Apparatus for improving the hydration reaction and / or reducing the particle size of a product and method of using the apparatus - Patents.com

Abstract

A method and apparatus for improving hydration and / or reducing particle size of a product or reagent is provided, the method comprising applying a pulsed electromagnetic field to the product or reagent for a period of time sufficient to allow increased hydration of the product or reagent and / or reduction in particle size of the product or reagent.

Description

[Technical field]

[0001] The invention to which this application relates is directed to an apparatus for improving the hydration of a product and / or reducing one or more particle sizes of the product, and methods of using the apparatus. [Background technology]

[0002] Although the following description refers almost exclusively to a particular example of the device of the invention being used to improve the reconstitution of a previously lyophilized product, in particular the blood protein Factor VIII, those skilled in the art will appreciate that the device may be used for the reconstitution of any freeze-dried product, the hydration of any product or the reduction of its particle size, and / or the like.

[0003] Freeze-drying of products is a well-known process that allows the products to be stored for much longer periods than other methods while retaining the properties of the product. Freeze-drying of proteins is also well-known and has become an important process for the preservation of biological products. There are various protocols for freeze-drying of proteins that are typically followed to ensure that the freeze-dried protein products can be stored, transported to a location for subsequent use, and reconstituted in a form that allows such use.

[0004] Freeze-drying, also known as lyophilization, is the act of removing water from a frozen product by sublimation and desorption. Although there are known problems with the use of freeze-drying, it is generally considered a useful and preferred method for extending the shelf life of protein products. The process relies on the control of pressure and temperature in a freeze-dryer to remove liquid from formulations of heat-sensitive or hydrolytically unstable active pharmaceutical ingredients (APIs) or formulation components. The resulting solid or powder has superior stability and can be stored at higher temperatures for longer periods than aqueous solutions before being freeze-dried.

[0005] During the process of freeze-drying, moisture in the form of water present within the protein is removed to a large extent to avoid protein collapse, and the water is typically replaced with a cryoprotectant, often one or more sugars, which provide an outer layer on the protein.

[0006] It has been found that when freeze-dried product needs to be reconstituted, the need to remove sugar and replace it with water to reconstitute protein does not occur to the desired extent.Therefore, this means that even if proteins can be partially reconstituted, they do not return to the form they were in before the freeze-drying process.Therefore, this may mean that the effectiveness of reconstituted protein is greatly reduced compared to original protein product, and furthermore, protein may act differently from what is expected before the freeze-drying process.

[0007] In particular, one problem is the continued presence of sugars in the reconstituted product, which means that the particles can effectively adhere to each other, causing them to aggregate into larger shapes. For example, in certain products, such as the blood protein factor VIII, when this product is reconstituted, the aggregation of particles (usually in the form of pentamers) means that when it is injected into the body, the body's immune system will react adversely to the aggregated particles as a threat to the body and will try to destroy or reject factor VIII because it will produce neutralizing antibodies against it. This problem is so severe that up to 30% of hemophilia patients who currently need to have factor VIII introduced into their bodies on a regular basis are believed to reject factor VIII because they actually build up immunity to the reconstituted factor VIII [1]. Thus, the life-saving therapeutic benefit of introducing factor VIII to patients is not obtained [2]. Despite the adverse medical effects on patients, factor VIII is an expensive product, resulting in a large expenditure on material that may not ultimately be used.

[0008] A conventional process for reconstituting protein materials such as factor VIII involves providing an end user with a vial containing freeze-dried factor VIII that is effective in powder form. The end user is instructed to add an amount of water to the vial, then manipulate the vial to mix the water and powder together by swirling them in the vial, removing the sugar with the water and replacing the sugar with water to reconstitute the factor VIII particles. This conventional process has generally been found to be insufficient to allow complete reconstitution of factor VIII, thus significantly reducing the effectiveness of the reconstituted factor VIII. This means that the end user may need to use and inject factor VIII more frequently than usual to obtain the required beneficial medical effect.

[0009] Although the above example only relates to the reconstitution of freeze-dried Factor VIII, it is estimated that more than 60% of biologics on the market today would not be possible without lyophilization. Furthermore, as more biosimilar or novel biologics are developed, the market demand for lyophilization technology will only increase. Thus, there is a need in the market to ensure efficient and effective reconstitution of lyophilized products.

[0010] An additional problem associated with intravenous (IV) delivery of therapeutic products is the occurrence of Complement Activation Associated Pseudoallergy (CARPA). This is a harmful non-immune anaphylactic or hypersensitivity reaction characterized by the independence of an antigen-specific immune response to a specific nanoprotein contained in the infusion exposed to the patient's blood. CARPA is generally harmless to most patients, but is a concern in IV delivery of therapeutic products because in some patients the reaction can be fatal. One example of a reagent associated with the occurrence of CARPA is Tween 80 or polysorbate 80. This reagent is often used as a surfactant dispersant in IV formulations. In some patients, exposure to IV-delivered Tween 80 leads to the occurrence of excipient aggregates (micelles or vesicles) that are thought to cause CARPA.

[0011] Another additional problem associated with large particle size is in the transfection process. Transfection is a technique that can deliver extracellular material, such as nucleic acid, into one or more eukaryotic cells. One example of transfection is the delivery of nucleic acid using DNA plasmids. In some cases, DNA plasmids are too large to be used in a single transfection. Therefore, it is necessary to use several transfection steps using smaller DNA plasmids. As a result, the process is time-consuming and significantly more expensive to perform. In addition, the overall transfection frequency is lower. Summary of the Invention [Problem to be solved by the invention]

[0012] It is therefore an object of the present invention to provide an apparatus for improving the reconstitution process of a freeze-dried or lyophilized product, such that agglomeration of product particles during the reconstitution process can be reduced or eliminated and / or the efficacy of the reconstituted product can be enhanced.

[0013] It is a further object of the present invention to provide a method for improving the reconstitution process of freeze-dried or lyophilized products.

