Cryopreservation vial apparatus and methods

The The present invention relates to methods and apparatus for the cryopreservation of biopharmaceutical products, especially the cryopreservation of biopharmaceutical products using a vial and using methods for cryopreservation.

Description of related Technology:

The Cryopreservation and Cryogenic processing of biopharmaceuticals Products of this is in the manufacture, use and when selling Products important. However, to many of these products work can, cryopreservation or low temperature processing must be applied uniformly and controls take place, because otherwise lost quality and value of products go. If when processing cells for cryopreservation, the cells For example, to quickly frozen with a high water content, so can The cells intracellular Ice crystals develop. Thus, the cells may rupture and unusable. Another example is the freezing of Protein solutions, the for pharmaceutical use are assembled. Ideally, finds the freezing of these solutions throughout the frozen volume evenly instead. By uniformity the frozen volume results in the frozen volume, Tendency towards concentrations of dissolved Substances, which are similar to those of Eiskristallmustern, as well as to similar glass states the frozen matrix (uniformity of trapped Moisture level, of glass transition temperature, or the local glass-ice Volumenvexhältnisses, and of glass composition). These properties are desirable in order throughout the volume uniform product attributes to achieve and to reduce a product reduction. It is desirable, regardless of the Freezing capacity similar freezing conditions maintain. Reproducibility of freezing in large and small samples permits rehearse the expansion of the operation and testing of small product in freezing conditions, which later biopharmaceutical during freezing of large volumes may choose products.

The Cryopreservation and cryogenic processing of large volumes is particularly desirable in terms of biopharmaceutical products A machining large scale For example, in the manufacture of biopharmaceutical products to be useful. A processing in such large scale is described in US patent no. 5,964,100 and in Patent Nos. WO-A-98 34 078 and US-A-6,079,215 described. In the development of production processes for biopharmaceuticals Products, however, researchers may not have a lot of the biopharmaceutical product in hands. This makes the process development and optimization difficult. It is quite possible that at this time not enough is present from the product to a vessel with a volume of ten to fill or hundreds of liters. Therefore "Reduction" techniques are required to freezing and thawing (ie cryopreservation) in a large scale, for example, on a production scale, with a very small volume, for example, on a laboratory scale, to simulate.

A solution is that cryopreservation or cryogenic processing on a large scale to simulate using small volume containers. The inventor However, when freezing small volume, the biopharmaceutical containing products, discovered a problem. When external cooling, a supercooled small volume of a medium comprising a biopharmaceutical product contains, first in liquid Form (reaches thermodynamic disequilibrium), and solidifies then quickly. The temperature drops first up to a supercooled State in a liquid from (the supercooling place in the whole volume of the liquid instead). Then solidifies the small volume after nucleation is fast and takes the heat of solidification. heated The small volume therefore fast to the solidification temperature (at this time carried a small plateau, followed by a temperature drop (the solidified small volume is cooled by external cooling)).

at the rapid solidification of the supercooled small volume could freeze all small volume quickly while ice crystals quickly "Shoot" in the (and by) solidifying volume. Typically, such shoot Crystals of the coldest Points on the inner surface the small volume. Such rapid crystal growth can for the biopharmaceutical product be harmful. This is particularly the case if the rapid growth produces very fine crystals, resulting in a large Boundary layer of ice and biopharmaceutical product, etc. results. also is the supercooling effect in small volume pronounced, than in larger volumes. Such small volume can therefore cryopreservation and cryogenic processing of larger volumes of biopharmaceuticals not exactly replicate.

it Therefore, there is a need for methods and apparatus for cryopreservation and cryogenic processing of biopharmaceutical products, with which the aforementioned shortcomings be resolved.

The US Pat. No. 5,059,399 discloses a vial for receiving a biological sample <?page 3?>while a cryogenic freezing process. The vial includes a container, the wall of a material having a predetermined coefficient of thermal conductivity K₁ exists, and an internal cavity defined for receiving the sample, and a pin by itself the outside of the container in the roundabouts of the internal cavity extends. The pin has a coefficient of thermal conductivity K₂ , which is substantially greater, as K₁, And defines a path of increased thermal conductivity from the inside to the outside of the container a sample contained therein cool.

The US Pat. No. 4,799,358 describes an apparatus for cooling and Freezing of samples of biological substances. The cooling area which the closed plastic vessels with the Sample contains, is between two parallel, plate-like layered cooling elements locked in.

The European Patent application no. 0642828 A1 discloses a disposable arrangement of reaction containers of the same Shape and the same amplitude for performing temperature cycling of a liquid mixture, located in the reaction vessels, each reaction vessel a first, conically shaped wall portion and a second, cylindrical having shaped wall portion to the opening of the reaction container a End forms, with the wall thickness is the first wall region is smaller than the wall thickness of the second Wall portion, and wherein the opening the reaction vessel can accommodate a closure that gas-tight to the reaction vessel to lock , when the closure is placed on the opening of the reaction vessel becomes.

SUMMARY OF THE INVENTION:

According to a Aspect, the invention relates to a cryopreservation biopharmaceutical Products for cryopreserving biopharmaceutical products, with a Kryokonservierungsbehälter, a cryopreservation, which is located in the Kryokonservierungsbehälter, and a vial for the cryopreservation of biopharmaceutical products, which in itself the Kryokonservierungsbehälter is surrounded by the cryopreservation, wherein the vial for cryopreserving biopharmaceutical products comprising a body the horizontally between the top and the bottom of the vial an elongated having cross-section of the proximal and distal ends Body defined and at least one nucleating structure, the one with least connected the distal end of the body , said at least one nucleating structure, the cryopreservation contacted, and wherein the body includes a low-temperature-stable material with biopharmaceutical products is compatible.

After In another aspect, the invention relates to a method for cryopreserving biopharmaceutical products comprising: Providing a Kryokonservierungsbehälters, placing a vial for the cryopreservation of biopharmaceutical products in the Kryokonservierungsbehälter wherein the vial for cryopreserving biopharmaceutical products comprising a body the horizontally between the top and the bottom of the vial an elongate cross-section having defining the proximal and distal ends of the body, and at least one nucleating structure, the one with least distal end of the body is connected, and wherein the body includes a low-temperature-stable material with biopharmaceutical is compatible products, introducing a cryopreservation in a space outside the Kryokonservierungsfläschchens, However, within the Kryokonservierungsbehälters, and extracting heat from the Kryokonservierungsbehälter, whereby the cryopreservation is frozen.

