Vitrification plant cryopreservation method, vitrified and lyophilised microgranules from biopharmaceutical products, and biopharmaceutical preparation

Abstract

The invention relates to a vitrification plant for producing microgranules from biopharmaceutical products (10) and to an associated cryopreservation method. The vitrification plant comprises an assembly for producing product droplets (13) by means of a vibration-excited application cannula (21) and an assembly for vitrification with a cooling unit (50), in which the product droplets (13) are transformed into the vitrified microgranules by contact cooling on a cryogenic vitrification medium (51). High freezing rates allow crystallisation to be avoided, enabling gentle cryopreservation without, or with minimal amounts of, cell-damaging antifreeze additives. The technology is preferably used in cell and enzyme therapy for patient-specific individual doses of the microgranules; for example, in the form of a biopharmaceutical, functional mitochondria-comprising preparation produced according to the cryopreservation method.

Description

[0001] Vitrification plant, cryopreservation process, vitrified and lyophilized microgranules from biopharmaceutical products and biopharmaceutical preparation

[0002] The invention relates to a vitrification plant for the production of microgranules from biopharmaceutical products, a cryopreservation process achievable using the vitrification plant, vitrified microgranules produced by the cryopreservation process, a process for revitalizing a biopharmaceutical product, a process for producing lyophilized microgranules from a biopharmaceutical product, lyophilized microgranules produced by the cryopreservation process, and a biopharmaceutical preparation comprising functional mitochondria for use as a drug. The technology serves to convert biopharmaceutical products containing therapeutic cells, therapeutically active cell components, microorganisms, and / or viruses into vitrified, free-flowing, and storable microgranules.The vitrification system and the cryopreservation process carried out with it are suitable for the vitrification of individualized biopharmaceutical single doses for direct medical application.

[0003] The state of the art in the cryopreservation of biopharmaceutical products encompasses various established methods for the long-term storage of live cells, enzymes, antibodies, and other biologically active components. Vitrification is one such method for cryopreserving live cells and biologically active cell components in aqueous media. The unique aspect of vitrification is that the aqueous medium—a product fluid containing the biopharmaceutical products, i.e., a product suspension and / or a product solution—is transformed from a liquid to a solid state without the formation of a crystalline structure (i.e., ice crystal formation), resulting in an amorphous, glassy structure.This prevents damage to biological structures from accompanying effects of ice crystal formation, such as water loss, increased concentration of dissolved components in the product fluid, or direct damage to biological structures by sharp-edged ice crystals.

[0004] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 A biopharmaceutical product in the present sense is a mostly liquid formulation, for example, a solution or a suspension, that contains one or more biological components with biopharmacological or therapeutic effects. The biological components with biopharmaceutical or therapeutic effects can belong to three different product classes. The first product class includes therapeutic cells. This encompasses all living cells with therapeutic effects, for example, autologous or allogeneic stem cells, mature somatic cells, in particular various immune cells such as natural killer cells, as well as genetically modified cells for therapeutic purposes, for example, CAR-T cells. The second product class comprises therapeutically active cell components (subcellular therapeutics).This includes all active components of living cells with therapeutic effects that are secreted by the cells or can be obtained from the cells in unaltered or modified form, for example, chemically or genetically engineered. This includes all cell organelles, in particular mitochondria or ribosomes, as well as components of the cellular secretome, such as exosomes and microvesicles for anti-aging treatments and other therapeutic purposes, and antibodies, such as monoclonal antibodies for therapeutic applications, for example, in cancer therapies, for immunosuppression in inflammatory diseases, or for passive immunization, as well as all enzymes with therapeutic effects. The third product class comprises microorganisms and viruses.This includes all prokaryotic or eukaryotic single- or multi-celled microorganisms such as bacteria, fungi or algae, and in particular unmodified or chemically or genetically modified viruses with therapeutic effects, for example bacteriophages for phage therapy or viral vectors for gene therapies.

[0005] To achieve vitrification under practical conditions, the temperature range between the freezing point and the glass transition temperature of the product fluid must be traversed extremely quickly to prevent the formation of crystallization nuclei. If this is not possible, highly concentrated additives, so-called antifreeze additives (also known as cryoprotective additives or cryoprotectants), must be added to the product fluid to prevent crystallization.

[0006] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 to prevent. Essential for classical vitrification processes are cryoprotectant additives such as glycerol and dimethyl sulfoxide (DMSO), which are able to cross biological membranes by diffusion and exert a cryoprotective effect inside membrane-bound cells and cell components.

[0007] These substances are known as permeating cryoprotective agents (pCPAs). They are often used in high concentrations for the vitrification of biopharmaceutical products to raise the glass transition temperature. This reduces the critical temperature range that the biopharmaceutical product must pass through during cooling, where unwanted ice formation can occur and potentially lead to product degradation. Thus, the cooling rate required for successful vitrification is reduced by several orders of magnitude with the addition of high pCPA concentrations, for example, 20% to 60% DMSO, compared to pCPA-free media. This allows the vitrification process to be carried out using simple technical means.At temperatures above freezing, however, the aforementioned penetrating antifreeze additives themselves have a damaging effect on the biological components of the biopharmaceutical products and therefore must be painstakingly removed after thawing. This is difficult or even impossible with a ready-to-use therapeutic formulation that is intended for immediate use after thawing according to its therapeutic purpose. Therefore, it is highly advantageous to avoid the use of pCPA during vitrification.

[0008] To avoid the addition of antifreeze additives, vitrification can be achieved by rapidly cooling small samples containing the product fluid. An established method is the direct immersion of small sample volumes in liquid nitrogen, using ultrathin supports such as capillary tubes or specially prepared carrier films to ensure homogeneous and rapid cooling. This technology is demonstrated, for example, in

[0009] WO 2007 / 120829 A2. Alternatively, microscopically small droplets can also be generated by atomization or microfluidic technologies and transferred into cryogenic media. Such microfluidic-based methods enable controlled droplet generation, but are usually technically complex and require

[0010] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 precise control mechanisms. Various methods of droplet generation and their subsequent deep freezing in a cryogenic medium are known, among other things, from US 2012 / 0251999 A1.

[0011] German patent application DE 102012 109406 A1 discloses a system and a process for the rapid, energy-efficient, and quasi-continuous production of pharmaceutical lyophilisates in the form of free-flowing, spherical particles. For this purpose, droplets of a defined size are generated from an active ingredient-containing solution using a droplet generator, introduced vertically into a freezing tube, and frozen there by heat transfer to a cooled gas flowing in the direction of fall. The frozen droplets are then collected in a loading chamber and subjected to primary and secondary drying (vacuum freeze-drying) in an evacuable drying tunnel before the dried particle product is transferred to sterile containers under aseptic conditions. DE 102012 109406 A1 describes the known freeze-drying process but does not mention vitrification.

[0012] DE 69433251 T2 discloses a method and an apparatus for producing frozen particles from a liquid product, in which droplets of the product are introduced into a housing by means of an atomizing nozzle and brought into contact there with a cryogenic liquid that circulates around the droplets to cause freezing. The cryogenic liquid is guided in such a way that the droplets are essentially enclosed and do not come into contact with solid, cold surfaces. The frozen particles are then collected and can be further processed.

[0013] EP 0478 118 A1 describes a device for producing frozen particles in which a material to be frozen is sprayed into a gas-tight, essentially adiabatic vessel by means of a material atomizer, while a coolant, in particular liquefied nitrogen, is introduced in a tangential direction via several atomizers arranged in a ring in the upper part of the vessel.

[0014] This creates a swirling stream of cold gas within the vessel, in which the sprayed material droplets freeze through heat transfer and form frozen particles. The frozen particles are transported downwards along the vessel wall in the gas stream and discharged through a lower outlet.

[0015] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026, whereby the tangential introduction of the coolant is intended to prevent uniform freezing and the particles remaining in the vessel.

[0016] Despite the progress described and the existence of devices and methods for freezing liquid droplets, challenges remain in the industrial implementation of vitrification. These include process scalability, the reduction or elimination of toxic antifreeze additives, ensuring the biological integrity of the product over long storage periods, and the possibility of direct medical application without complex purification or reprocessing procedures. While existing methods offer a variety of solutions, the development of efficient, scalable, and biocompatible vitrification methods remains a key objective.

[0017] The object of the invention is to provide a vitrification system and a cryopreservation process that enable the preservation of biopharmaceutical products by uniform and reliable cooling, avoiding undesirable ice crystal formation and without the need for high concentrations of penetrating antifreeze additives (pCPA), thereby ensuring the preservation of the functionality of the biological components contained in the biopharmaceutical products. A further object of the invention is to further process the products manufactured using the cryopreservation process and to make the resulting preparations available, particularly for medical applications.

[0018] This problem is solved by a vitrification plant for producing microgranules from a biopharmaceutical product with the features of claim 1, and by a cryopreservation process for producing vitrified, free-flowing microgranules from a biopharmaceutical product according to claim 14. Advantageous further developments of the vitrification plant are set out in claims 2 to 13, and embodiments of the cryopreservation process are set out in claims 14 to 18. The vitrified, free-flowing microgranules produced by the cryopreservation process are described in claim 19; an advantageous embodiment of the vitrified,

[0019] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Claim 20 is supplemented by a process for revitalizing the cryopreserved biopharmaceutical product. Claim 21 discloses a process for producing lyophilized microgranules from the vitrified, free-flowing microgranules. Claim 22 describes the lyophilized microgranules produced thereby; an advantageous embodiment of the lyophilized microgranules is supplemented by claim 24. Claim 25 further describes a biopharmaceutical preparation comprising functional mitochondria for use as a drug; an advantageous embodiment in this respect is supplemented by claim 26.

[0020] According to the invention, the vitrification system for producing microgranules from a biopharmaceutical product, which may include biological components with biopharmaceutical or therapeutic effects in the form of therapeutic cells, therapeutically active cell components, microorganisms and / or viruses, comprises two main assemblies, namely an assembly for generating product droplets from a product fluid containing the biopharmaceutical product and an assembly for vitrifying the product droplets, wherein the assembly for generating product droplets comprises a syringe pump, an application syringe, an application cannula and a vibration excitation unit, and the assembly for vitrifying the product droplets comprises a cooling unit and a collection vessel for collecting the microgranules.

[0021] For the purposes of this description, biopharmaceutical products are understood to be the formulations already presented which contain biological components according to the three product classes described, namely therapeutic cells (first product class), therapeutically active cell components (second product class) and microorganisms and / or viruses (third product class).

[0022] The product fluid contains the biopharmaceutical product in a liquid, aqueous medium. The product fluid can be a product suspension and / or a product solution. Within the product suspension, the biopharmaceutical product is present in the form of product particles suspended in the liquid, aqueous medium.

[0023] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 are dispersed. The size of the product particles can vary depending on whether the biopharmaceutical product contains therapeutic cells, therapeutically active cell components, microorganisms and / or viruses. In a product solution, the biological components of the biopharmaceutical product are present in dissolved form within the liquid, aqueous medium; this can be the case, for example, with enzymes or antibodies.

[0024] The application syringe, which is filled with the product fluid when used as intended and serves to convey the product fluid, is connected to the syringe pump in order to convey the product fluid in a controlled manner via the application syringe.

[0025] The application syringe is equipped with an application cannula, through which the product fluid, conveyed by the application syringe and syringe pump, is directed to generate a continuous jet of product fluid exiting the application cannula vertically, i.e., from a cannula outlet opening of the application cannula. For this purpose, the application cannula is also aligned vertically. The application cannula has at least one base cannula.

[0026] Preferably, the base cannula is elongated, where "elongated" refers to a geometric configuration of the base cannula in which the axial length of the cannula shaft is several times its largest outer diameter, in particular at least five times, preferably at least ten times, the outer diameter. If the application cannula comprises only the base cannula, the cannula exit opening is located directly on the base cannula.

[0027] Preferably, the application cannula is designed as a compound cannula comprising a base cannula and a cannula tip with the cannula outlet opening, wherein the inner diameter of the cannula tip at the cannula outlet opening is narrower than the inner diameter of the base cannula at the transition to the cannula tip. Particularly preferably, the inner diameter of the cannula tip tapers in the direction of product fluid flow, i.e., from the point where the cannula tip joins the base cannula to the cannula outlet opening.

[0028] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The cannula tip is designed here as a convergence nozzle for the product fluid conveyed through the application cannula.

[0029] The vitrification plant, that is, its apparatus for generating the free-falling product droplets, is further equipped with the vibration excitation unit, which includes a vibration exciter, wherein the vibration exciter is, for example, a linear actuator for generating a linear alternating stroke motion. The vibration exciter serves to excite a high-frequency vibration, the waveform, amplitude, and frequency of which are preferably adjustable. For the purposes of the invention, a high-frequency vibration is understood to be a vibration with a frequency in the kilohertz range or above.

[0030] Furthermore, the device for generating the free-falling product droplets of the vitrification system comprises a coupling with which the high-frequency vibration, preferably a sinusoidal vibration, is transmitted from the vibration exciter to the product fluid jet exiting the application cannula. This leads to the formation of monodisperse product droplets (microdroplets) that bud off from the product fluid jet, forming a uniform droplet chain of product droplets that fall freely in the vertical direction.

[0031] The generation of product droplets by vibration excitation is achieved, in particular, by utilizing the Plateau-Rayleigh instability according to the principle of a moving orifice. For this purpose, a high-frequency signal is generated using the vibration exciter and transmitted via the coupling to the application cannula to set it into vibration. This vibration then also acts on the product fluid jet exiting the application cannula. Devices and methods for generating droplets from a liquid, which are ejected as a continuous jet from a fine cannula, are generally known. The liquid jet is selectively destabilized by mechanical vibrations to form a droplet chain. This principle for generating monodisperse droplets and a suitable device for this purpose are described, for example, in Kosch, S., Ashgriz, N.: A simple vibrating orifice monodisperse droplet generator using a hard drive actuator arm, Rev. Sei. Instrum.

[0032] 1. April 2015; 86 (4): 046101, https: / / doi.Org / 10.1063 / 1.4916703.

[0033] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The production of product droplets, in particular monodisperse product droplets, is not limited to the vibration excitation technology described above. Rather, the product droplets can, in principle, be generated by any technology suitable for causing a periodic flow separation of the product fluid exiting the product-carrying application cannula. This includes technologies for the targeted generation of a Plateau-Rayleigh instability, as well as all other technologies, provided they also lead to a periodic destabilization of the product fluid jet with subsequent reproducible droplet formation. The vibration excitation unit and the coupling of the device for generating the free-falling product droplets can be designed accordingly.

[0034] The product droplets can be generated electromechanically, for example, by exciting an oscillating motion using the vibration exciter in the form of the linear actuator already described, such as a piezoelectric linear drive or an electric motor with oscillating motion. The oscillation can be transferred directly or indirectly to the application cannula, the product fluid contained within it, or the application syringe. In particular, a motor with translational or rotary motion can be used as the electric motor, for example, a motor with an oscillating motion known from hard disk drives or a voice coil motor (VCM) that generates linear motion.

[0035] Alternatively or additionally, droplet generation can be achieved by electrostatic polarization of the product fluid in the area of ​​the outlet opening of the product-carrying application cannula. For this purpose, a high-frequency alternating electric field, preferably a high-voltage alternating field, can be applied, with the cannula tip of the application cannula serving as an electrode and interacting with a counter electrode. The electric field periodically influences the product fluid in the area of ​​the cannula outlet opening, resulting in the controlled, recurring detachment of individual product droplets.

[0036] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Furthermore, droplet formation can be achieved by generating periodic pressure pulses within an application syringe or other product container that is fluidically connected to the product-carrying application cannula. The pressure pulses can be generated, for example, by means of a piezoelectric actuator or an electromechanical air compressor, such as those used in inhalation devices. The periodic pressure changes cause a recurring acceleration and destabilization of the product fluid exiting the application cannula, resulting in droplet formation.

[0037] Furthermore, it is possible to generate periodic pressure pulses by inducing a periodic cross-sectional narrowing of the product-carrying application cannula. Such a cross-sectional change can be achieved, in particular, using a piezoelectric crystal operated according to the piezoelectric inkjet principle. The time-controlled deformation of the piezoelectric crystal periodically alters the flow cross-section of the application cannula, thereby generating pressure pulses in the product fluid. These pulses lead to periodic flow separation and thus to the formation of individual product droplets.

[0038] For further processing of the product droplets, the vitrification system includes a cooling unit positioned below the freely falling product droplets. This cooling unit contains a cryogenic vitrification medium, by means of which the product droplets are vitrified by contact cooling, i.e., converted into product granules (microgranules) from the vitrified product fluid. For the purposes of this disclosure, "cryogenic" is understood to mean a temperature as low as possible, at least below the glass transition temperature of the product fluid. In particular, this refers to temperatures below -150 °C.

[0039] The collection vessel serves to collect the vitrified product granules. The temperature of the collection vessel and storage area is set to be in the cryogenic range, i.e., below the glass transition temperature of the product fluid. Advantageously, it is also below the temperature of the vitrification medium.

[0040] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 According to the preferred embodiment of the device for generating free-falling product droplets based on the moving-orifice principle, the vibration exciter is designed to generate a high-frequency mechanical vibration. The vibration exciter is attached to the application cannula via the coupling for vibration transmission. The mechanical coupling to the application cannula is such that it performs a lateral oscillation relative to the longitudinal extension of the application cannula, resulting in a lateral oscillation of the cannula outlet opening (moving-orifice principle). The following descriptions of the device for generating free-falling product droplets refer to this embodiment.

[0041] A prerequisite for generating a stable droplet chain from product droplets according to this design of the device for generating free-falling product droplets is the rigid connection of the application cannula to the vibration exciter to ensure lossless signal transmission. The coupling is designed accordingly. According to the prior art, the couplings in devices that generate droplets from a liquid jet according to the described principle are material-fit. This typically requires connecting the application syringe via a tubing system. This, in turn, leads to assembly effort, product loss due to dead spaces, and hygiene problems, since a system with a rigid connection between the vibration exciter and the application cannula is difficult to clean.

[0042] Preferably, the coupling between the vibration exciter and the base cannula is designed as a magnetic coupling that can be repositioned along the base cannula. This allows for quick syringe changes, including the product-carrying application cannula, and thus enables continuous, hygienic verification of many product doses without intermediate cleaning.

[0043] The cryopreservation process according to the invention for producing a vitrified, free-flowing microgranulate from a biopharmaceutical product can be carried out with the described vitrification system, or is carried out with it. It comprises the process steps described below.

[0044] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026According to the main components of the vitrification system, the cryopreservation process comprises two main process sections, namely first the generation of product droplets and then the vitrification of the product droplets.

[0045] The production and provision of the product fluid containing the biopharmaceutical product constituted a preliminary manufacturing step before these two main process sections.

[0046] In the first main step of the cryopreservation process, i.e., the generation of product droplets, a continuous product fluid jet is generated by conveying the product fluid using the application syringe and syringe pump. This jet exits the application cannula in a vertical direction. For this purpose, the product fluid is conveyed through the application cannula at a predetermined flow rate using the application syringe and syringe pump.