[0014] It is yet a further object of the present invention to provide an apparatus and / or method for improving product hydration such that agglomeration of product particles can be reduced or eliminated.

[0015] Another further object of the present invention is to provide an apparatus and / or method for mitigating CARPA reactions in IV delivered products or formulations.

[0016] It is yet a further object of the present invention to provide an apparatus and / or method for reducing the size of DNA plasmids to allow for improved transfection frequencies. [Means for solving the problem]

[0017] According to a first aspect of the present invention there is provided a method of improving hydration and / or reducing particle size of a product or reagent, the method comprising applying a pulsed electromagnetic field to the product or reagent for a period of time sufficient to allow increased hydration of the product or reagent and / or a reduction in particle size of the product or reagent.

[0018] The methods of the present invention have the advantage of reducing the particle size of a product or reagent and / or mitigating or reducing the formation of aggregates in the product or reagent, thereby increasing the effectiveness of the product or reagent and improving its function and / or efficacy.

[0019] It will be understood that references to application of a pulsed electromagnetic field also include exposure to a pulsed electromagnetic field and the like.

[0020] In one embodiment, the period during which the product or reagent is exposed to the pulsed electromagnetic field is a predetermined period of time, more preferably, this predetermined period of time is 10-15 minutes ±5 minutes.

[0021] In one embodiment, the product or reagent is culture medium or cell culture medium.

[0022] In one embodiment, the product or reagent is a lyophilized or freeze-dried product or reagent. Preferably, the method is performed during the reconstitution of a lyophilized product or reagent.

[0023] In one embodiment, the product or reagent is a powdered or granular product or reagent.

[0024] Preferably, the method of reconstituting a lyophilized or freeze-dried product or reagent comprises the steps of adding water or a liquid to the lyophilized or freeze-dried product or reagent and applying a pulsed electromagnetic field to the mixture of water or liquid and the lyophilized or freeze-dried product or reagent for a period of time sufficient to allow increased hydration of the product or reagent and / or reduction in particle size of the product or reagent.

[0025] In one embodiment, in addition to applying a pulsed electromagnetic field to the mixture, product or reagent, the mixture, product or reagent may be agitated at any time prior to, during and / or after exposure to the pulsed electromagnetic field to cause mixing of the lyophilized or freeze-dried product or reagent with water or a liquid, or agitation of the product, liquid or mixture.

[0026] Preferably, the stirring step is carried out using a stirring means or device such as, for example, a magnetic stirrer and / or a vibrating mechanism.

[0027] In one embodiment, the pulses of the pulsed electromagnetic field are generated in a predetermined manner, and such pulses may include or consist of successive pulses all of the same duration and frequency, and / or may include or consist of successive pulses of different durations and / or different frequencies.

[0028] In one embodiment, the mixture is exposed to a pulsed electromagnetic field for a period of time for reconstitution of products or reagents in the mixture.

[0029] In one embodiment, the addition of water or liquid to the product or reagents to form the mixture occurs while the water, liquid and / or mixture is exposed to the pulsed electromagnetic field.

[0030] In one embodiment, the water or liquid used for reconstitution is exposed to a pulsed electromagnetic field prior to addition to the lyophilized or freeze-dried product or reagent.

[0031] In one embodiment, the product, reagent or mixture is disposed in a container which may be integral to the device in which the pulsed electromagnetic field is generated, or which is separate from the device in which the pulsed electromagnetic field is generated, and which is placed therein or adjacent thereto.

[0032] In one embodiment, the container is one in which a freeze-dried or lyophilized product or reagent is initially provided and to which water or liquid is added.

[0033] Typically, the pulsed electromagnetic field generated is sufficient to cause rotation of water particles, the effect of the rotation being to assist in the removal of one or more sugars that may be present in the freeze-dried or lyophilized product or reagent, thereby increasing the level of removal of the sugars from the product and replacement by water particles, thereby improving the efficacy of the reconstituted product or reagent and reducing the tendency of the product or reagent to aggregate during the reconstitution process.

[0034] In one embodiment, increasing the effectiveness of a reconstituted product or reagent according to the present invention may reduce the frequency of use of the reconstituted product or reagent and / or reduce the amount of reconstituted product or reagent during each use.

[0035] In one embodiment, the reduction in aggregation of the reconstituted product or reagent compared to conventional processes reduces the propensity of the reconstituted product or reagent to be rejected.

[0036] In one embodiment, the product or reagent is a protein product, such as, for example, a monoclonal AB, a hormone, a fusion protein, and / or a protein construct.

[0037] In one embodiment, the product or reagent is any one or any combination of trastuzumab, pembrolizumab, infliximab, daxibotulinum toxin, immunoglobulin, omalizumab, abatacept, secukinumab, interferon beta 1a, and / or bortezomib, and the like.

[0038] In one embodiment the product, reagent, lyophilized or freeze-dried product or reagent is DNA or a DNA plasmid.

[0039] In one embodiment the product, reagent, lyophilized or freeze-dried product or reagent is Factor VIII.

[0040] In one embodiment, the product or reagent is or forms part of an intravenous (IV) formulation, product or reagent. Preferably, the product or reagent is one previously known to be associated with the CARPA reaction.

[0041] In one example, the product or reagent is a dispersing agent such as Tween 80 or Polysorbate 80.

[0042] In one embodiment, the product, mixture or reagent is exposed to the pulsed electromagnetic field during the process of infusion or injection into a patient. Preferably, the part of the process exposed to the pulsed electromagnetic field may be the in vitro part and / or the in vivo part.

[0043] In one embodiment, the product or reagent is exposed to a pulsed electromagnetic field during or prior to being placed into an IV bag or IV delivery container.

[0044] Preferably, the step of applying the pulsed electromagnetic field or signal is carried out at room temperature (such as, for example, 20° C.) or in an incubator which may be set to a temperature higher than room temperature (such as, for example, 37° C.).

[0045] Preferably, the pulsed electromagnetic field or signal is generated by one or more electronic devices, apparatus and / or circuits.