After a further aspect, the invention relates to a vial for cryopreserving biopharmaceutical products comprising a body, the horizontally between the top and the bottom of the vial an elongate cross-section having defining the proximal and distal ends of the body, at least one nucleating structure, the one with least distal end of the body is connected, and wherein the body includes a low-temperature-stable material with biopharmaceutical is compatible products.

BRIEF DESCRIPTION OF THE DRAWINGS:

<figref idrefs="S35">1</figref> shows a cross section of Kryokonservierungssystems invention.

<figref idrefs="S36">2A</figref>-<figref idrefs="S36">D</figref> show Cross-sections of inventive cryopreservation.

<figref idrefs="S37">3A</figref>-<figref idrefs="S37">C</figref> show Cross-sections and an elevation of inventive cryopreservation.

<figref idrefs="S38">4A</figref>-<figref idrefs="S39">V</figref> show Cross-sections of inventive nucleating Structures.

<figref idrefs="S40">5A</figref>-<figref idrefs="S40">H</figref> show Cross-sections of inventive Fläschchenfokussierspitzen.

<figref idrefs="S41">6A</figref>-<figref idrefs="S41">e</figref> show Cross-sections of inventive Fläschchenablenkspitzen.

<figref idrefs="S42">7A</figref>-<figref>D</figref> show an elevation and cross-sections inventive cryopreservation&lt;?page 4?&gt;vials.

<figref idrefs="S43">8A</figref>-<figref idrefs="S43">B</figref> show Views of cryopreservation-Nests invention.

<figref idrefs="S44">9</figref> shows a cross section of Kryokonservierungssystems invention.

<figref idrefs="S44">10</figref> shows a cross section of Kryokonservierungssystems invention.

DETAILED DESCRIPTION OF THE INVENTION

Of the Inventor has unexpectedly discovered that the problems in the aforementioned Art by using a vial in the cryopreservation of biopharmaceutical dissolved products can be, comprising a body comprises of a low-temperature-stable mate rial which the is compatible with biopharmaceutical products, wherein the body horizontal between the top and the bottom of the vial an elongated having cross section and ice crystal-nucleating structures at opposite ends of the elongate Section are.

Typically , as in <figref idrefs="S35">1</figref> As shown, the inventive bottle <figref>102</figref> to Cryopreservation of biopharmaceutical products within the system <figref>100</figref> to Cryopreservation of biopharmaceutical products placed. Operational approaches a Geftier Front <figref>106</figref> in the Kryokonservierungsbehälter <figref>112</figref>. by freezing the cryopreservation in the Kryokonservierungsbehälter <figref>112</figref> defined is a nucleating end structure <figref>114</figref> the Kryokonservierungsfläschchens <figref>102</figref>, During the Progression of freezing front <figref>106</figref> If the wall temperature of the nucleating structure <figref>114</figref> below 0 ° C, for example, under the reimbursement temperature of aqueous Solutions. This is because in the solid front, a temperature gradient is (Falling temperature of the liquid-solid interface against the cooled wall <figref>104</figref> the Kryokonservierungsbehälters). Consequently, the nucleating structure <figref>114</figref> in the solid front <figref>106</figref> flat embedded.

Of the elongated Cross-section of the body the Kryokonservierungsfläschchens reduces or prevents undercooling of media in the cryopreservation, when the longitudinal axis the elongate Cross-section is oriented at an angle to the solid front. A Such guidance shall ensure the tendency of Heat transfer surface area between the vial and liquid cryopreservation (This liquid Phase starts at near 0 ° C) while the initial is freezing process larger, as the heat transfer surface area between the vial and the frozen cryopreservation. Thus remains the medium containing the biopharmaceutical product in near 0 ° C (for example, the temperature of the outer liquid phase) and falls only near the nucleating structure from (this wall is through the surrounding outer solid Front-cooled). This local temperature drop area occupies a region <figref>108</figref> of internal product volume of Kryokonservierungsfläschchens on.

it While is not desired is to be tied to a specific action mechanism, because such knowledge is not necessary for practicing the invention he seems, but it is obvious that the formation of ice crystals in the cryopreservation is increased by the presence of a nucleating structure the formation of relatively immobile clumps of media molecules, preferably Water molecules, allows a macroscopic scale. This can be achieved by using a nucleating structure are to the boundary layer of liquid biopharmaceutical Product near the interior walls the Kryokonservierungsfläschchens to immobilize, thereby reducing or convection be excluded. additionally causes the nucleating structure lowering the local temperature the relatively immobile clumps of media molecules.

<figref idrefs="S35">1</figref> shows furthermore, as the inventive nucleating Structure may accomplish these goals.

On Temperature gradient forms in the area <figref>108</figref> (In the liquid phase) leading to local undercooling in or near the nucleating structure <figref>114</figref> leads. The Volume of the mass of the biopharmaceutical product that obtained by supercooling is affected in the entire volume of Kryokonservierungsfläschchens, is over containers substantially reduced without nucleating structure. Preferably the so influenced amount of mass of the product is essentially the left in and around the nucleating structure around pharmaceutical Product restricted. Indeed show readings of temperature magnitude of the mass within the Kryokonservierungsfläschchens no supercooling effects. Consequently, the liquid temperature remains in the cryopreservation at near 0 ° C, similar to the Temperature of cryopreservation contained in Kryokonservierungsbehälter.

Of the promotes temperature gradient the formation of ice crystals on the cold wall and their growth in the form of dendrites. The dendritic front forms inside the Kryokonservierungsfläschchens, and its position is consistent with the position &lt;?page 5?&gt;the solidification front outside the Kryokonservierungsfläschchens match. Both fronts then move together along the Kryokonservierungsfläschchens, until the end of Kryokonservierungsfläschchens achieved (the all in the vial located product solidifies). The cryopreservation can between two approaching placed solid fronts and then freezing in the cryopreservation of place more than one nucleating structure and instead takes place in about at a central point of Kryokonservierungsfläschchens. The solidification fronts can at central points outside and meet within the cassette. In this way the inventive cryopreservation system, the Freezing in a large Liquid volume under Using very little product replicate.