[0047] Simultaneously, a high-frequency vibration—preferably a sinusoidal oscillation with a predetermined frequency—is generated by the vibration exciter. For vibration transmission, the vibration exciter is coupled to the product fluid conveyed through the application cannula via the coupling. The vibration is thus transmitted to the product fluid stream via the coupling, resulting in the formation of a droplet chain of vertically free-falling, monodisperse product droplets (microdroplets).

[0048] By transferring vibrations (preferably sinusoidal oscillations) to the product fluid stream, product droplets of nearly identical size are pinched off. The droplet size can be adjusted via the flow rate, the size of the cannula exit orifice at the tip of the application cannula (i.e., the opening diameter of the cannula exit orifice), and the applied vibration frequency to completely encapsulate the biopharmaceutical product with the smallest possible droplet volume. The smaller the droplet volume, the higher the achievable freezing rate and the associated vitrification success.

[0049] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The droplet size of the product droplets can be adapted to the particle size of the product particles of the biopharmaceutical product contained in the product fluid by suitable process engineering measures, provided the product fluid is a product suspension. This enables complete embedding of the product particles at the smallest technically achievable droplet size or the one that is advantageous in terms of process time. This prevents the product particles from drying out and simultaneously ensures the necessary properties for the

[0050] For successful vitrification, the required freezing rate must be achieved. For most biopharmaceutical products, vitrification with droplet diameters between 50 pm and 500 pm is advantageous. A droplet diameter between 75 pm and 200 pm is particularly advantageous for biopharmaceutical products containing therapeutically active cell components, and a droplet diameter between 75 pm and 250 pm is advantageous for biopharmaceutical products containing therapeutic cells. Smaller droplet diameters, down to approximately 50 pm, can also be particularly advantageous, especially for product droplets produced from product solutions.

[0051] In the second main stage of the cryopreservation process, namely the vitrification of the product droplets, the monodisperse product droplets fall freely into the cooling unit. Here, the freely falling, monodisperse product droplets are cooled individually by contact with the cryogenic vitrification medium. That is, after forming from the product fluid jet, the product droplets come into contact with the cryogenic vitrification medium directly from free fall and are thereby transformed into vitrified product grains. Through contact cooling, the vitrified product grains of nearly uniform size form from the monodisperse product droplets. The grain size or diameter of the vitrified product grains, as well as the droplet diameter, is typically in the range of 50 pm to 500 pm.

[0052] Finally, the vitrified product granules are separated from the vitrification medium using a suitable process and collected in the collection vessel as vitrified, free-flowing microgranules and stored until use or further processing, for example by transferring them into a freeze-dried container.

[0053] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Product (lyophilisate), cryogenically stored in the collection container or after transfer to a transport or storage container.

[0054] Preferably, the cryopreservation process is carried out using the described preferred embodiment of the device for generating the free-falling product droplets according to the principle of the moving opening. For this purpose, in a preparatory step before the first main process stage of the cryopreservation process, i.e., the generation of product droplets, the vibration exciter, which is preferably designed as a linear actuator, is coupled to the application cannula by attaching the coupling, in particular the magnetic coupling, in a predetermined position along the base cannula, i.e., at a predetermined height.

[0055] Typically, the coupling, especially the magnetic coupling, is fixed to the application cannula at a height of 10 mm to 25 mm above the cannula outlet. The exact position of the fixation depends on the weight distribution and stiffness of the application cannula and the size of its outlet. When excited with a predetermined vibration frequency from the exciter, this position is crucial for a stable droplet generation process. The optimal coupling position on the application cannula can and should be determined experimentally for each type of application cannula. A high-speed camera or an oscilloscope with a fork coupler, which can be used to visualize the undisturbed droplet chain, are suitable tools for determining the optimal coupling position.

[0056] This process variant is suitable for producing and vitrifying product droplets with a diameter between 50 pm and 500 pm and a volume between 65 pl and 65 nl using cannula orifice diameters in the size range between 15 pm and 450 pm and vibration frequencies between 1.25 kHz and 20 kHz. It is particularly suitable for producing product droplets with a diameter between 50 pm and 500 pm and a volume between 65 pl and 65 nl using preferred cannula orifice diameters in the size range between 15 pm and 175 pm and vibration frequencies between 2.5 kHz and 20 kHz.

[0057] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 To produce diameters between 50 pm and 300 pm and a volume between approximately 65 pl and approximately 15 nl and to vitrify them nucleation-free or with low nucleation while fully or largely preserving product quality.

[0058] The proposed vitrification system and the described cryopreservation process enable the conversion of biopharmaceutical products into a vitrified, free-flowing, and storable microgranulate. The vitrified, free-flowing microgranulate according to the invention, derived from a biopharmaceutical product, is thus produced using the described cryopreservation process; the biopharmaceutical product comprises therapeutic cells, therapeutic cell components, in particular mitochondria, or microorganisms or viruses.Compared to established vitrification methods, including those known from fertility monitoring and reproductive medicine, the vitrification system according to the invention and the cryopreservation process carried out with it enable extremely high freezing rates that effectively suppress or completely prevent the formation of crystalline ice. This allows for successful verification with low doses or entirely without the addition of permeable or penetrating cryoprotectants (pCPAs) with membrane-damaging and cytotoxic effects, in particular dimethyl sulfoxide (DMSO) or glycerol. The generation and vitrification of the monodisperse product droplets with a defined size enable rapid, efficient, and reproducible processing of single-dose syringes for individualized therapeutic approaches.

[0059] This combination of product droplet generation using the vibration-stimulated product fluid exiting the application cannula and subsequent rapid cooling using the cryogenic vitrification medium of the cooling unit, unlike other known droplet-based vitrification devices and methods, enables the production of a fine-grained microgranulate consisting of product particles that are virtually identical to the biopharmaceutical product in terms of size and vitrification. This, in turn, allows for activity- and vitality-preserving thawing, i.e., revitalization, of the biopharmaceutical product.

[0060] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The technology according to the invention allows for simple, reproducible and very low-loss vitrification of small product quantities from the regularly sterile or sterilized application syringe. It is therefore particularly suitable for processing patient-specific single doses, especially valuable cell therapeutics, advanced therapy medicinal products (ATMPs) and related therapeutics.

[0061] The cryopreservation process requires minimal time. Single doses containing therapeutic cells or cell components can be transferred into the vitrified microgranules in less than two minutes, enabling even sensitive biopharmaceutical products, whose processing is very time-critical, to be cryopreserved with minimal loss.

[0062] The cryopreservation method enables the vitrification of suspended cells with typical droplet volumes between 200 pl and 8 nl with freezing rates

[0063] Up to 6.5 million K / min and beyond for the vitrification of living cells. For the vitrification of biopharmaceutical products with smaller particle sizes, for example, therapeutically active cell components, especially those that can be formulated as a product solution, smaller droplet volumes down to 65 pl and freezing rates up to 15 million K / min and beyond are physically achievable.

[0064] The size of the product droplets can be tailored to the size of the biological components contained in the biopharmaceutical product in order to achieve an optimal compromise between sufficient embedding of the biological components in the product droplet to prevent dehydration and the maximum physically achievable freezing rate.

[0065] Due to its fine-grained nature, the microgranules produced by the cryopreservation process according to the invention can be thawed at extremely high thawing rates of 15,000 K / min to 30,000 K / min using the simplest means, for example by drawing a sterile buffer solution into a deep-frozen syringe filled with a single dose of the microgranules.

[0066] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The high freezing and thawing rates thus allow, in many cases, the complete dispensing with permeable or penetrating antifreeze additives (pCPAs), such as DMSO or glycerol, which are membrane-damaging, cytotoxic, and undesirable for therapeutic applications at higher concentrations. DMSO is not prohibited as a component of biopharmaceutical products, but due to its undesirable effects, such as its potential to damage the product, its potential adverse effects in the context of the therapeutic use of the vitrified product, or its negative influence on the structure of vitrified products further processed by freeze-drying, it is highly advantageous to avoid its use or to use it only in very small doses.

[0067] The vitrified microgranules, especially from a mitochondria-containing biopharmaceutical product, can be stored at liquid nitrogen temperature for a period of at least 20 days, for example 30 days, 60 days or 90 days.

[0068] If a general avoidance of antifreeze additives is not possible, DMSO can be substituted by biologically compatible and medically safe, non-permeable or non-penetrating cryoprotective agents (npCPA) from the group of mono- and disaccharides (simple and double sugars) and their derivatives and / or from the group of sugar alcohols and / or other polyols and their derivatives and / or from the group of amino acids and their derivatives and / or from the group of organosulfur compounds.

[0069] According to one embodiment of the cryopreservation process, the product fluid containing the biopharmaceutical product further comprises at least one antifreeze additive selected from the group consisting of: one or more non-penetrating antifreeze additives that do not impair the biological function of the biopharmaceutical product, one or more penetrating antifreeze additives, and / or a combination thereof. The non-penetrating antifreeze additives that do not impair the biological function of the biopharmaceutical product are selected from

[0070] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 of the group consisting of: mono- and / or disaccharides and / or their derivatives, sugar alcohols and / or other polyols and / or their derivatives, amino acids and / or amino acid derivatives, organosulfur compounds, and combinations of two or more of these substances. The other polyols or their derivatives are preferably substances that are present in solid form at the intended storage and / or application temperature of the vitrified and lyophilized microgranules. The addition of the non-penetrating antifreeze additives (npCPA) to the product fluid enables external freeze protection of the therapeutic cells or therapeutically active cell components, for example, the mitochondria.

[0071] It is advantageous from a procedural standpoint to restrict the analysis to medically safe non-protonated chemical compounds (npCPAs) from the named substance groups that function as extremolytes or osmolytes. Extremolytes are all substances produced by extremophilic organisms to protect biological structures, such as proteins and lipid membranes, from heat, cold, dryness, high salt concentrations, or UV radiation. Osmolytes are all substances capable of regulating pressure in cells and protecting biological structures, such as proteins and lipid membranes, from heat, cold, dryness, high salt concentrations, or UV radiation, even if they were not produced by extremophilic organisms specifically for this purpose.

[0072] The procedure is particularly advantageous if it restricts the use to medically safe, non-permeable or non-penetrating materials.

[0073] Antifreeze additives (npCPA) from the named substance groups, which are also compatible solutes. Compatible solutes are all extremolytes and osmolytes that do not interfere with biological functions, such as cellular metabolism or protein functions, even at high concentrations and therefore exhibit good biocompatibility and can be added to the product medium in high concentrations.

[0074] Examples of process-advantageous and particularly advantageous npCPAs include the disaccharides trehalose and sucrose or a combination thereof, the amino acids and amino acid derivatives glutamic acid, L-proline, ectoine.

[0075] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 and hydroxyectoin or a combination thereof, as well as the sugar alcohols arabitol, inositol and mannitol or a combination thereof.

[0076] In the vitrification of a biopharmaceutical product fluid, trehalose, especially at a concentration of at least 300 mM or 350 mM, is particularly advantageous, as is its combination with other disaccharides such as glucose and / or sucrose or with amino acids, especially proline, for example L-proline, and / or proline derivatives.

[0077] According to a further embodiment of the cryopreservation process, it is provided that the biological components with biopharmaceutical or therapeutic effects contained in the biopharmaceutical product are brought into contact with one or more non-penetrating cryoprotectant additives that do not impair the biological function of the biopharmaceutical product prior to the production and provision of the product fluid containing the biopharmaceutical product, in order to enrich these additives within the biological components of the biopharmaceutical product for the purpose of cryoprotective preconditioning.The non-penetrating cryoprotectant additive(s) that do not impair the biological function of the biopharmaceutical product are selected from the group consisting of: mono- and / or disaccharides and / or their derivatives, sugar alcohols and / or other polyols and / or their derivatives, amino acids and / or amino acid derivatives, organosulfur compounds, and combinations of two or more of these substances. The other polyols or their derivatives are preferably substances that are present in solid form at the intended storage and / or application temperature of the vitrified and lyophilized microgranules. Cryoprotective preconditioning is preferably carried out in a cell culture, in particular a living cell culture.The biological components with biopharmaceutical or therapeutic effects contained in the biopharmaceutical product are, for example, therapeutic cells that form the cells of the cell culture, or they are therapeutically effective cell components contained in the cells forming the cell culture, such as membrane-bound organelles, for example mitochondria.

[0078] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 According to this variant of the cryopreservation process, which includes cryoprotective preconditioning with non-permeable or non-penetrating cryoprotectant additives (npCPA) prior to the production and provision of the product fluid, the aforementioned npCPA are thus brought into contact with the living therapeutic cells or with the living cells from which the therapeutically active cell components, such as organelles or exosomes, are obtained in a preliminary process step before the vitrification process. This can be achieved, for example, by incubating these substances in a cell culture for 6 to 48 hours.This process, via membrane transport by membrane-bound transport proteins, builds up a cryoprotective concentration of these non-penetrating cryoprotectant additives (npCPA) inside the biological components of the biopharmaceutical product enclosed by a biological membrane. The purpose of incubation in cell culture is to generate internal protection. A sufficiently long incubation period is advantageous to ensure the accumulation of a cryoprotective concentration of npCPA within the cell and / or its therapeutically used internal structures, particularly therapeutically used organelles such as mitochondria, by means of membrane transport by membrane-bound transport proteins that fully preserves the structure and function of the biological membrane.An enrichment of non-penetrating antifreeze additives (npCPA) inside the cell is particularly important for subsequent lyophilization, since, for example, trehalose must replace the removed water inside the cell to ensure the stability of the proteins.

[0079] According to this variant of the cryopreservation process, the recovery, purification, and vitrification of the live therapeutic cells, or the recovery, purification, and vitrification of the therapeutic components from the live cells, takes place immediately following the 6- to 48-hour incubation of the live cells with the non-permeable or non-penetrating cryoprotectant additive(s) used. The cryoprotective concentration of the non-penetrating cryoprotectant additive(s) (npCPA) can advantageously be maintained throughout the subsequent processing by adding them to the product fluid, as previously described, in order to prevent a secondary reduction in the

[0080] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 to prevent the cryoprotective concentration of npCPA enriched in the therapeutic cell or the therapeutically active components obtained from it between incubation and vitrification.

[0081] In another embodiment of the cryopreservation process, where the biopharmaceutical product contains isolated functional mitochondria, the isolated functional mitochondria are brought into direct contact with non-permeable or non-penetrating cryoprotectant additives (npCPA) for cryoprotective preconditioning. This can be achieved, for example, by directly incubating the mitochondria in non-permeable or non-penetrating cryoprotectant additives (npCPA) for less than 2 hours, such as 20 or 30 minutes. The cryoprotective concentration in the product fluid is advantageously maintained throughout the entire subsequent processing procedure.

[0082] The microgranules produced according to the invention from the biopharmaceutical products can – subject to the required product approval – be administered directly to a patient by injection after thawing, without the need to remove critical excipients.

[0083] Preferably, the base cannula of the application cannula, which is designed as a compound cannula, is a blunt cannula of suitable length and has an inner diameter of at least 250 pm (for example, a 26G cannula with a 26 gauge). Metallic materials, in particular stainless steel, have proven suitable for the base cannula. If the application cannula comprises only the base cannula, a narrower cannula inner diameter is required to produce the advantageous droplet sizes according to the process, for example, 108 pm for a 32G cannula with a 32 gauge or 133 pm for a 31G cannula with a 31 gauge. The preferred length of the base cannula is in the range of 30 mm to 50 mm.

[0084] The cannula tip of the compound cannula application can be made of glass. The preferred length of the cannula tip is approximately 5 mm ± 2 mm. The inner diameter tapers to a nozzle shape.

[0085] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 A convergence nozzle, formed by a glass material, achieves a predetermined cannula exit diameter at the cannula opening, preferably in the range of 15 pm to 175 pm. A cannula tip formed from a glass material with the geometry described above, forming a convergence nozzle, can be produced, for example, by heating one side of a glass capillary having a suitable inner diameter. This glass capillary is then cut to the desired length, for example, 5 mm, and bonded to the tip of the base cannula, which is preferably designed as described above.

[0086] An advantage of the application cannula constructed as a composite cannula according to the invention is the possibility of selecting a base cannula with a large diameter, which makes it robust against operational damage and resistant to clogging with product particles. The cannula tip, designed as a convergence nozzle and manufactured as a glass tip as described above, is also very break-resistant and resistant to operational damage. If the cannula outlet opening located on the cannula tip designed as a convergence nozzle becomes clogged, the application cannula can be easily cleaned by flushing, for example by repeatedly drawing it up with a syringe or in an ultrasonic bath, due to the short length of the cannula tip and thus the short distance with a narrow inner diameter. The application cannula can therefore be reused.

[0087] According to one embodiment of the coupling, which is a magnetic coupling that can be positioned along the base cannula in a repositionable manner, it comprises a base magnet that can be permanently connected to the vibration exciter and a holding magnet that can be magnetically attached to it within a magnetic coupling zone. Within the magnetic coupling zone is a cannula guide groove designed to securely and positively engage and retain the base cannula between the base magnet and the holding magnet. The magnetic coupling is suitable as a quick-connect and quick-release system for easily and quickly connecting and disconnecting the vibration exciter to and from the application cannula.

[0088] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026In addition, the magnetic coupling allows for movement along the base cannula and thus for stepless height adjustment of the coupling on the base cannula.

[0089] The base magnet and the holding magnet are preferably neodymium magnets with strong adhesive strength, for example, neodymium magnets of grade N50 or N52. Furthermore, they preferably have the same size. Advantageously, the base magnet and the holding magnet are particularly cylindrical, with a length in the range of 5 mm to 10 mm and a diameter of

[0090] exhibiting dimensions of 5 mm ± 1 mm.

[0091] For use, the base magnet is securely connected to the vibration exciter, the application cannula is inserted into the cannula guide groove, and the holding magnet is placed onto the base magnet so that the application cannula is held or clamped between the holding and base magnets. With appropriate dimensional design of the cannula guide groove, a firm connection is created between the vibration exciter and the application cannula, i.e., a positive-locking and force-locking connection within the coupling, which is suitable for transferring the vibration energy to the application cannula without loss. To change the syringe, the holding magnet is lifted off with a single movement, and the application syringe can be removed.

[0092] According to one embodiment of the magnetic coupling with the cannula guide groove, this groove is located in the magnetic coupling zone of the magnetic coupling on the base magnet, and the cannula guide groove has a rectangular or semicircular cross-sectional shape. That is, the base magnet contains a straight, rectangular or semicircular cannula guide groove whose depth corresponds to the outer diameter of the application cannula. The holding magnet, on the other hand, does not have a groove.