[0046] Preferably, the one or more electronic devices, apparatus and / or circuits comprise a transmission means or device for generating and / or transmitting the pulsed electromagnetic field or a signal therefrom, in use.

[0047] Preferably, the transmission means or device comprises one or more electronic transmission chips which, in use, are configured to generate, emit and / or transmit one or more pulsed electromagnetic signals.

[0048] In one embodiment, reference to a transmission means or one or more electronic transmission chips may include one or more transmitters, at least one transmitter and at least one receiver, or one or more transceivers. Thus, in one example, a pulsed electromagnetic field or signal may be transmitted from a central location or master transmitter and received by one or more remote and / or slave receivers and / or transceivers for subsequent retransmission or emission therefrom.

[0049] In one embodiment, the electronic device comprises a single transmission means or electronic transmission chip. Such a single transmission means or electronic transmission chip is sufficient to provide a pulsed electromagnetic field or signal to the product or reagent. In one exemplary embodiment, the single transmission means or electronic transmission chip is provided attached to or integral with a container for containing the product or reagent in use.

[0050] In one embodiment, the electronic device comprises two or more transmission means or electronic transmission chips, preferably arranged at a predefined distance from each other, for example within the electronic device.

[0051] Preferably, the predetermined separation distance is such that it provides the desired effect on the product or reagent that is pulsed with the pulsed electromagnetic field or signal (i.e., reducing particle size and / or improving hydration) and / or provides a uniform or substantially uniform distribution of electromagnetic radiation / signal in use.

[0052] Preferably, the electronic device comprises a plurality of transmission means or electronic transmission chips arranged in a predetermined pattern and / or array.

[0053] It has been found that while a single transmission means or electronic transmission tip is sufficient to provide the advantageous properties of the present invention, having multiple transmission means or electronic transmission tips allows the pulsed electromagnetic field or signal to be delivered over a larger surface area while still maintaining maximum effectiveness. 2 It has been found that distributing the transmission means or electronic transmission chips evenly so that there is at least one chip per area provides sufficient coverage for optimum effect.

[0054] In some embodiments, the electronic device, circuit or apparatus includes one or more transmission means or electronic transmission chips, hi some embodiments, the apparatus includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more transmission means or electronic transmission chips.

[0055] In some embodiments, the surface of an apparatus, device housing, or article defined herein is about 105-115 cm 2 and preferably about 110 cm of the surface of the housing of an apparatus, device or surface of an article as defined herein. 2 There is one transmission means or electronic transmission chip for each.

[0056] In some embodiments, the surface of an apparatus, device housing, or article defined herein is about 50-60 cm 2 and preferably about 55 cm of the surface of the housing of an apparatus, device or surface of an article as defined herein. 2 There is one transmission means or electronic transmission chip for each.

[0057] In some embodiments, the surface of an apparatus, device housing, or article defined herein is about 25-30 cm 2 and preferably about 27.5 cm 2 of the surface of the housing of an apparatus, device or surface of an article as defined herein. 2 There is one transmission means or electronic transmission chip for each.

[0058] In some embodiments, the surface of an apparatus, device housing, or article defined herein is about 15-20 cm 2 and preferably about 18.5 cm 2 of the surface of the housing of an apparatus, device or surface of an article as defined herein. 2 There is one transmission means or electronic transmission chip for each.

[0059] In some embodiments, the surface of an apparatus, device housing, or article defined herein is about 10-15 cm 2 and preferably about 12.2 cm 2 of the surface of the housing of an apparatus, device or surface of an article as defined herein. 2 There is one transmission means or electronic transmission chip for each.

[0060] In an exemplary embodiment, six transmission means or electronic transmission chips are provided within the apparatus, device and / or circuit.

[0061] In one embodiment, if more than one transmission means or electronic transmission chip is required, the spacing of the multiple transmission means or electronic transmission chips needs to be optimized. To achieve the optimal predetermined space between each transmission means or electronic transmission chip, the transmission means or electronic transmission chips should be placed at a distance equal to or substantially equal to half the wavelength of the electromagnetic radiation frequency used. Preferably, this distance should be considered in relation to any orientation surface or two or more transmission means or electronic transmission chips used together as part of an apparatus or device. For example, if the wavelength is 12.4 cm, the transmission chips need to be placed approximately 6.2 cm apart to generate an optimal electromagnetic field in use.

[0062] In one example, the predetermined separation distance=wavelength / 2.

[0063] In one example, the predetermined separation in the X-axis and / or Y-axis is half a wavelength between each transmission means or electronic transmission chip of the equally spaced grid. Such a configuration minimizes the risk of destructive interference.

[0064] In one embodiment, the electronic device includes a housing, with one or more transmission means or chips located within the housing.

[0065] Preferably, the housing includes at least one flat or planar surface to allow for stable positioning of the housing relative to another item or container that receives the pulsed electromagnetic field or signal in use. Alternatively, the housing may include one or more curved or non-planar surfaces to allow for stable positioning of the housing relative to one or more items or containers that receive the pulsed electromagnetic field or signal in use.

[0066] In one example, at least one surface of the housing includes one or more recesses for positioning one or more items that, in use, receive the pulsed electromagnetic field or signal.

[0067] In one embodiment, the housing includes a base surface to allow the housing to be supported directly or indirectly on a surface in use. More preferably, the housing includes a top surface opposite the base surface. Preferably, the top surface is a surface on which one or more items that receive the pulsed electromagnetic field or signal in use may be placed.

[0068] In one embodiment, the electronic device and / or housing is attachable to an exterior surface of a container and / or reaction vessel, etc. For example, the electronic device and / or housing may be attachable by one or more attachment means or devices including one or more screws, nuts and bolts, magnets, strings, clips, straps, interengaging members, adhesives, and / or welding, etc., or any combination thereof.