Lots different cross-sectional geometries are for the cryopreservation invention suitable. The<figref idrefs="S36">2A</figref>-<figref idrefs="S36">D</figref> show various embodiments inventive cryopreservation, having: a Kryokonservierungsbehälter Wall <figref>202</figref>a Frozen Front <figref>204</figref>And cryopreservation <figref>208</figref>. <figref>210</figref>. <figref>212</figref> and <figref>214</figref>. the nucleating structures <figref>218</figref>. <figref>220</figref>. <figref>222</figref>. <figref>224</figref>. <figref>226</figref> and <figref>228</figref> contain. In these embodiments each of the cryopreservation vial <figref>208</figref>. <figref>210</figref>. <figref>212</figref> and <figref>214</figref> in the freezing front <figref>204</figref> embedded that the cooling effects by the Kryokonservierungsbehälter Wall <figref>202</figref> is produced. The nucleating structures <figref>218</figref>. <figref>220</figref>. <figref>222</figref>. <figref>224</figref>. <figref>226</figref> and <figref>228</figref> serve to the inventive local supercooling initiate.

cryopreservation with cross-sectional geometries, the more than one nucleating structure exhibit, can in the realization of this embodiment, useful be. Multiple nucleating structures or more points where vials walls locally adjacent nearby are able be used to control the freezing process in the cryopreservation vial to accelerate. In a preferred embodiment, the case of a cryopreservation invention to disposal Related number of nucleating structures from one to about one hundred, respectively, more preferably from about two to about 10. For example, the in the <figref idrefs="S36">2A</figref>-<figref idrefs="S36">D</figref> shown Geometries are used. An illustration of this is in the<figref idrefs="S37">3A</figref> shown. The cryopreservation <figref>300</figref> has a cross-section corresponding to the cross section of the Kryokonservierungsfläschchens <figref>210</figref> is similar, wherein the nucleating structures <figref>302</figref> and <figref>304</figref> in the freezing fronts <figref>306</figref> and <figref>308</figref> are embedded, viewed in the by arrows <figref>310</figref> and <figref>312</figref> given Directions to move forward. Like in the <figref idrefs="S37">3B</figref> is shown, the length of the particular embodiment, the Kryokonservierungsfläschchens <figref>300</figref> in the generally greater than the width. Further, the interior angle alpha, the angle the nucleating structure <figref>302</figref> is formed, preferably less than about 90 °. The<figref idrefs="S37">3C</figref> shows an isometric elevation of Kryokonservierungsfläschchens <figref>300</figref> With the in the <figref idrefs="S37">3A</figref>-<figref idrefs="S37">B</figref> shown Cross-section.

Of the Body, from the cryopreservation vial according to the invention there should horizontally between the top and the bottom of the vial an elongated have cross-section, eg, the length of the body axes substantially different be such that a proximal end and a distal end defining becomes. The distal end is from a center of Kryokonservierungsfläschchens distally (eg the ends of an oval or the points of a star). The elongate Shape of the body generated over the internal volume of Kryokonservierungsfläschchens a large wall surface area. This surface area is for heat transfer used the internal liquid temperature stable and close to the outside liquid temperature holds (no supercooling the inner liquid). The aspect ratio of the axes is preferably in the range of about 1.3: 1 to about 450: 1, more more preferably between about 8: 1 and about 26: 1st Distances between the inner walls along the longer Axis move preferably between about 0.1 mm and about 500 mm, more preferably between about 5 mm and about 35 mm. The aspect ratio of nucleating structure to the longitudinal axis the Kryokonservierungsfläschchens located preferably in a range of about 1: 1 (for example, a triangular Shape of the vial) for about 1: 2 (for example, a diamond shape) to about 1: 500,000 (for example, light waves on the end wall of the vial). A more preferred Range of this ratio is between about 1: 2 and about 1: 800th

The inventive nucleating structures can have a variety of forms. In general, the inventive nucleating Structures coupled to at least a distal end of the body. For example can they designed nucleating structures in a convex extension from the body and preferably arranged an incoming solid front opposite be. The extensions can have a variety of forms. For example, the nucleating structures in the form of single or multiple narrow grooves, preferably, be present with convex corners. These corners can have various angles, preferably from about 0.01 ° to about 90 °, yet more preferably from about 5 ° to about 40 °. In a preferred embodiment should a basic structure of the nucleating structure a Point of local proximity create (up to and including a contact point with the &lt;?page 6?&gt;two surfaces meet, at Contact point an essentially describe acute angle) where still some medium can be supercooled locally without supercooling of Mass of the volume of the medium in the cryopreservation trigger.

at certain embodiments can they nucleating structures may be configured such that the inner walls of nucleating structures are positioned in the local vicinity of the tip cavity. In a preferred embodiment, the nucleating structure one or more points on the local area, more preferably two or more points of local proximity. Such areas of local proximity may have a Source of freezing nuclei with relatively very small local supercooling in the surrounding liquid are while the rest of the media is not supercooled is, for example, the supercooling only in the nucleating structure of Kryokonservierungsfläschchens take place. More specifically, the sub-cooling can locally near the point concentrated near will. The points of local proximity can be provided in that the inner sides of the vial wall in extensions (nipples) located opposite to each other, be molded, preferably so that the tips of their inner surfaces between about 0.001 mm and about 5.0 mm, more preferably between about 0.04 mm and about 0.5 mm, apart. In a preferred embodiment can the walls so close together that if during the embedding of the nucleating Structural bending of the walls of the Kryokonservierungsfläschchens into the solid front, the walls, resulting in local proximity are to each other, in actual Contact can be moved together.

The heat conduction through the walls caused that these nipples are cold spots and the proximity of their Tips causes the formation of locally supercooled microscopic Range between the peaks. This area is a nucleating place for the first ice crystals. In the formation of these first ice crystals fills the nucleating structure with ice crystals, which continue to grow and the temperature gradient formed between a distal part of the body and the rest of the vial volume is formed, followed. The ice crystals form a dendritic Crystal Front, which together with the dendritic crystal front outside the Kryokonservierungsfläschchens moved (the vial is externally solidifying substances embedded).

exemplary geometries for nucleating structures having points of local proximity, are in the <figref idrefs="S38">4A</figref>-<figref idrefs="S39">V</figref> shown. The <figref idrefs="S38">4A</figref>-<figref idrefs="S39">K</figref>. <figref idrefs="S39">M</figref> and <figref idrefs="S39">P</figref> show inventive nucleating Structures that are only connected to the distal ends of a body from which a cryopreservation consists. In the<figref idrefs="S38">4A</figref>-<figref idrefs="S38">K</figref> have the nucleating structures at least one point of local proximity. at a preferred embodiment, for example, when in the <figref idrefs="S38">4F</figref> embodiment shown, comprises the nucleating structure comprises two or more points of local Close up. In various embodiments, lace details can the waves and nipple one or more contact points form, for example, two nipples which of itself near the top Cassette end is to form the first nucleation zone, as in the <figref idrefs="S38">4L</figref>-<figref idrefs="S38">N</figref> shown is. Various embodiments multiple nucleating structures coupled to individual body are, are in the <figref idrefs="S38">4N</figref>-<figref idrefs="S39">O</figref> and <figref idrefs="S39">4Q</figref>-<figref idrefs="S39">V</figref> shown.

