: 10.1016 / j.xphs.2017.08.011]. The results show that the amount of visible (sub-visible) particles in formulations containing surfactants is much lower than in formulations without PS80. The BMI results also appear to correlate with the amount of surfactant and the amount of particles. This is expected, as surface tension measurements show lower surface tension at higher PS80 concentrations (Figure 8), which is consistent with previous studies [Das, T. 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, because the protein concentration of mAb1 is nearly half the target concentration (72 mg / ml) in the 0.06% PS80 formulation due to calculation error, the light obscuration measurements do not follow the same trend.

[0049] It should be noted that light obscuration is a noisy method and can result in significant discrepancies between identically processed samples. For this reason, product quality analysis is not limited to subvisible analysis and visual inspection; other stability-indicating methods, such as size-exclusion HPLC, are also used. Because light obscuration produces variable results, light obscuration results only show particles up to 10 μm in size. Background film imaging, a method also used to measure subvisible particles, offers several advantages over light obscuration but also disadvantages [Vargas, SK et al., 2020, DOI: 10.1016 / j.ijpharm.2020.119072]. Therefore, it was used in conjunction with light obscuration in this study.

[0050] This study showed interesting results, such as an increase in (sub)visible particles in two-dimensionally shaken liquid-formulated biopharmaceuticals, but also demonstrated that the addition of surfactants significantly reduced the formation of (sub)visible particles, thus proving effective for formulation developers to take precautions against aggregation and particle formation. Using the two-dimensional shaker disclosed herein can be extremely useful for formulation scientists and pharmaceutical companies to understand how their biopharmaceuticals are shaken during shipping and how these conditions can best be replicated in-house for testing pharmaceuticals during development.

[0051] Example 2 range A product quality impact (PQI) study is conducted on large molecule products, such as antibody drugs, to evaluate the impact of commercial shipping conditions on the quality attributes of the formulation through a representative shipping route. The primary objective of this study is to record and analyze the vibrations and shocks that occur during real-world shipping and how they propagate throughout stacked products on pallets. These shipping stresses are recorded by accelerometers along a supply chain representative of EU / US distribution.

[0052] This study provides a better understanding of long-term vibrations and their orientation inside packaging. From simulations and experiments, it is known that horizontal and vertical vibrations have different effects on fluid movement and therefore different contributions regarding drug stress (Zubiaga et al., 2019).

[0053] Three different test drugs, Active 1, Active 2, and Active 3, will be shipped with accelerometers and analyzed for potential shipping quality impacts on the drug product (DP). This allows for direct correlation of the recorded rocking stress profiles with their respective product quality impacts. The goal is to compare existing model systems for product quality impacts and develop a novel, lab-based approach to replace real-world shipping studies.

[0054] This collected knowledge allows products to be tested under more realistic, i.e., representative shipping conditions in a laboratory. Such lab-based model systems help save costs, resources, and the environmental footprint of real-world shipping studies. Further insight into the damping and resonance effects of realistic shipping configurations (e.g., stacked boxes) allows those skilled in the art to account for these effects by adjusting the resulting model system conditions and parameters. Furthermore, a data base is also collected for studying the shaking of drug product vials in secondary packaging.

[0055] Selection of formulation This study evaluates the impact of commercial shipping conditions on the quality attributes of drug formulations. Next to the water-filled vials for filling boxes and placing accelerometers, three different drug products are used in this shipping study (Table 5).

[0056] [Table 4]

[0057] Rationale for formulation selection These formulations have different sensitivities to changes in product quality upon shaking stress. Fill volumes and vial configurations were kept the same for all products to eliminate headspace-to-fill volume ratios and surface-to-fill volume ratios as potential influencing factors. Active 1 and Active 2 are known to be sensitive to interface-related aggregation. In contrast, Active 3 is expected to be more stable upon shaking. The Active 1 and Active 2 formulations described herein contain only minimal amounts of surfactants. Therefore, these formulations are more likely to exhibit related quality changes upon shaking.

[0058] The purpose of shipping these formulations is to evaluate the potential impact of rocking stress during a representative shipment on DP quality. Interpretation of the study results is performed by comparing the stress of shipped and control samples. Therefore, the material will be subdivided into shipped samples and unshipped control samples. Control samples will be stored in a controlled environment at 2-8°C under recommended storage conditions. All samples and controls will be visually inspected for any visible particles before shipping. DPs will be analyzed upon return. Water-filled vials serve as fillers for the shipping box and allow for realistic positioning of the accelerometer and DP. Water-filled vials will not be analyzed.