[0069] Preferably, the distance between the top surface of the housing and / or one or more items, containers, products or reagents which receive the pulsed electromagnetic signal when located on, in or against the housing or electronic device in use, and the transmitting means is about 25cm or less, 20cm or less, 15cm or less, 10cm or less or 5cm or less. More preferably, the distance is about 1cm.

[0070] Preferably, the pulsed electromagnetic field or signal is provided in a predetermined sequence of pulses.

[0071] In one embodiment, the pulsed electromagnetic signal or field is provided at a frequency in the range of about 2.2-2.6 GHz, more preferably the pulsed electromagnetic signal is transmitted at a frequency of about 2.4 GHz ±50 MHz, more preferably 2.45 GHz ±50 MHz.

[0072] In one embodiment, the pulsed electromagnetic signal or field is provided at a frequency within the Industrial, Scientific and Medical radio frequency band (ISM band) of 2.4 to 2.4835 GHz, preferably 2.45 GHz ±50 MHz.

[0073] Preferably, the pulsed electromagnetic signal or field is pulsed at a frequency of about 50 Hz or less, more preferably about 25 Hz or less, and even more preferably about 15 Hz or less.

[0074] Preferably, each pulse of the pulsed electromagnetic signal or field lasts for about 1 ms to 20 ms, more preferably each pulse lasts for about 1 ms.

[0075] Preferably, the time between pulses (also called the "rest time" or "relaxation time") is about 66 ms or less.

[0076] Preferably, the pulsed electromagnetic signal has a duty cycle of less than 2%.

[0077] In one embodiment, the transmission power provided by each transmission means or chip in the electronic device is between +2 dBm and +4 dBm, about 1 mW, about 2 mW or about 2.5119 mW.

[0078] In one embodiment, the predetermined frequency of the pulsed electromagnetic field or signal is about 2.2-2.6 GHz, 2.4 GHz±50 MHz, or 2.45 GHz±50 MHz, the predetermined pulse rate is about 15 Hz or has a duty cycle of less than 2%, and the predetermined power is +2 dBm-+4 dBm, 1 mW, 2 mW, or 2.5119 mW.

[0079] Preferably, the pulsed electromagnetic field or signal is transmitted using 0.45 to 0.55 Gaussian Frequency Shift Keying (GFSK).

[0080] Preferably, the pulsed electromagnetic field or signal is a radio frequency (RF) data signal.

[0081] Preferably, the pulsed electromagnetic field or signal is a digital continuous pulsed electromagnetic signal.

[0082] Preferably, the radio frequency fields or signals utilise the advertising functionality of the Bluetooth LE (BLE) protocol. Preferably, the advertising RF signals are on channels 37, 38 and 39 corresponding to frequencies 2402 MHz, 2426 MHz and 2480 MHz respectively.

[0083] Preferably, the pulsing electromagnetic signal is directed to one or more DNA plasmids for transfection, lyophilized products or reagents for or to be reconstituted, IV formulations, products or reagents, and / or the like.

[0084] In one embodiment the electronic device comprises power supply means for powering the device in use. Preferably the power supply means comprises a line power source, one or more batteries, a power cell, one or more rechargeable batteries and / or power generation means etc.

[0085] In one embodiment the electronic device comprises control means for controlling the operation of the electronic device and / or the transmission means in use.

[0086] In one embodiment, the electronic device comprises one or more circuit boards. Preferably, the transmission means may be provided on one or more circuit boards, typically in the form of an integrated circuit, and / or on which other components such as, for example, memory means are located.

[0087] In one embodiment, the electronic device includes memory means, such as a memory device and / or a data storage device.

[0088] Preferably, the other components of the electronic device include one or more components necessary for the selected operation of the apparatus and for the controlled operation to generate a pulsed electromagnetic signal when in operation. For example, user selection means, display means for displaying one or more setting and / or selection options etc. may be provided on the device to enable a user to select one or more conditions, operation and / or one or more parameters of the device in use.

[0089] In one embodiment, the further components or power means include one or more power batteries, which may all be contained within the housing.

[0090] In one embodiment, a housing for an electronic device is provided in a form such that the housing can be engaged with and / or positioned relative to a container that holds a material and / or one or more items that will be exposed to electromagnetic signals during use.

[0091] In one embodiment the control means includes options allowing a user to select either or any combination of the signal frequency, signal strength, signal power, signal pulse rate and / or signal pulse time etc. of the pulsed electromagnetic signal. In one embodiment the selection of frequency, strength, power, pulse rate, pulse time and / or other parameters etc. can be made for a particular form of reagent or product and / or one or more containers, amounts of product or reagent, dimensions of the container in which the device is placed for use and / or other parameters that are exposed to the pulsed electromagnetic field or signal during use.

[0092] In one embodiment the apparatus comprises an agitation means or device which allows agitation of the product, mixture or reagent during use.

[0093] In one embodiment, the electronic device generating and / or emitting the pulsed electromagnetic field includes a means or device for measuring the turbidity and / or particle size of a product or reagent exposed to the pulsed electromagnetic field. For example, the device may include light scattering means and / or nephelometer means, etc. The light scattering means and / or turbidity measuring means may be used as an indication of the presence of aggregates, micelles, vesicles and / or particles, etc. in the product, mixture or reagent.

[0094] In one example, the electronic device may include a laser or columnar light source located on one side of the positioning means where the product or reagent is located in use, and a photodetector may be located on the opposite side of the positioning means that detects the amount of light passing from the light source through the product, mixture or reagent as a means of determining the turbidity and / or particle size of the product, mixture or reagent in use.

[0095] In a further example, the second photodetector can be positioned laterally, perpendicularly or substantially perpendicularly to the light source and / or the first photodetector. A comparison of the light detected from the first and second photodetectors can be used to determine the turbidity and / or particle size of the product, mixture or reagent in use.

[0096] Typically, the means for measuring the turbidity and / or particle size of a product, mixture or reagent exposed to a pulsed electromagnetic field may be integral to the electronic device, or may be attached or removably attached to the electronic device, or may be coupled to the electronic device.