The Construction of the nucleating structures to the body can diverse be kind. For example, the outer surface of the nucleating structure by waves, angular projections, etc. be extended to the heat transfer between the nucleating structure and the external solidifying cryopreservation to increase.

The cryopreservation invention is designed such that it for testing proper conditions biopharmaceutical products provides. It can be sterilized be (conventional using steam or other Techniques) and, during remain sealed testing (ase ptical conditions may maintain be), the biological product reduction, for example by to prevent proteases or pollution.

The low temperature-stable material with biopharmaceutical products compatible and has compatibility with the cryopreservation in liquid and frozen state. Important properties, the compatibility of the low-temperature-stable influence material with biopharmaceutical products, for example, a lack of leaching biotoxischer compositions, minimal biodegradation, minimal surface interactions with the product at the molecular (example: formation of hydrogen bonds, molecular adsorption), microscopic (example: Adsorptionsbindungserleichterung by surface roughness) and macroscopic (example: jumps, collection of biological Substances) levels, and chemical resistance (to cleaning agents, Solution buffers, etc.). The absence or substantial absence of certain ingredients (Such as copper or heavy metals) is preferable to biological Moleküldenaturierung prevent or chelation.

Various polymers can as tieftem&lt;?page 7?&gt;temperature-stable material can be used, for example, polytetrafluoroethylene, Polystyrene, polyethylene or polypropylene. In a preferred embodiment, surface treatments on the surface the Kryokonservierungsfläschchens biopharmaceutical Products are applied, for example, the adsorption of biological molecules or reduce cell (eg, an RF plasma treatment at the surface the vial be applied). Hydrophilic coatings may be at the surface of vial the adsorption and denaturation of the biopharmaceutical product significantly reduced.

The inventive cryopreservation vial has a wall thickness (Other than the nucleating structure, which have a different wall thickness can), which is determined by the structural strength and vial integrity, lower limit has (eg a polymer layer, the functional one adequate structural strength and vial integrity has). Preferably, this thickness should at least about 0.001 mm, more preferably at least about 0.05 mm, be. The maximum strength the bottle wall is not strictly limited; Preferably, the maximum thickness of bottle wall about 150 mm, more preferably about 30 mm, respectively. The ratio of Strength the bottle wall the strength of bottle inside may be between about 20: 1 to about 1: 500,000, preferably between about 5: 1 and about 1: 50,000 are. The thickness of the walls of the nucleating structures may be lower, equal to or greater than the thickness the aforementioned remaining vial walls. If the thermal conductivity the vial material is lower, than those of the frozen media and / or cryopreservation, so are thinner walls of nucleating structures are preferred: Also, if the thermal conductivity the wall material RESIZE SSSR is, than those of the frozen media and / or cryopreservation, thicker walls be used.

In addition to biocompatibility, mechanical strength and has chemical resistance the vial material preferably specific properties required for its application with freezing and thawing are connected. thermal conductivity and / or specific heat the Kryokonservierungsfläschchens are preferably selected, that they essentially those of the frozen medium, the biopharmaceutical, the containing product, and / or the cryopreservation are similar. Frequently contains the cryopreservation mainly Water, so that the frozen cryopreservation has properties similar to those of ice (the thermal conductivity of ice is about 2.25 [W / m K] at 0 ° C and 3.94 [W / m K] at -95 ° C; the specific heat of Ice is 2,261 or 1,172 [kJ / kg K]).

The preferred specific heat for examples suitably low temperature-stable materials (all in [kJ / kg K]): Polyethylene (At 200 K): 1,11; Polypropylene (at 200 K): 1.132; Polystyrene (at 300K): 1.223; polytetrafluoroethylene (At 200 K): 0.6893; Nylon-66 (at 230 K): 1.139. Preferred thermal conductivities of suitable low temperature-stable materials are (respectively in [W / m K]): Nylon-12: 0.25; Nylon-6 (stampings): 0.24; Nylon 6.12: 0.22; polycarbonate: 0.20; Polyester (cast): 0.17; PEEK 0.25; PET: 0.15; PVC (rigid): 0.21; Teflon: 0.25. Stainless steel as a material for the vial wall has a thermal conductivity of about 16 [W / m K]. Composites, glass, ceramics and metals and their alloys be used as a low-temperature-stable material.

at one embodiment of the invention is the thermal conductivity the vial walls preferably not significantly higher, than that of the biopharmaceutical product and the cryopreservation. Is the thermal conductivity the vial walls is higher, can the side walls as thermally conductive shaped projections act that causes that the freezing of the sides walls far before the freezing fronts in the media and in the cryopreservation takes place. Such "projection effect" that fosters the inner solid front in the vial is pulled forward. This can stainless steel and metals in certain applications side walls the vial less desirable do.

The Tips of Kryokonservierungsfläschchens, the nucleating structure and a portion of the wall of the Kryokonservierungsfläschchens included near the nucleating structure may be made of the same material are prepared as the side walls, or may be made of a material of higher thermal conductivity (Eg similar of the frozen product and fluid or higher are produced). If the vial tips a frozen medium and cryopreservation similar thermal conductivity exhibit, the heat flux can through the vial tips carried out evenly. promotes Such an arrangement freezing the medium comprising the biopharmaceutical product with minimal wall effect, ie, the medium does not freeze substantially same speed as the cryopreservation The vial tips can for example, metals (such as stainless steel or titanium) or from Composites or filled Polymers (such as filled with stainless steel powder epoxide filled or coated with aluminum powder be prepared epoxy, or graphite fiber filled PET).