[0059] Packaging Configuration Shipping box (tertiary packaging) All vials and accelerometers are placed in a shipping box of a representative configuration, with one shipping box containing 105 secondary packaged vials.

[0060] Twenty vials of each lab-scale manufactured DP are included in each shipment, representing a total of 60 samples. The DP vials are distributed into boxes containing accelerometers. The remaining space in each of these shipping boxes containing DP samples is filled with water-filled vials. All other shipping boxes are packaged exclusively with water-filled vials.

[0061] Palette Configuration A total of 24 shipping boxes are placed on the pallet, with three layers of eight secondary packaging boxes (corresponding to the maximum allowable height of the pallet in the isolated container). Therefore, the pallet contains a total of 2510 secondary packaged vials (24 boxes of 105 vials each → 2520 vials minus 10 loggers = 2510). Of these, 2450 are water-filled vials and 60 are DP vials. The entire pallet is transferred to a thermal shipping system for air cargo, such as the RKN e1 or Skycell 1500C. The pallet is placed at the bottom of the aircraft.

[0062] Rocking Survey background Waves change as they pass through the medium, and accelerometers are useful for measuring vibration intensity at different locations within a pallet. A good practical example is a weighing table with a large marble block resting on an elastomer. This system results in a very low resonant frequency. For such a system, any waves below this resonant frequency undergo "rigid transmission," i.e., the waves are unchanged as they travel through the elastomer. If the excitation is at the resonant frequency, the vibration amplitude increases as it passes through the system (unless the system is beyond critical damping). The weighing table system is designed so that all vibration frequencies exceed the resonant frequency. In this case, the vibration intensity is greatly reduced and the measurement is somewhat isolated from external vibrations. This system can be considered a "mass-spring system," shown in Figure 10 for the three color regions (rigid transmission, vibration amplification, and wave attenuation).

[0063] Similarly, the shipping box can be considered a mass-spring system, but with essentially infinite degrees of freedom. It is expected that the packaging will affect the vibration strength of the vial, as it will most likely have a resonant frequency below or within our range (Figure 11).

[0064] When considering vibration during transport, there are usually two aspects to consider: shock and long-term vibration intensity. In many applications, shock is by far the most important because it exerts high stress on the structure and can lead to structural failure, while overall vibration creates long-term loads and causes fatigue.

[0065] When considering fluids, impact may be less important because there are no structures to destroy. Impact may result in short-term violent movement but may have little effect on the drug over time. On the other hand, long-term vibration can potentially cause constant movement and frequent splashing, thus stressing the drug and potentially causing its degradation. It is known that horizontal and vertical vibrations have different effects on fluid movement and therefore different contributions to drug stress (Zubiaga et al., 2019). Therefore, it is important to characterize stress input and propagation within our delivery system.

[0066] Oscillation Measurement The vibration measurements accomplish two distinct objectives. First, a complete time series of triaxial acceleration data at 1600Hz resolution is obtained throughout the entire shipment. Furthermore, the second objective is to understand the impact of packaging on vibrations down to the level of the secondary packaging where each vial is packaged.

[0067] The accelerometer mounted at the lowest level serves as a reference sensor that measures the pallet's excited vibration with sufficient accuracy for the purposes of this study. Laboratory studies have shown that measurements at the lowest level within a packaging configuration are comparable to the vibration of the underlying pallet. Due to the limited battery life and storage capacity of each logger, five reference accelerometers are started sequentially, covering a maximum of 15 days.

[0068] To reach the second objective, for example, to quantify the change in vibration intensity, accelerometers are placed at different locations within the pallet. The reference measurements are compared to the measurements of the remaining probe sensors, placed at five different locations within the pallet. The rationale for taking multiple measurements is that the wave path is significantly different in the secondary packaging near the bottom compared to the secondary packaging near the top. Comparing the data from the reference to the probe sensors helps to understand and determine the vibration transmission from the shipping container to the secondary packaging through the pallet. This vibration transmission is characterized by a frequency-dependent vibration intensity ratio, which is the amplitude ratio of the probe measurement to the reference measurement.

[0069] The probe sensor records for 2 minutes with an 8 minute pause to cover the same span, which is sufficient to characterize the vibration transmission as the frequency spectra are compared.