[0097] In one embodiment, the electronic device is configured to generate and / or emit a pulsed electromagnetic field for a period of time sufficient to reduce a measured turbidity and / or particle size of the product, mixture or reagent below a predetermined threshold level.

[0098] In one embodiment, the electronic device may include audio, visual and / or kinesthetic means for signaling a user when the required turbidity and / or particle size of a product, mixture or reagent being exposed to the pulsed electromagnetic field falls below a pre-defined threshold level, such that the user knows that the product or reagent is safe for injection and / or infusion etc.

[0099] For example, if the product or reagent detects that the turbidity and / or particle size of the product, mixture or reagent exceeds a predetermined threshold level, the device can cause a red light to be displayed and should not be used by the user, and if the electronic device detects that the turbidity and / or particle size of the product, mixture or reagent after exposure to a pulsed electromagnetic field is below a predetermined threshold level, the device can cause a green light to be displayed and may be used by the user.

[0100] In one example, one or more audio signals, such as a "ping" or alarm, may be emitted when a reagent, mixture or product is detected to be safe for use after exposure to the pulsed electromagnetic field.

[0101] In one example, the device may generate one or more vibrations when a reagent, mixture or product is detected as safe for use after exposure to a pulsed electromagnetic field.

[0102] According to a second aspect of the present invention there is provided an apparatus for improving the hydration of a product or reagent and / or reducing its particle size in use, the apparatus configured to generate and radiate a pulsed electromagnetic field which may be directed towards the product or reagent for a time sufficient to enable increased hydration of the product or reagent and / or reduction in particle size of the product or reagent.

[0103] Preferably, the apparatus comprises a means or device for detecting particle size and / or turbidity of the product or reagent.

[0104] According to one aspect of the invention, there is provided a product or reagent that has been reconstituted using the application of a pulsed electromagnetic field, the product or reagent having had the pulsed electromagnetic field applied to it for a period of time.

[0105] According to one aspect of the invention, there is provided an intravenous product or reagent suitable for intravenous delivery, to which a pulsed electromagnetic field is applied for a period of time.

[0106] According to one aspect of the invention, there is provided a DNA plasmid to which a pulsed electromagnetic field has been applied for a period of time.

[0107] According to a further aspect of the invention there is provided a method of reconstituting a lyophilized or freeze-dried product or reagent, the method comprising the steps of adding water to the lyophilized or freeze-dried product or reagent to create a mixture, operating one or more devices to generate a pulsed electromagnetic field, and placing the mixture in the pulsed electromagnetic field for a period of time to allow reconstitution of the product or reagent.

[0108] According to a further aspect of the present invention there is provided a method of preparing an intravenous product or reagent suitable for intravenous delivery to a patient in use, the method comprising applying a pulsed electromagnetic field to the intravenous product or reagent for a period sufficient to reduce or prevent the formation of excipient aggregates.

[0109] Preferably, the excipient aggregates are micelles and / or vesicles.

[0110] According to yet another aspect of the present invention, there is provided a method of reducing the size of a DNA plasmid, the method comprising applying a pulsed electromagnetic field to the DNA plasmid for a period of time sufficient to reduce the size of the DNA plasmid.

[0111] Specific embodiments of the present invention will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0112] [Figure 1a] 1 illustrates diagrammatically an apparatus according to one embodiment of the invention; [Figure 1b] 1 illustrates diagrammatically an apparatus according to one embodiment of the invention; [Diagram 2] 1 illustrates result data obtained from the use of a conventional reconstitution process (control) and a reconstitution process according to one embodiment of the present invention for Factor VIII. [Diagram 3] 1 illustrates result data obtained from measuring the size of DNA plasmids with and without exposure (control) to pulsed electromagnetic fields according to one embodiment of the present invention. [Figure 4a] Illustrating the result data obtained for the use of a pulsed electromagnetic field on a culture medium according to one embodiment of the present invention, FIG. 4a shows a control where the culture medium was not exposed to a pulsed electromagnetic field. [Figure 4b] Illustrating the result data obtained for the use of pulsed electromagnetic fields on culture media according to one embodiment of the present invention, FIG. 4b shows the invention when culture media is exposed to pulsed electromagnetic fields. [Diagram 5] FIG. 13 is a top view of the experimental setup of the pulsed electromagnetic field device for the active sample in experiment 6. [Figure 6a] Figure 6 shows result data obtained from using a conventional reconstitution process (control) and a reconstitution process according to an embodiment of the present invention for Factor XI. Figure 6a shows all particle sizes. [Figure 6b]Figure 6b shows result data obtained from the use of a conventional reconstitution process (control) and a reconstitution process according to an embodiment of the present invention for Factor XI. Figure 6b is an enlarged view showing the smaller sized particles of Figure 6a. [Figure 7] Illustrates Factor XI result data in Experiment 7 along with particle size versus number of particles per ml. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] Referring initially to FIG. 1, an apparatus according to one embodiment of the present invention is illustrated for use in reconstituting freeze-dried products or reagents.

[0114] The device comprises a container, in this case in the form of a vial 2, having a cavity in which a freeze-dried product 3, such as Factor VIII, is provided and housed. In one embodiment, the vial can be used to transport the Factor VIII in a freeze-dried state from the location where the freeze-drying is performed to the location of the end user, which may be a location in the home, where, as will be appreciated, there is limited or no equipment available to the end user. Thus, according to the present invention, a device is provided that can be used by non-skilled personnel to allow an improved reconstitution of the freeze-dried product. In the illustrated embodiment, the vial 2, in which the freeze-dried product 3 is located, is opened and placed on a plate 6 having a positioning recess 8 in which the vial is placed. A water source 10 is then provided that allows water to be poured into the vial to a predetermined extent, which may provide a physical mixing of the contents of the vial if necessary, or the plate may be provided with stirring means that allow the plate to effectively vibrate and perform a mixing action between the water and the product in the vial. Simultaneously with, prior to, or after mixing, a pulsed electromagnetic field generator 11, in this embodiment formed in combination with plate 6, is operated to generate a pulsed electromagnetic field, indicated at 14, which radiates from the device and enters the container through the plate as shown. This therefore ensures that the water particles in the container and the product in the container are exposed to the pulsed electromagnetic field, which causes the water particles to rotate, which in turn can act on the sugar coating of the freeze-dried protein and effectively remove the sugar from the surface, thereby allowing for more replacement of the sugar by the water particles, and thus allowing for a higher level of reconstitution of the product to its pre-freeze-dried shape. This reduces the possibility and chance that product particles will stick to each other, thereby reducing the possibility of product aggregation.