The Use of materials with low &lt;?page 8?&gt;Thermal conductivity in the vial tips , as compared with the frozen cryopreservation, increased thermal resistance the bottle wall rise and so the growth of the solid front in the cryopreservation affect. The configuration of the bottle top not only simplifies the Eiskistall nucleation, but may also heat flow from the outer freezing fluid on the end wall of the vial focusing to compensate for the lower thermal conductivity produce the vial wall. Furthermore, the tips are designed so that they, if desired, the heat flow detract from the cooling surfaces. In this way, the vial tips be configured so that the differences in the thermal conductivity between the cryopreservation, the wall of Kryokonservierungsfläschchens, the Tips of Kryokonservierungsfläschchens, the nucleating structures and the media, the biopharmaceutical, the containing product, be compensated partially or substantially.

Examples for Fläschchenfokussierspitzen that serve the flow of heat (Preferably on the nucleating structures) to focus, in the <figref idrefs="S40">5A</figref>-<figref idrefs="S40">I</figref> shown. The <figref idrefs="S40">5A</figref>-<figref idrefs="S40">B</figref> show Fläschchenfokussierspitzen, having external heat transfer protrusions, a radius (<figref idrefs="S40">5A</figref>) Or an angle (<figref idrefs="S40">5B</figref>) describe larger, as one of the inner walls the Kryokonservierungsfläschchens educated interior angles. The difference between the external radius or angle and the internal angle serves to direct the heat flux to focus. The<figref idrefs="S40">5C</figref> shows a Flächchenfokussierspitze, wherein a portion of the tip wall has a thermal conductivity greater than or is equal to that of the surrounding cryopreservation. This configuration results to focus the heat flux. The in <figref idrefs="S40">5D</figref> embodiment shown has a similar Structure as that in the <figref idrefs="S40">5C</figref> shown embodiment, except that in the vial tip small gap or intermediate space is, the gap with the Interior of Kryokonservierungsfläschchens communicates. The heat flux is focused when tip wall thicknesses <figref>502</figref> and <figref>504</figref> are larger, than the peak intensity <figref>506</figref>, The<figref idrefs="S40">5E</figref> shows another configuration, in which the external heat transfer protrusions for Focusing the heat flux be used. The<figref idrefs="S40">5F</figref> shows a configuration used in the internal heat transfer protrusions are to the flow of heat to focus. The<figref idrefs="S40">5G</figref>-<figref idrefs="S40">H</figref> show, how external heat transfer projections to can serve, heat flow to focus. The incoming solid front<figref>508</figref> is moving at a predetermined speed. move between the focusing projections located the fronts <figref>510</figref> and <figref>512</figref> However, faster than the solid front <figref>508</figref>So that the heat flux is focused. The<figref idrefs="S40">5I</figref> shows, how external and internal focusing projections into a single Fläschchenfokussierspitze can be combined.

External projections can be used to provide additional to the possible, explained above Focusing role, the heat flux distracting. examples for Vial deflecting tips, which serve to heat flow distract, are in the <figref idrefs="S41">6A</figref>-<figref idrefs="S41">e</figref> shown. In the <figref idrefs="S41">6A</figref>-<figref idrefs="S41">e</figref> describe the vial deflecting tips external Wärmeüberttagungsvorsprünge that from the incoming solid front <figref>602</figref> away and from the cryopreservation outward show. This configuration serves to heat flow, the firm with the front <figref>602</figref> is associated, from the rest of Kryokonservierungsfläschchens, with which the vial deflecting tip is connected to distract.

how in the <figref idrefs="S42">7A</figref> is shown, in a preferred embodiment the cryopreservation <figref>700</figref> a edge <figref>702</figref> exhibit at the bottom edge thereof to an air space form when it first down in the cryopreservation is immersed. This feature has a tendency to heat transfer reducing from the bottom upwards. The airspace is also taking out the Kryokonservierungsfläschchens facilitate upward, because the ground in the course of the freezing process, adheres less to the underlying solidified mass. The<figref idrefs="S42">7B</figref>-<figref idrefs="S42">C</figref> show a further preferred embodiment, wherein the removal of the Kryokonservierungsfläschchens <figref>704</figref> out the frozen cryopreservation by a tapered shape the vial - for example, a cross-sectional widening in the direction of taking out desired - relieved can be. This taper is from the <figref idrefs="S42">7B</figref> , the side elevation of a the Kryokonservierungsfläschchens <figref>704</figref> represents, wherein said lower cross-section <figref>706</figref> is smaller than the upper cross-section <figref>708</figref>, The<figref idrefs="S42">7C</figref> shows a cross section of Kryokonservierungsfläschchens <figref>704</figref> with a tapered shape, as well as the position of the medium <figref>710</figref> and cryopreservation <figref>712</figref>, The cryopreservation of <figref idrefs="S42">7D</figref> shows a combination of the embodiment with edge and the embodiment with tapered Form in the cryopreservation <figref>716</figref>, The cryopreservation <figref>716</figref> containing a medium <figref>718</figref> and located in the cryopreservation <figref>720</figref>, Of the edge <figref>702</figref> describes an airspace <figref>714</figref> with the above specified function.

at some embodiments of the present invention, the cryopreservation vial from the frozen outer material taken out and after sampling a &lt;?page 9?&gt;Part of the frozen Medium containing the biopharmaceutical product, even be used again. In such embodiments can the cryopreservation using the aforementioned tapered shapes and / or surface treatment the outer surfaces of the Kryokonservierungsfläschchens, related to the frozen cryopreservation medium in contact, are formed (for example by applying a Teflon coating, Applying RF plasma treatment, etc.). The vial can also be used in a nest, as in the <figref idrefs="S43">8A</figref>-<figref idrefs="S43">B</figref> shown is. The<figref idrefs="S43">8A</figref> shows the nest <figref>802</figref>. made of a similar or other material may be prepared as the cryopreservation vial. The <figref idrefs="S43">8B</figref> shows the nest <figref>802</figref> operational. The nest<figref>802</figref> shall in the cryopreservation <figref>804</figref> used. The cryopreservation <figref>806</figref>. which the medium <figref>808</figref> contains, is in the nest <figref>802</figref> used. The nest<figref>802</figref> can in the frozen cryopreservation <figref>804</figref> embedded stay while the vial <figref>806</figref> removed and later back in the nest <figref>802</figref> can be used. The inner Shape of the nest <figref>802</figref> preferably corresponds substantially with overall dimensions the vial <figref>806</figref> agree what a tight insertion of Kryokonservierungsfläschchens <figref>806</figref> in the nest <figref>802</figref> allowed and unwanted air isolation rooms, etc. reduced.