[0070] Figure 12 conceptualizes the experimental setup, where the reference measurement is taken at the lowest level (black) and the remaining probe sensors are at different positions within the transport box. The accelerometer (sensor, logger, and battery within the device) is shown on the right side of the figure. It is important to treat the pallet and box as representatively as possible while still remaining within the accelerometer's recording period. Any special handling may result in misinterpretation of the obtained data.

[0071] Accelerometer and logger holder A total of 10 MSR data loggers are used to record vibration stress throughout a real-world shipping process. The accelerometers are housed in a custom-made logger holder with the same external dimensions as the secondary carton packaging of 6ml vials. The holder is milled from solid aluminum, and the accelerometers are fastened to it with three M3 machine screws. The accelerometer holder is designed to allow for "rigid" transitions within the frequency spectrum of interest. This means there are no resonant frequencies below 500Hz. This allows for the measurement of external vibrations only, without the holder itself affecting the measurement.

[0072] Formulation sample and sensor location Based on the positioning rationale above, the reference accelerometers are placed as close as possible to the center of the pallet, without being in the corners of the box or directly touching each other. The probe accelerometers are distributed to cover the entire pallet and its edges. The DP vials are added to the same box as the accelerometers to first directly measure vibrations within that box, and then take into account the packaging procedure and operational aspects of the accelerometer operation. This results in the following allocation of accelerometers and DP vials:

[0073] Boxes 8, 10, 13, 23, and 24 contain three vials for each DP and one probe accelerometer. Box 7 contains five vials for each DP and two reference accelerometers. Boxes 2, 3, and 6 contain one reference accelerometer.

[0074] The holders with the accelerometers contained therein are arranged in the same manner as in regular secondary packaging with vials. The exact location of each sensor within the pallet is defined (Figures 13 and 14). Figure 13 shows an exemplary diagram of the accelerometers in the holders within a shipping box packaged with secondary packaged vials.

[0075] Treatment in real-life shipping studies All vials containing accelerometers placed in dedicated locations were subjected to actual shipping conditions. All DP samples should be stored at 2-8 °C before the start of shipping studies.

[0076] Unshipped control samples were stored at 2-8°C during the shipping process. Samples were stored at 2-8°C after return until analysis, and aliquots could be frozen at -70°C ± 20°C for future analysis.

[0077] The shipping plan was intended to represent a typical shipment of pharmaceutical products. A certified thermal shipping system was used to avoid temperature-related effects on the product. Product temperature must be maintained at 2-8°C before shipment, throughout the shipping lane, and after shipment until analysis. Each leg of the proposed route covers the following: Section 1: Transport by truck, duration: approximately 45 minutes. Section 2: Transport by truck, duration: approximately 5 hours. -Segment 3: Transportation by flight, duration: approximately 9 hours. Section 4: Transport by truck, duration: approximately 11 hours. Section 5: Transport by truck, duration: approximately 57 hours.

[0078] Product quality evaluation and acceptance criteria Post-release and respective control evaluation of DP quality is based on quantitative testing and qualitative comparison. [Table 5]

[0079] Because the DP formulation and configuration used in this study were specifically designed for this study, no acceptance criteria were given. Therefore, results are evaluated according to the acceptance criteria of no significant change (within assay precision) for each test method. Results of shipped samples are directly compared to unshipped vials as controls.

[0080] The chromatographic (SE-HPLC and IE-HPLC) profiles of the non-shipped control and shipped samples are compared and evaluated for a) the absence of novel peaks and b) overall profile comparability (within assay precision). Additionally, the results of the shipped samples are compared directly to non-shipped vials as a control to assess only the impact of shipping on product quality.

[0081] result The temperature was monitored during shipping, and after initial cooling of the sensor and packaging, the temperature remained stable within the expected range (Figure 14).

[0082] Vibration Monitoring Vibrations were monitored to understand real-world stress conditions, measuring the overall vibration input to the pallet and also determining the distribution of vibration through the stack of packages.

[0083] Long-term vibration Vibrations were recorded by measuring triaxial acceleration at 1600 Hz, resulting in 1600 data points per second. Five reference loggers at the bottom of the pallet recorded continuously to cover the transport.

[0084] Total Vibration Energy Grms (g-force root-mean-square), defined as the area under the PSD curve, measures the overall energy input through random vibrations.