[0115] Once the product is mixed, it can be removed from the container for human use, such as by injection, etc. The injected product more closely resembles the state required by the patient's body, in which case the level of immune rejection by the user of the product is greatly reduced, and the product effectively achieves a state of immune silencing not previously achievable using conventional reconstitution processes. EXAMPLES

[0116] Experiment 1 – Reconstitution of freeze-dried factor VIII Experiments were conducted to determine the effect of using the device of the present invention on the particle size of reconstituted freeze-dried Factor VIII.

[0117] The particle size of the blood protein product Factor VIII (FVIII) was measured using a dynamic light scattering instrument, in this example a Zetasizer Ultra™ (Malvern Panalytical Ltd, UK).

[0118] The experiments were carried out on control samples in which no pulsed electromagnetic field was applied to FVIII and on samples according to the invention in which a pulsed electromagnetic field was applied to FVIII.

[0119] The method steps were as follows: 1. Reconstitute a vial of freeze-dried Factor VIII with Water for Injection (WFI) according to the vial's instructions (gently swirl until all visible material has dissolved and the liquid appears clear). 2. For each control sample and inventive sample, 1 ml of sample was added to a clean cuvette (part numbers PCS8501, PCS1115 or DTS0012, Malvern Panalytical, UK). 3. Samples of the invention were treated with a pulsed electromagnetic field (using a PulzFector™ device that generates a pulsed electromagnetic field, St. Andrews Pharmaceuticals Technology Ltd, UK) by placing the cuvette in a PulzFector™ and turning the device on for 10-15 minutes. 4. The control cuvette and the cuvette of the invention were placed in sequence in a Zetasizer Ultra™ (Malvern Panalytical, United Kingdom) to measure the particle size of the reconstituted FVIII.

[0120] In one example, the PulzFector (trade name) device used to conduct the experiments is an electronic device or apparatus containing six electronic chips capable of emitting pulsed electromagnetic fields at frequencies in the range of 2.2 to 2.6 GHz, with a pulse frequency of 50 Hz or less, with each pulse lasting for 1 ms to 20 ms, with the time between each pulse being approximately 66 ms or less, and with an output power of 2 dBm to +4 dBm.

[0121] The experimental results are shown in Figure 2.

[0122] Figure 2 is a graph showing particle diameter in nanometers (nm) on the X-axis and volume percent on the Y-axis. The particle size distribution is shown as a solid black line for the sample of the present invention and a dotted black line for the control sample. In the control sample, two large peaks of particles with diameters of 37.3 nm and 173.5 nm are observed. These are typically pentameric aggregates that are known to be immunogenic. In the sample of the present invention, peaks of particles with diameters of 26.3 nm and 122.4 nm are observed.

[0123] It can therefore be concluded that the particle size of reconstituted FVIII is significantly reduced and aggregates are dispersed when exposed to a pulsed electromagnetic field compared to when no pulsed electromagnetic field is applied, and thus the present invention mitigates and reduces the formation of immune-induced protein aggregates.

[0124] The observed reduction in particle size in the reconstitution method using pulsed electromagnetic fields of the present invention is believed to occur due to increased uniformity of the hydration of the product, which creates a smaller hydration layer, which manifests as smaller particles when using light scattering equipment.

[0125] It will be appreciated that water (WFI) used to reconstitute a freeze-dried product or reagent may be exposed to a pulsed electromagnetic field in addition to or instead of exposing the freeze-dried product or reagent. A pulsed electromagnetic field may be directed at the mixture of water and freeze-dried product or reagent during or after reconstitution.

[0126] Experiment 2 - Determining the efficacy of reconstituted freeze-dried Factor VIII The effectiveness of reconstituted FVIII in forming thrombi can be analyzed using a one-step chromogenic assay. The method was as follows. 1. An equivalent volume (most commonly 0.1 ml) of test or reference plasma was incubated at 37° C. In addition, an equivalent volume of plasma deficient in factor VIII was incubated at 37° C. Note: The reference plasma was used to derive the concentration / activity of the test plasma by comparing clotting times. 2. Platelet poor plasma (PPP) was incubated with phospholipids at 37°C and contact activator was added followed by calcium (to initiate clotting). Note: All reagents were pre-warmed to 37°C. 3. Clotting time was measured.

[0127] Based on the data results obtained in Experiment 1, it is expected that samples of the invention exposed to pulsed electromagnetic fields will have significantly reduced clotting times compared to control samples. It can therefore be concluded that the application of pulsed electromagnetic fields to a product or reagent results in improved protein functionality and / or efficacy.

[0128] Experiment 3 - Reduction of CARPA responses in IV delivered products This experiment was performed to determine the effect of using a device of the present invention on the formation of excipient aggregates (such as micelles or vesicles) on a product or formulation delivered IV.

[0129] The particle size of aggregates in the IV product or formulation was measured using a dynamic light scattering instrument (in this example, a Zetasizer Ultra™ (Malvern Panalytical Ltd, UK)) following the protocol in Experiment 1.

[0130] Based on the data results of Experiment 1, it is expected that exposure to pulsed electromagnetic fields in accordance with the present invention will significantly reduce the formation of excipient aggregates in the IV product or formulation. The efficacy, functionality and / or potency of the IV product or formulation is likely to be significantly improved as a result of applying a pulsed electromagnetic field to the IV product or reagent.