at a preferred embodiment, can the tips (ie, the end walls) of the cavity to be designed such that the heat flux similar to that described as in the above heat flux focusing Fläschchenendstrukturen is focused. Each intermediate space between the inner wall of the cavity and outer wall the Kryokonservierungsfläschchens can with a thermally conductive composite (For example, a container filled with metal powder silicone grease) are filled. In a preferred embodiment can the dimensions of the vial and its nest by using very close dimensional tolerances adapted to each other be that are so narrow that the heat-conductive composite material not needed is to provide a substantially uniform heat flux through the vial and the nest walls to obtain.

instead a nest with cantilevered walls there are other versions of inserts in the frozen outer material remain embedded while the vial can be removed and replaced. For example, a sealed bag or a Teflon, polyester Polyamide layer is made and in which the vials sealed surface embedded (there are no air bubbles or liquid droplets between the bottle wall and the wall of this pocket layer), in the outer freezing fluid used. Since the pocket assumes the shape of the bottle, is produced in the frozen outer material of the bottom part of the vial, the coated with the polymer layer is (the pocket walls), a Cavity. The vial can thus be taken out of this cavity and, if necessary, again be used. The heat flux and the temperature deformation to the vial around only by this Polymer layer are generated, can, considering of the essential absence of air bubbles or frozen liquid drops, be insignificant. The layer used may also consist of a material be made, the low at the applied temperatures in the process (Up to -80 to -90 ° C) flexible remains, for example a silicone elastomer. An elastomer for single use can be used when there is no re-insertion of the vial in the frozen outer material takes place.

The similarity of the dendritic crystal growth outside and inside the vial depends some extent on the temperature gradients in the cryopreservation and in the Kryokonservierungsbehälter in general from. Preferably, the temperature gradients are within the cryopreservation vial volume and within the space, but to be within the Kryokonservierungsbehälters, outside the Kryokonservierungsfläschchens is defined similarly. This similarity the temperature may be further increased so that the thermal conductivity the low-temperature-stable material on the thermal conductivity of the frozen cryopreservation is adjusted and the frozen medium substantially. The essential Customize the thermal conductivities allows it that the freezing fronts inside and outside the Kryokonservierungsfläschchens even without significant deformations near the walls of the Kryokonservierungsfläschchens move together. In an alternative preferred embodiment, the thermal conductivity be of the low-temperature-stable material is lower than the thermal conductivities the frozen cryopreservation and frozen Medium.

The Containers described herein found in Kryokonservierungsvorgängen diverse uses, in particular in simulating much larger processes small scale. One of the areas where the vial is inserted is the freezing of biological substances in freezing containers / -behältern that many large have internal heat transfer surfaces. In such refrigeration systems must, dependent the particular use, the relationships between the speeds the freezing fronts, the interdendritic spacing, the temperature gradient temperature change the active (and passive - by Heat conduction) cooled Surfaces, the &lt;?page 10?&gt;Distance between the cooled Heat transfer surfaces and the product composition (transition points, Temperature values ​​of eutectic and glass states, solid mass content and soluble concentration, etc. are retained).

A interesting control variable in carrying out the freezing process in these chambers and in the vial For example, the removal of heat from the Kryokonservierungsbehälter (ie the heat flux from the Kryokonservierungsbehälter). In a preferred embodiment, this heat using cooling surfaces controlled manner and / or be deprived variable. varying the heat extraction can the spacing between dendrites within the Kryokonservierungsbehälters (the the cryopreservation vial according to the invention of one or more may include volume occupied) vary, the at the leading edge a fixed front or be formed within a solid front. Varying the dendritic spacing can for reasons that are discussed at another point of the present application, useful be. In a preferred embodiment is made of heat the Kryokonservierungsbehälter withdrawn at a speed which varies to the interdendritic to vary spacing at an edge of, or within a solid front, wherein the solid front located within the Kryokonservierungsbehälters is.

at a preferred embodiment, of the invention may, as in the <figref idrefs="S44">9</figref> shown is a control system of Kryokonservierungssystems <figref>900</figref> biopharmaceutical Products work so that an increase in intensity <figref>912</figref> the solid Fronts of frozen media <figref>910</figref> and / or the cryopreservation <figref>906</figref> With increased Heat flow through the cooling surfaces <figref>904</figref> (What again elevated to a Temperature driving force leads - the temperature difference between refrigerants, which are used in the Kryokonservierungsbehälter steeper temperature gradient to chill out) from the Kryokonservierungsbehälter coupled. These changes the heat flux from the Kryokonservierungsbehälter out can to are used, a substantially constant temperature driving force across the maintain solid front. In a preferred embodiment is heat withdrawn at a rate which is substantially a temperature driving force maintains within the Kryokonservierungsbehälters to a substantially constant rate of freezing solid fronts <figref>906</figref> and or <figref>910</figref> within the Kryokonservierungsbehälters to promote. This can be a substantially constant speed of the freezing fronts promote, just as substantially uniform conditions for undisturbed dendritic Ice crystal growth, independently by removal of the cooled Heat transfer surface within the freezing volume. The arrow<figref>908</figref> indicates the direction of Progress of solid fronts. The flow rate of the cooling liquid<figref>914</figref> can elevated and / or the temperature can be lowered to the heat flow through the cooling surfaces <figref>904</figref> to increase.

For example follows when the distance between the cooling surfaces of 10 cm and be retained Temperature gradient 10 ° C / cm is, the temperature drop of the pattern from 0 ° C to -50 ° C during the freezing, following the movement the freezing fronts (eg, when the fronts of about 3 cm from the cooling surfaces of the Kryokonservierungsbehälters are removed, is the temperature of these surfaces about -30 ° C, and if the fronts meet is, the temperature of the cooling surfaces about -50 ° C.