[0085] This time series shows that vibrations occurred during transport legs (1-5). Vibrations were stronger during truck transport (legs 4 and 5) compared to flight (leg 3). Furthermore, loggers at different heights within the pallet show that the overall energy had a decreasing order, highest at the top (M5) and down to the reference logger (R) at the bottom of the pallet (Figure 15). This indicates that vibrations are amplified through the stack of packaging.

[0086] The overall vibration intensity was lower than recommended by typical vibration testing guidelines such as ASTM D4169-16.

[0087] Vibration Spectral Density To analyze the power spectral density (PSD) of the vibration, two-minute intervals were combined and subjected to a fast Fourier transform (FFT). The PSD curve describes the random vibration spectral density, i.e., what level of energy occurs at what frequency during that two-minute period. The PSD curves for all combined two-minute intervals were plotted as a density. The density indicates the distribution of the intervals. From the density, quantiles and corresponding PSD curves can be derived, e.g., the 10% or 5% worst-case vibrations, as per the ASTM D4169 guidelines.

[0088] The PSD distribution is plotted, and the color gradient indicates the quantile or frequency at which such a PSD curve occurs. The darkest areas are those that occur most frequently, such as average vibration during transport. Yellow is less likely to occur, such as very low or high vibration. Similar to the ASTM D4169-16 guidelines, the corresponding assurance levels are plotted in solid black. Assurance Level: Level I: 99% of vibrations occurring above the line Level II: 95% of vibrations occurring above the line Level III: 90% of vibrations occurring above the line

[0089] For comparison, the relevant ASTM D4169-16 guideline profile is plotted as a blue dotted line.

[0090] The vibrations between the sections were clearly different. Air transport had the lowest vibration intensity, with the black single PSD assurance level clearly lower than the blue dotted line. Therefore, during this transport, less stress was detected than typically tested during laboratory-based ASTM D4169-16 testing. The reasons for this could be a smoother flight, vibration absorption by the thermal shipping system, or general improved aviation technology and larger aircraft.

[0091] During truck transport, the overall intensity was stronger than during air transport. Some frequencies between 8 and 20 Hz and below 3 Hz were stronger compared to typical test guidelines. In general, the measured vibrations were within or below expected values ​​for most frequencies. The results are shown in Figure 16.

[0092] Transport route and format To compare the different sections of the transport, data from the reference loggers was compared. Similar amplification patterns were observed for all transport routes. Frequencies between 10 and 60 Hz were amplified throughout the packaging stack, while frequencies above 200 Hz were attenuated. The results are consistent with laboratory-scale studies, where the same effect was observed.

[0093] impact Most of the impacts were recorded during loading and unloading of the cargo and during truck transport (sections 1, 2, 4, and 5) (Fig. 17).

[0094] Product quality No effect on product stability was detected. The stress conditions did not result in measurable degradation of the selected model drug. Subsequently, no effect of the position of the formulation container within the pallet was detected.

[0095] No effect of stress conditions was found on the following quality attributes: size variant, charge variant, colour, turbidity, visible particles, subvisible particles.

[0096] Consideration vibration The comprehensive vibration data set represents real-world stress conditions during transport. New data on lateral vibration was collected, and the data can be useful for multi-axial vibration testing. Overall energy input was lower than current laboratory test procedures. The effect of location within the pallet was observed. PSD frequencies between 10 and 60 Hz were highest at the top of the pallet, which is higher than current laboratory test guidelines. Therefore, a worst-case profile combining the highest PSDs across the pallet can serve as a worst-case test profile for future testing.

[0097] Product quality impact No significant degradation or impact on quality attributes was detected. Neither the stable standard nor the less stable formulations showed any degradation. The addition of a small amount of surfactant (0.01% w / v) PS20 appears to protect the protein from aggregation (Figure 20). Therefore, the vibration intensity and stress conditions were less intense than those typically tested during formulation development. It was assumed that transportation would be less intense than expected, and the formulation was stable enough to withstand these conditions.

[0098] During development, data on SvP and aggregate formation were observed in a shaking model system, so real-world transport is not as robust.

[0099] Embodiment Below, further specific embodiments of the present invention are listed.