[0131] Experiment 4 - Size reduction of DNA plasmids during transfection This experiment was performed to demonstrate the reduction in size of DNA plasmids used in the transfection process using pulsed electromagnetic fields according to the present invention. 1. The transfection process was performed using a DNA plasmid of interest that had not been exposed to pulsed electromagnetic fields. 2. The success of the transfection process was evaluated. 3. According to the present invention, a further transfection process was carried out using DNA plasmids exposed to pulsed electromagnetic fields for 10-15 minutes. Note that no transfection reagent was present with the DNA plasmids during exposure to pulsed electromagnetic fields. 4. The success of the transfection process was evaluated: The size of the DNA plasmid was measured using a Zetasizer Ultra (Malvern Panalytical, United Kingdom).

[0132] Figure 3 illustrates the result data obtained in measuring the size of DNA plasmids exposed and not exposed (control) to pulsed electromagnetic fields according to experiment 4. It can be seen that the diameter of the particles of DNA plasmid not exposed to pulsed electromagnetic fields was measured to be 15.03 nm, whereas the diameter of the particles of DNA plasmid exposed to pulsed electromagnetic fields was measured to be 12.19 nm.

[0133] Thus, the size of DNA plasmids can be reduced by exposure to pulsed electromagnetic fields compared to without exposure to pulsed electromagnetic fields, which will likely result in higher transfection frequencies.

[0134] Experiment 5 – Reduction of particle size in cell culture medium This experiment was performed to demonstrate that exposing cell culture medium to pulsed electromagnetic fields in accordance with the present invention reduces the size of particles in the cell culture medium.

[0135] The particle size of the aggregates in the medium was measured using a dynamic light scattering instrument, in this example a Zetasizer Ultra™ (Malvern Panalytical Ltd, UK), following the protocol in Experiment 1.

[0136] Dynamic light scattering data for the control medium (not exposed to a pulsed electromagnetic field) is shown in Figure 4a. Dynamic light scattering data for the medium exposed to a pulsed electromagnetic field according to the present invention is shown in Figure 4b. It can be seen that the particle size in the medium exposed to the pulsed electromagnetic field is reduced and more uniformly distributed compared to the particles in the control medium. This may improve cell growth in the medium exposed to the pulsed electromagnetic field compared to the control without the pulsed electromagnetic field. It may also improve the transfection speed and its efficiency and frequency when the cell culture medium is available.

[0137] Experiment 6 - Effect of pulsed electromagnetic fields on the reconstitution of lyophilized rhFVIII A chromogenic assay was performed to measure the effect of application of pulsed electromagnetic fields using the device of the present invention on the activity levels of reconstituted lyophilized rhFVIII (Advate®, Takeda, USA).

[0138] The chromogenic assay for factor VIII allows the quantitative determination of factor FVIII in a sample. Factor X is first converted to factor Xa (the rate of activation of factor X is linearly related to the amount of factor FVIII). The quantification of factor Xa activity is then measured using a synthetic chromogenic substrate. Factor Xa hydrolyzes the chromogenic substrate releasing paranitroaniline (pNA), which is kinetically monitored at 405 nm and is proportional to the FVIII in the sample [3].

[0139] Three separate control experiments were performed. The control experiments were not exposed to pulsed electromagnetic fields. These control experiments included the following method steps: a) A vial of 1000 IU Advate™ (Takeda, USA) was reconstituted with 5 ml of water for injection and left on the bench at room temperature and pressure for 15 minutes. b) An intermediate stock was prepared by adding 10 microliters of reconstituted Advate® to 90 microliters of FVIII deficient plasma sample (HemosIL® FVIII deficient plasma-Instrumentation Laboratory, USA). c) 50 microliters of the intermediate stock was then taken and added to 950 microliters of the FVIII deficient plasma sample.

[0140] The activity value of the final neat solution prepared (to measure how pure the sample was) was expected to be 100% in the control experiment, however, after the chromogenic assay test was performed in triplicate on three different Advate® vials, the values ​​obtained in the control experiment were 77.9%, 86.4% and 89.4%.

[0141] Three separate "active" experiments using pulsed electromagnetic fields in accordance with the present invention were carried out. The active experiments included the following method steps: a) A vial 18 of 1000 IU Advate (Takeda, USA) was reconstituted with 5 ml of water for injection and placed on a PulzFector (a device that generates a pulsed electromagnetic field, St. Andrews Pharmaceuticals Technology Ltd, UK) 20 as shown in Figure 5 and surrounded by four Pulzar Pi devices 22 for 15 minutes at room temperature and pressure. The Pi devices 22 were positioned equidistant from each other and from the vial of Advate 18. b) An intermediate stock was prepared by adding 10 microliters of reconstituted Advate® to 90 microliters of FVIII deficient plasma sample (HemosIL FVIII deficient plasma-Instrumentation Laboratory, USA). c) 50 microliters of the intermediate stock was then taken and added to 950 microliters of the FVIII deficient plasma sample.

[0142] Again, the final neat solution activity value in the activity experiment was expected to be 100%. Values ​​obtained after chromogenic assay testing in triplicate on three different Advate® vials were 98.7%, 97.1% and 108.1%.

[0143] After performing a t-test, it was found that the activity of a 1000 IU Advate vial was significantly increased by applying a pulsed electromagnetic field to the active sample for the reconstitution process in accordance with the present invention. Table 1 Neat chromogenic assay results [Table 1] α=0.05 t-Test: Means of two paired samples [Table 2]

[0144] Experiment 7 - Reconstitution of freeze-dried Hemoleven® (Factor XI) Experiment 1 was repeated, but using Hemoleven® (Factor XI) instead of Factor VIII.

[0145] This experiment was performed to determine the effect of using the device of the present invention on the particle size of reconstituted freeze-dried Factor XI and to confirm the surprising observation about the particle size of reagents other than Factor VIII.