[0093] According to another embodiment of the magnetic coupling with the cannula guide groove, the magnetic coupling has a guide disc containing the cannula guide groove, which is inserted in the area of ​​the magnetic coupling zone between the base magnet and the holding magnet. The guide disc is made of a material that does not shield the magnetic field, i.e., a

[0094] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 non-magnetic or magnetically transparent material, preferably made of a plastic, in particular polytetrafluoroethylene (PTFE). A magnetically transparent material is understood to be a material with low magnetic permeability. The guide disc can, for example, be made of a self-adhesive material, such as a film, wherein the thickness of the guide disc or film corresponds to the outer diameter of the application cannula. This self-adhesive guide disc is applied to the end face of the base magnet located in the area of ​​the magnetic coupling zone. A particularly preferred embodiment is the formation of the guide disc from a self-adhesive PTFE film.

[0095] The vibration exciter is preferably a linear actuator, by means of which a high-frequency, preferably sinusoidal longitudinal vibration can be generated, the axial vibration amplitude of which is in the range of a few micrometers. That is, the linear actuator is designed such that it can convert a high-frequency, preferably sinusoidal, control signal into a continuous linear alternating stroke movement with an amplitude of a few micrometers. The vibration exciter, designed as a linear actuator, is attached to the application cannula transversely to its longitudinal extent with respect to its linear alternating stroke movement, via the coupling, in particular the coupling designed as a magnetic coupling. The transmission of the longitudinal vibration of the linear actuator to the application cannula thus results in a transverse vibration of the application cannula.

[0096] For the described design of the application cannula, operation of the vibration exciter designed as a linear actuator with a vibration frequency in the frequency range of 1.25 kHz to 20 kHz is preferred.

[0097] According to one embodiment of the invention, the vibration frequency adjustable on the vibration exciter is controllable, in particular finely controllable, for example in steps of a maximum of 50 Hz, in order to adapt the vibration frequency adjustable on the vibration exciter to the characteristics of the application cannula used, the selected opening diameter of the cannula outlet and the selected flow rate of the product fluid.

[0098] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 enable. The vibration frequency required to generate monodisperse product droplets decreases with increasing cannula exit diameter.

[0099] It may also be provided that the vibration exciter, designed as a linear actuator, has a vibrating head with a threaded bore. The coupling intended for transmitting the vibration to the application cannula, in particular the coupling designed as a magnetic coupling, can be detached but firmly connected to the linear actuator by means of a fastening screw that can be screwed into the threaded bore. The fastening screw is also firmly connected to the coupling, for example, by a material bond or by a positive and force-fit connection. If the coupling is designed in the form of the magnetic coupling described above, which includes the base and holding magnets, the fastening screw can, for example, be materially bonded to the base magnet.

[0100] The vibration exciter, designed as a linear actuator, can be, for example, a piezoelectric actuator. Multilayer piezoelectric actuators in a stacked configuration with integrated mechanical preload and a resonant frequency in the range of 12 kHz to 22 kHz have proven particularly suitable.

[0101] According to the invention, two embodiments are preferred for the assembly for vitrifying the product droplets and for the second main process step of the cryopreservation process carried out therein, i.e., the vitrification of the product droplets: firstly, fluid vitrification, in which a vitrification fluid forms the cryogenic vitrification medium, and secondly, surface vitrification, in which a rotating vitrification roller is used as the cryogenic vitrification medium. This type of surface vitrification, which takes place on the outer surface of the rotating vitrification roller, is also referred to in the present case as roller vitrification.

[0102] If the product droplets are vitrified by fluid vitrification, the cooling unit comprises the vitrification fluid as the vitrification medium, which in one process variant is contained in a vitrification vessel of the cooling unit that is open at one end. The vitrification fluid is preferably in liquid form.

[0103] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026. The vitrification vessel is positioned vertically below the application cannula on the opening side, so that the vertically falling product droplets from the product droplet generation assembly fall directly into the vitrification fluid in the vitrification vessel. The vitrification fluid can remain static in the vitrification vessel or be pumped through it.

[0104] The vitrification fluid must be selected to possess suitable physical and chemical properties for the application. It should have no adverse effects (toxicity, inhibition of activity) on the biopharmaceutical product being vitrified, i.e., it should be inert to the biopharmaceutical product and should be practically or immiscible with the product fluid or with water. The melting point of the vitrification fluid must be significantly below the glass transition temperature of the product fluid so that it exists as a liquid within the temperature range of the cryopreservation process. Furthermore, its density within the temperature range of the process application should be significantly lower than that of the product fluid so that the product droplets sink in the vitrification fluid.The entry of the product droplets into the vitrification fluid and their subsequent settling within it serve to create the largest possible contact area between the biopharmaceutical product and the vitrification fluid, as well as to facilitate convective heat transfer. Both are essential for efficient heat removal from the product droplets and achieving the freezing rate required for vitrification. Furthermore, the boiling point of the vitrification fluid at atmospheric pressure should be significantly higher than the temperature range of the process application. This ensures that the vitrification fluid can dissipate the heat from the product droplets without being heated above its boiling point at the contact surface, thereby avoiding the Leidenfrost effect and becoming an insulator. Preferably, the boiling point of the vitrification fluid is at least 50 K above its melting point.

[0105] When one of the freely falling product droplets comes into contact with the surface of the cryogenic vitrification fluid, it sinks due to the density difference and displaces the vitrification fluid upwards. The full-surface contact between the product droplet and the vitrification fluid results in efficient vitrification.

[0106] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Transfer of heat energy from the product droplet to the vitrification fluid, which acts as a coolant. The transferred heat energy is dissipated to the environment by cooling the vitrification fluid using a vitrification vessel cooling unit of the cooling unit. The vitrification vessel cooling unit can, for example, include a cooling block in which the vitrification vessel is housed. Throughout the entire vitrification process, the vitrification fluid is cooled to a temperature above its melting point and below the glass transition temperature of the product fluid. The lowest technically feasible temperature above the melting point of the vitrification fluid is advantageous.Under normal operating conditions, the product droplets completely penetrate the vitrification fluid and solidify instantly into vitrified product granules, which collect as free-flowing microgranules at the bottom of the vitrification vessel after settling. The vitrification vessel typically also serves as the collection vessel for the vitrified microgranules.

[0107] The vitrified microgranules can be separated from the vitrification fluid after completion of the vitrification process, either thermally, i.e. by evaporating the vitrification fluid, or mechanically, e.g. by filtration.

[0108] The vitrification vessel is a cold-resistant vessel of suitable size. The amount of vitrification fluid held in the vitrification vessel should be at least 15 times, preferably 30 to 50 times, the volume of the microgranule dose to be vitrified.

[0109] Cylindrical, i.e., tube-shaped, vitrification vessels have proven suitable, provided they can withstand temperatures down to -180 °C and have a suitable volume and diameter. A conical bottom is advantageous, as it allows the vitrified microgranules to collect. For example, the vitrification vessel could be a screw-top laboratory tube or a centrifuge tube with a volume of 15 ml or 50 ml, made of polypropylene (PP) or a polypropylene copolymer, with a conical bottom.

[0110] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The vitrification vessel cooling unit for cooling the vitrification vessel and the vitrification fluid contained therein is designed to maintain the temperature of the vitrification vessel and the vitrification fluid within the intended temperature range during droplet application. The vitrification vessel cooling unit may, in particular, include a cooling block in which the vitrification vessel is housed. This cooling block may, for example, be an externally insulated aluminum block with a central recess or bore for receiving the vitrification vessel. The cooling block may also be equipped with integrated temperature control. Cold gas, obtained, for example, by heating liquid nitrogen, may be used as the cooling medium for the cooling block.The cold gas can be supplied from a thermally insulated pressure vessel (Dewar) via a control heater and passed through the cooling block at a constant pressure. Temperature control is achieved, for example, by an electric resistance heater integrated into the cooling block.

[0111] It may be possible to move the vitrification vessel with a uniform motion, in particular a circular or orbital motion. For this purpose, the cooling unit may include a shaking device, in particular an orbital shaker. The vitrification vessel, or the vitrification vessel housed in the cooling block, is installed on the shaking device so that the vitrification vessel performs the intended motion.

[0112] This results in a relative motion between the impacting product droplets and the surface of the vitrification fluid, which in turn promotes the individual deposition of the product droplets on the surface of the vitrification fluid and prevents them from coalescing into agglomerates with unfavorable cooling behavior. When using an orbital shaker as the shaking device, the rotational speed and orbital diameter (i.e., the diameter of the orbital motion) can be adjusted to the droplet frequency (i.e., the droplet or impact rate of the falling product droplets) and the resulting droplet diameter to achieve the most complete possible separation of the product droplets. In addition to separating the product droplets, the suction effect of the orbitally moving vitrification fluid also supports convective heat dissipation.

[0113] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 the heat dissipation within the vitrification fluid. The mass of the vitrification vessel cooling unit must be matched to the shaking device in such a way that the operation of the cooling unit is enabled when using the vitrification vessel cooling unit on a suitable shaking device, for example a conventional orbital shaker.

[0114] The vitrification vessel can also be integrated into a closed-loop system in which the vitrification fluid is continuously circulated. After vitrification in the vessel, the vitrified product particles carried in the circulating fluid are separated by filtration at a suitable point in the system.

[0115] Hydrocarbon compounds from the group of alkanes and alkenes are particularly suitable as vitrification fluids.

[0116] Ethene is a particularly suitable vitrification fluid because it is an inert hydrocarbon that is practically immiscible with aqueous media and non-toxic to biological cells and cell components. Within the application range of the process, it exhibits a low density of approximately 0.60 g / cm³. 3 up to 0.65 g / cm³ 3 The vitrified hydrocarbon has a very low melting point of -169 °C under normal pressure and exists as a liquid within a temperature range of 65 K. Its boiling point of -104 °C is particularly advantageous, as it is significantly below the boiling point of other hydrocarbons and allows for thermal separation from the vitrified product by evaporation. In a preferred process embodiment operating in the application temperature range between -165 °C and -160 °C, the difference between the application temperature and the boiling point is sufficiently large to prevent film boiling under favorably chosen process conditions.

[0117] Propene is also suitable as a vitrification fluid, with a melting point of -185 °C and a density in the range of 0.64 g / cm³. 3 up to 0.69 g / cm³ 3 It exhibits similarly advantageous properties in the process application area. Furthermore, due to its comparatively high boiling point of -48 °C, it lies within an extremely wide temperature range of 137 K.

[0118] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 as a liquid, which effectively prevents film boiling even under unfavorable process conditions. Since the boiling point of -48 °C is significantly above the glass transition temperature of aqueous-suspended biopharmaceutical products, thermal separation of the microgranules from the vitrification fluid without product damage, i.e., devitrification, is hardly possible when using propene as the vitrification fluid. Gentle separation can be achieved mechanically, for example, by filtration through a suitable sieve, such as a cell strainer of suitable pore size made of nylon or PTFE.

[0119] Ethane, with a melting point of -183 °C and a density of approximately 0.54 g / cm³, is also suitable as a vitrification fluid. 3 and a boiling point of -88.6 °C, propane with a melting point of -187.7 °C, a density of approximately 0.58 g / cm³ 3and a boiling point of -42.1 °C, 2-methylpropane with a melting point of -160 °C, a density of approximately 0.59 g / cm³ 3 and a boiling point of -12 °C, as well as but-1-ene with a melting point of -185.4 °C and a density of approximately 0.62 g / cm³. 3 and a boiling point of -6.3 °C. Since all four vitrification fluids have a significantly higher boiling point than ethene, mechanical separation of the vitrified microgranules from the vitrification fluid is also advantageous.

[0120] Fluid vitrification generally requires subsequent separation of the vitrified microgranules from the vitrification fluid. For vitrification fluids whose boiling point is below or near the glass transition temperature of the vitrified microgranules, the vitrification fluid can be separated by a thermal separation process, i.e.,

[0121] Evaporation allows the microgranules to be separated from the product without damage. During thermal separation, the temperature reached by the microgranules may briefly exceed the glass transition temperature of water or aqueous-formulated biopharmaceutical products, which is approximately -135 °C to -137 °C. Temperatures above -80 °C should also be avoided, even briefly, to prevent devitrification of the product particles.

[0122] Another method for separating the microgranules from the vitrification fluid is mechanical separation, for example by filtration. In this process, the vitrification vessel is emptied into a collection vessel through a pre-cooled sieve, such as a cell strainer, with a suitable pore size. The pore size must be lower than the vitrification fluid.

[0123] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 as the diameter of the product particles; typical, suitable pore sizes are in the range of 70 pm to 85 pm. The vitrified microgranules remain on the sieve. With continuous cooling, especially under a cold gas atmosphere, for example in a polystyrene box containing excess nitrogen, they can be transferred to a cooled transport or storage container, such as a storage tube or product syringe, and stored below their glass transition temperature in a cryogenic storage container until further processing, for example by freeze-drying, or medical use. The mechanical separation method by filtration is advantageous for all vitrification fluids. It is particularly suitable for vitrification fluids with high boiling points, for example ethane, propane, propene, 2-methylpropane, or but-1-ene.

[0124] Does the vitrification of the product droplets occur through surface or...

[0125] In roller vitrification, the cooling unit comprises a rotatable vitrification roller as the vitrification medium. The roller's cryogenic surface serves to rapidly cool the product droplets. Vitrification occurs upon contact of the product droplets with the cryogenic surface of the vitrification roller. To achieve vitrification, the temperature of the vitrification roller's surface must be below the glass transition temperature of the aqueous biopharmaceutical product. Temperature control of the surface below -150 °C is preferred. Temperature control of the surface below -160 °C, for example, within a temperature range of -165 °C to -170 °C, is particularly preferred. The heat released is absorbed by the vitrification roller and dissipated by cooling it.The vitrification roller is preferably cooled internally using a fluid cooling medium, for example, a cold gas. This means that the outer surface is cooled from the opposite side to the temperature required for the vitrification of the product droplets. To ensure that the freely falling product droplets strike the vitrification roller, its outer surface is positioned vertically below the stationary application cannula within the vitrification system.

[0126] When performing surface or roller vitrification with the vitrification system designed as an internally cooled vitrification roller, the monodisperse product droplets fall in the form of a droplet chain onto the surface of the continuously

[0127] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 A rotating, internally cooled, preferably metallic vitrification roller, usually on the upper side of the roller. Due to the relative movement between the droplet chain and the surface of the vitrification roller, the monodisperse product droplets are individually applied to the surface of the roller and vitrify instantaneously through heat transfer to the roller, forming the loosely adhering product granules that constitute the vitrified microgranules. Verification is completed after a maximum of half a rotation of the vitrification roller. During the cryopreservation process, the temperature of the internally cooled vitrification roller is above the temperature of the cooling medium used, but below the glass transition temperature of the product fluid.

[0128] For this purpose, the vitrification roll is cooled to the working temperature before the start of the process and kept at this temperature throughout the entire vitrification process.

[0129] On the vitrification roller, the vitrified product granules are mechanically detached from the roller by a suitable scraper tool of the cooling unit, for example, a cooled wire or a cooled blade, usually at the front or underside of the roller. After detaching from the roller's surface, they fall by gravity into the collection vessel positioned at an angle in front of or below the roller. The collection vessel is continuously cooled by a suitable cooling unit, for example, a temperature-controlled cooling block.

[0130] Once the intended microgranule dose has been verified, the collection vessel can be removed from the collection vessel cooling unit and transferred to a cryogenic storage container under continuous cooling. Here, the collection vessel, which can also be used as a transport and storage container, can be kept below its glass transition temperature until further processing or medical use.

[0131] The collection vessel used for the described surface or roll vitrification can be significantly smaller than that used for fluid vitrification, as it only needs to hold the

[0132] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The container serves to collect the vitrified, dry microgranules and does not need to contain vitrification fluid. It can therefore also be used directly as a transport or storage container for transfer to a cryogenic storage tank. Screw-top cryovials made of polypropylene (PP) or a polypropylene copolymer, with a volume of 0.5 ml to 5 ml, have proven suitable as collection containers for the described surface or roll vitrification using the vitrification roll as the vitrification medium. The collection container can also be a sterile product syringe made of a cold-resistant material, sealed with a cap on the cone or cannula adapter, which is then closed with the corresponding plunger after verification.

[0133] The vitrification roller is preferably made of a metallic material to ensure rapid heat dissipation. Suitable materials include aluminum, copper, and their alloys; stainless steel, for example X5CrNi18-10 (V2A), or similar temperature-, water-, and chemical-resistant steels or iron alloys are particularly suitable.

[0134] The outer surface of the vitrification roller can be treated to optimize product adhesion and achieve preferred wetting behavior. Contact angles between approximately 75° and 110°, and particularly between approximately 80° and 90°, have proven suitable, as these angles ensure both good thermal contact of the product droplets and good release of the vitrified product particles. This can be achieved, for example, by constructing the vitrification roller from stainless steel with an uncoated outer surface. Furthermore, coatings, such as CrN or SiOx coatings, can also be applied to optimize wettability.

[0135] The vitrification roller preferably has a roller diameter of at least 5 cm.

[0136] To ensure that the product droplets individually impact the outer surface of the rotating vitrification roller, the roller must be set to a sufficiently high rotational speed. The vitrification roller can be driven via a drive shaft.

[0137] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 with a speed-controlled motor. By selecting a suitable roller diameter and a suitable rotational speed, the peripheral speed of the vitrification roller must be chosen high enough so that, for the respective set droplet size and droplet frequency, the product droplets are deposited separately on the circumferential surface of the vitrification roller. This can be achieved by considering the following relationship:

[0138] TT ■ Roller diameter ■ Roller speed > Droplet diameter ■ Droplet frequency can be achieved.

[0139] The singulation of the product droplets can be supported by a floating mounting of the vitrification roller, which allows for an axial pendulum motion, for example, by using axially displaceable or axially movable radial bearings. The superposition of the rotational movement with the axial pendulum motion creates a two-dimensional relative motion between the droplet chain and the vitrification roller, thereby increasing the application distance of the product droplets. The individual product droplets follow a meandering path on the roller's surface. This design is particularly suitable for very small product droplets and high drip frequencies.

[0140] The collection vessel cooling unit is designed to accommodate the cryogenic collection vessel and maintain it within the specified temperature range during the cryopreservation process to prevent devitrification of the product. The collection vessel cooling unit can be designed as a cooling block. Advantageously, it is an externally insulated aluminum cooling block with a central bore for the collection vessel and is equipped with integrated temperature control, similar to the previously described vitrification vessel cooling unit. As with the vitrification vessel cooling unit, cold gas, for example, cold nitrogen gas, can be used as the cooling medium. This gas is supplied via a control heater from a thermally insulated pressure vessel (Dewar) and circulated through the cooling block at a constant pressure.The temperature is controlled, for example, by a solenoid valve which regulates the flow of cold gas.