[0100] 1. In an embodiment, a method for simulating the quality effects of transportation on a liquid is disclosed, the method comprising: a) selecting a set of instructions including: i) one or more PSD profiles including a plurality of frequencies and their corresponding amplitudes in at least two perpendicular axes (e.g., X and Z); and ii) a timetable specifying a duration for each of the PSD profile(s); b) inducing vibrations in the liquid according to said PSD profile(s); c) evaluating and comparing the quality of the liquid before and after performing step b; Includes:

[0101] 2. In an embodiment, a method according to embodiment 1 is disclosed, wherein vibrations in two axes are induced simultaneously.

[0102] 3. In an embodiment, a method according to embodiment 1 or 2 is disclosed, wherein the liquid comprises a pharmaceutical agent.

[0103] 4. In an embodiment, a method according to any one of embodiments 1 to 3 is disclosed, wherein the liquid comprises a biological product.

[0104] 5. In embodiments, the method of embodiment 4 is disclosed, wherein the biological product is a protein (e.g., an antibody), a nucleic acid, a sugar, or conjugates and combinations thereof.

[0105] 6. In an embodiment, a method as in any of the previous embodiments is disclosed, where the set of instructions similar to embodiment 1a is different for different axes.

[0106] 7. In an embodiment, a method according to any of the previous embodiments is disclosed, wherein the amplitude of vibration in the first axis is proportional to the amplitude of vibration in the second axis.

[0107] 8. In an embodiment, a method as in any of the preceding embodiments is disclosed, wherein the PSD profile is designed based on measurements of vibrations in one or more real shipments.

[0108] 9. In an embodiment, a method according to any one of embodiments 1 to 8 is disclosed, wherein the frequency range in the PSD profile is 1 to 300 Hz.

[0109] 10. In an embodiment, a method according to any one of embodiments 1 to 8 is disclosed, wherein the frequency range in the PSD profile is 15 to 80 Hz.

[0110] 11. In an embodiment, a method according to any of embodiments 1-7 is disclosed, wherein the PSD profile is as presented in Figure 16(AG).

[0111] 12. In an embodiment, a method according to any one of embodiments 1-7 is disclosed, wherein the PSD profile complies with an ASTM D4169 standard, e.g., aviation level I, II, or III, railway level I, II, or III, or truck level I, II, or III.

[0112] 13. In an embodiment, a method according to any of the preceding embodiments is disclosed, wherein the quality assessment as in embodiment 1c is carried out using an analytical method, such as size exclusion chromatography (SEC), ion exchange chromatography (IEC), analytical ultracentrifugation, visible or subvisible particle analysis.

[0113] 14. In an embodiment, a two-dimensional shaker adapted to perform the steps of the method of any of the preceding embodiments is disclosed.

[0114] 15. In an embodiment, the two-dimensional shaker of embodiment 14 is disclosed, wherein the shaker includes a temperature-controlled chamber.

[0115] 16. In an embodiment, a two-dimensional shaker according to embodiment 15 is disclosed, wherein the temperature of the chamber is maintained at 2 to 60°C, for example, 2 to 8°C.

[0116] 17. A two-dimensional shaker according to any of embodiments 14-16 is disclosed, wherein in an embodiment, the shaker further comprises vial holder(s) for syringes.

[0117] 18. In an embodiment, the two-dimensional shaker of embodiment 17 is disclosed, wherein the holder is adapted to function at various tilt angles.

[0118] 19. In an embodiment, an apparatus is disclosed for inducing vibrations in a liquid simultaneously in at least two perpendicular directions (eg, X-axis and Z-axis).

[0119] 20. In an embodiment, an apparatus according to embodiment 19 is disclosed, wherein the apparatus comprises at least one independently controlled linear motor for each axis.

[0120] 21. In an embodiment, an apparatus described in embodiment 19 or 20 is disclosed, wherein the apparatus induces vibrations based on a set of instructions received by the apparatus, the set of instructions including: i) one or more PSD profiles including multiple frequencies and their corresponding amplitudes in at least two perpendicular axes (e.g., X and Z); and ii) a timetable specifying a duration for each of the PSD profile(s).

[0121] 22. In an embodiment, a device according to any one of embodiments 19-21 is disclosed, wherein the liquid comprises a pharmaceutical agent.

[0122] 23. In an embodiment, a device of any of embodiments 19-22 is disclosed, wherein the liquid comprises a biological product.

[0123] 24. In an embodiment, a device according to embodiment 23 is disclosed, wherein the biological product is a protein (e.g., an antibody), a nucleic acid, a sugar, or conjugates and combinations thereof.

