A method and system for cryopreservation to achieve uniform viability and biological activity

Abstract

A method and system for controlled rate freezing and nucleation of biological materials is provided. The presently disclosed system and method provides the ability to rapidly cool the materials contained in vials or other containers within a cooling unit via forced convective cooling and optionally simultaneous pressure drop using uniform and unidirectional flow of cryogen in proximity to the plurality of vials disposed within a cooling unit. The rapid cooling of the biological materials is achieved by precisely controlling and adjusting the temperature of the cryogen being introduced to the system as well as the chamber pressure as a function of time.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present invention is a continuation-in-part application of U.S. patent application Ser. No. 12 / 266,760 filed Nov. 7, 2008 and also claims priority from U.S. provisional patent application Ser. No. 61 / 480,647 filed Apr. 29, 2011 the disclosure of both applications are also fully incorporated by reference herein.FIELD OF THE INVENTION[0002]The present invention broadly relates to a cryopreservation process, and more particularly, to a method and system for providing controlled rate freezing and nucleation control of biological materials to minimize cell damage resulting from intercellular ice formation and solute effects that arise during the cryopreservation process.BACKGROUND[0003]Cryopreservation is a process used to stabilize biological materials at very low temperatures. Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or mo...

AI Technical Summary

Benefits of technology

This patent is about a method for freezing biological materials in multiple containers to achieve uniform cooling and preservation of viability and biological activity. The method involves using a cooling area with parallel porous surfaces and delivering a unidirectional flow of cryogenic gas through one of the porous surfaces to cool the materials in the containers. The gas is quickly exhausted to prevent recirculation, resulting in a uniform temperature profile and nucleation of freezing for each container. This method can be used for large-scale commercial applications and provides biological materials with uniform viability and desirable biological activities.

Problems solved by technology

Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or more loss of cell activity and viability.

Cell damage during cryopreservation usually occurs as a result of intracellular ice formation within the living cell during the freezing step or during subsequent recrystallization.

Rapid cooling often leads to formation of more intracellular ice since water molecules are not fully migrated out of the cell during the short timeframe associated with the rapid cool-down rates.

If too much water remains inside the living cell, damage due to initial ice crystal formation during the rapid cooling phase and subsequent recrystallization during warming phases can occur and such damage is usually lethal.

On the other hand, slow cooling profiles during cryopreservation often results in an increase in the solute effects where excess water is migrated out of the cells.

Excess water migrating out of the cells adversely affects the cells due to an increase in osmotic imbalance.

Thus, cell damage occurs as a result of osmotic imbalances which can be detrimental to cell survival and ultimately lead to cell damage and a loss of cell viability.

Such equipment, however, is only suitable for relatively small volume capacities and is not suitable for commercial scale production and preservation of biological materials such as therapeutic cell lines.

Such existing controlled rate freezers, including the Kryo 1060 series, also suffer from the non-uniformity in cooling vial to vial due, in part, to the non-uniform flow of cryogen within the freezers and the requirement for close packing of the vials within the freezer.

The size of individual conventional freezers is limited due to these non-uniform effects.

As conventional controlled rate freezers are scaled up in size, the non-uniformities in cooling increase.

Consequently, the size of conventional controlled rate freezers must be limited to prevent non-uniform sample-to-sample properties due to non-uniform cooling of each sample.

Such convection based cooling or freezing systems cannot achieve temperature uniformity as the vials are often located at various distances from the internal fan or packed in the shadow of other vials or trays.

However, it is impossible to provide a uniform conductive surface area on the bottom of each glass vial since most glass vial bottoms are concave.

Therefore, temperature variations during the freezing process from vial to vial are the biggest drawback for these types of equipment.

Furthermore, the cooling rate can be painfully slow due to the very small conductive surface of the vial that remains in contact with the cold shelves.

However, many cryoprotectants such as DSMO are toxic to human cells and are otherwise not suitable for use in whole cell therapies.

Disadvantageously, cryoprotectants also add a degree of complexity and associated cost to the cell production and preservation process.

Also, cryoprotectants alone, have not eradicated the problem of loss of cell activity and viability.

Another problem associated with the above mentioned systems is a lack of control with respect to the uniformity of the nucleation temperature between the multiple vials.

This variability in the nucleation temperature of the multiple vials can lead to non-uniform vial-to-vial properties.

Furthermore, the drying stage of the freeze-drying process must be excessively long to accommodate the range of ice crystal sizes and structures produced by the natural stochastic nucleation phenomenon.

These additives are not typically acceptable or desirable for FDA regulated and approved freeze-dried pharmaceutical products.

These additives also do not provide control over the time and temperature during which the vials nucleate and freeze.

The “ice fog” method does not control the nucleation of multiple vials simultaneously at a controlled time and temperature.

When the freeze-dryer shelves are continually cooled, the time difference between when the first vial freezes and the last vial freezes creates a difference in the temperature between vials, which will also increase the vial-to-vial non-uniformity in the final freeze-dried products.

As with the other prior art methods, vial pre-treatment also does not impart any degree of control over the time and temperature when the individual vials nucleate and freeze, but instead only increases the average nucleation temperature of all vials.

In the transient or inertial cavitation regime, the gas bubbles rapidly grow and collapse, causing very high localized pressure and temperature fluctuations.

Drawbacks associated with an electrofreezing process in typical lyophilization applications include the relative complexity and cost to implement and maintain, particularly for lyophilization applications using multiple vials or containers.

Also, electrofreezing cannot be directly applied to solutions containing ionic species (e.g., NaCl).

A major drawback for implementing this ‘vacuum induced surface freezing’ process in a typical lyophilization application is the high risk of violently boiling or out-gassing the solution under stated conditions.

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