[0141] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Preferably, the temperature of the cooling block is regulated so that it is 10 K to 15 K below the temperature of the vitrification roller. For a particularly preferred vitrification roller temperature, the temperature of the cooling block is, for example, in the range of -175 °C to -180 °C. Due to the temperature difference between the vitrification roller and the cooling block, the uninsulated surface on the top of the cooling block acts as a cold trap for any moisture released from the product fluid or the freely falling product droplets, thus preventing frost from forming on the vitrification roller. A cooling block design with a large surface area is therefore advantageous. Furthermore, the uninsulated surface of the cooling block can be ribbed or studded, or provided with cooling fins or studs, to increase its efficiency as a cold trap and thus its surface area can be increased.These designs of the cooling block for the collection vessel cooling unit are equally applicable to the cooling block of the vitrification vessel cooling unit in fluid vitrification.

[0142] The stripping tool for mechanically removing the vitrified product grains from the surface of the vitrification roller can be designed as a cooled wire, for example, a pre-stressed, tensile-resistant tension wire made of a steel material with a preferred thickness of 0.17 mm to 0.32 mm, which, due to the pre-stress, lies close to the vitrification roller, preferably on its underside. Such a tension wire can, for example, be string wire. The necessary cooling of the thin tension wire is achieved through contact with the rotating vitrification roller. The tension wire is clamped between two clamping brackets by means of a clamping screw or swivel, which are firmly connected to the cooling block of the collection vessel cooling unit. This provides additional cooling to the free wire ends to prevent lateral heat input.Upon contact with the cold tension wire, the frozen product granules are detached from the vitrification roller and fall under the influence of gravity into the collection vessel below.

[0143] An alternative embodiment of the scraper tool comprises a blade angled steeply against the vitrification roller, preferably narrow or with a perforation, which is mounted on the cooling block of the collection vessel cooling unit via a blade holder. Ideally, the blade is spring-loaded.

[0144] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The blade is mounted and, due to spring pressure, remains firmly in contact with the vitrification roller even in the event of concentricity deviations. Additional cooling of the blade may be provided. For this purpose, the blade holder can, for example, be connected to the cooling block of the collection vessel cooling unit by means of copper strips (cooling strips) attached to the rear.

[0145] For internal cooling of the vitrification roll, it can be cooled by flowing through it with the cooling medium, in particular with a cold gas. For example, cold nitrogen gas is suitable for cold gas cooling; this is supplied via a control heater from a thermally insulated pressure vessel (Dewar) containing liquid nitrogen.

[0146] To support internal cooling, the vitrification roll can be designed with continuous flow channels for the passage of the cooling medium. These flow channels extend axially through the roll body, allowing the cooling medium to flow unidirectionally through them during the process, thus dissipating heat from the vitrification process.

[0147] According to one embodiment of the cooling unit with the vitrification roller, the roller is internally cooled by cold gas and integrated into a hollow shaft assembly designed for driving the roller and for supplying and discharging the cold gas. Preferably, the hollow shaft assembly is floatingly mounted by means of axially displaceable radial bearings. The vitrification roller is held within the hollow shaft assembly between two hollow shafts, one of which serves as the drive shaft. The cold gas, in particular nitrogen, which serves as the cooling medium, is supplied via the interior of one hollow shaft, then flows through the vitrification roller along the continuous flow channels, and exits the hollow shaft assembly via the other hollow shaft.

[0148] As an alternative to temperature control of the vitrification roll with cold nitrogen gas, cooling can also be achieved via a closed refrigeration circuit with a compression chiller. A suitable refrigerant mixture with an operating temperature within the range of the process execution must be used as the cooling medium, which provides the vitrification roll with a liquid temperature.

[0149] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The refrigerant is supplied from the evaporator of the refrigeration circuit and expanded in this form via an expansion valve. The evaporated refrigerant is then returned to the refrigeration circuit via a return line. In this design variant, the vitrification roller is a hollow cylinder, closed at one end and rigidly connected to the drive shaft. On the opposite end, the vitrification roller is rigidly connected to a hollow shaft, which is movably locked in place by a bearing block with a plain or ball bearing. The refrigerant supply and return lines to the vitrification roller are routed via the hollow shaft.For the purpose of expanding the refrigerant, a stationary, cylindrical valve head is installed in the rotating vitrification roller via a gas-tight bearing, which has a supply opening with expansion valve and a return opening for returning the expanded refrigerant mixture to the refrigeration circuit.

[0150] According to one embodiment of the invention, the vitrification system comprises a vitrification chamber formed by a hermetically sealed housing, in which the assembly for generating the product droplets and the assembly for vitrifying the product droplets are located. The purpose of the vitrification chamber is to establish and maintain a dry gas atmosphere by excluding humid ambient air and removing moisture resulting from partial evaporation of the product fluid. The housing of the vitrification chamber may have feedthroughs through which, for example, the shafts for receiving the vitrification roller or the lines of the cooling medium from the cooling units are routed. Preferably, a pre-chamber is formed in the upper region of the vitrification chamber, which is separated from the main chamber formed in the lower region of the vitrification chamber by a perforated plate or a grating.The pre-chamber is supplied with dry inert gas via a gas supply line. A suitable inert gas is, for example, dry nitrogen gas of purity class 5.0 from a liquid gas cylinder. The supplied inert gas is filtered of dust and germs by a sterile filter mat located on the floor of the pre-chamber, guided through the main chamber as a laminar flow, and discharged through outlet openings in the chamber floor and / or in the lower area of ​​the chamber's rear wall.

[0151] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 For the purpose of sample temperature control in the vitrification process, the chamber temperature can be regulated via the supplied inert gas flow. If cold nitrogen gas is used to temperature-control the vitrification roller, the cooling block of the collection vessel cooling unit (in surface or roller vitrification), or the cooling block of the vitrification vessel cooling unit (in fluid vitrification), a portion of the cold gas can be diverted and mixed with the inert gas flow via a temperature-controlled solenoid valve. The temperature measurement in the vitrification chamber is advantageously performed at the level of the droplet chain of the product droplets formed from the product fluid. A chamber temperature of 4 °C should not be undercut if possible, in order to reliably prevent the product droplets from freezing before contact with the vitrification medium.

[0152] The vitrification chamber can be an integral part of the vitrification system, regardless of whether it is used for surface or roller vitrification with a vitrification roller as the vitrification medium, or for fluid vitrification with a vitrification fluid as the vitrification medium. Frost formation, for example on the outer surface of the vitrification roller, can be effectively prevented by the vitrification chamber. The suppression of such frost formation—even during extended operation—can be further enhanced by using the cooling block of the collection vessel cooling unit as a cold trap, as described above.

[0153] According to a further aspect of the invention, the vitrified microgranules of the biopharmaceutical product are converted into a lyophilisate by freeze-drying. Thus, a process for producing a lyophilized microgranule from a biopharmaceutical product is provided, in which the vitrified, free-flowing microgranules produced by the described cryopreservation process are lyophilized using a freeze dryer. The lyophilized microgranules according to the invention are produced from a biopharmaceutical product by this process; the biopharmaceutical product comprises therapeutic cells, therapeutic cell components, in particular mitochondria, or microorganisms or viruses. A major advantage of the further processing of the vitrified microgranules of a biopharmaceutical product is that

[0154] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The advantage of freeze-drying products is their long shelf life under moderate temperature conditions, for example in air-conditioned rooms at approximately 20 °C or refrigerated at 4 °C to 6 °C, whereas vitrified biopharmaceutical products, especially those containing therapeutic cells and therapeutically active cell components such as mitochondria, require cryogenic storage at temperatures below their glass transition temperature for long-term storage, preferably below -150 °C in the gas phase over liquid nitrogen or in a suitable ultra-low temperature freezer.The lyophilized microgranules obtained from the vitrified microgranules produced according to the invention, the starting material of which is in particular a mitochondria-comprising biopharmaceutical product, can be stored after its lyophilization for a period of at least 1 month, for example 6 or 12 months, at a temperature of 4 °C or higher, for example at 20 °C or at room temperature.

[0155] While some biopharmaceutical products containing microorganisms and viruses or soluble subcellular components such as antibodies or enzymes can be freeze-dried using conventional freeze-drying methods while preserving their therapeutic effect, freeze-drying therapeutic cells and complex, therapeutically active cell components such as mitochondria and ribosomes that maintains their life- or function-preserving properties is not possible with conventional freeze-drying methods. In contrast, the vitrified microgranules produced by this process, due to their small particle size and extremely large volume-specific surface area, are ideally suited for the rapid and gentle freeze-drying of highly sensitive biopharmaceutical products that would otherwise be inaccessible to successful processing with conventional freeze-drying techniques.

[0156] According to the state of the art, freeze-drying of sensitive pharmaceutical products is carried out in the form of thin frozen product layers with a thickness of 5 mm to 10 mm on trays, in vats, or preferably in sterile glass ampoules on the shelf of the freeze dryer. A 10 mm high frozen product layer in a typical glass ampoule with an inner diameter of 11.28 mm has a volume-specific surface area of ​​approximately...

[0157] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.20260,2 mm' 1 The microgranules produced according to the process already have a volume-specific surface area of ​​more than 150 mm² at an average grain size of 145 pm. 1Since the volumetric heat transfer rate is proportional to the volume-specific surface area, the thermal efficiency is approximately 800 times higher than that of a conventional pharmaceutical freeze-drying process. Sublimation resistance also decreases proportionally to the volume-specific surface area, which drastically increases the possibilities for gentle process control. The low sublimation resistance of a microgranularly vitrified biopharmaceutical product makes it technically possible to process even products with high sugar and protein content very gently at extremely low drying pressures, without exceeding their low product-specific melting or boiling points by increasing the volume of vapor produced.

[0158] to risk collapse temperatures.

[0159] In the freeze-drying process, high concentrations of the process-permissible npCPA, in particular from the group of mono- and disaccharides (simple and double sugars) and their derivatives and / or from the group of amino acids and their derivatives, which are added to the product fluid and / or are brought into contact with the living therapeutic cells or with the living cells from which the therapeutically active cell components, such as organelles or exosomes, are obtained by means of a 6- to 48-hour incubation before vitrification, serve as water substituents and bulk-forming carriers for the therapeutically active biological components of the biopharmaceutical product after freeze-drying.

[0160] Particularly suitable are non-pharmaceutical chemotherapy compounds (npCPAs) that are compatible solutes with extremolytic (water-substituting) and osmolytic (hyperosmolar-balancing) properties, especially the disaccharides sucrose and trehalose and / or the amino acids and amino acid derivatives L-proline and ectoine. At a sufficient total concentration of the added npCPAs, for example 300 mmol / l, 500 mmol / l, or 600 mmol / l, the size and structure of the product granules can be largely preserved during the freeze-drying process, resulting in a dry, free-flowing microgranulate of the biopharmaceutical product with largely or completely retained therapeutic activity.

[0161] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The cryoprotective preconditioning within the framework of the cryopreservation process according to the invention is preferably carried out using non-permeable or non-penetrating cryoprotectant additives (npCPA); in principle, cryoprotective preconditioning using permeable cryoprotectant additives (pCPA) is possible, in particular if these are used in low concentrations.

[0162] For the use of the biopharmaceutical product in a therapeutic application, the vitrified microgranules must be revitalized prior to use. Revitalization of the biopharmaceutical product is achieved through a controlled thawing process. Under practical conditions, complete vitrification is rarely attainable. Even at extremely high freezing rates, isolated crystallization nuclei form in the vitrified product granules, but these have no effect on their quality with regard to viability or activity.

[0163] However, as soon as the glass transition temperature of the vitrified product fluid is exceeded during thawing, the formed crystallization nuclei can grow into product-damaging ice crystals. This effect is known as

[0164] This process is called devitrification or cold crystallization. The slower the thawing rate, the more pronounced the damage caused by cold crystallization. A high thawing rate is therefore essential for maintaining the quality of the thawed biopharmaceutical product. This precludes the common practice of indirectly heating the vitrified microgranules produced from the biopharmaceutical product by immersing the collection or storage containers, such as cryovials, in a warm water bath.

[0165] The method for revitalizing the biopharmaceutical product, which is in the form of vitrified, free-flowing microgranules produced by the described cryopreservation process, is carried out according to one aspect of the invention such that the revitalization of the biopharmaceutical product is effected by directly introducing the vitrified, free-flowing microgranules into a preferably warm, excess thawing medium. "Warm" here refers to a temperature in the range of approximately 30 °C to 40 °C. "Excess" means that the

[0166] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The defrosting medium is present in a quantity greater than the minimum amount required for the complete absorption and / or revitalization of the microgranules. The defrosting medium is, for example, a cell culture solution, a buffer solution, or deionized water.

[0167] The defrosting medium can be placed in a suitable receiving vessel, such as a 50 ml centrifuge tube, and rapidly circulated using appropriate tools, such as a vortex mixer. The vitrified, free-flowing microgranules are then quickly and completely added to the defrosting medium. Due to the extremely large contact area between the defrosting medium and the microgranules, the vitrified biopharmaceutical product thaws instantly. The desired temperature of the biopharmaceutical product can be reproducibly set by adjusting the chosen ratio of defrosting medium to microgranules and the temperature of the defrosting medium. A volume of defrosting medium four to six times the amount of microgranules to be defrosted is advantageous. The receiving temperature of the defrosting medium should be between 20 °C and 40 °C.The resulting temperature of the biopharmaceutical product can thus be adjusted between approximately 5 °C and 20 °C. The storage temperature of the vitrified microgranules has no significant influence on the resulting temperature of the biopharmaceutical product and should be between approximately -180 °C (for cryogenic storage over liquid nitrogen) and approximately -80 °C (for short-term storage in an ultra-low temperature freezer or on dry ice). The product concentration before vitrification should be chosen high enough so that, after mixing the defrosting medium and the vitrified microgranules, the desired target concentration of the therapeutic cells, the therapeutically active cell components, or the contained microorganisms and viruses is achieved.

[0168] One variant of the method for revitalizing the biopharmaceutical product by thawing in the thawing medium, i.e., by contact with the medium, involves placing the concentrated, vitrified microgranules in a sealed, sterile syringe. According to the invention, the frozen syringe is opened and fitted with a large-diameter aspiration needle. An inner needle diameter of 0.5 mm (21 gauge) to 2.7 mm (10 gauge) is advantageous.

[0169] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 permits rapid dispensing of the de-icing agent. Such rapid dispensing of the warm de-icing agent with the product syringe effectively mixes the microgranules and de-icing agent in the syringe cylinder due to the resulting turbulence.

[0170] In addition to the described method for revitalizing the biopharmaceutical product by thawing in the thawing medium, other thawing methods can also be used to achieve the desired high thawing rate. These include thawing methods using direct energy input via radiation, for example, focused light radiation (laser) or microwave radiation, also in combination with radiation-absorbing media components such as metal or carbon nanoparticles. Thawing methods using direct energy input via sound, especially ultrasound, or magnetic fields are also applicable, also in combination with metallic nanoparticles, for example, through nanowarming.

[0171] The physical challenge lies in the homogeneous spatial input of a very high energy density and the homogeneous conversion of the introduced radiant energy within the product volume to be heated. For this purpose, the vitrified product granules, which may contain, for example, cells or isolated mitochondria, can be exposed to a precisely calibrated electromagnetic energy input (radiant energy) to revitalize the biopharmaceutical product through controlled thawing at a heating rate of 10 4 K / min up to 10 6K / min to induce radiation. The radiation parameters are optimized depending on the type of electromagnetic energy input used, resulting in a uniform energy conversion within the product granules. This minimizes structural damage and preserves the functional integrity of the biological components of the biopharmaceutical product, such as cells or mitochondria. When using a light source, e.g., a laser, as the radiation source, reflection and refraction effects at the surface of the vitrified microgranules can be used to generate a uniform energy distribution. When using microwave radiation or ultrasound, the distribution and conversion of electromagnetic energy into heat can be optimized.

[0172] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026, for example, by using a suitable coupling medium and / or by incorporating radiation-absorbing nanoparticles into the product. A person skilled in the art can readily recognize how to select a suitable energy source, how to determine the optimal wavelength, and how to calibrate the intensity and temporal resolution of the application to achieve the desired homogeneous melting of the vitrified microgranules without local overheating of the biological components of the biopharmaceutical product or other harmful interactions with the therapeutically active structures of the biological components of the biopharmaceutical product, such as cells or mitochondria.

[0173] The revitalization of the biopharmaceutical product, which is in the form of a dry, free-flowing microgranulate produced by the described cryopreservation process in combination with subsequent freeze-drying, is carried out according to one aspect of the invention such that the revitalization of the biopharmaceutical product is effected by directly introducing the lyophilized microgranulate into a preferably temperature-controlled, excess reconstitution medium, i.e., by reconstitution with the reconstitution medium. "Excess" in the context of this application means that the reconstitution medium is present in an amount greater than the minimum amount required for complete wetting and / or reconstitution of the lyophilized microgranulate. The reconstitution medium is a pharmaceutically acceptable carrier, for example, a cell culture solution, a buffer solution, or deionized water.

[0174] A particularly advantageous variant of the method for revitalizing the biopharmaceutical product by dissolving it in the reconstitution medium, i.e., by media contact, involves placing the concentrated, lyophilized microgranules in a sealed, sterile product syringe. According to the invention, the cooled or uncooled product syringe is opened and fitted with an aspiration cannula of any desired diameter. By drawing the reconstitution medium into the product syringe, the lyophilized microgranules and the reconstitution medium mix, thereby dissolving the dry microgranules contained in the syringe barrel.

[0175] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 dissolves microgranules in the reconstitution medium, thereby forming the ready-to-use biopharmaceutical product.

[0176] The revitalized biopharmaceutical product obtained from the vitrified microgranules produced according to the invention, the starting material of which is in particular a mitochondria-comprising biopharmaceutical product, can be stored on ice for a period of at least 4 hours, for example 6, 8, 10, 12, 14, 16, 18, 20 or 24 hours, after its revitalization.

[0177] Another aspect of the invention relates to a biopharmaceutical preparation comprising functional mitochondria for use as a medicinal product, which is obtainable from the vitrified, free-flowing microgranules or the lyophilized microgranules according to the invention. The biopharmaceutical product from which the vitrified, free-flowing microgranules or the lyophilized microgranules were produced comprises functional mitochondria. This biopharmaceutical preparation according to the invention is intended for administration to a human or animal patient.

[0178] In the case of vitrified, free-flowing microgranules, administration takes place after revitalization of the biopharmaceutical product contained therein by directly introducing the vitrified, free-flowing microgranules into a defrosting medium, wherein the defrosting medium is water, a buffer solution or a cell culture solution, or by energy input in the form of electromagnetic radiation, mechanical waves or alternating magnetic fields.

[0179] In the case of lyophilized microgranules, administration takes place directly in lyophilized form or after reconstitution with a pharmaceutically acceptable carrier, i.e. by revitalization in a reconstitution medium forming the pharmaceutically acceptable carrier.

[0180] This biopharmaceutical, functional mitochondrial preparation is specifically intended for the treatment of acute kidney injury (AKI). Acute kidney injury, abbreviated as AKI, refers to a form of kidney failure characterized by a rapid decline in kidney function.