The Kryokonservierungssysteme invention biopharmaceutical Products are within the temperature and solidification rate areas operated, the uniform ice crystal growth and a uniform concentration solidifying resolved promoting substances between ice crystals in the Kryokonservierungsflächchen. This can be achieved by controlling the dendritic ice crystal growth will. The controlled dendritic ice crystal growth depends on the Temperature gradient from the direction of heat flow and the limited Hypothermia at the dendritic tip off. The uniform growth of dendritic ice crystals the mass of the volume of the medium, the biopharmaceutical product contains, depends on maintaining the temperature gradient at the speed of heat extraction from the cryopreservation (Or the cryopreservation system for pharmaceutical products, whichever is applicable), depending of the growing strength the solidified material from.

On such controlled growth provides a similarity of conditions between dendritic ice crystals sure where solutes (including the biopharmaceutical product that contained in the Kryokonservierungsflächchen is) are dehydrated and solidify in the form of glass. The similarity of solidification conditions includes an interdendritic "mushy" zone where the dissolved Substances are concentrated; the temperature in the interdendritic Zone decreases until the conditions of the glass state reached are. Such a glassy state is determined by the glass transition temperature the medium and the water level determined in the glassy state. At a preferred embodiment, the dwell time of the dissolved Material (including a biopharmaceutical product), when the dendritic front velocity on egg&lt;?page 11?&gt;ner is maintained substantially constant speed over the entire volume of the Kryokonservierungsfläschchens maintained while the dendritic Front (and the associated inter-dendritic zones) over this Volume moved. The significance of the similarity of the standing time of solutes in the interdendritic zone prior to the solidification into a glassy state is that the dissolved Material (including the biopharmaceutical product) is preferably similar conditions during the transition from dilute liquid starting product are exposed to the glass state - for example, the local history the biopharmaceutical product similar (time, temperature, concentration, etc.), regardless from its position in the cryopreservation vial volume. Similar or substantially similar Conditions of dendritic ice crystal growth can in the Kryokonservierungsbehälter as in a cryopreservation system on a large scale (ie at process level) be maintained.

The Speeds of invention solid front can from about 1 mm / h to about 800 mm / h, preferably of about 6 mm / h to about 140 mm / h, more preferably of about 12 mm / h to 70 mm / h, move. Preferably, the temperature gradient in the solid moves Front from about 1 to about 120 ° C / cm, more preferably from about 5 to about 25 ° C / cm. The Operating temperatures for Kryokonservierungssysteme the invention and - procedures biopharmaceutical Products range from about -1 to about -200 ° C, preferably between about -20 to about -200 ° C.

On Advantage of Kryokonservierungsbehälter invention is, that it included in the research and development work minimize product volume. The use of the "Reduction" of the present invention -Kryokonservierungssysteme allows rapid testing the variety of biopharmaceutical products under changing conditions. In a preferred embodiment reflects the Kryokonservierungsbehälter the case of containers for freezing and cryopreservation large scale existing geometries again.

how in the <figref idrefs="S44">10</figref> is shown which can Kryokonservierungsbehälter <figref>1000</figref> the Form of a simple elongated rectangular chamber with cooling surfaces <figref>1008</figref> exhibit, which serve the cryopreservation <figref>1006</figref> and the medium <figref>1004</figref> (Which in the cryopreservation <figref>1002</figref> contain is) to cool and freezing. In other embodiments, the other can Kryokonservierungsbehälter have shapes such as a square or a round / cylindrical Shape, or a shape as described in US Pat. No. 5,964,100 and in U.S. Patent Application No. 08 / 895.777. 08 / 895.782; 08 / 895.936 and 09 / 003,283 discloses. Such configurations can be selected to similarity of the freezing geometry to the chamber used in the process in large scale is and being replicated maintain a similar heat flow as in the Chamber of the method in large scale present similar Endbetriebstemperaturen and similar Temperature control schemes / arrangements can (these include the controller the temperature driving force such that it can be higher, while the solid front by the Kryokonservierungsbehälter of the actively cooled surfaces in the Kryokonservierungsbehälter moved away). The configurations of the placement of the vial in the container great scale can the temperature gradient along the frozen product and the seasonal temperature drop at the short end / the short ends the vial to ensure.

Of the Kryokonservierungsbehälter preferably forms the freezing and thawing of biopharmaceutical Products by, such as occurs in the process of large volume Generally can the Kryokonservierungsbehälter active (by the coolant have cooled) heat transfer surfaces, around the outer heat flow generate (withdrawal of latent heat of solidification). Cooling the Kryokonservierungsbehälters is preferably by one or more cooling surfaces reaches In a preferred embodiment have the one or more cooling surfaces a or more of the internal surfaces the Kryokonservierungsbehälters on. The distances between the cooling surfaces of the Kryokonservierungsbehälters hanging from the applied field from the temperature gradient. preferably vary the distances between two or more cooling surfaces between about 0.1 mm and about 1.500 mm, more preferably between about 1 mm and about 700 mm, most preferably between about 8 mm and about 500 mm.

The cryopreservation is typically comprised of a number of binders, stabilizers product and protective compounds composed. In a preferred embodiment, the cryopreservation substantially equal to the composition of the cryopreservation vial in Medium contained without the biopharmaceutical product. such promoting composition the similarity of the dendritic ice crystal growth within the volume of Kryokonservierungsfläschchens. This is preferable because similarity the dendritic ice crystal formation inside and outside the Kryokonservierungsfläschchens leads to improved reproduction results that the method for large-scale, using the inventive Kryokonservierungssystems biopharmaceutical &lt;?page 12?&gt;Products have been developed that are applicable. Kryokonservierungsflüssigkeiten used in the invention include, for example, biological cell cooling retardants (both penetrating, for example, glycerol, dimethyl sulfoxide, ethylene glycol, etc., and not penetrating, eg hydroxyethyl starch, dextran, Polyvinylpyrrolidone, etc.), vitrifying agents or components biopharmaceutical drug compositions (for example, surface treatment agents, PEG, Carbohydrates, polyols, amino acids or even proteins differently than the biopharmaceutical Product intended to be cryopreserved). The cryopreservation may have the same liquids contain (in compositions) that are found in the medium, which contains the biopharmaceutical product, but without the biological Component (cells, cell fragments, biopharmaceutical active Component), or it may only be water (eg distilled, deionized and / or high purity water). Between these two Borders, a plurality of compositions are used, eg water and salts (buffers) (like NaCl and water, ammonium sulfate and water, etc.), water and carbohydrates (like sucrose in water, or trehalose in water), water and salts and carbohydrates (such as Water, NaCl and sucrose), water and PEG, water and detergent / surface treatment agent, and / or water and buffer and carbohydrate and surface treatment agent. The substances used can provide temperature transitions, similar to the temperature transitions, in the biological product composition appear (eg can similarity the glass transition temperatures be maintained).