[0146] The particle size of Factor XI exposed to pulsed electromagnetic fields is shown in Figures 6a and 6b as the blue line (24) in comparison to the particle size of the Factor XI control (orange line (26)) not exposed to pulsed electromagnetic fields. It can be seen that the sample exposed to pulsed electromagnetic fields has a significantly reduced volume percentage of large particles compared to the control. This mirrors the results found in experiment 1. This indicates that the benefits of the present invention are not limited to Factor VIII but can be seen with other blood clotting factors as well.

[0147] Figure 7 shows data from Experiment 7 with particle size on the X-axis and number of particles per ml on the Y-axis. The blue dots with a cross represent the particle size of Factor XI exposed to a pulsed electromagnetic field, and the orange dots without a cross represent the particle size of the Factor XI control. This shows that the benefits of the present invention are not limited to Factor VIII, but are seen with other blood clotting factors as well.

[0148] References [1]-“Native-like aggregates of Factor VIII(FVIII)are immunogenic von Willebrand Factor deficient and hemophilia A mice”- J Pharma Sci.2012 Jun:101(6):2055-2065 [2]-“Molecular aggravation of marketed recombinant FVIII products:Biochemical Evidence and Functional Effects”- TH Open 2019 Apr;3(2):e123-e131 [3]-“Diagnostics Directorate,North Glasgow Sector,Department of Hematology,Chromogenic FVIII”- LAP-GRI-COA-076 - Revision No.1,page 3-11(NHSGGC)

Claims

1. A method for improving the hydration reaction of a product or reagent and / or reducing its particle size, the method comprising applying a pulsed electromagnetic field to the product or reagent for a period sufficient to enable an increase in the hydration reaction of the product or reagent and / or a reduction in the particle size of the product or reagent.

2. The period during which the product or reagent is exposed to the pulsed electromagnetic field is a predetermined period, and optionally, The predetermined period is 10 to 15 minutes ± 5 minutes, according to the method of claim 1.

3. The product or reagent is any one or any combination of a culture medium, a cell culture medium, a lyophilized or freeze-dried product or reagent, a powdery or granular product or reagent, a protein product, a monoclonal antibody, a hormone, a fusion protein, a protein construct, trastuzumab, pembrolizumab, infliximab, diphtheria toxin, an immunoglobulin, omalizumab, abatacept, secukinumab, interferon beta 1a, bortezomib, DNA, a DNA plasmid, factor VIII, an intravenous preparation, a product or reagent, known to be associated with the CARPA reaction or forming part thereof, a dispersant, Tween 80, polysorbate 80, according to the method of claim 1.

4. The product or reagent is a lyophilized or freeze-dried product or reagent, and the method comprises reconstituting the lyophilized or freeze-dried product or reagent to form a mixture by adding water or a liquid thereto, and applying the pulsed electromagnetic field to the mixture for the period or for the period of reconstitution of the product or reagent in the mixture, according to the method of claim 1.

5. The addition of the water or the liquid to the product or reagent for forming the mixture is carried out while the pulsed electromagnetic field is applied to the water, the liquid and / or the mixture, or, The method according to claim 4, wherein the pulsed electromagnetic field is applied to the water or the liquid used for reconstitution before the addition to the freeze-dried or freeze-dried product or reagent.

6. The method according to claim 1, comprising the step of stirring the product, reagent or mixture at any point in time before applying the pulsed electromagnetic field, during the step of applying the pulsed electromagnetic field and / or after the step of applying the pulsed electromagnetic field.

7. The method according to claim 1, wherein the pulsed electromagnetic field is applied to the product, reagent or mixture during the intravenous drip or injection process to the patient and / or during or before placement in the intravenous bag or intravenous delivery container.

8. The method according to claim 1, wherein the pulsed electromagnetic field is generated by one or more electronic devices, electronic apparatuses, circuits, transmission means or devices, and / or one or more electronic transmission chips.

9. A plurality of transmission means or electronic transmission chips arranged at a predetermined separation distance from each other and / or in a predetermined pattern or array are provided to provide the pulsed electromagnetic field, and / or one transmission means or electronic transmission chip exists per 105 to 115 cm2, or 50 to 60 cm2, or 25 to 30 cm2, or 15 to 20 cm2 of the surface of the electronic device or circuit. The method according to claim 8.

10. The pulsed electromagnetic field is provided at a frequency of 2.2 to 2.6 GHz and any one or any combination of frequencies within the industrial, scientific and medical frequency band of 2.4 to 2.4835 GHz. The pulsed electromagnetic field is pulsed at a frequency of about 50 MHz or less, 25 Hz or less, or 15 Hz or less. Each pulse of the pulsed electromagnetic field lasts for about 1 to 20 ms or about 1 ms. The time between pulses is about 66 ms or less, or The duty cycle of the pulsed electromagnetic field is less than 2%. The method according to claim 1.

11. The method according to claim 8, wherein the transmission power of the transmission means or the chip is 2 dBm to 4 dBm, about 1 mW, about 2 mW or about 2.5119 mW.

12. An apparatus for improving the hydration reaction of a product or reagent and / or reducing its particle size during use, the apparatus being configured to generate and emit a pulsed electromagnetic field that can be directed towards the product or reagent for a time sufficient to enable an increase in the hydration reaction of the product or reagent and / or a reduction in the particle size of the product or reagent.

13. Stirring means for stirring the product, reagent or mixture during use, turbidity and / or particle size measuring means or devices for measuring the turbidity and / or particle size of the product, reagent or mixture during use, and any one or any combination of audio, visual and / or kinesthetic means for sending a signal to the user when the product, reagent or mixture exposed to the pulsed electromagnetic field reaches a specific level of hydration reaction, turbidity and / or particle size. The apparatus according to claim 12.

14. The apparatus according to claim 12, wherein the pulsed electromagnetic field is provided at a frequency of 2.2 to 2.6 GHz and / or pulsed at a frequency of about 50 MHz or less, 25 Hz or less, or 15 Hz or less.

15. A reconstituted product or reagent, an intravenous product or reagent, or a DNA plasmid reconstituted using the method according to claim 1.

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