[0181] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026. The abrupt decline in kidney function in a patient or subject suffering from AKI leads to the retention of urea and other nitrogenous waste products, as well as dysregulation of extracellular volume and electrolytes. AKI is characterized by a sudden loss of kidney function, resulting in elevated creatinine levels in the blood, an important indicator of kidney health and function. AKI is a clinical syndrome that can have various causes, including reduced blood flow to the kidneys, direct kidney damage, or urinary obstruction.

[0182] The biopharmaceutical, functional mitochondria-comprising preparation according to the present invention can be administered via various routes: enterally (e.g., orally, rectally, sublingually), parenterally (e.g., intravenously, intramuscularly, subcutaneously), intranasally, or topically. The biopharmaceutical, functional mitochondria-comprising preparation used to treat or alleviate AKI is preferably administered via a blood vessel, for example, a renal artery.

[0183] The production of the vitrified microgranules, which comprise therapeutically active cell components in the form of mitochondria as a biopharmaceutical product, is carried out using the cryopreservation process according to the invention and the vitrification system according to the invention. The product fluid, which is a product suspension, contains the mitochondria, i.e., therapeutically active cell components, in a liquid, aqueous medium. The droplet diameters of the product droplets comprising mitochondria are typically in the range of approximately 50 pm to approximately 500 pm, preferably in the range of approximately 75 pm to approximately 200 pm, and particularly preferably in the range of 100 pm to 150 pm.

[0184] A product droplet with a diameter of approximately 50 pm has a volume of approximately 65 pl. A product droplet with a diameter of approximately 75 pm has a volume of approximately 0.2 nl. A product droplet with a diameter of approximately 114 pm has a volume of approximately 0.8 nl. A product droplet with a diameter of approximately 145 pm has a volume of approximately 1.6 nl. A product droplet with a diameter of

[0185] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Approx. 500 m³ has a volume of approx. 65 nl. A product droplet with a droplet diameter of approx. 1 mm has a volume of approx. 524 nl. The smaller the inner diameter of the cannula outlet, the lower the flow rate of the product fluid required by the process.

[0186] In application cannulas for generating product droplets from product fluids containing mitochondria, the inner diameter of the cannula exit opening is regularly in the range of approximately 15 pm to approximately 175 pm, preferably in the range of approximately 25 pm to approximately 100 pm, for example at approximately 42 pm, 65 pm or 95 pm.

[0187] The generation of product droplets from product fluids containing mitochondria is not limited to a specific combination of parameters, such as the inner diameter of the cannula outlet, the product fluid flow rate, or the vibration frequency; various combinations are possible. For example, the product droplets containing mitochondria have a droplet diameter of approximately 145 pm, e.g., between 144 pm and 147 pm, when a compound cannula with a glass tip and an outlet diameter of approximately 65 pm is used, the product fluid is conveyed at a flow rate of approximately 596 pl / min, and the cannula is excited at a vibration frequency of approximately 6250 Hz, e.g., between 6000 Hz and 6400 Hz. To produce product droplets containing mitochondria, whose droplet diameter is approximately 195 pm, e.g.If the oscillation frequency is between 190 pm and 210 pm, an application cannula designed as a composite cannula, whose cannula tip is designed as a glass tip and has a cannula exit opening of about 95 pm inner diameter, can be used if the product fluid is conveyed at a flow rate of about 1145 pl / min and the application cannula is excited with a vibration frequency of 4950 Hz, e.g. between 4700 Hz and 5100 Hz.

[0188] Mitochondria are biological components of biopharmaceutical products that are particularly sensitive and whose processing is time-critical. The cryopreservation process according to the invention enables the production of vitrified microgranules without undesirable effects when processing mitochondria.

[0189] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Ice crystal formation. The vitrified microgranules containing mitochondria can be cryogenically stored until use. The vitrification system and cryopreservation process according to the invention are therefore suitable for the production of a biopharmaceutical, functional mitochondria-containing preparation in single doses for direct medical use. The mitochondria contained in the vitrified, free-flowing microgranules can be easily revitalized, for example, by directly introducing the vitrified, free-flowing microgranules into a warm, excess thawing medium, such as a buffer solution or a cell culture solution, and can be ready for use for various purposes, which may include, among others, research, diagnostic, and therapeutic applications.The revitalization of the vitrified microgranules obtained by the cryopreservation process according to the invention is particularly low-loss. Furthermore, it is possible to freeze-dry or lyophilize the vitrified microgranules obtained according to the invention, which contain mitochondria. Such lyophilized microgranules can be stored at a temperature above -80 °C, for example -20 °C, or even at a temperature of 0 °C or above, for example about 4 °C or 6 °C in a refrigerator, or at 20 °C.

[0190] According to one embodiment of the invention, the product fluid containing the mitochondria-containing biopharmaceutical product may further comprise one or a combination of two or more of the following components, namely a buffering agent, a calcium chelating agent, an ionic component and albumin.

[0191] The buffering agent is preferably selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), piperazine N,N'-bis(2-ethanesulfonic acid) (PIPES), 4-morpholinethanesulfonic acid (MES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), N,N-bis(2-hydroxyethyl)glycine (Bicine), potassium phosphate, sodium cacodylate, tris(hydroxymethyl)aminomethane hydrochloride (Tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris-propane), sodium acetate, or a combination thereof. The buffering agent in the aqueous medium exhibits a

[0192] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Concentration from 0.5 mM to 50 mM. Preferably the buffering agent comprises HEPES.

[0193] The calcium chelating agent is preferably selected from the group consisting of ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2,2',2",2'"-(ethane-1,2-diyldinitrilo)-tetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), or a combination thereof. EGTA and / or EDTA are particularly preferred as the calcium chelating agent. The calcium chelating agent has a concentration of 0.1 mM to 10 mM in aqueous medium.

[0194] The ionic component is preferably selected from the group consisting of salts, acids or bases containing Mg 2+ , N / a + , K + , CI" or HCO3" contain, such as MgCl2, MgSO4, KCl, KH2PO4, NaHCO3, Na2HPO4, formate anions, e.g.

[0195] C₂H₂MgO₄ (magnesium formate), pyruvate anions, e.g., C₃H₃NaO₃ (sodium pyruvate), acetate anions, e.g., C₂H₃NaO₂ (sodium acetate), malate anions, oxaloacetate anions, glutamate anions, α-ketoglutarate anions, succinate anions, or a combination thereof. The ionic component has a concentration of 0.1 mM to 100 mM in aqueous medium.

[0196] Albumin is, for example, bovine serum albumin (BSA) and / or human albumin (HSA). Albumin has a concentration of 0.01% (w / v) to 10% (w / v).

[0197] According to another embodiment, the product fluid containing mitochondria further comprises: (a) 20 mM Tris, 2 mM EDTA and 10 mM MgCl2(pH 7.4);

[0198] (b) 5 mM MOPS, 10 mM BAPTA and 5 mM sodium pyruvate (pH 7.25); or

[0199] (c) 10 mM HEPES and 1 mM EGTA (pH 7.2). The aqueous product fluid contains 10 mM HEPES and 1 mM EGTA (pH 7.2).

[0200] Furthermore, the product fluid may contain one or more mitochondria in addition to mitochondria.

[0201] Contains non-permeable or non-penetrating antifreeze additives, such as trehalose, glucose, sucrose, proline, such as L-proline, and / or proline derivatives.

[0202] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 preferably trehalose, sucrose, glucose and / or L-proline. Optionally, the product fluid may also contain penetrating antifreeze additives, such as DMSO, in addition to mitochondria. Optionally, the product fluid may also include a buffering agent.

[0203] The use of antifreeze additives in the cryopreservation process according to the invention preferably involves the use of non-permeable or non-penetrating antifreeze additives; however, it is also feasible using permeable antifreeze additives. For example, in one embodiment, antifreeze additives selected from the group consisting of propylene glycol, ethylene glycol, glycerol, and / or dimethyl sulfoxide (DMSO) can be used. In one embodiment, the product fluid containing mitochondria comprises, for example, DMSO. Here, DMSO is present in a concentration of less than 5% (v / v), e.g., 2.5% (v / v) or 1% (v / v).

[0204] According to another embodiment, the non-permeable or non-penetrating cryoprotectant additives suitable for the cryopreservation of mitochondria include extremolytes. Extremolytes include: (i) ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) and its derivatives, such as hydroectoine, e.g., 5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, or homoectoine, e.g., 4,5,6,7-tetrahydro-1H-[1,3]-diazepine-4-carboxylic acid; (ii) pipecolic acid, i.e., piperidine-2-carboxylic acid; (iii) Heterosides, including but not limited to (iii.a) mannosylglycerate, i.e. [(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] 2,3-dihydroxypropanoate, and / or mannosylglyceramide, i.e. (2S)-3-hydroxy-2-[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxypropanamide, and (iii.b) glucosylglycerin, i.e.(3R,4S,5S,6R)-6-(Hydroxymethyl)-2-(1,2,3-trihydroxypropyl)oxane-2,3,4,5-tetrol, and / or glucosylglycerate; (iv) polyol phosphates, including but not limited to cyclic 2,3-diphosphoglycerate (cDPG), diglycerol phosphate, glycerol phosphomyo-inositol and / or di-myo-inositol phosphate (DIP).

[0205] According to another embodiment, the non-permeable or non-penetrating antifreeze additives suitable for the cryopreservation of mitochondria include, among others, mono-, di-, tri-, oligo- and polysaccharides,

[0206] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 preferably mono- and / or disaccharides. Saccharides may be selected from the group consisting of trehalose, maltose, lactose, fructose, sucrose, glucose, dextran, melezitose, raffinose, nigerotriose, maltotriose, maltotriulose, kestose, cellobiose, chitobiose, lactulose, or a combination thereof. Preferably, the saccharide is trehalose, sucrose, glucose, or a combination thereof. Preferably, the saccharide is trehalose alone or in combination with one or more saccharides, for example, trehalose in combination with sucrose and / or glucose. Even more preferably, trehalose alone is the non-permeable or non-penetrating antifreeze additive. The saccharide(s) can have a total concentration of at least 150 mM, e.g., at least 300 mM. Trehalose alone can have a concentration of at least 150 mM, preferably 300 mM or 500 mM.

[0207] In another embodiment, amino acids are non-permeable or non-penetrating cryoprotectant additives suitable for the cryopreservation of mitochondria. The amino acids include, but are not limited to, leucine, isoleucine (e.g., L-isoleucine), proline (e.g., L-proline), methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridine carboxylic acid, azetidine carboxylic acid, pipecolic acid, oxaproline, thiaproline, valine (e.g., L-valine), alanine (e.g., L-alanine), glycine, asparagine (e.g., L-asparagine), aspartic acid (e.g., L-aspartic acid), glutamic acid (e.g., L-glutamic acid), serine (e.g., L-serine), histidine (e.g., L-histidine), cysteine ​​(e.g., L-cysteine), tryptophan (e.g., L-tryptophan), tyrosine (e.g., L-tyrosine), arginine (e.g., L-arginine). L-Arginine), Glutamine (e.g. L-Glutamine), Lysine, Threonine, Selenocysteine, Methionine, Phenylalanine, Creatine, Taurine (e.g. L-Taurine), Betaine, Ectoin, Dimethylglycine, Ethylmethylglycine, an RGD peptide or a combination thereof.

[0208] Preferably, the amino acid(s) is selected from the group consisting of proline, methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridine carboxylic acid, azetidine carboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof. Preferably, the amino acid is proline. Even more preferably, the amino acid proline, e.g., L-proline, in combination with trehalose and one or more additional saccharides, for example...

[0209] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 one or more additional saccharides selected from the group consisting of sucrose, glucose or a combination thereof. Even more preferred is the amino acid proline, e.g. L-proline, in combination with trehalose alone. The amino acid has a concentration of at least 160 mM, for example at least 180 mM, 200 mM, 300 mM, 500 mM or 600 mM.

[0210] Furthermore, the antifreeze additives may include trehalose in combination with one or more amino acids, e.g. L-proline, possibly further combined with one or more saccharides, such as glucose and / or sucrose.

[0211] In the vitrified microgranules, lyophilized microgranules, and biopharmaceutical preparation comprising mitochondria according to the invention, the mitochondria are preferably isolated functional mitochondria. These isolated functional mitochondria have an intact outer and inner mitochondrial membrane and retain their membrane potential as well as their ability to synthesize ATP (adenosine triphosphate). The mitochondria of the present invention are respirable mitochondria. For example, the mitochondria are able to synthesize ATP by oxidative phosphorylation, wherein the oxidation of carbon substrates and the generation of a proton gradient across the inner mitochondrial membrane contribute to ATP production.

[0212] The mitochondria used according to the invention refer to functional mitochondria that are essentially free of eukaryotic cell material, such as foreign eukaryotic cell material, wherein the mitochondria have been isolated or purified from tissues, cells, or a cell culture. Thus, only minimal amounts of other cell components besides mitochondria are present in the mitochondria used according to the invention or in a composition thereof. Preferably, no other cellular components besides mitochondria are present in a composition of mitochondria used according to the invention. In this sense, the terms "mitochondria" and "isolated mitochondria" are used synonymously in the context of this description of the invention. Any known technique can be used for the isolation of mitochondria. The terms "isolated" and "partially purified" in this context

[0213] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Mitochondria refers to mitochondria that have been purified or at least partially purified from other cell components. The total amount of mitochondrial proteins in isolated or partially purified mitochondria is between 10% and 90% of the total amount of cellular proteins in the sample.

[0214] A mitochondrion is a double-membrane-enclosed organelle found in most eukaryotic organisms. Accordingly, a mitochondrion of the present invention can be a mitochondrion from any eukaryotic organism. A mitochondrion can be a mitochondrion from an animal, preferably a mammal, a plant, yeast, or a fungus. A mitochondrion can be a human mitochondrion. A mitochondrion of the invention can be obtained by any means, such as cell culture. Accordingly, a mitochondrion of the invention can be obtained by in vitro cell culture. Preferably, a mitochondrion can be obtained from an in vitro 2D or 3D cell culture. A mitochondrion of the present invention can also be obtained from tissue. A mitochondrion obtained from tissue can be obtained from any tissue of a eukaryotic organism.

[0215] Accordingly, a mitochondrion can be obtained from cells or tissue of a eukaryotic organism that is kept in culture. A mitochondrion can be obtained from an animal, plant, yeast, or fungal cell kept in an in vitro cell culture. Preferably, a mitochondrion is obtained from a human tissue or cell culture. Even more preferably, a mitochondrion is obtained from a human in vitro cell culture.

[0216] In preferred embodiments, a mitochondrion is obtained from animal tissue or a cell culture, in particular from murine tissue or a murine cell culture. Preferably, a mitochondrion is obtained from an in vitro cell culture of a mouse. A mitochondrion can be obtained from mouse embryonic fibroblasts (MEFs). A mitochondrion can be obtained from MEFs maintained in an in vitro cell culture.

[0217] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Mitochondria can be obtained from MEFs maintained in an in vitro cell culture using Dulbecco's Modified Eagle Medium (DMEM). In some embodiments, a mitochondrion is obtained, for example, from human cardiac fibroblasts (HCF). A mitochondrion can be obtained from HCFs maintained in an in vitro cell culture. A mitochondrion can be obtained from HCFs cultured in an in vitro cell culture using Fibroblast Medium-2 (ScienCell, Carlsbad, CA, USA; Cat. No. #2331). In further embodiments, a mitochondrion can be obtained from HepG2 cells. A mitochondrion can be obtained from HepG2 cells cultured in an in vitro cell culture. A mitochondrion can be obtained from HepG2 cells cultured in an in vitro cell culture using Roswell Park Memorial Institute (RPMI) medium.

[0218] A mitochondrion of the present invention can also be obtained fresh by isolating the mitochondrion from a cell culture or tissue, e.g., from eukaryotes. A mitochondrion obtained from tissue can be derived from placental, liver, muscle, or porcine tissue.

[0219] Accordingly, a mitochondrion can be obtained by fresh isolation from an animal, plant, yeast, or fungal cell culture, or from tissue. Preferably, a mitochondrion can be obtained by fresh isolation from a human cell culture or from human tissue. A mitochondrion can be obtained by fresh isolation from HCFs or HepG2 cells. A mitochondrion can be obtained by fresh isolation from HCFs or HepG2 cells maintained in an in vitro cell culture. A mitochondrion can be obtained by fresh isolation from HCFs maintained in an in vitro cell culture containing Fibroblast Medium-2 (ScienCell, Carlsbad, CA, USA; Cat. No. #2331). A mitochondrion can be obtained by fresh isolation from HepG2 cells maintained in an in vitro cell culture using RPMI medium. A mitochondrion can be obtained by fresh isolation from placenta, e.g.,from human placental tissue or cells, which are maintained in an in vitro cell culture using a typical xeno-free cell culture medium, wherein the cell culture medium consists exclusively of human components and with.

[0220] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Growth factors are added to support the growth of mesenchymal cells. Furthermore, a mitochondrion can be obtained by fresh isolation from a murine cell culture or from murine tissue. A mitochondrion can be obtained by fresh isolation from a mouse in vitro cell culture. A mitochondrion can be obtained by fresh isolation from MEFs. A mitochondrion can be obtained by fresh isolation from MEFs maintained in an in vitro cell culture. A mitochondrion can be obtained by fresh isolation from MEFs maintained in an in vitro cell culture containing DMEM.

[0221] Isolated mitochondria exhibit a preferred concentration of at least 0.02 pg protein / pl and at most 100 pg protein / pl in the product fluid.

[0222] A mitochondrion can be a modified mitochondrion. The mitochondrion can be associated with a drug, such as a pharmaceutical agent, a diagnostic agent, an imaging agent, a therapeutic agent, or another biocompatible agent. Alternatively, the mitochondrion can be associated with an antibody or an antigen-binding fragment, wherein the antigen-binding fragment comprises at least part of an antibody or a T-cell receptor (TOR) or recombinant variants thereof. In one embodiment, the mitochondrion is directly or indirectly associated with a drug, an antibody, or an antigen-binding fragment via a covalent or non-covalent bond (e.g., an electrostatic bond).In another embodiment, the active substance, antibody, or antigen-binding fragment is embedded in the mitochondrion, embedded in the mitochondrial membrane, substantially enclosed within the mitochondrion, or completely enclosed by the mitochondrion. In yet another embodiment, the mitochondrion is a genetically modified mitochondrion or comprises exogenous mtDNA.

[0223] In a further embodiment, the mitochondrion comprises one or more payloads that are directly or indirectly bound to the outer membrane of the mitochondrion via a covalent or electrostatic bond. The payload(s) includes, among other things, one or more of the following components: i) a

[0224] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Nucleic acid molecule; ii) a polypeptide; iii) a drug substance; or iv) a combination of one or more of the components (i) to (iii). If the payload and the mitochondrion both have a net negative charge, the payload can be bound to the outer membrane of the mitochondrion via positively charged species, such as a polycationic polymer, positively charged particles, and / or nanoparticles. The mitochondrion described herein can furthermore be associated with and / or partially or completely enveloped by an additional protective layer.