Inventive biopharmaceutical products include all conventional biopharmaceutical or pharmaceutical substances. In preferred embodiments can biopharmaceutical products include: biological macromolecules such as Proteins / enzymes, peptides, DNA, RNA, amino acids, nucleic acids, growth factors, Coagulation factors, antibodies, etc .; biological cells or cell fragments / Acquainted, including bacteria, Fungi, yeast, Einzellenorganismen, mammalian (especially human) Cells, animal cells, plant cells, organelles, cell membranes, inclusion bodies, or parts of tissue, and the like; Viral substances; organic or inorganic molecules or ions including stabilizing Salts or carbohydrates, antibiotics; or cell growth agents. Specific examples include blood and blood products (red and white blood cells, Plasma, human serum albumin, etc.) and two or more phase emulsions, biological or pharmaceutical substances.

The cryopreservation is preferably placed in the external freezing system that the heat flux of the actively cooled surfaces of this System approximately parallel to the longitudinal axis the vial is. This configuration reduces the supercooling of the bulk of the product in the vial and promotes similar in the vial Freezing conditions, such as during freezing in a cryopreservation large scale occur. The freezing fronts move inside and outside the Kryokonservierungsfläschchens with similar Front-speed and the like dendritic ice crystal. These conditions promote similar Conditions of product freezing (eg similarity of the standing time between Dendrites prior to solidification, similarity of concentration of dissolved Substances and the temperature distribution among dendrites (in the "mushy" zone), etc.) within and outside of the vial. The frozen final product preferably has a similar Distribution of dissolved Substances over distance and temperature gradients in both the cryopreservation vial as well as in the Kryokonservierungsbehälter / bins large scale.

The relationship of vial volume to Kryokonservierungsbehälter volume can be small, for example, to freezing in the cryopreservation almost as freezing with "infinite look at volume ". The depth at which the biopharmaceutical product in the vial and in the cryopreservation is, however, preferably remains substantially similar, to thermal conductivity effects in the vial walls to reduce (vertical heat flux effects in the bottle walls could occur if the outer and internal liquid level substantially vary).

at Alternatively, can the cryopreservation the length the Kryokonservierungsbehälters adjusted (eg, the length the vial about be equal to the distance between the cooling surfaces of the container), or they may be shorter be. Shorter vials can be placed in a position in which its center with the placement coincides in the center between the actively cooled ends of the container in a large scale. Then, the aptly freezing fronts outside the vial and within the vial both in the middle of the vial as well as in the middle of the container. The vial can also be placed so that its one short end with the Center of the container large Scale matches, and the other end is near one of the actively cooled surfaces of container large scale is. In this configuration the vial may preferably approximately half of the distance between the actively cooled surfaces of container large scale cover. Of the&lt;?page 13?&gt;last freezing can still be in the middle of Kryokonservierungsbehälters, is in the vial However it at one of the ends - a Such a configuration is due to the symmetry of the freezing process, possible (When two freezing fronts move towards each other).

The cryopreservation preferably along the heat flow path positioned, for example also substantially parallel to the directional pattern dendritic ice crystals (and substantially perpendicularly to the advancing solidification front). The position of Kryokonservierungsfläschchens parallel to the dendritic crystals promotes similarity of crystal growth inside and outside the Kryokonservierungsfläschchens. The controlled dendritic ice crystal growth, the optimum for a Cryopreservation of biological material is useful, not only can outside the vial in the cryopreservation take place (freezing front conditions such as parallel heat flux, Temperature gradient and front velocity may be maintained here, to promote the controlled dendritic crystal growth). Freezing in the interior of the vial preferably follows closely the freezing pattern in the exterior, for example, there is a Wär meflussrichtung, a similar Temperature and the like Front-speed, and therefore, the parallel dendritic Crystal growth maintained. The cryopreservation may be positioned in the Kryokonservierungsbehälter such that it is of an active cooling surface to a other covers what a simulating freezing over the entire freezer volume allows - then make the freezing fronts inside and outside near the center of Kryokonservierungsfläschchens. The similarity the freezing conditions over allows the entire freezer volume it also shorter cryopreservation to use, which cover only a part of the external freezing path in the Kryokonservierungsbehälter - the product is frozen as the part of the product with the length of the Kryokonservierungsfläschchens in the Kryokonservierungsbehälter would freeze.

at a preferred embodiment, can several cryopreservation are in the Kryokonservierungsbehälter. A preferred configuration is when the longitudinal axes of vial are substantially perpendicular to the solid-liquid boundaries of the external freezing front. In certain embodiments can the vials for rectangular container parallel can be arranged, or fan-shaped when the Kryokonservierungsbehälter is round or the shape of a wedge / section of a circle, is or triangular, for example, with a corresponding angle with the formation walls adjacent vial. Such configurations promote a nearly parallel growth of ice crystal dendrites inside and outside the vial. The distances between the vial be preferably about 0.1 times to about 200 times the vial width, more preferably the about 1 times to about 50 times the vial width.

at certain embodiments, where the Kryokonservierungsbehälter example a round or quadratic shape, the shape of the freeze-vial body have cross-shaped or star-shaped with multiple arms, coupled the nucleating structures to the distal ends of the body are. In such embodiments can the freezing fronts (circular or square) of all vial ends go to the center. After embedding the vial ends move the freezing fronts together inside and outside the body the Kryokonservierungsflächchen. The freezing fronts may , starting from all arms, hitting the center of the vial. This freezing pattern, the convergence of freezing fronts in the cylindrical or square Kroykonservierungscontainer simulate. Angle between the bodies can in these cases between about 5 to about 90 ° are, more preferably between about 30 and about 90 °. The number of bodies in a vial according to the invention may be between about one and are about twelve. In more preferred embodiments , the vial between about two and about eight bodies on. In the most preferred embodiments , the vial two or six bodies on.

it is for the skilled man evident that various modifications and variations in the Aufheizgeräten, systems and procedures the present invention can be made without departing from the spirit or scope the invention departing. Thus, it is intended that the present Invention amendments and variations of this invention encompassed when within itself the scope of the appended claims and their equivalents . are