[0124] 25. In an embodiment, the apparatus of any of embodiments 19-24 is disclosed, wherein the set of commands similar to embodiment 21a is different for various axes.

[0125] 26. In an embodiment, an apparatus is disclosed according to any of embodiments 19 to 25, wherein the PSD profile is designed based on measurements of vibrations in one or more real shipments.

[0126] 27. In an embodiment, a device according to any one of embodiments 19 to 26 is disclosed, wherein the frequency range in the PSD profile is 1 to 300 Hz.

[0127] 28. In an embodiment, a device according to any one of embodiments 19 to 26 is disclosed, wherein the frequency range in the PSD profile is 15 to 80 Hz.

[0128] 29. In an embodiment, an apparatus of any of embodiments 19-25 is disclosed, wherein the PSD profile, i.e., frequencies and their corresponding amplitudes, is that presented by the solid or dotted lines in Figures 16(A-G) or is within 9 dB in amplitude thereto.

[0129] 30. In an embodiment, an apparatus according to any of embodiments 19-25 is disclosed, wherein the PSD profile complies with ASTM D4169 standards, e.g., aviation level I, II, or III, railway level I, II, or III, or truck level I, II, or III.

[0130] 31. In an embodiment, a device according to any one of embodiments 19-30 is disclosed, wherein the device comprises a temperature-controlled chamber.

[0131] 32. In an embodiment, the device of embodiment 31 is disclosed, wherein the temperature of the chamber is maintained at 2 to 60°C, for example, 2 to 8°C.

[0132] 33. A device according to any of embodiments 19-32 is disclosed, wherein in an embodiment, the device further comprises vial holder(s) for a syringe.

[0133] 34. In an embodiment, the device of embodiment 33 is disclosed, wherein the holder is adapted to function at various tilt angles.

[0134] 35. In an embodiment, the device of any of embodiments 19-34 is disclosed for use in the method of any of embodiments 1-13.

Claims

1. 1. A method for simulating the quality effects of transportation on a liquid, comprising: a) selecting a set of instructions including: i) one or more PSD (power spectral density) profiles including a plurality of frequencies and their corresponding amplitudes in at least two perpendicular axes (e.g., X and Z); and ii) a timetable specifying a duration for each of the PSD profile(s); b) inducing vibrations in said liquid according to said PSD profile(s); c) evaluating and comparing the quality of the liquid before and after performing step b; A method comprising:

2. The method of claim 1 , wherein the vibrations in the two axes are induced simultaneously.

3. The method of claim 1 or 2, wherein the liquid comprises a pharmaceutical agent.

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

5. 5. The method according to claim 1, wherein the set of instructions according to claim 1a is different for different axes and / or the amplitude of vibration in the first axis is proportional to the amplitude of vibration in the second axis.

6. The method of claim 1 , wherein the PSD profile is designed based on measurements of vibrations in one or more real shipments.

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

8. 6. The method of any of claims 1 to 5, wherein the PSD profile, i.e., the frequencies and their corresponding amplitudes, is that presented by the solid or dotted lines in Figures 16(A-G) or is within 9 dB in amplitude thereto.

9. 6. The method of claim 1, wherein the PSD profile complies with ASTM D4169 standards, such as Aviation Level I, II, or III, Railway Level I, II, or III, or Truck Level I, II, or III.

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

11. A two-dimensional shaker adapted to carry out the method steps of claim 1b or any of claims 2 to 9 dependent thereon.

12. 1. An apparatus for inducing vibrations in a liquid simultaneously in at least two perpendicular directions (e.g., X-axis and Z-axis), optionally wherein the apparatus induces vibrations based on a set of instructions received by the apparatus, the set including: i) one or more PSD profiles including a plurality of frequencies and their corresponding amplitudes in at least two perpendicular axes (e.g., X and Z); and ii) a timetable specifying a duration for each of the PSD profile(s).

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

14. 14. The two-dimensional shaker of claim 11 or the apparatus of claim 12 or 13, further comprising a temperature-controlled chamber, optionally wherein the temperature of said chamber is maintained at 2 to 60°C (e.g., 2 to 8°C).

15. 15. A two-dimensional shaker according to claim 11 or 14 or an apparatus according to any of claims 12 to 14, further comprising vial holder(s) for syringes, optionally said holder adapted to function at various tilt angles.

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