[0225] Regarding further details and embodiments of the invention, reference is made to the German patent application with application number 102025 112090.0, the contents of which are hereby incorporated into this patent application.

[0226] The invention is explained in more detail below with reference to exemplary embodiments and the schematic drawings, wherein identical or similar features are provided with the same reference numerals; to this end, the following are shown:

[0227] Fig. 1: the process principle of the process carried out with the vitrification plant for the production of microgranules from biopharmaceutical products in perspective view,

[0228] Fig. 2: the process principle when carrying out the process with a first embodiment of the cooling unit in perspective view, Fig. 3: the process principle when carrying out the process with a second embodiment of the cooling unit in perspective view, Fig. 4: the components of the vitrification plant for producing the product droplets in perspective view,

[0229] Fig. 5: the first embodiment of the cooling unit in perspective view, Fig. 6: the vitrification roller of the second embodiment of the cooling unit in perspective view,

[0230] Fig. 7: Installation of the vitrification roller of the second embodiment of the cooling unit in a hollow shaft arrangement in longitudinal section view, Fig. 8: An embodiment of the collection vessel cooling unit with scraper tool in perspective view,

[0231] Fig. 9: Results of a viability test in diagram form,

[0232] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Fig. 10: Integrity and function of mitochondria after vitrification using the cryopreservation method in diagram form,

[0233] Fig. 11: Integrity and function of mitochondria after vitrification using the cryopreservation method in diagram form,

[0234] Fig. 12: Blood urea nitrogen and serum creatinine after administration of a biopharmaceutical, functional mitochondria-containing preparation in diagram form.

[0235] Fig. 13: Evaluation of the histopathological analysis of the kidneys after administration of a biopharmaceutical, functional mitochondria-containing preparation in diagram form.

[0236] Fig. 14: Stability assessment of vitrified and lyophilized microgranules after storage at elevated temperatures,

[0237] Fig. 15: Blood urea nitrogen and serum creatinine after administration of a biopharmaceutical, functional mitochondria-containing preparation in diagram form.

[0238] Fig. 16: Evaluation of the histopathological analysis of the kidneys after administration of a biopharmaceutical, functional mitochondria-containing preparation in diagram form.

[0239] The exemplary embodiments described below are preferred embodiments of the invention. In these exemplary embodiments, the described components each represent individual features of the invention, which can be considered independently of one another and which further develop the invention independently. Therefore, the disclosure is intended to include combinations of features of the embodiments other than those shown. Furthermore, the described embodiments can also be supplemented by further features of the invention already described above.

[0240] The representation of the vitrification plant according to Fig. 1 shows in the upper area the assembly for producing the product droplets 13 from the biopharmaceutical product 10 and in the lower area the assembly for vitrifying the product droplets 13.

[0241] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026The assembly for generating product droplets 13 comprises the (not shown) syringe pump, the application syringe 20, the application cannula 21 and the vibration exciter 31 designed as a linear actuator for generating high-frequency sinusoidal vibrations, which is height-adjustable by means of the coupling designed as a magnetic coupling 40 on the application cannula 21 in order to realize the vibration transmission from the vibration exciter 31 of the vibration exciter unit 30 to the application cannula 21.

[0242] The application cannula 21, designed as a composite cannula, is divided into the elongated base cannula 22, made of stainless steel, and the cannula tip 23, which is made of a glass material. For the sake of simplicity, the nozzle-shaped narrowing of the inner diameter of the cannula tip 23 is not shown in Fig. 1, Fig. 2 and Fig. 3.

[0243] To apply the monodisperse product droplets 13, the application syringe 20 is filled with the product fluid 11, fitted with the application cannula 21, and inserted into the syringe pump (not shown). The application cannula 21 is then connected to the vibration exciter 31 at a suitable height above the cannula outlet opening, i.e., the outlet opening of the application cannula 21 at the cannula tip 23, using the magnetic coupling 40.

[0244] To generate the product droplets 13, which is carried out using the Plateau-Rayleigh instability according to the principle of the moving opening, a high-frequency, sinusoidal, linear signal with low amplitude is generated with the help of the vibration exciter 31 and transmitted via the magnetic coupling 40 to the application cannula 21 in order to set it into vibration.

[0245] The product fluid 11 is then forced through the cannula opening using the application syringe 20 and the syringe pump at a suitable flow rate. The flow rate must be selected to be sufficiently high, depending on the diameter of the cannula outlet, to generate a stable product fluid stream 12. The product fluid stream 12 is stable when it

[0246] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 without vibration excitation only after a beam length corresponding to 15 to 20 times its diameter.

[0247] Under vibration excitation, the product fluid jet 12 breaks down into the monodisperse product droplets 13, which fall freely in the vertical direction, i.e.

[0248] Product droplets 13 of identical size. The number of product droplets 13 released per unit of time, i.e., the droplet frequency, corresponds to the vibration frequency of the vibration exciter 31. The droplet diameter and volume of the product droplets 13 are defined by the set flow rate and vibration frequency of the vibration exciter 31.

[0249] The assembly for vitrifying the product droplets 13 is arranged directly below the product droplets 13, which fall freely in the vertical direction. The released product droplets 13 fall as a droplet chain onto the preferably moving vitrification medium 51 of the cooling unit 50. The distance between the cannula outlet opening and the contact point with the vitrification medium 51, i.e., the fall distance, should not be too great in order to avoid disturbances to the droplet chain of the monodisperse product droplets 13. A free fall distance of 10 mm to 60 mm has proven advantageous.

[0250] Figures 2 and 3 illustrate fluid vitrification using the vitrification fluid 60 as the vitrification medium 51 in Figure 2 and the surface area or...

[0251] Roller vitrification using the vitrification roller 70 as vitrification medium 51 in Fig. 3. After the product fluid 11 exits the cannula tip 23 of the application cannula 21 in the form of the product fluid jet 12, it breaks down into the droplet chain of the monodisperse product droplets 13.

[0252] In fluid vitrification – see Fig. 2 – the product droplets 13 fall into the cryogenic vitrification fluid 60 located in the vitrification vessel 61. The vitrification vessel 61 simultaneously forms the collection vessel 52 for collecting the product granules 14 formed from the product droplets 13 in the vitrification fluid 60, which accumulate at the bottom of the collection vessel 52 or vitrification vessel 61 as the vitrified microgranules.

[0253] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 In the area treatment carried out with the vitrification roller 70,

[0254] In roller vitrification – see Fig. 3 – the falling product droplets 13 strike the cryogenic surface of the vitrification roller 70 and vitrify there, forming product granules 14. The product granules 14 are removed from the cryogenic surface of the vitrification roller 70 by the scraper 79, fall into the collection vessel 52 below the vitrification roller 70 and collect there as the vitrified microgranules.

[0255] The embodiment of the assembly for generating the product droplets 13 according to Fig. 4 shows the application syringe 20 with a conventional syringe design: A syringe cylinder of the application syringe 20 serves to receive the product fluid 11; with the syringe plunger, which is actuated by the syringe pump (not shown), the product fluid 11 can be conveyed at the specified flow rate through the application cannula 21 attached to the application syringe 20, which consists of the base cannula 22 and the cannula tip 23.

[0256] The vibration exciter unit 30 includes the vibration exciter 31, which is designed as a linear actuator and is installed on the mounting carriage 35 by means of the vibration exciter bracket 34 (or actuator bracket). The vibration exciter 31 can be easily adjusted relative to the application cannula 21 using the mounting carriage 35.

[0257] The coupling of the vibration exciter 31 to the application cannula 21 is formed by the magnetic coupling 40, also shown in Fig. 1, which comprises the base magnet 41, the holding magnet 42, and the guide disc 43. The design of the cannula guide groove in the guide disc 43 is clearly visible in Fig. 4. With the magnetic coupling 40 installed, the base cannula 22 is inserted into this groove in a force-fit and form-fit manner. The fastening screw 33 serves to connect the magnetic coupling 40 to the vibration exciter 31. The screw head of the screw is firmly attached to the base magnet 41, for example, by a metal-fit connection. In normal use, the base magnet 41 and the fastening screw 33 are screwed into the oscillating head 32 of the vibration exciter 31, which is equipped with a threaded bore, and thus firmly connected to the vibration exciter 31.

[0258] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The embodiment of the cooling unit 50 of the vitrification system for fluid vitrification shown in Fig. 5 comprises the tubular vitrification vessel 61, which receives the vitrification fluid 60 serving as the vitrification medium 51. The vitrification vessel 61, which simultaneously forms the collection vessel 52, is inserted into a recess in the cooling block 62, which in this embodiment forms the essential component of the vitrification vessel cooling unit. The core of the cooling block 62 consists of an aluminum alloy and is surrounded on its side surfaces by an insulating shell. The temperature sensor receptacle 63, which is integrated into the recess for the vitrification vessel 61 in the cooling block 62, serves to insert a temperature sensor for temperature-controlled cooling of the cooling block 62.

[0259] The cooling of the cooling block 62 in the vitrification vessel cooling unit is achieved by means of cold gas, which is supplied to the cooling block 62 via the cold gas supply line 65 and, after cooling the cooling block 62, is discharged again via the cold gas outlet 66.

[0260] A commercially available circular or orbital shaker serves as the shaking device 67. The peripheral speed should be selected to be high enough so that, for a given droplet size and droplet frequency, separate product droplets 13 are deposited on the circularly moving surface of the vitrification fluid 60. To adjust a suitable orbital motion, i.e., one that supports droplet separation, the following relationship can be used:

[0261] TT ■ Orbital diameter ■ Shaker speed > Droplet diameter ■ Dropping frequency can be used.

[0262] The two cooling block holders 64 serve to mount the vitrification vessel cooling unit, i.e., essentially the cooling block 62, on the shaking device 67. Receptacles for fixing multiwell plates, which many conventional orbital shakers have, can be used as cooling block holders 64; if necessary, these must be adapted to the cooling block dimensions by means of adapters.

[0263] The embodiment of the vitrification roller 70, which serves as the vitrification medium 51, shown in Fig. 6, of the cooling unit 50, which can be used for surface or roller vitrification, is made of stainless steel and has a SiOx-

[0264] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 coated surface with a contact angle of 85°. The continuous flow channels 71 serve to convey the cooling medium.

[0265] An embodiment of the cooling unit 50 for surface or roller vitrification, with the vitrification roller 70 integrated into the hollow shaft arrangement, is shown in Fig. 7: As in Fig. 6, the vitrification roller 70 has the continuous flow channels 71 and is rigidly connected on both axial sides via screw threads (locking fit 76) to two hollow shafts, which are guided in two bearings within the (not shown) vitrification chamber. The vitrification roller 70 is driven by the movably mounted hollow shaft (drive shaft 74) attached to the left of the vitrification roller 70 in Fig. 7, which is driven by a motor via the drive pulley 75, designed as a belt pulley. The supply of cold gas (here cold nitrogen gas) is provided by the cold gas supply line 72, designed as a rigid supply pipe.The two gas-tight radial bearings 77, designed as plain or ball bearings, hold the cold gas supply line 72 coaxially within the drive shaft 74. The cold gas is discharged directly through the hollow shaft (discharge shaft 73) mounted axially opposite the drive shaft 74 on the vitrification roller 70. A temperature measuring device (not shown) with a temperature sensor is located on the inside of the vitrification roller 70 and serves to control the temperature of the vitrification roller 70. The signal cables of the temperature measuring device are routed out of the moving drive train via a rotary feedthrough 78 at the end of the discharge shaft 73.

[0266] To achieve singulation of the product droplets 13 upon impact with the vitrification roller 70, the bearings supporting the hollow shafts are designed as floating bearings, for example, as floating bearing blocks, so that the entire drive train, i.e., the hollow shaft assembly with vitrification roller 70, can perform a horizontal oscillating motion. This increases the contact distance between the vitrification roller 70 and the impacting product droplets 13 many times over, and singulation of the product droplets 13 requires significantly smaller roller diameters and rotational speeds.

[0267] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The embodiment of the collection vessel cooling unit according to Fig. 8 comprises the multi-part cooling block 53. The side plates of the cooling block 53 serve for the inlet and outlet of cold gas; fins are incorporated into the top surfaces of these plates, which function as a cold trap, to increase the surface area. Below the central opening on the top of the cooling block 53 is the collection vessel 52, which comprises a cell strainer, i.e., a sieve, and a collection plate. The collection vessel 52 can be inserted into and removed from the cooling block 53 via the frontal opening.

[0268] The collection vessel cooling unit shown in Fig. 8 is designed for use in combination with the vitrification roller 70 for the production of microgranules by sheet or roller vitrification. The tension wire, forming the stripping tool 79, serves to strip the product granules 14 from the outer surface of the vitrification roller 70. This wire is attached between the two side plates of the cooling block 53. The tension of the tension wire can be maintained or adjusted by means of the tensioning screw or swivel on the right side plate of the cooling block 53. Two co-cooled springs in the cooling block 53 press the tension wire upwards against the outer surface of the vitrification roller 70 (not shown). After the product granules 14 are stripped from the outer surface of the vitrification roller 70 by the tension wire, they fall onto the collection plate and slide from there onto the cell strainer.

[0269] The execution of the cryopreservation process according to the invention is explained below with reference to exemplary embodiments carried out under laboratory conditions. Vitrification was performed by fluid vitrification using a vitrification system, the assembly of which for generating the product droplets 13 corresponds to the setup shown in Fig. 4 and the assembly of which for vitrifying the product droplets 13 corresponds to the setup shown in Fig. 5.

[0270] The first embodiment carried out under laboratory conditions illustrates the production according to the invention of vitrified microgranules from biopharmaceutical products containing living cells.

[0271] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 A product fluid 11 was provided, which contained dermal human fibroblasts obtained by abtrypsinization of an adherent cell culture as a biopharmaceutical product to be vitrified.

[0272] The aim was to produce two single doses of a ready-to-use cell suspension, each containing 150,000 cells in 1.2 ml of ready-to-use cell suspension. For this purpose, microgranules produced according to the invention from dermal human fibroblasts were thawed directly after vitrification for the purpose of process verification and seeded in a multiwell plate. Three wells of the multiwell plate were prepared from a vitrified single dose of microgranules.

[0273] 50,000 cells per well were seeded and cultured for 72 hours. Viability was then evaluated by fluorescence photometry. The control sample was seeded and cultured under identical conditions directly after cell collection, i.e., without verification.

[0274] To prepare for vitrification, fibroblasts in adherent cell culture were incubated for 24 hours in a suitable cell culture medium with an elevated glucose concentration of 54 mmol / l, an elevated pyruvate concentration of 27 mmol / l, and the addition of 75 mmol / l trehalose to promote intracellular accumulation of these substances. Subsequently, the pretreated fibroblasts were trypsinized from the surface of the cell culture vessel and resuspended in a suitable medium with the addition of 300 mmol / l trehalose to form product fluid 11. Before processing using the application syringe 20 and the application cannula 21, the product fluid 11 was filtered through a sieve with a pore size of 40 pm to remove cell aggregates and prevent clogging of the application cannula 21. The cell density was adjusted to 750,000 cells / ml.

[0275] To prepare for vitrification, the cooling block 62 was pre-cooled to a temperature of -165 °C and a centrifuge tube was inserted into the cooling block 62 as a vitrification vessel 61. The centrifuge tube used had a conical bottom and a capacity of 15 ml, which was filled with 14 ml of liquid ethene as the vitrification fluid 60.

[0276] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 For vitrification, approximately 500 pl of the product fluid 11 were drawn into a sterile glass syringe with a volume of 1.0 ml, forming the application syringe 20, using a short cannula of suitable diameter (aspiration cannula). After drawing up the application syringe 20, the aspiration cannula was replaced with the sterile application cannula 21, which has a cannula exit orifice with a cannula exit diameter of 65 µm, and carefully vented. The completed application syringe 20 was inserted into the syringe pump such that the cannula exit orifice was located centrally above the vitrification vessel 61 at a height of 50 mm above the liquid level of the vitrification fluid 60.Subsequently, the base cannula 22 was fixed at a height of 15 mm above the cannula exit opening with the magnetic coupling 40 on the vibration exciter 31 (piezo linear actuator) and the linear actuator was driven by a suitable frequency generator with a sinusoidal oscillation.

[0277] Frequency 6250 Hz applied.

[0278] Subsequently, the vitrification vessel 61 located below the application cannula 21 was set into circular motion with an orbit of 6 mm and a rotational speed of 3000 rpm by activating the shaking device 67, which is an orbital shaker. The syringe pump was then started with a flow rate of 600 pl / min. This generated a stable product fluid jet 12. The vibration excitation resulted in a stable droplet chain of monodisperse product droplets 13 with a droplet frequency of 6250 product droplets 13 per second. The droplet diameter was determined to be 145 pm based on the flow rate of the product fluid 11 and the number of applied droplets. This corresponds to a droplet volume of 1.6 nl.

[0279] Since the mathematical product of orbital circumference and shaker rotational speed (943 mm / s) resulting from the relationship described above is larger than the mathematical product of droplet diameter and droplet frequency (906 mm / s), individual product droplets 13 were deposited on the surface of the vitrification fluid 60, which was tempered to -165 °C. As they settled, these droplets vitrified into separate product grains 14 of identical size. A CFD simulation of the vitrification process on a 3D model of the product droplet 13, including heat conduction and convective heat transfer, was used to determine the following parameters:

[0280] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 the aforementioned embodiment determines an average freezing rate of 1.23 million K / min.

[0281] After an application time of exactly 20 seconds, 200 pl of product fluid 11 were vitrified and the syringe pump was switched off. After switching off the orbital shaker, the vitrification vessel 61 containing the first vitrified single dose of microgranules was removed from the cooling block 62 and replaced with a new vitrification vessel 61 containing liquid ethene at the same temperature as the vitrification fluid 60. The orbital shaker was then switched on again and the vitrification process was repeated to obtain the second vitrified single dose of microgranules.

[0282] After verification was completed, the vibration exciter 31 and the syringe pump were switched off. The application syringe 20 was unlocked by opening the magnetic coupling 40 and removed from the syringe pump. The remaining product fluid 11 was discarded.

[0283] The respective vitrification vessel 61 containing the vitrified microgranules was transferred to a fume hood immediately after removal from the cooling block 62. Deep-frozen 50-ml centrifuge tubes with 85 pm pore size sieves were placed in a polystyrene box containing liquid nitrogen. The vitrification fluid 60 containing the microgranules was poured through the sieve from the vitrification vessel 61 into the deep-frozen centrifuge tube. The vitrified microgranules remained on the sieve and were transferred to a deep-frozen storage vessel (storage tube) and then to a cell tank for cryogenic storage. The entire process, from the start of verification to the storage of the vitrified microgranules in the storage vessel, took less than two minutes.

[0284] To verify the process, the vitrified microgranules were thawed immediately after production and seeded into a multiwell plate. For this purpose, 1000 pl of warm cell culture medium at a temperature between 30 °C and 40 °C was placed in a 50 ml centrifuge tube (feed tube) as a thawing medium. Each feed tube was then pressed onto a touch-operated vortexer set to maximum speed.

[0285] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The microgranules were completely emptied from the frozen storage tube into the feed tube. Due to the large contact area between the microgranules and the thawing medium, the microgranules thawed rapidly. This resulted in 1.2 ml of ready-to-use cell suspension with the desired cell concentration of 125,000 cells / ml, which was divided among three wells of a multiwell plate for verification of the procedure. After the addition of cell culture medium, the multiwell plate was recultured for 72 hours and evaluated against the control sample using fluorescence microscopy for viability testing.

[0286] Since ice nucleation during the vitrification process can be almost completely suppressed by establishing intracellular cryoprotection through 24-hour incubation of the cells with an increased concentration of cryoprotective mono- and disaccharides, the use of a suitable vitrification medium 51 in the form of the selected vitrification fluid 60, and the achievement of a high freezing rate of >1 million K / min, the resulting thawing rate of approximately 30,000 K / min is sufficient for low-loss revitalization of the vitrified cells. A viability retention of more than 95% was reproducibly demonstrated after vitrification.In experiments carried out according to the exemplary embodiment with human dermal fibroblasts in a trehalose-containing product fluid 11 with 300 mM trehalose, from which product droplets 13 with a diameter of 145 pm and a volume of 1.6 nl were vitrified by fluid vitrification at a freezing rate of approximately 1.2 million K / min (determined by numerical simulation of the vitrification process), the viability of the cells vitrified in duplicate was 95% and 97% of the value of a non-vitrified control sample, respectively. Fig. 9 shows the results of the non-cryopreserved control sample (reference) and the two vitrified single doses of the cell suspension (Test 1 and Test 2). The viability test was performed fluorescence photometrically using a plate reader after four hours of incubation of three populated cavities of a multiwell plate with the redox dye resazurin.The graph shows the mean and standard deviation of the measurement results from three colonized cavities each. The reported viability values ​​are dimensionless (AU for "Arbitrary Units"). The viability of the vitrified cells, expressed as a percentage, refers to identical samples.

[0287] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 treated, non-vitrified cells as reference, whose viability is defined as 100%.

[0288] Another embodiment carried out under laboratory conditions illustrates the production according to the invention of vitrified microgranules from biopharmaceutical products 10 containing mitochondria.

[0289] Mitochondria were isolated from cryopreserved dissociated human placental cells that had previously been stored at -80 °C in 10% v / v DMSO in fetal calf serum (FCS). The placental cells were thawed for 2 minutes in a water bath at 37 °C and then placed on ice. The cell suspensions were then transferred to pre-chilled phosphate-buffered saline (PBS) and homogenized by gentle inversion. The cells were centrifuged for 5 minutes at 300 x g and 4 °C, then washed in PBS and centrifuged again under identical conditions. The resulting cell pellet was resuspended in a solution of 300 mM trehalose and 10 mM HEPES with the addition of 0.6 U / ml neutral protease (metalloprotease from Clostridium histolyticum), homogenized by careful inversion and incubated on ice for 10 minutes.Subsequently, 0.5 M EGTA was added up to a final concentration of 1 mM to inactivate the neutral protease by chelating divalent cations.

[0290] Samples were pre-texed for 2 minutes and then centrifuged for 5 minutes at 1000 x g and 4 °C. 10 pm nylon sieves were pre-moistened; the supernatants were then filtered under vacuum.

[0291] The filtrate was divided into 1-ml aliquots, and the mitochondria were pelletized by 5-minute centrifugation at 9500 x g and 4 °C. The supernatant was separated from the pellet. The control pellets were resuspended in 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA, while the remaining aliquots were resuspended in the respective product fluids listed in Table 1.

[0292] Examples of product fluids 11 in the form of liquid product suspensions, consisting of an aqueous medium in which the components forming the biopharmaceutical product 10 are contained.

[0293] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The product fluids 11 contained mitochondria suspended in an aqueous medium at a concentration between 0.20 mg / ml and 1.50 mg / ml, for example, between 0.25 mg / ml and 1.30 mg / ml or between 0.30 mg / ml and 0.40 mg / ml. Furthermore, the product fluids 11 contained antifreeze additives such as trehalose, proline and / or proline derivatives, such as L-proline, and / or DMSO, as specified in Table 1.

[0294] Table 1: Product fluids 11 (product fluid formulations)

[0295]

[0296] The vitrification of the mitochondria, which were suspended in the product fluid 11 as described above, was carried out according to the cryopreservation method of the invention using the vibration-excited, oscillating application cannula 21, by means of which the generated product droplets 13 were either introduced into the vitrification fluid 60, which was present as a liquid, or applied to the outer surface of the vitrification roller 70 (surface or roller vitrification; see, for example, samples V2.1, V2.2, V2.3, V5.1, V5.2, V5.3 in Table 2). For comparison, the product droplets 13 were frozen by conventional snap freezing by direct contact of the sample containers with dry ice (samples V2.F, V2.C, V5.F, V5.C in Table 2).

[0297] The droplet diameters of the product droplets containing mitochondria 13 were 145 pm at a volume of 1.6 nl and 114 pm at a volume of 0.8 nl, respectively. For example, a droplet diameter of 145 pm (1.6 nl) was achieved.

[0298] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 by applying the product fluid 11 at a flow rate of 596 pl / min and a vibration frequency of 6250 Hz through an application cannula 21 with an inner diameter of the cannula outlet opening of 65 pm. The freezing rate of the product droplets 13 using the vitrification system according to the invention was determined by numerical simulation of the cooling process to be 2 million K / min for a droplet diameter of 114 pm and

[0299] determined to be 1.2 million K / min for a droplet diameter of 145 pm.

[0300] Table 2 shows, using the product fluid formulations of sample V2 and sample V5 from Table 1 as examples, the type of freezing process that was carried out for all five product fluid formulations shown in Table 1, namely samples V1 to V5. Each combination of freezing method and product fluid formulation was performed three times. For example, the verification of the product fluid formulation according to sample V2 or V5 from Table 1 was tested three times (V2.1, V2.2, V2.3 or V5.1, V5.2, V5.3 according to Table 2) for each vitrification method, i.e., using vitrification fluid 60 and using vitrification roller 70.

[0301] Table 2: Freezing methods using samples V2 and V5 as examples from Table 1

[0302]

[0303] The post-freezing treatment for all product fluid formulations (samples V1 to V5 according to Table 1) was carried out as explained below.

[0304] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The vitrified, mitochondria-containing microgranules or product granules 14 were stored at a temperature below -80 °C. Subsequently, samples of the vitrified microgranules were thawed, for example in a water bath heated to 37 °C, and then revitalized to obtain a revitalized biopharmaceutical product 10, wherein the sample contained mitochondria in a solution of 300 mM trehalose, 10 mM K-HEPES and 1 mM K-EGTA at a pH of 7.2.

[0305] The volume of the aqueous mitochondrial suspension obtained by thawing the vitrified microgranules was adjusted as needed by adding buffer to ensure a final concentration of 300 mM trehalose.

[0306] The samples obtained in this way were subsequently analyzed using ATP assays to determine the integrity and function of the mitochondria after vitrification, measuring both ATP content and ATP production. Mitochondrial integrity was demonstrated by measuring ATP content and ATP production, which are only maintained with intact mitochondrial membranes. The ATP content, relative to the amount of protein used in the thawed microgranule suspension, serves as an indicator of the ATP present in the mitochondria. Mitochondrial functionality was determined based on ATP production, which requires the mitochondria's ability to oxidize substrates, a functioning electron transport chain, and ATP synthesis.The ATP production per amount of protein in the thawed microgranule suspension serves as a measure of the functional activity of the mitochondria, since intact outer and inner mitochondrial membranes as well as functionally preserved proteins of the mitochondrial electron transport chain are required for ATP synthesis.

[0307] Both vitrification in the vitrification fluid 60 and vitrification using the vitrification roller 70 according to the cryopreservation process according to the invention yield vitrified product granules 14 containing mitochondria that retain their functional activity, in particular their ATP content and ATP production (see Fig. 10 and Fig. 11). In the [various] shown in Fig. 10 and Fig. 11

[0308] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 In the diagrams, the abbreviation "AU" in the specification "AU / 10 pl" stands for "Arbitrary Unit", that is, for a device-dependent relative measured value output by the measuring instrument, which is based on a sample volume of 10 pl.

[0309] Fig. 10 shows an example of an ATP assay for determining the ATP content and ATP production of mitochondria. The product fluid 11 used in the experiment shown in Fig. 10 consisted of mitochondria and 500 mM trehalose as an antifreeze additive (product fluid formulation according to sample V2 in Table 1).

[0310] The replicates 1, 2, and 3 shown in Fig. 10 represent three biological replicates of sample V2 according to Table 1 and Table 2 (i.e., replicate samples V2.1, V2.2, and V2.3, which were vitrified in vitrification fluid 60). The mitochondria were vitrified using the cryopreservation method according to the invention. For this purpose, product droplets 13 were generated using a cannula tip 23 of the application cannula 21 designed as a convergence nozzle, wherein the cannula outlet had an inner diameter of 65 pm and the product droplets 13 had a droplet diameter of 145 pm corresponding to a droplet volume of 1.6 nl. The product droplets 13 generated by means of the oscillating application cannula 21 were subsequently introduced into a liquid vitrification fluid 60, for example ethene, with verification being carried out with a freezing rate of approximately 1.2 million K / min determined by numerical simulation of the freezing process.

[0311] Formulation control sample V2.F refers to a product fluid 11, comprising mitochondria in 500 mM trehalose in an aqueous medium, frozen on dry ice. Freezing method control sample V2.C refers to a product fluid 11, also frozen on dry ice, comprising mitochondria in a conventional buffer of 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA at a pH of 7.2.

[0312] The samples were subsequently thawed in a water bath at 37 °C, resulting in a revitalized biopharmaceutical product 10 containing mitochondria in 300 mM trehalose, 10 mM HEPES and 1 mM EGTA at a

[0313] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 contained a pH value of 7.2. In the case of a deviating trehalose concentration in the vitrified samples, this was subsequently adjusted to a final concentration of 300 mM by adding an aqueous buffer containing 10 mM HEPES and 1 mM EGTA with a pH value of 7.2.

[0314] The ATP content in 0.9 pg of protein from each mitochondrial preparation was measured using the ATPIite assay (Revvity). ATP content and ATP production were determined in the presence of 1 mM ADP, 5 mM pyruvate, and 2 mM malate. Statistical analysis was performed using the Kruskal-Wallis test followed by a Dunn post-hoc test.

[0315] Figure 10 shows triplicate measurements for three vitrified biological replicates 1, 2, and 3 of the test samples according to sample V2, as well as for the formulation control sample and the freezing method control sample (see Table 2 for the three replicates V2.1, V2.2, and V2.3, as well as for the formulation control sample V2.F and the freezing method control sample V2.C). The points shown in the diagrams in Figure 10 represent measurement points of the triplicated measurements for the respective sample; the respective bar indicates the sample mean. Partial view a) of Figure 10 shows the ATP content, and partial view b) of Figure 10 shows the ATP production.

[0316] The ATP content did not differ significantly (Kruskal-Wallis test, p = 0.2140). ATP production was no lower than that of the control group treated with the alternative freezing method. This demonstrates that mitochondrial function was preserved during the vitrification process in the vitrification fluid 60.

[0317] Fig. 11 shows another example of an ATP assay for determining the ATP content and ATP production of mitochondria. The product fluid 11 used in the experiment shown in Fig. 11 comprised mitochondria in 300 mM trehalose (product fluid formulation according to sample V5 in Table 1).

[0318] The replicates 1, 2 and 3 shown in Fig. 11 represent three biological replicates of sample V5 according to Table 1 and Table 2 (i.e. replicate samples V5.1, V5.2 and V5.3, which were vitrified on a Vitrification Roll 70).

[0319] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Mitochondria were vitrified using the cryopreservation process according to the invention. For this purpose, product droplets 13 were generated with a cannula tip 23 of the application cannula 21 designed as a convergence nozzle, wherein the cannula outlet opening had an inner diameter of 65 pm and the product droplets 13 had a droplet diameter of 145 pm corresponding to a droplet volume of 1.6 nl. The product droplets 13 produced by means of the oscillating application cannula 21 were subsequently vitrified on the outer surface of a stainless steel vitrification roller 70 rotating at 450 U / rnin, the outer surface of which had a contact angle of 85°, the vitrification being carried out with a freezing rate of approximately 1.25 million K / min determined by numerical simulation of the freezing process.

[0320] Formulation control sample V5.F refers to a product fluid 11, comprising mitochondria in 300 mM trehalose, frozen on dry ice. Freezing method control sample V5.C refers to a product fluid 11, also frozen on dry ice, comprising mitochondria in a conventional buffer of 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA at a pH of 7.2. RT control sample refers to a sample comprising mitochondria in 300 mM trehalose in an aqueous medium, which was stored in a reservoir at 20 °C for a period corresponding to the duration of the vitrification carried out concurrently within the cryopreservation process according to the invention and frozen on dry ice at the time of completion of the concurrently carried out vitrification process. In the following descriptions, RT stands for room temperature.

[0321] Subsequently, the samples were thawed and revitalized with a solution of 300 mM trehalose, 10 mM HEPES and 1 mM EGTA, resulting in a revitalized biopharmaceutical product 10.

[0322] The ATP content in 0.9 pg of protein from each mitochondrial preparation was measured using the ATPIite assay (Revvity). ATP content and ATP production were determined in the presence of 1 mM ADP, 5 mM pyruvate, and 2 mM malate. Statistical analysis was performed using Kruskal-Wallis tests followed by a Dunn post-hoc test.

[0323] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Fig. 11 shows triple measurements for three vitrified biological replicates 1, 2, and 3 of the test samples according to sample V5 from Table 1 and Table 2, as well as for the formulation control, the RT control, and the freezing method control (see Table 2 for the three replicates V5.1, V5.2, and V5.3, as well as for the formulation control V5.F, the RT control V5.RT, and the freezing method control V5.C). The points shown in the diagrams in Fig. 11 represent measurement points of the triple measurements for the respective sample; the respective bar indicates the sample mean. Partial view a) of Fig. 11 shows the ATP content and partial view b) of Fig. 11 shows the ATP production.

[0324] The ATP content did not differ significantly (Kruskal-Wallis test, p = 0.2036). ATP production was no lower than that of the control group treated with the alternative freezing method. This indicates that mitochondrial function was preserved during the vitrification process.

[0325] The therapeutic efficacy of the biopharmaceutical preparation according to the invention, comprising functional mitochondria, which was produced from microgranules vitrified and subsequently lyophilized using the described cryopreservation process, is demonstrated by means of the following exemplary embodiment.

[0326] To investigate the therapeutic efficacy of the inventive biopharmaceutical preparation comprising functional mitochondria, an established mouse model for acute kidney injury (AKI) was used. For this purpose, renal ischemia was induced in the animals by bilateral clamping of the renal arteries for a period of 35 minutes, followed by reperfusion. Immediately after ischemia, reconstituted mitochondria were injected into the renal artery. Ten animals were used per experimental group. The experimental groups comprised three treatment groups, a comparator group (reference therapy), and a control group (vehicle).

[0327] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 The starting point for the production of the inventive biopharmaceutical preparations comprising functional mitochondria tested in the treatment groups was the production of a vitrified microgranulate according to the cryopreservation process according to the invention. The vitrified microgranulates were based on product fluids 11, which contained the following antifreeze additives:

[0328] - Formulation A: 300 mM trehalose and 600 mM L-proline, according to the product fluid formulation according to sample V1 in Table 1;

[0329] - Formulation B: 500 mM Trehalose, according to the product fluid formulation according to sample V2 in Table 1.

[0330] The vitrified microgranules were subsequently lyophilized and stored at -80 °C. The lyophilized microgranules of formulation AB were then reconstituted in 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA at pH 7.2, yielding the biopharmaceutical, functional mitochondria-containing preparations of formulation AB. These were then injected into the renal artery of mice.

[0331] For the comparator group (reference therapy), a preparation containing mitochondria in 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA at pH 7.2 was used, which was frozen in dry ice and thawed before injection. The control group (vehicle) received a corresponding injection of the reconstitution buffer without mitochondria.

[0332] Figure 12 shows the functional evaluation of kidney damage in the treatment groups receiving biopharmaceutical preparations containing functional mitochondria (formulations A and B), the comparator group (reference therapy), and the control group (vehicle). Figure 12a shows serum creatinine levels, and Figure 12b shows blood urea nitrogen (BUN) levels in peripheral blood at baseline and 1, 3, and 7 days after injection.

[0333] The results are shown as boxplots in Fig. 12. The boxes show the interquartile range of the observed values, with the center line representing the median.

[0334] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026. The whiskers correspond to 1.5 times the interquartile range. Individual dots indicate measurements outside this range.

[0335] Statistical analysis was performed using two-sided analysis of variance, taking into account the factors treatment group and time. A Tukey HSD post-hoc test was used to determine pairwise differences between the groups.

[0336] The treatment group (formulation A) showed a significant reduction in both serum creatinine and BUN compared to the control group. The adjusted p-values ​​in the Tukey HSD test for serum creatinine were...

[0337] p = 0.0000001 and for BUN p = 0.0017. The values ​​measured in the treatment group (formulation A) were at a comparable level to those of the comparison group, so no significant difference was found between these two groups.

[0338] The other tested treatment group (formulation B) is also shown. Figure 12 illustrates that, in particular, the biopharmaceutical, functional mitochondria-encompassing preparation according to formulation A leads to a significant reduction in the function-related parameters of kidney damage compared to the control group and achieves a protective effect comparable to the reference therapy.

[0339] Fig. 13 shows the histopathological evaluation of the kidneys of the animals examined in the treatment group (formulation A), the comparison group (reference therapy) and the control group (vehicle).

[0340] Following completion of the experiment, the kidneys were removed during autopsy, embedded in paraffin, and histological sections were prepared. The sections were stained with hematoxylin and eosin using routine procedures. The evaluation was performed in collaboration with a pathologist.

[0341] The extent of acute tubular necrosis (ATN) in the cortex – see partial view a) – and in the outer medulla (OM) – see

[0342] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Partial view b) - as well as the extent of inflammatory changes - see partial view c). The assessment was carried out using an ordinal scale from 0 to 4 (AKI score, inflammation), where 0 corresponded to no damage and 4 to the highest level of damage. The highest level of damage was defined as:

[0343] - in the cortex: very severe acute kidney injury with > 50% ATN,

[0344] - in the outer medulla: very severe acute kidney injury with > 90% ATN, - in case of inflammation: very severe inflammation with > 20 cells per field of view.

[0345] Ten fields of vision were evaluated for each animal and included in the statistical analysis.

[0346] To assess the differences between the groups, the linear association test according to Agresti was used. A p-value of < 0.05 was considered statistically significant.

[0347] Treatment with the biopharmaceutical, functional mitochondria-encompassing preparation according to formulation A led to a significant reduction in acute tubular necrosis in the cortex compared to the control group (p = 7.077 x 10⁻⁶). 15 ) as well as in the outer medulla (p = 4.902 xw 16 Furthermore, the inflammatory response was also significantly reduced (p = 2.797 xw). 9 ).

[0348] The results shown in Fig. 13 thus demonstrate that the biopharmaceutical, functional mitochondria-comprising preparation according to the invention, according to formulation A, not only causes a functional improvement in kidney parameters (see Fig. 12), but also leads to a significant reduction in structural tissue damage and inflammatory changes in kidney tissue.

[0349] In one aspect of the study, the stability of the vitrified and subsequently lyophilized microgranules after storage at elevated temperatures was investigated; the results are shown in Fig. 14. These lyophilized microgranules were stored for 23 days at either 20 °C or 4 °C and compared with samples stored at -80 °C. After the respective storage periods, the lyophilized microgranules were reconstituted in 300 mM trehalose, 10 mM HEPES, and 1 mM EGTA at pH 7.2.

[0350] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Subsequently, the protein concentration, particle concentration, and ATP production were determined. The protein concentration was measured by qubit fluorometry using the qubit protein assay reagent according to the manufacturer's instructions. To determine the particle count, 1 pl of the reconstituted sample was diluted in 10 ml of isotonic solution, and the measurement was then performed using a Multisizer 4e Coulter counter. To assess mitochondrial function, ATP production was determined as previously described, starting from 1 pg of each sample.

[0351] The results shown in Fig. 14 using formulation A as an example demonstrate that both the protein concentration and the particle concentration remained constant across all tested storage conditions and showed no significant differences compared to the reference sample stored at -80 °C.

[0352] Regarding mitochondrial function, a reduction in ATP production of approximately 37–44% was observed compared to the -80 °C reference sample. However, no significant difference was found between the samples stored at 20 °C or 4 °C. The results shown in Fig. 14 thus demonstrate that the structural integrity of the vitrified and lyophilized microgranules produced according to the invention is maintained even when stored above -80 °C, while the functional activity in the form of ATP production, although moderately reduced, shows no temperature dependence within the range of the tested storage temperatures.

[0353] Based on the stability assessment shown in Fig. 14, the microgranules or product grains 14, stored for 23 days at 20 °C and subsequently reconstituted, were investigated in the previously described mouse model for acute kidney failure under identical experimental conditions as in Fig. 12 and Fig. 13; the results are shown in Fig. 15 and Fig. 16.

[0354] Fig. 15 shows the functional evaluation of kidney damage after administration of the biopharmaceutical, functional mitochondria-containing preparation according to formulation A, which was stored as lyophilized microgranules at 20 °C for 23 days prior to administration and subsequently reconstituted (formulation A - RT). The investigation was carried out under identical experimental conditions as before.

[0355] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 described. The control group received a corresponding injection of the reconstitution buffer without mitochondria.

[0356] Renal damage was assessed based on serum creatinine and peripheral blood urea nitrogen (BUN) levels at baseline and 1, 3, and 7 days post-injection. The results are shown as boxplots in Figure 15, where the box represents the interquartile range of the observed values ​​and the midline represents the median. Whiskers represent 1.5 times the interquartile range, while single dots indicate values ​​outside this range.

[0357] Statistical analysis was performed using a two-factor analysis of variance (ANOVA) considering treatment group and time, followed by a Tukey HSD post-hoc test to determine pairwise differences. The biopharmaceutical, functional mitochondria-containing preparation of formulation A, stored as lyophilized microgranules at 20 °C for 23 days prior to administration and subsequently reconstituted, resulted in a significant reduction in kidney damage compared to the control group. The adjusted p-value for BUN was p = 0.0007338, and for serum creatinine, the adjusted p-value was p = 0.0056905. These results demonstrate that the functional protective effect is maintained despite storage at room temperature.

[0358] Figure 16 shows the histopathological evaluation of the kidneys of the animals treated in this experiment. The kidneys were removed during autopsy, embedded in paraffin, histological sections were prepared, and stained with hematoxylin and eosin using routine procedures. The evaluation was performed in collaboration with a pathologist.

[0359] The extent of acute tubular necrosis in the cortex was assessed – see partial view a), in the outer medulla (OM) – see

[0360] Partial view b) – as well as inflammatory changes – see partial view c) – based on an ordinal scale from 0 to 4 (AKI score, inflammation), where 0 corresponded to no damage and 4 to the highest level of damage. The highest level of damage was defined as > 50% ATN in the cortex, > 90% ATN in the

[0361] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 outer medulla or > 20 inflammatory cells per field of view. Ten fields of view were evaluated for each animal.

[0362] To statistically assess the differences between the groups, the linear association test according to Agresti was used, with p < 0.05 considered significant. In mice treated with the biopharmaceutical, functional mitochondria-containing preparation of formulation A, which was stored as lyophilized microgranules at 20 °C for 23 days prior to administration and subsequently reconstituted, acute tubular necrosis was reduced in both the cortex (p < 2.2 xw). 16 ) as well as in the outer medulla

[0363] (p < 2.2 x 10' 16 ) significantly reduced. Likewise, a significant reduction in inflammation was observed (p < 2.2 xw). 16 ) compared to the control group.

[0364] The results shown in Fig. 15 and Fig. 16 thus demonstrate that the mitochondria in the biopharmaceutical preparation according to the invention, comprising functional mitochondria, according to formulation A, retain their functional and structural protective effect in the mouse model for acute renal failure even after storage at room temperature.

[0365] KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Reference List

[0366] 10 biopharmaceutical product 11 product fluid

[0367] 12 Product fluid jet

[0368] 13 product drops

[0369] 14 product grains

[0370] 20 application syringes

[0371] 21 application cannula

[0372] 22 Base cannula

[0373] 23 cannula tips

[0374] 30 vibration excitation unit

[0375] 31 vibration exciters

[0376] 32 Swivel head with threaded hole 33 Mounting screw

[0377] 34 Vibration exciter bracket 35 Mounting carriage

[0378] 40 Magnetic coupling

[0379] 41 Base magnet

[0380] 42 Holding magnet

[0381] 43 Guide disc

[0382] 50 cooling unit

[0383] 51 Vitrification medium

[0384] 52 Collection container

[0385] 53 Cooling block

[0386] 60 Vitrification fluid

[0387] 61 Vitrification vessel

[0388] 62 Cooling block

[0389] 63 Temperature sensor recording

[0390] KAUFMANN ® Patent and Trademark Attorneys K 43 321 / 8.6 26.03.20264 Cooling block holder

[0391] 65 Cold gas supply line

[0392] 66 Cold gas discharge

[0393] 67 Shaking device

[0394] 70 Vitrification roller

[0395] 71 flow channels

[0396] 72 Cold gas supply line

[0397] 73 Exit wave

[0398] 74 Drive shaft

[0399] 75 Drive pulley

[0400] 76 Locking fit

[0401] 77 Radial bearing (axially movable) 78 Rotary feedthrough

[0402] 79 Stripping tool

[0403] KAUFMANN ® Patent and Trademark Attorneys K 43 321 / 8.6 26.03.2026

Claims

1. Patent claims 1. Vitrification plant for the production of a microgranulate from a biopharmaceutical product (10), comprising biological components with biopharmaceutical or therapeutic effects in the form of therapeutic cells, therapeutically active cell components, microorganisms and / or viruses, wherein the vitrification plant comprises: - a device for generating free-falling product droplets (13) from a product fluid (11) containing the biopharmaceutical product (10) and - an assembly for vitrification of the free-falling product droplets (13), the assembly for vitrification of the free-falling product droplets (13) comprising: - a cooling unit (50) positioned below the freely falling product droplets (13) exiting the device for generating the freely falling product droplets (13) for the formation of vitrified product grains (14) from the product droplets (13) by contact cooling with a cryogenic vitrification medium (51) integrated into the cooling unit (50), wherein the cooling unit (50) comprises the cryogenic vitrification medium (51), and - a collection vessel (52) for collecting the vitrified product granules (14) in the form of microgranules, characterized in that the device for generating the freely falling product droplets (13) comprises: - an application syringe (20) for conveying the product fluid (11), a syringe pump connected to the application syringe (20) for controlling the conveyance of the product fluid (11), and an application cannula (21) attached to the application syringe (20) for generating a continuous product fluid jet (12) exiting the application cannula (21) in a perpendicular direction from the product fluid (11) conveyed by means of the application syringe (20) and the syringe pump, wherein the application cannula (21) has at least one elongated base cannula (22), - a vibration excitation unit (30) with a vibration exciter (31) for exciting a high-frequency vibration, - a coupling for transmitting the high-frequency vibration from the vibration exciter (31) to the fluid exiting the application cannula (21) KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Product fluid jet (12) for the formation of vertically free falling product droplets (13) from the product fluid jet (12).

2. Vitrification system according to claim 1, characterized in that the application cannula (21) is designed as a compound cannula comprising the elongated base cannula (22) and a cannula tip (23) designed as a convergence nozzle.

3. Vitrification system according to claim 1 or 2, characterized in that the vibration exciter (31) is designed to excite a high-frequency mechanical vibration, wherein the vibration exciter (31) is attached to the application cannula (21) for vibration transmission by means of the coupling.

4. Vitrification system according to claim 3, characterized in that the vibration exciter (31) is a linear actuator for generating a linear alternating stroke movement, wherein the coupling between the vibration exciter (31) and the base cannula (22) is a magnetic coupling (40) that can be positioned variably along the base cannula (22), wherein the vibration exciter (31), designed as a linear actuator, is attached to the application cannula (21) by means of the coupling designed as a magnetic coupling (40) with respect to its linear alternating stroke movement transversely to the longitudinal extent of the application cannula (21), wherein the magnetic coupling (40) comprises a base magnet (41) that can be rigidly connected to the vibration exciter (31) and a holding magnet (42) that can be magnetically attached to the base magnet (41) in the region of a magnetic coupling zone.and wherein a cannula guide groove is formed in the magnetic coupling zone for force- and form-locking reception and retention of the base cannula (22) between the base magnet (41) and the magnetically adhering holding magnet (42).

5. Vitrification system according to claim 4, characterized in that the cannula guide groove is provided in the area of ​​the magnetic coupling zone of the magnetic coupling (40) on the base magnet (41), wherein the KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Cannula guide groove has a rectangular or semicircular cross-sectional shape.

6. Vitrification system according to claim 4, characterized in that the magnetic coupling (40) has a guide disc (43) included in the area of ​​the magnetic coupling zone between the base magnet (41) and the holding magnet (42), wherein the guide disc (43) is made of a magnetically transparent material.

7. Vitrification system according to one of claims 1 to 6, characterized in that the cooling unit (50) comprises a vitrification fluid (60) as the vitrification medium (51), which is contained in a vitrification vessel (61) open at one end, wherein the vitrification vessel (61) is arranged vertically below the application cannula (21) on the opening side.

8. Vitrification plant according to claim 7, characterized in that the cooling unit (50) further comprises a cold gas-cooled cooling block (62) in which the vitrification vessel (61) is received, and a shaking device (67), wherein the cooling block (62) is installed on the shaking device (67).

9. Vitrification plant according to claim 7 or 8, characterized in that the vitrification fluid (60) has a melting temperature below the glass transition temperature of the product fluid (11) at normal pressure and a boiling temperature that is at least 50 K above the melting temperature of the vitrification fluid (60), wherein the vitrification fluid (60): - has a lower density in the liquid state and at the same temperature than the product fluid (11), - is not or hardly miscible with the product fluid (11) and / or water in its liquid state and - is inert towards the biopharmaceutical product (10) contained in the product fluid (11). KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.202610. Vitrification plant according to one of claims 7 to 9, characterized in that the vitrification fluid (60) is a hydrocarbon compound from the group of alkanes and alkenes.

11. Vitrification plant according to one of claims 7 to 10, characterized in that the vitrification fluid (60) is ethane, ethene, propane, propene, 2-methylpropane or but-1-ene.

12. Vitrification plant according to one of claims 1 to 6, characterized in that the cooling unit (50) comprises as vitrification medium (51) a cold gas internally cooled, rotatable vitrification roll (70) made of a metallic material, the outer surface of which is arranged perpendicularly below the application cannula (21).

13. Vitrification plant according to claim 12, characterized in that the vitrification roller (70) is integrated into a hollow shaft arrangement designed for driving and for supplying and removing cold gas to the vitrification roller (70).

14. Cryopreservation process for producing a vitrified, free-flowing microgranulate from a biopharmaceutical product (10), characterized in that, after preparation and provision of the product fluid (11) containing the biopharmaceutical product (10), the production of the vitrified, free-flowing microgranulate is carried out by means of a vitrification plant according to one of claims 1 to 13 according to the following process steps: - Generating the continuous product fluid jet (12) exiting the application cannula (21) in a vertical direction by means of the application syringe (20) and the syringe pump, wherein the product fluid (11) is conveyed through the application cannula (21) at a predetermined flow rate by means of the application syringe (20) and the syringe pump, - Coupling the vibration exciter (31 ) to the product fluid (11) conveyed through the application cannula (21 ), - Excitation of the high-frequency vibration by means of the vibration exciter (31), wherein the high-frequency vibration is transmitted by means of the coupling to the product fluid jet (12) exiting the application cannula (21) and a KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026 Droplet chain of vertically free falling, monodisperse product droplets (13) is formed from the product fluid jet (12), - Contact cooling of the monodisperse product droplets (13) in the cooling unit (50) by individual contacting of the freely falling, monodisperse product droplets (13) with the cryogenic vitrification medium (51), whereby vitrified product grains (14) are formed from the monodisperse product droplets (13) by contact cooling, - Collecting and separating the vitrified product granules (14) as vitrified, free-flowing microgranules.

15. Cryopreservation method according to claim 14, characterized in that the production of the vitrified, free-flowing microgranules is carried out using a vitrification system according to one of claims 3 to 13, wherein, for coupling the vibration exciter (31) to the product fluid (11) conveyed through the application cannula (21), the vibration exciter (31) is connected to the application cannula (21) by attaching the coupling in a predetermined position along the base cannula (22), wherein the high-frequency mechanical vibration is excited by means of the vibration exciter (31) in the form of a sinusoidal vibration with a predetermined vibration frequency.

16. Cryopreservation method according to claim 14 or 15, characterized in that the contact cooling takes place in the cooling unit (50): - either by means of the cooling unit (50) according to one of claims 7 to 11 by introducing the monodisperse product droplets (13) into the vitrification fluid (60) acting as the vitrification medium (51), wherein the separation of the product grains (14) from the vitrification medium (51) is carried out by filtering or evaporating the vitrification fluid (60), - or by means of the cooling unit (50) according to claim 12 or 13 by applying the monodisperse product droplets (13) to the outer surface of the rotating vitrification roller (70) acting as the vitrification medium (51), wherein the separation of the product grains (14) from the vitrification medium (51) is carried out by stripping the product grains (14) from the vitrification roller (70) by means of a stripping tool (79). KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.202617. Cryopreservation method according to one of claims 14 to 16, characterized in that the product fluid (11) containing the biopharmaceutical product (10) further comprises at least one antifreeze additive selected from the group consisting of: (a) one or more non-penetrating antifreeze additives; (b) one or more penetrating antifreeze additives; and / or (c) a combination thereof, the non-penetrating antifreeze additives are selected from the group consisting of: (i) Monosaccharides; (ii) Disaccharides; (iii) Derivatives of mono- and disaccharides; (iv) sugar alcohols; (v) other polyols; (vi) Polyol derivatives; (vii) Amino acids; (viii) Amino acid derivatives; (ix) Organosulfur compounds; and / or (x) Combinations of two or more of the substances mentioned under (i) to (ix).

18. Cryopreservation method according to any one of claims 14 to 17, characterized in that, prior to the production and provision of the product fluid (11) containing the biopharmaceutical product (10), the biological components with biopharmaceutical or therapeutic effects contained in the biopharmaceutical product (10) are brought into contact with one or more non-penetrating cryoprotectant additives that do not impair the biological function of the biopharmaceutical product (10) and are cryoprotectively preconditioned by their accumulation within the biological components, wherein the non-penetrating cryoprotectant additives that do not impair the biological function of the biopharmaceutical product (10) are selected from the group consisting of: (i) Monosaccharides; (ii) Disaccharides; (iii) Derivatives of mono- and disaccharides; KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026(iv) Sugar alcohols; (v) other polyols; (vi) Polyol derivatives; (vii) Amino acids; (viii) Amino acid derivatives; (ix) Organosulfur compounds; and / or (x) Combinations of two or more of the substances mentioned under (i) to (ix).

19. Vitrified, free-flowing microgranules of a biopharmaceutical product (10) produced by a cryopreservation process according to any one of claims 14 to 18.

20. Vitrified, free-flowing microgranules according to claim 19, characterized in that the biopharmaceutical product (10) comprises functional mitochondria.

21. Method for revitalizing a biopharmaceutical product (10) in the form of a vitrified, free-flowing microgranulate, characterized in that the vitrified, free-flowing microgranulate was produced by a cryopreservation method according to one of claims 14 to 18, wherein the revitalization of the biopharmaceutical product (10) is carried out by directly introducing the vitrified, free-flowing microgranulate into a thawing medium, wherein the thawing medium is water, a buffer solution or a cell culture solution.

22. Method for producing a lyophilized microgranulate from a biopharmaceutical product (10), characterized in that a vitrified, free-flowing microgranulate produced by a cryopreservation process according to one of claims 14 to 18 is lyophilized using a freeze dryer.

23. Lyophilized microgranules from a biopharmaceutical product (10), characterized in that it is produced according to the method of claim 22. KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.202624. Lyophilized microgranules according to claim 23, characterized in that the biopharmaceutical product (10) comprises functional mitochondria.

25. Biopharmaceutical preparation comprising functional mitochondria for use as a medicinal product, obtainable from a vitrified, free-flowing microgranule according to claim 20 or from a lyophilized microgranule according to claim 24, wherein the biopharmaceutical preparation is intended for administration to a human or animal patient: a) in the case of the vitrified, free-flowing microgranules after revitalization of the biopharmaceutical product contained therein (10) - by directly introducing the vitrified, free-flowing microgranules into a defrosting medium, wherein the defrosting medium is water, a buffer solution or a cell culture solution, or - through energy input in the form of electromagnetic radiation, mechanical waves or alternating magnetic fields, and b) in the case of lyophilized microgranules - either in lyophilized form or - after reconstitution with a pharmaceutically acceptable carrier.

26. Biopharmaceutical preparation according to claim 25 for the treatment of acute renal failure. - 16 pages of drawings follow - KAUFMANN ® Patent and Trademark Attorneys K 43321 / 8.6 26.03.2026

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